Advances in Genetics Volume 17

Contributors to This Volume Rolf Blakh Karl Esser G. 1. Fowler Philip E. Hartman George 1. Gabor Miklos N. M. Nayar W. J. Peacock John R. Roth Henry M. Sobell

ADVANCES IN GENETICS VOLUME 17 Edifed by E. W. CASPARI Department of Biology University of Rochester Rochester, New York

1973 ACADEMIC PRESS

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CONTRIBUTORS TO VOLUME 17 Nunibers in parentheses indicate the pages on which the authors’ contributions begin.

ROLFBLAICH(107) , Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitlit Bochum, Bochurn, Germany KARLESSER(107) , Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat Bochum, Bochum, Germany G. L. FOWLER*(293), Department of Biology, University of Oregon, Eugene, Oregon PHILIPE. HARTMAN ( l ) , Department of Biology, The Johns Hopkins University, Baltimore, Maryland GEORGE L. GABOR MIICLOS (361) Research School of Biological Sciences, Australian National University, Canberra, A.C.T., Australia N. M. NAYART(153), lnstitut fur Pjlanzenzuchtung, Universitat Gottingen, Gottingen, West Germany W. J . PEACOCK (361), Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra, A.C.T., Australia )

JOHN R. ROTH ( 1 ) ) Department of Molecular Biology, University of California, Berkeley, California HENRYM. %BELL (411), Department of Chemistry, The University of Rochester, Rochester, N e w York; Department of Radiation Biology and Biophysics, The University of Rochester School of Medicine and Dentistry, Rochester, New York

* Present address: Institut fur Allgemeine Biologie, Universitat Dusseldorf, Dusseldorf, Germany. t Present address: Central Plantation Crops Research Institute, Regional Station, Vitta1-574243, Mysore State, India. vii

MECHANISMS OF SUPPRESSION Philip E. Hartman and John R. Roth Deportment of Biology, The Johns Hopkins University, Baltimore, Morylond, a n d Deportment of Molecular Biology, University of California, Berkeley, California

I. Introduction. . . . . . . . . . . , . A. Suppressors in Genetics . . . . . . . . B. Nomenclature . . . . . . . . . . . 11. Intragenic Suppression (“Internal” Suppression) . . A. Different Letter of a Codon . . . . . . . B. Active Conformation . . . . . . . . . C. New Initiators and Elimination of Polarity. . . D. Elimination of a Toxic Polypeptide . . . . . E. Double Frameshifts. . . . . . . . . . 111. Intergenic Suppression (“External” Suppression) . . A. Informational Suppression (“Direct” Suppression). B. Reconstruction of Active Enzyme Conformation . C. Substitute Protein Activity . . . . . . . D. Elimination of a Deleterious Accumulation . . . E. Effective Dosage of a Limiting Gene Product . . IV. Other Interesting Cases . . . . . . . . . References . . . . . . . . . . . . .

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2

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5

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1 4

. 6 . 14

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17

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37 58 60 66 69

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. 18 . 19 . 20 . 28

I. Introduction

A. SUPPRESSORS IN GENETICS Suppressor mutations are one class of secondary mutations (“modifiers”) that modify the phenotype in the presence of the originally mutant gene. In contrast to “enhancers” that make the mutant phenotype more extreme, suppressor mutations yield organisms phenotypically more like the wild type: the mutant phenotype is “suppressed.” I n some cases the gross wild-type phenotype is completely restored; in other cases, restoration is only partial. Thus, suppressors are mutations that elicit a revertant or partially revertant phenotype. But suppressor mutations can be genetically separated, by recombination, from the mutation (s) that they suppress. 1

2

PHILIP E. HARTMAN AND JOHN R. ROTH

The first cases of genetic suppression were interpreted as gene duplications (Bridges, 1919; Morgan et al., 1925) and, indeed, some were duplications (Morgan et al., 1925; Schultz and Bridges, 1932). Later experiments showed that suppression also could result from interaction between nonallelic genes (Bonnier, 1927; Plough, 1928; Bridges, 1932; Schultz and Bridges, 1932). Biochemical analyses, coupled with microbial genetic methods, in the 1950s began to shed light into the mechanisms underlying particular nonallelic suppressor activities. Brief critical reviews of suppressor action appeared (Wagner and Mitchell, 1955, 1964 ; Yanofsky and St. Lawrence, 1960; Campbell, 1963; Gorini and Beckwith, 1966). The present review is intended as a supplement to these condensed reviews and more recent extensive summaries restricted to aspects of informational suppression (Garen, 1968; Davies, 1969; Gorini, 1970). Our intent here is to describe systems that seem to us particularly informative and/or illustrative of types of suppressor activity. Our summary of these few selected examples also calls attention to the vast potential offered by the study of suppressor activities. Suppressor analysis can yield insight into arrays of problems not readily subject to more classic genetic experimentation. The examples described below indicate that suppressors can supply basic information on unsuspected interactions as well as providing probes so that direct selection for reversion through suppression may allow ready isolation of mutations of primary interest. For example, much of the critical experimentation in the E . coli lactose system depends in one fashion or another on mutants recognized by their abilities in suppression under defined sets of conditions (cf. Reznikoff, 1972). We believe that the analysis of suppressor mutations will find expanding and increasingly important use as the techniques of genetics and molecular biology are applied to more complicated biological situations. Some ways in which suppressors act are summarized in Fig. 1. The figure and our outline show that we attempt to describe suppressor actions in terms of biochemical mechanisms. Contributions to genetics and the resolving power of suppressor studies also will be touched upon.

B. NOMENCLATURE In Drosophila the classical symbols for suppressors are su or Su for recessive or for dominant suppressors, respectively, followed by more specific designation (e.g., Su-S for a dominant suppressor of S, star) (Lindsley and Grell, 1968). In bacteria the standardized nomenclature of Demerec et al. (1966) has been supplemented to include compatible symbols such as sup for suppressor (Sanderson, 1970; Taylor, 1970),

3

SUPPRESSORS

x-Y-z

W

FIG.1. General modes of suppression of enzyme defects. Metabolites are designated by capital letters, and genes and enzymes by numerals. A mutation in gene 2 the structural gene for enzyme 2, may be suppressed by a second mutation which: 1. Allows production of some wild-type or effective enzyme 2 from mutant gene 2 messenger RNA via an alteration in the protein-synthesizing system (“Informational” suppression). 2. Occurs in the same gene (intragenic suppression) but rectifies the effect of the first mutation by: (a) a change in a second letter in the same triplet codon affected by the first mutation, allowing insertion of an amino acid more compatible with functioning of enzyme 2 ; (b) a second, genetically separate mutation in gene 2 that leads to a “doubly mutant” enzyme 2: (1) enzyme 2 has regained functional activity, (2) reinitiation of messenger RNA or polypeptide synthesis relieves polar effects of nonsense codons or defective promotors, or (3) a toxic polypeptide is eliminated. 3. Increases the amount of partially defective enzyme 2 through increased gene dosage or through altered regulation of enzyme 2 production. 4. Releases an inhibition of mutationally altered enzyme 2 by ions, metabolites, or macromolecules (the wild-type enzyme 2 may be inhibited to some extent by the same agents or the mutationally altered enzyme 2 may be uniquely sensitive to these factors). 5. Increases substrate B by affecting the amount or the regulation of enzyme 1 activity. 6. Allows catalysis by an alternate protein that mimics enzyme 2 in its function (“duplicate gene”). To be effective in suppression the duplicate gene may be placed under new regulation or it may assume its new role through mutational alteration affecting substrate specificity. 7. Supplies metabolite C from a second pathway which may be parallel (X = B, Y = C) or which may be unique (X and Y distinct from B and C ) by mutational alterations affecting accumulation of Y (defective enzyme 4) or conversion of Y to C (altered control or constitution of enzyme 5 ) . 8. Relieves inhibition of other reactions by accumulated compound B through : (a) limit in synthesis of B via altered regulation or decreased efficiency of enzyme 1; (b) Lowered sensitivity of the inhibitory site, for example, enzyme 3, through an increase in the amount of enzyme 3, a decrease in its sensitivity to inhibition, or more ready availability of substrate D. Numerous additions, rearrangements, and modifications of the above examples can be envisioned; they merely serve to point out some of the highlights of suppressor action.

4

PHILIP E. HARTMAN AND JOHN R. ROTH

suf for suppressor of frameshift mutations (Riddle and Roth, 1970), sbc for suppressors of red3 and re& mutations (Barbour and Clark, 1970; Barbour et al., 1970). I n some cases the special phenotypic attributes imparted by the suppressor mutation (e.g., crr for catabolite repression resistant in bacteria) or knowledge of its biochemical mode of action (e.g., pyr-3 for suppressors of arg mutants in Neurospora) have suggested special symbols for designation of loci that sometimes have other alleles which behave differently. I n all cases the symbol stands for the wild type which, most often, does not contain the suppressor activity in question. This usage has been distorted in the case of some papers on suppressors in bacterial systems where conveys the idea that the suppressor strain has the particular suppressor and suis used for the strain lacking suppressor activity. While this unique usage is something to be wary of when reading the literature, our usage here will adhere to SU+ or an analogous symbol for wild type, in keeping with worldwide usage for a variety of genetic markers in a variety of organisms over a span of many years.

+

+

II. lntragenic Suppression (“Internal” Suppression 1

We consider a gene as a polynucleotide stretch from which a functional segment of an RNA molecule is transcribed. Genes sometimes serve t o dictate the base sequence, and thus the structure, of RNA molecules directly functional in the cell, for example, transfer RNA (tRNA) and ribosomal RNA (rRNA) . Genes also serve as templates “transcribed” into messenger RNA (mRNA), which then guides synthesis of polypeptide chains. This “translation” into polypeptide product is achieved through initiation of the N-terminal amino acid a t the proper site on the mR,NA, reading of the subsequent base sequence in strict sets of triplets, each coding for an amino acid, and termination a t a nontranslatable “nonsense” triplet a t the C-terminal end of the polypeptide. Figure 2 shows the coding triplets (codons on mRNA) for the amino acids and the chain-terminating nonsense triplets. Mutations occur through changes in the sequence of base pairs in the DNA. These may be ( a ) addition or deletion of one or more base pairs that disturb triplet reading a t the site of mutation and, if not additions or deletions of sets of 3 base pairs, also affect subsequent reading ( “frameshift” mutations), and (b) base substitutions leading to replacement in the polypeptide of one amino acid for another (“missense” mutations) or to premature chain termination (“nonsense” mutations: amber or UAG, ochre or UAA, and UGA) .

5

SUPPRESSORS

Second letter

First letter

U

C

A

G

U

Phe Phe Leu Leu

Ser Ser Ser Ser

TYr TYr Ochre nonsense Amber nonsense

C

Leu Leu Leu Leu

Pro Pro Pro Pro

A

Ile Ile Ile Met

G

Val Val Val Val

Third letter

CYS CYS Nonsense Trp

U C A G

His His Gln Gln

Arg Arg -4% A%

U

C A G

Thr Thr Thr Thr

Asn Asn LYS LYS

Ser Ser -4% Arg

U C A G

Ala Ala Ala Ala

ASP ASP Glu Glu

GlY GlY GlY GlY

U C A G

FIG.2. The genetic code. The abbreviated names of amino acids are given in the body of the table. For example, Met = methionine. Coding triplets (codons) on messenger RNA are read 5’ to 3’ using the first letter (left-hand ColUmh) and then the second letter (top heading) and thence the third letter (right-hand column). For example, the only codon for methionine is AUG. The three triplets that predominantly lead to polypeptide chain termination also are shown : these are UAA (“ochre”), UAG (“amber”),and UGA.

Below are examples of suppressors which have their effect as a consequence of a secondary change within the original mutant gene. A. DIFFERENT LETTEROF

A

CODON

a. Yanofsky and co-workers found two missense mutations that recombined at very low frequency to yield wild type and contained different amino acid substitutions a t residue 210 of Escherichia coli tryptophan synthetase A protein (cf. Yanofsky e t al., 1967). These substitutions were Gly (GGA) to Arg (AGA) in mutant A23 (Helinski and Yanofsky, 1962) and the same Gly (GGA) to Glu (GAA) in mutant A46 (Henning and Yanofsky, 1962a). Various full and partial (weak enzyme activity) revertants were next described containing at position 210: Val (GUA) ,

6

PHILIP E. HARTMAN AND JOHN R. ROTH

Ala (GCA), and Gly (GGA) from mutant A46 and Gly (GGA), Ser (AGE) from mutant A23 (Henning and Yanofsky, 1962b; Allen and Yanofsky, 1963; Yanofsky, 1963; Carlton and Yanofsky, 1963). Later, additional substitutions at the same residue yielding ten different amino acids as well as studies of double frameshift mutants served to verify the in vivo codon assignments listed above (Yanofsky, 1965; Yanofsky et al., 1966, 1969; Berger and Yanofsky, 1967; Berger et al., 1968a). The substitutions are summarized on page 144 of Hartman and Suskind (1969). The collective data conclusively show that any one of a variety of amino acids at residue 210 is compatible with enzyme catalysis while a few amino acids lead to inactive protein. Some revertants that are phenotypically wild type as judged by growth properties, accumulations, and even by some general properties of the enzyme in extract are in fact pseudo-revertants. They contain an amino acid coding triplet that is mutationally altered in two different base pairs, recombinationally separable a t low frequency. Analogous observations have been made in yeast (Sherman et al., 1970; Sherman and Stewart, 1971). b. Studies similar to those described above have been performed in analyses of revertants of chain-terminatingJ nonsense mutations (Fig. 3 ) . A particular nonsense triplet may arise by mutation of a number of different coding sequences in the DNA. Similarly, the nonsense coding sequence may revert either back to the original form (true reversion) or to any one of a number of other sequences that lead to the insertion of an amino acid compatible with functioning of the protein involved. These latter mutations often involve an alteration in a nucleotide pair not involved in the original mutational event. Figure 3 presents a compilation of the mutational changes found in several bacterial systems (Weigert et al., 1966, 1967; Sarabhai and Brenner, 1967; Brenner et al., 1967) and in yeast (Sherman et al., 1970). These studies contributed to deduction of the in vivo genetic code and, today, lend caution in analyses of the action of chemical mutagens in cases where protein primary structure is not examined.

B. ACTIVECONFORMATION Here we summarize cases where the deleterious effects of a mutation on the macromolecular gene product are partially or completely rectified by a balancing change at a second place in the same molecule. This applies both in cases involving polypeptide gene products and those involving RNA end products such as transfer RNA. a. An amino acid substitution at one position in a polypeptide chain

7

SUPPRESSORS

sometimes can be compensated for by a second substitution at some distance away in the same polypeptide (“second-site reversion”). Thus, a Gly to Glu substitution a t position 210 of the tryptophan synthetase A protein resulted in inactive enzyme but activity was present if a second substitution, Try to Cys, occurred a t position 174 (Fig. 4). Both T ~ P UEG

Ser

I Gln

c A-A Glu GAA

((%)

Ser

-UuA

or

Leu

UAU UAG

FIG. 3. Messenger RNA sequences due to base-substitution mutations leading to and from amber and ochre nonsense codons in Eschem’chia coli. Nonsense mutations (circled) were elicited. Where the amino acid originally present in the wild-type protein is known or can be strongly inferred, an arrow points from that amino acid with its appropriate codon to the nonsense codon. Revertants of the nonsense mutants have been obtained, and the particular amino acid replacement in each revertant has been determined or inferrred from genetic experiments. Reversions are indicated by arrows pointing from the nonsense codons to the respective amino acids. The compilation shows only those changes actually observed ; in each case, changes in both directions are theoretically possible.

mutations were required for activity; the Tyr to Cys substitution alone led to inactive enzyme (Helinski and Yanofsky, 1963). The two mutations suppressed each other. A similar situation was found for a mutant, A187, which contained Val both at residue 120 and a t residue 212 and lacked enzyme activity. Mutant A187 could revert by mutations leading to substitution of Ala a t position 210, Ala a t position 212, or by a

8

PHILIP E. HARTMAN AND JOHN R. ROTH

Leu to Arg substitution a t position 176 (Yanofsky et al., 1964; Carlton and Yanofsky, 1965). The arrangement of these compensating changes led Yanofsky et al. (1964) to suggest that residues 174-176 interact with residues 210-212 in the folded protein (Fig. 4 ) . While the necessity for such direct interaction of different parts of the polypeptide chain is conjectural, there is no doubt that suppression of one genetic defect in a gene can be achieved by compensating amino acid substitution elsewhere in the same structural gene. Brockman (1968) describes second-site reversion of an adenine-SB mutant in Neurospora that appears analogous to the cases just mentioned. I

H,N-Me1

I I

173 174 175

176

-----:----thr-Tyr-Leu-Leu-

177 Ser

I

0

I

:----Gln- Gfy209 210

j - - - - - J

Phe- G l y - Ser 211 212 213

II -------___Ser-C-OH

267

FIQ.4. Possible relationship between different regions of the folded wild-type tryptophan synthetase A protein molecule suggested by second-site reversion analysis. The amino acids changed in primary mutational events are shown in italics and those changes by second-site reversion are shown in bold letters. An amino acid replacement a t residue 210 is compensated for by a second replacement in residue 174, and vice versa. Similarly amino acid substitutions at residues 176 and 212 compensate for each other. The spatial arrangement of compensating changes in the polypeptide chain had led to the speculation that the two segments of the chain interact when the protein achieves its final tertiary structure.

Mills and Ellingboe (1969) describe eight hydroxylamine-induced reversions of an arginine mutant (arg-d) in Schizophyllum commune. All eight grow on minimal medium but are recessive when combined in heterokaryons with arg-6. That is, arg-6 possecses a dominant effect in complementation, as sometimes occurs in tests with mutant missense proteins when the enzyme is a multimer (e.g., Fincham, 1966; Foley et al., 1965; Nashed et al., 1967; Zimmermann et al., 1969). Seven of the “suppressors” could not be separated from arg-6,but another (su-1) was separable. In crosses of arg-6 su-1 to wild type, six progeny identical in properties to arg-6 were recovered along with a new phenotype in 13 others (= arg-6+ su-1 1 ) out of 87 progeny tested. These latter recombinants were arginine-requiring, like arg-6, but were effective in weak complementation with arg-6. Complementation is often found for widely spaced missense mutations in a gene which exhibits complementation. The easiest interpretation is that su-1 is a new missense mutation in the arg-6 gene. It also, however, could be defective in zt gene coding

SUPPRESSORS

9

for a distinct protein species that aggregates with the product of the arg-2 gene to form an active multimer. The other seven “suppressors” are either changes in the mutant codon of arg-2 or are closely allied second-site intragenic missense mutations. Heterokaryons between arg-2 su-1 and the other suppressed strains (e.g., “arg-2 su-2” = altered arg-2 codon or nearby base-pair change) grow on minimal medium, indicating that the dominant blocking of complementation by arg-2 has been alleviated by the intragenic “suppressor.” Mills and Ellingboe (1969) were perplexed by the high percentage of recombination between su-1 and arg-2. However, an entirely analogous situation was more adequately investigated by Morgan (1966) in Coprinus where a spectrum of recombination values was obtained in a series of 41 suppressor mutations. And, in part, the observation of “allelic suppression” was previously described in Aspergillus for paba-92 revertants by Luig (1962). So this seems something to be wary of in diploids and heterokaryons, namely, a second intragenic change that allows complementation under certain circumstances. Such situations mimic exactly the behavior expected of an extragenic suppressor. Material selected for in such studies, however, could supplement more conventional, randomly obtained mutants for studies of genetic map position and complementation behavior in relation to protein tertiary structure (cf. Fincham, 1966; Gillie, 1966, 1968). b. An additional method potentially useful for studies of possible interactions between different parts of polypeptide chains in vivo may be regarded operationally as analogous to the cases of intragenic suppression just described. The method relies first upon reversion a t a site in one protein that engenders a “semi-acceptable,” but not optimal, amino acid sequence. One then screens for new mutants that again have lost the function in question. One can picture three types of mutants arising from the strain now “sensitized” by its possession of an altered polypeptide chain; the proportion of each type will be dictated by the remaining nucleotide sequence, the position of the initial change with regard to the active center of the enzyme, and on interactions of polypeptide chains (different segments of the same chain as well as interactions between independent chains): (a) mutants with new alterations at or near the “semi-acceptable” site, (b) mutations in parts of the gene dictating amino acid sequence in the portion of the polypeptide that interacts with the defective site (intrachain associations), and (c) similar interactions as in (b) but involving two different polypeptides (in cases where the polypeptide is part of an oligomeric complex). I n an apparent analysis of this type in Salmonella, Riyasaty and Dawson (1967) found a partial revertant of a tryptophan-requiring mu-

10

PHILIP E. HARTMAN A N D JOHN R. ROTH

tant, trpA.47, that gave rise to an unusually high frequency of mutants that again were tryptophan requiring. These comprised: (1) mutations that either were “silent” in wild-type genetic background or mapped at or very close to the original site of mutation, (2) mutations in a t least five different nucleotide pairs more distantly located and presumed to lie within the trpA gene (but which actually could lie in the trpB gene and merely elicit a “trpA” phenotype), and ( 3 ) mutations in one (or more) nearby trp genes. Mutant trpA47 is a frameshift mutation with defective anthranilate synthetase (ASase) specified by the trpA gene (Bauerle and Margolin, 1966). ASase function is activated by association of the trpA gene product with the polypeptide product of the trpB gene that, in addition, catalyzes the next step of tryptophan biosynthesis (Bauerle and Margolin, 1966; Ito and Yanofsky, 1966, 1969; Zalkin and Kling, 1968; Smith and Bauerle, 1969; Ito et aZ., 1969; Tamir and Srinivasan, 1969; Henderson et al., 1970; Nagano and Zalkin, 1970; Nagano et aZ., 1970). Thorough analysis of this system might supplement other information (Hwang and Zalkin, 1971; Yanofsky et aZ., 1971; Grieshaber and Bauerle, 1972) not only in revealing associations of different parts of one polypeptide chain but also how this polypeptide interacts with another to form an active complex (cf. Stuttard and Dawson, 1969). Chemical cross-linkage (Myers and Hardman, 1971) would seem a nongenetic method of potential usefulness in confirmatory study of protein tertiary and quaternary structure. A second case illustrating the genetic approach is that studied by Koch and Drake (1970). They started with a leaky, “sensitizing” mutation in the bacteriophage T4 rIIA gene and isolated mutants with complete rIZ phenotype. Some of the newly isolated rIZ mutations were cryptic, exhibiting a pure rII phenotype only in the presence of the original sensitizing mutation. Cryptic mutations were found a t certain regions of the rZIA gene as well as in the rIIB gene, indicating that the rIIA and rIIB proteins interact in viva In other systems, previously unsuspected interactions could well be revealed or useful mutants isolated by these and accessory genetic methods (see page 35). c. Jinks (1961b) described interesting suppressors for mutants of the T4 bacteriophage h gene region. If the suppressors are internal suppressors, the mutations suppressed must be missense mutations or in-phase (triplet) deletions since the h gene product is involved with tail fibers, phage structures essential to phage maturation and adsorption. Of 129 revertants for the several host-range ( h ) mutants examined (Jinks, 1961a) all were found to be due to suppressors, and all but three mapped in the h region of the phage chromosome close to the original sites of mutation (Jinks, 1961b). This is the region where all but one of the

SUPPRESSORS

11

tail fiber protein and assembly genes are located (cf. Epstein et al., 1963; Wood et al., 1968). Some of the suppressors suppressed more than one h mutant, but none of the suppressors could be isolated from these original sites of mutation (i.e., they had a wild-type phenotype or were lethal when isolated). The suppressors primarily were selected on the basis of reversion of phage thermal sensitivity and secondarily for suppression of altered host-range phenotype. Therefore, they probably represent a unique class of revertants. Because they map close to the original sites of mutation, the suppressors could be second-site revertants of the type described above for tryptophan synthetase. On the other hand, their explanation may lie in the intricacies of phage tail fiber assembly (cf. King and Wood, 1969; Ward et al., 1970; Takata and Tsugita, 1970). At least some suppressors may involve changes in one tail fiber component that compensate, through protein-protein interactions, for alterations in a separate constituent (see page 66). d. Genes for tRNA molecules also can undergo second-site reversion. Studies of multiply mutant tRNAs are yielding information pertinent to the in vivo conformation of tRNA and its interaction with other macromolecules. A gene, su-3 (or S U I I I ) in E. coli, can mutate to a form active in suppression of chain-terminating nonsense mutations of the amber (UAG) type leading to the insertion of tyrosine a t the amber triplet (Weigert et al., 1965; Kaplan et al., 1965; Garen et al., 1965). This informational suppression is achieved when the gene product, a tyrosine transfer RNA (Andoh and Ozeki, 1967; Landy e t al., 1967), contains an altered anticodon capable of “recognizing” UAG sequences of messenger RNA (Goodman et al., 1968). Most recently, it has been demonstrated that intragenic secondary mutations that render the suppressor tRNA nonfunctional can be detected and the tRNA analyzed; further intragenic mutations that again result in active informational suppression also can be assessed. Such studies have provided evidence pertinent to in vivo conformation of this tRNA, its function and stability (Abelson et al., 1970; Smith et al., 1970), on the structure of the gene region involved in suppression (Russell et al., 1970), and on some steps in the maturation of tRNA following transcription (Altman, 1971). E. coli mutants with nonfunctional “suppressor” tRNA and revertants therefrom were isolated by setting up alternate conditions under which amber suppression is either lethal or is required for growth. Bacteria are poisoned by exogenous galactose if they possess functional galactokinase but no epimerase (see also page 58). Therefore, strains were constructed with an amber mutation in the galactokinase gene and a nonsuppressible mutation in the epimerase gene. Mutants whose suppres-

12

PHILIP E. HARTMAN AND JOHN R. ROTH

sor had been rendered nonfunctional lacked galactokinase and thus could not accumulate the toxic intermediate, galactose phosphate. I n the absence of galactose, selection was made for suppressor function by virtue of an amber mutation located elsewhere in the chromosome. Most mutations causing a loss of suppression cause a severe reduction in the amount of suppressor tRNA. This may be due to improper folding and premature degradation of the mutant tRNA (Abelson et al., 1970). Possibly many base substitutions have little or no effect on activity

PG CG UGGG-

Aon C C A C CW A C C C

-

A-U @, G C @‘/vG A-ILA 0’0 C 40 U A* x.C u A

-

FIQ.5. Cloverleaf model of suIII tRNATVr of Escherichiu coli. The nucleotide Bequence is from Goodman et d. (1968). Residue numbers are referred to in the text and in Table 1.

but can alter the structure so as to cause gross instability. Mutants with temperature-sensitive tRNA, functional a t 32OC but nonfunctional a t 42OC, contain simple base substitutions in the amino acid acceptor arm of the standard “cloverleaf” arrangement (Fig. 5 ) . One such temperature-sensitive mutation (A2) causes a G + A change a t residue 2. This base substitution prevents the formation of the G :C base pair which normally occurs between residues 2 and 80, and leads to a temperature-sensitive tRNA. Among the revertants of A2 are second-site revertants which have a C + U change a t residue 80. This change permits residues 2 and 80 to pair again, although the pair is now an A:U rather than the G:C in wild-type tRNA. With either pair in place, the tRNA

13

SUPPRESSORS

can function a t both 42O and 32OC. The fact that this second-site revertant permits 2 and 80 to pair again, suggests strongly that bases 2 and 80 are in contact and that a cloverleaf arrangement (cf. Cramer, 1971; Arnott, 1971) has at least partial validity in v i m . A second case of such restored pairing is provided by temperature-sensitive mutant A25, a G +-A change at residue 25. In the “cloverleaf” arrangement, base 25 is paired with base 11 in the arm of the dihydrouracil loop (Fig. 5). Among the revertants of A25 is a second-site TABLE 1 Summary of Second-Site Reversions Affecting SUIII Transfer RNAe Genotype of tRNAb

Base change@)

Function alteration

A2 U80 A2, U80

G -+ A C -+ U -+ A -+ U

(residue 2) (residue 80) (residue 2) (residue 80)

Temperature sensitive Temperature sensitive

A25 A25, U l l

G -+ A +A -+ U G -+ A -+ U

(residue 25) (residue 25) (residue 11) (residue 25) (residue 19)

Loss of suppressor activity Fully restored suppressor activity Partially restored suppressor activity

A25, U19 A15 A15, D19 A15, D20

I I

{(-+A (residue 15)

A (residue 15) D” (residue 19) G - + A (residue 15) -+ Do (residue 20) -+ -+

I

Normal activity

Loss of suppressor activity Partially restored suppressor activity Partially restored suppressor activity

Data from Abelson et al. (1970) and Smith et aE. (1970). All tRNAs carry the anticodon change G -+ C (residue 35) which permits reading of the amber codon. D = dihydrouracil; this is probably formed by modification of uracil. b

revertant having a C + U replacement at residue 11. This change would restore a base pair between residues 11 and 25. This restored pairing apparently permits a functional tRNA at 42°C and 3OoC even though a U :A pair replaces a C :G pair a t that point in the structure. Smith and co-workers (1970) have presented three other examples of second-site mutations restoring function to tRNA. These last three examples do not involve restoration of base pairing, but rather must cause other sorts of structural changes which compensate for the defect or the original mutant. These examples are presented in Table 1 (see Anderson and Smith (1972) for additional examples).

14

PHILIP E. HARTMAN AND JOHN R. ROTH

C. NEWINITIATORS AND ELIMINATION OF POLARITY a. Sherman and co-workers (Stewart e t al., 1969; Sherman et al., 1970) found second-site revertants of a yeast iso-1-cytochrome c mutant that lacked a normal polypeptide initiator codon (AUG = Met). The base substitutions in the second-site revertants created AUG codons effective in initiation of the cytochrome polypeptide chain a t either of

-

Wild type

(Met)- Thr Glu - Phe - Lys - Ala - Gly -N N N-A U G-A C N-GA R-U U Y-AA G-G C N-G G N-

Revertant No. 1

Met - Ile - Thr Glu - Phe - Lys - Ala - Gly -A U G-A U A-A C N-G A R-U U Y-A A G-G C N-C; G N-

Revertant No. 2

Met - Leu - Thr - Glu - Lys -Phe Ala - Gly -A U G-Y U G-A C N - G A R - U U Y-AA G-G C N-G G N-

Revertant No. 3

Met - Arg - Thr - Glu - Phe - Lys Ala Gly -A U G-A G G-A C N-G A R-U U Y-A A G-G C N-G G N-

Revertant No. 4

(Met)- Val - Thr - Glu - Phe - Lys - Ala - Gly -A U G-G UG-A C N - G A R - U U Y-A A G-G C N-G G N-

Revertant No. 5

(Met)- Ala Gly A U G - G C N-G G N-

-

-

-

-

-

-

-

FIG.6. N-terminal amino acid and presumed messenger RNA sequences of yeast iso-1-cytochrome c. The sequences shown are for wild type and for 5 different secondsite revertants of a mutant with a base-substitution in the normal AUG initiator ( A U G in wild-type line). Methionine residues initiating polypeptide chains in the wild type and in revertants 4 and 5 were not detected owing to presumed elimination post-synthesis and are placed in parentheses. N = any of the four ribonucleotides, R = either of the two purine ribonucleotides, and Y = either of the two pyrimidine ribonucleotides. The complete amino acid sequence of the protein from yeast (Narita and Titani, 1969) also is given in Sherman et al. (1970).

two new sites, allowing synthesis of functional protein (Fig. 6). The studies elegantly demonstrate the initiator function of the AUG triplet in a eukaryote and the presen’ce of an untranslated messenger sequence preceding the normal AUG initiation point. The studies also point out that in very special cases the corrective action of second-site suppressors operates on the translational level, not a t the level of protein tertiary structure. Finally, the studies demonstrate an effect of the site of polypeptide initiation on protein level; Revertant 5 (Fig. 6) with an initia-

SUPPRESSORS

15

tion removed only 12 bases distally produces only about half of the normal iso-1-cytochrome c level. b. Where several genes form a transcriptional unit, or operon, mutations in genes coding for proteins sometimes exert two effects: (1) an inactive gene product is made, and (2) the mutation exerts a “polar” effect. That is, the mutation limits the expression of distal genes in the same operon. This is a phenomcnon apparent in a wide array of bacterial and phage operons (e.g., Franklin and Luria, 1961 ; Jacob and Monod, 1961; Ames et al., 1963; I t o and Crawford, 1965; Henning et al., 1965; Yanofsky and Ito, 1966; Bauerle and Margolin, 1966; Stahl et al., 1966; Jordan and Saedler, 1967; Levinthal and Nikaido, 1969; Robertson e t al., 1970; Cordaro and Roseman, 1972). External suppressors can relieve polarity (e.g., Beckwith, 1963, 1964a; Jordan and Saedler, 1967; Morse and Prirnakoff, 1970; Carter and Newton, 1971). I n addition, intragenic second-site mutations of two types also can suppress. The first type merely reverses the polar effects; the second type not only reverses polarity to some extent, but, in rare instances, also permits restoration of function to the product of the now doubly-mutant gene. Polar mutations are nonsense mutations (Newton et al., 1965; Henning et al., 1965; Martin et al., 1966; Yanofsky and Ito, 1966; Sambrook e t al., 1967; Jordan and Saedler, 1967; Zipser, 1967) or frameshift mutations (Whitfield et al., 1966; Malamy, 1966) that place nonsense triplets in the phase of reading (Martin, 1967). I n polarity a normal number of messenger RNA molecules is made, but many lack information distal to the general region of the nonsense mutation (Contesse et al., 1966; Imamoto et al., 1966; Imamoto and Yanofsky, 1967a,b). It is not clear whether polarity is due to a termination of transcription a t or near intragenic nonsense triplets (Imamoto, 1970; Imamoto and Kano, 1971), to a low density of ribosomes beyond the nonsense triplet, allowing extremely rapid degradation of distal messenger RNA (Morse and Yanofsky, 1969b; Morse et al., 1969; Morse and Primakoff, 1970; Morse, 1971; Morse and Guertin, 1971; Kuwano et al., 1971), or to both factors. The extent of polarity often is a function of the distance of the intragenic nonsense triplet from the nucleotide sequence responsible for initiation of the next protein on the messenger RNA (Newton et al., 1965; Yanofsky and Ito, 1966; Fink and Martin, 1967; Michels and Reznikoff, 1971). Revertants with restored levels of activity for distal genes may be selected without requiring return of function in the mutant gene. For example, mutations in the E . coli lactose operon that drastically decrease lactose permease levels by polar effects also are melibiose-negative at 4OoC since melibiose enters only by lactose permease a t this temperature. Revertants which are still lactose negative

16

PHILIP E. HARTMAN AND JOHN R. ROTH

but have lost polarity may be selected on melibiose (Beckwith, 1964a,b). In the case of the tryptophan system, regained ability to grow on a biosynthetic intermediate is selected (Balbinder et al., 1968). One mode of suppression brings the terminator triplet closer to the initiator for the subsequent protein by deletion of the intervening genetic material, thus reducing polarity and allowing gene function (Beckwith, 1964a,b; Newton, 1966; Zipser and Newton, 1967; Balbinder et al., 1968). In some cases the site of the polar mutation is excised and polarity is eliminated by in-phase deletions (Beckwith, 1964a,b; Balbinder et al., 1968; Elseviers et al., 1969). Rechler and co-workers (Rechler and Bruni, 1971; Rechler et al., 1972; Bruni et al., 1972) report an interesting case where mutation affecting a polar restrictive site results in fusion of two genes to yield a bifunctional enzyme. A second way polarity effects are relieved is through the introduction of a new polypeptide initiation site near and distal to the polar mutation. Such translational reinitiation mutations eliminate strong polarity and allow function of the distal portion of the mutant gene as well as distally located genes (Grodeicker and Zipser, 1968; Michels and Zipser, 1969; Newton, 1969) . There are indications that structural genes sometimes already contain valid initiating sequences that are detected when chain termination occurs earlier in that gene (cf. Newton, 1966, 1969; Michels and Zipser, 1969; Zipser, 1970; Zipser et al., 1970; Yanofsky et al., 1971; Platt et al., 1972). Second-site reversions that allow polypeptide reinitiation also can restore function to the gene in which they occur. I n these rare situations the original nonsense triplet must occur in a noncritical portion of the gene, and the resulting polypeptide fragment(s) also must be able to assume an active conformation. A situation of this type was described by Sarabhai and Brenner (1967) in the bacteriophage T4 rIIB gene. Suppression eliminating polarity also can be achieved by mutations giving rise to new transcriptional start signals (“promoters”) . These restore functional transcription of distally located bacterial genes after polar chain-terminating mutations and in mutants lacking the normal promotor (Margolin and Bauerle, 1966; St. Pierre, 1968 ; Fankhauser, 1971; Arditti et al., 1968; Morse and Yanofsky, 1969a; Wuesthoff and Bauerle, 1970; Atkins and Loper, 1970; Callahan and Balbinder, 1970). Some of the mutations to new promoters lead to inactivation of the products of the genes in which the mutations lie (Morse and Yanofsky, 1969a; Atkins and Loper, 1970) and others are “silent,” i.e., probably are acceptable missense mutations (St. Pierre, 1968; Wuesthoff and Bauerle, 1970). Some operons already contain “internal” low-level promoters that seemingly are not under specific regulatory control ( t r p operon: Margolin and Bauerle, 1966; Bauerle and Margolin, 1967;

SUPPRESSORS

17

Morse and Yanofsky, 1968; Jackson and Yanofsky, 1972; gal operon: Jordan et at., 1968; arg operon: Cunin et al., 1969; his operon: Atkins and Loper, 1970). E. coli and Salmonella “promoter” mutants possessing low enzyme levels and low messenger levels also can be suppressed by very closely linked mutations (Scaife and Beckwith, 1966; Friedman and Margolin, 1968; Silverstone et al., 1970; B. Ely, T. Kasai, D. B. Fankhauser, and P. E. Hartman, unpublished). These second-site mutations seem to alter the susceptibility of the nucleotide sequence involved in transcriptional control. They do not seem to give rise to entirely new “promoters” or to delete transcriptional “stop” signals and allow transcriptional “read-through” from another operon (cf. for lac system, Reznikoff, 1972). Deletions fusing operons and relieving polar effects also are known, however (Ames et al., 1963; Beckwith, 1964a,b; Jacob et al., 1964, 1965; Margolin and Bauerle, 1966).

D. ELIMINATION OF A TOXIC POLYPEPTIDE Among rII mutants of bacteriophage T4 a frameshift mutant (r238) produces a peptide fragment that is toxic to phage growth in a particular host. One manner in which the toxic effects may be suppressed is by intragenic mutations of the nonsense and frameshift types that lead to earlier chain termination and, thus, elimination of the toxic polypeptide (Barnett et al., 1967). A somewhat similar case of suppression occurs in Satmonella. Function of isopropylmalate isomerase, the product of the leuC gene, requires activation either by the product of the leuD gene or by the product of another gene, sup& (also see page 48). The leuC gene product can not be activated by the product of sup& if the leuC product is complexed with a defective, mutant product of gene leuD that has lost activating function. Kemper and Margolin (1969) demonstrated that elimination of the inhibitory leuD product through additional intragenic mutation or by deletion of the leuD gene restores the ability of the bacteria to grow in the presence of sup&. Finally, some semidominant lactose-negative mutants in E. coli are mutant in the i gene and have repressor proteins with decreased affinity for inducer and, sometimes also an increased affinity for operator. This results in a drastic inhibition of enzyme synthesis dictated by the lactose operon. One mode of reversal is by an additional mutation in the i gene that eliminates the hyperactive regulatory protein (Jacob and Monod, 1961; Willson et at., 1964; Bourgeois et al., 1965; Bourgeois and Jobe, 1970). Some dominant mutations in higher organisms might also lead t o pro-

18

PHILIP E. HARTMAN AND JOHN R. ROTH

duction of toxic macromolecules. Two possible examples are K - p n (Killer of prune), discussed in Section 111, D, c, and N s (Nasobemia) in Drosophila. I n both cases, reversion frequencies are high and include deletion mutations (Lifschytz and Falk, 1969a,b; Denell, 1972).

E. DOUBLE FRAMESHIFTS Crick et al. (1961) proposed that deletion or addition of base pairs in other than multiples of three leads to alterations in the “reading frame” during translation of the “triplet” genetic code on messenger RNA (also see Barnett et al., 1967). Alteration in the number of nucleotides leads to translation of the subsequent code letters in new multiples of three, as detected by alterations in the amino acid sequences of proteins. A second frameshift can compensate to restore the proper reading frame and lead to production of active protein so long as the amino acids inserted between the two frameshift sites are acceptable to catalytic activity and the nucleotide sequence does not lead to termination of the polypeptide chain. An example is given in Fig. 7. Data on double frameshift mutations come predominantly from studies on bacteriophage T 4 rIZ mutants (op cit), lysozyme (Terzaghi et al., 1966; Okada et al., 1966, 1968; Streisinger et al., 1966; Inouye et al., 1967), E. coli tryptophan synthetase A protein (Brammar et al., 1967; Berger et al., 1968a,b),and L-histidinol dehydrogenase of S. typhimurium (Yourno and Heath, 1969; Tanemura and Yourno, 1969; Yourno, 1970, 1971, 1972; Kohno et al., 1970). The collective data indicate that frameshifts preferentially occur in sequences of repeating nucleotides or lead to repetitions (Streisinger et al., 1966) although the mechanisms engendering frameshift mutations still remain unclear. Thus, “reversions” toward the wild-type phenotype may occur either by reversal of the original mutation or, frequently, by intragenic suppressor mutations (by deletions and insertions near the original mutation) leading to a protein that is catalytically active but contains one or more amino acid substitutions, additions, or deletions (Fig. 7 ) . Double frameshifts also can be detected in eukaryotes (Brink et al., 1969). Malamy (1966, 1970) reported a case slightly different from those described above. Spontaneous mutants with absolute polar effects in the E. coli galactose and lactose operons are due to small insertions of genetic material (Jordan et al., 1968; Shapiro, 1969; Michaelis et al., 1969; Malamy, 1970). Reversion may be accomplished either by deletion of the inserted sequence (Shapiro, 1969; Malamy, 1970) or by a frameshift mutation (Malamy, 1966, 1970). The frameshift either disrupts a termination signal or creates a new promoter sequence. These two possibilities

19

SUPPRESSORS m i n o m i d -Val -Thr - A 1 0 - L e u - A r g 4 0 1 Wildtype

triplet codons

-

Mulonl

Revertmil

R5

GU A!G

GU

ACA GCG

C

UA CGC GUC

dhr-Pro ACC CCU

h,r030/8

A GA G

GA

-110A G

U AUC A

UGA (terminole1

-c

CGc UAC GCG UCA CCC CCU

CCC CCU

u

-MI -Ly% - A ~ Q-Tyr -A10

-Glu -GI“

-Ser -Pro -Pro

-Pro -Pro

GAA GAP’ AU! .Reverlanl G G R95 A -GI” -GI” - 1 l a -

+ AG

Reverton1 R52

GU

ACA GAG CGc

-Val -Thr -GI”

UAC GCG

UCACCC

- A ~ Q-Tyr -A10 - % - P r o

ccu -Pro

GA‘ -GI”

G A ~A$ -GI” -110-

FIQ. 7. Amino acid alterations and proposed genetic code in double frameshift mutants of Salmonella typhimurium L-histidinol dehydrogenase. The amino acid sequence and corresponding triplet codons for a portion of histidinol dehydrogenase from wild-type bacteria are shown a t the top. Mutant hisD3018 is assumed to have lost a G/C base pair in the DNA and a C in the messenger RNA (center) as based upon analysis of proteins produced by different double frameshift revertants (lower portion of figure). Frameshift mutant 3018 protein is not detected; presumably the protein is terminated at the UGA triplet whose “recognition” was created by the shift in reading frame. Revertant R5 has an altered string of 6 amino acids due to a -1 frameshift that compensates for the $1 frameshift originally present. The +1 frameshift also can be compensated by repetition of an AG sequence (revertant 52), again restoring in-phase translation and resulting in a protein one amino acid longer than the wild type, or by deletion of a CGCG sequence (revertant R95), resulting in a functional protein that is one amino acid shorter than the wild type. Additional classes of revertants, including true wild types, also have been described (Yourno and Heath, 1969; Yourno, 1970; Kohno et al., 1970).

might be differentiated were it known whether the regained synthesis of enzyme (about 2-7% of wild-type activity) is constitutive or under normal repression control. 111. lntergenic Suppression (“External” Suppression)

Many classes of suppressor mutations occur outside of the gene that carries the primary mutation. These are termed “extragenic” or “external” suppressors not because they occur outside of genes, but, rather, because they affect the structure of a second functional unit of the genetic material, i.e., are due to intergenic suppression. The external suppressors are sometimes subclassified. One subclass of intergenic suppressors “corrects” the amino acid sequence in the mutant protein. ‘LCorrection”is exerted through modifications in the protein-synthesizing system (“informational” or “direct” suppression). A second, more heterogeneous, sub-

20

PHILIP E. HARTMAN AND JOHN R. ROTH

class of intergenic suppressors allows continued production of the gene product in mutant form but compensates indirectly to allow expression of the wild-type phenotype by other means (“indirect” suppression).

A. INFORMATIONAL SUPPRESSION (“DIRECT” SUPPRESSION) One commonly encountered class of suppressors in microorganisms is the group that alters the fidelity with which the genetic message is translated, a possibility first suggested by Yanofsky and St. Lawrence (1960) and by Benser and Champe (1962). I n these cases, the mutant gene still provides “mutant” messenger RNA but the mutant region of the message is occasionally “misread” in suppressor strains so that a functional protein is formed, These informational suppressors include external suppressors of missense, nonsense, and frameshift mutations. Informational suppressors contributed to our knowledge of the genetic code and still serve today to pinpoint particular kinds of mutational alterations. Furthermore, the allele specificity of informational suppressors allows differentiation of homoalleles that appear identical by other tests including recombination tests (cf. Barben, 1966). Extensive prior reviews of this area exist (Gorini and Beckwith, 1966; Garen, 1968; Davies, 1969; Gorini, 1970), so we discuss here only some recent aspects of informational suppression not previously covered in detail. Our discussions focus on microorganisms, but we are convinced that higher organisms will shortly receive just as concerted attention. Nonsense suppressors may occur in Drosophila, for example, suppressor of Hairy wing. Suppressor of Hairy wing acts on some mutations a t many loci (Lindsley and Grell, 1968) and, when homozygous, results in female sterility due to autonomous pathology in ovarian nurse cells (Klug et al., 1968). In these cells protein synthesis is inhibited and RNA is labile (Klug e t al., 1970). Perhaps a minor tRNA species completely altered in the suppressor homosygote is used only once during development. Or, “oversuppression” might occur in genes uniquely used in nurse cells analogous to the host-dependent phage mutants uniquely sensitive to particular nonsense suppressors of bacteria (Horiuchi and Zinder, 1967). That is, other Drosophila genes may have double termination signals, but the suppressor-sensitive genes may have only one (cf. Lu and Rich, 1971). Also in Drosophila, “Minute” mutations have been associated with positions of deletions possibly involving redundant genes (Ritossa et al., 1966a; Tartof and Perry, 1970) specifying tRNAs (Steffensen and Wimber, 1971). While no correlation of allele-specific suppression has been made with any of the “Minute” gene regions, Green

SUPPRESSORS

21

(1946) found that each of three Minute mutations enhanced the expressivity of various recessive vg (vestigial) mutations. The increased mutant expression was related to a prolongation of the third larval instar, characteristic of Minutes. Larval tumors also are enhanced by elongation of larval life (see Section IV, a ) . Perhaps these characteristic responses could be applied to selection for interesting kinds of suppressors for the Minute phenotype. 1. t R N A Suppressor Mutations External to Anticodons

Transfer RNAs can gain activity in suppression of nonsense (Goodman et al., 1968, 1970; Gopinathan and Garen, 1970; Altman et al., 1971) and probably of missense (Carbon and Curry, 1968; Carbon et al., 1969; Squires and Carbon, 1971) codons through base substitutions occurring directly in the anticodon of the tRNA. However, in Section 11, B, d tRNA mutants are described which lose and then regain ability of informational suppression through mutations scattered a t other particular points in the tRNA gene. Several recent lines of evidence suggest that creation of nonsense suppressors from wild-type strains also may involve base substitutions in the tRNA outside of the anticodon or, alternatively, in some modification (So11, 1971) of the tRNA molecule. A strong UGA nonsense suppressor (Sambrook et al., 1967) leads to production of an abnormal tryptophan tRNA (Hirsh, 1970, 1971; Hirsh and Gold, 1971 ; Chan et al., 1971). Both wild-type and suppressor tRNAs have the same anticodon (CCA) which should, by the predictions of the “wobble” hypothesis (Crick, 1966), read only the tryptophan codon, UGG. The fact that the suppressor tRNA differs from wild-type by an A to G change elsewhere than in the anticodon suggests that a tRNA sequence alteration far from the anticodon region may affect codon-anticodon pairing, allowing the CCA anticodon of the tRNA to violate wobble rules and recognize the UGA codon as well as UGG. A second sort of UGA suppressor seems to involve a defect in tRNA modification. Reeves and Roth (1971) found a UGA suppressor which is recessive in contrast to most bacterial nonsense suppressors, which are dominant mutations. The recessive UGA suppressor elicits undermethylated tRNA (Reeves and Roth, unpublished). Although it is uncertain which undermethylated tRNA is responsible for suppressor activity, it would seem likely that one undermethylated species occasionally miscodes, and thereby suppresses the mutant phenotype. An additional recessive suppressor ( s u f F on pages 26,27, and 28) may represent another case of miscoding by unmodified tRNA. Both recessive suppressor genes control vital functions since temperature-sensitive lethal mutations of these genes are known.

22

PHILIP E. HARTMAN AND JOHN R. ROTH

Wild-type enteric bacteria exhibit a low-level reading of the UGA codon (Model et al., 1969; Roth, 1970; Ferretti, 1971), but UGA triplets are predominantly chain terminating (Khorana et al., 1966; Brenner et al., 1967; Sambrook et al., 1967; Zipser, 1967; Model et al., 1969). The relationship of this low-level UGA suppression to the above UGA suppressors is not clear, but some of the activity may be due to miscoding by wild-type tRNAT1’P. Recessive allele-specific suppressors have been described on several occasions in Aspergillus (for ade-26 by Pritchard, 1955; and for meth-2 by Luig, 1962). Other recessive nonsense suppressors have been found in yeast (Inge-Vectomov, 1965; Magni et al., 1966). The mechanism of action has not been determined for either suppressor. 2. Lethal Suppressors

A recessive lethal amber suppressor (su-7) has been described in E. coli by Soll and Berg (1969a). Cells carrying this suppressor can survive only if they also carry a wild-type copy of the affected gene. This suppressor has been shown to insert gln in response to the amber codon (Soll and Berg, 1969b). Miller and Roth (1971) have described lethal amber and UGA suppressors in Saliizonella which are allelic and map at a position analogous to that of su-7 in E. coli. Tryptophan transfer RNA which reads the UGG codon should be capable of mutating so as to read UAG or, by a different mutation to read UGA. Thus the finding of an allelic UGA suppressor did not fit with the gln insertion of the E. coli lethal suppressor, su-7. Recently this discrepancy has been resolved by Yaniv, Soll, and Berg (personal communication; discussed in Berg, 1972), who found that the glutamine-inserting tRNA from su-7 is actually an altered tryptophan tRNA. Apparently one mutation affecting the anticodon region can cause tRNATrp to read UAG and be mischarged with the wrong amino acid, gln. The lethality of the su-7 suppressor is probably due to the loss of the single trp transfer RNA species present in E. coli. 3. Substituted Proteins

The prevalence of nonsense mutations and the specificities of insertion of different amino acids by nonsense suppressors in E . coli has allowed the in vivo synthesis of proteins with particular amino acid substitutions at any one of a large number of locations. The effects of such substitutions on the properties of p-galactosidase have been described (Langridge, 1968a,b,c; Langridge and Campbell, 1968). Originally, suppressors with altered tRNAs and effective in inserting serine, glutamine, and tyrosine a t amber codons and tyrosine a t amber or a t ochre codons

23

SUPPRESSORS

were described (reviewed by Garen, 1968). More recently, suppressors eliciting insertion of leucine a t amber codons (Chan and Garen, 1969; Gopinathan and Garen, 1970), tryptophan a t UGA codons (Chan and Garen, 1970; Hirsh, 1970, 1971; Hirsh and Gold, 1971; Chan et al., 1971), and lysine at UAA and UAG codons (Kaplan, 1971) have been added to the substitution repertory. These suppressors should prove useful for construction of particular sorts of proteins in the study of enzyme action.

4. Nonsense Suppressors in Eukaryotes Suppressors with properties of nonsense suppressors are present in yeast (“supersuppressors” : Hawthorne and Mortimer, 1963 ; Mortimer and Gilmore, 1968) and Neurospora (Seale, 1968; Case and Giles, 1968; Chalmers and Seale, 1971). The following evidence supports the identity of supersuppressible mutations of yeast and the nonsense mutations, amber and ochre, found in bacteria: a. The supersuppressors act on some, but not on all, alleles of many loci (Hawthorne and Mortimer, 1963; Mortimer and Gilmore, 1968; Gilmore et al., 1971). For example, in the tr-5 locus, approximately 40% of the known mutations are supersuppressible (Manney, 1964) ; in the his-Q locus, 35% of the classified mutations are supersuppressible (G. Fink, personal communication). Additional frequency data are cited in Mortimer and Hawthorne (1969). This frequency is comparable to that of nonsense mutations in several his genes of Salmonella typhimurium (Whitfield et al., 1966; Hartman et al., 1971) and in the lac2 gene of E . coli (Langridge and Campbell, 1968). b. As expected for organisms carrying chain-terminating mutations, supersuppressible mutants are not “leaky” or temperature-sensitive, nor does intragenic (intracistronic) complementation occur in most cases. The exceptional supersuppressible mutations exhibiting “intragenic” complementation show a polarized pattern explicable by participation of incomplete polypeptide chains in the complementation reaction (Manney, 1964; Fink, 1966, 1971). c. Data on several systems indicate that supersuppressible mutations lead t o gene products whose molecular size is decreased relative to native wild-type enzyme. This could be due either to premature chain termination, to polarity, or to effects on protein-protein interactions in aggregates of different polypeptide chains (for critical discussion, see Fink, 1971). Native wild-type Neurospora and yeast tryptophan synthetase complexes have molecular weights close to 150,000 and catalyze two successive half-reactions: (I) indole-3-glycerol phosphate + indole glyceraldehyde-3-phosphate, and (11) indole serine + tryptophan. The Neurospora enzyme (Bonner et al., 1965; Lacy, 1965; also see

+

+

24

PHILIP E. HARTMAN AND JOHN R . ROTH

Lacy, cited in Hartman and Suskind, 1969, p. 66) and the comparable yeast enzyme (Duntze and Manney, 1968; Manney et al., 1969) each are dictated by a contiguous genetic region predominantly concerned with elaboration of activity I from one subregion and activity I1 from a second subregion. The Neurospora enzyme may be composed of two different polypeptide chains (Carsiotis et al., 1965), but neither it (Ensign et al., 1964) nor the yeast enzyme (Duntze and Manney, 1968) readily dissociates into subunits under mild conditions. Supersuppressible yeast tryptophan synthetase mutants most often lose both half-reactions, but some contain an enzyme which has lost activity I1 but retained a substantial part of activity I. The molecular weight of the mutant enzyme (activity I) is reduced to approximately 35,000 as estimated by Sephadex gel chromatography (Manney, 1968). Similarly, supersuppressible mutations in the yeast his-4 (Fink, 1965, 1966, 1971; Shaffer et al., 1969) and Neurospora arom (Case and Giles, 1968, 1971) genetic regions lead to enzyme aggregates considerably diminished in molecular weight from the wild-type enzyme complex. d. Two supersuppressible mutations in the iso-l-cytochrome c gene of yeast (Sherman et al., 1966) carry a UAA (ochre) codon a t the mutant site (Gilmore et al., 1968, 1971; Sherman et al., 1970). Both mutant sites studied have a glutamic acid residue in the wild-type protein (Glu = GAA or GAG). Revertants specify mutant proteins carrying Gln (CAA), Lys (AAA), Leu (UUA), Tyr (UAU or UAC), or Ser (UCA), but none which carry Trp (UGG). Although it is not known whether cytochrome with a Trp substitution a t either of these sites is functional, this region of the protein is tolerant of amino acid substitutions and thus the data favor UAA as the mutant codon (compare Fig. 3, page 7 ) . A second type of supersuppressible mutation has been shown to be of the amber UAG type. This type was first identified by the fact that it can be converted to ochre (UAA) by mutagenesis with hydroxylamine and ethylmethanesulfonate (Hawthorne, 1969a,b). This test, along with use of the informational suppressors mentioned below substantiate the UAA codon assignment made in the previous paragraph. This identification of the amber mutation was confirmed more recently by reversion studies analogous to those outlined above (Sherman and Stewart, 1971). In addition to the amber and ochre mutations, UGA mutations have also been identified by Hawthorne (personal communication). Three general types of nonsense suppressors have been described in yeast : (a) amber-specific suppressors, (b) ochre-specific suppressors, and (c) suppressors which act on both amber and ochre mutations (Gilmore, 1967; Mortimer and Gilmore, 1968; Hawthorne, 1969a,b). Re-

SUPPRESSORS

25

cently Hawthorne (personal communication) has found a fourth class of suppressors which are UGA-specific. He has also found that the amber-ochre suppressors (type c above) are able to suppress UGA mutations as well. The ochre-specific suppressors, not found in E. coli, may reflect the presence of inosine in tRNA of yeast whereas inosine is absent in tRNA of E. coli. The ‘Lwobble’’rules, suggested by Crick (1966; consult Jukes and Gatlin, 1971, for recent discussion) to describe codon-anticodon base pairing, predict that inosine in the first position (5’ end) of the anticodon will pair with U, C, and A in the third position (3’ end) of the codon. Thus, a tRNA with the anticodon IUA would pair with UAC (T y r), UAU (Tyr) , and UAA (ochre). Several tyrosine-inserting nonsense suppressors have been described (Gilmore et al., 1968, 1971). Other explanations of ochre-specific suppressors are possible if the general structure or modified bases of particular tRNAs can permit exceptions to the “wobble” pairing rules (discussed in Gilmore et al., 1971). It is surprising to note that no yeast supersuppressor has been described corresponding to the ochre suppressors of E. coli, which suppress both ochre and amber mutations. The supersuppressors which act on UAG, UAA, and UGA may prove to be of the ribosomal type (cf. Gorini, 1970). A second distinctive feature of yeast nonsense suppressors is the large number of suppressor loci. Gilmore (1967) described eight classes of nonsense suppressors in Saccharom yces based on their ability to suppress five different ochre mutations. Several of these suppressor classes were further divided into subsets based on the strength of the suppressive effect, leading to 21 phenotypic classes of ochre-specific and amberochre-UGA (see above) suppressors. Hawthorne ( 1969b) classified another group of suppressors into ten classes of which five were ochre-specific, three were amber-ochre-UGA, and two were amber-specific ; only three of these classes overlap the suppressor classes described by Gilmore (1967). Sixteen map positions, scattered over eleven linkage groups, have been found for nonsense suppressors in Saccharomyces; three gave rise to amber-specific, three to ochre-amber-UGA, and eleven to ochre-specific suppressors (Hawthorne and Mortimer, 1968). A group of over 20 ochre-specific suppressors (Class I subunit 1 ; Gilmore, 1967) behave identically in suppression of a series of ochre mutations but are distributed among eight clearly genetic loci; each suppressor leads to insertion of Tyr a t ochre codons but is ineffective in suppressing an amber mutation (Gilmore et al., 1968, 1971). Of several hundred suppressors acting on five ochre mutations, all map in these same eight loci (Striimnaes and Mortimer, cited in Gilmore et al., 1971). Combination in a haploid yeast cell of two Class I11 (inefficient)

26

PHILIP E. HARTMAN AND JOHN R. ROTH

suppressors increases suppressor activity and has no overt effect on general growth properties (Mortimer and Gilmore, 1968), but combinations of two Class I (Tyr) suppressors is lethal or results in impaired and abnormal growth in nonrestrictive medium (Gilmore, 1967; Mortimer and Gilmore, 1968). Some strong ochre-specific suppressors are lethal in haploids containing the extrachromosomal factor, $ (Cox, 1971), a factor that stimulates suppression by very weak ochre suppressors (COX, 1965). I n E . coZi, a gene dosage effect on efficiency of nonsense suppression (Hoffman and Wilhelm, 1970) and a deleterious effect of ochre suppressors (Gallucci and Garen, 1966) also have been noted. It seems plausible that one cause of these deleterious effects is interference with normal chain termination a t essential positions (cf. Lu and Rich, 1971; discussion in Gilmore et al., 1971). In addition, withdrawal of tRNA genes from their normal function through suppressor mutations may cut down tRNA gene redundancy (Schweizer et al., 1969) essential to synthesis of adequate tRNA (cf. Mortimer and Gilmore, 1968; Mortimer, 1969). The mutagen ethylmethsnesulfonate, which predominantly elicits G/C + A/T base pair substitutions, causes suppressor mutations of the ochre-specific and amber-specific suppressor type (Hawthorne, 1969b). It has been suggested that yeast (Magni and Puglisi, 1966; Magni et al., 1966) and SaZmoneZZa (Whitfield et al., 1966; Hartman et aZ., 1971) nonsense suppressors also can arise through additions and deletions of base pairs since the ICR compounds (Ames and Whitfield, 1966) can induce suppressors. The nature of these suppressors and their relationship to other nonsense suppressors remains unexplored. This finding may not be surprising in the case of yeast, since there is some evidence that the mutagen ICR-170 causes largely base substitution mutations in that organism (cited in von Borstel, 1969; F. Sherman, personal communication). Barben (1966) describes seven nonsense suppressor loci in Schizosaccaromgces, and five nonsense suppressor loci have been located in Neurospora (Seale, 1972). 6. Suppressors of Frameshifts

Additions or deletions of base pairs other than in multiples of three throws the organized triplet reading out of phase (see pp. 4, 18). The linear messenger RNA molecule is translated into a normal polypeptide chain up to the point of the mutation, but reading proceeds beyond the mutation in an improper phase. The protein-synthesizing apparatus merely translates consecutive triplet codons in the order they appear

SUPPRESSORS

27

on the mRNA (Crick et al., 1961). Extended missense protein is made until a nonsense codon terminates translation (cf. Martin, 1967; Barnett et al., 1967). Extensive analyses of frameshift mutations were first carried out in bacteriophage T4 (Crick et al., 1961; Streisinger et al., 1966; Barnett et al., 1967). The fact that most of the early work on frameshift mutants was done in phage may be one reason why intergenic, informational suppressors of frameshift mutations were not found earlier. A second explanation may stem from the apparent preponderance of base-pair deletions, as opposed to base-pair additions, in frameshift mutants unless they have been induced by intercalating agents or selected in back-mutation tests (discussed in Hartman et al., 1971); we see below that all suppressible frameshifts are probably +1 frameshifts. Indications of the presence of external suppressors for frameshift mutations (Riyasaty and Atkins, 1968; Oeschger and Hartman, 1970) were independently noted by Yourno et al. (1969), who next clearly demonstrated external suppression of the frameshift mutation hisD3018 (Yourno and Tanemura, 1970). Comparison of the amino acid sequences of wild-type protein and those produced by various revertants showed that the suppressible mutation carries one extra G/C base pair (Yourno and Heath, 1969; Tanemura and Yourno, 1969; Yourno, 1970; Kohno et al., 1970). Additional suppression was found for thirteen of twenty-one tested frameshift mutations in the Salmonella histidine operon when the mutants reverted to prototrophy (Riddle and Roth, 1970). Forty-eight suppressor mutations were placed into six groups on the basis of map location (Fig. 8 ; Riddle and Roth, 1972a). Two general sorts of suppressors exist. One type (sufA, B, and C ) never suppress mutations suppressible by the second type (sufD, E, F ) . The first suppressor type may correct the phase of +1 frameshift mutations located in runs of C in the messenger RNA since (1) three mutations suppressible by sufA, B, and C are known to lie in runs of C (Yourno and Heath, 1969; Yourno, 1971; Yourno and Kohno, 1972) ; (2) one supprcssor of this type inserts proline a t low frequency (Yourno and Tanemura, 1970) ; (3) mutations of the sufA and sufB genes affect different species of proline tRNA (Riddle and Roth, 1972b). Suppressors of the second general type (sufD, E, F ) may restore proper reading phase to frameshift mutations in runs of G, since one suppressor of the sufD locus affects a glycine tRNA (Riddle and Roth, 1972b) which normally reads the codon GGG (Carbon et al., 1970). This supposition agrees with the amino acid sequence data of Yourno (1972). Recently, a four-letter anticodon sequence has been found in a sufD tRNAo’y (Riddle and Carbon, 1973).

28

PHILIP E. HARTMAN AND J O H N R. ROTH

Thus, one suppressor tRNA contains a 4-base anticodon very likely capable of reading 4-base codons. I n addition, tRNAs may have difficulty maintaining proper phase when monotonous base sequences undergo translation. Atkins et al. (1972) have demonstrated that ribosomal alterations can affect the frequency of mistakes in phase maintenance. The combined results suggest a critical role for tRNA in determining the distance traveled by the ribosome in the translocation step of protein synthesis and implicate ribosomal structure in the fidelity of this process.

suf F

XY I

pur E

suf A

ser A

wfD

his W

suf B

FIQ.8. Suppressors of frameshift mutations in Salmonella typhimurium. Genetic map of 8. typhimurium chromosome showing locations of frameshift suppressor genes (suf)described in the text. Based on Riddle and Roth (1972a).

B. RECONSTITUTION OF ACTIVEENZYME CONFORMATION Common among mutants are those that form polypeptide chains that are unusually sensitive to thermal denaturation. The thermal sensitivity often is expressed by the native protein but in some cases is restricted to the polypeptide at the time of synthesis, for example, while it exists as a monomer before joining with other molecules to form a “native” enzyme with coenzyme or other proteins attached. Temperature-sensitive tRNA mutants also are known. I n addition, mutants sensitive to low temperature have been described; this last class is most often concerned with highly allosteric, multimeric proteins that undergo great conformational changes during union with various small or large ligands. In view of the “plasticity” of protein molecules as exemplified by the examples just cited, we should expect to find it common that “inac-

SUPPRESSORS

29

tive” enzyme can be “reactivated” under appropriate environmental conditions. These conditions may vary, in separate cases, from what we might consider as quite general conditions of pH or ionic concentration to changes in the concentrations of more specialized molecules, effector molecules. Effector molecules are those that influence catalytic activity of an enzyme through inhibition or activation and thus serve to regulate its activity. While, in this general sense, almost anything could be specified as an effector if high enough concentrations were examined, we restrict our definition here to effectors that are physiologically significant in intracellular metabolism. Effector molecules are extremely important in bringing about the balanced regulation that dictates the flow of metabolites in normal microbial, plant, and animal systems (e.g., Atkinson, 1966; Umbarger, 1964, 1969; Kornberg, 1965; Koshland and Neet, 1968; Denburg and DeLuca, 1968; Tuli and Moyed, 1966; Whitehead, 1970). I n fact, mutants contain defective proteins that are nominally active under the proper environmental conditions. Unfortunately for the cell, however, these conditions are special and do not mimic the intracellular environment; in vivo the protein is relatively inactive and leads to a mutant phenotype. Often, suppressor mutations lead to an adjustment of the intracellular milieu so that catalytic activity is restored to the defective protein. Here we discuss cases where the suppressor mutation serves to remove or modify normal molecules and thus releases a mutant protein from inhibition. On pp. 17-18, we mentioned cases where the original mutant protein itself has a deleterious effect on normal cell metabolism and where this effect is suppressed by removal of the toxic polypeptide by second-site mutation. 1. Inactivating Zon or Environment

Suskind and co-workers found that a temperature-sensitive Neurospora tryptophan synthetase mutant, tdZ4, synthesized an enzyme protein abnormally sensitive to inhibition by zinc. (The wild-type enzyme is described on pp. 23-24.) Active enzyme was recovered once it had been purified away from other cellular constituents, including an inhibitor. Enzyme from a strain carrying an allele-specific suppressor mutation (Suskind and Yanofsky, 1961) retained the properties of the defective tdZ4 protein (Suskind, 1957a,b; Suskind and Kurek, 1957, 1959; Suskind and Jordan, 1959). This led to the suggestion that the suppressor gene acted to alter the intracellular environment to allow adequate function of defective enzyme. The suppressor mutation might lead to an alteration in the concentration or location of zinc ion.

30

PHILIP E. HARTMAN AND JOHN R. ROTH

A number of presumed missense mutants in fungi form enzymes that allow growth only on special media, for example, of high osmotic strength (“osmotic remedial” mutants: Hawthorne and Friis, 1964; Kuwana, 1961; Nakamura and Gowans, 1967; Lacy, 1968; Esposito, 1968; Meteenberg, 1968) or a t high pH (Stokes et al., 1943; B. S. Straws, cited in Emerson, 1952). One surmises that the external environment has impact on the intracellular milieu sufficiently to allow the defective enzymes to assume active conformations, for example, by adequate binding of pyridoxal phosphate to apoenzyme in the latter instances cited above (cf. Guirard et al., 1971). In these special cases it should be possible to obtain among “revertants” suppressor mutants of high interest, for example, organisms with alterations in intracellular osmolality or production of cofactor. The same line of reasoning applies to a case in which temperature or osmotic conditions circumvent a genetic block imposed by nonsense mutations and deletions in the ade-3 locus of Saccharomyces (Jones, 1972). 6. Inactivating Metabolite One would expect to find cases where a mutant enzyme for a limiting reaction was (hyper)sensitive to normal levels of a metabolite; mutations that cut down the rate of synthesis or increase the rate of utilization of the metabolite might then serve to suppress. At this time, however, we are unable to pinpoint cases of suppression that fall into this category. 3. Inactivating Macromolecule

a. t R N A . Mutations in one of the earliest suppressor genes analyzed (Schultz and Bridges, 1932) eliminate the presence of a species of transfer RNA that drastically inhibits particular mutant proteins (Jacobson, 1971; Jacobson and Grell, 1971; Twardzik et al., 1971). Drosophila mutants a t the vermilion ( v ) locus lac,k the brown constituent of eye pigment, accumulate nonprotein tryptophan (Green, 1949), and are deficient in the activity of an inducible enzyme (Kaufman, 1962), tryptophan pyrrolase (Tryptophan peroxidase) (Baglioni, 1959, 1960; Kaufman, 1962; Marzluf, 1965a,b; Tartof, 1969). Various v mutants fail to complement; i.e., the locus is a single cistron (Green, 1954; Barish and Fox, 1956). Tryptophan pyrrolase catalyzes the first step in the conversion of tryptophan to ommochrome pigment (Fig. 9 ) . Kyurenine (“v substance”) is not made by v mutants (Beadle and Ephrussi, 1936, 1937; Butenandt et al., 1940; Tatum and Haagen-Smit, 1941; Kikkawa, 1941) although v mutants contain an excess of kynurenine formamidase (Glassman, 195s) and can use kynurenine to form eye pigment.

31

SUPPRESSORS

Kynurenine is hydroxylated to 3-hydroxykynurenine (Ghosh and Forrest, 1967a), and both compounds are able to react with products of tyrosinase activity to yield brown ommochrome pigments (Glassman, 1957). Sex-linked mutations partially restore production of eye pigment (Schultz and Bridges, 1932), kynurenine (Beadle and Ephrussi, 1936, 1937), and tryptophan pyrrolase activity. The latter is detected in extracts of suppressor-sensitive (Green, 1952) v mutants (Baglioni, 1960; Kaufman, 1962; Marzluf, 1965a,b; Tartof, 1969). These suppressible mutants also produce some brown pigment and presumably possess slight enzyme activity under certain starvation conditions in the absence of the suppressor (Tatum and Beadle, 1939) whereas nonsuppressible v alleles fail to do so (Green, 1954; Shapard, 1960). Kynurenine production in the fat body (Beadle, 1937) is stimulated in the presence of tryptophan

anthronflic acid

/

tryptophon pyrrolose Tryptophan+-Formylkynurenme

A" su-er?

/

~ ~ 6 -Kynurenins

~

,/" ~

kynurenine : ~ ~ hydoxylase

~

e

--

4 +-3-

O H -Kynurenme-

Ommochrome

(Brown eye pqment)

SU-I"

7

oxygen

er

Phenylolonine -Tyrosine

-DOPA

-DOPA-Quinone

-Melanins

FIG. 9. Some aspects of tryptophan metabolism in Drosophila melanognster.

or of the suppressor in suppressor-sensitive strains (Rizki, 1961b, 1963, 1964; Rizki and Rizki, 1968), and the suppressor alters the intracellular distribution of 420 A halolike particles in fat body cells (Rizki et al., 1970). The suppressors are recessive (Schultz and Bridges, 1932; Tartof, 1969), and even deletion of the suppressor locus results in suppression, suggesting that the su+ locus is responsible for the synthesis of a product toxic to v mutants (Shapard, 1960). However, partial fractionation and mixtures of extracts failed to reveal the presence of inhibitors (Marzluf, 1965a,b; Tartof, 1969). Recently, it has been found that treatment of extracts with ribonuclease leads to activation of latent tryptophan pyrrolase activity in suppressible mutants. Addition of a particular species of uncharged tRNATyr (tyrosine transfer RNA) inactivates mutant enzyme (Jacobson, 1971; Jacobson and Grell, 1971). Suppression of v is accompanied in Drosophila homozygous for suppressor by the disappearance of the inhibitory species of tRNATyr and an increase in another tRNATyr isoaccepting fraction. The

32

PHILIP E. HARTMAN AND JOHN R. ROTH

recessive nature of the suppressor and behavior of the tRNATyr species indicate that the suppressor locus does not code for the primary structure of tyrosine tRNA “but that it may control an enzyme that modifies the tyrosine tRNA” (Twardzik et al., 1971). Mutants of four other genes involved in pigment formation (sable, speck, purple, and black: Bridges, 1932; Green, 1954; Lindsley and Grell, 1968; Rizki and Rizki, 1968) also are suppressed by a t least one of the v suppressors. One speculation is that tRNATy” plays a normal role in coordinating the two pathways that combine to elicit brown eye pigment (ommochromes) in wild-type Drosophila (Fig. 9 ) . That is, the suppressible v mutant enzymes may be hypersensitive to normal effector molecule (Baille and Chovnick, 1971). Furthermore, there are many indications of coordination between the pathways of ommochrome synthesis and those involved in synthesis of red eye pigment. There are parallel decreases in red (pteridine) and brown (ommochrome) eye pigments in flies homozygous and heterozygous for various w (white) alleles (Morita and Tokuyama, 1959) and in flies grown under various conditions (cf. Ephrussi and Herold, 1945). There also seems to be some coordination between the synthesis of the pigments and the pigment “core granules” themselves (cf. Caspari, 1964) although the pigments are distributed differently in two distinct granule types (Shoup, 1966). These and other scattered data (Ziegler, 1961; Ziegler and Harmsen, 1969; Nolte, 1959; and others) as well as “intuition” lead to the assumption that coordination must exist between these “separate” reaction sequences ; the question is: How? One possibility for coordination of eye pigment pathways has been indicated ; tryptophan pyrrolase is noncompetitively inhibited by certain pteridines (Ghosh and Forrest, 1967b). Quite possibly many “morphological” mutants are affected in coordination rather than in catalytic activity per se. For example, mutations often result both in a reduction of, say, red pigment and simultaneously an increase of brown pigment (Nolte, 1955) or lead to accumulation of a new spectrum of pteridines (McIntire and Gregg, 1966). Close analyses of these situations and cases of suppression should assist to unravel the interrelationships of various genes and help define gene products and control mechanisms. b. Mukai and Margolin (1963) present evidence indicating that a particular mutant of Salmonella is sensitive to an abnormal inhibition of enzyme synthesis. The mutant (leu-500) lacks activity for all three of the leucine biosynthetic enzymes that are dictated by the four adjacent leu genes in Salmonella (Fig. 10). Mutation leu-500 is believed to be a single base-pair substitution in the promoter region of the leucine operon and essential for regulation of the cluster of genes (Margolin, 1963, 1971; Mukai and Margolin, 1963; Burns et al., 1966). The leu-500

33

SUPPRESSORS

mutant may revert by back-mutation or it may grow on medium lacking leucine due to mutations in a particular suppressor gene (su leu500 = supX) that maps a t an entirely different region of the Salmonella chromosome. Among effective suppressor mutations are singlebase transition mutations as well as a number of deletions of the entire supX gene region. Suppression is a recessive trait. Thus, elimination of an inhibitory and dispensable molecule restores gene function in mutant Zeu-500 (Mukai and Margolin, 1963; Dubnau and Margolin, 1972). The supX mutations also affect functioning of other operons; the pleiotropic effects of supX mutations favor the hypothesis that supX+ allows production of a molecule that directly or indirectly is toxic to transcription initiation of sensitive promoters (Dubnau and Margolin, 1972).

-

Acetyl Cop.

isomerose

a-lsopopylmalote

dehydrcqenose a-ketoisocaproale

=B-lsopropylrmloie

a-Keioisovoleraie

Neurosporo

-

leu 4

leu - 2 and feu 3

-

-

leu I

L- leucine

FIG. 10. Pathway of leucine biosynthesis in Salmonella and Neurospora and its genetic control.

I n addition to this mode of suppression, the leucine operator region may undergo secondary suppressor mutation that, under certain growth conditions, prevents it from serving as a sensitive receptor to the “foreign” protein (Friedman and Margolin, 1968). c. Dawson and Smith-Keary (1960, 1963 ; Smith-Keary, 1960) have described a curious system of suppression that could involve elimination of a “toxic protein” that exerts direct inhibition. Mutations in a short genetic region closely linked to the SaZmoneZla leucine operon (Margolin, 1963) suppress only two ZeuA mutants out of 26 leu mutants tested (including an unknown number of ZeuA mutants). Although these suppressors have not been disproved as second-site reversions, one of the suppressor mutations may be a deletion, indicating the possibility of a distinct suppressor locus adjacent to the ZeuA gene (Dawson and Smith-Keary, 1960, 1963). Unfortunately, the map position of the suppressor region is not known in relation to the operator-promoter region of the leucine operon, also adjacent to ZeuA (Margolin, 1963; Burns

34

PHILIP E. HARTMAN AND JOHN R. ROTH

et al., 1966; Calvo et al., 1969a,b). The product of the suppressor locus could be a feedback regulatory subunit that specifically inhibits altered proteins of the two suppressible mutants. The product coded by the wild-type l e d gene, isopropylmalate synthetase (Fig. lo), is highly labile in the absence of substrate and is feedback-sensitive to leucine (Jungwirth et al., 1963; Burns et al., 1966). Loss of feedback inhibition is deleterious (Calvo and Calvo, 1967), and its loss might be selected against, leading to mutational instabilities (cf. Dawson and SmithKeary, 1963; Smith-Keary and Dawson, 1964). Reinvestigation of the suppressors from a biochemical point of view would seem worthwhile, and material for a more definitive genetical analysis also is currently available (e.g., Calvo et al., 1969a,b, 1971). d. Cell division is impaired on media containing high levels of carbon source in Salmonella containing mutations that engender excessive amounts of the histidine biosynthetic enzymes (Roth and Hartman, 1965; Murray and Hartman, 1972). Also, bacteria with high enzyme levels cannot grow at 42OC on minimal medium (Voll, 1967). The pleiotropic effects have been traced to overproduction of normal products of the hisH and hisF genes. These two proteins appear to act in concert, and neither presence of substrate nor ability to carry out the normal catalytic activity is requisite to their inhibitory effects (Murray and Hartman, 1972). Mutations in the histidine operon that lower levels of either hisH or hisF proteins suppress these pleitropic effects. Some of these suppressor mutations lead to a histidine requirement (Fink et al., 1967) while others do not (Voll, 1967); all are in genes H or F or exert polar effects on one or both of these genes. Suppressors with similar effects on enzyme levels (and on cell division) also occur in the gene specifying histidyl-tRNA synthetase (Wyche, 1971). A second type of suppression is exerted, namely, by mutations that do not alter the levels of the inhibitory proteins but, rather, affect some other cell process (smo mutations: Z. Ci6sla and T. Klopotowski, cited in Sanderson, 1970). Perhaps suppressors a t the smo loci alter the sensitive cellular site(s) where inhibition is exerted. One smo class is tightly linked to mutations in the histidyl-tRNA synthetase gene, indicating that this gene product may be part of the inhibitory complex (Wyche, 1971). On two occasions mutants have been detected with abnormal cell division in the presence of normal histidine enzyme levels (Fankhauser, 1971; Savic, 1972). The retarded cell division can be alleviated by mutations that lower histidine enzyme levels below the normal (Fankhauser, 1971; B. Ely, personal communication). Analysis of these and other mutants of generally similar nature (wrk mutations; T. Klopotowski,

SUPPRESSORS

35

cited in Sanderson, 1970) may assist in analyses of the bacterial cell division mechanism. e. Nash (1965, 1970) observed that the dominant mutation Hairless (H) in Drosophila is suppressed by deletion of a gene, S u ( H ) , and enhanced by duplication, E ( H ) , of the same gene. Both H and S u ( H ) are lethal when homoaygous. Suppression is effected through deletion of only one of two homologs of the S u ( H ) gene and enhancement also has dominant effects. Interpretation is further restricted by the observation that homoaygous E ( H ) / E( H ) ; H+/H+flies are phenotypically wild type. One possibility is that H specifies a protein that has lost normal regulation (Nash, 1965) or has widened specificity allowing production of an illicit metabolite. The S u ( H ) +gene, then, would be involved in a dosage-dependent role in control of H activity or in the metabolism of the illicit product to a compound active in causing the Hairless phenotype. A study of the interactions of S u ( H ) with various mutants already known to interact with Hairless (Lindsley and Grell, 1968) may furnish some clues as to the role of the toxic molecule produced through action of the S u ( H ) +gene. Falk (1963) cites experiments indicating that scute (sci) suppresses hairy ( h ) in a manner indicating that scute is involved with synthesis of a structural component whereas hairy exerts a regulatory function. Other cases of dominant suppression of dominant mutations in Drosophila occur (Lindsley and Grell, 1968). f. I n the lactose system of E. coli, certain ‘Lpromoter” mutants are thought to be unable to bind RNA polymerase and initiate transcription efficiently. The effects of these mutations can be partially suppressed and result in increascd enzyme levels by mutations in the i gene, which eliminate repression of thc lac operon (Seaife and Beckwith, 1966) or by mutations in a gene, crp (Plenge and Beckwith, cited in Reznikoff, 1972), that is concerned with dictating the structure of a protein thought to interact directly with a portion of the promoter nucleotide sequence (reviewed in Reanikoff, 1972).

4. Compensating Proteins I n Section I1 (pages 6-10), we mentioned in passing some methods potentially useful for studies of tertiary protein structure, that is, for analysis of interactions between different segments of one polypeptide chain. Since interactions between different macromolecules involve similar forces, the methodology would also seem to be applicable to studies of intracellular macromolecular organization, of cell regulation (examples in Calvo and Fink, 1971; Frieden, 1971) and in dissection of stages in the assembly of complex cell structures.

36

PHILIP E. HARTMAN AND JOHN R. ROTH

I n the section below we cite studies indicating that a type of suppression occurs which also has particular potential in analysis of macromolecular architecture in vivo. I n these cases, modification in one protein (,‘A”) serves to suppress a modification in another protein (“B”). Suppression occurs because functional interaction of modified A and modified B can occur whereas wild-type A protein is unable to form a functional complex with modified B. a. A system where protein-protein interactions are highly critical in assembly and function is the ribosome (for reviews, see Spirin, 1969; Nomura, 1970, 1972; Kaji, 1970). The effects of foreign molecules and of mutations on ribosome function and on the fidelity of protein synthesis have been extensively reviewed (Gorini and Beckwith, 1966; Davies, 1969; Schlessinger and Apirion, 1969; Nomura, 1970; Gorini, 1970; Pestka, 1971). Two examples can be briefly cited regarding genetic suppression. Two proteins, P13 and P5, exhibit cooperative binding to ribosomal RNA during ribosome assembly; a mutant P5 protein can suppress the defect in a mutant P13 protein by assisting in its binding and allow proper ribosome “maturation” (Nomura, 1970). I n the second case, a mutant P4a protein can rectify restraints placed upon ribosome function by mutant P10 protein. P10 protein, dictated by the strA gene, is involved with the interaction of aminoacyl tRNA with the ribosome and facilitates inhibition by various antibiotics such as streptomycin. Mutations at various positions in the strA gene lead to functional resistance and even to dependence on streptomycin or other antibiotics (Momose and Gorini, 1971). Drug dependence is suppressed by mutation in the gene, rum ( = ribosomal ambiguity), specifying a second ribosomal protein, P4a (Birge and Kurland, 1970; Deusser et al., 1970; Kreider and Brownstein, 1971). Analysis leads to the conclusion that mutant P4a protein engenders a high level of misreading (mistakes in translation) and releases some restriction on translation imposed by the defective P10 (strA) protein (Bjare and Gorini, 1971; Zimmermann e t al., 1971a,b). Mutations in another gene, for ribosomal protein P4, also suppress streptomycin dependence (Kreider and Brownstein, 1972). Genetic studies may be supplemented by other methodology, for example by chemical cross-linking of pairs of ribosomal proteins (Chang and Flaks, 1972) as well as reconstitution experiments (Nomura, 1970, 1972). [Also consult Davies and Stark (1970) .] b. Mutants of the gene for the lactose repressor counterbalance operator-constitutive mutations (J. Sadler, cited in Reanikoff, 1972). Presumably, the normal repressor protein has lowered affinity for the altered operator nucleotide sequence, but this affinity can be regained by modification of the attaching repressor protein. This is one method of deter-

SUPPRESSORS

37

mining which portions of the repressor polypeptide are involved in the DNA-protein interaction, complementing other genetic methods (cf. Platt et al., 1972; Pfahl, 1972). c. The suppression by interacting gene products may influence both ease of assembly of phage-specific proteins and the “specificity” of the resulting mature product, the intact phage particle. Mutations at many loci scattered over the phage genome were early shown by Baylor and co-workers (Baylor et al., 1957; Baylor and Silver, 1961) to influence adsorption of T2 phage. They concluded that many ((separate protein components . . . incorporated in different positions in the external phage coat influence host range of the phage although the main specificity resides in the product of the h gene.” Further instances of suppression of h mutants are described on pages 1&11 and 66. d. Genes 0 and P are suspected jointly to control a n endonuclease activity in lambda phage (Freifelder and Kirschner, 1971). In a cleverly designed experiment specifically set up to see whether the two gene products cooperate directly, Tomizawa (1971) provided genetic evidence that there is indeed interaction in the formation of a functional molecule. Tomizawa found that the temperature-sensitivity of lambda replication due to a mutation in gene 0 was eliminated by a mutation in gene P . Presumably, the altered P product interacted with the thermolabile 0 product to yield a temperature-resistant complex. e. Ito (1972) has provided analysis of interactions between compensating and noncompensating mutations in the genes for tryptophanyl tRNA synthetase ( t r p S ) and the tryptophan repressor protein ( t r p R ), indicating that these two proteins may interact directly in vivo. Obviously, analyses of the types mentioned above form a powerful adjunct to other methodology in the detection and study of macromolecular interactions involved in the formation of aggregates, structures, and the performance of cellular functions.

C. SUBSTITUTE PROTEIN ACTIVITY Alternate pathways and protein components are one commonly postulated mechanism of suppressor gene action. The suppressor mutation opens up an alternative biochemical pathway or alters a protein to widen its function so that it may serve in a capacity it is unable to serve in the wild-type organism. Or, regulation of enzyme production may be altered so that new functions may be served without actual alteration of catalytic activity. In such cases one would expect that ordinarily many mutant alleles of the suppressor locus would be effective in suppressing many or all alleles of the locus whose mutant phenotype is suppressed. Even deletions of the locus might be effectively suppressed.

38

PHILIP E. HARTMAN AND JOHN R. ROTH

The suppressors are thus expected to be “locus-specific” in their mode of action, and in fact the ability to be suppressed has constituted one criterion of allelism (Houlahan and Mitchell, 1947; Finck et al., 1965). This characteristic may not be unique to bypass situations; for example, it is also possible to obtain ‘Llocus-specific” suppressors that decrease deleterious accumulations (see Section 111,D) . The following examples illustrate some cases where metabolic lesions are overcome through the action of suppressor mutations that often are “locus-specific” and lead to a supply of substitute protein component (s) . I n terms of biochemical mechanisms, there are several quite different modes of action by which the phenotypic damage may be corrected. 1. Overflow in Channeled Pathways

The flow of metabolites often occurs in channeled pathways; that is, intermediates in the channeled pathway are not free to interact with other pathways. As a consequence of mutation the barrier between pathways may break down and thus lead to correction of the effects of genetic blocks. Analyses of these systems has proved valuable for the information they have given concerning channeling mechanisms and the evolution of distinctive control mechanisms. The discussion will focus on microorganisms, but it appears probable that similar phenomena exist in higher organisms, for example in Drosophila (see, e.g., Bahn et al., 1971). a. Arginine-Pyrimidine Pathways in Fungi. Studies on the mode of action of one series of suppressor mutations has afforded an especially well-documented example described below (also reviewed by Davis, 1967; Reissig et al., 1967; O’Donovan and Neuhard, 1970). I n these studies, analysis of the first suppressor mutation found in Neurospora crassa (Houlahan and Mitchell, 1947) has afforded interesting insights into metabolic flow in vivo (see Davis, 1972). N . crassa contains two enzymes active in the synthesis of carbamyl phosphate (CAP). The first is an enzyme responsible for CAP used in arginine synthesis (Davis, 1963, 1965a,b), and the second is a CAP synthetase specific for pyrimidines (Davis, 1967; Reissig et al., 1967; Williams and Davis, 1970). Figure 11 shows that mutations in the arg-W and arg-J loci block formation of CAP used in arginine biosynthesis ( CAPARG) and certain mutations ( p y r - 3 and pyr-Ja) in the pyr-3 gene block formation of CAP used in pyrimidine biosynthesis (CAPPYR). Since the respective mutants require only arginine or only pyrimidines, respectively, the two pathways must be channeled; CAP made for one pathway is not used in the other pathway. The mechanism of channeling is unclear but could involve as one com-

39

SUPPRESSORS

ponent the bifunctional nature of the pyr-J gene product. Gene pyr-3 dictates the structure of an enzyme that carries out two consecutive steps in pyrimidine biosynthesis: the synthesis of CAPPyRand the synthesis of ureidosuccinate from CAPPYRand aspartate (aspartate transcarbamylase = ATCase in Fig. 11). Pyr-Sa mutants lack CAPPYReynthetase (Finck et al., 1965; Davis, 1967; Reissig et al., 1967; Williams and Davis, 1970) and have a kinetically altered ATCase (Hill and Woodward, 1968) ; pyr-Jd mutants are partially or totally defective in ATCase (Davis, 1960; Davis and Woodward, 1962; Reissig, 1963a,b; Woodward and Davis, 1963) ; and mutants designated pyr-3 lack both activities. Perhaps in vivo (Bocterio) AOose

..**

’..** a*

... ........ . ...

GLUTAMATE>ORN>I ocetylglulamote OGTose (Fungi)

.

‘,

ocetylqlutarnic-ysernioldehyde AOTose ocetylorniihine

-

Pro-3”

,;?ern

qlutomic ioldehyde

*&Aose

prol- I pyrroline- 5 - -PROL corboxylote

........................................................

arg- I2 org- I OTCose~it ru Iline -orginosucc

AOose

I NE qrginose

....

i nate-AKINlNE

(bacteria)

CAPARG

t

HCOAT P

ATCase

ureidosuccinote---URI Pyr- I

DY LATE

AS PA^ FIQ. 11. Some aspects of proline, arginine, and pyrimidine metabolism in Neurospora and bacteria. Gene loci are italicized (e.g., p d - l ) , and enzyme names are abbreviated (e.g., AOase). Heavy dotted arrows indicate routes of overflow accounting for suppression in particular cases (consult text).

much of CAPPYRis enzyme-bound and can thus be channeled specifically into pyrimidine biosynthesis by rapid interaction with aspartate (Davis, 1965b, 1967 ; Reissig et al., 1967). Genetic studies on mutants of the pyr-3 gene indicate that i t dictates the structure of a single species of polypeptide chain with ATCase activity localized in the C-terminal portion (Radford, 1969a,b, 1970a,b, 1972). The two enzyme activities copurify (Williams e t al., 1970). Mutations affecting CAP utilization in one pathway can serve t o suppress mutations that decrease CAP synthesis in the second pathway. This genetically controlled suppression operates by creating a CAP “overflow,” connecting the otherwise channeled pathways. Suppressors of pyr-Ja mutants (Houlahan and Mitchell, 1947) occur in the arg-18 gene and have reduced ornithine transcarbamylase (OTCase in Fig. 11)

40

PHILIP E. HARTMAN AND JOHN R. ROTH

activity (Davis, 1961, 1962a,b; Davis and Thwaites, 1963; Woodward and Schwarz, 1964). Davis (1961, 1962a,b) showed that one suppressor mutation in arg-12 leads to a 98% reduction in OTCase activity with no change in growth rate in the absence of the end product, arginine. The residual OTCase activity is sufficient to maintain a supply of arginine adequate for growth but leads to an accumulation and overflow of CAPARQ into the pathway of pyrimidine biosynthesis. Similarly, mutations in the pyr-J gene of the pyr-Jd type lead to accumulation and overflow of CAPPYRand thus serve as a dechannelizing mechanism, suppressing arg-2 and arg-3 mutants (Reissig, 1960, 1963a,b ; Davis and Woodward, 1962; Reissig et al., 1965; McDougall et al., 1969). Reissig et al. (1965) showed that pyr-Jd mutants are effective in suppression if they contain 60% or less of the normal ATCase activity (and retain CAPPYRsynthetase activity), whereas a growth requirement for pyrimidine is not expressed until mutation has reduced the ATCase level to below about 25% of wild-type specific activity. A differential in minimum enzyme level leading to suppression and to pyrimidine requirement also can be inferred by the ability of an arg-lf?/arg-12 pyr-bd heterokaryon to grow on minimal medium (Reissig, 1958). The degree of suppression is a function both of the amount of reduction of ATCase level and the maintenance of CAPPYRactivity of the mutant bifunctional enzyme protein (Williams and Davis, 1970). Alterations in the K , for aspartate of the altered ATCase proteins (Jobbhgy, 1967) also probably are important. A most important factor in channeling appears to be compartmentation of the duplicate enzymes (Bernhardt and Davis, 1972), Histochemical tests show that OTCase is located in the mitochondria while ATCase may reside in the nucleus. Since each enzyme is associated with its respective CAP synthetase, intracellular segregation of the two pathways serves to allow distinctive metabolic flow. This factor, plus the possible sequestering of CAP as an enzyme-bound intermediate also compartmentalized in the cell (Williams et al., 1971) certainly must be a promi.nent factor in channeling. A number of accessory observations is consonant with the above interpretations of suppressor action. Suppressed pyr-3u mutants are hypersensitive to arginine inhibition (Houlahan and Mitchell, 1947) since CAPARQsynthetase activity is repressed by arginine (Davis, 1965b; Thwaites, 1967). One arginine-insensitive derivative has been indicated as having a reduced rate of arginine assimilation (Thwaites, 1967; Thwaites and Pendyala, 1968), presumably due to mutation in a transport system for basic amino acids (cf. Roess and De Busk, 1968).

41

SUPPRESSORS

Similarly, suppression of citrulline sensitivity (which gives rise to arginine, see Fig. 11) appears to be due to mutations affecting permeation of citrulline by a permease system primarily serving to transport neutral and aromatic amino acids (Thwaites et al., 1970). These examples illustrate how new classes of mutations of primary interest may be obtained as suppressors for other systems. There is a small amount of normal overflow in the direction CAPPYR to CAPARG,as evidenced by slight residual growth (“leakiness”) of arg-S mutants. This residual growth is eliminated by inclusion of uridine in the medium (Charles, 1962, 1964; Davis, 1963; Reissig et al., 1967). Exogenous uridine also serves to decrease the strength of suppression of arg-2 by pyr-Sd mutations (Reissig et al., 1967). These effects are due to repression and feedback inhibition of the pyr-S bifunctional enzyme protein by uridine triphosphate (Donachie, 1964a,b; Davis, 1965b; Caroline and Davis, 1969; Williams and Davis, 1970). Overflow can be secured in the absence of suppressor mutations in high CO, atmospheres (Charles, 1962, 1964). The bifunctional ATCase of the pyr-S gene is induced by CO, (Nazario and Reissig, 1964), and suppression of arg-2 by pyr-Sd mutants is dependent upon the presence of CO, (Reissig et al., 1967). Thus, this form of phenotypic suppression, like genotypic suppression, appears to rely upon mechanisms inducing overflow. Similarly, a-aminobutyrate supports growth of pyr-Sa mutants by inhibition of OTCase activity (Fig. 11),again leading t o dechanneling (Fairley, 1954; Fairley and Wampler, 1964; Charles, 1964). There are further suppressor genes for pyr-Sd mutants aside from arg-12 (McDougall and Woodward, 1965) ; all may act by inducing CAPARGoverflow. One class restricts ornithine production, limiting its coupling to CAPARG and thus releasing CAPARG to overflow. For example, mutants arg-4 and arg-7 suppress pyr-Sa mutants (Mitchell and Mitchell, 1952a; McDougall and Woodward, 1965) and are deficient in ornithine-glutamate transacetylase activity (Vogel and Vogel, 1965) (OGTase in Fig. 11). In mutants of this type, addition of ornithine to the medium inhibits growth (Mitchell and Mitchell, 1952a; McDougall and Woodward, 1965). Still other suppressor pyr-Sa combinations are stimulated by exogenous ornithine (McDougall and Woodward, 1965) ; these strains could have readily inducible ornithine transaminase (OTAase in Fig. 11) so that the actual intracellular ornithine level is lower than normal. In addition, it is possible that some suppressors affect other control mechanisms. ATCase is repressible (Donachie, 1964a,b; Davis, 1965b; Caroline and Davis, 1969; Williams and Davis, 1970) and is influenced by CO, tension (Charles, 1962, 1964; Nazario

+

42

PHILIP E. HARTMAN AND JOHN R. ROTH

and Reissig, 1964; Reissig et al., 1967) while the later enzymes in pyrimidine synthesis may be substrate inducible (Caroline and Davis, 1969) ; a t least some enzymes involved in ornithine formation appear to be repressible (Vogel and Vogel, 1965). Coprinus radiatus also contains a bifunctional ATCase, channeled CAP pools, and suppressor mutations similar in action to the Neurospora arg-13 mutations (Cabet et al., 1965; Hirsch, 1968; Gans and Masson, 1969). This could be the case also in Drosophila (Bahn et al., 1971). Saccharomyces cerevisiae contains an arginine-specific CAP synthetase and a bifunctional pyrimidine CAP synthetase-ATCase with regulation similar to that of Neurospora (Lacroute, 1964, 1968; Lacroute et al., 1965; Kaplan et al., 1967, 1969) ; however, there is only weak channelling of CAP. The only Saccharomyces mutant described to be specifically deficient in CAPPYRsynthesis was originally isolated as a double mutant, grows on minimal medium when separated from the second mutation, and is only slightly inhibited by arginine (Lacroute et al., 1965; Lacroute, 1968). Thus, CAP overflow appears to occur in the absence of suppressor mutations in Saccharomyces although some metabolic compartation occurs (Lue and Kaplan, 1970). CAP synthetases of other organisms are reviewed elsewhere (O’Donovan and Neuhard, 1970; Williams et d., 1970; Gots, 1971; Jones, 1971). I n Section 111, C, 2, b, we discuss suppression of CAP synthetase by an entirely different mode of action in E. coli. b. Arginine-Proline Pathways in Fungi. Channeling in the arginine and proline pathways (Fig. 11) also may be connected by overflow and consequent creation of a “new” pathway that circumvents a genetic block. I n wild-type Neurospora, proline is synthesized from glutamate without appreciable flow of ornithine into proline biosynthesis (Vogel and Bonner, 1954; Abelson and Vogel, 1955; Yura and Vogel, 1955, 1959; Yura, 1959; Vogel and Kopac, 1959; Davis, 1968). Mutations in the arg-8 and arg-9 loci (now termed pro loci: Barratt and Radford, 1970) lead to inability in the conversion of glutamate to glutamic-ysemialdehyde (Vogel and Bonner, 1954; Vogel and Kopac, 1959) and those a t pro-1 are blocked in the last step of proline biosynthesis (Yura and Vogel, 1955, 1959; Yura, 1959). Suppressor mutations a t the arg-12 locus that are effective in suppressing pyr-Ja mutants also suppress arg-8 and arg-9 mutants (Mitchell and Mitchell, 1952a; Barry and Marsho, 1968). The accumulated ornithine in arg-12 mutants induces an enzyme, ornithine transaminase (OTAase in Fig. l l ) , that converts ornithine to glutamic-y-semialdehyde (Fincham, 1951, 1953; Fincham and Boulter, 1956; Vogel and Kopac, 1959; Castafieda et al., 1967; Davis, 1968). The arg-8 and arg-9 mutants are slightly “leaky” and

SUPPRESSORS

43

this leakiness as well as suppression by mutations in the arg-12 gene is eliminated in mutant,s lacking OTAase activity (Davis and Mora, 1968; Davis, 1968). Thus, accumulation and subsequent induction of a normal nonessential catabolic enzyme (Castaiieda et al., 1967; Davis and Mora, 1968; Davis, 1968) serve to join two pathways that are well-channeled in wild-type Neurospora. A suppressor mutation in the arg-12 gene can simultaneously suppress a pyr-Sa and an arg-8 mutation (Mitchell and Mitchell, 1952a), the former by CAP overflow and the latter by ornithine overflow. One important mechanism behind channeling is the intracellular compartmentation of enzymes and substrates. AOase, endogenous ornithine, and OTCase (Fig. 11) in wild type are predominantly located in the mitochondria whereas OTAase is a soluble enzyme located outside of the mitochondria (Weiss and Davis, 1972). This compartmentation probably plays a major role in the channeling of ornithine described above and explains some accessory observations reported below. The arg-8 and arg-9 blocks (pro-3) can be phenotypically circumvented by addition of exogenous ornithine, citrulline, or arginine, accounting for their designation as “arg” loci (Srb et al., 1950). Both exogenous ornithine and arginine induce OTAase activity, and arginine also induces an arginase that hydrolyzes arginine to ornithine and urea (Mora et al., 1966; Castaiieda et al., 1967; Davis e t al., 1970; Morgan, 1970). Arginine also probably inhibits OTCase (Messenguy and Wiame, 1969) leading to further accumulation of ornithine. Exogenous ornithine is used for proline synthesis even in wild-type Neurospora (Abelson and Vogel, 1955; Vogel and Kopac, 1959) owing to induction of this very active OTAase activity that utilizes ornithine before it can be converted to citrulline (Davis and Mora, 1968). Ornithine may also inhibit the glutamate to glutamic-7-semialdehyde reaction (AnderssonKotto and Ehrensvard, 1963) and thereby assist flow of ornithine into the proline pathway. Arginase :pro-3 double mutants can be suppressed, but it is not known whether these suppressed mutants have constitutive or hyperactive OTA activity, are leaky arg-12 mutants as discussed above, or are mutants having increased synthesis of ornithine (Morgan and Shaw, 1970). Other aspects of the channeling of ornithine in Neuiosporn are discussed by Davis (1967, 1968, 1972). Aspergillus nidulans mutants analogous to the N . crassa arg-8 and arg-9 mutants carry mutations a t the closely linked pro-1 and pro-3 loci (Forbes, 1956; see also Forbes, cited in Kafer, 1958; Weglenski, 1966). Mutations at these two proline loci are suppressed by mutations at a number of suppressor loci (Forbes, 1956; see also Forbes, cited in Kafer, 1958; Weglenski, 1966, 1967). One suppressor locus is involved

44

PHILIP E. HARTMAN AND JOHN R. ROTH

with the specific activity of OTCase and the mechanism of suppression is like that of the Neurospora arg-12 suppressors mentioned above (Weglenski, 1967). Mutations a t six other suppressor loci do not affect OTCase activity but produce altered regulation of arginase and of OTAase activity (Weglenski, 1967; Piotrowska et al., 1969). The effect of these suppressor mutations appears to be exerted by the constitutive or increased presence of catabolic enzymes that divert ornithine (and arginine, via ornithine) into the proline pathway. c. Arginine-Proline Pathways in Bacteria. The arginine and proline pathways of E . coli (cf. H. J. Vogel, 1953; R. H. Vogel et al., 1971) and S. typhimurium have been joined by suppressor mutations to circumvent genetic blocks in proline biosynthesis, but the mechanism of overflow differs from that just described in Fungi. I n the case of the bacteria, a deficiency in acetylornithine transaminase (AOTase in Fig. 11) leads to accumulation of acetylglutamic-y-semialdehyde,and this is deacylated by acetylornithinase (AOase) to yield glutamic-7-semialdehyde, a precursor of proline. Bacon and Vogel (1963a) found that mutants blocked in conversion of glutamate to glutamic-y-semialdehyde [comparable to the pro-3 (arg-8 and arg-9) mutants of Neurospora] require proline for growth whereas a second genetic lesion eliminating acetylornithine transaminase activity (AOTase) restores growth on minimal medium. The interpretation of the mechanism of suppression is that the double mutant accumulates acetylglutamic-y-semialdehyde and that this overflows to supply glutamic-y-semialdehyde sufficient for proline biosynthesis. Arginine is supplied a t a slow rate by the low activity of some other transaminase with AOTase activity (Bacon and Vogel, 1963b; Albrecht and Vogel, 1964). This second transaminase is now suspected to be an enzyme that is present at low levels in wild-type strains but is induced by arginine to high levels in other strains (Vogel et al., 1963, 1967; Bacon and Vogel, 1963b; Jones et al., 1965,1966). The Salmonella proA and proB loci are concerned with the synthesis of glutamic-y-semialdehyde (Kanazir, 1956; Miyake and Demerec, 1960; Itikawa and Demerec, 1968). Suppressor mutations have been detected that suppress mutations both in proA and in proB loci, including deletions that encompass both of these adjacent genes; the suppressed mutants are hypersensitive to inhibition by arginine (Itikawa et al., 1968; Kuo and Stocker, 1969). The arginine-sensitivity can be attributed to four effects. First, in some strains arginine induces a protein with AOTase activity (Vogel et al., 1963; Bacon and Vogel, 1963b; Jones et al., 1965, 1966) ; this would tend to eliminate the pile-up of the precursor, acetylglutamic-y-semialdehyde, that occurs when the normal, biosynthetic AOTase is absent. Second, arginine represses the arginine biosynthetic

SUPPRESSORS

45

enzymes, including those responsible for the synthesis of acetylglutamicy-semialdehyde (Baich and Vogel, 1962; Vyas and Maas, 1963; Vogel et al., 1963). Third, arginine use is partially channeled (Sercarz and Gorini, 1964; Tabor and Tabor, 1969). Finally, it appears that one of the arginine biosynthetic enzymes, acetylornithinase (AOase in Fig. 11) , is responsible for the conversion of the accumulated acetylglutamic-ysemialdehyde to the proline precursor, glutamic-7-semialdehyde.AOase acts on acetylglutamic-y-semialdehyde a t about 3% of the rate that it acts upon its normal substrate, acetylornithine (Itikawa et al., 1968). Since AOase is repressed by arginine (Vogel, 1957, 1961; Maas, 1961; Vogel et al., 1963; Itikawa et al., 1968), suppressed strains that require extended activity of this enzyme would be sensitive to arginine repression. Mutations to constitutivity of the arginine enzymes relieve the arginine sensitivity (Itikawa et al., 1968) ; suppression of this arginine sensitivity thus allows a direct selection for arginine regulatory mutations. The experiments cited above, and additional ones (Bacon and Vogel, 1963a) support the view that suppressor mutations retarding arginine biosynthesis create a new flow of metabolites that circumvent a genetic block early in the pathway of proline biosynthesis. 2. Alternate Proteins

I n the cases discussed above, “locus-specific” suppressor mutations lead to an altered flow of metabolites and essentially create new metabolic pathways carried out by enzymes common to wild-type organisms. Sometimes the pathways are quite novel as major pathways; sometimes they mimic the same sequence of biochemical steps normally functioning. I n each instance, suppression results from substitute proteins, similar to cases described below. And again, in the cases we describe next, there is some locus specificity in suppression. Morgan et al. (1925) and Schultz and Bridges (1932) were first to show that some cases of suppression in Drosophila result from shifts in gene arrangement; a wild-type locus translocated to a new position merely serves to “suppress” recessive alleles of that same locus in the proper genetic background. Muller and Oster (1957; Oster et al., 1958) describe observations suggesting that a particular suppressor of forked (Pih) also is due to actual translocation of the forked genes, although other explanations could be suggested (see p. 68). As pointed out by Wagner and Mitchell (1955, 1964), cases of “duplicate genes,” so often found in plant hybrids, could have origins stemming from translocations that occurred during the evolutionary separation and divergence of the strains involved.

46

PHILIP E. HARTMAN AND JOHN R. ROTH

In addition to transposed nucleotide sequences of reasonably recent vintage of separation, evolutionarily separate but functionally “duplicate” genes and “isozymes” are exceedingly common in microorganisms (reviewed by Datta, 1969; Umbarger, 1969; Mortimer, 1969; Mortimer and Hawthorne, 1966), plants (citations in Wagner and Mitchell, 1955, 1964) and animals (Markert, 1968; Harris, 1969; Wagoner, 1969; Ohno, 1970; Manwell and Baker, 1970). Most often, these “duplicate genes” are not truly duplicate; during evolution their substrate specificity, enzyme kinetics, or regulation has diverged (examples in Vessel1 et al., 1968). Sometimes the substrate specificity overlaps fairly precisely; in other instances, there is a slight or potential overlap that can be broadened by mutation. Sometimes one of the enzymes functions catabolically and the other anabolically, the two functions being separated by different control mechanisms and possibly also by channeling mechanisms. I n such cases, gene mutations may alter the control patterns or the binding of metabolites so that the one enzyme may serve in place of the other enzyme under conditions where such replacement of function does not normally occur. Such divergence of nonallelic genes is superimposed upon allelic differences which contribute substantially to enzyme polymorphisms (cf. Fincham, 1972). One wonders whether sudden substitution of a homologous protein, perhaps protected and allowed to evolve without the normal mode of selective pressure (cf. Boyer et al., 1969, 1971), is not an important factor in evolution (cf. Ohno, 1970). Additionally, suppressor mutations that channel the flow of metabolites through a second pathway may become fixed in evolution and account for some metabolic differences among species (cf. Tanaka et al., 1967). a. Two types of suppression have been described for mutants of E . coli defective in the enzyme-mediated process of genetic recombination (Clark, 1971). One class of suppressors are defective in an enzyme that appears to interfere with a secondary mode of genetic recombination. A second class of suppressors involves an activation o i derepression of one or a series of enzymes which either directly replace the missing function or participate in an alternate pathway of minor import to wildtype bacteria. In E. coli several genes have been identified whose alteration leads to a deficiency in recombination (Willetts et al., 1969; Willetts and Mount, 1969; Clark, 1971). Mutants of two of these genes, recB and recC, contain a low residual recombination ability and lack an ATP-dependent DNase activity (Wright and Buttin, 1969; Oishi, 1969; Barbour and Clark, 1970; Goldmark and Linn, 1970; Wright et al., 1971; Gold-

SUPPRESSORS

47

mark and Linn, 1972; Nobrega et al., 1972; Tanner et al., 1972). One class of suppressor mutations (sbcA) restores high recombination ability and accessory phenotypic properties (e.g., resistance to ultraviolet light and to mitomycin C) to all strains tested carrying recB or recC mutations as well as to re&-recC double mutants (Barbour e t al., 1970). The sbcA revertants still lack the ATP-dependent DNase but now contain a high level of an ATP-independent DNase, presumably a substitute protein involved with their enhanced ability t o perform genetic recombination. The low residual recombination ability of recB and recC mutants is greatly enhanced by a second class of suppressor mutations (sbcB) that lead to defective DNA exonuclease I activity (Kushner et al., 1971, 1972). The elimination of exonuclease I activity serves in some manner to release otherwise cryptic activity in genetic recombination and in resistance to irradiation. Amplification of the secondary pathway (s) by sbcB suppressors has allowed Clark and co-workers to obtain exonuclease I mutants, which are difficult to identify by direct search. Isolation of further recombination-deficient mutants from recB-re&-sbcB suppressed strains have revealed mutations in genes hitherto unanalyzed from the point of view of the enzymology of the recombination process (Clark, 1971). Thus, the discovery of indirect suppression of rec- mutations greatly broadens the base for study of an involved physiological process critical in genetics. Other instances of suppression and epistasis involving interactions of phage genes or of phage genes with the bacterial rec genes are reviewed by Clark (1971). One case is worth mentioning here, since it superficially resembles the bacterial system described above. Bacteriophage T4 DNA ligase-defective mutants are unable to grow on wild-type host bacteria ; however, suppression (growth) occurs if the phage also carries a mutation in the r I I genes (Berger and Kozinski, 1969; Karam, 1969). The suppressed mutants rely on host ligase for phage growth and exhibit an increase in genetic recombination that is due to alteration of some parameter of the recombination process rather than to alterations in DNA synthesis (Krisch et al., 1971, 1972). It is possible that the rII genes specify a nonessential protein that is a nuclease or affects susceptibility to nuclease activity. T4 ligase-defective mutants also are suppressed by mutations eliminating phage-induced endonuclease I1 (Warner, 1971). b. Action of an alternate protein may explain cases of suppression of pyrA mutants in Salmonella. The pyrA locus of S. typhimurium and the corresponding locus of E . coli dictate the structure of a single enzyme,

48

PHILIP E. HARTMAN AND JOHN R. ROTH

carbamyl phosphate (CAP) synthetase. CAP synthetase, in contrast to fungal systems, supplies CAP for biosynthesis both of arginine and of pyrimidine (CAPARGand CAPPYRin Fig. 11). While a variety of phenotypes are engendered by mutations in the p y r A locus (reviewed by O’Donovan and Neuhard, 1970; Gots, 1971, Toshima and Ishidsu, 1971), the most common is an absolute requirement for the products of the two pathways requiring CAP, namely, arginine and pyrimidine. Yan and Demerec (1965) found suppressors for such mutants, allowing slow growth on arginine or citrulline in the absence of pyrimidine (Fig. 11). Such suppressors were found for 5 of the 15 mutants tested, and crosssuppression was observed between the two mutants tested (Yan and Demerec, 1965). It is possible that the suppressors allow growth when supplied with arginine through the synthesis of CAP adequate for pyrimidines by a second enzyme, carbamate kinase (acetokinase) (cf. Brzozowski and Kalman, 1966). Fungi lack carbarnate kinase and possess . duplicate CAP synthetases (see pp. 3 8 4 0 ) so this suppression mechanism would not be found in Neurospora. On the other hand, the structure and regulation of CAP synthetase activity is extremely complex; it is inhibited by UMP, especially in the presence of glutamine, and it is activated by ornithine and by ATP (Abd-El-A1 and Ingraham, 1969; Trotta et al., 1971; for reviews, see Anderson et al., 1970; O’Donovan and Neuhard, 1970) so that alternate mechanisms of suppression can be visualized. Clearly, additional studies on these mutants would be of interest since the prediction of suppressor activity cited above should be locus specific rather than allele specific. c. Kemper and Margolin (1969) have supplied evidence suggesting that suppressor mutations which arise in certain Salmonella leucine-requiring mutants supply a substitute component that replaces the function of the leuD gene product. In Salmonella and Neurospora, leucine biosynthesis proceeds by the pathway outlined in Fig. 10 (Calvo et al., 1962; Gross et al., 1962, 1963; Jungwirth e t al., 1963; Burns et al., 1963). I n Salmonella isomerase activity is coded for by two adjacent cistrons, EeuC and D [termed leu111 and IV in Margolin (1963) and Gross et al. (1963)], while in Neurosporu two unlinked genes code for the isomerase (Gross, 1962, 1965; Gross et al., 1963; Gross and Webster, 1963). There is strong evidence that the isomerase of Neurospora is a multimeric enzyme composed of two different polypeptide subunits whose structures are dictated by the leu-2 and l e u 3 genes, respectively (Gross, 1962, 1965; Gross and Webster, 1963). A similar situation is likely to exist in Salmonella. It can be proposed that the product of the l e d gene carries significant isomerase activity only when in combination with a polypeptide produced by the leuD gene, a common type of occurrence

SUPPRESSORS

49

in multimeric proteins (cf. Ginsburg and Stadtman, 1970; Trotta et al., 1971). Kemper and Margolin (1969) found that suppressor mutations affecting Salmonella leuD mutations map a t a specific locus, sup& (cf. Sanderson, 1970). Different mutations in sup& suppress the leucine requirement to varying degrees; deletions of the entire sup& locus do not suppress. The suppressors are effective with mutations that lead to the most defective leuD gene products, namely, with deletions of parts or all of the 1euD gene as well as with leuD nonsense mutations. Thus, sup& function replaces leuD function. The suppressors are not effective on mutants of other leu genes, nor are the suppressors active with leuD mutants that produce a protein that is only slightly altered. Failure of suppression in these cases is attributed to the formation of a tightly bound, eneymatically inactive complex of the 1euC polypeptide with defective leuD polypeptide. [Subunit dissociation and reassociation do not appear to take place readily in mixed extracts of leuC and leuD mutants for they exhibit no isomerase activity (Gross et al., 1963).] Sup&, then, could serve to elicit, through mutation, a protein product that replaces the function of normal leuD protein in activating previously uncomplexed leuC polypeptide to functional isomerase. This activation could occur either directly through protein-protein interactions (Kemper and Margolin, 1969) or, perhaps, indirectly by supplying an active metabolite. Due t o the nature of sup& mutations, Kemper (1971) argues that the sup& gene product is normally tied up in a complex with a protein elicited by another gene, gene W , and is released for suppression when W is deleted or inactivated or by particular missense mutations in sup& that alter protein-protein interactions. d. Suppressors of cysA mutants of Salmonella typhimurium appear to supply a novel component active in sulfate and thiosulfate transport across the bacterial cell membrane. Mutants of gene cysA can grow on cysteinc, sulfide, or sulfite as sources of sulfur but cannot grow on sulfate or thiosulfate (Mizobuchi et al., 1962; Dreyfuss and Monty, 1963). The cysA region is comprised of three adjacent cistrons (Mizobuchi et al., 1962; Ohta et al., 1971). Mutants with lesions in each of the three cysA cistrons contain all the enzymes necessary for sulfate assimilation into cysteine (Dreyfuss and Monty, 1963) but lack a component of a transport system necessary for accumulation of sulfate and thiosulfate from the medium (Dreyfuss, 1964). Mutants defective in sulfate transport may be isolated either as cysteine-requiring mutants (Mizobuchi et al., 1962) or as chromate-resistant mutants (Pardee et al.,, 1966; Ohta et al., 1971) since chromate is toxic and taken up by the sulfate permease.

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PHILIP E. HARTMAN AND J O H N R. ROTH

Suppressor mutations isolated for any of a number of cysA mutants partially eliminate the mutant phenotype in all nine cysA mutants tested, including the deletion cysA20 that has lost all three cistrons (Howarth, 1958). The suppressors are specific for cysA mutants. Mutations in the other cys loci tested [the cysBa, Bb, Bc, C, D, Ea, Eb, G, H , I , J loci of Mizobuchi et al. (1962) and Demerec et al. (1963)l are not suppressed (Howarth, 1958 ; Flatgaard and Hartman, 1962 unpublished) ; the suppressors are not linked by P22-mediated transduction with these other cys loci (Howarth, 1958; Flatgaard and Hartman, 1962 unpublished). The suppressors allow cysA mutants to grow either with sulfate or with thiosulfate as sulfur sources, and the suppressors can be selected for on either compound. The suppressor mutations have a deleterious effect in that they approximately double the generation time of wild-type bacteria growing in minimal medium both in the presence and in the absence of cysteine and of cysA mutants growing in the presence of cysteine (Howarth, 1958). Sulfate is unable to passively diffuse into wild-type enteric bacteria at the concentrations normally used in culture media (Pardee, 1957), as also evidenced by the inability of the transport mutants to grow on sulfate. One might hypothesize that the cysA suppressor mutations nonspecifically allow more ready passage. This seems unlikely since some suppressor mutations allow better growth of cysA mutants on sulfate than on thiosulfate while the opposite is true of other suppressors (Flatgaard and Hartman, 1962 unpublished). Some transport-negative cysA mutants, including the cysA20 deletion, still bind sulfate to a specific protein that is located in the cell surface. The protein is repressed by cysteine, is regulated by the same genes active in regulating the cysteine biosynthetic pathway, and is thought to be involved in sulfate transport (Dreyfuss and Pardee, 1965, 1966; Pardee and Prestidge, 1966; Pardee et al., 1966; Pardee and Watanabe, 1968; Ohta et al., 1971). This binding component has been crystallized and some of its properties studied (Pardee, 1966, 1967, 1968). Other cysA mutants, including three nonsense mutants, each defective in one of the cysA complementation groups, exhibit little or no binding activity for sulfate (Ohta et al., 1971). S. V. Shestakov, Pardee, and Hartman (1967 unpublished) found that the four suppressors tested (Howarth’s su-2,-6, -7, and -8) fail to suppress two transport-negative cysA mutants (SP-25 = cysAllSO and SP30 = cysA1131) that contain but low levels of binding protein (Pardee e t aZ., 1966; Ohta et aZ., 19711, whereas these same suppressors are active on deletion cysA20 and a number of other cysA mutants (Howarth, 1958; Flatgaard and Hartman, 1962 unpublished). The cysA

SUPPRESSORS

51

suppressors must restore sulfate transport (cf. Kaback, 1970) by supplying a component that can take the place of the products of the three cysA cistrons. It seems likely that the new component elicited by the suppressor gene (s) cooperates with the sulfate-binding protein in transport to form a novel transport system. e. Mutations blocking transport of organic molecules also are readily reversed by suppressor mutations. One mechanism of suppression can be due to the revelation of alternate, cryptic permemes with incidental activity on a second substrate (cf. Schaefler, 1967; Schaefler and Maas, 1967; Arditti et al., 1968; Lin, 1970; Hofnung and Schwartz, 1971; Saier et al., 1972). I n a second mechanism of suppression, the K , and V,,, of existing permeases may be altered to accommodate the excluded molecule. For example, Neurospora mutants with genetic blocks in the normal tryptophan transport system are suppressed by mutations that increase the affinity for tryptophan of a second, parallel transport system (Stadler, 1967; Brink et al., 1969). I n a third type of situation, a mutant of Salmonella defective in a phosphorylating system for “sugar transport” regained ability to utilize D-mannose as sole carbon source by a suppressor mutation. The secondary mutation resulted in a 25-fold elevation of a mannokinase that was ordinarily present at very low constitutive levels. It was suggested that phosphorylation of mannose activated a latent transport process by facilitating release of mannose from a membrane carrier into the cytoplasm (Saier et al., 1971). I n a somewhat analogous case, Aerobacter mutants were discerned to lack components of the sugar transport system involved in the conversion of mannitol to mannitol l-phosphate. Mannitol l-phosphate is substrate for the only mannitol-specific dehydrogenase in this species of bacteria (Tanaka et al., 1967; Tanaka and Lin, 1967). Growth on mannito1 is restored by a mutation leading to constitutive production of D-arabitol dehydrogenase (DAD). DAD is an enzyme with loose specificity and able to convert mannitol to fructose. The synthesis of DAD is induced by D-arabitol, not by D-mannitol, so that constitutive synthesis is necessary for enzyme production in the presence of mannitol (Tanaka et al., 1967). Penetration of mannitol appears adequate to permit its use as a carbon source once the ‘(new” dehydrogenase is present whereas in the absence of the dehydrogenase, no detectable mannitol accumulation occurs (Tanaka and Lin, 1967). Indirect effects of sugar permease mutants are overcome by still further types of mechanisms. Saier and Roseman (1972) found one type of suppressor mutation in Salmonella that released inhibition of sugar uptake, allowing operation of an alternate mode of transport. I n the case of glycerol utilization, Lids research group has found three classes

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PHILIP E. HARTMAN AND JOHN R. ROTH

of suppressors that specifically allow growth on glycerol of an E . coli phosphotransferaseless mutant unable to grow because of failure t o induce proteins necessary for glycerol uptake and catabolism: (1) mutations eliciting constitutive high production of glycerol kinase and glycerophosphate dehydrogenase (Berman et al., 1970), (2) a suppressor that increases the levels of glycerol kinase and of a protein mediating facilitated diffusion of glycerol yet allows normal repression of these enzymes (Berman-Kurtz et al., 1971), and (3) mutations in the structural gene for glycerol kinase that engender an enzyme no longer sensitive to feedback inhibition by fructose 1,6-diphosphate ; this leads to increased production of glycerophosphate, which then induces even higher levels of the catabolic enzymes (Berman and Lin, 1971). Finally, defects in permease systems allow direct selection for bacterial suppressor mutants with defects in the cell envelope allowing a more ready penetration of substrate across this barrier (Lazdunski and Shapiro, 1972). Such “nonspecific” suppressor mutations should allow analysis of the constitution of the permeability barrier itself. g. Guespin-Michel (1971b) describes two suppressor loci in Bacillus that restore wild-type polymyxin resistance to pleiotropic polymyxinsensitive, sporulation-defective mutants. All mutants blocked early in spore-formation and representing five different loci are suppressed by each of the suppressors. While ability to sporulate is not returned, other secondary manifestations of the pleiotropic mutations (esterase, protease, and antibiotic production ; regulation of nitrate reductase) also are suppressed to varying discrete degrees in a neatly ordered sequence. The various pleiotropic effects of the original mutations also are arranged in this same hierarchy. It is proposed that the suppressors restore lost membrane functions and affect catabolite repression, partially compensating for functions lacking in the primary mutants (Guespin-Michel, 1971b). Indeed, membrane transport systems are intimately tied to the commitment to sporulation (Freese et al., 1970). There are at least two other loci effective in suppressing some of the pleiotropic effects in sporulation mutants (Guespin-Michel, 1971a). h. Giles (1951) described a suppressor locus in Neurospora that mutates to partially relieve the methionine requirements of two nonallelic methionine mutants. The suppression is exerted on alleles of three genes, me-& me-3, and me-7 but not on me-6 (Murray, 1960; Tokuno et al., 1962). Mutants of each of the four genes are leaky (Tokuno et al., 1962; Kerr and Flavin, 1970). It was originally thought that the suppressors, which alone lead to partial methionine requirement, eliminate secondary accumulations by creating a partial block a t the early reaction specified by m e 4 (Tokuno et al., 1962) (see Fig. 12). However, the

53

SUPPRESSORS

suppressor gene maps away from me-6 (linkage group IVR: Murray, 1960) and on linkage group I near albino-2 (Giles, 1951) as does me-6 (Barratt et aZ., 1954; Murray, 1960; Barratt and Radford, 1970). The me-1 and -6 mutants peculiarly appear defective in two steps required for methionine biosynthesis (Selhub et al., 1969; Kerr and Flavin, 1970). Because all me-8, -3, and -7 mutants are leaky and because of the pattern of suppression, it was suggested that the suppressor strain contained an activity (now known as sulfhydrylase utilizing, S= in Fig. 12) that opened up a minor “side pathway” of methionine biosynthesis circumventing the genetic blocks (Flavin, 1963). However, sulfhydrylase H,PteGlu

me-6 H4PieGlun+

+,

me- I 5.1 0-CH2-H4PteGIun+ 5-CH3-H4PteGIun 7

- cystolhionose

cysteine ketoglutorote

Q

cysteine

-

P-cystothionose

homoserine~O ocetylCoA me-5 (me -6)

FIG. 12. Proposed main pathway (heavy arrows) of methionine biosynthesis and accessory reactions (light arrows) in Neurospora crassa.

activity of methionine-grown Neurospora is not significantly increased in me-2 carrying the suppressor, so the suggestion was discarded (Kerr and Flavin, 1970). On the other hand, Wiebers and Garner (1967a,b) found an enzyme activity that could be the same as sulfhydrylase and which was repressed and feedback-inhibited by methionine. If native sulfhydrylase is indeed feedback-sensitive to methionine or to S-adenosylmethionine, as opposed to Kerr and Flavin’s (1970) findings, then the suppressor could act to elicit feedback-resistant enzyme and open up the side pathway (Kerr, 1971). The sulfhydrylase pathway has been suggested as th2 normal major pathway in yeast (Cherest et al., 1969), but this is questioned (Kerr and Flavin, 1970; Savin and Flavin, 1972). Alternatively, the suppressor could release for biosynthesis cryptic y-cystathionase and p-cystathionase (cystathionine-p-synthase in Fig.

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PHILIP E. HARTMAN AND JOHN R. ROTH

12), activities that normally appear, for example, only on low sulfur medium. Such a 7-cystathionase activity was found by Flavin and Slaughter (1967) ; this and several other enzymes of sulfur metabolism are jointly under the control of two regulatory genes, cys-S and scon (Burton and Metzenberg, 1972). I n addition, Fischer (1957) found both y- and p-cystathionase activities “returned” in suppressed mutants. The observation of sulfur flow from S-methylcysteine into cysteine in the presence of high sulfate when the suppressor gene is present but not in the absence of the suppressor (Wiebers and Garner, 1964) and the failure of cys-S mutants to grow on S-methylcysteine (Burton and Metzenberg, 1972) also are consistent with this idea. One simple prediction is that me-S, -7, and -2 mutants will be leakier on low than on high sulfate or cysteine media. In any event, a “side shunt” seems to operate to circumvent and thus suppress genetic blocks in methionine biosynthesis. Analysis of methionine suppressors (as well as the sfo suppressors described in Section 111, D, d) should allow better discernment of sulfur flow in Neurospora. The reduction of selenite (Zalokar, 1953) would seem a “marker” useful in the genetical portions of these analyses. Five recessive methionine suppressor loci have been detected in Coprinw (Lewis, 1961) and a t least six suppressor loci, only one of which yields dominant suppressors, in Aspergillus (Lilly, 1956; Gaj ewski and Litwifiska, 1968). The opening of side shunts could be a common phenomenon in eukaryotes. There are duplicate pathways (“conditional” pathways) that often are opened by mutation or unique cultural conditions (cf. Ulane and Ogur, 1972). i. Gots and Gollub (1963) discovered a suppressor that relieves the purine requirement of all purl3 (adenylosuccinase) mutants in Salmonella (cf. Gots, 1971). The suppressor strains produce a substitute deacylase of unknown primary function that is immunologically distinct and fractionates differently from wild-type adenylosuccinase. The same research group (Benson et at., 1972) also found evidence for two phosphoribosyltransferases in Salmonella. Deletion of the gene, gxu, coding for the transferase most specific for guanine and xanthine could be suppressed by mutation in the gene coding for the adenine-hypoxanthine specific transferase, allowing for its more efficient utilization of guanine. j . Slonimski and co-workers (Sherman and Slonimski, 1964; PBre et al., 1965) describe a yeast mutant, cy 1-1, unable to grow on lactate even though containing lactate dehydrogenase, but completely devoid of the normally preponderant cytochrome c, iso-l-cytochrome c (iso-1) . Mutation cy 1-1 is an extensive deletion of the structural gene for iso-1

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(Parker and Sherman, 1969; Clavilier et al., 1969) yet cy 1-1 readily mutates to growth on lactate. Among a small sample of revertants examined, mutations a t five unlinked loci were detected. Each leads to growth on lactate and contains greatly increased amounts of a protein homologous to iso-1, namely iso-2-cytochrome c (PBre et aZ., 1965; Clavilier et al., 1966, 1969). Each of these “compensator genes” is considered to be an independent locus, but a t least one is probably due to a translocation of the C Y 2 gene plus a mutation within the gene (Clavilier et al., 1969). Perhaps all three loci, whose members are infrequently found in diploid strains and are dominant or semidominant, are translocations of this type. The other two suppressor genes, selected for in haploids where suppressor mutations are found with high frequency and which are recessive, may involve mutations in “regulator genes” (Clavilier et al., 1969). In any event, i t is clear that in all the above cases a normally minor protein species, iso-2, has come to substitute in activity for the normally predominant species, iso-1. This is not surprising, since Mattoon and Sherman (1966) showed that electron transport and oxidative phosphorylation in cytochrome c-deficient yeast mitochondria can be restored by either type of cytochrome in vitro. I n addition, both cytochromes contain a methylated lysine residue (DeLange et al., 1970) that may be critical to activity and respiratory control (see below). Analysis of further cy 1-1 revertants able to grow on lactate but containing no increase in cytochrome c (LAC mutants of Clavilier et al., 1969) should reveal further modes by which the cytochrome c deficiency can be suppressed. Coupled oxidation of lactate in yeast proceeds exclusively by way of, and requires higher levels of, cytochrome c than does oxidation of substrates linked through cytochrome b (references in Sherman and Stewart, 1971). The LAC suppressors may circumvent this peculiar high-level requirement by opening a new channel or by adjusting electron flow through facilitated binding of other cytochromes c present in low concentration. Suppression of cytoplasmic mi-1 (“poky”) mutants in Neurospora might operate through action of substitute systems affecting protein methylation. “Poky”-type strains, of which there are several different isolates (Mitchell and Mitchell, 1952b; Garnjobst et al., 1965; Diacumakos et al., 1965; Griffiths et al., 1968; Bertrand and Pittenger, 1969, 1972), are deficient in cytochromes b and a as, cytochrome oxidase, and contain a great excess of nonparticulate cytochrome c (Haskins et al., 1953; Tissieres et al., 1953; Bertrand and Pittenger, 1969) and fatty acids (Hardesty and Mitchell, 1963). The fatty acid accumulation seems merely a secondary effect (Silagi, 1965). The content of other

+

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PHILIP E. HARTMAN AND JOHN R. ROTH

mitochondrial enzymes has been reported as altered (Haskins e t al., 1953; Tissieres e t al., 1953; Tissieres and Mitchell, 1954; Woodward and Munkres, 1967; Woodward, 1968). Many of these observed differences could be due to secondary effects such as increased lability in poky strains (Edwards and Woodward, 1969; Eakin and Mitchell, 1970). The slow-growing “poky” phenotype is reversed in some strains during continuous culture (Haskins e t al., 1953; Silagi, 1965) or through action of a chromosomal suppressor gene, f = fast (Mitchell and Mitchell, 1956; Griffiths e t al., 1968; Bertrand and Pittenger, 1972). Outgrowth also restores the wild-type cytochrome and respiratory patterns, but the suppressor has no marked effect on the cytochrome content of either poky or wild-type. The suppressor does n o t enhance growth in a second type of cytoplasmic mutants, mi-3, that has a different cytochrome spectrum (Mitchell et al., 1953; Tissieres and Mitchell, 1954; Mitchell and Mitchell, 1956 ; Bertrand and Pittenger, 1972) . Chromosomal suppressor f does not suppress two chromosomal mutations that influence cytochrome content, nor does a suppressor for one of those mutations wppress pokp; in fact, p o k y blocks suppression of the nuclear mutation (Mitchell and Mitchell, 1956). Strains mi-3 and poky do not complement in common cytoplasm but certain strains of the “poky” phenotype do complement (Pittenger, 1956; Gowdridge, 1956; Bertrand and Pittenger, 1972). P o k y also fails to complement a slow-growing cytoplasmic mutant, SG, that has normal cytochrome content (Srb, 1958,1963). An amino acid substitution was reported in a supposedly major protein species of mitochondria, a “membrane structural protein” (Woodward and Munkres, 1966, 1967; Woodward, 1968). The observation of a single major structural protein species in which such a change could be detected has been seriously challenged (Sebald et al., 1968; Ashwell and Work, 1970), and the claim was retracted (Zollinger and Woodward, 1972). Alternative suggestions have been made that p o k y mitochondria lack several minor protein species in normal concentration (Sebald et al., 1968), exhibit an imbalance in synthesis of mitochondrial ribosome subunits (Rifkin and Luck, 1971), and contain altered transfer RNAs (Brambl and Woodward, 1972). Let us simply postulate that poky mitochondria do not bind cytochromes with normal efficiency and contain altered cytochrome oxidase and other undermethylated molecules. This then triggers a multitude of secondary effects. Wild-type Neurospora cytochrome c and cytochrome c in ‘(old” (normal growth) “poky” cultures contain an unusual amino acid, P-N-trimethyllysine, a t amino acid residue number 72 that is present only as lysine in “young” (abnormal) “poky” cultures (Scott and Mitchell, 1969;

SUPPRESSORS

57

DeLange et al., 1969). It is suggested that methylation of cytochrome c changes its binding affinity for proper sites on mitochondria; this binding could modify an otherwise normal respiratory chain and its regulation (Eakin and Mitchell, 1970). Methylation also might protect cytochrome oxidase from damage (see discussion in Edwards and Woodward, 1969) and could be critical to normal cytochrome oxidase function (discussion in Scott and Mitchell, 1969). Suppressor f, then, could merely serve to facilitate modification of cytochromes and other molecules to enhance proper binding and function. An altered route of methylation in suppressor f strains is a possibility. More direct evidence of interaction of cytoplasmic and nuclear factors involved in one-carbon metabolism has been found in yeast (Lowenstein, 1971). The combined tools of genetics and biochemistry appear capable of presenting a detailed picture of factors involved in organelle enzyme content and function (for reviews, see Coen et al., 1970; Linnane and Haslam, 1970; Sherman and Stewart, 1971; King, 1971). k . A case of suppression in Neurospora appears to be due to induction of a catabolic enzyme that replaces the function of its constitutive biosynthetic counterpart. Case, Giles and Doy (1972) found that arom-1 mutants, blocked in the conversion of 5-dehydroshikimic acid (DHS) to shikimic acid and lacking DHS reductase activity could revert by suppressor mutations in a second gene, qa-4. The qa-4 mutations prevent metabolic destruction of accumulated DHS. Accumulation of high levels of DHS leads to induction of a normally catabolic enzyme, shikimic acid dehydrogenase, which supplies shikimic acid from DHS, fulfilling the function of the constitutive reductase. 1. Sometimes suppressors merely operate to create a more efficient flow of metabolites rather than truly circumventing the genetic block. One typical example is cited here. E. coli and Salmonella mutants lacking thymidylate synthetase require 10-fold the expected level of thymine for growth (Cohen and Barner, 1954). Secondary mutants are readily selected with about one-tenth of this requirement (Harrison, 1965; Alikhanian et al., 1966; Okada, 1966; Eisenstark et al., 1968). This partial suppression is due to genetic blocks affecting either of two enzymes in the catabolism of deoxyribose l-phosphate (dR1P) on the degradative pathway for deoxynucleosides (Breitman and Bradford, 1967; Beacham et al., 1968). The second genetic block hestows an enzyme normally used in catabolism, thymidine phosphorylase, with a ready supply of substrate, dRlP, for use in the reverse reaction, that is, in the conversion of thymine to thymidine and prevents the destruction of thymidine. The suppressed strains are sensitive to deoxynucleosides if the genetic block in d R l P catabolism allows forma-

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PHILIP E. HARTMAN AND JOHN R. ROTH

tion of deoxyribose 5-phosphate (dR5P) since dR5P is itself inhibitory (Lomax and Greenberg, 1968) or induces excess phosphorylase and leads to accumulation of another toxic compound, possibly d R l P (Becham et al., 1968; Bonney and Weinfeld, 1971). Further mutants resistant to deoxynucleosides were isolated. These suppressors actually fit into the next section of this review, for they are mutations in earlier reactions that shut off the toxic accumulation (Robertson et al., 1970).

D. ELIMINATION OF A DELETERIOUS ACCUMULATION I n Section 11, D we discussed situations in which mutant proteins act as inhibitors of cellular processes. Second-site mutations eliminate the toxic protein. Below we cite examples of inhibitions by metabolites and the various means by which these inhibitions may be relieved. The main phenotypic manifestations of mutations often s r k e simply from deficit of a metabolite beyond a genetic block; effects of accumulated metabolic intermediates, etc., often are secondary. However, cases are known where an end-product deficit does not cause the most readily observed or extreme phenotypic change. Rather, a slowed reaction results in accumulation behind a partial or complete genetic block of a metabolite in toxic concentration or of an excess of a metabolite that subsequently is converted to a toxic compound. Relief of the deleterious accumulation or of its effects through suppressor mutations can restore, or partially restore, the wild-type phenotype even though the initial genetic block persists. a. Phosphate esters of sugars are toxic to bacteria when accumulated in large amounts such as behind a genetic block. If an alternate source of carbon is available, toxicity is overcome and growth can ensue due to suppressor mutations that prevent the accumulation of the toxic phosphate ester. The first such case analyzed in detail was the accumulation of galactose l-phosphate by E . coli mutants blocked in the utilization of the phosphate ester (Yarmolinsky et al., 1959; Nikaido, 1961; Fukasawa and Nikaido, 1961). Suppression is achieved by creation of a second block in galactose metabolism preventing accumulation of the phosphate ester, namely a block in galactokinase (Yarmolinsky et al., 1959; Nikaido, 1961; Sundararajan et al., 1962; Fukasawa et al., 1963). Suppression of galactose sensitivity is widely used in selection of particular mutants of high interest (see pp. 11-12; also see Ippen et al., 1971). L-Arabinose mutants accumulating L-ribulose l-phosphate are arabinose sensitive, and this sensitivity is relieved by mutations leading to loss of L-ribulokinase (Englesberg et al., 1962). Similarly, E . coli defective in 2-keto-3-deoxygluconate-6-pho~phate(KDGP) aldolase cannot grow on gluconate although an intact alternate pathway for glu-

SUPPRESSORS

59

conate utilization is still present. Suppressor mutations allow growth on gluconate by channeling gluconate into the remaining pathway and eliminating its conversion to the toxic sugar phosphate ester, KDGP, which otherwise is accumulated behind the primary genetic block (Fradkin and Fraenkel, 1971; Fraenkel and Banerjee, 1972). This type of situation is not merely a test tube creation. Just as in the case of mutant bacteria mentioned above, some wild-type bacteria are sensitive to the presence of particular sugars. For example, Salmonella typhosu strains are sensitive to L-rhamnose and cannot utilize it as a carbon source. Rhamnose apparently is metabolized to toxic rhamnulose 1-phosphate but no further in this species. Mutations blocking conversion to the phosphate ester lead to rhamnose resistance (Englesberg and Baron, 1959; Englesberg, 1960). b. Studies with Neurospora were initially interpreted as indicating that a suppressor of acetate mutants opened up a secondary pathway of acetate production (Lein and Lein, 1952). The suppressor ( s p ) was active on all acetate mutants. Closer biochemical scrutiny, however, revealed that the suppressor acted by eliminating accumulation of an inhibitory substance (Strauss, 1953, 1955a,b ; Strauss and Pierog, 1954). Strauss found that the block in the acetate mutants leads to the funneling of accumulated pyruvate into toxic acetaldehyde and thence into ethanol. Acetate inhibits the accumulation of pyruvate from glucose. The requirement for acetate is partially alleviated by mutations in either of two nonallelic genes, s p and car, that lower pyruvate carboxylase activity and thus limit the conversion of pyruvate to acetaldehyde. c. Sturtevant (1956) described in Drosophila an autosomal dominant mutation, K-pn (Killer of prune), that is lethal when in combination with alleles of pn (prune) but has no phenotypic effect alone (i.e., pn+ suppresses K-pn). The pn mutations result both in a reduction of red (pteridine) pigment and an increase in brown eye pigment (cf. Nolte, 1955). Lifschytz and Falk (1969a,b) speculate that pn flies accumulate a pteridine pigment precursor that is converted t o a toxic substance in the presence of a single dose of K-pn; that is, the enzyme dictated by the K-pn locus carries out its normal or an analogous function but has either widened substrate specificity or altered regulation in flies carrying the K-pn mutation. An alternate possibility is mentioned on pp. 17-18. There is speculation that lethals are very common in populations of Drosophila but that many are suppressed ; recombination between the otherwise “silent” suppressors and the lethals reveal “synthetic lethals” such as in the instance just cited (Magalhiies et al., 1965). d. Sometimes suppressor mutations occur that do not drastically affect

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PHILIP E. HARTMAN AND JOHN R. ROTH

the overt phenotype but nevertheless lend a selective advantage under particular growth conditions. The suppressor mutations appear to shut off deleterious accumulations behind the primary genetic block and thus minimize secondary ramifications of this metabolic lesion. Therefore, in culture of a single mutant one sometimes ends up with a double mutant that retains the primary genetic block and, in addition, carries a new mutation affecting an earlier step in the same metabolic pathway. One example is the adenineless double mutant of Neurosporu (Mitchell and Mitchell, 1950), and another example is the cysteineless double mutants of Salmonella (Gillespie et al., 1968). We would guess that such occurrences are more common than reported and widely overlooked as potential adjuncts to other methods of genetic analysis. e. Emerson (1948, 1952) described reversions of a sulfonamide-requiring mutant (sfo) of Neurospora sensitive to normal intracellular levels of p-aminobenzoic acid (Zalokar, 1948; Emerson 1949). The revertants proved to be heterokaryons carrying the sfo mutation in all nuclei and an additional “suppressor” mutation in some nuclei. These suppressors were mimicked in an artificial heterokaryon carrying in some nuclei sfo and a mutation ( p u b ) blocking p-aminobenzoic acid Bynthesis (sfo pub) whereas other nuclei carried sfo pub+ (Emerson, 1948). Growth on minimal medium occurs in mycelia containing the properly balanced gene dosage (nuclear ratio) that allows p-aminobenzoic acid synthesis sufficient for growth but inadequate for inhibition. Zalokar (1950) showed that the sfo mutation could be phenotypically reversed by adjustment of the ammonium concentration or by the addition of threonine, and that sfo was hypersensitive to inhibition by methionine. Besides the pub suppression, Zalokar (1950) detected restoration of growth by suppressors that limit methionine biosynthesis (also see Emerson, 1952) . The sfo strain would appear an ideal tool for selection of new urom and met mutants but does not seem to have been used for this purpose. f. The growth requirement of two “pantothenicless” (pun) mutants of Neurospora was shown by Wagner and Haddox (1951) to be alleviated by either of two mutations affecting aromatic amino acid biosynthesis. The data of these workers implicated the production of an inhibitor in the pan mutants whose production was reduced in the suppressed strains. They stressed “the concept that many biochemical mutants are due to internal upsets in the balance of metabolic systems.”

E. EFFECTIVE DOSAGE OF A LIMITING GENEPRODUCT Several cases have been described where suppression results from an increase in a limiting gene product brought about through increased

SUPPRESSORS

61

effective gene dosage without apparent alteration in the product itself. Sometimes, the suppression occurs as the result of gene duplication (two chromosomal copies) or “gene magnification” (multiple chromosomal copies) and merely serves to make available more of the limiting gene product. No role of “gene amplification” (production of extrachromosoma1 gene copies: cf. Brown and Dawid, 1968, 1969; Gall, 1969) has been shown in suppression, but this mechanism remains a possibility. At other times suppression is achieved by elimination of a source of restriction of gene function, again making available more of the limiting gene product. Finally, mutation may alter the availability of another cellular component and suppress through restoration of a compatible balance between interacting molecules. a. The first example of suppression by gene duplication stems from the studies of Stern (1929), who demonstrated quantitative effects of different doses of various bobbed ( b b ) alleles in Drosophila and interpreted his data in surprisingly modern fashion. We now know that the “bobbed locus” contains a string of repeated gene sequences for ribosomal RNA (Ritossa and Spiegelman, 1965; Ritossa et al., 1966a,b; reviewed by Birnstiel et al., 1971). Various of the bobbed mutant alleles are deficient to various extents in ribosomal RNA gene sequences. The mutants are unstable, due to what appears to be disproportionate gene replication (i.e., “gene magnification” rather than unequal crossingover), yielding gene copies again adequate for wild-type levels of ribosomal RNA synthesis (Ritossa et al., 1966c, 1971; Ritossa, 1968; Atwood, 1969; Ritossa and Scala, 1969; Henderson and Ritossa, 1970; Tartof, 1971). The study of various bobbed mutants and their suppression promises to yield interesting information regarding regulation of ribosomal RNA gene loci, possibly pertinent to extrapolation with regard to other sequences of redundant DNA. b. Folk and Berg (1971) present evidence indicating that a glycine-requiring E . coli mutant (Folk and Berg, 1970a,b) containing a defective glycyl tRNA synthetase with increased K , reverts frequently (> to glycine independence through gene duplication. An increased gene dosage appears to allow increased production of synthetase subunits, albeit defective ones, to circumvent the metabolic block. Various genes surrounding the synthetase structural gene also were duplicated in some revertants, allowing indirect demonstration of the chromosomal location of the duplication. A further type of suppressor was also mentioned but not characterized by Folk and Berg (1971) ; its study might yield information pertinent to interaction of the synthetase with other cellular components. Carbon et al. (1966a,b) have provided an example of biochemical

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PHILIP E. HARTMAN AND JOHN R. ROTH

interactions leading to enhanced suppression. Suppression of a missense mutation resulting in a Gly for an Arg amino acid substitution (Brody and Yanofsky, 1963) was shown to be due to a genetically altered transfer RNA accepting glycine (Carbon et al., 1966a,b). Suppression by the altered tRNAG1ywas enhanced in a strain carrying a second genetic alteration leading to an approximately 7-fold enhanced level of GlytRNAG1y synthetase activity (Carbon et al., 1966b). It appears that the suppressor tRNAG'y is only sluggishly charged by the synthetase so that enhancement of synthetase activity assists in overcoming this metabolic bottleneck (Carbon and Curry, 1968). The genetic mechanism underlying enhanced synthetase levels remains to be determined, i.e., if due to increased effective dosage of synthetase genes or to an alteration in the synthetase itself. c. Unstable informational suppressors sometimes result from a duplication of a critical gene in a haploid organism and subsequent mutation of one of the two gene copies. Brody and Yanofsky (1963) found an unstable allele-specific suppressor in E . coli that contains a new tRNA species active in insertion of glycine instead of the usual arginine at the AGA codon (Carbon et al., 1966a,b). The instability of suppressors of this type has been shown to be due to the involvement of gene duplication. Two copies of a glycine tRNA gene plus mutation in one of the twin genes leads to a su+/su- genotype effective in suppression. Due to genetic homology of the duplicated chromosome region, one of the two su genes is readily eliminated by crossing-over (Hill et al., 1969, 1970; Carbon et al., 1969). The gene duplication is necessary since the presence of a t least one wild-type (su+)gene is needed for normal growth (Carbon et al., 1970). 2. Elimination of a Restrictive Site

In Section 11, C we surveyed instances where second-site mutations result in a partial or complete suppression of the mutant phenotype by circumventing the effects of a restrictive site. This may be a chainterminating nonsense codon, a promoter mutation, or a gene whose expression is blocked by repression of transcription. While these cases will not be reiterated here in detail, it must be realized that many of the papers cited in Section 11, C afford examples where polarity is relieved by mutations in genes other than those originally affected. We will briefly survey below some representative examples. a. Deletion or Modification of the Restrictive Site. Deletions of the normal promoter regions in the histidine and tryptophan operons of Salmonella essentially lead to failure of expression of the remaining,

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63

intact genes of the operon. This failure may be due to lack of a promoter site for binding of RNA polymerase and initiation of messenger RNA transcription, or to fusion with repressed operons. Selection for function of the remaining genes of the operons reveals one class of revertants in which the original deletion is extended (Ames et al., 1963; Margolin and Bauerle, 1966). These extended deletions may create new promoters, bring the remaining genes closer to existing promoters, or fuse the operons to entirely different operons which are not repressed. In the lactose operon of E . coli, expression is shut down by mutations in the i gene that lead to formation of “superrepressor.” One class of suppressors which again permit enzyme synthesis are operator constitutive mutations that have alterations in the DNA sequence with which the repressor molecules interact (Jacob and Monod, 1961; Willson et al., 1964; Jacob et al., 1964, 1965; Bourgeois et al., 1965; Bourgeois and Jobe, 1970). We might point out here the opposite type of situation. Pseudo-revertants of constitutive operator mutants have been detected as mapping in gene i (J. Sadler, cited in Reznikoff, 1972). One imagines that the normal repressor protein has decreased affinity for the mutant operator nucleotide sequence whereas particular mutant i gene products have regained recognition and regulatory ability. b. N e w Effective Promoters. Mutants which have lost an “activator” protein involved in enhancement of transcription of the arabinose or maltose operons of E . coli regain activity by suppressor mutations. These suppressors create new sites (promoters) a t which transcription may occur in the absence of “activator” (Englesberg et al., 1969; Englesberg, 1971; Gielow et al., 1971; Hofnung and Schwartz, 1971). Other cases of suppressor mutations engendering new promoters are discussed in Section 11,C. c. Chromosomal Transposition. For a long time it has been appreciated that “suppression” can occur merely by the combination in a diploid of a translocated wild-type gene and a mutant recessive gene (cf. Morgan et al., 1925; Schultz and Bridges, 1932). In such cases, the transposed gene and its control are not altered; the transposed gene carries out its normal function in a normal manner. Another possible instance of a similar effect, but where regulation is altered, is found in yeast with regard to dominant “suppressors” producing iso-2-cytochrome c (pages 54-55).

I n E. coli, a gene involved in arginine biosynthesis and necessary for growth on ornithine is restricted in function in particular mutants. Function is restored by duplication and transposition to a new chromosomal location away from the restrictive site (Glansdorff and Sand, 1968; Elseviers et al., 1969; Cunin et al., 1970). Three arginine biosyn-

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thetic enzymes are specified by three genes arranged in an operon in the sequence argC, B, H . The effects of a polar argB mutation in causing a drastic reduction in the expression of argH are eliminated in one revertant that carries an additional, transposed copy of argH. The functional argH gene maps close to the original arg gene cluster but is clearly located outside of it. The authors were quite careful in proving that the newly active gene was a newly arising duplication, not merely a previously present cryptic gene with overlapping function (Cunin et al., 1970). Duplications of this sort may also explain some of the unstable revertants of promoter deletions in the tryptophan operon of Salmonella (Margolin and Bauerle, 1966; Margolin, 1971) and also of the histidine operon (Ames et al., 1963; Levinthal and Yeh, 1972) as discussed below. d. Translocation to an Episome. Ames et al. (1963) described reversions of histidine-requiring Salmonella mutants wherein function was restored to intact but nonfunctional genes through duplication and translocation to an extrachromosomal site. The accessory genetic structure (pi, for piece) containing the histidine genes was unstable and frequently lost, was not linked by transduction to the chromosomal histidine genes, and did not require recombinational events for its transfer by transduction to other bacteria. Further investigations showed that p i was sometimes transferred along with the chromosomally located histidine genes during bacterial conjugation. Levinthal and Yeh (1972) found unstable recombinants from such conjugal crosses that now contain two chromosomal copies of the histidine gene region, namely, the original set plus the additional set formerly contained on the extrachromosomal element. The old and new sets of genes are now linked by transduction, and their instability may arise from crossing-over between the duplicated regions with elimination of one block of genes. 3. Increased Substrate

Mutations in the metG gene of Salmonella typhimum'um result in methionyl-tRNA synthetases with 100-fold or greater reduced affinity for methionine and a substantial growth requirement for this amino acid (Gross and Rowbury, 1969, 1971). Such synthetase K, mutants are suppressed by mutations in two distantly located genes, metJ and metK, that are involved in the regulation of the levels of the methionine biosynthetic enzymes (Lawrence et al., 1968; Chater et al., 1970; Chater, 1970; Smith, 1971). Although suppression could be achieved by protein-protein interactions, the mode of suppression proposed by these workers is that the secondary mutations result in an elevation in the intracellular methionine pool sufficient to saturate the defective synthetase (Chater et aE., 1970; Smith, 1971). Feedback inhibition of the first

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enzyme specific to methionine biosynthesis is incomplete in vivo except at excessive external methionine concentrations (cf. Lee et al., 1966; Chater and Rowbury, 1970) ; mutations to high levels of the biosynthetic enzymes thus expand the methionine pool and lead to methionine excretion. Apparently, mutations to full feedback-resistance alone do not increase the methionine pool sufficiently for suppression (Chater et al., 1970; Smith, 1971). The opposite situation applies with regard to histidine biosynthesis in the same organism. I n this case, histidyl-tRNA synthetase mutants with altered I<,,, for histidine are suppressed by mutations eliminating sensitivity to feedback inhibition (Sheppard, 1964; Hartman et al., 1971) and leading to a consequent increase in the internal pool of “free” histidine sufficient for suppression (Roth and Ames, 1966; Wyche, 1971).

4. Restoration of “Balance” Many cellular structures and enzymes are composed of dissimilar protein subunits which aggregate in a specific ratio. It is possible that aberrant structures are formed through protein-protein interactions if a balanced supply of the different subunits is not maintained. I n a theoretical example, formation of a functional structure of composition A,B, will be limited if either a short supply of B or an excess of A subunits diverts complex formation into nonfunctional A,B,, A,B,, and A,B, aggregates. This type of situation might be rectified, according to the case, by a decrease in formation of A or an increase in B, respectively. Here we are speaking of the quantity of a protein, not its quality. Particularly in cases where assembly involves partially defective subunits, an influence of “balance” would be expected to have a strong influence. Also, it is possible that imbalance at the level of protein subunits is one component of “genetic imbalance” associated with changes in chromosome number in higher organisms. a. Mutations in the classic w (white) locus of Drosophila serve as “dominant” suppressors of z (zeste). Gans (1953) showed that two functional doses of the w+ gene were necessary for expression of light eyecolor in zeste flies. Through the mapping of point mutations and use of various types of duplication and deficiency stocks, the w+requirement was later narrowed down to a dosage of just the right-most portion of w. This is a gene region encompassing two mutationally and recombinationally separable sites (Green, 1959a,b,c, 1963, 1969; Judd, 1959, 1964; Rasmuson, 1965). Deletions of this portion of the w “locus” also suppress (Green, 1959c, 1969; Judd, 1959, 1964). Detailed knowledge of cytological fine structure in the w chromosome region support the genetic analysis (Gersh, 1962, 1967; Lefevre and Wilkins, 1966; Rayle

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and Green, 1968). Abridged reports of immunological studies indicate that a protein (“w-1”) is the product of some locus other than w and is modified differently in the suppressing w mutants than in the wild-type or in nonsuppressing w mutants (Fuscaldo and Fox, 1962; Fuscaldo and McCarron, 1965, 1967; McCarron and Fuscaldo, 1968a,b). Owing to the genetic homology known between the zeste and white chromosomal regions, it has been speculated that the “modified” protein is a joint rnultimeric protein combining products of the z and w genes (McCarron and Fuscaldo, 1968a,b). The suppressing w mutants lack 3-hydroxykynurenine (Fig. 9) and contain but one-half the kynurenine hydroxylase activity of wild-type flies whereas the nonsuppressing mutants contain 3-hydroxykynurenine (Ghosh and Forrest, 1967a) . b. Floor (1970) has shown that mutations affecting bacteriophage T4 tail fiber production (gene 37) are suppressed by secondary mutations in genes affecting baseplate or head synthesis. Mutations in tail sheath gene 18 are suppressed by mutations affecting baseplate synthesis. Mutations affecting head structure (gene 93) are suppressed by mutations in gene 20 (another head gene) or in gene 96 (baseplate). The suppressive effects can be attributed to “balance” of functional components in phage assembly. Further cases of suppressors in phage assembly also may involve such “balances” (cf. pp. 1Ck11, and 37). IV. Other Interesting Cases

The compilation by Lindsley and Grell (1968) informs us of many situations in which analysis of suppression offers avenues for interesting exploration. The cases below are presented merely as examples of situations where suppressors offer a powerful probe into biochemical mechanisms. a. The origin and nature of various hereditary tumors in Drosophila (Bridges, 1916; Stark, 1919) remain to be defined in spite of intensive investigations [see reviews, larval tumors ( t u ): Burdette, 1959; erupt ( e r ) : Glass, 1957; Tumorous head ( t u - h ) : Gardner, 19701. I n each case tumor formation is dependent on a polygenic system; numerous cases of suppression and modification of tumor incidence and distribution have been found since the original observations of Stark and Bridges (1926). Tumor incidences are differentially influenced by the presence of tryptophan and certain related compounds (see Fig. 9), such as phenylalanine, kynurenine, anthranilic acid, and 3-hydroxyanthranilic acid (Glass and Plaine, 1954; Plaine and Glass, 1955; Kanehisa, 1956; Brooks, 1967; Burnet and Sang, 1968). I n some systems, effects of the feeding of tryptophan may have a component of “toxicity” (cf. Turner

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and Gardner, 1960), but i t seems unlikely nonspecific “toxicity” (for example, a mere lengthening of the normal larval period) rather than specific physiological effects could account for all the data. The response of tumor incidence to a given compound is influenced by particular modifier genes and metabolism by organisms in the larval food (cf. Burnet and Sang, 1968), probably accounting in part for the divergence of reports in the literature as to the relative efficiencies of these various compounds. Oxygen also stimulates tumor production (Glass and Plaine, 1952 ; Plaine and Glass, 1952, 1955 ; Plaine, 1955a; Brooks, 1967). Generally cysteine and methionine reverse the effects of tryptophan and related compounds (Plaine, 1955b; Burnet and Sang, 1968). The content of some free amino acids is altered in tu strains, but free tyrosine content remains constant (cf. Lewis, 1954; Yamazaki, 1968). These various responses led Glass (1957) and Brooks (1967) to suggest reactions affected by a suppressor of erupt (su-er) and by a suppressor of larval tumors (su-tu) as shown in Fig. 9. However, the proposed sites of action assume that effects are elicited directly by some metabolite rather than indirectly through regulation of another step in metabolism. Melanotic larval tumors in tu strains are due to a particular type of blood cell, the “lamellocyte” (Russell, 1940; Rizki, 1957, 1960). The circulatory lamellocytes are precociously produced in tu strains (Rizki, 1957) or appear when larval life is extended beyond its normal period (“overaged larvae”) such as in larval lethals unable to pupate (e.g., ZtZ, Kobe1 and van Breugel, 1967). Chemicals mimicking juvenile hormone (Bryant and Sang, 1969) and X-rays (Glass, 1944; Glass and Plaine, 1950; Hildreth, 1967) increase tumor incidence in particular suppressed tu stocks while ecdysone leads to repression (cf. Burdette, 1959). The spindle-shaped lamellocytes originate in the lymph gland, and some suppressors may influence their release from this source (Barigozzi, 1958 ; Barigozzi et al., 1960). The lamellocytes next lodge between tissues, preferentially encapsulating caudal fat cells, a step inhibited by the feeding of glucosamine (Rizki, 1961a). Perhaps the effect of glucosamine is exerted through alterations on specific sites involving intercellular adhesion (cf. Oppenheimer et al., 1969; Roth et al., 1971). Later, melanization of the tumors takes place. This latter step (important in tumor recognition) is possibly separately inherited (Barigozzi et al., 1960) and might accompany cell dissolution and release of stored tyrosinase substrates to enzyme action (cf. Henderson and Glassman, 1969), allow activation of DOPA oxidase (cf. Lewis and Lewis, 1963), or allow action of some other active phenoloxidase (cf. ParBdi and CsukGs-Szatl&zky, 1969). Preer (1971) reviews recent evidence that viruses form one component of certain melanotic tumors; similar claims from other labora-

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tories in the past have not been reproduced or followed up. Reports of cytoplasmic effects on tumor incidence are common, however. The above review, admittedly incomplete, suggests that tumor formation in Drosophila is a multistep process that can be stimulated by a wide variety of genes (Burdette, 1959) and which also can be blocked (suppressed) by mutations at many sites elsewhere in the genome. Probably different genes engender blocks a t different points in the sequence of events culminating in tumor formation. We would surmise that intense cytological and biochemical analyses of these interactions would reveal phenomena of real importance to students of developmental biology. b . A recessive temperature-sensitive suppressor, su (f) , shows interesting allele-specific effects on members of two loci that presumably are of diverse function. The suppressor acts to suppress a minority of mutations a t the forked (f) “locus” and enhances one mutation (w”)out of 14 . of 19 w mutatested a t the white locus (Green, 1955, 1956, 1 9 5 9 ~ )Out tions tested, the wamutation also is the only allele suppressed by another recessive suppressor, su-wa. Of two partial back mutants (presumably intra-locus) of wa,the phenotype of one was unchanged by either suppressor while the second still behaved as wa.That is, it was enhanced by su-f and suppressed by su-wa (Green, 1959c). c. A suppressor, tol, overcomes growth restriction imposed by the content of both A and a mating type loci in duplication strains of Neurospora (Newmeyer, 1968). Most often, restriction in growth is overcome by transitions to homozygosity or hemizygosity rather than to suppressor mutation (Newmeyer and Taylor, 1967). The occurrence of suppressors, however, may allow a more direct analysis of the biochemical basis of mating type incompatibility. ACKNOWLEDGMENTS Ordinarily one feels embarrassed a t dedicating a paper to anyone, much more so to the Editor. However, we feel Professor Ernst Caspari is such a staunch person and his Editorial Pen has covered such a broad spectrum in this series, in Genetics, and elsewhere that his modesty now might be prepared to withstand even a dedication to himself. So, we do fondly dedicate this review to Professor Caspari, a warm and inspiring person with a special interest in young scientists and an Editor who has contributed vastly to communication of scientific data and ideas. The origins of this paper go back to a conversation with Professor Caspari a t the time Gorini and Beckwith must have been planning their own review of the same subject (1966) , a subject not exclusively covered before that time (although citations in his papers suggest that such

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an undertaking was contemplated by C. B. Bridges in his latter years). Dr. Sigmund R. Suskind deserves credit for keeping the idea of this review alive. We thank a number of our colleagues for responding graciously to our requests for updated information and for comments on certain sections during the preparation of this review. Our own work cited herein has been supported by U.S.Public Health Service Research Grants A101650 (P. E. H.) and AM12115 (J. R. R.). Contribution No. 684 of the Department of Biology, The Johns Hopkins University.

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Yura, T., and Vogel, H. J. 1959. Pyrroline-5-carboxylate reductase of Neurospora crassa: Partial purification and some properties. J . Biol. Chem. 234, 335-338. Zalkin, H., and Kling, D. 1968. Anthranilate synthetase. Purification and properties of Component I from Salmonella typhimurium. Biochemistry 7, 3566-3573.

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Zalokar, M. 1948. The p-aminobenzoic acid requirement of the “sulfonamide-requiring” mutant strain of Neurospora. PYOC.N u t . Acad. Sci. U.S. 34, 32-36. Zalokar, M. 1950. The sulfonamide-requiring mutant of Neurospora : Threoninemethionine antagonism. J. Bacteriol. 60, 191-203. Zalokar, M. 1953. Reduction of selenite by Neurospora. Arch. Biochem. Biophys. 44, 330-337. Ziegler, I. 1961. Genetic aspects of ommochrome and pterin pigments. Advan. Genet. 10, 349-403. Ziegler, I., and Harmsen, R. 1969. The biology of pteridines in insects. Advan. Insect Physiol. 6, 139-203. Zimmermann, F. K., Schmiedt, I., and ten Berge, A. M. A. 1969. Dominance and recessiveness at the protein level in mutant x wild type crosses in Saccharomyces cerevisiae. Mol. Gen. Genet. 104, 321-330. Zimmermann, R. A., Garvin, R. T., and Gorini, L. 1971a. Alteration of a 30s protein accompanying the ram mutation in Escherichia coli. Proc. N a t . Acad. Sci. U.S. 68, 2263-2267. Zimmermann, R. A., Rosset, R., and Gorini, L. 1971b. Nature of phenotypic masking exhibited by drug-dependent streptomycin A mutants of Escherichia coli. J . Mol. Biol. 57, 403-422. Zipser, D. 1967. UGA: a third class of suppressible polar mutants. J . Mol. B w l . 29, 441-455. Zipser, D. 1970. Polarity and translational punctuation. In “The Lactose Operon” (J. R. Beckwith and D. Zipser, eds.), pp. 221-232. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Zipser, D., and Newton, A. 1967. The influence of deletions on polarity. J . Mol. Bi01. 25, 567-569. Zipser, D., Zabell, S., Rothman, J., Grodzicker, T., Wenk, M., and Novitski, M. 1970. Fine structure of the gradient of polarity in the z gene of the lac operon of Escherichia coli. J . Mol. Biol. 49, 251-254. Zollinger, W. D., and Woodward, D. 0. 1972. Comparison of cysteine and tryptophan content of insoluble proteins derived from wild-type and mi-1 strains of N e w rospora crassa. J . Bacteriol. 109, 1001-1013.

HETEROGENIC INCOMPATIBILITY I N PLANTS AND ANIMALS Karl Esser and Rolf Blaich Lehrstuhl firr Allgemeine Botanik, Ruhr-Universitat Bochum, Bochum, Germany

I. Introduction . . . . . . . . . . . . . . . . . . 11. Genetic Basis of Heterogenic Incompatibility in Podospora . . . . . 111. Further Genetic Parameters Which Control Sexual or Parasexual Processes A. The Basic Control of Propagation by Various Breeding Systems . . B. Sterility Genes. . . . . . . . . . . . . . . . . C. Cross Sterility Due to Irregularities during Meiosis . . . . . . D. The Species Concept . . . . . . . . . . . . . . . IV. Compilation and Analysis of Data from the Literature with Respect to Heterogenic Incompatibility . . . . . . . . . . . . . . A. Fungi . . . . . . . . . . . . . . . . . . . B. Higher Plants . . . . . . . . . . . . . . . . . C. Animals. . . . . . . . . . . . . . . . . . . V. Correlations between Heterogenic Incompatibility and Histoincompatibility . . . . . . . . . . . . . . . . VI. Conclusions. . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

107 109 113 113 114 114 115 115 115 135 138 141 142 145

I. Introduction

Incompatibility is usually defined as restriction of the mating competence controlled by genes other than those determining sexual differentiation. However, it has been recognized recently that incompatibility concerns not only the sexual phase, but also the vegetative phase. The latter becomes apparent, especially in fungi, as the so-called heterokaryon or protoplasmic incompatibility. I n both sexual and vegetative incompatibility the action of the genetic factors involved prohibits the exchange of genetic material. This inhibition of recombination is achieved in the first instance by a lack of karyogamy and in the second by an inability of the incompatible nuclei or cytoplasmic factors to coexist in a common cytoplasm and to undergo those processes necessary for somatic recombination. Since recombination is a prerequisite for evolution, the biological significance of incompatibility as a factor controlling recombination becomes apparent. 107

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Nature has evolved two different systems to effect the genetic control of incompatibility. According to the mode of their genetic determination they have been called homogenic and heterogenic incompatibility, respectively (Esser, 1962). The genetic basis of the homogenic system consists in sexual incompatibility of nuclei carrying identical incompatibility factors. In contrast to this, in the heterogenic system genetic material in order to be incompatible requires a t least a genetic difference of one single gene or one cytoplasmic factor. From these definitions it follows that homogenic incompatibility enhances outbreeding and favors through recombination the evolution of the species as a whole. Heterogenic incompatibility, however, restricts outbreeding and favors the evolution of isolated groups within a species. This may create a basis for further speciation within a single species. From these reflections it becomes evident, that both systems, in spite of controlling recombination in a different way, are integrated constituents of evolution. Homogenic incompatibility has been known since the times of Darwin, and its various mechanisms have now been analyzed in detail both in higher plants and in fungi. Its action and its distribution have been the subject of numerous reviews (see below). However, heterogenic incompatibility, although discovered recently and studied in detail in the ascomycete Podospora anserina (for references, see below) has not yet attracted much attention. This is understandable, because heterogenic incompatibility, on account of its genetic background, never occurs within true-breeding laboratory strains but only between races differing in their genetic equipment. In addition, this phenomenon often has been overlooked and sometimes interpreted as “sterility” merely because of failure of mating. In addition to considering the general significance of heterogenic incompatibility for evolution, we have analyzed the relevant literature in order to illustrate the concept. Furthermore, we discuss the common features of those cases of heterogenic incompatibility which have been interpreted in other ways as a result of inadequate comprehension of the genetic basis of the phenomenon. Detailed information on incompatibility in general is provided in the following books and reviews : the concept of homogenic and heterogenic incompatibility (Esser and Kuenen, 1967; Esser, 1971) ; incompatibility in fungi (Raper and Esser, 1964; Esser and Raper, 1965; Raper, 1966; Caten and Jinks, 1966; Esser, 1967) ; homogenic incompatibility in higher plants (Linskens and Kroh, 1967; Lewis, 1954; Brieger, 1930; East, 1940; Arasu, 1968; Townsend, 1971).

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II. Genetic Basis of Heterogenic Incompatibility in Podospora

For simplicity and as a starting point, it seems necessary to present a t least in an abbreviated form the general principles of heterogenic incompatibility with Podospora anserinu as an example (Rizet and Esser, 1953; Esser, 1954, 1956, 1959a,b; Beisson-Schecroun, 1962; Bernet, 1965; Blaich and Esser, 1970, 1971; a fairly complete compilation of the Podospora literature may be found in Esser (1973). I n P . anserina the mating competence of different strains is basically controlled by the bipolar mechanism of homogenic incompatibility,* i.e., and - mating types which are compatible, there exist within each race X and the - X - combinations are incompatible. whereas the Heterogenic incompatibility occurs only between strains of different mechanism. The two mechageographical origin and overlaps the nisms of the heterogenic system will be exemplified by explaining the action of incompatibility genes from two geographic races s and M (Fig. 1). The allelic mechanism caused by alleles of the t or the u locus does not interfere with sexual compatibility. If strains differ a t either one or both loci, a vegetative incompatibility is provoked, i.e., an inability of nuclei of the mating partners to coexist after heterokaryosis in a common cytoplasm. This leads to a prevention of somatic recombination. The formation of the unpigmented barrage zone (diameter 1 mm) is the result of the ‘‘fight” of the incompatible nuclei, leading finally to a decrease of the hyphal compartments concerned. The nonallelic mechanism depends on the interaction of two specific alleles of two different loci. The incompatibility of a,/b and cl/v results not only in vegetative incompatibility, but also in a nonreciprocal sexual incompatibility, which restricts fruiting body formation and therewith the possibility of an exchange of genetic material. By recombination of the four unlinked loci one may easily obtain strains in which the two mechanisms overlap. They exhibit a total incompatibility in both the sexual and the vegetative phase, which cuts down the chance of somatic and of meiotic recombination to zero. The genetic analysis of more geographical races has confirmed the results obtained with the two races as described above and given more

+ +

+

+/-

* The fact that bipolar incompatibility is masked by pseudocompatibility need not be treated in detail in this connection, because this phenomenon caused by the specific nuclear arrangement in binuclear ascospores may be overcome easily by using uninucleate spores. For details, see Esser and Kuenen ( 1967).

........ tuabcv

tlUl albl c1v1 interrace cross S X M

overlapping of various mechanisms

........ ........ ........ ........ ................ t

tl

U

U1

allelic mechanism vegetative incompatibility

bl

aEl

cm

m . 1

nonallelic mechanism vegetative and sexual incompatibility

rnblCJvl

2 T1

l? q

td

r

g

apJq.1

X

overlapping o f the two no nallel ic mechanisms

FIG.1. Scheme of the action of the genes responsible for the heterogenic incompatibility between races s and M of Podospora anserina. The different alleles are symbolized by small letters, nonallelic genes which interact are framed with squares. The two parallel lines symbolize the vegetative incompatibility which leads to the macroscopically visible, so-called barrage, which occurs in the zone of contact between two incompatible mycelia. The black dots along the barrage indicate the formation of fruiting bodies. In all combinations and - strains are confronted, but for reasons of clarity we have omitted the mating type symbols. For furthm explanation, see text.

+

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information about the overall incompatibility picture within the species P . anserina. I n addition to the above-mentioned genes a new locus for the allelic mechanism has been found. Another interaction with the b locus in the nonallelic mechanism has been identified. Furthermore, for some of the loci multiple alleles were established. For some combinations of nonallelic genes, a temperature dependence of the incompatibility reaction and a partial reduction in the number of fruiting bodies has been observed. I n Fig. 2 a scheme of the mating relations in all possible combinations of 19 geographical races is presented; it demonstrates that in interracial crosses only 13 (7.6%) exhibit normal compatibility, whereas 158 (92.4%) show either or both vegetativc and sexual incompatibility. Hence within the species P. anserina the exchange of genetic material is drastically reduced by heterogenic incompatibility acting as an isolation mechanism for evolution. The physiological action of the incompatibility genes in either of the t,wo mechanisms could be attributed to a protein reaction leading to the formation of a specific protein, formed when incompatible nuclei coexist for a short period in n common cytoplasm (Esser, 195913). However, as preliminary experiments have shown (Blaich and Esser, 1971), this protein seems not to be the cause of heterogenic incompatibility, but a consequence of this phenomenon indicating the disintegration of the protoplast. I n accordance with Williams and Wilson (1966, 1968; see also p. 123), who suggest “that membranes are somehow involved in the incompatibility reaction,” we also have observed that peptidases which are enclosed in normal hyphae in vesicles are liberated either by mechanical destruction of the protoplast or by the incompatibility reaction after hyphal fusion (for working hypotheses cf. Section IV, B). As a result of the detailed information obtained from genetic studies with Podospora, we are now able to present a general definition of heterogenic incompatibility as follows: Heterogenic incompatibility is brought about b y genetic elements which cannot exist i n close proximity to each other, i.e., neither in one nucleus nor in a common cytoplasm, when located in different nuclei of a heterokaryon. When these factors are brought together by natural or artificial means, the nuclei concerned either degenerate or segregate depending on the presence of the incompatibility genes in one or different nuclei. Therefore the first indication of heterogenic incompatibility is the failure of zygote formation or, if the vegetative phase is concerned, failure to establish stable diploids or heterokaryons, respectively (Fig. 3). I n this connection it should be noted that heterogenic incompatibility

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FIQ.2. Scheme of the mating reactions between various races of Podospora anserina isolated from different localities in France and Germany. Meaning of the symbols: +/- = mating type; capital letters = designation of races; black squares = compatible in both vegetative and sexual phase; black squares with inserted open squares = sexual compatibility and vegetative incompatibility; hatched squares = vegetative incompatibility and reduced fruit body formation (partial sexual incompatibility) ; open squares = incompatibility in both, vegetative and sexual phase. (Adapted from Esser, 1971.)

as will be shown later (see pp. 128, 138, 139), is controlled not exclusively by nuclear genes but also by cytoplasmic genetic elements. However, the mechanisms concerned are covered by the above general definition, because a mixture of genetically different cytoplasm also exhibits a failure of coexistence leading to disintegration or cellular death.

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FIG.3. Examples of heterogenic incompatibility concerning the vegetative phase of Podospora anserina. The monokaryotic strains (1 and 2), on the one hand, and 3, on the other hand, differ (apart from a marker gene, mycelial color) by a single incompatibility allele. Heterokaryons between the incompatible strains show a sectoring (4 and 6) due to the mutual action of the incompatibility factors resulting in internal barrage formation or to elimination of one nuclear component (5).

Ill. Further Genetic Parameters Which Control Sexual or Parasexual Processes

Before analyzing the literature for further cases of heterogenic incompatibility, it is necessary to consider some other genetic parameters which also may provoke the failure of zygotic or somatic union of nuclei and thus will mask or phenocopy heterogenic incompatibility. These have been described and discussed in detail elsewhere (Esser, 1971).

A. THEBASICCONTROL OF PROPAGATION BY VARIOUS BREEDING SYSTEMS The prerequisite for studying heterogenic incompatibility is a proper knowledge of all other genetic factors controlling the sexual and asexual

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behavior within a species; e.g., in Podospora, identical mating types (controlled by the bipolar mechanism of homogenic incompatibility) are not able to react sexually but do form heterokaryons. A similar effect in other organisms may be caused by physiological dioecism.

B. STERILITY GENES Sterility genes may phenocopy heterogenic incompatibility, especially if the sexual structures of the organisms concerned are only visible microscopically or seem to have been disrupted a t some stage of internal development (e.g!, during formation of sexual spores or during development of fertilized eggs). This phenomenon is relatively easy to distinguish from heterogenic incompatibility since after heterokaryosis or vegetative hybridization (e.g., grafting) no antagonistic reaction occurs. These deficiencies may be canceled, especially in fungi, by complementation leading to normal fruiting body production.

C. CROSSSTERILITY DUETO IRREGULARITIES DURING MEIOSIS Such cross sterility caused by gross chromosomal or genomic diversities is sometimes difficult to distinguish from true incompatibility. I n order to exclude this type of sterility as being responsible for the failure of progeny production, two criteria need to be considered: (1) Cross sterility often does not inhibit the formation of zygotes but never leads to viable products of meiosis, i.e., it gives sterile spores in haplonts, sterile offspring in diploids. (2) Two self-fertile strains or races A and B may never cross with each other; however, if they are both fertile with a third strain C, chromosomal divergence, etc., must be excluded and the failure in fertilization between A and B has to be attributed to specific genic differences, i.e., to heterogenic incompatibility. This leads to the question of the distinction between sterility based on gross genetic differences (e.g., cross between elephant and mosquito) and true heterogenic incompatibility based on small genetic differences. The most convincing evidence for true heterogenic incompatibility is the demonstration of a nonreciprocal incompatibility as occurs in Podospora interracial crosses (see Fig. 1) and which may be found in some other instances (e.g., Cules, see p. 139). Otherwise one has to resort to the above-mentioned A-B-C crossing scheme.

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D. THE SPECIESCONCEPT Although there is no generally adopted definition of a species in quantitative terms, one of the most widely accepted rules is that the representatives of one species should be able to fertilize each other. This may be a valuable criterion if one is aware of all other factors that may influence this procedure, such as the breeding systems and sterility genes mentioned above. But in many lower organisms which are difficult to propagate in laboratory conditions these parameters have not been studied in detail. This holds true especially for pathogenic organisms. I n addition, the delimitation of species in these organisms is based very often on their host specificity which is controlled in some cases only by one or a few Mendelian factors. Last but not least, species delimitation based on morphological differences may be completely wrong, for in a number of examples (especially in fungi) it has been shown that single-gene mutations may lead to gross alterations of the phenotype without having any influence on the mating capacity of the organisms concerned (Esser and Kuenen, 1967). This again stresses the necessity for a thorough genetic study of all possible parameters controlling fusion as a preliminary t o meiotic or somatic recombination in order to understand not only speciation but also propagation in general. IV. Compilation and Analysis of Data from the literature with Respect to Heterogenic Incompatibility

The published data indicate that heterogenic incompatibility is found not only in fungi, but also in higher plants and in some animals, as summarized in Table 1. The particular cases will be treated in detail below. I n the literature there is a great diversity of names, definitions, and gene symbols for effects which, we think, belong to one basic phenomenon : heterogenic incompatibility. Resulting from this diversity and owing to the fact that the authors of this paper are not experts on the whole field of biology, the attempt to present a single phenomenon in its huge versatility will risk occasional misinterpretations for which we apologize in advance. 1. Myxoinycetes

A. FUNGI

I n a number of true slime molds, the basic genetic control of the life cycle is exerted by physiological dioecism, i.e., haploid myxarnoebae

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TABLE 1 A Survey of the Occurrence of Heterogenic Incompatibility in Various Genera of Fungi, Higher Plants, and Animals as Interpretable from Symptoms Described by Investigators“ 1.

Fungi

Myxomycetes Didymium Ph ysarum

Phycomycetes Phytophthora Ascomycetes Neurospora

Aspergillus spp.

Cochliobolus Microsporum Saccharomyces Sordaria Erysiphe Podospora

Basidiomycetes Ustilago Sistotrema Rhizoctonia

Polyporus spp. Fomes a

Collins and Clark (1968) ; hlukherjee and Zabka (1964) Carlile and Dee (1967); Poulter and Dee (1968) ; Collins and Haskins (1970); Henney and Henney (1968); Wheals (1970)

Incompatibility of plasmodia, due to special genes

Savage et al. (1968)

Restriction of mating competence

Heterokaryon incompatibility, caused by distinct parts of the mating type genes Heterokaryon incompatiWilson and Garnjobst bility, caused by known (1966) ; Williams and genes, not identical with Wilson (1968) ; Pittenger the mating type genes (1964) Grindle (1963a,b); Jones Failure of heterokaryon formation (1965); Butcher (1968, 1969) ; Jinks et al. (1966) ; Kwon and Raper (1967); Caten et al. (1971) Restriction in mating Nelson (1970) Restriction in mating Padhye and Carmichael (1971) “Killer” reaction Somers and Bevan (1969) Olive (1956); Esser Heterokaryon incompatibility (unpublished) Morrison (1960) Cf. p. 3 Newmeyer and Taylor (1967)

Bauch (1927); Vandendries Uni- or bilateral sexual in(1927, 1929) compatibility Lemke (1969) Killing reaction after Whitney and Parmeter formation of anastomoses (1963); Garza-Chapa and Anderson (1966) j Stretton et al. (1967); McKeazie et al. (1969) Demarcation line Macrae (1967) ; Barrett ( = barrage) and Uscuplic (1971) Adams and Roth (1967); Barrage

Only the main references are listed; additional citations are given in the text.

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TABLE I (Continued)

Cyathus Peniophora Uatilago

Neuhauser and Gilbertson (1971) Fulton (1950); Brodie (1970) McKeen (1952)

Nonreciprocal sexual incompatibility Inhibition of clamp connections Hankin and Puhalla (1971) "Killer "-phenomenon

II. Higher Plants

Petunia Antirrhinum L yeoperaicon

Leavenworthia Rubus Solanum Oenothera

Cilia Agmstis and Anthoxanthum Clarkia Lotus

Zea

111.

Mather (1943) Harrison and Darby (1955) Gunther and Juttersonke (1971); Martin (1967); Hardon (1967); Lewis and Crowe (1958) Lloyd (1968) Keep (1968) Pandey (1967, 1968); Grun and Aubertin (1966) ; Martin (1967) Arnold and Fellenberg (1965) Lewis and Crowe (1958) ; Steiner (1961); Schulta (1962) Grant (1965) McNeilly and Antonovics (1968) Parnell (1968) ; Miller (1969) Paterniani (1969) Nelson (1952)

Unilateral incompatibility, due to modified S genes (sometimes due to additional factors)

Incompatibility gene, independent of S genes Unilateral incompatibility, due to unknown factors

Genes, independent from S genes

Animals

Protozoa, Ciliata

Chordata, Rana sp.

Sonneborn (1957) ; Siege1 (1956); Reiff (1968) Jeon and Lorch (1969); Makhlin and Yudin (1970) Cuellar (1971)

Insects, Culex

Laven (1957, 1959)

Coelenterata

Theodor (1970)

Protozoa, Amoeba

Failure of conjugation; death of exconjugants Incompatibility of nuclei in alien cytoplasm Nonreciprocal incompatibility between different races Reciprocal and nonreciprocal sexual incompatibility Transplantation incompatibility; not due to a n immune system

originating from Sporangiospheres (= Meiospores) and belonging to different mating types fuse with each other to give rise to zygotes and diploid plasmodia (for literature, see review of Ling and Collins, 1970). I n the most thoroughly studied species, Didymium iridis and Physarum

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FIQ.4. See legend on opposite page.

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polycephalum, the various mating types seem to be determined by multiple alleles of a single locus (Dee, 1966; Collins, 1963). Mukherjee and Zabka (1964), however, claim that in a strain of D. iridis two loci are responsible for the existence of the various mating types. I n addition to the fusion of haploid amoebae the fusion of diploid plasniodia is under genetic control. In D. iridis this fusion is entirely prevented when both plasmodia differ a t one or more fusion loci (Collins and Clark, 1968). A similar phenomenon occurs in P. polycephalurn, and in addition fusion between genetically different strains identical a t the fusion locus leads to a destruction of the plasmodia (Carlile and Dee, 1967; Carlile, 1972). I n both cases, genetically different nuclei are prevented from coexisting in close proximity in a common cytoplasm. Therefore both these phenomena have to be attributed to heterogenic incompatibility. a. D i d y m i u m iridis. I n Didymium iridis two different races have been analyzed for the existence of incompatibility factors (Collins and Clark, 1968, Ling and Collins, 1970; Collins and Ling, 1972). I n both races, a t least 11 incompatibility loci were detected. Since no interracial crosses have been performed, it is not known whether these loci are identical or not. If they are, this would bring up the question of the existence of multiple allelism. I n order to understand the action of the incompatibility ( = “fusion”) genes, we have to consider that the plasmodia1 nuclei are diploid and may be heterozygous for these genes. According to the definition of heterogenic incompability, (p. 111), one should expect these genes when brought under heterozygous conditions to interact and cause leaky mycelia as exemplified in Podospora and Neurospora (p. 122). Surprisingly enough, no such interaction has been found in the heterozygous nuclei of D. iridis. From the data presented in Table 2, it follows that the incompatibility genes behave like dominant and recessive alleles, the former always determining the phenotype, i.e., the type of incompatibility reaction of the plasmodium. As a consequence, the same incompatibility phenotype may be due to homo- or heterozygosis a t the locus concerned. Concerning the physiological action of the incompatibility genes one has to assume that: (1) they are not yet active in the stage of developFIG.4. Heterogenic incompatibility in the Myxomycete Physarum polycephalum. In contrast to the normal contact between two compatible strains (A) where the two plasmodia have established veinlike cytoplasmic connections, the contact of two incompatible strains is characterized by the absence of fusion between the two plasmodia (B). (Originals from M. Carlile and J. Dee, unpublished.)

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TABLE 2 Genetic Control of Heterogenic Incompatibility Occurring between Diploid Plasmodia of Didymium iridis by the Action of a Pair of Dominant and Recessive Alleles" Plasmodium I Genotype Phenotype

D/D D/D D/d D/d a

D D D D

Plasmodium I1 Reaction Compatible Incompatible Compatible Incompatible

Phenotype Genotype D d D d

D/d d/d D/d d/d

Compiled from data of Collins and Clark (1968).

ment where karyogamy occurs; (2) when plasmodia come into contact, only the activity of one allele is turned on; this allele is therefore called the dominant allele, and the recessive allele remains inactive in presence of the dominant. Unfortunately there is no experimental proof of this working hypothesis. There are also indications that the fusion genes are not exclusively responsible for the heterogenic incompatibility between plasmodia of D. im'dis. The mating type genes controlling the fusion of amoebae and thus determining the physiological dioecism of this organism seem also to participate in the regulation of heterogenic plasmodium incompatibility; i.e., different mating type alleles promote the fusion of amoebae but inhibit the fusion of plasmodia (Clark and Collins, 1968). This phenomenon can be explained if one admits that in analogy to Neurospora (p. 122) the mating type locus is bipartite and consists of two subunits, one being instrumental in the control of physiological dioecism and the other in heterogenic incompatibility. I n contrast to the case of Neurospora, this assumption lacks experimental proof. b. Physarum polycephalum. In Physarum polycephalum the genetic control of the inhibition of the plasmodia1 fusion and of the lethal reaction in the zone of contact is not fully understood. Poulter and Dee (1968) found a multiple-allele, single-locus system for this type of heterogenic incompatibility, Wheals (1970) reported a second locus with a dominant and recessive allele involved, and Collins and Haskins (1970) favor a polygenic system from their findings with another isolate of the same species. All workers, however, found no involvement of the mating type locus. There is also indication that heterogenic incompatibility might affect not only the fusion of plasmodia, but also the fusion of amoebae. Henney and Henney (1968) describe in Physarum jlavicomum a failure of fusion

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between amoebae from different geographical races. Unfortunately genetic data are not available.

R. Phycornycetes Phycomycetes have been used very little for genetic studies. This may be due in part to the fact that in contrast to the Ascomycetes and Basidiomycetes, the location and mode of meiotic division and nuclear distribution after meiosis have not been elucidated in detail in this taxon. I n addition, there are numerous indications (see Sansome, 1966) that the large order of Oomycetales is diploid, thus complicating genetic analysis. Therefore it is not surprising that our knowledge of genetic control of breeding systems, and especially proof for heterogenic incompatibility, is very meager. The only indication for the existence of heterogenic incompatibility in Phycomycetes concerns the Oomycete Phytophthora, which causes the false mildew in higher plants (for reference, see Savage et al., 1968). I n these studies the mating relations of almost 350 isolates, representing 30 of the 42 species and varieties of the genus have been analyzed. With the exception of one strain which consistently failed to produce oospores, strains could be distributed into two different groups according to their mating competence: (1) compatible monoecists, which are selffertile (15 species) ; (2) incompatible monoecists, i.e., the bisexual strains, which produced only mature oospores owing to the bipolar mechanism of homogenic incompatibility with a difference in the two mating type alleles (A' and A * ) .No multiple alleles of the mating type locus have been observed (14 species). I n the possible combinations between the different mating types of the incompatible strains, fertilization did not take place in 6 combinations, and in 12 combinations no oospores developed after fertilization. Although no further genetic analysis of this phenomenon has been performed, this failure of spore formation can be attributed to heterogenic incompatibility, since in all incompatibie cases the A-B-C scheme (see p. 114) is instrumental, i.e., the two incompatible partners have both been compatible with a t least one other strain. This result renders it unlikely that the phenomenon might be caused by inter-species crosssterility; moreover, the validity of the species concept in this parasitic genus is very questionable (see p. 115). 3. Ascornycetes

Probably because this class includes some of the genetically most thoroughly studied genera (e.g., Neurospora, Aspergillus, Podospora) , there are numerous data pointing to the existence of heterogenic incom-

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patibility. Apart from Podospora, which has been already discussed in detail above (p. log), it is with Neurospora that most data have been accumulated.

Meurospora As early as 1933 Moreau and Moruzi reported “cross sterility” between opposite mating types in interracial crosses of N . sitophila. They tested the offspring of 5 A and 5 a mating types (obtained from the “Dodge strains”) with the opposite mating types of their own isolates in all possible combinations and found that 3 of the Dodge strains reacted with the other strains according to the A-B-C-scheme (Moreau and Moruzi, 1933). A comparable phenomenon of cross “sterility” has also been reported by Lindegren (1934) for N . crassa. I n both cases genetic analyses have not been performed. A first indication for the occurrence of vegetative heterogenic incompatibility has been described in the classical “heterokaryon-paper” of Beadle and Coonradt (1944) : strains of N . crassa must ordinarily be of the same mating type in order to form a vigorous and stable heterokaryon. Whereas the genetic basis of heterogenic incompatibility concerning the sexual phase still remains to be elucidated in detail, the vegetative incompatibility has attracted the attention of numerous authors and is largely understood in its genetic details. There are evidently several loci involved, all of which do not concern the sexual phase and act according to the allelic type (see Fig. 2). a. T h e Mating T y p e Genes. Quite recently Newmeyer (1968, 1970) was able to explain the above-mentioned observation of Beadle and Coonradt, familiar at least to every neurosporologist, that different mating types of N . crassa in spite of being the prerequisite for the completion of the sexual cycle in some cases are not able to form normal heterokaryons. In order to eliminate the influence of loci other than mating type genes, which in general can be achieved only in isogenic strains, chromosomal duplications for the mating type region have been produced by the use of a specific chromosomal inversion (Newmeyer and Taylor, 1967). The duplications initially exhibited a drastic inhibition of growth, thus proving that the heterokaryon incompatibility is caused by the mating-type alleles (Turner et al., 1969). After a few days the A / a heteroeygosity is usually eliminated via somatic events and thus the normal growth is restored. This phenomenon is very reminiscent of the observations about the incompatibility of the allelic and nonallelic factors in Podospora in a heterokaryon or in one single nucleus (see p. 109).

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Since the gene toZerant (tol, linkage group IV) which is not linked with the mating type genes only suppresses the vegetative incompatibility and exhibits no influence on the sexual reaction, Newmeyer supposes that the mating type locus consists of two genetic entities. I n crosses between different mating types one unit is instrumental only in the homogenic system as “true” mating type and causes sexual compatibility, whereas the other genetic trait controls heterokaryon formation and causes vegetative incompatibility. Newmeyer and Howe (personal communications) have failed to separate the two functions of the mating type region by recombination, in experiments involving large numbers of progeny selected for recombination between flanking markers. b. The CD Genes. Using nutritional mutants it has been proved that two unlinked allelic pairs ( C / c and D / d ) also control heterokaryon formation in N . crassa (Garnjobst, 1953, 1955; Garnjobst and Wilson, 1956, 1957; Williams and Wilson, 1966, 1968; Wilson, 1961, 1963; Wilson et al., 1961). A heterokaryon will result only if the partners carry like alleles a t both loci. If one or both factors are heterogenic, there will be an incompatibility reaction leading to a destruction of the hyphal parts which have fused. The C D genes are neither linked with the mating type genes nor suppressed by the to1 gene of the mating type complex, nor do they interfere with the sexual compatibility of the different mating types. I n addition, Wilson and Garnjobst (1966) have found a third locus ( E / e ) which resembled in its effects the C D genes. I n analyzing different geographical races, Perkins (1968) has detected multiple alleles of the C-locus. Thus the CD loci can be considered as determinants of an allelic mechanism which exactly corresponds to the action of the (‘barrage genes” in P. anserina as quoted above (p. 109). This similarity extends also to the physiological action of these genes, since Williams and Wilson (1966, 1968), having used an ingenious method of microsurgery and performed biochemical experiments, suggest that the active principle for vegetative heterogenic incompatibility is “closely associated with the membrane’’ and becomes effective only after membrane damage. The investigations of Gross (1952), Holloway (1955), and De Serres (1962) confirmed the view that, in Neurospora crassa, failure of heterokaryon formation is controlled by a number of genes which are not identical to the factors determining homogenic incompatibility. Whether or not these are the same loci analyzed by Garnjobst (1953, 1955) was not determined. c. The I Locus. Pittengcr and Brawncr (1961) and Pittenger (1964) found an allelic pair ( I / i ) which controls the capacity of nuclei in the heterokaryon to divide.

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When the proportion of i nuclei in an ( I + i ) heterokaryon is less than 70%, the capacity of the i nuclei to divide is decreased to such an extent that the number of I nuclei increases and the heterokaryon becomes an I homokaryon. By making corresponding tests with homokaryons, it was shown that cytoplasmic differences or a differential division frequency of the two kinds of nuclei could not account for the incompatibility between the I and i nuclei. By the use of different marker genes in the nuclear components of the heterokaryons, the I / i incompatibility was shown to be generally independent of the genetic background. Sexual incompatibility (as in the case of the CD genes) is not influenced by the I / i alleles. Tests in which strains carrying the I locus were compared with the C and D loci of Garnjobst (1953, 1955) indicate that different genes are involved. This is confirmed by the fact that phenotypic expression of vegetative incompatibility differs in the two cases.

Aspergillus The failure of heterokaryon formation between various natural isolates of Aspergillus has been described by Gossop et al. (1940) for A . niger and by Raper and Fennel1 (1953) for A . fonsecueus. Since both species are imperfect the genetic background of this phenomenon remained obscure until comprehensive studies initiated by Jinks with the perfect species A . nidulans had been performed (Grindle, 1963a,b; Jinks et al., 1966; Butcher, 1968, 1969). A synopsis of the observations and experiments done by the Birmingham group, including numerous related and unrelated isolates from all over the world, has led to the following conclusions : 1. Heterokaryon incompatibility is very common between different isolates of this self-compatible fungus. It is not due to geographical isolation since the various “compatibility groups” comprise isolates from adjacent regions as well as from distant areas. It is furthermore not linked with minor morphological differences of the isolates. 2. This vegetative incompatibility does not prevent the formation of anastomosis. After nuclear exchange the heterokaryon of incompatible combinations seems to have a selective disadvantage and is supposed to be overgrown by the monokaryons. 3. The sexual reaction leading to perithecial formation is in general not inhibited by the vegetative incompatibility. In most cases, however, the number of fruiting bodies is reduced as was also noticed in Podospora. 4. Concerning the genetic basis, extrachromosomal traits can be excluded as responsible for controlling heterokaryon formation. Unfortunately, a detailed gene analysis is not available, but statistical calcula-

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tions have led to the assumption that incompatible strains differ by a t least 5 genes. 5. The physiological action of these genes is not fully understood, since a “killing reaction” like that in Neurospora seems not to take place. However, some sort of interaction seems to be present since the progeny of crosses from vegetative incompatible partners are less vigorous than their parents. Despite the fact that there are some open questions the existence of true heterogenic incompatibility cannot be neglected. Studies of some more imperfect species A . versicolor, A . terreus (Caten, 1971), and A. amstelodami (Caten et al., 1971) have in general confirmed these results obtained with A. nidulans. Thus in A . glaucus and in its imperfect relatives, heterogenic incompatibility inhibiting heterokaryon formation is caused by the presence of different alleles a t one or several specific loci. As with the barrage phenomenon in Podospora (p. 109) even a single allelic difference concerning one locus is sufficient to suppress heterokaryosis. Heterogenic incompatibility has also been observed by Jones (1965), who studied heterokaryon formation among 35 isolates of A . glaucus. In addition Jones (1965) observed that the exchange of nuclei in some combinations is unilateral, a very interesting parallel to the semiincompatibility of Podospora, but a well defined genetic mechanism is also lacking in this case. I n Aspergillus heterothallicus, the only representative of this genus exhibiting bipolar incompatibility, heterokaryosis is controlled by factors other than the mating type genes (Kwon and Raper, 1967). This has been revealed by a study of 11 isolates. In order to obtain information on the number of genetic loci controlling heterokaryon formation, a more detailed analysis of two of these isolates has been performed, since failure of heterokaryon formation does not inhibit the sexual reaction. Among the offspring of 165 mycelia a proper 1 :1 segregation for the mating type factors was observed, but in none of the possible combinations of the F, members could a heterokaryon be established, this being due to failure of anastomosis. Assuming that heterokaryon formation is controlled by heterogenic incompatibility, as in N . crassa it requires heterogeneity of all loci concerned. Kwon and Raper propose on the basis of statistical calculations that both parents should differ by a t least 5 unlinked loci. The authors state that an alternative explanation would be to admit the action of 8 “cytoplasmic factor” which cannot be definitively excluded by the experiments “at hand.” Which one of these two interpretations might be true is not important in the present context, since it is of minor

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importance whether the vegetative heterogenic incompatibility is caused by chromosomal or extrachromosomal genetic diversity. In completing this review of the Aspergillus data we shall mention one more indication of heterogenic incompatibility within this genus. Prasad (1970) reports that in some cases mutants of conidial color of Aspergillus nidulans, which had been induced by UV or chemical mutagens, failed to form heterokaryons because of an inability to undergo anastomosis. I n spite of the absence of further genetic data the author suggests that this kind of vegetative incompatibility “is under genetic control.”

Cochliobolus The genus Cochliobolus includes plant parasites causing leaf and inflorescence diseases in Gramineae. The imperfect stage is known as Helminthosporium. Delimitation of species in this genus was originally performed according to host specificities, origin of isolates, and slight morphological diversity. I n contrast to Neurospora and Aspergillus, in this fungus data have been accumulated which point to the existence of sexual heterogenic incompatibility, whereas vegetative incompatibility haa not yet been described. Nelson and his collaborators have studied very intensively the genetics of the mating reactions within this genus (for references, see Nelson, 1963, 1965a,b, 1966, 1970; Nelson and Kline, 1964; Webster and Nelson, 1968). The detailed analysis of the mating reactions of nearly 10,000 isolates from North and South America, comprising more than 40,000 matings, has led to the following results: 1. The bipolar mechanism of homogenic incompatibility is responsible for the basic control of mating; i.e., only the combination of the alleles A and a leads to fruiting body formation. 2. The fertility in crosses between A and a mating types of strains from different hosts or origin is about 29%. Most of the infertile crosses produce perithecia with immature or sterile ascospores. The rest show no fruit body formation. 3. Several sterility genes blocking the normal ontogenesis a t different stages could be identified and were predominantly responsible for the formation of sterile perithecia. 4. The fact that some strains exhibiting incompatibility in certain combinations were compatible in all others can only be explained by the action of heterogenic incompatibility, despite the fact that the genes have not yet been identified. The objection that this phenomenon might be provoked by gross genetic diversity or be the result of species differences is refuted by the following experiments.

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Seventy-nine different isolates with similar conidial morphology, undoubtedly not belonging to different species, were crossed in all possible combinations. For most isolates there were combinations that yielded fertile perithecia, sterile perithecia, or no perithecia. I n crossing 7 strains differing in conidial morphology in various combinations, it was possible to abolish the incompatibility barrier by isolating compatible recombination types. This excludes unequivocally the likelihood that chromosomal diversity or aberrations are instrumental in the failure of fruit body production. Cochliobolus is a good example of the necessity to check the criteria mentioned above in order to understand not only the mating relations within one genus, but also to establish a species concept which considers the evolutionary events. Furthermore it points to the role that heterogenic incompatibility has played and still plays as an isolation mechanism in evolution. I n this connection it seems worthwhile to acknowledge that Nelson has been able to assign 99.2% of his strains to 5 distinct morphological types which might correspond to single species. This classification takes into account also the genes that are responsible for pathogenicity.

Micros porum Within the dermatophytic fungi of the genus Microsporum (perfect state, Nannizzia) several authors have observed a failure of cleistothecial production in some crosses of opposite mating types (for literature, see Padhye and Carmichael, 1971). Padhye and Carmichael have tested the mating reactions of 17 isolates of Microsporuin cookei (syn. Nannizzia cajetani) originating from different sources. I n all possible combimating types and the 6 - mating types, only nations between the 11 19 combinations were fertile and 31 yielded no fruiting bodies. Although no genetic analyses were performed, these data indicate that within this species genetic factors other than the ones for the mating type genes may lead to a n incompatibility that probably belongs to the heterogenic type.

+

Saccharomy ces As heterogenic incompatibility does not allow for the coexistence of unlike genetic material, whether located in the nucleus or in the cytoplasm (see the Culez case, p. 139), and leads in the most thoroughly analyzed examples to an antagonistic reaction, the “killer phenomenon” detected in Xaccharomyces cerevisiae (Bevan and Somers, 1969; Somers and Bevan, 1969) can be regarded as an example of this breeding system. This conclusion is also substantiated by the observation t h a t the killer reaction ultimately prevents the exchange of genetic material.

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There are three different phenotypes: the killer cells release an unstable macromolecular protein into the medium which kills the sensitive cells, and there are also neutral cells that do not kill sensitive cells and are not killed by the killers. These relations are controlled by three genetic elements: The allelic genes M and the cytoplasmic elements killer [ k ] and neutral [ n ]. Both cytoplasmic determinants are maintained only in cells containing the gene M ; they disappear in presence of the recessive allele m. From this it follows that one can attribute to the three phenotypes the following genotypes: Killer M [ k ] , Neutral M [ n ] ,and Sensitive M [ o ] or m [o] where o stands for absence of the cytoplasmic particles [ k ] and [ n ] . The killer phenomenon may serve as an example of a cooperation of nuclear and extranuclear genetic elements in causing heterogenic incompatibility and thus demonstrates that exchange of the genetic material is under a multiple hereditary control. However, from a very preliminary note of Berry and Bevan (1970), one may deduce that the killer effect in yeast may have something in common with the killer effect in Paramecium rather than with heterogenic incompatibility, These authors have established “that both killer and neutral contain a unique species of RNA which is absent in sensitive strains.” This RNA is supposed to be double stranded, and thus points to the presence of viruslike particles.

Sordaria Heterokaryon incompatibility has been observed in both species of this genus, S. fimicola and S. macrospora, which have been submitted to detailed genetic analysis (Olive, 1956; Carr and Olive, 1959; Esser, unpublished) . Since in these self-compatible species which lack male sex organs and male gametes crossings between different strains are initiated by heterokaryosis, this kind of heterokaryon incompatibility causes indirectly a sexual incompatibility. Even though relevant genetic analyses have not been performed, the existence of heterogenic incompatibility may be inferred since the races in question reacted according to the A-B-C scheme (see p. 114).

Blysiphe A detailed examination of 44 clones of Erysiphe cichoracearum isolated from various flowering plants has shown that the basic breeding system in this species is controlled by a single locus, the two mating types of which react according to the bipolar mechanism of homogenic and incompatibility (Morrison, 1960). However, in some cases the

+

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- mating types failed to react with each other. Since this phenomenon has not been analyzed genetically, i t can be regarded only as an indication of heterogenic incompatibility.

4. Basidiomycetes I n this taxonomic group of fungi many cases of failure of fruit body production (apart from homogenic incompatibility) have become known since the publications of Bauch (1927) and Vandendries (1927, 1929). I n Ustilago violacea Bauch found that within each race of this species, which show bipolar incompatibility, both mating types are compatible with each other. In interracial crosses, in contrast, opposite mating types are either (as in Podospora p. 112) semi-incompatible or incompatible. Bauch explained this [‘abnormal” mating behavior by the assumption that “secondary sex factors” are involved. These findings of Bauch have been later confirmed by Grasso (1955) , who studied interracial crosses of two other species, U . avenue and U. laevis, originating in Italy and the United States. The phenomenon designated by Vandendries as sterility in interracial crosses of Coprinus micaceus, the basic mating system of which is tetrapolar incompatibility, can also be interpreted as heterogenic incompatibility. I n view of the lack of precise knowledge of the existence and genetic basis of the various mating systems (physiological dioecism, homogenic incompatibility, heterogenic incompatibility of the sexual or vegetative phase) and their interaction with true sterility genes, all phenomena resulting in inhibition of fructification were poorly understood or referred to as crossing barriers in the older publications. No attempts have been made to undertake detailed genetic analyses until very recently. Thus it is more than understandable that even in a quite recent review (Burnett, 1965) all these restrictions of mating in 17 species of Basidiomycetes due to either sterility or heterogenic incompatibility or both, could be treated only under the general heading of “restriction of outbreeding.” Our own efforts to analyze the data a t our disposal have made it evident that many of those mating restrictions can be attributed to heterogenic incompatibility, but the data concerning the basic genetic mechanisms are very poor. We will therefore present in detail only the better analyzed cases, and examples merely indicative of heterogenic incompatibility are referred to in Table 3. Additional handicaps are the numerous synonyms under which the Basidiomycetes have been published by the taxonomists. These cause much confusion for those geneticists and biochemists who are not mycolo-

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gists and have also led to parallel research work with strains of the same species under different names. Therefore we have attempted to present all synonyms which have come to our attention.

Sistotrema I n Sistotrerna brinkrnanni (syn. Corticiuin coronilla) strains obtained from different geographical locations exhibit three types of basic control of sexuality: (1) compatibility, i.e., mycelia grown from monokaryotic basidiospores produce fruiting bodies with normal viable spores ; (2) homogenic incompatibility characterized by the bipolar mechanism, i.e., one mating type locus with multiple alleles; (3) homogenic incompatibility characterized by the tetrapolar mechanism, i.e., two incompatibility TABLE 3 Fungi in Which Mating Restrictions Have Been Observed Which Might Be Caused by Heterogenic Incompatibility Organism

Tilletia controversa Mywcalia denudata Mycocalia caatanea Auricularia auricula-judae Gloeocystidium h u e Coprinus micaceus Coprinus macrorhizus f . microsporus Coprinus callinus Coprinus subimpatiens a

References Hoffmann and Kendrick (1969) Burnett and Boulter (1963) Barnett (1937) Boidin (1950) Kuhner et al. (1947) Kimura (1952) Lange (1952) Lange (1952)

The references are partially derived from Burnett (1965).

factors with multiple alleles (Biggs, 1937). Recently Lemke (1969) has confirmed and extended the work of Biggs by analyzing the interstrain relations of 11 isolates from different parts of the world. He used the technique of forced heterokaryosis between auxotrophic mutants and established that there are fertility barriers within each of the above-mentioned three basic classes as well as in interclass crosses. This phenomenon is interpreted by Lemke as heterogenic incompatibility for two reasons: (1) I n the incompatible interracial crosses the two auxotrophic partners form unbalanced mycelia with poor vegetative vigor and no clamp connections. This points to an antagonistic reaction similar to that in Podospora heterokaryons. (2) In one compatible interracial cross, recombinant types were obtained with an altered compatibility pattern. This excluded the presence of sterility genes, which were sometimes found in other combinations.

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Rhizoctonia I n the plant parasite Rhizoctonia solani (syn. Thanatephorus cucumeris; syn. Ceratobasidium filamentosuva), the basic mating system is determined by monoecism, i.e., single basidiospore isolates taken from nature are able to form fruiting bodies. However, some of these isolates fail to form viable heterokaryons despite the occurrence of hyphal fusions. This phenomenon seems to be under the genetic control of one locus with multiple alleles (for references, see Whitney and Parmeter, 1963; Garza-Chapa and Anderson, 1966). Stretton et al. (1967) showed that cultures which are genetically identical anastomose successfully, whereas anastomosis of genetically nonidentical strains “frequently results in the death of participating and neighbouring cells.” McKenzie et al. (1969) have studied this disturbance of heterokaryosis in detail. From one natural isolate, 4 induced and 2 spontanous sterile morphological mutants have been obtained. With the exception of two cases, in all possible combinations between these 6 mutants a killing reaction occurred immediately after anastomosis leading to cell death. However, a t 25OC still a few anastomosing cells survive and thus allow the establishment of a heterokaryon which remains stable and leads to the formation of fruiting bodies through a complementation of the nonallelic sterility genes. A 5OC no anastomosis survives and no heterokaryons are formed. There is not yet an explanation of this peculiar phenomemon of lethal interaction which also occurs in Myxomycetes (see p. 119), but there is an assumption of differential gene activity. The authors state that only the sterility genes, the existence of which could be proved by genetic analysis of the fertile heterokaryons, must be responsible for this initial heterokaryon incompatibility, since all strains have been derived from one uninucleate isolate. This is not necessarily so, since it is known that spontaneous mutations can occur at incompatibility loci (Bernet and Belcour, 1967).

Polyporus Macrae (1967). has studied the mating reactions of 31 single-spore isolates of the tetrapolar Polyporus abietinus (syn. Hirschioporus abietinus) originating in different places in eastern and western North America and Europe. All strains were much alike in general appearance and similar in microscopic characters. From differences in the morphology of their hymeneal surfaces the isolates have been assigned to three morphological groups. Within one of these groups (called the poroid form) isolates of different mating type failed to pair. The North Ameri-

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can strains could be subdivided into two classes A and B which are incompatible with each other, but both of which are compatible with a third class C which included the European strains. Geographic isolation could be excluded as responsible for the incompatibility between A and B, since collections from the same host and from both eastern and western parts of North America have yielded members of both groups. After discussing the possibility that the incompatibility between A and B might be due to the presence of identical incompatibility alleles (i.e., to homogenic incompatibility) Macrae comes to the conclusion that this interpretation is not valid and that “the explanation must be found elsewhere.” Despite the fact that there are no genetical analyses of the compatible combinations from A/C and B/C the control of the mating behavior seems likely to depend on genic diversity, i.e., heterogenic incompatibility (see the A-B-C scheme on p. 114). In Polyporus schweinitzii, the cause of root- or butt-rot in conifers, a zone of “aversion” is very often observed in crosses of geographical races. I n this zone dark pigments are formed which are apparently the result of disruption of hyphae after anastomosis (Barrett and Uscuplic, 1971). This phenomenon, the genetic basis of which is unknown, is reminiscent of the vegetative incompatibility of Neurospora and Podospora and the formation of a demarcation line in Fomes (see below). Fomes

Paired isolates of wood-destroying fungi very often form a so-called line of demarcation, also sometimes called barrage (see literature in Adams and Roth, 1967). These dark lines may appear between compatible as well as between incompatible matings. Adams and Roth have made a first attempt to define the regularity of occurrence of this phenomenon in terms of the genetic relationship of the participating strains. Four dikaryotic strains of Fomes cajanderi, characterized by bipolar incompatibility, have been isolated from trees of one area. Two of the isolates did not form a demarcation line, whereas in all the other combinations the black zone appeared. Various crosses between monokaryons, obtained from the dikaryotic isolates, and di-mon mating between monokaryons allow the following generalizations : 1. I n any combination exhibiting a demarcation line there is no dikaryotization leading to the formation of clamp connections and fruit body production. 2. The intensity of the demarcation line may be diminished by using recombinant strains which are “more closely genetically related.” The authors conclude that the demarcation line in the wood-destroying fungi may be used as an indication of genetically distinct mycelia to

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provide evidence of separate biological entities. Unfortunately, there are no analyses of the genetic factors responsible allowing the assignment of this phenomenon with certainty to heterogenic incompatibility. These observations have been confirmed recently by Neuhauser and Gilbertson (1971) with different natural isolates of F. cajanderi from all over the United States. Since their main interest was the identification of incompatibility alleles of the bipolar mechanism, no new informations about the demarcation line was obtained. The formation of a demarcation line has also been found in other species of the genus Fomes, as published some time ago: A worldwide sample of geographical races of the bipolar F. pinicola (syn. F . marginatus) could be subdivided into three groups (Mounce, 1929; Mounce and Macrae, 1938). Groups A and B are confined to North America, whereas the members of group C originate from other parts of the world. There was no barrier to fertilization between isolates belonging to the same group. However, dikaryotization between members of group A and C was not observed, whereas formation of dikaryotic hyphae occurred readily between A and C and rarely between B and C. Unfortunately this typical example of the A-B-C scheme is not supported by genetic data. In F . ignarius Verrall (1937) distinguished three “ecological types” on the basis of their host origin, which differed also in their mating systems (bipolar or tetrapolar) and in minor morphological characteristics. There was no dikaryotization between strains of different groups and barrage formation occurred.

Cyathus The sexual processes in the “Bird’s Nest Fungi” belonging to the genus Cyathus are in general controlled by the tetrapolar mechanism of homogenic incompatibility (for literature, see Brodie, 1970). However, in two species (C. stercoreus, C. africanus) it became clear that these factors are not the only genetic traits controlling the process of dikaryotization (Brodie, 1948, 1970; Fulton, 1950). Thus both authors observed that in crosses of compatible mating types (e.g., AB X AIB,) originating from natural isolates a unilateral dikaryotization occurred, i.e., only one of the two paired monokaryons was able to receive nuclei from the partner strain in order to become dikaryotic. This phenomenon, which occurred in about 40% of all compatible matings, is reminiscent of the nonreciprocal sexual incompatibility in Podospora (p. 109). As regards the genetics of this failure of normal fertilization, it can only be stated that the genes responsible are not correlated with the mating type factors. So far this phenomenon can only be evaluated as one more indication for the occurrence of heterogenic incompatibility.

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Peniophora A comprehensive study of the mating relations among natural isolates of the three bipolar species P . heterocystidia, P . mutata, and P. populnea which do not differ in gross morphology have shown that, in crosses of monosporous mycelia of different mating types, the formation of clamp connections occasionally does not take place (McKeen, 1952). This phenomenon occurs as readily in intraspecies as in interspecies combinations and seems to be correlated with which host is involved (Populus or other trees, respectively). Genetic data are not available. Ustilago In the tetrapolar Ustilago maydis, an antagonism between different genetic strains which does not depend on the mating type genes has been observed (Puhalla, 1968; Hankin and Puhalla, 1971). This phenomenon, which is caused by the interaction of genes and cytoplasmic factors, is similar to the killer phenomenon in yeast as already described (p. 127). There are three phenotypes, the genetic constitutions of which have been revealed by tetrad analysis : 1. Antagonistic strains producing a heat-labile protein which inhibits the growth of the sensitive strains and does not interfere with its own growth. Genetic configuration: gene s or s+ and cytoplasmic elements [I], CSI; the s+ allele confers insensitivity, the s allele sensitivity which is suppressed by the cytoplasmic element [SI; the element [I1 is responsible for the production of the inhibitor substance.

2. Sensitive strains, which do not produce inhibitor protein but are sensitive to it. Genetic configuration: gene s, no cytoplasmic elements [OI.

3. Neutral strains, which do not produce the inhibitor protein and are insensitive to it. Genetic configuration:s+ [OI,s [SI, or s+ [Sl.

There are some differences in the genetic mechanism as compared with the yeast killers, e.g., two cytoplasmic elements [I] and [S] ; the gene s causing sensitivity is dominant over sc. However, the main difference is that in Ustilago there is no cell death or interference with the fusion of different mating types, and hence sexual propagation is not

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prevented. This reveals a similarity with the heterogenic vegetative incompatibility in Neurospora or Podospora (pp. 109,122).

B. HIGHERPLANTS The phenomenon of sexual incompatibility was first observed by Kolreuter as early as 1764 in higher plants and has been found to be widely distributed among them (East, 1940). It causes an inhibition of the development of the male gametophyte ranging from inhibition of pollen germination on the stigma through inhibition of pollen tube growth to an inability of the male nuclei to enter the ovule. This restriction of mating is controlled exclusively by various mechanisms of the homogenic system which, independently of their different genic action in homomorphic and heteromorphic plants, inhibit self- and cross-fertilization when like genetic determinants are present in pollen and stylar tissues. One of the most common mechanisms of homogenic incompatibility in higher plants, which acts only in homomorphic flowers, is the S-gene mechanism: There is one locus S of which multiple alleles exist. Incompatibility is caused by an interaction between haploid pollen grains and diploid stylar tissue containing the same S allele, e.g., S,and S2 pollen cannot penetrate with their pollen tubes S,S, styles, whereas S,S, . . . S,, pollen tubes will reach the ovule. I n contrast to the detailed knowledge available about the numerous genetic systems of higher plant incompatibility our knowledge of the physiological action of these genetic systems is confined to models or working hypotheses (for literature see the reviews quoted on p. 108). 1. Unilateral Incompatibility

During their studies of homogenic incompatibility various authors have observed that in some cases the incompatibility between two hermaphrodite plants is restricted to one of the two reciprocal crosses. This phenomenon is reminiscent of the heterogenic incompatibility found in interracial crosses of the Ascomycete Podospora anserina (see Fig. 1 and p. 109)) and was called “unilateral incompatibility” (for literature, see review of Townsend, 1971). The fact that in unilateral incompatibility only one of the two possible combinations of male and female nuclei is inhibited excludes the presence of sterility factors or the action of lethal factors which may act as crossing barriers as found by Chu and Oka (1968) and Chu et al. (1969) in Oryza spp.

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From the more thoroughly genetically analyzed cases of unilateral incompatibility, it is obvious that there exist a t least three different genetic mechanisms. a. Unilateral Incompatibility Controlled b y the S Genes. I n some plants in which the S-gene mechanism is present, it has been observed that in interracial or interspecies crosses between self-incompatible (SI) and self-compatible (SC) plants the pollen tubes are inhibited only in the cross 9 SI X 8 SC whereas in the reciprocal cross 9 SC X 8 SI fertilization will take place, e.g., in Petunia (Mather, 1943) or in Antirrhinum (Harrison and Darby, 1955). On the basis of studies in some genera of the Crucijerae, Onagraceae, and Solanaceae in which inhibition of the SC pollen tubes in the style of the SI plant was similar to that after self-fertilization by SI pollen, Lewis and Crowe (1958) and Lewis (1965) postulated that the S genes are bipartite, consisting of one component active only in the pollen tube and another component active only in the stylar tissue. This would mean that in the self-compatible plants the stylar component either has not been developed or has been lost by mutation during evolution. This working hypothesis, that unilateral incompatibility is caused solely by the S genes of the homogenic system, has been supported by experiments of Gunther and Juttersonke (1971) in Lycopersicon peruvianum. These authors found that self-incompatibility in L. peruvianum can be brought about either by inhibition of the pollen tubes or by degeneration of the endosperm, whereas the unilateral incompatibility between L. esculentum and L. peruvianum is caused exclusively by the first mechanism. This has led to the suggestion that unilateral incompatibility is a part of the S-gene mechanism. Similar results were found by Lloyd (1968) in Leavenworthia and by Keep (1968) in Rubus. b. Unilateral Incompatibility Controlled b y S Genes and Additional Genes. Pandey (1967, 1968) has studied in a very comprehensive manner the cross relations between 16 self-compatible and 1 self-incompatible species and races of Solanum originating from South America and Australia, which were crossed in every possible combination. Despite the fact that some species differed in their chromosome numbers in all compatible crosses the pollen tubes reached the ovule and in most cases set fruit, whereas in the incompatible combinations an inhibition of pollen tubes in the style was observed. Unilateral incompatibility was found not only in crosses between the SC and SI groups of plants (in both reciprocal combinations), but also in crosses within the SI and SC groups. I n all four types the unilat,eral incompatibility was caused by an inhibition of pollen tube growth on

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the stigma, whereas the SI in intrastrain crosses was due to an inhibition in the style. An evaluation of these data allowed the grouping of the 17 hermaphrodite strains into 7 ‘Lfemale”and 7 “male” classes according to their properties in interstrain crosses. Since most of the hybrids were fertile, tests of some of the descendants have been performed. The multiplicity of results could not be explained solely by the S-gene theory; therefore Pandey concluded that the properties of the S-gene complex “under certain conditions may be modified by other genes of the genome.” This hypothesis is also supported by data on unilateral incompatibility obtained earlier by Grun and Aubertin (1966) in Solanum and by Martin (1967, 1968) and Hardon (1967) in Lycopersicon. c. Unilateral Incompatibility Controlled by Factors Other than S Genes. Arnold and Fellenberg (1965) detected a factor called H50 located within the complex cl of Oenothera campylocalix which causes unilateral incompatibility in crosses between 0. campylocalix and other species of the subgenus Euoenothera. Since in one of the strains the H50 factor was found to be mutated to the recessive factor H57 which restored normal bilateral compatibility, the conclusion that these factors display their action independently of the S genes seems to be justified. I n summing up the various data describing the “unilateral incompatibility” in higher plants, it becomes evident that this phenomenon coincides with our definition of “heterogenic incompatibility,” because in all cases the block of fertilization is caused by genetic differences, not by genetic identity as in the homogenic system. This conclusion is not in contradiction even with the first mechanism considered, where it is assumed the S genes of the homogenic system are instrumental in the realization of unilateral incompatibility, because a genetic difference of the complex genes is required. A certain parallel with Didymium and Neurospora (see pp. 119 and 122) , where homogenic incompatibility (mating types) is overlapped by the heterogenic mechanism seems to be present in the second class of mechanisms, as in the Solanaceae. The third class (Oenothera) is so far the only instance of genetic traits which cause heterogenic incompatibility independently from any mechanism of the homogenic system. Further research may show whether these three genetically analyzed examples of heterogenic incompatibility represent the only mechanisms or whether other cases of incompatibility as yet analyzed (e.g., in Oenothera, Steiner, 1961 ; Schultz, 1962 ; in Gilia, Grant, 1965; in Agrostis and Anthoxanthum, McNeilly and Antonovics, 1968 ; in Clarlcia, Parnell, 1968; in maize, Paterniani, 1969) are controlled by other hitherto unknown mechanisms.

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8. Plastome Incompatibility

Since in Oenothera plastids are transferred to the ovule by the pollen tubes, it is possible to produce plastid hybrids. This kind of hybridization mostly leads to the formation of chimeras exhibiting zones of genetically different plastids which can be observed macroscopically if the plastid species are distinguishable by the intensity of chlorophyll formation. Kutzelnigg (personal communication) observed a whitish zone of about 2 mm in width on the leaves of an Oenothera chimera between areas containing dark green wild-type plastids and light green mutant plastids (type 18). Cytological observations have shown that in this zone both growth and the formation of chlorophyll in the mutated chloroplasts are inhibited by the presence of the wild-type chloroplasts. I n addition, the cytoplasm is degenerate and the cell walls exhibit irregular thickenings. This phenomenon reminds strongly of the barrage formation in Podospora and of the vegetative incompatibility in other fungi. Since genetic influences of nuclear genes or of the cytoplasm could be excluded, the incompatibility of plastids is caused solely b y their own genetic factors. This proves that heterogenic incompatibility is a phenomenon which concerns the genetic information in general regardless of its localization in chromosomes, cytoplasmic units, or plastids.

C . ANIMALS In comparison to the predominantly hermaphrodite plants, sexual incompatibility of the homogenic type does not play an important role as a breeding system in animals. Its effect of increasing the outbreeding capacity is in general achieved in animals by dioecism. The occurrence of homogenic incompatibility in some hermaphrodite Protozoa and other lower animals (for reference, see Grell, 1968) may be regarded as an exception. However, there are some examples known in which karyogamy between female and male nuclei is prevented by genetic differences not identical with sex factors. These phenomena can be regarded as heterogenic incompatibility. 1. Protozoa

In some Ciliata, the basic mating system of which is the bi- and tetrapolar mechanism of homogenic incompatibility, some irregularities in the coexistence of genetically different nuclei have been observed which suggest similar phenomena of sexual and vegetative incompatibility as those described above for fungi.

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a. I n Paramecium (Sonneborn, 1957) , some Ozytricha (Siegel, 1956), and Uronychia (Reiff, 1968), a combination of opposite mating types of different geographical races (syngens, Sonneborn, 1957) may lead either to a failure of conjugation or to a lethality of the exconjugants after sexual reaction and nuclear exchange (sexual incompatibility). b. I n Amoeba proteus, Jeon and Lorch (1969) were able to demonstrate a vegetative incompatibility. Since these animals are not able to mate or to conjugate, nuclear transplantation from different strains was used to give “heterokaryons” which usually died whereas the “homokaryons” from control transplantations survived. The authors suggested that a protein factor might be responsible for this lethal effect (Lorch and Jeon, 1969). There is the objection that this experiment is not relevant to the problem of heterogenic incompatibility because in nature amoebae do not form heterokaryons. However, the data are of interest with respect to the evolution of incompatibility systems, because both incompatible strains originated from one strain and were kept for only two years in different laboratories. This period, though relatively short, is clearly sufficient to produce genetic differences that cause strong symptoms of heterogenic incompatibility. A comparable lethal reaction occurring immediately after nuclear transplantation was observed by Makhlin and Yudin (1969, 1970) between Chaos chaos and Amoeba proteus. 2. Insects

Culex The most thoroughly analyzed example of heterogenic incompatibility in animals has been published by Laven (1957, 1959). By crossing mosquitoes (Culex pipiens) of 17 geographical races originating from Europe, Africa, Asia, and North America, he found 9 groups which reacted in various (Fig. 5) ways, reminiscent of the heterogenic incompatibility of Podospora (see also Fig. 2 ) . a. Reciprocal compatibility : no crossing barrier between females and males of the two races concerned. b. Nonreciprocal incompatibility : after copulation with a male of the other race, females of the one race laid eggs which did not produce larvae whereas normal egg development occurred in the reciprocal cross. c. Reciprocal incompatibility: a crossing barrier was present in both directions. The similarity between the heterogenic incompatibility of Culex and Podospora concerns only the phenomenon itself, not the genetic mechanism, because in Culex no specific nuclear genes were identified, but only cytoplasmic differences. In a series of more than 50 successive backcrosses, Laven was able to replace gradually the genome of one race

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by the genome of another race without altering the incompatibility reactions of the strains concerned. By these experiments, the existence of “lethal genes” (as in the Protozoa) was also excluded. Thus it became evident that the heterogenic incompatibility in Culex is caused by cytoplasmic factors being transferred both by the egg and the sperm and capable of self-replication. These factors, the nature of which is still unidentified, are neither of viral nor bacterial origin as

FIO.5. Scheme of mating reactions between various races of Cubx pipiens from different localities in Europe, Africa, and Asia. Meaning of the symbols: designation of races is given by the abbreviations used by the author; black squares = compatibility; open squares = incompatibility; open squares with dash = crosses not performed. Note: In some crosses between females and males of different races, incompatibility is nonreciprocal. (Adapted from Laven, 196713.)

has proved to be the case in comparable instances of “cytoplasmic inheritance” (e.g., killer phenomenon in Paramecium, Sonneborn, 1937; CO, sensitivity in Drosophila, L’HBritier, 1951), because it is very improbable that the 9 different plasmon types and their complicated interactions (see Fig. 5) are the result of different microbial infections (Laven, 1957).

Mormoniella In some strains of the hymenopteran Mormoniella, similar phenomena to those in Culex were observed: Females with the eye color mutants

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p e , ti, st, and oy produce few or no female offspring when mated with wild-type males, whereas the reciprocal cross yields the normal percentage of females. p e and ti are completely incompatible with each other (i.e., both reciprocal crosses yield no females), whereas st and o y are incompatible only with p e but not with ti. The incompatibility factor in each stock is unrelated to the eye color mutation and seems to be of cytoplasmic origin, which in contrast to Culex is transmitted through the egg alone. Cytological investigations showed that sperm and eggs have no defects and fertilization is normal. After fertilization, however, the male nucleus forms a tangled mass of chromatin instead of chromosomes. The eggs then develop parthogenetically and hence only males are produced (Saul and Lohrmann, 1968; Ryan and Saul, 1968). The cytoplasmic “factorsf’ arose spontaneously. Their incompatibility behavior as described above cannot be explained by a n infection and must therefore be due to cytoplasmic mutation. To what degree nuclear genes are involved is unknown.

V. Correlations between Heterogenic Incompatibility and Histoincompatibility

From our definition (see p. 177) that heterogenic incompatibility is caused by a n inability of different genetic elements to coexist in close proximity both heterogenic incompatibility and histoincompatibility occurring after tissue transplantations (see literature in Rapaport, 1966) in mammals are very similar phenomena. Despite the fact that the latter is caused by a complicated immune response mechanism and heterogenic incompatibility requires a contact of genetically different structures, the gap between both phenomena seems not to be a fundamental one. There is, as described above, no unique mechanism for heterogenic incompatibility but a great variety of mechanisms ranging from single genes to unidentified constituents of the cytoplasm which provoke heterogenic incompatibility. Moreover, there are studies which demonstrate effects intermediate between the two phenomena. In Coelenterata (Gorgonaceae) contact between transplants of different species results in a mutual destruction. The same occurs in allogenic combinations of two individuals of the same species, which may happen naturally after close contact. If the grafts differ in size (1:8) the destruction is restricted to the small one (the “target”), whereas the large one (“killer”) survives. This is prevented by treatment of the target, but not the killer with inhibitors of protein synthesis. If a classical immunological system was involved the opposite would be expected. An

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active autodestruction of the target, involving protein synthesis and triggered by the killer-explant, is strongly suggested for even a relatively short contact is sufficient to bring about the*“suicide” of the target. Theodor (1970) considers this system a precursor of true immune mechanisms. On the other hand, since “cell membrane bound action . . . triggered by a diffusible substance” is suggested as a result of similar experiments with mouse lymphocytes (Granger and Kolb, 1968) it may represent an intermediary type between histo- and heterogenic incompatibility. In this context the review of Amos (1966) in which nonimmunological histoincompatibility in mammals is discussed is of interest. Observations of tissue incompatibility in sponges, cnidarians, molluscs, and earthworms cited by Theodor (1970) will not be considered, as no experimental analysis is available, although immune mechanisms are apparently not involved. It seems justifiable to conclude that both heterogenic incompatibility and histoincompatibility which seem to have convergently developed during evolution exhibit one and the same effect, i.e., inability of genetically different material to cooperate in a common physiological machinery. VI. Conclusions

The above compilation and analysis of the literature has shown that in many cases the occurrence of heterogenic incompatibility has been unequivocally demonstrated by comprehensive genetic data. However, in a number of examples the phenomenon is inferred from circumstantial evidence only. The main reason for this is that effects properly attributed to heterogenic incompatibility either have been considered as having other causes or else have been merely mentioned as inexplicable secondary effects. However, in spite of these difficulties there are some general points which need to be amplified in a more general manner. 1. Distribution. Heterogenic incompatibility has to be acknowledged as occurring in all groups of Eukaryotes and thus must be regarded as a basic phenomenon controlling the coexistence of different genetic determinants. This clearly differentiates it from homogenic incompatibility which is mainly restricted to plants and within this group, is analogous to the effect of dioecism occurring mainly in animals. 2. Nature of genetic determinants. It is also indicative of the general importance of heterogenic incompatibility that it may be caused either by single genes, by cytoplasmic determinants, or even by genetic traits localized in the plastids leading to identical consequences as may be seen from a comparison of Podospora and Culex (Figs. 2 and 4) and Oenothera.

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3. Efficiency in sexual and vegetative phases. Since there is no fundamental difference between recombinational events in the sexual and the parasexual cycle it is not surprising that heterogenic incompatibility influences both. This also applies to the initiation of plasmogamy which may occur either by sexual processes or, in many fungi, by heterokaryosis. 4. General action. The general effect of heterogenic incompatibility is 2-fold, (a) As already stated in the Introduction it has to be considered as a breeding system which in contrast to homogenic incompatibility favors inbreeding by restricting the exchange of genetic material to within races. This mode of isolating strains or races leads to further speciation. Thus heterogenic incompatibility must have been and still is one of the genetic factors acting in evolution. (b) On the other hand, this isolation may have also a second effect which should not be overlooked. The suppression of cell fusion stops also the transfer of harmful cytoplasmic components, such as viruses or mutant suppressive mitochondria, from one individual to another and thus inhibits the distribution of cell diseases and favors the survival of uninfected cells or tissues. This is especially useful for organisms without true cellular compartmentation, such as fungi. For example, the transmission of infective cytoplasmic factors, which in the ascomycete Podospora anserina cause senescence and finally lead to cell death (Marcou, 1961), may be prevented by vegetative heterogenic incompatibility. Whereas the control of genetic recombination has to be attributed mainly to sexual incompatibility, protection from harmful cytoplasmic factors results predominantly from vegetative incompatibility. 5. Demarcation versus diffusible inhibitory substance. Toxic or inhibitory diffusible substances, not identical to or comparable with antibiotics, are produced in some microorganisms (for reference, see Burnett, 1968). They may even be effective between closely related organisms (e.g., antagonism within coprophilous fungi, Ikediugwu and Webster, 1970a,b). These phenomena must be sharply delineated from heterogenic incompatibility, because they do not require protoplasmic contact in order to be realized. The same holds true for the kappa particles of Paramecium and other similar infective agents. 6. Biochemical basis. There are no clear-cut theories regarding the physiological action of the genes that bring about heterogenic incompatibility. One may speculate that in Podospora the heterogenic incompatibility could be brought about by lysosomes. It is known (see reviews of De Duve, 1963; Allison, 1967; Matile, 1969) that lysosomes may fuse with each other on randomly coming in contact and also that the membranous envelopes of these cell organelles have different structures.

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One might assume that the incompatibility genes are responsible for the structure of lysosomal membranes. After cytoplasmic contact, the accidental fusion of lysosomes from the two partners will be initiated but not completed, owing to the structural differences, and thus these “suicide bags” burst and decompose the cells concerned. Since lysosomes come in contact only by chance, this would also solve the recognition problem of the different genetic elements, which is not understood a t all even in the much better analyzed homogenic incompatibility. Since many of the genetic mechanisms that lead to heterogenic incompatibility seem to have developed independently, it may well be that there are a variety of mechanisms a t the molecular level. 7. Relations with histoincompatibility. Both phenomena have the same effect: hostile interaction of different genetic material originating from closely related organisms. Therefore physiological and biochemical analysis of such phenomena in relatively simple systems, for instance in the fungi (e.g., Podospora or Neurospora) should lead to a basic model which might be used as a starting point for understanding histoincompatibility, which is more relevant for humanity than any other example of heterogenic incompatibility. 8. Consequences for taxonomy. I n imperfect fungi heterokaryosis is often used as a criterion of taxonomic relationship (e.g., Garber et al., 1961; Dhillon et al., 1961). Because of heterogenic incompatibility, this parameter should be handled with great care to avoid creating taxonomic distinctions which are not realistic and may depend on only a single gene difference. 9. Practical consequences. Heterogenic incompatibility has a practical relevance, e.g., in pest eradication and control. Since in Culex the block caused by heterogenic incompatibility affects the development of fertilized eggs (i.e., males copulate with incompatible females and inject their spermatozoa) and since a female is able to copulate only once in her life, the idea of eradicating a Culex population by overloading it with males incompatible with the females has arisen. By competition with the few native males they should reduce the offspring. A repetition of the process in the next generation might lead to an elimination of the whole population (Laven, 1966). With the support of the World Health Organization, large-scale experiments of this kind have been successfully performed in Asia (Laven, 1967a, 1968), thus practicing a genetic method of pest control. With this review we intend to acquaint biologists not only with the existence and versatile genetic basis of heterogenic incompatibility, but also with its general importance as a basic biological phenomenon. If this stimulates a t least some investigators, from taxonomists to geneti-

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cists, to consider in their future research work that there is a phenomenon called heterogenic incompatibility, the purpose of this paper will have been achieved.

ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, and the Landesamt fur Forschung NRW, Dusseldorf. We are very much indebted to Drs. M. Carlile (London), D. D. Perkins (Palo Alto), and L. S. Olive (Chapel Hill) for critical reading of the manuscript.

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Garber, E. D., Wyttenbach, E. G., and Dhillon, T. S. 1961. Genetics of phytopathogenic fungi. V. Heterocaryons involving formae of Fusarium oxysporum. Amer. J . Bot. 48, 325-329. Garnjobst, L. 1953. Genetic control of heterocaryosis in Neurospora crassa. Amer. J . Bot. 40, 607-614. Garnjobst, L. 1955. Further analysis of genetic control of heterocaryosis in Neurospora crassa. Amer. J . Bot. 42, 444448. Garnjobst, L., and Wilson, J. F. 1956. Heterocaryosis and protoplasmic incompatibility in Neurospora crassa. Proc. Nat. Acad. Sci. U.S. 42, 613-618. Garnjobst, L., and Wilson, J. F. 1957. Heterokaryosis and protoplasmic incompatibility in Neurospora crassa. Cytologia, Suppl. Vol. pp. 539442.

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Garza-Chapa, R., and Anderson, N. A. 1966. Behavior of single-basidiospore isolates and heterokaryons of Rhizoctonia solani from flax. Phytopathology 56, 1260-1268. Gossop, G. H., Yuill, E., and Yuill, J. L. 1940. Heterogeneous fructifications in species of Aspergillus. Trans Brit. Mycol. SOC.24,337-344. Granger, G. A., and Kolb, W. P. 1968. Lymphocyte in vitro cytotoxicity: Mechanisms of immune and non-immune small lymphocyte mediated target L cell destruction. J . Zmmunol. 101, 111-120. Grant, V. 1965. Evidence for the selective origin of incompatibility barriers in the leafy-stemmed Gilias. Proc. N a t . h a d . Sci. U.S.54, 1567-1571. Grasso, V. 1955. Studio sulla genetica dei carboni dell’avena: Ustilago avenae e U. levis. Boll. Sta. Patol. Veg., Roma 12, 115-1Q.6. Grell, K. G. 1968. “Protozoologie,” 2. Aufl. Springer-Verlag, Berlin and New York. Grindle, M. 1963a. Heterokaryon compatibility of unrelated strains in the Aspergillus nidulans group. Heredity 18, 191-204. Grindle, M. 196%. Heterokaryon compatibility of closely related wild isolates of Aspergillus nidulans. Heredity 18, 397405. Gross, S. R. 1952. Heterokaryosis between opposite mating types in Neurospora crassa. Amer. J. Bot. 39, 574-577. Grun, P., and Aubertin, M. 1966. The inheritance and expression of unilateral incompatibility in Solanum. Heredity 21, 131-138. Giinther, E., and Jiittersonke, B. 1971. Untersuchungen iiber die Kreuzungsinkompatibilitat zwischen Lycopersicon peruvianum und Lycopersicon esculentum und den reziproken Bastarden. Biol. Zentralbl. 90, 561-574. Hankin, L., and Puhalla, J. E. 1971. Nature of a factor causing interstrain lethality in Ustilago maydis. Phytopathology 61, 50-53. Hardon, J. J. 1967. Unilateral incompatibility between Solanum pennellii and Lycopersicon esculentum. Genetics 57, 795-808. Harrison, B. J., and Darby, L. A. 1955. Unilateral hybridization. Nature (London) 176, 982.

Henney, M. R., and Henney, H. R. 1968. The mating-type systems of the myxomycetes Physarum rigidum and P. flavicomum. J. Gen. Microbiol. 53, 321-332. Hoffmann, J. A., and Kendrick, E. L. 1969. Genetic control of compatibility in Tilletia controversa. Phytopathology 59, 79-83. Holloway, B. W. 1955. Genetic control of heterocaryosis in Neurospora crassa. Genetics 40, 117-129. Ikediugwu, F. E. O., and Webster, J. 1970a. Antagonism between Coprinus hep54, 181-204. temerus and other coprophilous fungi. Trans. Brit. Mycol. SOC. Ikediugwu, F. E. O., and Webster, J. 1970b. Hyphal interference in a range of coprophilous fungi. Trans. Brit. Mycol. SOC.54, 205-210. Jeon, K. W., and Lorch, I. J. 1969. Lethal effect of heterologous nuclei in amoeba heterokaryons. Exp. Cell Res. 56,233-238. Jinks, J. L., Caten, C. E., Simchen, G., and Croft, J. H. 1966. Heterokaryon incompatibility and variation in wild populations of Aspergillus nidulans. Heredity 21, 227-239. Jones, D. A. 1965. Heterokaryon compatibility in the Aspergillus glaucus link group. Heredity 20, 4936. Keep, E. 1968. Incompatibility in Rubus with special reference to R. idaeus L. Can. J . Genet. Cytol. 10, 253-262. Kimura, K. 1952. Diploidization in the Hymenomycetes. I. Preliminary experiments. Bwl. J . Okayama Univ. 1, 72-83.

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Kuhner, R., Romagnesi, H., and Yen, M. C. 1947. Diffhrences morphologiques entre plusieurs souches de Corprins de la section Micacei et confrontation de leurs haplontes. Bd1. SOC. Mycol. Fr. 63, 169-186. Kwon, K. J., and Raper, K. B. 1967. Heterokaryon formation and genetic analyses of color mutants in Aspergillus heterothallicus. Amer. J. Bot. 54, 49-60. Lange, M. 1952. Species concept in the genus Coprinus. Dan. Bot. Ark. 14,7-162. Laven, H. 1957. Vererbung durch Kerngene und das Problem der ausserkaryotischen Vererbung bei Culex pipiens. 11. Ausserkaryotische Verelrbung. Z. Zndukt. Abstamm.-Vererbungsl. 88, 478-516. Laven, H. 1959. Speciation by cytoplasmic isolation in the Culex pipiens-complex. Cold Spring Harbor S y m p . Quant. Biol. 24, 166-173. Laven, H. 1966. Genetik gegen Schiidlinge. Bild Wiss. pp. 447-453. Laven, H. 1967a. Eradication of Culex pipiens fatigans through cytoplasmic incompatibility. Nature (London) 216, 383-384. Laven, H. 1967b. Speciation and evolution in Culex pipiens. In “Genetics of Insect Vectors of Disease” (J. W. Wright and R. Pal, eds.) pp. 251-275. Elsevier, Amsterdam. Laven, H. 1968. Genetische Methoden zur Schadlingsbekhmpfung. Anz. Schaedlingskunde 41, 1-7. Lemke, P. A. 1969. A reevaluation of homothallism, heterothallism and the species concept in Sistotrema brinkmanni. Mycologia 61, 57-76. Lewis, D. 1954. Comparative incompatibility in angiosperma and fungi. Advan. Genet. 6, 235-285. Lewis, D. 1965. A protein dimer hypothesis on incompatibility. Genet. Today, Proc. Znt. Congr., l l t h , The Hague, 1968 3, 657-663. Lewis, D., and Crowe, L. K. 1958. Unilateral interspecific incompatibility in flowering plants. Heredity 12, 233-256. L’Hbritier, P. 1951. The CO, sensitivity problem in Drosophila. Cold Spring Harbor S y m p . Quant. Biol. 16, 99-112. Lindegren, C. C. 1934. The genetics of Neurospora. VI. Bisexual and akaryotic ascospores from N. crassa. Genetica 16, 315-320. Ling, H., and Collins, 0. R. 1970. ContTol of plasmodia1 fusion in a Panamanian isolate of Didymium iridis. Amer. J Bot. 57, 292-298. Linskens, H. F., and Kroh, M. 1967. Inkompatibilitat der Phanerogamen. In “Handbuch der Pflanzenphysiologie” (W. Ruhland, ed.), Vol. XVIII, pp. 506-530. Springer-Verlag, Berlin and New York. Lloyd, D. G. 1968. Partial unilateral incompatibility in Leavenworthia (Cruciferae). Evolution 22, 38!2-393. Lorch, I. J., and Jeon, K. W. 1969. Character changes induced by heterologous nuclei in amoeba heterokaryons. E x p . Cell Res. 57,223-229. Macrae, R. 1967. Pairing incompatibility and other distinctions among Hirschioporus (Polyporus) abietinus, H. fusco-violaceus and H. laricinus. Can. J. Bot. 45, 1371-1398.

McKeen, C. G. 1952. Studies of Canadian Thelephoraceae. IX. A cultural and taxonomic study of three species of Peniophora. Can. J . Bot. 30, 764-787. McKenzie, A. R., Flentje, N. T., Btretton, H. M., and Mayo, M. J. 1969. Heterokaryon formation and genetic recombination within one isolate of Thanatephorus cucumeris. Aust. J. Biol. Sci. 22, 895-904. McNeilly, T., and Antonovics, J. 1968. Evolution in closely adjacent plant populations: barriers to gene flow. Heredity 23, 205-218.

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Matile, P. 1969. Reaktionsraume der Pflanzenzelle : Lysosomen und Peroxysomen. Fortschr. Bot. 31, 64-75. Miller, J. D. 1969. Crom-compatibility of birdsfoot trefoil, Lotus corniculata L. Crop Sci. 9, 552-555. Moreau, F., and Moruzi, C. 1933. Sur de nouvelles irrdgularitb de la bipolaritd sexuelle chez les Ascomycetes du genre Neurospora. Bull. SOC. Bot. Fr. 80, 574-576.

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151

Newmeyer, D. 1970. A suppressor of the heterokaryon-incompatibility associated with mating type in Neurospora crassa. Can. J . Genet. Cytol. 12, 914-926. Newmeyer, D., and Taylor, C. W. 1967. A pericentric inversion in Neurospora, with unstable duplication progeny. Genetics 56, 771-791. Olive, L. S. 1956. Genetics of Sordaria fimicola. I. Ascospore colour mutants. Amer. J. Bot. 43, 97-107. Padhye, A. A,, and Carmichael, J. W. 1971. Incompatibility in Microsporum cookei. Sabouraudia 9, 27-29. Pandey, K. K. 1967. Origin of genetic variability: Combinations of peroxidase isoaymes determine multiple allelism of the S gene. Nature (London) 213, 669. Pandey, K. K. 1968. Compatibility relationships in flowering plants: role of the 5-gene complex. Amer. Natur. 102, 475-489. Parnell, D. R. 1968. Reproductive barriers in Clarkia deflexa. Brittonia 20, 387-394. Paterniani, E. 1969. Selection for reproductive isolation between two populations of maize, Zea mays L. Evolution 23, 534-547. Perkins, D. D. 1968. Multiple alleles at a heterokaryon-compatibility locus in Neurospora. Genetics 61, 47. Pittenger, T. H. 1964. Spontaneous alterations of heterokaryon compatibility factors in Neurospora. Genetics 50, 471484. Pittenger, T. H., and Brawner, T. G. 1961. Genetic control of nuclear selection in Neurospora heterokaryons. Genetics 46, 1645-1663. Poulter, R. T. M., and Dee, J. 1968. Segregation of factors controlling fusion between plasmodia of the true slime mould Physarum polycephalum. Genet. Res. 12, 71-79. Prasad, I. 1970. Genetic analysis of the coloured mutants of Aspergillus niger. Genetica 41, 388-398. Puhalla, J. E. 1968. Compatibility reactions on solid medium and interstrain inhibition in Ustilago maydis. Genetics 60, 461474. Rapaport, F. T., ed. 1966. Seventh International Transplantation Conference. Ann. N.Y. Acad. Sci. 129, 1-884. Raper, J. R. 1966. ‘‘Genetics of Sexuality in Higher Fungi.” Ronald Press, New York. Raper, J. R., and Esser, K. 1964. The fungi. I n “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 6, pp. 139-244. Academic Press, New York. Raper, X. B., and Fennell, D. L. 1953. Heterokaryosis in Aspergillus. J. Elkha Mitchell Sci. SOC. 69, 1-29. Reiff, I. 1968. Die genetische Determination multipler Paarungstypen bei dem Ciliaten Uronychia transfuga (Hypotricha, Euplotidae). Arch. Protistenk. 110, 372-397. Rizet, G., and Esser, K. 1953. Sur des phhnomhes d’incompatibilit6 entre souches d’origines diffhrentes chez Podospora anserina. C. Rend. Acad. Sci. 237, 760-761. Ryan, S. L., and Saul, G. B. 1968. Post-fertilization effect of incompatibility factors in Mormoniella. Mol. Gen. Genet. 103, 29-36. Sansome, E. 1966. Meiosis in the sex organs of the Oomycetes. Chromosomes Today, Proc. Oxford Chromosome Conf., Ist, 1964 1, 77-83. Saul, G. B., and Lohrman, S. 1968. Action of incompatibility factors in Mormoniella. Genetics 60,221.

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ORIGIN AND CYTOGENETICS OF RICE

. .

N M Nayar* Inrtitut fur PtlanzenzGchtung. UniverritBt Gottingen. Gottingen. West Germany

I . Introduction . . . . . . . I1. Classification . . . . . . . I11. Origin . . . . . . . . . . A . AsianRice . . . . . . . B . African Rice . . . . . . IV . Subspeciation in Cultivated Rices . A . Asian Rice . . . . . . . B . African Rice . . . . . . V . Species Relationships . . . . . A . Subgeneric Classification . . . B . Interrelationships . . . . . C . Genome Relationships . . . VI . Chromosome Complement . . . A . Early Studies . . . . . . B . Chromosome Morphology . . C . Chromosome Number . . . VII . Other Studies . . . . . . . A. Secondary Association . . . B . Nucleolus Number . . . . C . Chiasma Frequency . . . . D . Asynapsis and Desynapsis . . E . Interchange Heterosygotes . . F. Nature of Rice Genome . . . References . . . . . . . .

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157 157 178 196 196 203 204 204 206 225 233 233 233 240 255 255 258 259 259 260 261 264

.

I Introduction

Rice and wheat are the most important food plants of man . Out of the world production of cereals of 1198 million metric tons in 1970. wheat accounted for 311 million tons and paddy for 307 million tons (200 million tons rice. when converted at 65%) (FAO. 1971) . Maize came next with 267 million tons. but most of it is used for industrial purposes or as livestock feed . The importance of rice is much more

* Present address: Central Plantation Crops Research Institute. Regional Station. Vittal-574243. Mysore State. India . 153

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N. M. NAYAR

than the production figures would indicate. About 90% of the rice is produced and consumed in south and southeast Asia (Chandler, 1969). It forms the staple food of 54% of all humans, against 34% of wheat and only 4% of maize (which ranks third). Rice supplies 21% of the total calorie intake of man, and wheat supplies 20% (Brown, 1963). Compared to its importance, much less is known about the genetics of rice than of other crop plants. Besides, a number of papers, particularly until about the 1950s, were being published in periodicals with limited circulation and in more than 10 languages. A symposium organized by the International Rice Research Institute, Los Banos, Philippines, in early 1963, helped to bring together many of the prevalent viewpoints on origin, species relationships, and genetics of rice (International Rice Research Institute, 1964). Ramiah and Rao (1953), Chandraratna (1964), and Chang (1964) have reviewed, though briefly, the literature pertaining to the origin and cytogenetics of rice. Shorter reviews have also been published by Vasconcellos (1946, 1963), Kihara (1959), Ghose et al. (1960), T. C. Katayama (1963b), Bianchi (1965), Grist (1965), Angladette (1966), and Russo (1967a,b).

11. Classification

A brief account is given here because it will help in discussing and understanding the origin and species relationships better, and this information is probably not available in any single publication. Rice, Oryza spp., belongs to the tribe Oryzeae of the family Gramineae. This tribe is small and its affinities with other tribes are not well established. Some of the most successful grasses of wet lands belong to it. It combines several primitive and advanced features. The genus most closely related to Oryza is Leersia. The absence of glumes (sterile glumes or sterile lemmas) is best used to separate Leersia from Oryza. The genus Oryza is itself small, consisting of about 26 taxa (Table 1, last column). The actual number of species will be less. A few other names like formosanu, paraguayensis, balunga, and cubensis have also been used in some literature as synonyms of rufipogon (annual and perennial forms). Two of the taxa, ubangensis and schlechteri, have not been collected since they were first described. Tateoka (1963), who was the last to revise the genus, was unable to locate the original herbarium sheet of ubangensis, but he inferred from the description that it was related to the Asiatic and African latifolia complex. The second species,

TABLE 1 Enumeration of Oryza Species* Prodoehl (1922)

Roschevicz (1931)

Sampath (1962)

Tateoka (1963)

saliva fatua

saliva saliva var. f a t u a

saliva rufipogon

sativa f. aquatica longistaminafa

saliva rufipogon perennas

= barthii = perennis

perennis

barthii

barthii

latifolia

barthii grandiglumis

dewildemanii grandiglumis

8. punctnta

punctata

ssp. longistaminata barthii latifolia var. grandiglumis oilicinalis = manuta ssp. punctata

perennis grandiglumis

As used in the text saliva (annual) rufipogon (perennial) ruyipogon (perennial) longistaminata longzstamtnata grandaglumzs

punctata

punctata

punctata

eichingeri glaberrima sap. stapfii stapfii breviligulata breviligulata ausfraliensis australiensis glaberrima glaberrima latifolia latifolia alto minuta sap. punctata punctata oficinalis punctata = minuta ssp. oilicinalis

eichingeri breviligulata breuiligulafa australiensis glaberrima latifolia latifolia eachingeri oficinalis

eichingeri breviligulata breviligulata australtensis glaberrzma latifolia alta punctata oficinalis ssp. officinalis

eichingeri stapfii barthii australiensis glaberrima latifolia alta schmein furthiana oficinalis

malampuzhensis

minuta

minuta ubhangensis

granulnta granulata abromeitiana abromeitiana

granulata granulata meyeriana

granulata

granulata

meyeriana

meyeriana

schlechteri

schlechteri

schlechleri

schlechteri

schlechteri

ridleyi coarctata

rid 1??ti coarctata brach yantha

ridleyi coarctata brachyantha

ridleyi coarctata brachyantha

subulata

subulatu

subulata perrieri tisseranti

subulata perrieri tisseranti

ridleyi coarctata brachyantha angustifolia subulata perrieri tisseranti

ofictnalis ssp. malampuzhenszs minuta nomina nuda: no latin description jeyporensis: nomina nuda meyeriana ssp. granulata meyeriana ssp. meyeriana meyeriana ssp. abromeitiana schlechteri longiglumis ridleyi coarctata brachyantha angustifolia Rhynchoryza subulata perrzeri tisseranti

mnlampuzhensis

minuta

9.

10. 11. merii 12. 13. glaberrima 14. latifolia

stapfii breviligulata australiensis glnberrima latifolra

16. schweinfurthiana 17. oficinalis

schweinfurthiann officinalis

15 -l.

18. 19. minuta 20. 21. 22. granulata 23. meyeriana 24. abroma'tiann

a

Chatterjee (1948)

sativa saliva f . spontnnea

6. 7. grandiglumis

25. 26. 27. 28. 29. 30. 31. 32. 33.

Chevalier (1932)

minuta

+

See text for details.

punctata

minuta ubhangensis jeyporensis granulatn meyeriana abromeitiana

0

2 2 Z

H 0 1

E M

schlechteri longiglumis ridleyi Sclerophyllum coarctuta brachyantha Leersia angustifolia Rhynchoryza subulata Leersia perrieri Leersia tisseranti

c.

cn cn

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schlechteri, was described by Pilger (1915) from a specimen collected by Schlechter in 1907 from northeast New Guinea. The first monographic treatment of the genus was done by Prodoehl (1922), and 17 species were then described. Subsequently, the genus has been treated in full by Roschevicz (1931), Chevalier (1932), Sasaki (1935), Chatterjee (1948), Sampath (1961, 1962, 1964a), and Tateoka (1962a, 1963, 1964a). Roschevicz’s work remains, even now, the most comprehensive treatment of the genus. Chevalier (1932) followed Roschevicz’s treatment. He suggested some nomenclatural changes and added two species. Chatterjee (1948) also based his work on that of Roschevicz. He made a few nomenclatural changes and added two species that were subsequently described. For the first time in the genus, Sampath (1962) used cytogenetic data to suggest some modifications to Chatterjee’s treatment of the genus. He also added three species described subsequent to Chatterjee’s classification. Tateoka (1963), whose revision of the genus is the latest, had the advantage of visiting important herbaria around the world to study Oryza sheets and also of examining large accessions of living material made during the several collection trips organized by the National Institute of Genetics, Japan. He removed subulata from the genus, added a new species described in 1953 and cited two other new taxa invalidly published. Tateoka’s work was expected to clear a t least most of the areas of disagreement, but from the literature subsequently published, it does not appear to have done so. The taxa as recognized by different authors are listed in Table 1. There have been two notable changes subsequent to the publication of Tateoka’s (1963) revision. One was the removal of 4 more species from the genus (Launert, 1965; Tateoka, 1964b, 1965~).The second was the proposal of Clayton (1968), based on the International Code of Botanical Nomenclature, that the species known all along as breviligulata should be called barthii, and the one known generally as barthii (but longistaminata to Roschevicz) should be known as longistaminata. These modifications are incorporated in Table 1, last column. The area of greatest disagreement is in the delimitation and nomenclature of Asian wild rices-a complex of wild and also weedy forms that occur in greatest abundance in the monsoon areas of Asia, and, to a lesser extent, in Central and South America and north Australia. Weedy rices known as red rice occur also in rice fields of Europe and North America. They have been reported by earlier workers (Roschevicz, 1931) from Africa also. Roschevicz (1931) actually recognized two forms of wild rices related to the cultivated rice, sativa, viz., spontunea and aquatica. The taxon aquatica seems to have been overlooked by all later taxonomists who revised the genus, possibly because it was not

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mentioned in the English summary. I n retrospect, it appears that much of the controversy about the taxonomy of Asian wild rices would not have taken place if this taxon had been taken note of by later workers. Ill. Origin

The origin of rice has been speculated upon from early times, but it is only since about 1950 that cytogenetic work has been taken up in this direction. Three papers on the origin of rice presented a t a symposium in Delhi formed the basis of most of the subsequent experimental work (Chatterjee, 1951; Ramiah and Ghose, 1951; Sampath and Rao, 1951). For this reason, the work up to this period will be reviewed chronologically. Later it will be dealt with under the following topics: time and place of origin, taxonomy of wild rices, progenitor of rice, and mode of speciation. The cultivated rices of the world belong to two species, sativa and glaberrima. Commonly known as Asiatic rices, sativa is the species cultivated throughout the rice-growing areas of the world. However, it is in south and southeast Asia, where most of the rice is grown, that both saCiva and wild rices occur in great profusion and variability. Most workers have simply been unable to comprehend this high degree of variability (e.g., Watt, 1892; Roschevicz, 1931 ; Chatterjee, 1951). 0. glaberrima is called the African rice. It is generally known from tropical west Africa only, but Porthres (195513, 1960) has reported its occurrence in Guyana and El Salvador in Central America.

A. ASIANRICE 1. Early Work I n 1886 de Candolle cited two authors, Bretschneider and Stanislav Julien, as mentioning a religious rite, instituted by a Chinese emperor Chin-nong (2800-2700 BC), in which seeds of five crop plants were ceremonially sown. They were rice, sweet potato, wheat, and two kinds of millet, and these were considered as indigenous to China (pp. 4 and 385-387). I n this ceremony, rice was sown by the emperor himself and the others by lesser members of the royalty. This story has been so widely quoted (e.g., Watt, 1892; Roschevice, 1931) that we will refer to it again later. He said that Indians used rice after the Chinese and possibly beginning from the Aryan invasion of India because of the occurrence of names for rice in the Sanskrit language. He thought that the names for rice in various Indian languages, ancient Greek, and

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Arabic originated from Sanskrit. While he did not find any reference to wild rices occurring in China, he mentioned that it was widely present in India and that people harvested them for grain in the Circars (southeast India). George Watt, author of a six-volume monograph on economic products of India, wrote a t great length on rice (Watt, 1892). He observed that philological data on the origin of rice were not in accordance with botanical data. He mentioned also the wide use of rice in various Indian ceremonies. He concluded from his studies that rice originated in peninsular India and that it spread out from there. It spread to China in about 3000 BC from south and southeast Asia. Watt classified wild rices (in which he included rufipogon, annual and perennial forms, and Porteresia coarctata) , into four botanical varieties and believed that different forms of cultivated rices originated from them. Further, some of the panicle characters present in certain varieties were assumed to have been derived from officinalis through hybridization. Vavilov (1926, 1951) assigned rice to his Indian center of origin and felt that it might have been introduced into China later from India. Roschevicz (1931) believed that while the center of origin of section Sativa (which included the two cultivated species and about ten other most nearly related taxa) was in Africa, because of the presence there of more species than in Asia, rice cultivation itself might have arisen in the region of India, China, and Indochina. He proposed a polyphyletic origin for rice, with the majority of sativa varieties arising from f. spontunes (rufipogon annual), some of the small-seeded varieties from minuta, and some of the West African cultivated forms, which could be grouped under glaberrima, arising from breviligulata. He. concurred with Watt about the role of oficinalis in the production of certain cultivated varieties. The remaining publications until 1951 are of passing interest only. Gustchin (1938) proposed that rice might have originated on the slopes of the Himalayas, both on the Indian and Chinese sides. Chatterjee (1947, 1948, 1951) also believed that it originated in India. He reasoned on philological grounds that the Arabs were the first to know about rice from Indians. In support of an Indian origin, he mentioned the existence of an extensive terminology for various rice and rice products in the Indian languages. According to him, rice was evolved by man from sativa var. fatua (rufipogon, annual) (cf. also Darlington, 1947). On the other hand, Hamada (1949) and Burkill (1953) considered Indochina to be the center of origin of rice. Hamada based his proposition on the observation that rice showed the widest taxonomic differentiation in southeast Asia.

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Ting (1949, 1957, 1958) strongly believed that rice originated in China. According to him, rice was first mentioned in the Chinese classical literature of the time of Shen-nung (3000 BC) and also during the periods of Hwangti, Yu, and Chi (2600-2200 B C ) . Rice culture was finally established a t the time of the Chou dynasty (1122-274 B C ) . A phonetic consideration of the character for rice as used during the Chou dynasty indicated to him that it was related to certain dialects of the southeastern coasts of China, Indochina, and Siam. From these, and also from the presence of certain wild forms of rice-‘‘the cultivated form of 0. minuta” (presumably small-grained rice varieties)-he concluded that rice originated in China. Ramiah and Ghose (1951) supported Watt’s (1892) theory that rice evolved in peninsular India. They mentioned the antiquity of the Dravidian culture and the designation of separate rice varieties for use a t specific religious occasions in ancient Tamil (a south Indian language). While they considered fatua (rufipogon, annual) to be the progenitor of most cultivated varieties, they proposed for the first time that the wild species perennis (rufipogon, perennial, longistaminata) could have contributed to the development of some forms of sativa. They disagreed with the proposals of Watt (1892) and Roschevicz (1931) about the role of minuta and officinalis in the evolution of rice by pointing out that minuta is a tetraploid species of possibly amphdiploid origin and officinalis has larger chromosomes. I n his paper, Chatterjee (1951) referred t o the philological work of Mahdihassan (1950), in which the author had argued that the generic name Oryza, the Greek work Oruza, and the Tamil word Arisi have all originated from the word Oze-li-zz, meaning rice in the Nengpo dialect of Chinese. This, along with the story of the rice-sowing ceremony performed in China (2800 B C ) , made him modify his earlier stand slightly (Chatterjee, 1947, 1948). The whole region covering south and southeast Asia was considered by him as the area where rice originated. He believed also in a polyphyletic origin with var. fatua (rufipogon, annual) and officinalis as the putative parents. Sampath and Rao (1951), on the other hand, suggested that the perennial wild rice perennis (rufipogon perennial and longistaminata) was the sole progenitor of both Asian and African rices. They pointed out that this taxon has the widest distribution of all Oryza species, that it has the same chromosome number as the two rice species, and also that it gives fertile hybrids with sativa. They mentioned also that some of the segregants from perennis-sativa crosses closely resembled the annual wild rices, which were termed “spontanea” (rufipogon annual). I n addition, the spontaneas were heterogeneous in nature, and the authors

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concluded from these observations that “such a complex and artificial group as spontanea paddies are not likely to be a species ancestral to 0. sativa.” Several textbooks and authors have referred to the account of the seed-sowing ceremony performed by a Chinese king as quoted by de Candolle, and have concluded from it that rice originated in China. Even if the story is a fact, it does not prove that the crops sown by the king originated there, as de Candolle assumed. In fact, according to present-day evidence, of these five crop plants some millets are probably Chinese in origin and the others originated in India and tropical Africa (Vavilov, 1951). Wheat is almost certainly of Middle Eastern origin (Helbaek, 1964; Harland and Zohary, 1966; Zohary, 1969), and sweet potato originated possibly in South America (Vavilov, 1951 ; Yen, 1970). Thus, there appears to be more legend and fiction in it than history. 2.

Place and Time of Origin

Inferences on the origins of crop plants are derived from archeological, historical, philological, and botanical evidence. We have seen that evidence obtained from philology and history has not yielded any positive information about the origin of rice. Archeology can give direct information. From the foregoing review, we could see that the Asian rice originated somewhere in south and southeast Asia, including China. However, the warm and humid climates prevailing in these areas are hardly conducive to the preservation of biological materials over long periods of time. The political climate prevailing in most of these regions since about 1940 has also made archeological studies more difficult. Ting (1949) mentioned rice and rice leaf remains obtained from Yang-shao excavations which he dated to 2600 BC, and also the discovery of an oracle bone (1400-1122 BC) on which characters for rice were inscribed. The age ascribed to the Yang-shao period may be exaggerated, as we will see below. I n a subsequent paper, Ting (1960) reported on his studies of rice glumes and grains found from three sites within about 150 km of Uckan in the Yangtse River valley. They were ascribed to a period “much later than the Yang-shao and Lung-shan cultures.” The glumes were mixed with clay and were probably used as binding material for house construction. The grains measured 6.97 cm in length and 3.47 cm in width. From the grain size, the glume pubescence, and the presence of awns, Ting inferred that they were related to “0. sativa f. spontanea ssp. Keng Ting” (possibly meaning thereby a primitive form of the Chinese botanical variety Keng) .

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There is no absolute or even developed relative chronology for the neolithic period in China (K. C. Chang, 1968; Watson, 1969). The earliest established neolithic cultural stage of China is the Yang-shao period, named after the original site of Yang-shao-bun in western Honan. T. G. Andersson, who discovered it, dated it a t 2200-1700 BC, but K. C. Chang (1968, 1970) thought that it was much older. This culture was based predominantly on millets, particularly Seturiu italics. Rice was obtained only once in the form of husks. Watson (1969) felt that climatic and soil conditions of the region made the possibility of rice cultivation problematic and therefore that rice culture in the context of the Yang-shao neolithic was yet to be proved. Rice remains have characterized some sites of the Lungshanoid culture, first found in eastern China. This is now generally considered to be a later cultural development compared to the Yang-shao culture. K. C. Chang (1968) has dated it a t the third or fourth millenium BC, and Watson (1969) thought that it was a little later-about 1650 BC. During this stage, the farmers began migrating rapidly toward east and southeast China and even beyond. The earliest cereal remains from the Indian subcontinent are of wheat and barley, dated to late fourth millenium BC (Allchin, 1969). The oldest direct evidence of rice are impressions of paddy on clay lumps and remnants of husks obtained from Lothal (District Ahmedabad, Gujarat) which have been dated as 2300 BC (Ghosh, 1961). Altogether 11 samples, all older than 2000 years (some more than 3000 years old), and consisting mostly of charred grains, have been reported from various sites in India (Chowdhury and Ghosh, 1953; Ghosh, 1961; Chowdhury, 1965; Allchin, 1969). The grains generally measured about 5 mm in length and 2.5 mm in width. The two oldest samples belong to the wellknown Harappan civilization (about 2200-1700 BC) (Allchin, 1969). It was Sauer (1952) who had probably first proposed that rice might have originated as a weed of Colocusiu fields (see also Burkill, 1953). Possibly as an elaboration of this idea, Barrau (1966) speculated that rice might have been domesticated in the western Indo-Pacific area (cf. also, Spencer, 1963). It is well known that wild rices occur in this region. The staple food of the local inhabitants consisted of various root crops in ancient times. Barrau considered that these people might not have felt the need and also not have had the know-how to domesticate wild rice. Then these areas received immigrants from Inner Asia moving southward. These people, possibly belonging to the Lungshanoid culture, and therefore already well accustomed to the cultivation and use of a cereal, the foxtail millet, would then have domesticated wild rices.

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K. C. Chang (1970) suggested that the rice samples obtained from the Lungshanoid sites of south China and the single sample of the late Yang-shao culture might have been secondarily derived from this region. Nair et al. (1964) advanced some phytogeographical evidence in support of considering peninsular India and more particularly the Malabar coast as the center of the origin of rice, The occurrence of as many as five wild taxa, including both annual and perennial rufipogon, the presence of high varietal diversity, and also the wide occurrence of several dominant genes in local rice varieties, such as red pericarp color, awning, bold grains, black husk color, were given as supporting evidence. They also pointed out that the warm and humid tropical climate (more than 3500 mm annual rainfall) was unlikely to preserve archeological specimens in this area. Thus, we see that archeological findings take us back to about 4500 years only as regards the antiquity of rice. A further consideration on the spatial and temporal aspects of rice will be made after the botanical evidence has been considered. 3. The Wild Rices The classification and nomenclature of wild rices immediately related to sativa are probably the biggest taxonomic problem of the genus. Together, the wild and cultivated rices exhibit an enormous amount of variability. a. Distribution. Wild rices occur extensively throughout the monsoon areas of Asia, which is also where most of the rice is grown. They occur commonly as weeds of paddy fields (often causing severe losses), on bunds and water channels, disused fields and waste lands, in roadside ditches and sides of railway embankments, and also in marshes and waterways. de Candolle (1886), Watt (1892), and Hooker (1897) recorded their wide presence in the Indian subcontinent. They have been reported from various regions of this area by several workers (Roy, 1921; Bhide, 1925; Bhalerao, 1928, 1930; Sampath and Rao, 1951; Ramiah and Rao, 1953; Chatterjee, 1947, 1948; Nayar, 1958; Nair et al., 1964; Sharma and Shastry, 1965a,b; Richharia et al., 1966). Beale (1927) reported it from Burma; Chatterjee (1948) and Senaratna (1956) in Ceylon; Backer (1946) and T. C. Katayama (1963a) from Malaysia and Indonesia; Capinpin and Pancho (1961) and T. C. Katayama (1963a) from the Philippines ; Chevalier (1932), Porthres (1956), and Oka (19644 from Thailand and Indochina; Kuang (1951) from southern China; Oka (1956a) from Taiwan; and Tateoka (1963) from northern Australia.

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Roschevicz (1931) recorded its presence from central Africa, but this has not been substantiated (but see Jachuck and Hakim, 1966). It is also known from the Americas (West Indies, Cuba, Panama, Hispaniola, El Salvador, Venezuela, Colombia, Ecuador, Guyanas, and Brazil) (Roschevicz, 1931; Chevalier, 1932; Hitchcock, 1935; Swallen, 1943; Oka, 1961). Wild rices, popularly called “red rices” also occu’r in the rice-growing areas of Japan, the Mediterranean countries and the’ United States (Nagai, 1959; Grist, 1965). b. Nature. Wild rices were an enigma to all early rice systematists. For instance, Watt (1892) and Roschevicz (1931) considered them as a complex of several species. Roy (1921) classified wild rices of central India alone into 24 groups. Early work on classification and nomenclature of wild rices has been summarized by several workers (PortBres, 1956; Nayar, 1958; Tateoka, 1962a,c, 1963; Sharma and Shastry, 1964, 1965a,b). I n the main, most authors recognize two types of wild rices. The first is a perennial, aquatic plant with variable habit, and erect and lax panicles that bear narrow, oblique, and beaked spikelets possessing awns. The anthers are about two-thirds or more as long as the spikelets. They correspond to m f i p o g o n , perennial, in Table 1. The second type is typically annual in habit with erect or decumbent habit, and bearing panicles of variable shapes and sizes. The spikelets also have variable length and shape, but are generally oblong, and are mostly awned. Their anthers are only about half as long as spikelets or even smaller. They correspond to rufipogon annual. The former grows in channels, marshes, tanks, and similar permanent habitats. The latter is more widespread. It occurs mainly as weeds of paddy crops (where it may cause considerable loss), on field bunds, roadside channels, disused fields, etc. The study of herbarium sheets of wild rices also showed a similar variation (Nayar, 1958; Sharma and Shastry, 1965a). Several workers have studied the nature of wild rices (Ramiah and Ghose, 1951; Oka and Chang, 1959, 1961, 1962, 1964b; Sampath and Rao, 1951; Oka, 1956a; Nayar, 1958; Sampath and Govindaswamy, 1958; Richharia, 1960; Morishima et al., 1961; Sharma and Shastry, 1964, 1965b; Govindaswamy et al., 1964; Richharia et al., 1966; Morishima, 1969; Chu and Oka, 1970b). Both types are characterized by heterozygosity and semisterility, but genetic studies of perennial forms have indicated that their variability is largely the result of their perennial habit. In experimental studies (Nayar, 1958), 17 out of 20 single plant progenies of annual forms segregated for various plant characters. Different lines showed varying degrees of heterozygosity. Two progenies segregated for high proportions of albinos. Meiosis was abnor-

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ma1 in 20-50% of the PMCs, the abnormalities being in the nature of translocations and irregular anaphase separations. Bulk samples of annual wild rices from other countries also showed similar characteristics. In a hybrid between sativa (variety Fukoku) and perennis ssp. ba2unga (rufipogon, perennial), the F, population showed the expected dominance of wild characters and the F, appeared typically like annual wild rices (Nayar et al., 1966). The various characters showed Mendelian inheritance with complementary, modifying, and duplicate modes of segregation. Mitra and Ganguly (1932) had also obtained similar results in a cross between sativa (var. Latisail) and sativa var. fatua (probably a perennial form). The annual wild rices are characterized by semisterility and heterozygosity, characteristics usually associated with hybridity. If they are hybrids, their putative parents may be cultivated rice and perennial wild rices. Several workers (Roy, 1921; Oka, 1956a) have observed that significant amounts of cross fertilization takes place in wild rices. This has been estimated by different methods (progeny tests for a marker gene, variance of certain characters, etc., generally after interplanting different genotypes) to vary from 20 to 40% (cf. Oka, 1964a). Small (up to about 5%) amounts of cross fertilization are also known to occur in cultivated rices. From the above considerations, we can see that the annual wild rices fulfill the various criteria generally adopted to prove the occurrence of introgression (cf. Heiser, 1949; Anderson, 1949, 1953). There is general agreement that natural hybridization between cultivated rices and perennial wild rices results in the production of annual wild rices. But some authors like Porthres (1956), Oka (1964a), Shastry (1964b) , and Sharma and Shastry (1965a,b) have maintained that not all annual wild forms are of hybrid origin. They assume that these are of direct descent. Either assumption is difficult to prove. Genetic studies have shown that a small proportion of the annual wild rices are homozygous, but these can result from hybridization and subsequent segregation also. Sharma and Shastry (1965a,b) contend that they are mainly an upland annual species growing wild and inhabiting seasonal ditches. They have even designated it by a specific epithet, 0. nivara. But their morphology and habitat preferences are characteristic of hybrid forms also. Perennial wild rices occur only less frequently or not at all in upland areas and as such these hybrid derivatives could have been carried there through natural spread and mechanical contamination. In such regions they can then hybridize with cultivated forms only. They have thus better opportunities to become homozygous. We will touch on this again later. It has been pointed out (Nayar, 1967) that these annual wild rices

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are typically of the nature of “companion weed races” (cf. Harlan, 1965). They also fulfill all the characteristics associated with successful weed races, whose function and strategy have in recent years been discussed by several workers (Harlan and de Wet, 1965; Baker, 1965; Grant, 1967; Zohary, 1965; de Wet, 1968; Harlan, 1970; cf. Vavilov, 1926; Baker and Stebbins, 1965). They hybridize intermittently in both time and place with cultivated rices. Extension officers in rice areas of India are only too familiar with the sporadic complaints of farmers (in some years) of infestation of their rice crops by weed rices. The situation may be similar in other countries also. That they retain the benefits of heterozygosity is evident from the observations of reciprocal translocations and albinos in their progenies (Nayar, 1958, 1967; Basak and Dana, 1967). There is but one difference in the situation existing here as compared to those in other weed-crop complexes. In rice, it is a three-tier system of wild species-weed-cultivated species complex. The wild taxon occupies a permanent and distinct habitat adjacent to that of the weed-crop complex (which are mostly sympatric), and shows variable amount of reproductive isolation. The perennial rufipogon, for instance, does not show the “pioneer aggressiveness” of Avena sterilis, a wild relative of cultivated hexaploid species (Ladizinsky and Zohary, 1971). The perennial habit and the comparatively LLsafe”habitat that perennial wild rices occupy may have made them more sedate and complacent in nature. c. Nomenclature of Wild Rices. Though the occurrence of natural crossing between wild and cultivated rices was known even earlier, it was only after about 1950, after the publication of Anderson’s (1949) and Heiser’s (1949) work, that the significance of introgressive hybridization was begun to be appreciated in rice. The position now is fairly clear and the dispute is one of taxonomic semantics: over the number of species or taxa to be recognized in this complex-2, 3, or &and the application of the names rufipogon and perennis. The perennial wild rices are pantropical in distribution. They are characterized by differences in form, breeding behavior, and sterility in interracial hybrids. They are generally caespitose in the Americas and have been a t various times known as cubensis, paraguayensis, or perennis. In Africa they are erect and are characterized by well-developed rhizomes and have been called barthii, longistaminata, madagascarensis, dewildemanii, or perennis. I n Asia, Oceania, and north Australia, they have a floating and procumbent habit and have been known in the past as rufipogon, fatua, sativa var. bengalensis, sativa f. aquatica, sativa f. spontanea, formosana, balunga, longistaminata, or perennis. The African taxon is characterized by prevalence of

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self-incompatibility, while it is only partial in others (Nayar, 1958, 1967; Sampath, 1961; Jachuck and Sampath, 1966a; Oka and Morishima, 1967; Chu et al., 1969b). Hybrids between geographical races also show varying degrees of sterility and reproductive barriers (Nayar, 1958; Morishima, 1969; Chu et al., 1969a). Exceptions to their habits are known from all continents; for example, floating forms from the Amazon basin (Oka, 1961), nonrhizomatous and also floating forms from central Africa (Oka and Chang, 1964a), and erect and also rhizomatous forms from Asia (Govindaswamy et al., 1964; Oka, 1964a). Chatterjee (1948) included the American, African, and Ceylonese perennial forms under perennis, and all the remaining Asian forms under sativa var. jatua. He was unable to decide about the correct specific name to be applied to the latter. He apparently included the annual wild rices under sativa sensu latiore. At the Los Banos symposium in 1963, it was decided to designate the perennial wild rices of different continents as subspecies of perennis, via. cubensis (American) , barthii (African), and balunga (Asian) International Rice Research Institute, 1964). Tateoka (1962c, 1963, 1964a) discarded the epithet perennis as of uncertain application. He then restored the specific status of 0. barthii for African forms and lumped all the rest, both perennial and annual forms, under 0. rufipogon. Sharma and Shastry (1964, 1965a,b) felt that three taxa are discernible in Asian wild rices, an annual upland form, the hybrid derivatives, and a perennial form. They designated them 0. nivara, field spontaneas, and 0. rufipogon, respectively. Thus they sought to restrict the use of 0. rufipogon to perennial forms only. Sampath (1964~) a t the same time contended that 0. rufipogon was correctly applied to the annual forms only, and then gave an amended description for 0. perennis to include the Asian and American perennial forms. Griffith had described 0. rufipogon as an erect and elegant plant. Watt (1892) apparently took it to mean an annual form, retaining the name sativa var. rufipogon for it, and named the floating forms sativa var. bengalensis. As mentioned earlier, Roschevicz (1931) had actually recognized two forms in wild rices, f. spontanea and f. aquutica. The former was termed an annual. The latter was described as 2.53.0 meters long, of which 1.5-2.0 meters remained under water. He gave a photograph (credited to Kikkawa, 1912, and taken in Cochin China) of two persons in a boat harvesting wild rice. Since the Asian perennial wild rices are morphologically distinguishable from annual wild rices (habit, extravaginal branches, spikelet shape, anther length) and occupy a distinct and stable habitat and are able to maintain genetic identity, a distinct specific name appears to be justifiable for them. At the 8ame time, the annual wild rices, even if they exist separately from the hybrid

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derivatives, are not morphologically distinguishable from the latter and also cannot be separated by genetic studies. No reproductive barrier exists between them. Such an entity may not, therefore, deserve a separate status. Since it seems impossible to know for certain in which sense Moench had intended the name 0. perennis, and there are also doubts about the sense in which Griffith had used 0. rufipogon and since type specimens are not available for either, it would appear best to reject both these names as of uncertain application. Thus, the perennial Asian and American wild rices are left without a proper name. The perennial, rhizomatous African wild rices have been designated 0. longistaminata (Clayton, 1968).

4. T h e Ancestral Species Some early botanists like Roxburgh, who worked in India during the middle of the 19th century, had considered this question but could only state that wild rices widely present there were the probable ancestors of Asian rice. Watt (1892) divided the Indian wild rices into four varieties of sativa, and proposed that each of them gave rise to different groups of rice varieties. Two of these, var. rufipogon and var. abuensis, probably correspond to rufipogon annual and a third, var. bengalensis, to rujipogon perennial. The last one, var. coarctata, corresponds most probably to Porteresia coarctata. He also suggested that officinalis might have contributed to the evolution of rice through hybridization. Roschevicz (1931) considered the possible role of all species included by him in Section Sativa and proposed that oficinalis and minuta might have contributed to the evolution of rice. While the wild rices (seemingly all rufipogon annual and part of rufipogon perennial) were considered the progenitor of the majority of cultivated forms, minuta was thought to have given rise to some of the small-grained varieties, and officinalis, by hybridization, t o some of the panicle characters of rice. Porteresis coarctata (formerly, 0. coarctata) is a tetraploid species ( 2 n = 48). It is confined in its distribution to the saline marshes of the Ganges delta in the Indian subcontinent. Earlier reports mentioned that it was also present in the river deltas of Madras (India), Irrawaddy river (Burma), and Indus river (W. Pakistan). These reports have not been substantiated, It was the high salt tolerance of this species and the saline resistance of some cultivated rice varieties which prompted Watt to propose an ancestral role to this taxon. All attempts to hybridize it with other Oryza species have been unsuccessful so far. It is now no longer considered to have contributed to the origin of rice. 0. minuta is a tetraploid species endemic in its distribution to four

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islands of the Philippines (Tateoka and Pancho, 1963; T. C. Katayama, 1963a). It is closely similar morphologically, and genetically to officinalis, a widely distributed Asian species. Based on cytological studies of species hybrids, Nandi (1938) proposed it to be an amphidiploid of sativa and officinalis. On the other hand, Morinaga (1940) found it even difficult to obtain dividing PMCs for analysis and meiosis was highly irregular in those that he could study. With its highly restricted distribution, tetraploid nature, and difficulties in producing hybrids with sativa, it is also no longer considered as a progenitor of rice (Morinaga, 1940; Ramiah and Ghose, 1951; Richharia, 1960; Kihara, 1959). 0. officinalis is a diploid species that is widely present in south and southeast Asia. I n fact, it may have the widest distribution after rufipogon, annual and perennial. It is closely related to minuta, malantpuzhensis, and collinu of Asia, punctata, eichingeri, and schweinfurthiana of Africa, and latijolia, grandiglumis, and alta of America. Geographical races show variation in morphology and varying degrees of hybrid sterility (Gopalakrishnan, 1965a; Hu and Chang, 1965a, 1967). It does not appear to be gregarious as wild rices. It may grow in the open or in shade, generally in moist lands and in streams, and at middle elevations (Backer, 1946; Bor, 1960; T. C. Katayama, 1963a). In gross morphology, it resembles rice and wild rices in panicle characters (which are the characters that have been proposed to have introgressed into rice), but differs in having a rhizome (sometimes absent) and smaller plant organs. Watt (1892), Roschevicz (1931), Chatterjee (1951), and Anderson (1951) attributed an ancestral role to officinalis on morphological grounds. Several workers have studied the hybrid between sativa and officinalis (Ramanujam, 1938c; Nandi, 1938; Gopalakrishnan, 1959; . hybrids Nezu et al., 1960; Bouharmont, 1962a; Li et al., 1 9 6 4 ~ )The are obtained with difficulty and are sterile. Meiosis is highly abnormal, with most cells showing 24 I at diakinesis and MI. Occasionally up to 3 I1 are observed. The subsequent course of division is also very irregular with haphazard distribution of univalents a t both divisions, formation of restitution nuclei, failure of septa formation, suppression of first division, degeneration of PMCs, etc. Seeds when formed produce triploid plants (Ramanujam, 1938c; Li et al., 1964c; Ho and Li, 1965). For these reasons, it has been argued that ofiicinalis could not have played any part in the evolution of sativa (Morinaga, 1940; Ramiah and Ghose, 1951; Nayar, 1958; Richharia, 1960). Shastry and co-workers (Shastry et al., 1960b, 1961; Shastry, 1962, 1964b) found that hybrids between sativa and a Ceylonese form (collina) of officinalis showed nearly complete pachytene pairing, as far as could be analyzed by them. The subse-

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quent course of meiosis was similar to what others had seen. They interpreted this as an instance of desynapsis and suggested that the chromosomes of the two taxa were fairly homozygous. They argued for a reexamination of the role of officinalis in the evolution of sativa on the grounds that this has been denied by others for inadequate reasons. However, the status of collina is in dispute now (see under Species Relationships, Section V ) , some workers claiming it to be oficinalis and others to be eichingeri, with Sharma and Shastry (1 9 6 5 ~ )themselves proposing a new name, 0. collina, for it on the ground that its characters fall beyond the range of variation available in both oficinalis and eichingeri. This takes the main brunt off their argument. I n another paper, however, Shastry et al. (1963) reported the recovery of two plants looking similar to oficinalis in some characters, out of four plants obtained from a highly sterile plant, chimeric for asynapsis. They isolated it from a population which they considered to be perennis with some introgression from sativa. The other two plants appeared to be similar to spontanea (rufipogon, annual). The officinalis-like plants were triploid, asynaptic, and sterile. The authors hypothesized that asynapsis in the parent diploid plant might have been responsible for the reshuffling of chromosomes which created a numerically balanced but genetically unbalanced gamete. This might have given rise to plants resembling officinalis. They, therefore, considered that the morphological differences between sativa and officinalis might be “due to dosage relationships at one or more compound loci controlling this character complex which differentiate panicle and grain morphology of perennis and sativa.” An analysis of the morphological characters of the four progeny plants shows that they segregated for a number of characters, thus indicating a hybrid origin. The original seed sample came from the Central Rice Research Institute, Cuttack, India, and natural interspecific crossings are of frequent occurrence in its genetic collections, and more particularly in semisterile cultures (Jachuck and Sampath, 196613). This adds to the possibility of a hybrid origin for their sample. Hybrids between sativa and officinalis are always sterile, with meiosis characterized by univalents and other abnormalities. Their progeny, raised from seeds that are obtained upon backcrossing to sativa, is triploid (Ramanujam, 1938c; Ho and Li, 1965). So there are at least good possibilities that the plant which gave rise to the officinalis-like plant was a natural hybrid between perennis and oficinalis. Further, pachytene analysis of a hybrid involving a genuine officinalis and sativa showed that there was no pairing in prediakinesis either (Li et al., 1964b). Detailed morphological and anatomical studies of T. C. Katayama (1969) have also shown that the two taxa possess distinctive characters. Thus, there is

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as yet no direct evidence to support the thesis that officinalis has been a progenitor of rice, or that it has, through introgression, contributed to the evolution of rice. 0. rufipogon, annual: When earlier workers like Roschevicz (1931), Hamada (1949), and Zukovsky (1950) stated that forms variously known as sativa var. fatua or sativa f. spontanea were the progenitor of cultivated rice, they included in it the whole spectrum of wild rices (rufipogon, annual and perennial). Chatterjee (1951), Ramiah and Ghose (1951), Portbres (1956), Kihara (1959), and Shastry (1964b) were aware of the phenomenon of introgression and the occurrence of rufipogon, perennial, when they considered the annual taxon as the species most responsible for the evolution of rice. A large volume of evidence is available by now to demonstrate the hybrid origin of most if not all annual wild rices (Sampath and Govindaswamy, 1958; Nayar, 1958; Oka and Chang, 1959, 1961, 1962; Yeh and Henderson, 1961b; Henderson, 1964a; Nayar et al., 1966; Govindaswamy et al., 1966; Richharia et al., 1966). These have already been reviewed. Oka, Morishima, and co-workers, who have made detailed and extensive studies on the nature of wild rices (Hinata and Oka, 1962a,b; Morishima et al., 1961, 1963; Oka, 1964a), have also shown that these forms do not possess the potentialities to have been the ancestral form of sativa. The annual wild rices are, however, very ubiquitous and, in their role as the companion weeds of rice, they may serve as reservoirs of germplasm for cultivated rices. 0. rufipogon, perennial: This leaves only the perennial wild rice as the putative ancestor of rice. In Chatterjee’s (1948) revision of the genus, he recognized the presence of perennial wild rices in Asia (in Ceylon). This fact apparently made it easier to recognize soon after (Chatterjee, 1951; Ramiah and Ghose, 1951; Sampath and Rao, 1951) its widespread occurrence in Asia. Then, Sampath and Rao (1951) proposed that perennis is the progenitor of all cultivated rices, both sativa and g2aberrima. Ramiah and Ghose (1951), Porthres (1956), and Kihara (1959) conceded only a collateral role to this taxon. However, perennis was at that time widely conceived. It was then proposed that sativa originated from the Asian perennial floating rice, here designated as rufipogon perennial (Nayar, 1958; Sampath and Govindaswamy, 1958; Richharia, 1960). The detailed studies conducted by Oka, Morishima, and their co-workers have amply confirmed that sativa has directly evolved from this taxon only and not from rufipogon annual (Morishima et al., 1961, 1963; Hinata and Oka, 1962a,b; Oka and Chang, 1962; Oka, 1964a; Morishima and Oka, 1960; Oka and Morishima, 1971). Considerable supporting

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evidence has been presented by other workers also (Nayar, 1958, 1966, 1967; Sampath and Govindaswamy, 1958; Richharia, 1960; Yeh and Henderson, 1961a,b; Henderson, 1964a; Nair et al., 1964; Nayar et al., 1966). These are: (1) their widespread occurrence in Asia; (2) it is also a diploid species like rice (2n = 24). They produce semifertile hybrids and show normal F, segregation; (3) the two species have similar chromosome morphology; (4) the populations of perennial wild rices store up considerable variability unlike the annual forms; (5) hybrids between geographical races of rufipogon perennial produce by recombination, the various characters of cultivated rice; (6) the actual process of evolution of rice can be observed in some areas even today. Perennial rufipogon is widely distributed throughout most of the areas of rice cultivation. It is gregarious in habit. As we will see presently, the transition from the progenitor species to cultivated species is slight compared to other important cereals, and the wild rices have been, and are even now, harvested by local people in southeast Asia (de Candolle, 1886; Watt, 1892; Kikkawa, 1912; Nayar, 1958,1966; Oka and Morishima, 1971). Thus early man in the monsoon area of Asia might not have felt the need or urgency to nobilize wild rices until comparatively late. With large stands of wild rices occurring, most of his needs might have been met by them. Thus all evidence overwhelmingly indicates the evolution of the Asian cultivated rice sativa from the Asian perennial wild rice, rufipogon perennial. 6. Evolution of Rice

a. Progression from Wild t o Cultivated Rice. Watt (1892) suggested that this progression was achieved by a reduction of size and alteration of receptacle shape, the loss of awns, and the shortening but widening of the combined outline of the inner glumes. Now that the progenitor species is more definitely known, the changes can also be defined more accurately. It consists of a change in habit from a prostrate or procumbent t o an erect and compact one, from shattering character of the grains to nonshattering habit, an increase in the number and length of panicles, from lax to compact panicles, an increase in the weight and number of spikelets per panicle, reduction in awns, from black lemma-palea t o brown or golden color, and from red to white pericarp (Nayar, 1958). Shattering refers to the falling off of seeds or spikelets mechanically by wind or other means, and before harvesting if it is a cultivated species. Two other significant changes have been from a perennial to annual habit and from a partly cross-fertilizing nature to

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predominant self-fertilization (Nayar, 1958, 1967; Yeh and Henderson, 1961b; Oka and Morishima, 1967,1971). i. Cytological changes associated with the evolution of rice. Meiosis and seed set in cultivated rice are normal (Kuwada, 1910; Rau, 1929; Selim, 1930), but occasionally 1 IV or 2-4 I may be seen (Sakai, 1935; Nandi, 1936; Nayar, 1958; Yeh and Henderson, 1961b). I n rufipogon perennial, pollen stainability is normal, and seed set is only a little lower, about 80% or more (Nayar, 1958; Yeh and Henderson, 1961b; Chu et al., 1969a). Meiosis is mostly normal, but single quadrivalents, 2 4 univalents and lagging chromosomes are sometimes seen. Basak and Dana (1967), however, reported considerable structural heteroaygosity in a few populations studied by them. Hybrids between sativa and rufipogon perennial (Asian) have been studied by several workers (Ghose et al., 1956; Nayar, 1958; Nezu et al., 1960; Yeh and Henderson, 1961b; Bouharmont, 1962a; Hinata and Oka, 1962a; Li et al., 1962; Oka, 1964a; Shastry, 196413; Henderson, 1964a; Chu et al., 1969a). The crossability is at best fair (10-30%), but pollen and spikelet fertility are good-approximately 60-90% and 50-80%, respectively. Nezu et al. (1960), Li et al. (1962), and Shastry (1964b) obtained normal pairing a t diakinesis and metaphase, but some abnormalities have been observed by others in a low percentage of cells, for instance, differential segments, and unequal and loostly paired segments a t pachytene (Shastry, 1964b), quadrivalents, univalents, and greater number of rod bivalents than are found in homozygous varieties (Nayar, 1958; Yeh and Henderson, 1961b; Chu et al., 1969a). The sterility and meiotic abnormalities are more pronounced in hybrids with rufipogon perennial (Cuba). The minor variations in results obtained by different workers may be due to the great variability present in the parental species. I n general, the nature of differentiation between rufipogon perennial and sativa is of the type of cryptic structural differences (cf. Stebbins, 1950, 1958a) resulting from small inversions (Nayar, 1958; Yeh and Henderson, 1961b; Hinata and Oka, 1962a; Shastry, 1964b; Henderson, 1964a) and duplications. The karyomorphology of the two taxa is also broadly similar, as we will see later. The presence of more than one secondary constriction in the chromosomes of balunga (rufipogon perennial, east Indian type) has been suggested to have arisen through repeated translocations (Ghosh, 1964). The nature of sterility barriers between wild and cultivated rice has been studied in some detail (Hinata and Oka, 1962a, b; Morishima et al., 1963; Chu et al., 1969a). No cytoplasmic effects on sterility have been observed. The sterility barriers present in Asian perennial wild rice are of both haplontic and diplontic types, with the latter predominat-

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ing. Both pollen and embryo sac sterility have been noticed. However, these are of an incomplete nature, as is obvious from the occurrence of natural crossing between wild and cultivated rices. ii. Genetic nature of differentiation. Mitra and Ganguly (1932) crossed an indica cultivated variety with sativa var. fatwc (probably rufipogon, perennial). The F, hybrid was dominant for all wild characters. In the F, generation, simple monogenic inheritance was observed for anthocyanin pigmentation in eight plant parts and for plant habit, epistasis of dominant genes in awning and pericarp color, complementary gene action for panicle shape and ripening spikelet color, and duplicate genes for auricle color. Nayar (1958) and Nayar et aE. (1966) studied the inheritance in a cross between a japonica variety and a southeast Indian form of rufipogon perennial and also in an interracial hybrid of rufipogon perennial (between a southeast Indian form and a Cuban form). I n the first cross, the F, hybrid was intermediate for habit, spikelet shape, and spikelet weight and dominant for “wild” characters. All F, and F, plants could easily have passed for rufipogon annual plants. A few F, plants were weak and unthrifty. The mode of inheritance of various characters was of the nature of complementary, modifying, duplicating, and polygenic types involving 2 or 3 genes. Of 12 F, lines, 3 bred true for all characters and a fourth segregated for only one character, showing “block transference” of characters (cf. Harland, 1936; Anderson, 1949). In the second cross involving two geographical races of perennial wild rice, interestingly, all characters associated with the cultivated species appeared in the F, populations in the eight characters studied, though neither of the parents showed this. This is possible only with the operation of duplicate genes, and in fact, such a mode of inheritance was obtained for three characters whose inheritance could be determined. The reason for the inability to obtain Mendelian ratios for the remaining five characters might have been due to the operation of a complex polygenic system, or to the operation of gametic and of zygotic lethality resulting in distortion of ratios. The recovery of recessive characters of cultivated rices signifies that wild rices have enough variability stored up within them and that they have the potentiality to produce cultivated rices by recombination and segregation (Nayar, 1958). A complementary type of inheritance in interspecific hybrids has been attributed to pseudo-allelic action of small repeats, and these have been suggested to be transition stages between duplicate and independent genes (Stephens, 1950,1951). The importance of major genes in crop plant evolution is well known. Hutchinson (1965) pointed out with regard to wheat that it was difficult to conceive of the persistence of an unstable polyploid long enough for

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meiotic stability of the kind demonstrated by Riley (and Sears and Okomoto) to be built up by selection of a constellation of genes of small individual effects. In maize, Mangelsdorf (1965) considered that it was the changes at the Tu-tu locus, more than any other, that set the plant on new evolutionary paths that led to its becoming one of the world’s most successful cultivated species. In barley, a complementary system of inheritance involving Bt and Bt, loci controls rachis brittleness (R. Takahashi, 1955). I n rice, there seems to have been no mutation to match in impact any of the above three cases. But the single most desired and effective change would have been from a highly shattering nature of the spikelets to less shattering and more persistent spikelets (Nayar, 1958, 1966). This character is controlled by a pair of complementary genes. A nonshattering nature could have been obtained either by recombination, or by mutation a t one of the two loci involved. With such a primary gain, selection for other desirable characters could be taken up in the course of time. iii. Other studies. Several authors have observed that wild rices show a continuous variation to cultivated forms (Watt, 1892; Roy, 1921; Ramiah and Ghose, 1951; Sampath and Rao, 1951; Sampath and Govindaswamy, 1958; Nayar, 1958; Oka and Chang, 1961, 1962). Roy (1921) did not find that wild rices evolved into cultivated forms when exposed to improved cultural practices. However, later studies have shown that responsiveness of wild and cultivated rices to cultural practices are directly correlated with their degree of domestication (Oka and Chang, 1959, 1964b; Hinata and Oka, 1962b; Oka and Morishima, 1971). They found that the partially allogamous nature, asexual reproduction, and pronounced seed dormancy of perennial wild rice enabled it to accumulate variation brought about by mutations and hybridization. When plants approached cultivated types, the probability of selfing increased and seed dormancy was weakened. When intermediate types of wild rices were grown in cultivated fields, they tended rapidly to become homozygous and show cultivated characters. Rices did not show any adverse effects of inbreeding. According to Morishima et al. (1961), the incipient cultivated genotypes might have arisen due to mutation and recombination, and the most essential factor in creating a cultivated type might have been the occurrence of niches in which such plants (incipient cultivated genotypes) had a selective advantage, for instance, a land cultivated or disturbed by man. We have mentioned already that some workers continue to believe that there exists an annual wild rice directly descended from perennial wild rice. The annual wild rices are ubiquitous in all rice-growing areas,

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they demonstrate polymorphism, and, more importantly, their features and characteristics are truly intermediate between perennial wild rices and cultivated rices. Obviously, during the progression from rufipogon perennial the ancestral species, to sativa the cultivated species, such forms would have constituted an intermediate phase. But the question to be considered is whether such intermediate types would exist, whether they still exist by themselves in nature in observable numbers, or whether they would have occurred only transiently in man’s cultivated fields during the course of domestication. The latter seems to be the more probable. Even today, people in certain parts of Asia (not primitive but poor people) consider it worthwhile to harvest the grains of perennial wild rice. Annual wild rices are also collected wherever large stands are available, but they are fewer and this does not detract from the argument here. There is overwhelming evidence to consider perennial wild rice as the species ancestral to rice. It would thus have constituted the starting material for cultivation. With mutations and recombination having continually taken place, and with the release of all the pent-up genetic variability in the niche of the protected environment provided by man, there would have been ample scope for him to select out the more desirable and uniform types. These early generations constituting, as it were, the prototypes of sativa would in all probability have looked like the annual wild rices of today. But these could have had an existence only in a man-made environment during a certain stage of ennoblement. This can be observed even today in parts of Asia (Govindaswamy and Krishnamurthy, 1958a; Kihara and Nakao, 1960; Oka and Chang, 1962, 1963). The annual wild rices which occupy intermediate habitats are then swarms of hybrid origin in most cases, if not in all. A direct descent from the ancestral type for them would also lead one to assume a more efficient mode of seed dispersal than is available today. This would be difficult indeed with the habitat preferences of perennial wild rices for deep marshes and their easy shedding nature of their spikelets. Last, with rufipogon perennial and sativa in contact over very wide areas, with improved cultural practices such as weed control and environmental sanitation not being practiced as thoroughly as in the subtropics, with the observed introgression between these two entities, and also with the availability of intermediate habitats such as fallow lands, in much of the monsoon areas of Asia, large hybrid swarms have only to be expected. I n areas such as river deltas and irrigation projects, where land is a t a premium and cultivation practices are more efficient, and also in such areas as uplands where perennial wild rices are not present, rufipogon annual occurs also, but less frequently. Massive stands of

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hybrid swarms are well known in other crops and plants in areas of similar contact between related species (Anderson, 1949, 1953 ; Heiser, 1949, 1965; Anderson and Stebbins, 1954; Harlan, 1965, 1970; Zohary, 1965, 1970). Thus the burden of proving the existence of an independent and direct origin of the annual wild rices still remains. b. M o d e of Speciation. Since all evidence favors an in situ origin of rice in the area of its cultivation and since the progenitor species occurs throughout most of the areas of greatest diversity of rice, speciation in rice has evidently been sympatric in nature. However, the concept of sympatric speciation is a long-standing, much-debated and unsettled question (Grant, 1971). But the objections raised against its occurrence (Mayr, 1963)-neglect of dispersal, overlooking ecological plasticity and polymorphism, bridging effects of sexual reproduction and heterozygotes-lose much of their relevance in plants, and more particularly in cultivated species where their dispersal and directions of evolution are regulated by man. Mayr (1963, p. 480) has observed that “the essential component of speciation, that of the genetic repatterning of populations, can take place only if these populations are protected from the disturbing inflow of alien genes.” In rice, this needed protection would have been provided not only by man, but also by the change brought about in the breeding system during the course of evolution, viz., from a partly cross-fertilized system to one that is predominantly selfpollinated (Nayar, 197313, in press), The gregarious nature of the two species and the fact that they are wind-pollinated and not insectpollinated can have provided even additional protection from an alien gene flow. The crucial step in sympatric speciation is said to be the establishment of stable polymorphism in a heterogeneous environment, and this stable polymorphism involving alleles adapting individuals to different ecological niches can be accomplished under two conditions only : (a) the density-dependent factors regulating population size must operate separately in the two niches; (b) the selective advantages must be large (Maynard Smith, 1966). These conditions are obviously fulfilled in rice also. Mayr (1963) listed four main models of sympatric speciation: disruptive selection, cytoplasmic sterility, mutation changing host specificity, and seasonal isolation. The work of Morishima, Oka, and their co-workers has shown that cytoplasmic factors are not a cause of sterility in the wild-cultivated rice complex. Speciation by a change in host specificity is relevant to parasitic organisms only. There is also no case for speciation by seasonal isolation in rice, as wild and cultivated rices flower almost synchronously (Nayar, 1958; Nair e t al., 1964). This leads us to disruptive selection. Basing largely on the theoretical

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considerations of Mather (1955), Thoday and co-workers have over the years shown that disruptive selection occurs when more than one type of phenotype or polygene complexes are selected from a population to provide the parents of each generation. In a widely quoted experiment, Thoday and Gibson (1962) practiced simultaneous selection for high and low sternopleural chaeta (bristle) number in Drosophila with random mating, and a partial reproductive isolation was achieved within just 12 generations. The probability of this taking place in nature is, however, low because of the severity and absoluteness of selection, the complete prevention of gene flow and elmination of mutual competition (Mayr, 1963; Scharloo, 1971; also see Thoday and Gibson, 1971). We will now see how this could have taken place in rice. The process of nobilization in rice brought about by man is comparable to the above-mentioned selection experiment of Thoday and Gibson (1962) (Nayar, 1973b, in press). The conditions would have been, however, even less favorable for random mating and increasingly adapted to assortive mating. And this could only have hastened the process of building up polymorphism and reproductive isolation and thereby contributing to speciation. People harvest grains from wild rices by going over the fields and swinging a basket over ripening ears. Those that collect in the basket will be those that have not been shed by wind or by man’s movement. A part of such a harvest may have been sown to raise the first generation. Competition between the two populations was naturally excluded and gene flow between them would also have been restricted initially due t o some degree of spatial isolation and, later, due to changes brought about in the breeding system. As we have seen, this gene flow between the two entities is not completely eliminated even now. This may be advantageous to both by contributing to their plasticity, adaptation, and continued success. The relevance of disruptive selection to crop plant evolution is being gradually realized (cf. Hutchinson, 1970, 1971). It has been best demonstrated in sorghum (Doggett and Majisu, 1968; Doggett, 1970). They have shown that cultivated sorghum was developed from EL single wild species of the subsection Arundinaceae. An intermediate population of hybrid origin from the wild and cultivated species occurring in intermediate habitats exists in this crop also. Postscript: We can now see that rice has had a diffuse origin (of. Harlan, 1965) both in space and time. Several centers of origin have been proposed for Asian rice: southern India (Watt, 1892; Ramiah and Ghose, 1951; Nair et al., 1964), Jeypore in southeastern India (a secondary center, Ramiah and Ghose, 1951), the Philippines (Ramiah and Ghose, 1951), China (de Candolle, 1886; Roschevicz, 1931 ; Ting, 1949),

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Indochina (Hamada, 1949) and the western Indo-Pacific area (Barrau, 1966). All these areas show today considerable varietal diversity. In addition, the northwestern and northeastern foothills of the Himalayas (Ramiah and Ghose, 1951) and the Guyanas (Porthres, 1949b) are known to show much variation. The difficulties resulting from considering centers of diversity as centers of origin are well known by now (cf. Zohary, 1970). With the ancestral species present in its original habitat in all these regions of variability and with the knowledge that this species is found good enough even today for harvesting, the changeover from rice gathering to rice culture could have been attempted in each of these regions and a t various times. This can be witnessed even today in some areas such as Jeypore in eastern India, as already mentioned. Further, the transition from the wild to the cultivated form in rice is of a much lower magnitude than that involved in at least the principal cereals. And this ennoblement could have been effected by the people inhabiting these regions even without the expertise of immigrants from other areas as Barrau (1966) proposed. B. AFRICANRICE

I. General African rice is botanically 0. glabem'ma. It is grown in tropical west Africa (from Senegal to north Cameroon and Chad). As already mentioned, it occurs to a very limited extent in Guyana and El Salvador also. Another cereal known from this region as hungry rice, fonio, or acha grass, is Digitaria e d i s . It forms a staple crop in some areas. Until about the early 1960s, practically all our information on African rice came from just two sources: from the extensive writings of Dr. Porthres (most of which, unfortunately, are not seen referred to in English language publications on rice), and from the few seed samples sent out by some of the research stations of this region. These seed samples were usually highly selected material possessing all the distinctive features attributed to them in the literature. This has had an unfortunate effect in creating a lopsided view of this rice. Then the National Institute of Genetics, Misima, Japan, organized two collection trips to west Africa. They assembled a large number of seed samples of both wild and cultivated rices of this region. The report of the second trip (Oka and Chang, 1,964a) gives considerable information on rice cultivation in west Africa and on the ecology and distribution of wild rice species there. It is importance to keep in mind that, unlike the case in the monsoon areas of Asia, rice is a crop of only minor importance in the region

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under discussion (northern half of tropical west Africa, i.e., Senegal to Chad including north Cameroons, also termed sub-Sahara ; this region, but without the peripheral parts of the lowlands and montane evergreen forest zones, is also known as the Sudan zone. It lies to the west of the country Sudan). It stands no comparison with the importance of rice in Asia. The subsistence economy of this region is primarily agricultural, or agricultural and pastoral, with fishing in some areas (Murdock, 1959; Clark, 1962). Throughout the huge Sudan zone of the interior, millets and sorghums stand virtually alone as the dominant staple food crops. Rice is important only in coastal western Africa, south of 'Gambia to the Bandama river in the Ivory Coast (Church, 1960). Even here, it has acquired this status only in this century (Johnston, 1958). And the rice here is sativa (Oka and Chang, 1964a). The caloric contribution of rice for the entire tropical west Africa (including subSahara, the coastal region, Angola and Congo, but excluding Cameroons) is only 8% of that provided by all food crops, as compared to 34% by millets and sorghum, 27% by manioc, 16% by yams, and 9% by maize (calculated from Table 4.1 of Johnston, 1958). The contribution from rice in the northern part of tropical west Africa will be even much less when the data for Cameroon are included (where it is a very minor crop) and that of Congo and Angola are excluded (where it is a major crop in some areas). Another characteristic of rice culture in tropical west Africa is that even in its original habitat, Asian rice is generally preferred to African rice because the former is considered higher yielding and more adaptable and is thought to possess better quality (PortBres, 1959; Angladette, 1966). Oka and Chang (1964a), however, did not think that it is so inferior as is generally supposed. Another peculiarity is that in the Sudanese zone, mixed crops of Asian and African rices are grown with the proportion of each varying in different regions. Mixed fields of the two rices show considerable variability. The farmers are unable to distinguish the two cultivated species. But they can be separated by the short ligules of glaberrima (Oka and Chang, 1964a). All other characters attributed to glaberrima, such as glabrous spikelets, rigid rachis and rachilla, are found in sativa, and all the characteristics associated with Asian rice are found in glaberrima also (PortBres, 1945). In fact, PortBres (1945, 1960, 1962), who has considerable experience with African rices, has repeatedly mentioned the close parallelism in variation between African and Asian rices. 2. The Wild Rices

Leprieur was the first to collect cultivated rice samples from tropical west Africa. He collected them from near Dakar in 1824-1829 and identi-

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fied them as sativa. Jardin again collected rice samples from the coast of Guinea in 1845-1848. Steudel described 0. glaberrima in 1855 from this material (Porthres, 1955a). Three related wild taxa occur in the area of cultivation of glaberrima. They are stupfii (also known as sylvestris), barthii (until recently known only as breviligulata: see Table 1), and Zongistaminuta (variously known also as barthii, perennis ssp. barthii, madugascaremis). After Roschevicz (1931) first described 0. stapfii, Chevalier (1932) proposed it as one of two series of glaberrima. H e assigned to this all glaberrima forms having hairy spikelets. But nowadays stapfii is considered a synonym of barthii (as is breviligulata) (Porthres, 1956; Sampath, 1962; Tateoka, 1963; Morishima et aZ., 1963; Bardenas and Chang, 1966). This taxon occurs widely throughout tropical Africa, but more commonly in the interior, in streams and ponds, as weeds in rice fields and in fallows (Chevalier and Roehrich, 1914; Roschevicz, 1931; Porthres, 1956; Oka and Chang, 1964a; Clayton, 1970). It is an annual with truncate ligules, panicles with tertiary branches bearing big, bold spikelets (about 10 mm long and 3 mm broad), and stiff awns. Its spikelet may be the biggest and heaviest in the entire genus. It shows about 3-20% crossfertilization (Morishima et al., 1963). The second taxon, longistaminata (most commonly known as perennis ssp. barthii and barthii) grows widely throughout tropical Africa and southward up to South West Africa, South Africa (Transvaal), and Malagasy (Chevalier, 1932; Porthres, 1949a; Schweickerdt and Marais, 1956; Tateoka, 1963 ; Oka and Chang, 1964a; Clayton, 1970). It has the widest distribution in Africa of all Oryza species. It is a perennial with extensive creeping and underground rhizomes, long ligules, long and slender spikelets (about 8 mm long and less than 2.5 mm broad) and possesses anthers about threefourths as long as the spikelets. It is predominantly self-incompatible. The seeds of both barthii and longistaminata are collected by local people in west tropical Africa for consumption. Porthres (1949a, 1959, 1962) has reported that more of longistaminata (his barthii) is harvested, whereas Oka and Chang (1964a) found that more of barthii (their breviligulata) was being collected. I n tropical west Africa, and more frequently in the Sudan zone, barthii and longistaminata sometimes occur sympatrically. These species may also invade rice fields. Because of the difficulty of weeding out the rhizomes of longistaminata, the rice fields may then have to be abandoned temporarily (PortBres, 1949b, 1959; Oka and Chang, 1964a). Natural crossing in wild and cultivated rices has been known from tropical west Africa also, but it appears to be on a much lower scale than in Asia. The chief reason may be that the African landscape has

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been subjected to less human interference than in Asia. Chevalier (1932) also observed cultivated forms which were intermediate between sativa and glaberrima, and he assumed them to be hybrids. He called them 0. glaberrima series stapfii. Chevalier and Roehrich (1914) might have been referring to these same types when they reported finding some cultivated rices different from true sativa and related to barthii (their breviligulata) in ligule and panicle characters. Nayar (1958) studied two samples of stapfii and found that they showed characteristics of hybridity. Jachuck and Hakim (1966) , who studied the range of variability in 43 collections of African wild and cultivated rices, considered that stapfii Constituted intermediate stages in the evolution of glaberrima from barthii (their breviligulata) and also segregants from natural crosses between these two taxa. They also studied five wild rice samples originating from Sudan, Nigeria, and Malagasy. They showed characteristics of Asian annual wild rices (rufipogon annual). The samples from Sudan and Nigeria were suggested to be natural hybrids between sativa and longistaminata, while that from Malagasy was taken as a rufipogon annual introduction from Asia. Jachuck and Sampath (1966a) also observed plants with varying degrees of barthii characters in the progenies of several longistaminata plants. However, they took this t o mean that barthii constituted intermediate stages in the evolution of glaberrima from longis taminata. More extensive observations on natural crosses in African rices were made by Oka and co-workers. During their field trips lasting about 3 months in the fall of 1963 (Oka and Chang, 1964a), they encountered only one population that appeared to be of hybrid origin involving sativa. At the same time, hybrid swarms of glaberrima and barthii and populations that were intermediate in appearance between these two species were frequently seen (also see Morishirna et al., 1963). Chu and Oka (1970b) also found evidence for natural crossing between longistaminata, and sativa, glaberrima, and barthii. The taxon longistarninata is known to be isolated from other Oryza species by strong sterility barriers (Nayar, 1958; Chu et al., 1969a; Chu and Oka, 1970a). Many of the apparently hybrid plants did not show the isolating barrier present in longistaminata. Chu and Oka (1970b) felt that a balance between reproductive isolation and “leakages in the isolating barriers” might have a role to play in the evolutionary dynamics of the species. 3. Time and Place of Origin The only observations made on these aspects are by Porthres (1945, 1950, 1959, 1962). H e postulated that African rice originated in the

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region of the central delta of the river Niger about 3500 years ago. The Bantus, who originally inhabited this area, only gathered wild rices, but the Negroes who followed them domesticated African rice, and the Mande people who came later improved it further. He gave several reasons in support of this. This area is characterized by many lakes, marshes, and smaller rivers, and glaberrima shows maximum variability here. This consists of both dominant and recessive characters, but more of the former. Further, barthii (as breviligulata), which he considered the ancestral species, was present here. According to him, the names for rice were not derived from Asian words for rice. These are malo, mano, and maro. The word ma is of Bantu origin and means a thick liquid, and lo, no, and TO mean food or nutrition. H e also found two secondary centers of variation. He was confident that it was an indigenous development and not the result of a borrowing of ideas from Asia. PortBres’ proposal received strong (but unintentional) support from Murdock (1959) in his masterly study of African ethnography. According to Murdock, Neolithic civilization appeared on the African continent in the fifth millenium BC, independently in two regions, in the lower Nile Valley and in the Western Sudan zone (i.e., the same area as that proposed by PortBres) . The people of the lower Nile borrowed agriculture from southwest Asia. This latter statement is generally accepted (cf. Clark, 1962; Dixon, 1969), and we will not go into it further. About the second center, Murdock concluded : “the invention of Agriculture in Negro Africa is most probably to be credited to the Mande peoples around the headwaters of the Niger . . . before it had diffused from Asia to lower Nile.” He ranked this as one of four major agricultural complexes evolved in the entire course of human history, the other three being the southwest Asian complex (developed by the Caucasoids), the southeast Asian complex (by the Mongoloids), and the middle American complex (by the American Indians). He then listed 25 crops as having been ennobled by the nuclear Mande people. Among cereals, he mentioned hungry rice (Digitaria erilis), millet (Pennisetum), and Sorghum. “While the book was in press,” he added glaberrima in a footnote, quoting Johnston (1958) as authority (Johnston, in his book, mentioned glaberhma as only indigenous to tropical west Africa and as cultivated since ancient times. There is no reference to its time and place of origin.) Murdock based his theory on linguistic grounds and on the distribution of crop plants. He argued that the inventors of so successful an art as agriculture should be expected to have spread out widely, carrying with them their spoken language. This condition does not prevail in either the central or the eastern Sudan area, but only in the western Sudan area, the languages of the far-flung Negritic stock and of the

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Mande subfamily having identities. From botanical evidence, he eliminated from the list of indigenously cultivated crops those that he knew were introductions. H e assumed that the remainder were developed locally. Regarding the time of development of this complex, he said that it could only have been before agriculture arrived in the lower Nile, as otherwise, the crops of this latter complex would have seeped into this area also. Actually, only minor adoptions of southwest Asiatic crops have been made in this part. Murdock seems to have overlooked climatic differences of the two regions while putting forth this argument. PortBres’ (1962) proposal came in for much comment a t the Third Conference on African History and Archaeology when it was presented (cf. Gray, 1962). Tucker, for instance, pointed out that it was not correct to draw philological deductions without making comparative philological studies. Clark (1962) noted that, on the basis of archaeological evidence, it appeared that even 2000 years ago Neolithic people occupied only a few parts of sub-Sahara and it was hunter-gatherers who occupied most of the remaining areas. Murdock’s (1959) proposal for a center of origin of cultivated crops in the upper Niger delta has also been severely criticized by Wrigley (1960) and Baker (1962). Wrigley pointed out that an equally good case could be made out for an early diffusion of agriculture including cultivated plants from the east coast of Africa to the west. The dispersal of Mande languages was more plausibly attributed to empire building and trading a t a very early date. Baker (1962) criticized Murdock’s theory on botanical evidence. He took one by one the crop plants said to have been ennobled in this area and pointed out that there was as yet no or insufficient botanical evidence for assuming the origin of most of them in the west Sudan area. These could have been either Asiatic in origin or developed elsewhere in Africa. Only some minor crops like Butyrospermum (shea butter tree), Telfairia (fluted pumpkin), Digitaria (fonio) , Kerstingiella (geocarpa groundnut) were probably West Sudanic in origin and most of these were either wild plants protected by man or plants not yet sufficiently differentiated from their immediate wild ancestors. He considered glaberrima of local origin, but of a later date. He cited it as an instance of a local addition of a crop to an agricultural system which might have originated elsewhere. Baker also remarked that a longstanding agricultural system will usually have an adapted and associated weed flora. No such separate weed flora existed for the west Sudan zone. That of the Savannahs appeared more indigenous than that of the forest and coastal areas of west Africa.

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Though the upper delta region of Niger may not have been a great and ancient center of agricultural origin, it is widely accepted that this region, as well as the area around Lake Chad to the east, have been areas of much human activity since ancient times (Murdock, 1959; Wrigley, 1960), mainly because these areas have been endowed with sufficient supplies of ground water. Considerable caravan trade also took place through the Saharas between the coastal Mediterranean region and sub-Sahara from very early times, even before the camel was domesticated in the early centuries of Christian Era (Murdock, 1959). And African rice could also have evolved here in the upper delta region of the Niger as suggested by Porthres in his publications. There is, however, no need, nor is there any evidence so far, to postulate an ancient origin of African rice. Rice is a crop of minor significance only in the entire sub-Saharan region, contributing less than 8% of caloric intake, as we have already seen. In the upper Niger delta area, the dependence on rice could have been even less, because the subsistence economy of this region is based on fishing (Map 7 in Murdock, 1959). The picture would no doubt have been different before 500 years ago, as maize, manioc, and sweet potatoes were introduced by the Portuguese only from the 16th century on (Johnston, 1958). But millets and sorghums have been grown there as dominant crops since ancient times, and so has it been with yams, both wild but edible and cultivated. This region lies just to the north of the present-day yam zone of tropical west Africa (Alexander and Coursey, 1969). The dependence on game would also have been much more pronounced in older times. Further, the pressure on land is minimal even today, and shifting cultivation is still in vogue. Only a small portion of the vast flood areas of the upper delta region of Niger is cultivated because of a shortage of labor (Oka and Chang, 1964a). A cultivated field is abandoned when it becomes less productive or when it is overrun by the wild rice longistaminata. There is also the fact that Zongistaminuta occurs extensively in the entire tropical west Africa, with some populations in the Sudan region covering large areas extending over a few kilometers (Oka and Chang, 1964a). Thus, with fish and also game constituting sources of protein, and sorghums and millets as dominant starch sources, and yams as supplementary sources of starch, any additional need for starch could most readily have been complemented by harvesting the large stands of longistamineta, a practice in vogue even today. Consequently, it becomes difficult to conceive that the people who inhabited the Niger’s upper delta region would have felt any need or even an inclination to evolve another cereal plant like glaberrima.

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4. T h e Ancestral Species Both the related wild Oryza longistaminata and barthii species have been proposed as the progenitor species of glabem'ma. Both occur throughout its area of cultivation and even beyond it. a. Longistaminata and barthii. Sampath and Rao (1951 ; also see Sampath, 1962) proposed that perennis gave rise to glaberrima by human selection. They reasoned that this species had the widest distribution of all Oryza species, and that it gave rise also to sativa in Asia. This viewpoint was supported by a number of other workers-Richharia (1960), Seetharaman (1962), Gopalakrishnan et aZ., (1964), and Gopalakrishnan (196613). Some among them (Sampath and Rao, 1951; Richharia, 1960; Gopalakrishnan, 1966b) also considered that barthii (their breviligulata) was of hybrid origin from glaberrima and Zongistaminata, an origin similar to that of rufipogon annual. This proposal about the origin of glaberrima was made soon after Chatterjee's (1948) revision of the genus Oryza, in which he had included under perennis all the perennial wild rices of America and Africa and some of Asia. Although hybrids between longistaminata and other closely related species are difficult to make (see above), those between glaberrima and rufipogon perennial of Asia (as perennis ssp. balunga) show low fertility (Nayar, 1958; Morishima et aZ., 1963; Gopalakrishnan, 1966b ; Chu et al., 1969a) and this apparently kept the proposition viable for some time afterward. It was gradually realized that longistaminata is characterized by strong isolating barriers, and the proposal about the origin of glaberrima from barthii has since lost most of its following. Sampath and co-workers (Jachuck and Hakim, 1966; Jachuck and Sampath, 1966a) have subsequently proposed that Zongistaminata might have given rise to forms intermediate between longistaminata and barthii (as breviligulata) and these forms by intercrossing might have given rise to barthii, which then gave rise to glaberrima. Most workers consider that barthii is the ancestral species of African rice. It has only a slightly smaller range of distribution than longistaminata, but still covers a much wider territory than glaberrima. It is more frequent in the Sudan zone. It probably does not grow in such extensive stands as Zongistaminata. It is an annual. It is propagated by seed. Its chief habitats are streams and seasonal swamps. It is also found in rice fields, irrigation ditches, old rice fields, and other disturbed habitats (Roschevicz, 1931; PortGres, 1956, 1959; Oka and Chang, 1964a). Porthres (1956, 1959) observed that it never develops a floating habit, is less tolerant of flooding conditions, and its habitat is marginal

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to that of longistaminuta. Bardenas and Chang (1966) made a detailed study of glaberrima and barthii comparing several strains in each for 22 characters. PortBres (1950, 1956, 1959) proposed that while barthii gave rise to glaberrima, some of the variability present in the latter could have come from longistaminata. Others (Chatterjee, 1951; Morishima et al., 1963; Angladette, 1966; Chu and Oka, 1967) have assured a monophyletic origin for glaberrima from barthii. Hybrids between glaberrima and barthii have been studied by several workers (Ghose et al., 1956; Neau et al., 1960; Morinaga and Kuriyama, 1960; Li et al., 1962; Yeh and Henderson, 1962; Morishima et aZ., 1963; Chu et al., 1969a). Morishima et al. observed that while intravarietal crosses in sativa and ru.fipogon annual and perennial (Asian), and also hybrids between them show a complete series of gradations in sterility (1-99% pollen stainability) , similar crosses in glaberrima and barthii are marked by high degrees of fertility (83-99% pollen stainability). Meiosis in the hybrid is normal. I n genetic studies of this cross (Nayar et al., 1966), the F, hybrid showed dominance for 9 characters of the wild parent, heterosis in 4 other characters and dominance of the glaberrima character in one case. I n the F, generation, hairiness of spikelets, grain shedding, and sensitivity to photoperiod segregated in a simple monogenic ratio and spikelet color showed a duplicate mode of inheritance involving 2 factors. Thus, cytological and genetic studies indicate a very close relationship between these two taxa. Natural populations of barthii (Oka and Chang, 1964a) show the presence of many intermediate types and populations. These variations are often continuous between the two taxa. Though sativa and longistaminata also occur sympatrically with it, no putative hybrids with sativa were observed, and those with longistaminuta were only rarely seen (Chu and Oka, 1970b). The authors felt that some of the types intermediate between glaberrima and barthii and occurring in disturbed habitats were comparable to stapfii. Some authors have compared the modes of evolution of African and Asian cultivated rices, considering as their progenitors barthii and rufipogon (PortBres, 1950; Morishima et al., 1963; Oka, 1964a; Chu and Oka, 19671, respectively. The cultivated species are distinguished from the wild species by the reduced grain shedding and seed dormancy, greater spikelet number per panicle, and narrow spikelets of the former. The analysis of intraspecific variations suggested that selection might have proceeded in a different manner in the evolution of the two cultivated rices. On the whole, barthii contained considerably less variability

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than rufipogon perennial. A greater proportion of the variability was due to interpopulational differences. This was attributed to the annual habit of barthii and the prevalence of less outcrossing in it. PortBres (1950) thought that the comparatively homogeneous habitat of barthii contributed to its lowered variability. He also observed that while the progenitor species of sativa was able to realize an “internal diversification,” this was only beginning to take place in barthii. In this respect, the level of differentiation attained by African rice was of a much lower magnitude than that of Asiatic rices. I n several characteristics, including habitat preferences, population structure, and longevity, barthii is comparable to the annual wild rices of Asia (Morishima et al., 1962, 1963; Oka, 1964a; Chu and Oka, 1967). Notwithstanding the fact that barthii possesses certain features that could make it the ancestral species of the African rice glaberrima (wide and sympatric occurrence, closest genetic relationships, a certain degree of variability), there are also some difficulties in assuming such an origin. Since rices, both Asian and African, have naturally been domesticated for their grain yields, it is difficult to conceive that during this process of domestication of African rice, man would have selected negatively for several yield components, for instance, smaller spikelets, lower tiller numbers, and absence of secondary branching in panicles. Some gains have, however, been made in spikelet number; but this is unlikely to compensate for the losses from the other yield components (Table 2 ) . Comparative yield data for barthii and glaberrima are not available. Although correlations are known for only a few characters in glaberrima, spikelet number per panicle shows a significant positive correlation with ligule length, panicle length, and rachis number per panicle, and a significant negative correlation with degree of shedding (Morishima et al., 1962). Even otherwise, it is hard to concede that early man would have let some of the already available assets from the progenitor species slip by during the process of ennoblement. The only other trait of any significant advantage possessed by the cultivated species over that of barthii is reduced shattering (Morishima et al., 1963; Bardenas and Chang, 1966). The rachis number per panicle is also higher in glaberrima, but this is covered by the increased number of spikelets per panicle. It is also open to question whether barthii really possesses the range of variation and potentiality that are required of a progenitor of a cultivated species. There are several points of similarity between barthii and rujipogon annual, as we have already seen, and the latter taxon has been shown not to possess the potentialities and characteristics of an ancestral species t o Asian rice (Sampath and Rao, 1951; Sampath

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and Govindaswamy, 1958; Nayar, 1958; Yeh and Henderson, 1961b; Morishima et al., 1963; Oka and Chang, 1962,1964b). The difficulties pointed out above can be met if the origin of glaberrima is assumed to derive from the Asian cultivated rice sativa (Nayar, 1973a). There is also good evidence to indicate that barthii evolved by hybridization between glaberrima and sativa, followed by backcrossing with glaberrima. This proposition will be examined in the next section. b. 0. sativa as a Progenitor of African Cultivated Rice. i. M,orphology. The distinguishing features of sativa, glaberrima, and barthii are given TABLE 2 Range of Variability in Yield Components of barthii and glaberrima Character and authority 1. Spikelet size (mm) Roschevicz (1931) Sampath (1962) Morishima et al. (1963) Bardenas and Chang (1966) 2. 100-grain weight (g) Nayar (1958) Sampath (1962) Morishima et al. (1963) Bardenas and Chang (1966) 3. Panicle-bearing tillers (number) Bardenas and Chang (1966) 4. Number of spikelets/panicle Nayar (1958) Sampath (1962) Morishima et al. (1963) 5. Protein percentage Bardenas and Chang (1966)

barthii

glaberrima

10.0-11.0 x 3 . 0 - 3 . 5 1 0 . 0 (variable) x 3 . 0 8.8 X 2.9 9.2-11.0 X 3.1-3.4

7 . 0 - 8 . 0 X 2.5-3.0 8.0 X 3.5 8.4 x 3.4 7.4-9.0 X 2.9-3.6

3.0 3.0 2.4 2.3-3.4

2.8 3.2 2.3 1.6-2.8

20-61

18-34

37 73 42

54 125 90

13.4-17.3

9.5-13.8

in Table 3. Typically, the spikelets of glaberrima are glabrous, while those of satiua are hairy. However, it is by now generally accepted that there are hardly any features that are characteristic of either taxon (Ramiah and Ghose, 1951; Portbres, 1956, 1962; Sampath, 1962; and others). The shorter ligule size of glaberrima may be an exception. When mixed stands of these two cultivated species occur in fields, as is frequent in the Sudan zone, cultivators are most often unable to distinguish them (Oka and Chang, 1964a). It has even been proposed that the two taxa be merged (Ramanujam, 1938a; Rarniah and Ghose, 1m).Morishima

TABLE 3 Distinguishing Characters of sativa, glaberrima, and barthii (Roschevicz, 1931) Character

5. 6. 7.

glaberrima

7.0-8.0 X 2.5-3.0 Orbiculate, 3 4 mm Secondary and tertiary Primary Ending in small beak Acute without beak Palea tip Anther length (mm) 2.5-2.6 1.5-1.8 Stigma color Yellowish brown Black purple Shape and length of upper empty glumes Narrow lanceolate, 1 . 5 - 3 . 0 Narrow lanceolate, 2 . 0 - 3 . 0 (mm)

1. Spikelet size (mm) 2. Ligule shape and size 3. Panicle branching 4.

sativa 6.0-8.0 X 4.0 Acute, up to 40 mm

barthii 10.&11.0 x 3 . 0 - 3 . 5 Oblong, 3 4 mm

Secondary and tertiary Ending in beak 1.8-2.0

Black purple Linear lanceolate, 4 . 0

z P

n

j

0

8 3e Era 0

EM

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et al. (1962) made a comparative study of the two species taking 17 characters, dividing each character difference into 11 classes and classifying a large number (50-80 of each) of varieties. Except for ligule length, the range of variation of the two species overlapped in all characters. The range of variation of glaberrima was narrower than that of sativa except for potassium chlorate resistance and grain shedding. Some differences between them were also observed in resistance to drought (glaberrima less resistant) and seed dormancy (glaberrima more dormant), but some sativa varieties can show extreme behavior for these characters. These studies indicate their close morphological and physiological similarities. ii. Crossability and fertility of hybrids. Crosses between the two cultivated species sativa and glaberrima have been made by several workers (Ramanujam, 1938a; Peltier, 1953; Morinaga and Kuriyama, 1957; Nayar, 1958; N e m et al., 1960; Bouharmont, 1962a; Morishima et al., 1962; Seetharaman, 1962; Yeh and Henderson, 1962; Richharia and Seetharaman, 1965; Jachuck and Hakim, 1966; Joseph and Raman, 1966; Chu et al., 1969a). Hybrids are obtained without any difficulty. The rate of success of crossing was not much lower than that between varieties of sativa (Morishima et al., 1962). I n extensive crossings, Chu et al. (1969a) obtained 51% success in intervarietal sativa crosses, 62% in intervarietal glaberrima crosses, 58% in intra-breviligulata crosses, 42-39% in sativa-glaberrima crosses, 3 0 4 2 % in sativa-breviligulata crosses, and 59-62% in glaberrima-breviligulata crosses. The germination of hybrid seeds is also normal, with usually more than 90% success. The F, plants grow normally and show hybrid vigor. Hybrids are generally sterile and only occasionally show slight fertility. It is this characteristic that has been most influential in attributing specific status and a “near-but-far” relationship between the two taxa. There are however points of evidence to indicate that their sterility is of the same nature, but of a higher magnitude, as that found in intervarietal crosses of sativa (Hu, 1960a). Oka (1968) has also inferred that the sterility is of a haplontic nature and is controlled largely by a complementary genic system. In these respects, the relationship between the two species can be compared to that of sibling species. Ramanujam (1938a) reported that the cross sativa-glaberrima was highly fertile, but this high fertility has not been corraborated by other workers. Morinaga and Kuriyama (1957) studied 13 hybrids and the percentage of stainable pollen grains ranged from 0.4 to 28.9 only. Nayar (1958) obtained less than 1% pollen stainability in five hybrids. The values obtained by Bouharmont (1962a) ranged from 2.4 to 14.0%. Morishima et al. (1962) crossed 21-39 varieties of glaberrima with five test strains of sativa and two of glaberrima. They obtained a maximum

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of 10% pollen stainability with most crosses setting no seed. At the same time, the hybrids show a fair amount of embryo sac fertility, which makes backcrosses possible (Seetharaman, 1962; Chu et al., 1969a). Such backcrosses have been in fact obtained by other workers (Morinaga and Kuriyama, 1957; Jachuck and Hakim, 1966). iii. Cytology of hybrids. Meiosis in hybrids between sativa, glaberrima, and barthii proceeds normally according to several reports (Morinaga and Kuriyama, 1957; Richharia, 1960; Nezu et al., 1960). Minor abnormalities of the nature of occasionally two univalents or lagging chromosomes have been reported by others (Peltier, 1953; Bourharmont, 1962a; Li et al., 1962; Chu et al., 1969a). However, Nayar (1958) observed single quadrivalents and up to 8 univalents in about one-fifth of cells studied (also see Jachuck and Hakim, 1966). Yeh and Henderson (1962) found univalents in high frequencies. The formation of univalents in higher frequencies in certain combinations was determined to be due to desynapsis and this might be genically controlled (Misra and Shastry, 1969). These extensive and independent studies show that the frequency and range of abnormalities obtained in these interspecific crosses (between sativa, barthii, and glaberrima) is not any more extensive than that found in interracial crosses of sativa (cf. Morishima et al., 1962; Chu et al., 1969a). The comparative studies made on chromosome morphology and meiotic pairing in haploids of the two cultivated species have given further evidence on their very close relationship. Hu (1960a) found their karyotypes to be similar and also that meiotic pairing in their haploids did not show any significant difference. Bouharmont (1962a), however, observed differences in chromosome length. The lengths of haploid sets of somatic chromosomes in rujipogon perennial was 16.7 =!Z 0.3 p, in sativa it was 17.0 k 0.4 p and in glaberrima it was 13.8 5 0.3 p. But variations of higher magnitude have been reported for varieties of sativa (Hu, 1964; Misra and Shastry, 1967). The number of chiasmata per bivalent at diakinesis was estimated to be 1.98 0.65 in sativa, 1.70 f 0.50 in sativa-rufipogon perennial hybrid, 1.83f 0.59 in glaberrima-sativa hybrid and 1.23 2 0.69 in sativa-barthii hybrid (Chu et al., 1969a). These data again show that the two cultivated species are closely related. The cytology of two amphidiploids glaberrima-sativa and sativa-barthii sativa-barthii (Gopalakrishnan et al., 1964) points out similar affinities between them. The F, plants were sterile but both the amphidiploids showed 75% pollen stainability. However, the seed set was different; the former gave 47% seed set and the latter 13%. Meiosis in the two amphidiploids was similar. Both showed 0-12 quadrivalents in their PMCs with two modes a t 6 and 8. This kind of sterility is considered

*

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to be haplontic resulting from abnormal segregation of chromosomes or genes, and it has been designated “segregational” by Stebbins (1958a, 1970). Oka (1968) studied a tetraploid hybrid of glaberrima and sativa produced by crossing their autotetraploids. The hybrid showed a lower number of quadrivalents than the parents (by 1.6 IV to 4.8 IV) . A similar degree of quadrivalent reduction was found in hybrids of autotetraploid sativa varieties (Oka et al., 1954). From genetic studies, Oka inferred the occurrence of preferential pairing, but it was difficult to estimate its extent because of gametic selection. He concluded that small structural differences may also be present in their chromosomes. iv. Genetic studies, Only a couple of genetic studies have been made in glabem‘ma. Richharia and Seetharaman (1962, 1965) found that the two species show allelic relationships for some characters, but not for some others. Morishima et al. (1962) studied the correlations between 12 characters in both the species. While the characters differentiating the two species such as ligule length and spikelet hair length, were correlated with other characters in a different manner, correlations for others were broadly similar in both species. The authors concluded that they have a similar genetic constitution. v. Conclusions. Thus, all evidence-morphological, physiological, cytological, and genetic-indicates that the two species sativa and glaberrima are intimately and directly related. Some earlier workers were also aware of a close relationship between them, but they explained it either as being the result of having a common ancestor-in perennis, a superspecies that included longistaminata of Africa and also the wild rices of America and Asia, or, as an instance of homologous variation. We have seen that there is no evidence to indicate that longistaminata could have been a direct ancestral species of g l a b e m h a . Many of those who took the latter explanation believed in the origin of glaberrima from barthii. This implies that with all the differences present in the two putative ancestral species of the respective cultivated rices, only a remote common ancestry can be inferred, and this would then mean stretching the law of homologous variation far and thin. An origin for the two cultivated species from an immediate common ancestor is another possibility. But since sativa has originated from the Asian perennial wild rice, rufipogon perennial, it would imply that the latter species is present in tropical west Africa. This implies then that it has either not been collected or has now become extinct. Since rufipogon perennial occupies a comparatively safe habitat and such habitats are widely present in tropical west Africa, and since a number of Oryza species are already present in tropical Africa, the possibility of this taxon having become extinct becomes improbable.

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A somewhat oblique evidence for the evolution of glaberrinaa from Asian rices, cultivated or wild, can be derived from the report of an invalidly published species, 0. jeyporensis, collected from the Jeypore tract of southeastern India (Govindaswamy and Krishnamurthy, 1958b). The Jeypore region was originally proposed as a secondary center of origin of Asian rice by Ramiah and Ghose (1951). Evidence for the evolution of sativa-forms and for their further differentiation into races has been obtained (Govindaswamy and Krishnamurthy, 1958a; Oka and Chang, 1962). According to Govindaswamy and Krishnamurthy (1958b), the taxon jeyporensis resembles glaberrima most. Sampath (1962) felt that it resembled barthii (his stapfii) and Tateoka (1963) considered that it “is close to the wild plants related to cultivated rice.” If the discovery of this taxon is substantiated, it may imply that under certain environmental conditions, forms resembling glaberrima can evolve from either or both of the primary taxa present there, vizi, rufipogon perennial and sativa. 6. Time of Origin: Reconsideration

It is generally believed that the Asian rice sativa was introduced into tropical west Africa by the Portuguese from the early 16th century on (PortBres, 1959; Oka and Chang, 1964a; Angladette, 1966; and others) ; but there is evidence that it arrived first at a much earlier time (Nayar, in press, a ) . Rice has not been discovered from any of the ancient Neolithic sites of the Middle East (cf. Ucko and Dimbleby, 1969). The conditions of the Lower Nile Valley are ideal for the culture of rice and growth of wild rices, but evidence for their presence in this region in ancient times is lacking. There is only one mention of rice in connection with Ancient Egypt: about rice straw having been used as a binder in plaster used on a bronze figure. Only the picture of this statuette is available now, and from this, neither it.s date nor the presence of rice straw can be determined (Dixon, 1969). Alexander the Great (4th century BC) introduced rice into Egypt after his invasion of the Indian subcontinent (Roschevicz, 1931). However, rice is generally thought to have been cultivated in Egypt by the Arabs beginning about the 7th century (Roschevicz, 1931; PortBres, 1959; Murdock, 1959). Angladette (1966) has given an earlier date for the introduction of rice into Egypt, vie, from 4th century BC to 1st century AD. The Neolithic culture that arrived in Egypt in the fifth millenium BC spread westward along the Mediterranean coast by the fourth millenium BC. A close association between Egypt and northern Africa has been maintained since then. By the third millenium BC, the Berbers,

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who were among the earliest inhabitants of northern Africa, had initiated trade across the Sahara with the south, Soon it developed into an active occupation, trading materials and accepting and exchanging culture in the process. The trade routes crisscrossed the entire Sahara, but four main caravan routes were recognized, and the southern terminus of one of these was Timbuctoo in Mali, near the headwaters of the river Niger. Rice was one of the crops grown in the oases on these routes. Therefore, the knowledge of Asian rice could have come to this region not much later than it came to be known in Egypt. Even if Angladette’s (1966) earlier chronology is not accepted, this could be a t about the 7th-10th century AD. I n addition, there is also evidence that the Indonesians and Malaysians had arrived on the coasts of tropical west Africa in the early centuries of our era carrying with them their food crops (Murdock, 1959; Jones, 1959; Christie, in Gray, 1962). Since rice is a staple food crop in southeast Asia, inferentially rice could also have been one of the crops that they introduced during this period. There is thus a good probability that Asian rice was introduced into tropical west Africa sometime before the 10th century AD, either from the north or the south or both. Some of the characteristics shown by African rice are not those of a crop of ancient origin. It does not, for instance, possess the adaptability and qualities expected of an indigenous crop plant of long evolutionary history even if it is granted that agriculture in tropical west Africa is less developed than in the monsoon area of Asia. The Asian rice is steadily replacing glaberrima because of the better adaptability, higher yield, and improved qualities of the former (PortBres, 1959, 1962; Oka and Chang, 1964a; Angladette, 1966). The African rice shows a significantly lower resistance to drought and a higher degree of seed dormancy than sativa, and the two rices do not show much difference from each other in certain other agronomic characters (Morishima et al., 1962). 6. Mode of Evolution

In its distribution, gross morphology, and hybrid sterility, glaberrima shows the characteristics of a sibling species with sativa (Nayar, in press, a ) . I n its limited area of distribution, narrower range of variability, lower adaptability, and diminution in certain plant organs (short anthers, short ligules, no secondary branches in panicles), it shows the features of a daughter species derived from sativa. Sibling species are “sympatric populations that are morphologically similar or identical, but are reproductively isolated” (Mayr, 1963). They are considered as valid species. Several instances of sibling species are known (cf. Dobzhansky, 1951, 1970; Mayr, 1963). The best known in-

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stance are of Drosophila pseudoobscura and D. persirnilis in animals and of the Gilia transmontana group in plants (cf. Grant, 1971). The mode of evolution of glaberrima may have been of the nature of quantum speciation, but the actual pathway will have to be determined. Quantum speciation is defined “as the budding off of a new and very different daughter species from a semi-isolated peripheral population of an ancestral species in a cross-fertilizing organism” (Grant, 1963, 1971). It is a concept that involves the blending of several parallel concepts introduced by earlier authors as haphazard local racial variation in species containing semi-isolated populations (Gulick) , genetic drift (Sewall Wright) , aberrant characteristics in peripheral populations and genetic revolution (Mayr) , macrogenesis (Jepsen), quantum evolution (Simpson), and catastrophic selection (Lewis). This mode of speciation takes place more rapidly than geographic speciation. When the concept is introduced into plants, the effects of inbreeding that is necessary for the isolation of the progenitors of the deviant daughter species can be easily, and therefore more often, achieved by self-fertilization. This phenomenon has been observed to take place in plants by many workers (Lewis, 1962, 1966; Runemark, 1970; Levin, 1970b; Grant, 1971). As we have seen already, the differentiation of glaberrima may have involved both genetic changes and cryptic structural differences of chromosomes. 7. Origin of barthii The best evidence for the proposed hybrid origin of this taxon from sativa and glaberrima comes from their artificial hybrids themselves. The taxon barthii is characterized by big, bold, hairy spikelets with stiff awns, easy shedding nature, short ligules, short anthers, and panicles with secondary and tertiary branches. The F, hybrids between sativa and glaberrima show invariably varying degrees of hybrid vigor, bolder and hairy spikelets with dense awns, easily shedding nature, ligules of intermediate length between the parental taxa, and panicles with secondary and tertiary spikelets (cf. Morinaga and Kuriyama, 1957; Nayar, 1958; Seetharaman, 1962; Morishima et al., 1962; Joseph and Raman, 1966). The dense awns and deciduous nature of spikelets are shown by hybrids even when the parents do not possess these characters. Morinaga and Kuriyama (1957) have given the most complete descriptions of parents and F, hybrids. The hybrids are completely or very highly pollen sterile, but show about 30-50oJo embryo sac fertility (Oka, 1968; Chu et al., 1969a). Consequently, back crosses are successful and sterile F, plants often set a few seeds under open pollinating conditions (Morinaga and Kuriyama, 1957; Seetharaman, 1962; Jachuck and

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Hakim, 1966). The near-complete fertility of crosses of barthii with glaberrima and its sterility with sativa lend support to the proposition that it developed as a result of repeated backcrosses of sativa-glaberrima hybrids to glaberrima. This could have been made possible by a preponderance of glaberrima and a rare occurrence or even an absence of sativa following its transformation (cf. Mayr, 1963) into glaberrima, and possibly before sntiva was reintroduced by the Portuguese in the 16th century AD. The significant amounts of interpopulational variability present in this taxon and the frequent occurrence of populations intermediate between barthii and glaberrima (Morishima et al., 1963; Oka and Chang, 1964a) suggest that this process is continuing. The role of hybridization in the production of new taxa is well known, as this has been discussed by several workers (Anderson, 1949, 1953; Heiser, 1965; Anderson and Stebbins, 1954; Stebbins, 1950, 1959, 1970; Levin, 1970a; Grant, 1963,1971). IV. Subspeciation in Cultivated Rices

Coincident with the great amount of variation shown in their morphology and adaptation, cultivated rices display also certain broad features in sterility relationships between varieties of different geographical regions; only that their distinctions are blurred because of man’s domineering influence in directing their evolution. The process is less defined and is also less worked out in African cultivated rice. A. ASIANRICE Two broad phases of work can be recognized, the first ending about the middle of the 1950s during which time the work was directed primarily toward studying the extent of this phenomenon and classifying it, and the second phase ending about a decade later, when the work was concerned with studying the nature of differentiation. 1. Classification

There have been attempts to classify varieties on the basis of morphology, sterility relationships, and geographic distribution. These have been briefly reviewed by several workers (PortBres, 1950; Ramiah and Rao, 1953; Morinaga, 1954; Nagai, 1959; Chandraratna, 1964; Angladette, 1966). The main classifications are summarized in Table 4. Koernicke in 1885 was the first to classify cultivated rices. He divided

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them into two classes, glutinous and nonglutinous, and subdivided them into 39 botanical varieties based on differences in spikelet size and color of pericarp and glume. The presence of sterility barriers between geographical races was first observed by Is0 in 1928, but the credit for the first comprehensive, and still the most widely accepted classification, should go to Kato and co-workers (1928, 1930). They studied the nature of sterility relationships and, on the basis of their data, divided the species into two subspecies, indica and japonica. They used about 100 TABLE 4 Subspecific Classification of sativaa Kato el al. (1928) Gustchin (1934) brevis communis Terao and Mizushima (1939) Matsuo (1952) Wagenaar et al. (1952), after Van der Stock and Van der Muelen Porthres (1950, 1956) brevis Series brevipilosa communis Series pilosa Series nuda Oka (1958) Center of distribution a

japonica

japonica Groups Ia, Ib

indica

Ic

indica Groups 11, 111

B bulu

C tjereh

breviindica

brevijaponica eujaponica nudijaponica Type Temperate Insular (IIb) Japan

indojaponica nudindojaponica Tropical Insular (IIa, IIab) Java (Indonesia)

euindica nudindica Continental (Ia, Ib) India

Adapted from Table XI11 of Angladette (1966).

cultivated varieties representing different geographical regions, upland and lowland rices, glutinous and nonglutinous rices, normal and longglumed rices, scented and nonscented rices, and also rices with red and white pericarp. Most varieties from India, Ceylon, Java (Indonesia), southern China, and Taiwan belonged to the subspecies japonica. Some varieties from central China, the United States, Taiwan, and Brazil could not be classified. The hybrids between varieties within japonica and within indica showed over 50% fertility. The japonica-indica hybrids were characterized by low levels of fertility, ranging from zero to 3376, the average values in two experiments being 5% and 14%.

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They found also some morphological differences between the two subspecies. The work in the next few years (Jones, 1930; Gustchin, 1934; Terao and Mizushima, 1939; Matsuo, 1952; Morinaga, 1954; Oka, 196413, and earlier papers) mainly confirmed the above findings, but it also showed that the distinctions are not as clear-cut as originally envisioned. Several varieties were intermediate between the two subspecies. Terao and Mizushima (1939) recognized, in fact, three main groups, I, 11, and 111. ,Group I was further divided into three subgroups, I a comprising varieties that were not interfertile with I1 or 111, I b containing varieties that were fertile with 11, and Ic containing varieties that showed fertility with both I1 and 111. Matsuo (1952) made extensive studies using 1409 varieties. Employing 22 morphological and ecological characters, he classified them into 43 types, which were then grouped under 3 plant types, A, B, and C. Grain shape was used as a distinguishing character. Type A forms (possessing short grains 7.00 mm long and 3.37 mm broad) came from Japan, Korea, Manchuria, north China., and Africa; type B forms (grains 8.30 mm long and 3.39 mm broad) came from Indonesia (Java), the Philippines, Europe, and the United States, and type C (grains 7.93 mm long and 2.97 mm broad) represented varieties from India, Indochina, and south and west China. I n a series of papers, Oka (1953a,b,c,d, 1954d, 1955a, 1956b, 1957a,b,c,d,e, 1958, 1960, 196413) tried to elucidate the nature of the phylogenetic differentiation in cultivated rice. He chose 120 varieties from throughout Asia and studied them for 12 characters which included some new responses as phenol reaction, resistance to potassium chlorate, and alkali damage to the endosperm. Nowadays, rice workers recognize three taxa in scxtiva: indica, japonica, and javanica; their status-whether subspecies or races-is undefined. Their chief characteristics are given in Table 5. 9. Nature of Differentiation

There was some interest in the 1950s to study the nature and inheritance of sterility in interracial hybrids, mainly because of the availability of a large amount of experimental material from an international rice hybridization project operated by the F A 0 for south and southeast Asian countries for developing varieties combining desirable features of different races. However, the results have not come up to the expectations. There are two broad lines of opinion on the nature of subspecific differentiation in sativa, chromosomal and genic. A number of reports have been published on the occurrence of sterility in F, hybrids of interracial crosses and also on their mode of segregation and heritability in succeeding generations (Hsu, 1945; Kuang and Tu,

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1949; Kuang, 1951; Wagenaar, van Schouwenberg and Siregar, 1952; Morinaga and Kuriyama, 1954; Subramaniam and Roy, 1956; Katsuo and Mizushima, 1958; Misro and Shastry, 1959-1960; Nagamatsu and Omura, 1958, 1960; Sampath, 1959, 196413; Richharia et al., 1961; Ru and Kung, 1963a; Henderson, 1964b; Shastry, 1964c; Sen, 1964a; Jennings, 1966a,b ; Rao, 1964, 1965, 1967a,b ; Raj, 1968). Their main findings are given below: (1) Sterility of F, hybrids ranges from 1-99%, (2) Pollen stainability and spikelet fertility show significant correlation. TABLE 5 Characteristics of sativa Races= Character 1. Grain shape 2. Length of 2nd leaf blade 3. Angle between 2nd leaf and stem 4. Texture of plant parts 5. Angle between flag leaf and stem 6. Flag leaf 7. Tiller number 8. Tiller habit 9. Leaf pubescence 10. Glume pubescence 11. Awns 12. Shattering 13. Panicle length 14. Panicle branching 15. Panicle density 16. Panicle weight 17. Plant height

A: japonica

B: javanica

C: indica

Short Short

Large Long

Narrow Long

Small Hard

Small Hard

Large Soft

Medium Short, narrow Large Erect None Dense Usually absent Difficult Short Few High Heavy Short

Large Long, wide Small Erect Little Dense Usually present Difficult Long Many Moderate Heavy Taller

Small Long, narrow Large Spreading More Sparse Usually absent Easy Medium Intermediate Moderate Light Tall

Modified from Matsuo (1952) and Chandraratna (1964).

(3) Cytoplasmic factors play no significant role in causing sterility. (4) F, hybrids show heterosis, but its expression varies with the character. ( 5 ) F, populations show wide and continuous segregation for fertility. (6) F, segregation ratios are generally normal and undisturbed by degree of sterility. (7) There is a high correlation between the fertility of the parent and its progeny beginning with the F, generation. (8) Selection for sterile lines is as effective as for fertile lines. (9) Gametes generally deteriorate at the first division stage of haploid nuclei. (10) Hybrid weakness may be seen in a small number of crosses. Con-

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trary findings have been reported for finding (3) by Sampath and Mohanty (1954), Nagamatsu and Omura (1958), Kitamura (1962), and Shastry (1964b), for (6) by Oka (1964b), for (7) by Ru and Kung (1963a), and for (8) by Hsu (1945). It is clear from these findings that sterility in interracial hybrids is of complex nature as it does not show the characteristics of a typical qualitative or quantitative character. a. Cytological Studies. Highly variable and contradictory results have been published by various workers. Kato et al. (1930) found that meiosis in interracial hybrids was normal. Kuang (1951), summarizing the work done in China, also reported normal meiosis except for some univalents. Hsieh (1957) and Hsieh and Oka (1958) found also no disturbance in chromosome pairing in several hybrids. Occasionally, univalents, stretched chromosomes, and anaphase bridges were found. The authors attributed them to precocious separation or reunion of sister chromatids after a break a t the diplotene stage. Mello-Sampayo (1952) and Sampath and Mohanty (1954) found low frequencies of bridges and fragments indicating the presence of paracentric inversions. Sampath (1959) and Venkataswamy (1963) found quadrivalents in certain crosses which showed the presence of reciprocal translocations. They were also able to isolate homozygous semisterile lines which showed one (and rarely two) quadrivalents in 18% of the PMCs. Velasco-Demeterio et al. (1965) obtained essentially similar results. They saw univalents, quadrivalents, and anaphase bridges in low frequencies in some crosses (cf. also Engle et al., 1969). Meiotic analyses in the pachytene stage were done by Yao, et al. (1958) for the first time. They observed loops, which were interpreted as resulting from inversions, in less than 10% of the cells examined in 5 out of 7 crosses. Diakinesis and M I were normal. I n further studies, Henderson et al. (1959) obtained bridges with fragments in 0.28% of A1 cells in 9 out of 12 combinations. Anaphase bridges without fragments were noticed in both parents and hybrids. The authors concluded that a genetic basis for the cause of sterility was improbable, and that instead it could be attributed to cryptic structural differences caused by inversion of an included type (Henderson, 1964b). Pachytene analyses were repeated by Shastry and Misra (1961a,b). Meiosis was highly abnormal, showing the presence of inversions, translocations, deletions, and differential segments. In three semisterile hybrids, up to 31% of the chromatin length was unpaired. M I and A1 stages were normal. They proposed that the main cause of sterility was cryptic structural differences of the chromosomes caused mostly by translocations (Shastry, 1962, 1 9 6 4 ~ ) . I n contrast to these findings, Wu et al. (1964) found a pairing abnormality only once during pachytene analyses of four hybrids. Basak and

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Ghosh (1966) also observed considerable pairing anomalies in hybrids involving indica and javanica varieties. Studies on the fertility and meiosis of autotetraploids and tetraploids of intra- and interracial hybrids have also provided evidence for structural differentiation of chromosomes in different races of sativa. The F, fertility becomes higher and chromosome pairing becomes more regular than in the autotetraploid parents (Cua, 1951b, 1952; Oka et al., 1954; Masima and Uchiyamada, 1955a,b). The frequencies of multivalents are also inversely correlated with fertility. Oka (1954c, 1955a,b) however, did not consider that preferential pairing took place in these polyploid hybrids because he obtained polysomic ratios for some characters. Further, the F, genotypic ratio for glutinous endosperm showed a significant deviation from the expected ratio. It was found to be due to certation in favor of nonglutinous pollen. He also thought that heterosis could account for this high fertility. b. Genetic Studies. Hsu (1945) proposed that complementary genes were responsible for sterility in interracial hybrids. Kuang and Tu (1949) found that the extent of F, sterility in japmica-indica varietal hybrids did not correspond with their degree of morphological and varietal differentiation. It showed some relationship with the date of heading. They were unable to determine the genetic basis of sterility. Oka (1953b,c, 195613, 1957a,b, 1964b) and Oka and Doida (1962) made extensive genetic studies and postulated four kinds of gene action: (1) complementary dominant lethals to account for the (rare) occurrence of weak F, hybrids, (2) gametic development genes to account for the (common) occurrence of F, sterility in which genes in complementary recessive conditions interfered with gamete development in F, hybrids, (3) complementary recessive lethals to account for the (occasional) occurrence of weak plants in segregating generations, and (4) duplicate fertility genes to account for the (widespread) sterility in segregating generations. Siddiq and Swaminathan (1968a,b) were of the opinion that disturbed genetic coherence, rather than chromosomal differences, was the cause of semisterility and skewed segregation ratios in indica-japonica hybrids. No simple explanation can then account for the wide occurrence of sterility in interracial hybrids. As several workers have proposed, cryptic structural differences of chromosomes may account for much of the sterility in interracial hybrids of sativa. The genetic basis of sterility “may represent rearrangements of chromosomal segments which are so small that they contain perhaps only one to five genes” (Stebbins, 1958a). Genetic and cytogenetic studies may have to be carried out in the same set of hybrids to determine the relative role of each factor in causing sterility.

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3. Origin of Differentiation

The races designated as japonica and indica represent, in a way, modes in a continuous spectrum of variation, and more varietal groups, corresponding roughly to ecotypes or ecological races, may be distinguished. For instance, rices of eastern India are known as aus, aman, and boro varieties, those of Indonesia are divided into bulu and tjereh varieties, and of China into Keng and Hsien (Sen) varieties. Earlier, it was assumed that japonicas were evolved from indicas, or that both originated independently (Jones and Longley, 1941; Chou, 1948). Later, when fertility relations among these varietal groups or ecotypes became better known, Morinaga and Kuriyama (1955, 1958) proposed that sativa rice might have differentiated near its center of origin into two main seasonal ecotypes by mutations, and that japonica rices might have developed from aus rices (also, Morinaga, 1968). An introgression into indicas of perennis present in southern China and Taiwan-which is insensitive to photoperiod-was suggested to have contributed to the thermo- and photoperiod responses of japonica rices (Sampath and Seetharaman, 1962). The isolation of chemically induced mutant types in a japonica and an indica cultivar possessing certain characters of the other race prompted Siddiq and Swaminathan (1968a) to suggest that differentiation did not involve a systemic or macromutation, but that it proceeded through a series of independent mutations affecting grain and plant characteristics, which were brought together probably under the influence of disruptive selection. The finding of Siddiq and Swaminathan may incidentally indicate that a t least some of the distinguishing features of the two races are controlled by duplicate or complementary genic systems. Rice types possessing varying degrees of japonica characters have been collected from the Jeypore tract in southeastern India (Govindaswamy and Krishnamurthy, 1958a; Oka and Chang, 1962), the foothill regions of Nepal (Hamada, 1956; Nakao, 1957) and Sikkim (Kihara and Nakao, 1960; Kihara and Katayama, 1964) and from northern Thailand (Oka and Chang, 1963). With the aid of discriminant formulas, Oka and Chang (1962, 1963) showed that wild forms from the Jeypore area and from Thailand are gradually differentiating into japonica and i n d i m types. This study also furnishes more definitive evidence for a monophyletic origin of sativa. Since responsiveness to temperature was not the primary selection force that differentiated the races, Oka and Chang assumed that a primitive cultivated form arose in India, Burma, or Thailand by some evolutionary force and from the genetic variability released by the adoption of self-fertilization, the

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203

japonica and indica rices further differentiated under hill and plains conditions in these regions. It has been indicated already that sativa may have evolved through disruptive selection, and that the origin was most probably diffuse both in a spatial and a temporal sense. Evolution might have resulted in the formation of only one dominant ecotype or race in some areas of origin. Whereas, in some other regions where more than one varietal group or ecotype coexist, each adapted to different conditions of photoperiod and land type [as aus, aman, and boro of India, or bulu and tjereh of Indonesia; cf. Chandraratna (1964) for their morphology and characteristics) 1, this ecotypic differentiation could have come from the accumulation of gene mutations affecting perhaps primarily their physiological characteristics such as thermo- and photoperiod responses. Selection would, of course, have been based on their form, yield, uniformity, and adaptation to the environment. This is being effected in the Jeypore tract (Govindaswamy and Krishnamurthy, 1958a ; Oka and Chang, 1962). Both japonica and indica varieties appear either together or, more commonly, in the same region, but a t different altitudes in the hills of the Jeypore area, Nepal, Sikkim, and northern Thailand (Hamada, 1956; Nakao, 1957; Govindaswamy and Krishnamurthy, 1958a ; Kihara and Nakao, 1960; Oka and Chang, 1962, 1963). It implies that racial differentiation is taking place simultaneously. Disruptive selection may bring about either isolation or polymorphism (Mather, 1955). Starting from a polymorphic population, subspeciation can take place in a heterogeneous environment with individuals adapted to different ecological niches, particularly if they are protected from an inflow of alien genes (Mayr, 1963; Maynard Smith, 1966). The mountainous terrain of these regions then obviously provides an ideal setting for the racial differentiation of sativa. The acquisition of self-fertilization (by a process yet to be determined) could have occurred earlier or even simultaneously and would have furnished a good measure of protection or isolation from alien genes.

B. AFRICANRICE Only little information is available on speciation processes and variability. Three centers of varietal diversity are known, a primary center of varietal diversification in the upper delta region of the river Niger, a secondary center of variation a t Nioro du Rip on either side of the Gambia River, and an incipient center in the Guinea mountains (Portbres, 1945, 1956, 1962). About 1500 cultivars have been collected in this cultivated species. These have been divided into 13 botanical varieties on

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the basis of morphological characters (Porthes, 1956). No evidence of sterility barriers within the species has as yet been obtained (Morishima et al., 1963; Chu et al., 1969a). V. Species Relationships

A. SUBGENERIC CLASSIFICATION Baillon classified the genus in 1894 (cf. Chevalier, 1932). It has subsequently been revised several times (cf. Table 6). Four of these revisions were based on morphological characters. Ghose et al. (1956) used cytogenetic data also (see also Richharia, 1960). Morishima and Oka (1960) followed the numerical taxonomic approach developed by Michener and Sokal, and the results are broadly similar to those of Ghose et al. I n the two latest classifications (Tateoka, 1962a; Sharma and Shastry, 1965d), the genus was subdivided into sections and series, but this may have lost much of its relevance after the deletion of several species from the genus (cf., Table 1 ) . Many authors continue to use Roschevicz’s (1931) classification, but after splitting his section Sativa into two groups or sections: sativa and officinalis. In the genus, three large groups of taxa can be recognized. The first consists of the two cultivated rices and five closely related taxa (Group Sativa, Morishima and Oka, 1960). It is pantropical in distribution, and the wild taxa are generally gregarious in nature. All are diploids (2n = 24). The second group (Officinalis or Latifoliae) has an almost equally wide distribution. They seem to occur singly or scattered in small populations. They are mostly tetraploids and show the basic features of officinalis, a diploid taxon with a wide distribution in Asia. They will be discussed in three subgroups according to distribution (Asian, African, and American). The remaining eight taxa comprise two groups of five species (Granulata and Ridleyi subgroups), two species of undetermined relationships, australiensis and brachyantha, and one, schlechteri, that is known from a single herbarium specimen only. Two more taxa, jeyporensis and ubhanghensis, are listed in Table 1 (last column). The standing of the former has been discussed already. The taxon ubhanghensis is known from its description and figures only (Chevalier, 1951). It is possibly related to the African taxa of the Officinalis group (Sampath, 1962; Tateoka, 1963). Fairly detailed information about the taxa belonging to the group Sativa is available on their distribution, occurrence, ecology, range of variation and breeding, and cytogenetic behavior to enable us to make tentative inferences about their interrelationships. Just the opposite is the case with those taxa included under the Miscellaneous group.

TABLE 6 Subgeneric ClaasScation of the Genus Oryza

Taxa

Chromosome number (2n)

1. sdiaa 2. mfipogon 3. rufpogon, annual 4. longietaminatu 5. glaberrima 6. barthii 7. Stapfii

24 24 24 24 24 24 24

8. auetraliasis

48

9. 10. 11. 12. 13. 14. 15. 16. 17.

latifolio alta grandiglumis punctata eiehinueri schweinfurthiana ofieinalis malampurhenms minuta

18. brachyantha

19. ridleyi

21. schlecYeri

Not known

22. meyeriana

24 24, 4%

Not known

c

d

Morishima and Oka

Section Sativa

Sativa Group

Series Sativa

Intermediate

Miscellaneous Group

Series Aust.ral-, ieneis

NS Section Officinalis NS NS

NS Offioinalia Group NS

(1960)

Section

NSd

Euoryza

NS NS

NS NS

NS

NS

Section Coarctata NS

Section Sclerophyllum NS

Section Granulata

= T. T. Chang (1964, 19701, 2n = 48. b

(1932)

Section Saliva

24,%3* 24, 48' 48 24 48 48 24

Chevalier

(1931)

AFI

20. longiglumis

24. abroneitiana

Roschevicz

48 48

24, 48* 48

23. wand a t a

Authority Ghose et d. (l960), Richharia (1960)

Van, in Seshu and Karibasappa (1960), 2n = 24. Abraham, in Sampath and Rao (1951), 2n = 48. NS not studied by author.

Section Padia

Intermediate Miscellaneous Group NS

NS

NS Section Granulata

NS

NS

NS Officinalis Group NS

Tateoka (19628)

Section Oryza

Sharma, Shastry (1964)

Series Sativae Section Oryza

Series Latifolia NS Section Augustifoliae

P

3

Series Latifoliae NS Section Angustifolia

Section Ridleyanae Section Schlechterianae Section Granulatae

Series Auatraliensea

Section Padia

Series Brachyanthae

0

Series Ridleyanae Series Schleche enanae

M

Seriem Meyerianae

r

E

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N. M. NAYAR

B. INTERRELATIONSHIPS 1. Sativa Group

Most of the work has been discussed already. Practically all recent cytogenetic and taxonomic work treat the African perennial wild rice as a separate species, which, as pointed out by Clayton (1968), should correctly be called 0. Zongistaminata. The American form is approximately intermediate between Asian and African perennial wild rice. Detailed studies by Morishima (1969) and several others have not shown any absolute distinctness. Sterility barriers are only rarely complete, as was found by Yeh and Henderson (1961b), and even this may have been largely due to environmental factors. Mostly it ranges from partial to complete fertility (Morinaga and Kuriyama, 1956; Nayar, 1958; Li et al., 1962; and others). The real differences are in their geographical distribution and to a certain degree in their morphology. Pairing a t pachytene and later stages is generally normal, and only minor abnormalities like loosely paired segments, univalents, etc., are usually encountered in hybrids between different geographical races of perennial rufipogon (Misra and Shastry, 1966; Chu et aZ., 1969a). Morishima’s (1969) study of 65 collections by cluster analysis and pattern analysis showed the presence of four geographical groups-Asian, African (now recognized as 0. Zongistaminata) , American, and Oceanian. This may indicate that subspeciation is probably taking place. However, it seems adequate for the present to recognize them as semispecies or subspecies of one species. The annual wild rices are a vexing problem. They are intermediate between sativa and rufipogon perennial in morphology and habitat preferences. Evidence for their hybrid origin from these species has already been given. During the course of evolution of rice from perennial rufipogon, the intermediate forms would also have appeared morphologically similar to these. Today we see such intermediate forms in some areas, e.g., the Jeypore tract in southeast India, where the process of ennoblement can be witnessed. But then, they may occur only in cultivated fields under the direction and care of man’s selection. The odds against disruptive selection (the probable process by which sativa has been evolved) taking place in nature to make possible the evolution of such forms are simply very high (cf. Mayr, 1963; Maynard Smith, 1966). It is, therefore, questionable if such forms, which originate either as natural hybrids or as transient forms in cultivated fields, are to be given any taxonomic status just because they are present ubiquitously and also because they can be distinguished morphologically in a majority

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of cases. They do not possess any reproductive barriers with either sativa or perennial rufipogon. The specific status of barthii has not so far been questioned. But, in view of its similarities with rufipogon annual in its probable mode of origin, in its habitat preferences (as weeds of one of its putative ancestral species and in disturbed, hybrid habitats), and in its absence of any kind of crossability barriers with glaberrima, one of its putative parent species, some doubts could be raised about its specific status also. Like rujipogon annual, it also possesses distinctive morphological features, but unlike the former, barthii occupies also primary and stable habitats, shows much less semisterility, and more important, it has spread beyond the area of occurrence of glaberrima. It occurs throughout tropical Africa, from Mauretania through the Sudanese region to the country of Sudan, and also in Tanzania and Zambia (Clayton, 1970). It has, therefore, established itself as a stable entity and has shown itself to be capable of existing independently of its ancestral species. There is a report of a hybrid between sativa and Pennisetum species studied in the F, generation (Wu and Tsai, 1963). It was almost completely male and female sterile. The chromosome number was very variable, viz., 1 2 4 2 in somatic cells and 12-20 in PMCs. Meiosis was irregular. Further details are not available. 1. Officinalis Group

The Officinalis group consists of 10 t a x a - 4 occurring in Asia (collina, officinalis, malampuzhensis, and minuta) , 3 in Africa (punctata, eichingeri, and schweinfurthiana) , and 3 in America (alta, latifolia, and grandiglumis) . a. Asian Taxa. Of these, officinalis and collina are diploids (2n = 24) and malampuzhenis and minuta are tetraploids. The taxon officinalis is widely distributed all over south and southeast Asia-in India (Gujarat, peninsular, central, and eastern India), Nepal, Burma, Malaysia, Indonesia, Thailand, Cochin China, New Guinea, and the Philippines (Roschevica, 1931 ; Backer, 1946; Chatterjee, 1948; Capinpin and Pancho, 1961 ; Tateoka, 1963 ; Tateoka and Pancho, 1963 ; T. C. Katayama, 1963a; and others). The forms described as minuta from Sikkim (Hamada, 1956) and China (Kuang, 1951) are also most probably officina2is (also see Hu et al., 1965, for occurrence of officinalis in China). It is so similarhorphologically to minuta of Asia, punctata and schweinfurthiana of Africa, and latifolia of America that they have all been considered as conspecific by one or the other taxonomist (Hooker, 1897; Chevalier, 1932; Backer, 1946; Bor, 1960). It is a rhizomatous perennial t,hat prefers partially shaded forest areas, but occurs

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also in the open, in and by streams, in swamps, and sometimes even in paddy fields (T. C. Katayama, 1963a). Geographical forms show variations in morphology, and hybrids between them show differences in fertility ranging from zero to near normal. Meiosis in these hybrids is generally normal. About 10% of the PMCs may show 2-4 Is. Some hybrids may show one multivalent in up to 50% of the PMCs (Morinaga and Kuriyama, in Kihara, 1963; Gopalakrishnan, 1965a ; Hu and Chang, 1967). This sterility has been attributed to genetic causes (Hu and Chang, 1967). Gopalakrishnan (1965a) proposed subspecific status for them. The taxon collina ( 2 n : 2 4 ) is confined to Ceylon. Its taxonomic status has been a subject of controversy in recent years. It was originally described as sativa var. collina by Trimen in 1889 and then as latifolia var. collinu (Hooker, 1897). Subsequently, it came to be included in officinalis (Roschevicz, 1931; Senaratna, 1956; PortBres, 1956; Karibasappa, 1962). The controversy began after Bor (1960) and Tateoka (1962b) included it under the tropical east African taxon eichingeri. Then Sharma and Shastry (1965~)proposed a specific status for it (0. collina) because they felt that it was different from both officinalis and eichingeri. It appeared to them more as a dimunitive form of eichingeri. But eichingeri was then considered as an amphidiploid or segmental allopolyploid of the constitution BBCC. Being a diploid taxon, collina would then have the constitution BC if it is taken as eichingeri. They argued that the high fertility shown by collina was incompatible with this assumed constitution. However, the identification and constitution of eichingeri has lately been disputed (see below) and Tateoka (1965b) has reidentified this material and also that used by several workers in India and Japan as tetraploid punctata. Eichingeri is now conceived of primarily as a diploid entity. Sharma and Shastry’s (19654 arguments have, therefore, become largely redundant. Hybrids of collina with the other geographical races of officinalis showed identical behavior (Gopalakrishnan, 1966a) . The characters of this taxon such as nonrhizomatous condition, thin culm, narrow leaves and absence of pigmentation a t spikelet base which had prompted Sharma and Shastry (1965~)to elevate collina to a distinct species, were found by him to be within the range of morphological variation shown by officinalis. Therefore, he contended that it should be retained within officinalis as a subspecies. Sampath and Subramanyam (1968) also studied the status of this Ceylonese form. Hybrids of collina with officinalis (Assam, India) and eichingeri (diploid form, Uganda) showed 10-200/0 fertility and similar meiotic behavior. They suggested that collina was intermediate between officinalis and eichingeri, but preferred

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to keep it as a subspecies of eichingeri without giving any reasons. Studies of hybrids of collina and officinalis with other taxa as minuta, latifolia, and alta with a view to determining their genomic constitution have shown also that collina and officinalis possess the same genome C (Richharia and Seshu, 1961; Morinaga, 1940, 1941, 1943, 1964). Incidentally, genome C is basic to all taxa of the Officinalis group. It appears that Bor (1960), Tateoka (1962b, 1963), and Sharma and Shastry (1965~)made their propositions on incorrect or incomplete assumptions. Bor, for instance, by including collina in eichingeri and considering the latter as only a tetraploid implied collina also to be a tetraploid. It is known so far as a diploid only. Tateoka (196213) might have possibly relied on Bor’s (1960) judgment, for he did not give any reasons for including collina under eichingeri. Tateoka (196513) has implied that spikelet dimensions are the most important character to distinguish minuta, officinalis, eichingeri, and punctata (cf. also Sastry and Subramanyam, 1966). Then, collina (cf. Sharma and Shastry, 1965c, for spikelet measurements) will have to be included in punctata as defined by Tateoka (1965b). And it is a more widely distributed taxon than eichingeri. At the same time, the spikelet dimensions of officinalis given by Tateoka (196213, 196513) are much smaller than those given by T. C. Katayama (1963a) for 43 samples collected from Indonesia, the Philippines, and New Guinea. The differences cannot be ascribed to measurements having been made on highly desiccated herbarium sheets as used by Roschevica (1931), for the measurements of freshly collected samples from the Philippines given by Tateoka and Pancho (1963) are also smaller. It can, therefore, be inferred that the morphological variation shown by collina is within the variability range of officinalis. Cytogenetics and geographical distribution point out the affinity of collina with officinalis. Its variations may be attributable to i t being a peripheral isolate. It could also be a link between the Asian and the African officinalis taxa. The taxa malampuzhensis and minuta are both tetraploids (2n = 48). They are closely related morphologically and genetically to officinalis ( 2 n = 24). Both occur in very restricted areas in two widely separated peripheral regions of officinalis. Of these, minuta has been known for a long time. It has been recorded from the islands of Luzon, Leyte, and Bohol in the Philippines (Capinpin and Pancho, 1961; T. C. Katayama, 1963a; Tateoka and Pancho, 1963). The second taxon, malampuzhensis, was first reported in 1957. It has been collected so far from only two localities on the Western Ghats, near Coimbatore, in peninsular India (Krishnaswamy and Chandrasekharan, 1957, 1958). Both share similar habitats with officinalis.

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0. minuta (2n = 48) has been widely used in cytological studies. Okura (1937) and Nandi (1938) studied its hybrids with sativa and officinalis and observed 12 I1 12 I at meiosis. They thought that the chromosomes of officinalis were bigger than those of sativa and that minuta had equal numbers of small and bigger chromosomes. Therefore, they proposed that minuta was an allopolyploid of sativa and officinalis. Hybrids between sativa, minuta, and officinalis have been subsequently studied by several workers (Morinaga, 1940, 1964; Capinpin and Magnaye, 1951; Neeu et al., 1960; Shastry et al., 1961; Li et al., 1962; Kihara, 1963; Katayama, 196513, 1966b). It is generally agreed that the genome of officinalis (CC) constitutes one of the two genomes in minuta. Hybrids between sativa and minuta are produced without much difficulty, but there is considerable degeneration of sporogenous tissues. Morinaga (1940) observed mostly 36 I, with 1-3 11s being formed occasionally. Nezu et al. (1960) observed 0-9 I1 with a mean of about 5 11. Li et al. (1962) observed 1-5 I1 (mean 3 11) and remaining univalents. The genome constitution of minuta is designated as BBCC (cf. Morinaga, 1959, 1964) . Cytogenetic studies of malampuzhensis show that it is closely related to both officinalis and minuta. Crosses of this taxon with officinalis (Gopalakrishnan, 1962), latifolia (Kihara, 1963) , and punctata (Hu and Chang, 1965b) showed that it contains the basic genome C of officinalis.The hybrid between minuta and malampuzherasis showed 93% pollen sterility with hardly any seed setting, but meiosis was fairly normal with 42 out of 55 PMCs showing 24 Is (Gopalakrishnan et al., 1965). They proposed that the genome constitution of malampuzhensis is the same as that of minuta, via. BBCC. At the same time, Morishima and Oka (1960) found in their numerical taxonomic analysis of the genus that malampuzhensis showed a maximum correlation coefficient with granulata (0.696)-a taxon that is incidentally present in the same region-followed by officinalis (0.687), and minuta (0.640). I n the same study, minuta showed the highest correlation coefficient with officinalis (0.851), malampuzhensis (0.640) and granulata (0.633) following. Sampath (1962) speculated that minuta might either have evolved from eichingeri “by dispersal and evolution” and that malampuzhensis “could be a link between eichingeri and minuta” (sic) with the B genome originating in Africa or that different geographical races of officinalis represented different species possessing B and C genomes. The eichingeri that Sampath had in mind was most probably the same as that identified by Tateoka (see above) as tetraploid punctata. The first pathway appears too contrived to be probable and is also not compatible with the distribution patterns of the taxa. The second alternative will be discussed later.

+

ORIGIN A N D CYTOGENETICS OF RICE

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It is simpler to assume, and consequently more probable, that both malampuzhensis and minuta have arisen in situ by polyploidy from peripheral populations of officinalis. The facts that both are very restricted in their occurrence, occurring only on the borders of the distribution range of officinalis, and also that they possess the C genome of the latter, support this contention. The B genome is considered as a variant of the basic C genome. As Mayr (1963) has pointed out, “when a new species evolves, it is almost invariably from a peripheral isolate” (p. 513). Many instances of such speciation are known (cf. Mayr, 1963; Grant, 1971). There is a report on the possible occurrence of a similar tetraploid form from China also (Sampath and Subramanyam, 1966). Both the taxa minuta and malampuzhensis are reproductively isolated from officinalis, but they can be distinguished from each other only by a combination of minor morphological characters. Thus, their interrelationships are of the nature of sibling species. Some additional clues on the origin of m k t a and malampuzhensis may be drawn from the studies on interracial hybrids of officinalisand their tetraploids. Hu and Chang (1967) crossed seven geographical races of officinalis in most of the possible combinations. Out of 31 hybrids obtained, 11 showed 50-66% pollen stainability, 5 showed 3-976 stainability, and the remaining 14 were completely sterile. Two of the sterile hybrids showed “48 or more chromosomes . . . in some of their PMCs,” showing thereby that polyploid gametes can sometimes be formed under some stimulus (here, hybridization). An autotetraploid and interracial 85%. tetraploid studied by Hu (1967) showed high fertility-about Meiosis was fairly normal with an average of about 19 11s in M I in the autotetraploid. Anaphase I was more regular. This is quite unlike the expected behavior of autotetraploids. These studies indicate that the origin of minuta and malampuzhensis can be visualized as due to autopolyploidy or interracial polyploidy. But it needs to be determined whether there has been an introgression of alien genetic material into these tetraploid taxa as appears probable from the correlation studies of Morishima and Oka (1960). The taxonomic status of the three taxa has also posed problems. After 0. officinaliswas described (Watt, 1892), Roschevicz (1931), Chatterjee (1948), and Tateoka (1963) retained officinalis and minuta as separate species. On the other hand, Merrill (1923), Chevalier (1932), Backer (1946), and Bor (1960) considered them conspecific. Morishima and Oka (1960) also obtained between minuta and officinalis the highest correlation between any two taxa in their study of 18 Oryza species. Tateoka (1963) kept them as separate species and treated malampuzhensis as a subspecies of officinalis. Tateoka and Pancho (1963) made

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detailed studies of 21 collections of minuta and 15 of officinalis. Although they showed the same mode for spikelet length, the main distinguishing character used by Roschevicz and Chatterjee, they felt that minuta and officinalis could be differentiated by two other characters, viz, spikelet breadth and arrangement of spikelets on the lower branches of inflorescence. A reexamination of their data shows that variation is continuous for spikelet breadth but with two modes, and the variation is overlapping with regard to the latter character (their Fig. 2 scatter diagram has only one variable). Even otherwise, it was somewhat arbitrary to treat minuta as a separate species and malampuzhensis as a subspecies. T. C. Katayama’s (1963a, 1971) data on spikelet dimensions and several other panicle characters also show that the two taxa cannot be distinguished by these characters. Considering the range of morphological variation, the pattern of distribution and the cytogenetic relations of the three taxa, it seems more appropriate to treat the two tetraploid taxa minuta and malampuxhensis as subspecies of the diploid species. The correct name of the latter will be 0. minuta. This proposal is in consonance with the current biosystematic treatment of polyploid races which are morphologically distinguished with difficulty only (Davis and Heywood, 1963; Lewis, 1967; de Wet, 1971; see also Love, 1964, for a dissenting opinion). A good example of this is the proposal of Dodds (1962) to include all the cultivated potatoes, diploids, triploid, tetraploid, and pentaploid, under one species Solanum tuberosum. b. African Tma. The three taxa are punctata, eichingeri, and schweinfurthiana. Another related taxon is ubanghensis. This has been discussed earlier. Only limited information is available on habitat, distribution, and breeding behavior of these taxa. They show considerable morphological and cytogenetic affinities among themselves and also with the Asian and the American taxa belonging to the Officinalis group. This has been responsible for some confusion in the cytogenetic literature. Two tetraploid samples which have been widely used in cytogenetic studies as eichingeri has been identified by Tateoka (1965b) to be tetraploid punctata. The taxa are distinguishable by a combination of characters only, consisting usually of differences in length of awns, ligules, and spikelets. Actually, published data show a continuous variation with one or more modes representing the three taxa. This is so between officinalis and latifolia also. After studying a number of samples, Tateoka (1965a) found that punctata could be distinguished from eichingeri by soft, whitish ligules (vs. hard and yellowish in eichingeri) , spreading panicle branches (vs. nonspreading) , and acute and triangular sterile lemmas (vs. acuminate and narrowly triangular). The culms are generally somewhat spongy in punctata and hard and thin in eichingeri,

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but this varies to some extent (Tateoka, 1965a). This is also known to be modified by environment, with hard stems developing in well drained soils and spongy stems developing in shallow, swampy and waterlogged soils. 0. punctata was described in 1854. It has the widest distribution of the three taxa. It has been collected from Ivory Coast, Ghana, Dahomey, Nigeria, Angola, Congo, Ethiopia, Sudan, Kenya, Uganda, Tanzania, Malagasy, Rhodesia (Roschevicz, 1931; Chevalier, 1932; Tateoka, 1963; Oka and Chang, 1964a; Clayton, 1970), Swaziland (Parker, in Sampath, 1969b), and Chad [a wild Oryza sample collected by Oka and Chang (1964a) and identified by Sampath (1969b) 1. Since schweinfurthiana had for some time been considered as a synonym of punctata (cf. Chatterjee, 1948; Tateoka, 1963), part of this distribution-probably from tropical west Africa, Congo, and Rhodesia-may represent the distribution of schweinfurthiana. Punctata has been reported to occur in Cochin China (Chevalier, 1932) and Thailand (Clayton, 1970), but this, like the reported occurrence of oficinalis in Dahomey (Roschevicz, 1931), is evidently attributable to misidentification resulting from the close morphological similarities existing among these taxa. Roschevicz (1931) has reported that in lean years its grains are harvested by people for consumption. Chevalier and Roehrich (1914) and Chevalier (1932) considered punctata to be conspecific with oficinalis of India and, consequently, designated it as a subspecies of 0. minuta. Subsequent taxonomists have treated it as an independent species. 0. eichingeri was first described in 1930. Roschevicz’s (1931) and Chevalier’s (1932) revisions of the genus do not include it. Later taxonomists have retained it as a separate species. Prodoehl (1922) first described 0. schweinfurthiana. Roschevicz (1931) maintained it as a separate species. Following Chevalier (1932), subsequent taxonomists considered it synonymous with punctata (Chatterjee, 1948; Tateoka, 1963; Clayton, 1970). Bouharmont (1962a,b) used it as a valid tetraploid species in his cytogenetic studies. Gopalakrishnan (1965b) and Sampath (1966, 1969b) have also urged the revival of this name to include all African tetraploid oficinalis forms. It is a rhizomatous perennial and resembles oficinalis (Roschevicz, 1931; Bouharmont, 1962a,b). Sampath (1962) first suggested that it resembled tetraploid punctata (as eichingeri) and later Sampath, 1966, felt that it was “recognizably a blend of the two plant types (punctata and eichingeri) , though the grains closely resemble those of punctata [sic].” The three taxa are thus apparently difficult to distinguish, and intermediate forms resulting possibly from natural crosses may also be present (see below). Cytological studies in this group have suffered from lack of material

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and misidentification of certain samples used in earlier years. Living materials of punctata became available only in recent years (cf. Tateoka, 1965a,b). Two tetraploid samples widely used in experimental studies under the name eichingeri (by Morinaga and Kuriyama, 1960; Morinaga, 1959, 1964; Nezu et al., 1960; Li et al., 1962; according to Hu, 1970a; possibly also by Sampath and Rao, 1951; Rao and Seetharaman, 1955; Sampath, 1962; Gopalakrishnan, 196513) have been identified by Tateoka (1965b) as tetraploid punctata. Pathak (1940) reported eichingeri as a tetraploid species. Rao and Seetharaman (1955) found up to 8 IV in eichingeri (2n = 48, possibly tetraploid punctata) with an average of 4.3 multivalents. They proposed that this taxon might have evolved by autopolyploidy or segmental allopolyploidy. Morinaga and Kuriyama (1960) designated its genome constitution as BBCC (see below). Hu and Chang (1965b) found almost regular meiosis in what appears to be the same material. Bouharmont (1962a,b) found 1-3 IV in about 45% PMCs. T. T. Chang (1964, 1970) showed both eichingeri and punctata as having diploid and tetraploid forms. Tateoka (1965b) found all seven samples of eichingeri that he studied to be diploids (one of these, No. W1524, has been reported by Bu, 1970a to be a tetraploid) . I n punctata, five collections were tetraploids and two were diploids. One reason for obtaining both diploid and tetraploid numbers for punctata may be that Tateoka (1963) considered schweinfurthiana as synonymous with punctata. Cytogenetic data indicate that different isolating mechanisms are operating among the three diploid taxa. H u (1970a) found that hybrids of eichingeri with both punctata and officinalis were sterile. While there was normal pairing in the eichingeri-officinalis cross with about 11.6 I1 out of a possible 12 11, the eichingeri-punctata cross showed mostly univalent5 a t meiosis. The maximum number of bivalent5 was 7, and the average only 2:3 11. H e proposed that eichingeri and officinalis contained the same genome C and that the genome of punctata was partially homologous to those of eichingeri and sativa. Sampath (1966) also found that punctataeichingeri hybrids were sterile. Attempts to obtain hybrids between punctata and officinalis have not been successful so far (Katayama, 1967b). Hybrids between diploid and tetraploid taxa have been studied mainly with the object of determining their genomic consititution. Katyama (1967b) proposed that the genome constitution of diploid punctata was BB, because he observed 10-13 I1 in hybrids between diploid punctata and minuta (BBCC) and was unable to obtain hybrids between diploid punctata and officinalis (CC) . H u (1970a) found that both the African diploid taxa punctata and eichingeri gave about 10-12 I1 in crosses with

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minuta. Studies on hybrids between different tetraploid taxa of this group belonging to Asia and Africa have shown that they all have essentially the same genome constitution. Morinaga and Kuriyama (1960) determined that the genome constitution of the African tetraploid taxon (as eichingeri) is identical to that of minuta (BBCC) (also Nezu et al., 1960; Hu, 1970a). Hybrids of tetraploid punctata with the above tetraploid form showed about 73% fertility. About one-fourth of PMCs showed single multivalents (Hu and Chang, 1965b). Hybrids of tetraploid punctata with minuta and malarnpuzhensis (tetraploid taxa belonging to Asian Officinalis Group) showed identical behavior (Hu and Chang, 196513). Pollen stainability was less than 4% in both hybrids. During meiosis, about 20% PMCs showed normal division, about 30% showed 2 4 I and in the rest up to 8 I and a single multivalent were observed. The authors concluded that all three tetraploid taxa have the same genome constitution BBCC, but were differentiated by reciprocal translocations. From the above accounts of morphological and cytogenetic studies, it is seen that while the African taxa, punctata, eichingeri, and schweinfurthiana, show almost continuous morphological variation for most characters indicating intimate relationships, cytogenetic studies show that they are separated by strong sterility barriers. These are possibly genetic in nature. Tateoka (1965a,b) was able to distinguish punctata (including tetraploid schweinfurthiana) and eichingeri only by a combination of six characters. He found also that 6/50 living and herbarium samples he studied possessed combinations of characters of the two taxa. He assumed that the two taxa might be hybridizing introgressively in nature, since they occupy similar habitats. H u (1970a) compared two of these living intermediate samples with a diploid punctata-eichingeri hybrid and found both similarities and differences between them. But he did not consider them to be natural F, hybrids because they showed high fertility, while the artificial F, hybrid was sterile. But the possibility still remains that these intermediate plants are F, hybrids backcrossed to either of the parents. Though F, hybrids may be completely pollen sterile, they may still possess some embryo sac fertility to permit backcross progenies, as has been observed in sativa-glaberrima hybrids. Natural selection sometimes favors formation of strong sterility barriers between closely related sympatric taxa. This is well documented in Gilia (cf. Grant, 1963, 1971). A system in which complete pollen sterility is combined with partial embryo sac fertility in F, hybrids may help either of the parents to “ingest” small amounts of genetic variability from the other taxon in a gradual manner. The two diploid taxa show certain features that may throw some

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light on the nature of their mutal relationships. The taxon punctata is widely distributed in tropical Africa while eichingeri is confined to tropical east Africa, Both prefer swampy soils, but eichingeri occupies forest shades and punctata occurs in the open, by stream banks and pond margins (Clayton, 1970). The latter apparently shows more natural variation (cf. Tateoka, 1962b, 1965a; Hu, 1970a). Roschevicz (1931) observed that grains of punctata are collected by people in lean years for consumption. It is not known whether punctata occurs in bigger stands to make this more feasible. Incidentally, this taxon possesses the largest spikelets among the Asian and African taxa of the Officinalis group. Recently, it has become a serious weed of paddy fields in Swaziland, east Africa (Parker, in Sampath, 1969b). About their origin, Sampath (1962, 1966) proposed that punctata arose first by “slow variation” from Zongistaminata and the Officinalis group arose from it (punctata) by “geographical variation and speciation.” Then eichingeri originated from punctata “by saltation in peripheral area in east Africa” and schweinfurthiuna (that is, all African tetraploid forms of the Officinalis group) by hybridization between the two taxa followed by polyploidy. Since information on distribution, habitat preferences, longevity, and breeding behavior is scanty, any proposition will have to be necessarily speculative. We do not yet know of any area where all the three taxa occur together, though there is a possibility that they occur together in the Congo (cf. Robyns and Tournay, 1955; Bouharmont, 1962a; Tateoka, 1965a; Clayton, 1970). It is probable that schweinfurthiana originated from either of the diploid taxa or from both by hybridization and polyploidy. Another possibility is that eichingeri was the progenitor of both schweinfurthiana and punctata. The taxa schweinfurthiana and eichingeri are rhizomatous perennials (Robyns and Tourney, 1955; Bouharmont, 1962a; Tateoka, 1965a; Clayton, 1970). Besides, eichingeri occupies a more stable habitat (forest swamps, cf. Stebbins, 195813). By all accounts, punctata is an annual (Roschevicz, 1931; Sampath, 1969b; Clayton, 1970) and occupies intermediate and less stable habitats. It shows also many characteristics of a weedy and colonizing taxon. This property of punctata may have been acquired by a shift in ecological adaptation from that of its ancestral taxon, possibly by increasing its phenotypic plasticity (cf. Mayr, 1965). Some of these properties are a toughness provided by its heavy seeds, short life cycle (cf. Sampath, 1969133, an ability to find new habitats, annual habit, and possibly polyploidy. The nomenclatural problems have been touched upon already. The difficulties in delimiting punctata from eichingeri and these from officinalis of Asia and Zatijolia of America have also been mentioned,

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as also the morphological similarity of the schweinfurthiana with officinalis and also its inclusion under punctata. It is in this context that the suggestions of Roschevicz, Bouharmont, Gopalakrishnan, and Sampath to designate all tetraploid African forms belonging to the Officinalis group as schweinfurthiana is to be considered. If this is done, it may also become possible to delimit punctata more easily. Sampath and Subramanyam (1969) even went so far as to suggest that punctata, eichingeri, and officinalis be reclassified as subspecies of a single species. The correct name may then be 0. punctata and not 0. officinalis as suggested by them (cf. Chatterjee, 1948; Tateoka, 1963). This suggestion is complementary to the proposal of Kihara (1963) to include all taxa possessing BBCC genomes (that is, schweinfurthiana, malampuzhensis, and minuta) in one species. However, such a lumping together may make both of them unwieldy superspecies. A more realistic proposition may be to have punctata, eichingeri, and schweinfurthiana as subspecies of a single species as has been proposed for the Asian Officinalis taxa. c. American Taxa. Four taxa are mentioned in the literature, paraguaensis, latifolia, alta, and grandiglurnis. Of these, paraquaensis originally included part of longistaminata and rujipogm perennial from America (Chevalier, 1932; PortBres, 1956). Roschevicz (1931), Chatterjee (1948), and Tateoka (1963) did not even cite it as a synonym. The form used under this name in cytogenetic studies by Nezu et al. (1960), Li et al. (1962), Kihara (1963), and Morinaga (1959, 1964) has the same genome constitution as the other American taxa (cf. Nezu et al., 1960; Li et al., 1962; Morinaga, 1964). Sampath (1961) observed that it is identical with alta. The three remaining taxa are all generally aquatic, occurring in swamps, margins of rivers, lakes, and ditches (Oka, 1961). All are perennial, 1 4 meters tall, erect, and possess big, open panicles. They have similar gross morphologies. They are distinguished from each other by differences in shape of sterile lemma and size of spikelets and awns. Tateoka (1962b), who has made the most detailed taxonomic studies to date of this group, showed that spikelet size (latifolia has smaller spikelets) and glume size (grandiglumis has larger glumes) are the best discriminating characters. The taxon latifolia was a t one time considered conspecific with the diploid taxa punctata (African) and officinalis (Asian). Roschevicz (1931) treated them as separate species and this distinction has been kept by later taxonomists. The spikelets of latifolia are less than 7.0 mm long while those of alta and grandiglumis are longer. It has the widest distribution among the three taxa, ranging from Mexico and the West Indies to Brazil and northern Argentina (Oka, 1961; Tateoka, 196213, 1963). 0. alta was described by Swallen

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(1936). Earlier it had been known as latifolia var. grandispiculis (Chevalier, 1932) , Its distribution is only slightly narrower than that of latifoliu (Oka, 1961 ; Tateoka, 1963). Prodoehl (1922) first described grandiglumis as a species. Its only difference from alta is that the structure and size of its sterile lemmas is nearly the same as that of its fertile lemmas (Tatoeka, 196213). It has been collected from Columbia, Guyana, Peru, and Brazil (Oka, 1961 ; Tateoka, 1963). Chevalier (1932) kept the three taxa as varieties of the same species latifolia. Swallen (1936, 1955) and Chatterjee (1948) considered them as three independent species. Tateoka (1962b, 1964a) observed that it was a “matter of choice” whether alta and grandiglumis should be regarded as species or subspecies (or varieties) of 0. latifolia. He chose to retain them separately. The three taxa have similar genome constitutions (Morinaga, 1964). Hybrids between them show mostly low degrees of fertility. Morinaga (1943) studied the cross between minuta (Asian tetraploid) and latifolia. Meiosis showed 12 I1 24 I, indicating that they have one genome in common. Later, a large number of hybrids involving latifolia, alta (as paraguaensis) , officinalis, minuta, were studied (Morinaga, 1959, 1964; Morinaga and Kuriyama, 1960) and CCDD was proposed as the genome constitution of the three American taxa, against BBCC of the African and Asian tetraploid taxa. Meiosis was described as normal in crosses among them with 24 I1 formed in MI. Nezu et al. (1960) and Kihara (1963) remarked that hybrids show only low fertility in spite of largely normal meiosis. They thought that the sterility was of the same nature as that observed in other species-hybrids of the genus and ascribed it to genic or physiological differences between the parental taxa. A hybrid between two geographic forms of latifolia (Guatemala and Cuba) showed only 29% seed setting (Morinaga, in Kihara, 1963). The chromosome configuration observed in the hybrids alta (CCDD)-oficinaZis (CC, Ceylon) (Richharia and Seshu, 1961) and latifolia (CCDD)-minuta (BBCC) (Shastry and Issar, 1965) were also in accordance with their genomic constitutions. Observing that only 21% PMCs showed the expected 12 11, Shastry and Issar (1965) remarked that chromosomal differentiation in the C genome in the two continuents might be responsible for the lower recovery of bivalents. Gopalakrishnan (1964) obtained more than 80% spikelet fertility in an alta-grandiglumis cross, while it was less than 10% with other crosses. He was also able to raise F, populations of all cross combinations, although a few degenerate plants were also obtained. During meiosis, most PMCs in alta-grandiglumis hybrids showed 24 I1 and occasionally multivalents were observed. The average number of bivalents in the alta-latifolia cross was about 21, and the remaining chromosomes appeared as uni-

+

ORIGIN A N D CYTOGENETICS O F RICE

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valents and multivalents. The formation of multivalents was attributed to homology between C and D genomes. Thus, all the three taxa are tetraploids (CCDD), perennial, and occupy similar habitats. Their ranges of distribution are approximately represented as three eccentric circles (cf. Oka, 1961; Tateoka, 1962b), grandiglumis having the smallest distribution and latifolia the widest. The taxa grandiglumis and latifolia appear to show some photoperiod sensitivity (Gopalakrishnan and Sampath, 1967; Hu, 1970b), but Oka (1961) does not think that this factor is very critical in the entire Officinalis group. Roschevicz (1931) speculated that the American taxa might have originated either by migration from Africa to America “through southern Atlantis” (possibly meaning the Atlantic Ocean), or that their seeds were carried there by wind or ocean currents. Seed dispersal by air or sea in this case appears to be unrealistic (Gopalakrishnan and Sampath, 1966, 1967). The latter authors proposed the theory that the tetraploid forms originated in situ by polyploidy following hybridization between two ecotypes carrying the C genome. According to them, either officinalis migrated from southeast Asia to America through the Siberian land bridge or another land bridge, or, following continental drift between Africa and America, some diploid punctata forms remained in America. Either of these diploid taxa then gave rise to tetraploid forms similar to grandiglumis (which they considered as the most primitive American form) by segmental allopolyploidy and from it alta and later latifolia evolved by ‘Lcrossing-over in homologous segments of chromosomes followed by segregation and recombination.” The diploid ancestors later became extinct because of competition from tetraploids. Continental drift is now considered a more acceptable theory than land bridges (Hawkes and Smith, 1965; Melville, 1966). However, it is more logical to assume that it was a tetraploid officinalis form, and not a diploid form, that was left behind on the American continent following continental drift. In the Officinalis group in Asia and Africa, tetraploids have been much less successful than diploids in extending their ranges of distribution, possibly because of the nature of polyploidy. There is no reason to assume that it would have been different in America had the diploids also been present there originally. As we will see later, the B genome of Asia and D genome of America are both considered to be variant forms of the basic Officinalis genome C. Besides, a tetraploid form is already known to exist in tropical west Africa (schweinfurthiana), the area that would have been contiguous with the American continent before the land split. I n fact, the success achieved by the tetraploid taxa in America may itself be ascribed to the fact that they did not have to compete with their diploid ancestor for establishment and spread. Morphological, cytogenetic and distribution data suggest that the three

220

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taxa are better considered as conspecific in agreement with Chevalier (1932). Roschevicz (1931), Swallen (19361, and Chatterjee (1948) treated them as separate entities. Tateoka (1962b, 1963, 1964a) retained them as separate species only because he felt it “unwise to create confusion by changing their status,” He noted that identification of taxa was possible in the northern ranges of their distribution, but it was difficult in the southern and eastern parts. Oka (1961), who surveyed the region, observed that distinction between alta and latifolia was often difficult. Richharia (1960), Sampath (1962), Kihara (1963), and Gopalakrishnan and Sampath (1967) have all argued on the basis of cytogenetic data for merging them into one species. 3. Miscellaneous Group

The relationships of the remaining eight taxa with Officinalis and Sativa groups are undetermined. They consist of two smaller groups of five taxa occurring in the same region and showing similarities in gross morphology, and three others occurring in different continents. These are (a) Granulata subgroup consisting of granulata, meyeriana, and abromeitiana, found in southern and southeast Asia including the Philippines; (b) Ridleyi subgroup consisting of ridleyi and longiglumis and occurring in southeast Asia including Burma; (c) schlechteri endemic to New Guinea; (d) australiensis known from north Australia only; and (e) brachyantha, widely distributed in tropical Africa. Of these, schlechteri is known only from a single herbarium sheet (Pilger, 1915). Interspecific hybrids have been reported using australiensis and brachyantha. Information about their distribution, habitat preferences and breeding behavior is limited. a. Granulata Subgroup. All the three taxa are short, with rhizomatous, perennial habit, occurring usually in well drained, shaded forest areas. They possess the smallest panicles in the genus (along with brachyantha), each bearing about 13-20 spikelets only (cf. Katayama, 1971). Among the three taxa, granulata has a wide distribution, ranging from Ceylon, peninsular and eastern India, Sikkim, Burma, Thailand, Indochina, Indonesia, to southern China (Tateoka, 1963). It is a diploid (2n = 24), but a tetraploid number has also been reported for this taxon from southern India (Abraham, in Sampath and Rao, 1951). The taxon meyeriana has a more restricted distribution, having been collected from Indonesia (Java and Borneo) and the Philippines (Backer, 1946; T. C. Katayama, 1963a). It is also a diploid. The third form, abromeitiana, is also known from the Philippines and Indonesia (Moluccas). Different authors have recognized two of these three taxa as valid species, with the third as a synonym of one of them. I n the latest classification of

ORIGIN AND CYTOGENETICS OF RICE

22 1

the genus, Tateoka (1962c, 1963) considered all three as subspecies of meyeriana, with each one delimited mainly on the basis of spikelet size and shape. Morishima and Oka (1960) found that granulata showed maximum correlation with alta, officinalis, and malampuxhensis. They did not study the other two taxa. Sampath (1962, 1966) proposed that granulata and meyeriana have the same genome and that granulata arose from officinalis “by systemic mutation.” Incidentally, both occur in southern and southeast Asia, are perennials and prefer similar habitats. b. Ridleyi Subgroup. Both the taxa ridleyi and longiglumis are rhizomatous perennials with vigorous growth habits. They prefer moist and shaded habitats, but longiglumis occurs in swamps also (Backer, 1946; T. C. Katayama, 1963a). Both are tetraploids with 2n = 48 chromosomes T. T. Chang, 1970). A Malaysian form of ridleyi has been reported by Van to be a diploid (Seshu and Karibasappa, 1960). The taxon ridleyi has a wide distribution in southeast Asia: in Burma, Thailand, Malaysia, Indochina, Indonesia, and New Guinea (Tateoka, 1963). 0. longiglumis was described in 1953. It is endemic to New ,Guinea. It is recognized from ridleyi by the presence of longer awns and empty glumes, and shorter ligules and spikelets. The sterile glumes in ridleyi are also longer than in most other Oryza taxa, being about half as long as the spikelets. Based on their natural distribution and spikelet morphology, Sampath (1962, 1966) assumed that ridleyi evolved by hybridization between granulata and meyeriana, with longiglumis being a genetic variant of ridleyi. c. Others, schlechteri, brachyantha, and australiensis. The first of these, schlechteri, is known only from a herbarium specimen. Schlecter collected it in 1907 from northeastern New Guinea from a rock about 300 meters above sea level (Pilger, 1915). It is a small plant 30-35 cm high with panicles only 4-5 cm long. The sterile glumes may be absent, or when present are only about 0.5 mm long. Its spikelets are characteristic in that the fertile glumes are only 1.50-1.75 cm long (Pilger, 1915). It has been suggested to be either an evolved form of minuta or derived by introgression from another genus like Leersia (Sampath, 1962). 0. brachyantha has a wide distribution in Africa, extending from Guinea to the Nile in the Sudan and southward to northern Rhodesia (Jacques-Fdlix, 1958 ; Tateoka, 1963). Chevalier and Roehrich (1914) originally described that its habitat was similar to that of barthii (their breviligulata), but it has now been observed t o grow more characteristically in depressions containing water in stony or gravelly areas. (Jacques-Fklix, 1958). It is typically an annual. Its culms are short

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N. M. NAYAR

and slender, with small panicles bearing long and slender spikelets that possess thick long awns. It is a diploid. The first hybrid of this taxon was obtained with alta (2n = 48, as paraguaensis) using embryo culture techniques (Li et al., 1961). This was also the first intersectional hybrid obtained in this genus. Most cells of the triploid hybrid showed only univalents during meiosis and only occasionally 1-3 loosely paired bivalents were seen. I n a sativa-brachyantha cross, nearly all cells showed 24 I . Single bivalents were seen in only 9/350 PMCs. In a minuta-brachyantha cross, 47/130 PMCs showed univalents only, and the remaining cells had 1-7 11. From size differences of chromosomes, the bivalents appeared to have resulted from intragenomic pairing (Wuu et al., 1963; Li, 1964; Yang, et al., 1965). Jacques-Fklix (1955) felt that brachyantha and tisseranti resembled the related genus Leersia more (tisseranti has since been transferred to Leersia; Launert, 1965). Li (1964) felt that cytogenetic studies favored keeping this taxon in section Sativa as Chevalier (1932; had suggested rather than in section Coarctata as had Roschevicz (1931). Morishima and Oka (1960) did not find any significant correlation between this taxon and any others of the genus. Sampath (1962) suggested that brachyantha arose from punctata “by systemic mutation” and from brachyantha “by evolution, the subspecies 0. augustifolia, as well as 0. tisseranti and 0. perrieri arose” (sic). He also suggested that it might have a possible relationship with coarctata. He felt that data on morphological characters and distribution could be advanced in support of his contention. All these four taxa have since been removed from the genus (Launert, 1965; Tateoka, 1965c), and there are as yet no experimental data to support his proposals. The areas of distribution of brachyantha and punctata overlap partially. The last taxon that remains to be considered, australiensis, is a tall erect rhizomatous perennial, diploid taxon that grows only in northern Australia. I n general morphology including panicle characters, i t shows features intermediate between the taxa of groups Sativa and Officinalis. Recent classifications of the genus have taken cognizance of this (Table 6 ) . Its chromosomes are bigger than those of sativa during metaphase (Karibasappa, 1957; ,Gopalakrishnan, 1959; Hu, 1961; Korah, 1963; Sampath in Shastry and Rao, 1961b; Li et al., 1963a). Shastry and Rao (1961a,b) found that the pachytene chromosome length of australiensis was not any greater than that of some sativa varieties, but because the chromosomes of the former were more heterochromatic, they reasoned that its chromosome complement might be undergoing less condensation.

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The cytological behavior of the sativa-australiensis hybrid has been the subject of a controversy after Gopalakrishnan (1959) found that it showed up to 8 I1 at meiosis. Using a japonica rice variety, Nezu et al. (1960) did not see more than one bivalent during meiosis in the hybrid. Shastry and Rao (1961b) studied the same material as used by Gopalakrishnan. According to them, true allosyndetic bivalents were not formed a t MI. The most frequent associations were nonchiasmatic, end-to-end pseudobivalents, and their number varied from 0 to 7. The meiotic cycle exhibited timing imbalance with earlier condensation of the chromosomes and migration of univalents belonging to australiensis. Li and colleagues (Li et al., 1963a,b, 1964a; Wu and Li, 1964; Ho and Li, 1966a; Wu et al., 1967; Ku et al., 1969) studied the cross again. They also found that austraziensis chromosomes were partially heterochromatic. They obtained about 2 I1 per PMC which they insisted were “authentically true” allosyndetically paired ones with 1-2 chiasmata each. According to them, 67% of the bivalents were formed due to pairing between sativa and australiensis chromosomes, 10% were formed due to pairing between australiensis chromosomes and 23% due to pairing among sativa chromosomes. These findings of different workers are somewhat difficult to reconcile with each other. A number of crosses between australiensis and taxa belonging to the Officinalis group have also been studied. Morinaga and Kuriyama (1960) observed only univalents in the triploid hybrid australiensis-minuta. They felt that australiensis did not possess either of the minuta genomes. Li et al. (1963a) found in the same cross 0-10 I1 per PMC with a mean of 4.7 11. They estimated that 72% of the bivalents were formed by autosyndetic pairing between chromosomes belonging to the B and C genomes of minuta and only 21% were genuine allosyndetic bivalents. Watanabe and Ono (1967) found that the amphidiploid of the cross minuta-australiensis studied by Morinaga et al. (1960) gave on an average 35 I1 and 2 I. The pairing was considered to be homogenetic. The hybrid showed 23% pollen stainability and 14% seed setting, whereas the amphidiploid minuta-sativa cross was male-sterile. In crosses with the American taxon alta (one cross as paraguaensis), Li et al. (1961) obtained about 7-8 I1 and the remainder univalents. They thought that pairing was predominantly allosyndetic, but later revised their opinion (Li et al., 1962, 1963a). They concluded that the genome of australiensis was different from the A, B, C, and D genomes (also see Morinaga, 1964). In the cross with the American taxon latifolia (CCDD) (Gopalakrishnan and Shastry, 1966), the hybrid showed irregular meiosis. Both bivalents and univalents were observed. The bivalent number ranged from 0 to 12, with a mean of 4 I1 per PMC. The authors at-

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N. M. NAYAR

tributed the bivalents to autosyndetic pairing. Based on the observation of Sharma in 1964 that australiensis resembled punctata in panicle and spikelet morphology, Hakim and Sampath (1968) hybridized these two taxa. The F, was sterile and generally only univalents were obtained. Since punctata characters showed dominance in F, hybrids, and considering the morphological similarities between them, they proposed that punctata was the “nearest taxon” to australiensis. They also attributed the increased size in plant parts of australiensis to high amounts of both chromatin and heterochromatin present in its nuclei. Gopalakrishnan (1959 ; also see Richharia, 1960) pointed out that aus traliensis was intermediate between the groups Sativa and Officinalis. Morishima and Oka (1960) did not find any significant correlation between australiensis and any of the other 15 taxa that they used in their numerical taxonomic study. Shastry and Rao (1961b) proposed that this taxon, far from being derived from the Sativa and Officinalis groups, might as well have been the progenitor of these two groups, since its karyotype is highly symmetrical and it contains excessive amounts of heterochromatin, both considered as primitive characters (also see Shastry, 1964b). Although our knowledge about the nature and function of heterochromatin is incomplete, present evidence does not lend support to the assumptions that the presence of higher amounts of heterochromatin either results in increased size of vegetative parts or represents a primitive condition. Heterochromatin is cytologically variable in its expression both within the same cell at different developmental times and between different cells in the same organism (Swanson, 1957; Brown, 1966). The heterochromatic regions show little or no crossing-over when compared to euchromatic regions (Brown, 1966), and this, rather than lack of homology, may explain the large number of pseudobivalents observed in a t least some of the crosses involving australiensis. It also needs to be confirmed that the bivalents found in crosses with tetraploid Officinalis taxa are all formed by autosyndetic pairing. Brown (1966) has asserted that the evolutionary and developmental significance of the heterochromatic state lies in its capacity for shutting off normal gene function. It appears that no other Oryza taxon is present in the area of distribution of australiensis. The rufipogons are known to be present in northern Australia, but whether the two occur close enough to make gene-flow between them a possibility is not known. Taking the Miscellaneous group as a whole, it is difficult to determine the relative positions of the different taxa in the genus, mainly for want of experimental data. I n the Ridleyi subgroup, longiglumis may have evolved to occupy a more aquatic habitat. Except for brachyantha, which is an annual with a wide distribution in tropical Africa, the remaining

ORIGIN AND CYTOGENETICS OF RICE

225

seven taxa are perennials that have New Guinea and certain Indonesian islands as the epicenter of distribution. The Asian cultivated rice sativa with its related taxa rufipogon and oficinalis also occur widely in this area. According to available information (Backer, 1946; T. C. Katayama, 1963a), they share similar habitats and often occur sympatrically. At least three of these seven taxa (schlechteri, longiglumis, and australiensis) and perhaps even as many as five (also meyeriana and abromeitiana) appear to have also only limited distributions and only the other two taxa, ridleyi and granulata, are widely distributed. It may be worth finding out whether some of these have been formed by a process termed “character divergence” by Charles Darwin and “character displacement” by Brown and Wilson more recently (cf. Wilson, 1965). This implies that a challenge from a related sympatric species constitutes a sufficient stimulus for divergence of local populations from a population of either species to enable them to overcome effects of competition. Such instances of species formation are often met with in the animal kingdom (cf. Mayr, 1963; Wilson, 1965; Grant, 1971), but in plants only a couple of instances have so far been described, in Gilia by Grant and in Phlox by Levin and Kerster (Levin, 1969; Grant, 1971). I n Phlox, P. argillacea is thought to have originated from P. pilosa via a saltational shift in adaptive mode in response to disruptive gene-flow from P. glaberrima to P. pilosa. The latter two species occur sympatrically in parts of the central and north-central United States. Gene-flow between them is restricted because of differences in flowering period. P. argillacea occurs as a geographical and ecological endemic beyond the northern limits of distribution of pilosa and glaberrima. Based on evidence obtained from geographical distribution, ecological preferences, breeding behavior, and population structure, it has been shown that argillacea arose from pilosa after incorporation of a few genes from glaberrima. Considering the fact that australiensis can be crossed with taxa belonging to both groups Sativa and Officinalis and that it is also morphologically somewhat intermediate between them, it is possible that this taxon might have evolved in a somewhat similar way, with perhaps oficinalis as the basic taxon involved. If this had taken place somewhere in New Guinea, where several Oryza taxa occur sympatrically, then the diverging entity might have sought and found new frontiers to the south of this area, viz., north Australia for colonization. C. GENOME RELATIONSHIPS Part of the work has been already covered in the preceding section. The groundwork on genome analysis was done by Morinaga and col-

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leagues. After studying hybrids between sativa, minuta, and latifolia, Morinaga (1943) designated their genomes as AA, BBCC, and CCDD, respectively. Later, the genome of officinalis was designated C (Morinaga and Kuriyama, 1959b). Subsequent studies led to determining the genome constitution of six other taxa (Morinaga, 1959; Morinaga and Kuriyama, 1960). Their results have been summarized by Morinaga (1964). Nezu et aZ. (1960) studied crossability of 17 Oryza taxa, and their results largely confirmed Morinaga’s proposals. They found two intrafertile, but largely intersterile, groups among taxa possessing the A genome. The two taxa minuta and eichingeri (identified as punctata by Tateoka, 1965b), although having the identical constitution BBCC, were also intersterile. The B genomes of these taxa were suggested to be partially homologous to A of Sativa group. Rao and Seetharaman (1955) had also earlier found 5 11, 3 111 and the rest univalents in the hybrid punctata (as eichingeri)-glaberrima (AA) . They considered that a basic complement of 5 chromosomes of glaberrima was common to punctata also. Gopalakrishnan (1959, 1962) studied a hybrid between the peninsular Indian tetrapoloid taxon malampuxhensis and officinalis and found that they shared one genome ( C ) , and soon the second genome was found to correspond to B (Kihara, 1963; Gopalakrishnan et al., 1965). Kihara et al. (1961, 1962) determined the genome formula of stapfii (now barthii) as AA and of alta and grandiglumis as CCDD. The rest of their work confirmed Morinaga and Kuriyama’s work. Kihara (1964) has discussed the advantages of different systems of genome designations. Richharia (1960) and Sampath (1962) proposed the use of an alternative system of designating genomes. In this, the basic genome is given the first letter of the original species containing it and deviant forms are distinguished by superscripts. Thus rufipogon perennial (as perennis) , sativa, and glaberrima were represented as P’, P2, and Pa, respectively, The B, C, D genomes of the Officinalis group were Os,O’, and 02, respectively. As Kihara (1964) also pointed out, it has the disadvantage of creating confusion following possible nomenclatural revisions. Yeh and Henderson (1961a,b, 1962; also see Henderson, 196413) used a combination of the above two systems: alphabets with subscripts. They represented the African taxa of the Sativa group by a genome symbol different from that used for their Asian relatives. Subsequent findings have not justified this proposal. The work of Li and colleagues on interspecific hybridization further confirmed the genome designations of Morinaga and colleagues. They assigned the symbol E for australienst (Li et al., 1963a) and F for brachyantha (Li et al., 1961). Afterward, Li (1964) changed the symbol of australiensis to G because Yeh and

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Henderson (1962) had earlier assigned this letter to the genome of glaberrima and related taxa. Bouharmont (1962a) used a combination of different systems for designating the genomes of 10 taxa. Although he used a different symbol for schweinfurthiana, he proposed a t the same time that it might be similar to those of ZatifoZia and related species. At a symposium on rice genetics and cytogenetics held a t the International Rice Research Institute, Los Banos, in early 1963, the participants agreed on the genome symbols for 17 taxa. They also laid down certain guidelines for the future (International Rice Research Institute, 1964). A comparative statement of various proposals is given in Table 7. Thus, various taxa have been suggested to contain six genomes, A, B, C, D, E (G), and F. No information is available about the genome constitution of five of the taxa included under the Miscellaneous group. At the present time, the work is mainly directed toward determining the interrelationships among the different genomes. It is generally agreed that A and C are basic genomes and also that B, C, and D genomes show some affinity to each other. The relationships of E (G) and F to the other genomes are not clear, but opinion is divided about the nature of the relationship between the A and B, C and D genomes, and also about the constitution of the two African diploid taxa punctata and eichingeri. Part of this ground has already been covered. Several workers have studied the hybrid between sativa (AA) and officinalis (OC). The results have been partly reviewed earlier (pp. 168170). Early workers observed lack of pairing between chromosomes of the two taxa with 1 4 I1 seen only occasionally (Ramanujam, 193%; Nandi, 1938; Gopalakrishnan, 1959; Nezu et al., 1960 ; Bouharmont, 1962a; Morinaga, 1964). Then, using the Ceylonese form of officinalis, Shastry et al. (1960b, 1961) reported observing complete pairing during meiotic prophase, and 1-2, and rarely 3-4, I1 during M I in 35% PMCs. They interpreted this as desynapsis. Although they (Sharma and Shastry, 1965c) considered the Ceylonese form as different from oficinaZis and even proposed a new specific status for it (cf. p. 208), Shastry (196413) maintained that the Officinalis group was more primitive than the Oryza group and that both were probably more closely related than was generally assumed. Katayama (1965b) found that temperatures of 20°-30°C had no effect on pairing in the hybrid. He obtained 1-5 I1 during MI (mean 1.1 11) and proposed that the chromosomes of sativa and officinalis were partly homologous. At the same time Li et al. (196413) did not find any pairing in the hybrid even a t pachytene. These seemingly contradictory findings are hard to explain except by assuming varietal differences in the parent material. This will be taken up again later.

I9 I9

TABLE 7 Genome Designation

Given name

Identification

sativa sativa sativa var. spontanea, rufipogon, annual fatm balunga, perennis, rufipogon, perennial Asian (Ah) CUbellsiS rufipogon, perennial (Cuba) lon~utaminata perennis eap. barthii sativa var. formosana rufipogon, perennial globemimu globerrirna breviligulafu barthii barthii stapfii minuta minuto malampurhensis malampulhensis punctata, diploid pundata, diploid eichingeri, diploid eichingeri, tetraploid schweinmrthiana ofidnalis mllina latifdia alta grandiglumis paragmyensis australiensis brachyantha

dchingen', diploid punctata. tetraploid echweinfurthiana ofinalis olJicinalis, Ceylon latifolia alta grandiglumis alta australiensis brachuanthn

Morinaga (1964); Morinaga and Kuriyama (1960)

Others

-

00

International Rice Research Institute (1964)

Richharia (1960): Sampath (1962)

Yeh and Henderson (19618): Henderson Bouharmont (19648) (19628)

AA AA

AA

AA AA

-

PIPI

AiAi AiAi

AA AA

AA

-

AA

PIP'

AiAi

-

AA

-

AcuAm

P'P'

A&

-

-

P'P'

AaAz AiAi EE EE

AA AA

-

AA (Nezu et al., 1960)

-

BBCC (Kihara, 1963) BB (Katayama, 1967b) A, C (Hu, 19708) CC (Hu. 19708)

BBCC

-

BBCC

-

cc

-

CCDD

-

CCDD CCDD Not A, B, or C

-

-

-

CCDD (Nezu et al., 1960)

-

EE, GG (Li et al., 19638) FF (Li et al., 1961)

AbAb

-

-

AcAr AsAa ArAs BBCC

PIP' P'XP' PlXP' O'O'M'M'O'O'O'O'

-

0'0'0'0' 0'0'

-

-

BBCC

cc -

CCDD CCDD CCDD CCDD

EE FF

O'OlOaOa O'O'?? 0'0'

-

O'O'O'Of

0'0'0'0' OLO'O'O'

-

-

-

AiAi AiAi AlAi BBCC

EE -

-

-

-

-

-

-

-

-

-

FFGG

00

CCDD OOEE

-

ORIGIN A N D CYTOGENETICS O F RICE

229

I n contrast, it is generally agreed that B, C, and D genomes show some relationships to each other. Rao and Seetharaman (1955) proposed that the African Officinalis taxon eichingeri [identified as tetraploid punctata by Tateoka (1965b), and possibly corresponding to schweinfurthiana] is an autopolyploid or segmental allopolyploid because they observed up to 8 IV during meiosis. Subsequent workers have concurred with the conclusions for various reasons, but none has observed such high frequencies of quadrivalents. Richharia (1960), by using the symbols O', Oz, and O3 to represent, respectively, C, D, and B genomes, implied their homology. Then Sampath (1962, 1966) suggested that they could be considered to be homologous as they might be different from each other only a t a few loci (also see Gopalakrishnan and Shastry, 1966). The regular occurrence of bivalents in low frequencies in hybrids between tetraploid taxa possessing BC or C D genomes on one hand and diploid taxa having A, E ( G ) , or F genomes on the other, have been attributed by several authors to autosyndetic pairing between chromosomes of genomes B, C, and D (Morinaga, 1941; Li et al., 1963b; Wuu et al., 1963; Gopalakirshnan and Shastry, 1966). But direct confirmation of this has been possible only in a few cases where there existed differences between parents in chromosome size. The pairing observed between diploid taxa containing the A genome (sativa, glaberrima, etc.) and tetraploid taxa containing BC (minuta, schweinfurthiana, etc.) or C D (alta, Zatifolia, etc.) genomes have been interpreted differently by various workers. The B and D genomes are known only from tetraploid taxa in combination with the C genome. The suggestion that diploids punctata and eichingeri contain B and D genomes remains to be corroborated. A partial homology of A with both B and C genomes has been suggested, and also that B and D genomes have differential effects on pairing of C genome chromosomes. Okura (1937) and Nandi (1938), who were the first to study the hybrid sativa (AA)-minuta (BBCC) found 12 I1 12 I during meiosis. They postulated that one of the component genomes of minuta was of sativa origin. Nandi went further to suggest that minuta was an allopolyploid deriving from sativa and officinalis. Subsequent studies of these hybrids have not supported this assumption, as we have already seen. Further, a sativa-officinalis allopolyploid was male-sterile (Watanabe and Ono, 1968). At the same time, Katayama (1966b,c, 1967a) studied a large number of hybrids a t pachytene and metaphase in four cross-combinations, viz.J A-BC, A-CD, (A-BC)-A and (A-CD)-A and reported results to support his (Katayama, 1965b) proposal based on pachytene pairing in the F1 hybrid sativa-officinalis, that their two

+

230

N. M. NAYAR

genomes A and C showed a t least partial homology. Katayama observed in these diploid-tetraploid hybrids chiasma formation a t pachytene and up to 1&11 I1 in M I (means 1-6 11).He believed that most of them were formed by allosyndetic pairing between A and C chromosomes. Other workers, who studied A-CD hybrids, observed either degenerated anthers and/or little or no pairing during meiosis (Gotoh and Okura, 1935; Hirayoshi, 1937; Morinaga, 1941, 1964). The 1 4 I1 obtained by Li et al. (1961, 1962, 196313) were attributed to autosyndetic pairing between chromosomes of C and D genomes. Kihara and colleagues (Neau et al., 1960; Kihara, 19641, on the other hand, have proposed that there is partial homology between the A and B genomes. They found that sativa (AA)-minuta (BBCC) and barthii (AA, as breviligulata)-tetraploid punctata (BBCC, as eichingeri) hybrids showed P 5 I1 in M I (range: 0-9 11). As already mentioned, Morinaga (1940, 1943, 1964) saw no or little pairing in similar combinations. Li et a2. (1961, 1962, 1963b) also obtained the same results, but attributed them to autosyndetic pairing. Discussing the results of the hybrid latifolia-australiensis, Gopalakrishnan and Shastry (1966) observed that B, C, and D genomes might be showing preferential pairing in the presence of alien genomes like A, E (G), or F. This appears to he a distinct possibility. Hu (1970b) put forth the suggestion of differential effects of C and D genome chromosomes on pairing of C genome chromosomes. According to him, B genomes could partly pair with C but did not suppress C-C pairing, whereas D chromosomes suppressed it (C-C pairing). The hybrid minuta-tetraploid officinalis (BCCC) gave about 18 I, 7 11, and 15 I11 (rarely quadrivalents) during MI, whereas grandiglumis-tetraploid officinalis hybrid (DCCC) showed a much higher number of univalents, via., 36 I and 6 11. Hu cited also the meiotic behavior of induced octoploid latifolia (cf. Watanabe and Ono, 1965) and amphidiploid latifolia-minuta (cf. Morinaga et al., 1964) in which quadrivalents were suppressed while such a suppression (of quadrivalents) was not found in induced octoploid minuta (cf. Watanabe and Ono, 1966). H e therefore proposed that the speciation mechanisms of taxa containing B, C, and D genomes involved “not only chromosomal but also genetic differentiation.” An examination of meiotic data of hybrids involving B, C, and D genomes shows that D genome does not always suppress C-C pairing and quadrivalent formation as compared to B. At MI, octoploid minuta (BBBBCCCC) gave about 33 I1 and 7 IV, and octoploid Zatifolia (CCCCDDDD) about 40 I1 and 4 IV. Katayama’s (1966a,b) data on pairing behavior in a number of A-BC and A-CD crosses also do not show any significant difference between them. The same was true in

ORIGIN A N D CYTOGENETICS OF RICE

231

meiosis of backcrosses of A-BC and A-CD hybrids to taxa containing the A genome (Morinaga and Fukushima, 1956; Katayama, 1967a). These backcrosses formed only tetraploid hybrids and had the genome formula (A-BC)-A and (A-CD)-A. Thus, they differed only with regard to the presence of the B or D genomes. Morinaga and Fukushima (1956) obtained 12 I1 12 I in both these backcrosses. Katayama (1967a) found 8-16 I1 (mean 12-13 11) in (A-BC)-A hybrids and 8-15 I1 (mean 11-12 11) in (A-CD)-A hybrids. The rest were mostly univalents and rarely trivalent5 and quadrivalents. A slight, but almost consistent, reduction in pairing is discernible in crosses of CD with A as compared to those of BC, but it seems unnecessary to attribute this to a suppression of pairing by the D genome. It is obviously difficult to make out any consistency in the pairing pattern of chromosomes involving A, B, C, and D genomes. First, there are the contradictory results obtained about pachytene pairing in sativa-officinalis hybrids by Shastry et al. (1960b, 1961) and Katayama (1965b), on one hand, and Li et al. (1964b) on the other. It needs to be determined whether nonhomologous pairing and crossing-over have taken place or if some other genetic mechanisms are involved. Since the C genome has a wide pantropical distribution alone and in combination with the B and D genomes, it could have undergone considerable cytogenetic modifications. High to complete sterility between geographical types of officinalis is already known (Morinaga and Fukushima, in Kihara, 1963; Gopalakrishnan, 1965a; Hu and Chang, 1967). Hu (1970a) found that the hybrid eichingeri-officinal& is sterile, but that pairing is almost normal. A similar behavior is shown by other hybrids also-in minutu-mulumpuzhemis (both Asian BBCC taxa) , in eichingeripunctata (both African CC taxa) , within Zatifolia (American CCDD taxon), and also within Zatifolia group (alta, latifolia, grandiglumis, all American CCDD taxa). These data indicate that B, C, and D are closely related, and may even be homologous, as suggested by Richharia (1960), Sampath (1962, 1966), and Gopalakrishnan and Shastry (1966). The D genome however appears to have become more differentiated than B from C. This can explain the observation by H u (1970b) of multivalents in a BC-CC hybrid (minuta-tetraploid officinal&) and of the increased number of univalents in a DC-CC hybrid (grandiglumis-tetraploid officinal&). This may also account for the higher number of quadrivalents and the much reduced fertility obtained by Watanabe and Ono (1965, 1966) in induced octoploid minuta (BBBBCCCC: about 7 IV and 33 11, 17% pollen stainability and 33% seed setting). The increased differentiation of the D genome when compared to B could have been brought about by the much wider distribution of taxa containing the D genome in America. The two Asian BC taxa

+

232

N. M. NAYAR

are endemic in their occurrence. Only little is known about the distribution of the African BC taxa, but they are not known to have a wide distribution. The C genome differs from A in certain characteristics. The first is the allotetraploid-like (or diploidlike) behavior of an autotetraploid officinalis studied by Hu (1967). It showed almost normal pairing and fertility-about 20 I1 1 IV others, mainly univalents, and 89% seed setting (only the true autotetraploid is considered here). Autotetraploids of sativa show about 8 IV and about 25-50% seed setting. Hu thought that certain genetic factors which regulate pairing were present in the C genome. The second characteristic is the high propensity of the C genome to form polyploids. Tetraploid taxa possessing A, E (G), or F genomes are not so far known in nature, although A has an even wider distribution than the C genome. Third, it appears probable that sterility barriers between geographical races develop more readily in taxa containing the C genome. A few bivalents are generally observed in sativa-minuta and sativatetraploid punctata (as eichingeri) hybrids. Occasionally 1-2 I11 are also seen. Autosyndetic pairing between chromosomes of the B and C genomes was offered as one of the explanations. But since only small numbers of quadrivalents have been found in induced polyploids of officinalis and minuta (cf. Hu, 1967; Watanabe and Ono, 1967), this explanation may not account for all the bivalents found in these hybrids. Another suggestion has been that A and B genomes are partially homologous (Nezu et al., 1960) or that there was introgression of officinalis into sativa (Watt, 1892; Anderson, 1951; and others). Introgression a t the diploid level seems improbable because of: (i) difficulties in hybridization between sativa and officinalis;(ii) high meiotic irregularity and complete pollen sterility of hybrids; and (iii) both the reported cases of backcrosses of sativa-officinalis to sativa (no backcrossing attempted with officinalis)produced only sterile triploids (Ramanujam, 1938c; Ho and Li, 1965). I n addition, when triploid hybrids of sativa-minuta (AA X BBCC = ABC) were backcrossed to sativa (AA), only tetraploids (AABC) were formed (Morinaga and Fukushima, 1956). Polyploidy is known also to make gene exchange possible between species that are isolated from each other at the diploid level (Lewis, 1967; de Wet, 1971). When a diploid-tetraploid hybridization takes place between taxa separated by strong isolating barriers, usually gene-flow takes place from diploid to tetraploid and a whole genome gets transferred. Since populations of sativa, officinaZis, and minuta occur sympatrically (distances within which cross-pollinations can take place) in the Philippines (cf. T. C. Katayama, 1963a; Tateoka and Pancho, 1963) oppor-

+

+

ORIGIN AND CYTOGENETICS OF RICE

233

tunities do exist there for incorporation and “ingestion” of sativa genomes into minuta populations even if it occurs rarely. Plants from such a population of minuta can then be reasonably expected to show increased pairing with sativa compared to other populations of minuta in which such a process did not take place. VI. Chromosome Complement

A. EARLY STUDIES Kuwada in 1909 (Kuwada, 1910) was the first to study microsporogenesis, megasporogenesis, and mitosis in rice, 0. sativa, and determine its somatic chromosome number as 24. He used six cultivars and described their meiosis in great detail. He is also credited with having made the first observation of the phenomenon that is now known as secondary association, but he himself has stated in his paper that “this peculiar behavior of chromosomes in homotypic division (“paired arrangement”) was first discovered by my friend Dr. M. Tahara in Morus.” However, both their reports appeared in 1909. His findings on number and pairing of chromosomes and course of meiosis have since been confirmed by several workers (Nakatomi, 1923; Rau, 1929; Selim, 1930; and several others later).

B. CHROMOSOME MORPHOLOGY I n spite of the small size of rice chromosomes (largest about 4-5 p long) , several authors have described their morphology employing both somatic and meiotic stages of haploid and diploid plants and also pollen mitosis. Some authors have optimistically stated that various descriptions agree generally, but it does not appear to be so. Differences exist in size, location of centromere, and number and position of satellites, nucleoli, and secondary constrictions. Stebbins’ (1958b) system of classifying karyotype symmetry has been used somewhat indiscriminately in rice. It is probably the most practical method for classifying karyotypes. Stebbins (1966, 1971) himself has cautioned that karyotype morphology by itself must never by regarded as of overriding importance (also see Moore, 1968; Lewis, 1969; Jones, 1970). And yet this is exactly what certain authors have done. Further, a karyotype (or ideogram) is normally taken to represent the morphology of a chromosome complement a t mitotic metaphase (cf. Rieger et al., 1968; Stebbins, 1971), but in rice some authors have used chromosome maps prepared a t pachytene

234

N. M. NAYAR

to classify taxa for karyotype assymmetry (Stebbins’ method). It is well known that absolute and relative lengths and morphology of chromosomes can vary greatly between pachytene and metaphase. For locating the position of centromere on chromosomes, certain workers have described them as median or submedian without indicating arm ratios (e.g., Korah, 1963), while some others have used different standards and terminologies (Sen, 1963, 1964b; Ghosh, 1964; Karunakaran, 1967; Chu, 1967), and, a t least in one instance, in the same paper (Das and Shastry, 1963, short-arm to long-arm ratio of 0.64 and 0.68 submedian, 0.58 and 0.61 median, and so on). Some of the conclusions of Korah (1963), Ghosh (1964), and Shastry and Rao (1961a; also see Shastry, 1962, 1964a) are based on wrong premises and arbitrary groupings (about longevity and ploidy of taxa, karyotype symmetry classification, grouping of taxa into two classes on the basis of haploid chromosome length, number of chromosomes in different taxa whose long arms are a t least twice as long as short arms, and so on). I n this paper, the nomenclature recommended by Levan et al. (1964) for centromeric position on chromosomes has been used for describing the morphology of rice chromosomes, and the data available in the literature have been recalculated. Rau (1929) observed that 5 pairs of sativa chromosomes were large, 4 were medium, and 3 small. The large chromosomes were twice as long as small ones. Nandi (1936, 1937) found that length of somatic chromosomes varied from 0.7 p to 2.8 p. The longest pair had a median constriction and the shortest pair was much shorter than the rest. Two pairs of chromosomes had satellites, and one pair was attached t o the nucleolus. The haploid complement consisted of 10 types of chromosomes, composed of 2 groups of 5 each. One chromosome was duplicated in each group. Nandi designated them as A, BB, C, D, and E and A,A,, B,, C1,D,, and El.Sethi (1937) observed varietal differences in size, morphology, and location of the centromere. The chromosome length varied from 1.2 to 2.7 p. According to Pathak (1940; also see Pathak and Tyagi, 1951), 4 pairs of chromosomes were median. Yasui (1941) used haploid somatic chromosomes to characterize all chromosomes of the complement. The total length of 12 chromosomes was about 40.5 p. The length of individual chromosomes ranged from 2.0 p to 5.0 p. The 10th chromosome was the only subterminal chromosome and it organized the satellite. Yasui’s studies were followed by those of Hu (1957, 1958a,b, 1960a,b, 1961, 1964) on haploids of both sativa and glaberrima, and of Ishii and Mitsukuri (1960) and Mitsukuri (1966). Hu found that the two cultivated taxa had identical karyotypes and that the various wild taxa also did not show much variation except for granulata. Regarding size, australimis and officinalis had bigger

ORIGIN A N D CYTOGENETICS O F RICE

235

chromosomes and brachyantha smaller ones. Variance analysis showed that the length differences of chromosomes within cells and between cells were highly significant. The rates of condensation of different chromosomes seemed to vary. B y considering both total length and arm ratios, he felt that all chromosomes except 5 and 6 could be distinguished, provided a number of cells were studied. Observations on chromosome length and morphology of cultivated and wild taxa were also made by Bouharmont (1962b), Korah (1963), and Ghosh (1964). Bouharmont observed that the length of a haploid set of chromosomes in 9 taxa ranged from about 12.3 p to 20.9 p and identification of all individual chromosomes was difficult because of their small size. He also found that the chromosome complement of brachgantha was the smallest (12.3 p ) and that of officinalis the longest (20.9 p ) . The remaining taxa showed intermediate lengths. The only description of a gametophytic karyotype in first microspore division is that of Sen (1963). It agreed with the pachytene chromosome morphology of the same variety that he studied. Earlier, Bhaduri et al. (1958) had described a technique for studying pollen mitosis in rice. However, the technique needs considerable improvement before it can be used advantageously. The data of various authors are summarized in Tables 8 and 9. The morpohology of the pachytene chromosome complement was first described by Shastry et al. (1960a) in a japonica variety. Shastry and colleagues subsequently studied five wild taxa, nine sativa cultivars, and one glaberrima variety (Shastry and Rao, 1961a; Shastry, 1962, 1964a; Das and Shastry, 1963; Misra and Shastry, 1967). Similar studies have also been made by Hu (1960b) in granulata, Sen (1963, 1964b) in a sativa cultivar and rufipogon perennial (India), Karunakaran (1967) in two javanica cultivars, and Chu (1967) in haploids of six japonica varieties. Misra and Shastry (1967) used five indica varieties, two javanica varieties, and two japonica varieties for pachytene analysis. All of them showed 1-2 big nucleoli and 2-5 smaller ones. The majority of small nucleoli lay free in the cytoplasm. I n 90 PMCs (10 PMCs per variety), the total chromatin length varied from 242 p to 454 p per PMC. The average values for nine cultivars ranged from 273 p to 374 p . The mean absolute lengths of the longest and shortest chromosomes were about 46 p and 15 p , respectively. There was no constancy with respect to the nucleolar organizing bivalent and long arms organized the nucleolus in the majority of cases. Sometimes the nucleolus was organized terminally without any visible satellite. The authors concluded that rice varieties showed considerable intraspecific variation in chromosome morphology. Chu (1967) studied pachytene morphology in haploids and saw no varietal differences in six japonica varieties. The total length

TABLE 8 Characteristics of Rice Somatic Chromosomes Range and tots1 length

Chromosome numbere Author: material

1

2

3

4

5

6

7

8

9

10

5.7 2.5:sm

5.0 2.7:sm

5.0-2.0 40.4

6.7

5.8

2.4-1.2 20.8 4.2-1.9 34.5 4.3-1.8 33.5

11

12

Yasui (1941), haploid root tip

12.4* 2.5:sm

10.9 10.1 2.0:sm 2.0:sm

9.4 2.0:sm

9.2 2.5:sm

8.7 2.5:sm

7.7 2.5:sm

7.4 3.0:sm

7.2 2.0:sm

Hu (1958b). haploid root tip

11.5

10.6

10.6

9.0

8.2

7.7

7.7

7.7

7.2

6.4 6.0:st SAT0 7.2

Ishii and Mitsukuri (1960). haploid root tip Hu (1964). haploid root tip

12.2

11.3

9.8

9.3

9.1

8.1

7.8

7.2

7.0

6.7

6.1

5.5

12.8 2.1:sm

11.0 1.5:m

9.8 1.3:m

8.7 1.l:m

8.7 1.7:m

11.7 1.l:m (3)SAT

10.8 1.5:m (2)

10.1 1.9:sm SAT

9.8 1.3:m

7.8 2.1:sm (6) 7.4 1.l:m

7.8 2.1:m (7)SAT 6.6 2.8:sm

6.9 1.6:m (10) 5.8

6.9 1.3:m (11) 5.5 1.2:m

6.0 3.0:sm (8)SAT 4.5 1.5:m

5.4 2.1:sm

15.5 1.8:sm

8.4 2.4:sm (9) 8.5 1.7:m

Sen (1963). pollen mitosis

6.0:st

3.8

1.3:m

(Ir)

3.34.9 23.2

Arranged according to size: numbera in parentheses indicate numbers assigned by authors wherever different. Upper figure is relative length (in percent); lower left figure is arm ratio (long arm:short arm); lower right is centromere position according t o h v a n et al. (1964). M = median point, arm rati0:l.O; m = median region, arm ratio: l.Cl.7; sm = submedian region, arm ratio: 1.7-3.0; st = subterminal region,arm ratio: 3.0-7.0; t = terminal region, arm ratio: 7.0-00; T = terminal point, arm ratio: m). 0 SAT: satellited chromosome.

?:

7$ a

TABLE 9 Characteristics of Chromosome Complements of Wild and Cultivated Rices

Author Bouharmont (1962b) Bouharmont (1962b) Korah (1963) Korah (1963) Korah (1963) Korah (1963 Bouharmont (196213) Korah (1963) Bouharmont (1962b) Bouharmont (1962b) Korah (1963) Ghosh (1964) Korah (1963) Bouharmont (1962b) Ghosh (1964) Bouharmont (1962b) Ghosb (1964) Ghosh (1964) Bouharmont (1962b) Korah (1963) Korah (1963) Korah (1963) Bouharmont (1962b) Bouharmont (1962h) Ghosh (1964) Shastry and Rao (1961a) Shaatry and Rao (1961a) Daa and Shastry (1963) Das and Shastry (1963) Das and Shastry (1963) Sen (1964b) Gopakumar, in Shastry (1964a) Gopakumar, in Shastry (1964a) Gopakumar. in Shastry (1964s) Gopakumar, in Shastry (1964s) Sharma and Shastry, in Sheetry (1964s)

Given name

Material identification

Stage studied

rufipogon, annual Root tip mitosia satiua Root tip mitosis satiua. adieu Root tip mitosis sativa, indica Root tip mitosis satiaa. japonicu Root tip mitosia satiua, iaponica Root tip mitosb glaberrima Root tip mitosis glaberrima Root tip mitosia rufipogon, annual Root tip mitosis rujipogon, perennial Root tip mitosis Probably rujipogon Root tip mitosis perennial perennis, India rufipogon,perennial Root tip mitosis oficinalis omnalis Root tip mitosis oficinalis, Burma ofiinalis Root tip mitosia oficinalis, India oficinalis Root tip mitosis longistaminata. Congo longistaminata Root tip mitosis barthii longistaminata Root tip mitosis madagascar ensis longiitaminata Root tip mitosis stapjii barthii Root tip mitosis australiensis australiensis Root tip mitosis eichingk pundata tetraploid Root tip mitosis minuta minuta Root tip mitosis brachyantha brachyantha Root tip mitosis schweinfurthiana schweinfurthiana Root tip mitosis ridleyi ridleyi Root tip mitosis australimais australiensis Pachytene stapfii. barthii Pachytene barthra longistaminata Paehytene perennis, Assam. India rufipogon. perennial Pachytene balunga, Orissa, India rufipogon, perennial Pachytene perennis, Cambodia rufipogon, perennial Pachytene rufipogon. annual Pachytene f.spontaneu, Assam. India rufipogon. annual Paehytene f.apontunea,Assam. India Paebytene f.spontanea, Andhra, India rujipogon. annual f.spontanea, Orissa, India rufipogon,annual Pachytene brariligulata barthii Pachytene

fatua 'R.66':sdiua 'Rupsail' 'Chinsurah Boro' 'Zuiho' 'Fukoku' 'Local Black Rice' glaberrima fatua, India perennis, India perennis

Haploid number (n)

Length o f gametic chromosome complement

(id

enge in length (p)

M

-

_ -- -- -_ - _ _

12 12 12 12 12 12 12 12 12 12 12

15.9 17.0 21.6 24.8 19.6 23.2 13.8 20.3 15.9 16.7 22.6

12 12 12 12 12 12 12 12 12 24 24 12 24 24 12 12 12 12 12 12 12 12 12 12 12

20.0 30.5 20.9 14.7 13.5

3.5-1.8 -

18.7 15.5 26.6 42.6 43.2 12.3 30.1 40.1 411 343 288 190 320 834

-

-

-

Chromosome and karyotype clsasification (after Levan et al., 1964: Stebbins. 1958b)

2.6-1.4 2.8-1.7 2.3-1.0 3.0-1.3

-

2.81.0 -

2.6-1.5

-

3.3-1.5 2.8-1.2 2.8-1.2

-

62-19 44-14 37-16 26-11 46-17 100-32 -

-

m

sm

at

- - - - -A - - - - -A - - - - -B - _- _- --- - B - - - - -B

_ _ - - _ - _ - - -

-

1

6

- 5

0

0

5

7

1 2 B

0 0

9 7

3 4

0

-A 2B -A

- _- _- ---

- - - - -

_ - - - -1 - - - -

1B 2 B -B

- - - - -B B - - - - --

_ - _ - _ 0 0 0

0 0

0

0 0 0 0 0 0

17 4 5 12 10 10 7 5 8 8 8 7

7 6 5 0 2 2 4

5 2 3 3 5

0 2 2 0

0 0

1 2 2 1 1 0

2C

-

-

-

-

-

-

TABLE 10 Characteristics of Rice Pachytene Chromosome& Chmmosome length

Chromosome number Material

1

5

3

4 9.6 2.0:sm Nuc.10.2 2.9:sm 9.9 1.4:m 9.1

7.6 2.1:sm

6.9 3.7:st

6.6

1.0:m

5.8 1.7:m

5.3 3.3:e.t

5.3 6.O:at

9.3 1.l:m 7.8 1.3:m 8.6

8.5 1.5:m 7.7 1.8:sm 7.9

7.5 1.4:m 7.7 1.3:m 7.5

7.5 1.l:m 7.1 1.4:m 7.1

6.8 1.0:m 6.3 1.2:m 6.5

6.1

6

7

8

2

9

10

(#I.

12

range

5.1 1.6:m

4.5 2.8:sm

79-18 398

Z

5.3 2.4:sm 4.8 2.1:sm 5.3

4.4 2.3:sm 3.9 1.6:em 4.8

-

F

-

-

5.5 2.2:sm

4.0 1.6:m

74-19 368 47-19 374 47-17 309

11

Norin 6 : sdiua. japonieac

19.8 1.7:m

11.9 2.2:sm

N.P. 130: S d i U U , indiead

13.1 2.1:sm 18.5 1.0:M 13.4

12.5 5.3:st 12.8 2.3:sm 12.4

11.8 1.2:m Nuc.11.2 1.4:m 12.8 2.3:sm 11.4

14.9 1.4:m

11.5 1.l:m (3)Nuc.

10.2 1.4:m (2)

9.8 1.4:m Nuc.LA

9.2 1.9:em

8.1 2.1:sm

7.4 1.0:M

7.3 3.1:st

6.2 3.9:st

1.6:m 5.8 1.0:M 5.9 NUC. 5.6 1.3:m

12.7 1.3:m

11.3 1.3:m

10.6 2.0:e.m

9.4 1.l:m

9.0 1.4:m

8.2 1.2:m

7.1 2.2:sm

6.6 2.0:sm

6.4 2.4:sm

6.1 2.5:sm

15.1 2.O:sm

11.3 1.5:m

10.2 1.8:sm

8.8 1.3:m

7.7 1.2:m

7.1 1.8:sm

6.6

6.0

1.7:m

1.6:m

11.7 1.5:m

10.4 1.6:m

9.5 1.8:sm

8.0 1.4:m

1.3:m

6.4 1.3:m

6.1 1.7:m

5.0 1.3:m

47-18 359

11.6 1.9:sm

10.8 1.6:m

9.5 2.1:sm

8.5 1.4:m

8.2 1.7:m

7.1 2.1:am Nuc.LA 7.5 l.7:am

6.6

12.8 1.5:m Nuc.-LA

7.6 1.2:m Nuc.SA 7.6 1.4:m

6.8 1.6:m Nuc.LA

5.0 1.7:m Nuc.LA 5.4 1.5:m

13.2 1.2:m

7.8 1.9:sm Nuc.LA 8.8 1.3:m

7.5 1.6:m Nuc.LA 7.2 1.2:m

6.7 1.3:m

6.3 1.5:m

5.7 1.4:m Nuc.LA

4.7 1.4:m

35-13 273

N. 32: sdwad sdiua varietyindicu variety'

S. 273:

satiuu, indiean

-

-

SA

-

-

-

-

-

-

-

-

T. 141; sativa, indicar

13.2 1.4:m

12.5 1.8:sm

10.3 2.0:sm

A. 31: saliva, japonicau

16.3 1.8:sm

12.7 2.0:sm

11.0 1.6:m

Norin. 20: sativa. japonicar

17.8 2.1:sm

14.8 1.3:m

10.6 1.2:m

9. 285: satiua. javanicar

17.2 1.6:m

11.4 1.8:sm

9.2 1.7:m

9. 134: sativa, javanicau

12.4 1.9:sm 15.3 2.9:sm 13.7 2.5:sm 14.2 1.5:m

12.2 1.2:m 12.1 2.8:sm 11.3 2.7:sm 12.5 1.5:m

11.9 1.3:m 10.4 2.4:sm 10.4 1.6:m 9.8 1.2:m

10.5 l.2:m 10.1 1.l:m 9.0 1.8:sm 9.1 1.3:m

9.5 2.2:sm 9.4 1.4:m 8.6 3.3:st

17.0 2.9:sm

11.4 1.9:sm

11.0 2.2:sm

8.5 2.9:m Nuc.LA

W. 108: d i v a . javanimh T. 2357: satiua, jauanicah Norin Nos. 8,22,25, Shin-ai, Senbonssahi, Saitamamochi: sativa, japonica (haploids) a plaberrim vsrietyi

7.8 2.O:sm Nuc.LA 10.0 8.5 1.3:m 1.3:m Nun.LA 8.4 8.2 2.6:am 1.6:m Nuc.SA 8.7 8.4 1.9:sm 1.4:m 8.5 2.1:sm

b

4.9 1.l:m

4.4 1.2:m

46-1 1 281

5.6 1.4:m

4.2 1.8:sm

55-13 317

5.5

5.4 1.3:m 4.7 3.8:st 5.9 2.0:sm 5.7 1.4:m

5.0 2.6:sm 4.3 1.3:m 5.1 3.0:sm 5.4 2.0:sm Nuc.LA

44-18 363 62-18 403 48-18 344 42-15 296

6.0 1.8:sm

5.1 l.7:m

5.1 3.0:m

40-12 235

7.2 2.2:sm

6.9 1.7:m

6.5 1.2:m

8.5 1.2:m

7.1 2.9:sm

7.0 1.6:m

5.9 1.l:m

6.8

6.2 1.4:m

6.1 2.6:sm

5.9 1.8:sm

5.5 1.2:sm Nuc.SA 5.8 1.l:m Nuc.LA 6.4 1.3:m

7.5 1.5:m

7.0 1.2:m

8.1 2.0:sm

7.7 1.6:m Nuc.SA 7.5 2.0:sm 8.1 1.8:sm 8.1 1.4:m 8.1 3.0:sm

6.9 2.5:sm Nuc.LA 6.9 7.2 1.9:sm 2.4:sm 6.7 7.4 2.0:sm 1.5:m 7.1 7.7 1.5:m 2.1:sm 7.1 7.4 1.0:M 1.6:m

6.4 I.9:sm 5.5 2.9:sm 6.8 2.1:sm 6.4 1.7:m

8.1 1.4:m

8.1 1.9:sm

7.2 1.3:m

6.0 1.2:m

6.8 1.l:m

Explanations as in Table 8. Nuc.LA and Nuc.SA mean nucleolus in long arm and short arm, respectively. Most values recalculated by author (see p. 234). c Data from Shastry et al. (1960a). d Data from Miatra, in Shsetry (1964a). * Data from Wu. in H u (1964). f Data from Sen (1963). u Data from Mkra and Shastry (1967). A Data from Karunakaran (1967). i Data from Chu (1967). i Data from Shastry and Rao (196la).

0

47-10 294

7.2 1.2:m

1.6:m

43-19 323

6.4 5.8 1.9:sm 1.4:m Nuc.LA 3.3 5.0 1.2:m 1.7:m

7.7 1.3:m

1,l:m 5.2 1.2:m 6.4 1.7:m 6.4 1.4:m Nuc.LA

240

N. M. NAYAR

of the complement was 296 p , and the length of individual chromosomes varied from 15 p to 42 p . The data are summarized in Table 10. There is considerable disagreement among authors about the characteristics of individual chromosomes and complements (of., Tables 8 and 9 ) . Although the data are fairly extensive, especially for a genus with small chromosomes, these have in most cases been gathered from a few cells only. The data presented by the same group of workers show understandably some correspondence. Also, the apparent similarity in relative length of the sativa chromosome complement is no more than what is expected for a set of 12 chromosomes which show an approximately continuous gradation in size differences ranging from about 4% to 14% in relative length. Many workers have expressed difficulties in locating the centromere, and this may explain the variations noted in arm ratios and also the disagreement in ordering of chromosomes experienced by the same worker in different studies. Sen (1963) and Misra and Shastry (1967) attempted to define chromomere patterns, but the presence and location of proper morphological markers on specific chromosomes is yet to be established. While Hu (1964) and Chu (1967) found constancy in the karyotypes of the sativa cultivars they studied, data of Misra and Shastry (1967) show significant variation in arm ratios, location of nucleolar organizers, etc., even among varieties belonging to the same area (northeast India). According to their data, the nucleoli in most case8 are located on long arms. They felt that subterminal translocations might account for the reshuffling of satellites in the chromosome complement and that other structural changes of the chromosome might have taken place in varietal differentiation. Since intervarietal hybrids (particularly those originating in the same region) hardly show any genetical or cytological evidence for reciprocal translocations and other gross structural changes, this seems to be an unlikely explanation. From the results of pachytene studies obtained so far, we can infer only that while the usefulness of prophase analysis for a qualitative determination of structural differences among different taxa has been established, following the work of Yao et al. (1958) and subsequently confirmed by several others, karyomorphological work in rice needs further refinement (cf. Sharma and Mukhopadhyay, 1965; Mitsukuri, 1966) before it can be used for the identification of individual chromosomes in applied cytogenetic work as has been done, for instance, in tomato and maize (cf. Rick, 1971). C. CHROMOSOME NUMBER 1. Haploidy

Haploids are so far known only from the two cultivated taxa. Morinaga and Fukushima (1931, 1932) obtained the first haploid plants

ORIGIN AND CYTOGENETICS O F RICE

24 1

in rice following hybridization between two japonica varieties Dekiyama and Bunketuto. Out of 13 seeds so obtained, one gave rise to a haploid plant. This was incidentally the second report of a haploid plant in cereals. Gaines and Aase (1926) had obtained a haploid of Triticum compactum after cross-pollination with Aegilops cylindrica. Soon after, Morinaga and Fukushima (1934) obtained six more haploids. Four of these were recovered from segregating populations (F,-F, generations) of intervarietal crosses, and two from fields grown with different varieties. They estimated the frequency of occurrence of spontaneous haploids as 0.0023%. As is generally the case with haploids, those in rice are reduced in size and sterile. There is a reduction in plant parts by about one-third to one-half. Tiller number, however, is increased. Rice (sativa) haploids can be maintained indefinitely by vegetative propagation. Morinaga and Fukushima ( 1934) propagated the haploids asexually and found that 37 panicles out of 13,800 produced 41 seeds under openpollinated conditions. The seed setting worked out to 0.82% (with 5009 spikelets in 37 panicles) and 0.0022% (133.25 spikelets per panicle in 13,800 panicles). When artificially pollinated, seed set increased to 1.06% after emasculation, and 2.41% without emasculation. Morinaga and Fukushima (1934) have described microsporogenesis and megasporogenesis of haploids in great detail. The course of cell division is similar to the general pattern of meiosis in haploids (cf. Kimber and Riley, 1963, for details of haploid meiosis). Univalents only were observed during M I in 98/135 microsporocytes. I n the rest, two, or rarely four, of I “appeared in contact making one or two loose pairs.” Most of these were taken to be artifacts or brought about by chance. The true bivalents were thought to have been formed as a result of a 0-12 distribution of univalents in A1 or by suspension of cell division. The A1 and subsequent stages showed many abnormalities, such as irregular separation, tripolar spindles, nonsynchronized second divisions, fusion of second division spindles, and so on. Since the seeds obtained from haploids gave rise to diploids only, the authors believed that only those megaspores having the full haploid complement of 12 chromosomes were functional. Several workers have subsequently obtained haploids-Ramiah et al. (1933b, 1934), Nakamura (1933), Takahashi (1936), by Beachell, Jodon, and Jones, in Jones and Longley (1941), Yasui (1941), Beachell and Jones (1945), Kuang (1951), Hsieh and Chang (1954), Katayama (1954), Takenaka et al. (1956), Hu (1957, 1958a,b, 1959, 1960a), Govindaswamy and Henderson (1956), Ho and Li (1966b3, and Chu (1967). The haploid isolated by Ramiah et al. (1933b, 1934) was the first one obtained by polyembryony in any species (Kimber and Riley, 1963). Ramiah et al., found a pure-line variety in which about 0.1%

242

N. M. NAYAR

of the seeds gave rise to two seedlings. I n one instance, one of the two seedlings turned out to be a haploid. The haploid of glaberrima reported by Hu (1960a) was also obtained by polyembryony. Here, both the seedlings were haploids. The haploids studied by others have been picked up either from field cultures of cultivars, or from progenies of semisterile cultures, or in segregating populations of intervarietal hybrids. Their modes of origin are therefore unknown, but they could be assumed to have arisen from some haploid component of the embryo sac. I n meiotic studies, Nakamura (1933) found only univalents. All others have corraborated Morinaga and Fukushima’s (1934) findings. Occasionally, 1-2 I are split into two or four half-univalents during MI (Jones and Longley, 1941; Hsieh and Chang, 1954). Hu (1960a) found also that haploids of glaberrima could not be maintained vegetatively, like their diploids. The appearance and behavior of glaberrima haploids were similar to those of sativa haploids. The average number of bi- and trivalents per PMC was 0.50. They also showed secondary associations. Out of 403 PMCs observed, 66% of the cells showed 12 I, 23% showed 10 I 1 11, 5% showed 8 I 2 11, 1% showed 6 I 3 11, 3% had 9 I 1 111, 1% had 7 I 1 I1 1 111, and the remaining 4 cells showed 7 I 2 I1 1 I11 (2 cells), 6 1 + 2 I11 (1 cell) and 4 I + 1 1 1 + 2 I11 (1 cell). Ho and Li (1966b) estimated that a haploid of sativa produced about 3.5% stainable pollen. Meiotic studies revealed that they were formed by unreduced gametes, which in their turn were produced either by a restitution nucleus or by nonsynchronization of nuclear division and cytokinesis. Data on prophase pairing during microsporogenesis of haploids can often provide more information on the genetic nature and origin of the taxon concerned. Chu (1967) attempted such a study in rice, but he presented only pooled data of association resulting from both chiasmata and crossing-over, and secondary pairing. The question of secondary association will be taken up in a later section. The formation of true bivalents, even if in low numbers, has been advanced as supporting evidence for the hypothesis of a secondary polyploid origin of rice. Bivalents and multivalents are regularly observed in low numbers even in such species as Secale cereale, Triticum monococcum, and Antirrhinum majus, which are considered to be genuine diploids. I n their review on haploid angiosperms, Kimber and Riley (1963) found that occasional bivalents or multivalents were formed during monoploid meiosis in 17 out of 34 truly diploid species. Levan (1942a), for instance, studied meiosis in six haploids of rye ( n = J: = 7). In one extreme case, one cell in pachytene showed complete pairing of chromosomes. The average number of chiasmata worked out to 0.33 per cell a t M I for

+

+ +

+

+

+

+

+

ORIGIN A N D CYTOGENETICS OF RICE

243

all haploids, and the maximum number of chiasmata was four per cell. The average number of chiasmata per cell ranged from 0.08 to 0.83 in different plants. Both genetically and environmentally determined differences may influence pairing. Chromosome pairing in haploids may be attributed to several causes-homology due to archaic polyploidy, the remote ancestors being extinct, aneuploid origin of the present chromosome number, or duplicated segments (Kimber and Riley, 1963). The probable reasons for the occurrence of univalents in low numbers in rice will be considered in the subsequent discussion on the basic chromosome number of rice. 6. Triploidy Since naturally occurring Oryza taxa are either diploids or tetraploids, new euploid types are formed either as spontaneous mutants or as products of hybridization. Autotriploids of sativa have been obtained both spontaneously and by hybridization between diploid and autotetraploid forms. Allotriploids in Oryza are produced in three ways, viz., from hybridization between a diploid and a tetraploid taxon, from backcrossing certain hybrids of diploid taxa to one of the parents, and from crossing an autotetraploid of one taxon with a diploid taxon. To the first category belong the hybrids obtained by crossing a diploid taxon like sativa to a tetraploid taxon like alta, minuta, or latifolia. Such hybrids have been discussed already. When F, hybrids, such as sativa-officinalis (AC) , sativa-brachyantha ( AF) , and sativu-australiensis (AE) , are backcrossed to sativa, allotriploids possessing two sativa genomes (AAC, AAF, and AAE) are formed. Ramanujam (1938~)obtained such a triploid with officinalis. He proposed that the F, hybrid produced unreduced diploid gametes by double division of the chromosome complement. The F, hybrid sativa-officinalis produces mostly univalents only a t MI. The triploid hybrid (sativa-officinalis) sativa showed 0-12 I1 (average of 7 11).Subsequent course of meiosis was highly irregular and only sterile gametes were produced. Ho and Li (1966a) found that the F, hybrid sativa-australiensis (AE) produced unreduced gametes by dyads omitting the second division. Since pollen stainability of F, hybrids was about 2747, this value was taken to represent the frequency of unreduced gametes formed. Upon backcrossing to sativa, triploid zygotes would be formed. The third category of allotriploids is produced by crossing an autotetraploid taxon with a diploid taxon. Two such cases are known, sativa-glaberrima (AA-As) (Oka, 1968) and sativa-longistaminata (AA-A”) . Results of the first one have been discussed earlier. The second hybrid was obtained by Chandrasekharan in 1959. Shastry and Misra

244

N. M. NAYAR

(1964) studied the frequencies of different types of multivalents in it. As already mentioned, longistaminata is almost self-incompatible and it is also difficult to produce its hybrids with other taxa. When crosses between longistaminata and other taxa were made, F, zygotes deteriorate 3-6 days after fertilization and this crossing barrier is controlled by a set of complementary dominant genes (Chu and Oka, 1970a). Hybrids of sativa and longistaminuta show some fertility. Meiotic abnormalities consist of univalents and lagging chromosomes only (Chu et al., 1969a). I n contrast, the allotriploid showed several abnormalities. The number of univalents ranged from 0 to 11 per PMC (mode about 3), of bivalents from 0 to 7 per PMC (mode about 3) and trivalents from 2 to 12 (mode about 7). Associations higher than quadrivalents were infrequent. Based on the predominance of ring bivalents and frying-pan-shaped trivalents, the authors proposed that chromosome complements of these taxa were only segmentally homologous, the differentiation having taken place through equal, unequal, and reduplicative translocations. Autotriploids of sativa have been studied by several workers. These have been obtained mostly as spontaneous mutants in segregating generations of intervarietal crosses or in established varieties. Triploids are also obtained along with haploids and tetraploids in populations raised from seeds or flowering panicles exposed to extreme temperatures and radiations (Ichijima, 1934; Beachell and Jones, 1945). Triploids are produced only with difficulty by diploid-tetraploid crosses. Okura (1940) obtained only diploid and tetraploid plants from such crosses. Morinaga and Kuriyama (1952, 1959a) obtained the highest success (1.09%) after 2x 0 X 4x 8 crosses when pollinations were done 24 hours after hot water emasculation. The seed set is usually better when tetraploids are used as female parents (Nagamatsu et al., 1964). It appears as though imperfect seeds give a higher recovery of triploid plants, especially when they are cultured in an artificial medium (Koga and Nagamatsu, 1967a,b). The primary interest in triploids lies in the fact that they give rise to trisomics. Nakamori (1932) obtained the first autotriploid of sativa in the eighth generation of an intervarietal cross. Autotriploids usually are characterized by broader and coarser leaves, thicker and taller stems, fewer tillers, larger spikelets, and short awns. The seed set is less than 4%, but some amount of parthenocarpic seeds are also formed. Autotriploids have also been obtained by Ramiah et al. (1963a), Ichijima (1934), Morinaga and Fukushima (1935), Capinpin (1938), Ramanuj am (1938b), Cua (1952b), Karibasappa (1961), Hu and Ho (19631, T. Katayama (1963), Hu et al. (1965), Sen (1965), Sahay and Sampath (1965), Nagamatsu et al. (19641, Koga. and Nagamatsu (1967a,b,c), Koga et al. (1967), and Watanabe et al. (1969).

245

ORIGIN AND CYTOGENETICS O F RICE

The most frequent type of chromosomal association obtained in autotriploids are trivalents, but their number varies in different instances. Ramiah et a2. (1933a) found usually 12 111, and univalents were seen only occasionally. Ichijima (1934) obtained 5 I11 10 I1 1 I. This means that some nonhomologous pairing has apparently taken place. Morinaga and Fukushima (1935) picked up more than 150 plants as possible triploids on the basis of their appearance. Of these, 107 plants turned out to be actually triploids. They studied both micro- and megasporogenesis. Usually 12 I11 were found a t M I , but sometimes up to 18 chromosomal bodies were observed. Second division proceeded more or less regularly, but the microspores mostly degenerated without developing into pollen grains. The lowest number of trivalents observed by Ramanujam (1938~)was 6, and the most frequent configuration was 10 I11 2 I1 2 I. One PMC showed a single hexavalent. The fryingpan type of trivalent was the most frequent. According to Hu and Ho (1963), about 10% of the PMCs showed nonhomologous chromosome pairing. They thought that this indicated partial homology between chromosomes within a genome. While about 24% of the cells showed exclusively trivalents, most commonly there were 10 I11 per PMC. The number of trivalents in two triploids studied by T. Katayama (1963) ranged from 8 to 12, and the remaining chromosomes appeared as bivalents and univalents. About 32% of the PMCs contained only trivalents. Sen (1965) could find w l y 3-7 I11 per PMC. Karibasappa (1961), who studied apparently the same material earlier, obtained, on an average, 9.4 I11 2.5 I1 2.5 I a t MI. A time lapse of 3-5 years cannot possibly account for this variation. Differences such as this, which are met with frequently in rice, may be due to either sampling error or to differences in personal judgment in analyzing clumped and crowded chromosome configurations. Sen also mentioned that there were a number of cases of nonhomologous pairing, but his data (in his Table 2) do not indicate this. Watanabe et al. (1969) obtained a high degree of trivalent pairing (mean 11 I11 1 I1 1 I ) . Trivalents alone were obtained in as much as 50% of the PMCs, and the lowest number of trivalents observed was seven (plus 5 I1 5 I ) . Autotriploids are expected to form predominantly trivalents, but in a group possessing small chromosomes like rice, their number may be lower because of reduced chiasma formation. [For a critique of this presumption, but with reference to autotetraploids, see Morrison and Rajhathy (1960) .] The average number of trivalents obtained in various studies ranges from 5 to 11, but it is mostly 9-10 111per PMC. Associations higher than trivalents were found by Ramanujam (1938~)and by Hu and Ho (1963), and between them they obtained only 3 VI. These results, by themselves, may be taken to mean that triploids of

+

+

+

+

+

+

+ +

+

246

N. M. NAYAR

sativa behave like true autotriploids. The question of nonhomologous pairing will be taken up later. 3. Tetraploidy

Two kinds of tetraploids will be considered here: (a) naturally occurring and induced tetraploids of cultivated rice and wild taxa, and (b) allopolyploids and intervarietal polyploids of japonica-indica hybrids. Two other types of tetraploids have been discussed already: (a) those obtained by crossing an autotetraploid of a taxon with a tetraploid taxon, e.g., the hybrid tetraploid officinalis-minuta, and (b) tetraploid plants obtained upon backcrossing a triploid interspecific F, hybrid (A-CD) such as sativa-ZatifoZia to sativa where a hybrid of constitution AACD is obtained. a. sativa Tetraploids. There have been several studies on tetraploids in Oryza, mainly of the cultivated species sativa, with the aim of practical utilization. Most studies have been terminated within 1-3 generations, and there are no published accounts of any observations made beyond about the tenth generation. Most of the work is descriptive in nature. Before the usefulness of colchicine to induce polyploidy came to be known, tetraploids were being recovered either spontaneously (Ramiah et al., 1935; Morinaga and Fukushima, 1937) or from progenies of intervarietal crosses (Nakamori, 1933; Morinaga and Fukushima, 1937) or after irradiation, heat treatment, or chloral hydrate treatment (Ichijima, 1934; Matusima, 1935; Morinaga and Fukushima, 1937). Nakamori (1933) obtained an autotetraploid plant in the F, generation of an intervarietal cross. It did not show any “remarkable abnormalities.”‘It had enlarged spikelets and strongly developed awns. It showed 27% spikelet fertility. The tetraploid obtained by Ichijima (1934) showed 5-6 IV and 10-12 I1 a t meiosis. Morinaga and Fukushima (1937) picked up several tetraploids from cultivators’ plots, in segregating generations of invarietal crosses, and after chloral hydrate treatment. They made detailed studies of their morphology, fertility, and micro- and megasporogenesis. The autotetraploids of sativa are usually shorter than their diploids. Their culms and leaves are thicker and coarser, the number of ear-bearing tillers is fewer, spikelets are bigger awned and more persistent, and pollen grains are larger. The pollen stainability was about 50%, and spikelet fertility about 22%. About 7-36% seeds were formed by parthenocarpy, and only about 45-75% of the seeds germinated. Micro- and megasporogenesis were generally similar. At MI, 8-9 IV and 6-8 I1 (range: 4 1 2 IV) were seen. Only rarely were an octovalent and univalents observed. The rest of meiosis was regular except for 1-2 lagging univalents a t AI. However about half of the microspores and 40% of the megaspores degenerated after the second division.

ORIGIN AND CYTOGENETICS OF RICE

247

Tang and Loo (1940) induced tetraploids of sativa by treating 2-dayold seedlings with colchicine solution. Subsequently, several workers described various modifications for inducing polyploidy by the use of colchicine (Beachell and Jones, 1945 ; Hedayatullah and Ghosh, 1946; Heyn, 1947; Ghosh, 1948, 1950; Tabata and Kuriyama, 1949; Cua, 1951c; Jacobs, 1952; Oka, 1953d; Nyst, 1959; Bouharmont, 1961a,b; Ru and Kung, 1963b). All these methods are similar to those that are generally available in literature. The development and morphology of induced tetraploids are similar to those of naturally occurring ones. They are somewhat slower than diploids in germination and show a lower germination rate (Cua, 1950, 1952b ; Oka, 1954b).Varieties respond differently to polyploidization with regard to various characters, such as period of maturity and spikelet fertility (Cua, 1951a; Kuriyama and Watanabe, 1952; Oka, 1954a,b). Progenies of tetraploids show a greater range of variation than those of their diploids. Tetraploidy also depresses their competitive ability (Sakai and Uchiyamada, 1957). The meiotic behavior of induced tetraploids is also similar to that of naturally occurring tetraploids (cf. Hsieh, 1952; Chang and Hsieh, 1954; Oka et al., 1954; Bouharmont, 1963rt; Richharia and Govindaswamy, 1963a ; Rangaswamy and Devasahayarn, 1971). Several developmental abnormalities have also been seen to affect the embryo and endosperm of tetraploids (Bouharmont, 1964; M. Takahashi, 1955). Bouharmont (1963a,b) observed that the number of chromosomes in rice associated together (63-78%) was much higher than that of most other diploid species. H e felt, however, that some of the associations were not true quadrivalents in that chiasmata might not have been formed between them. H e called them pseudoquadrivalents, and suggested that they, like pseudobivalents, were formed by a fusion of chromosome matrix. Richharia and Govindaswamy (1963a,b) found that autotetraploids of sativa varieties did not show any segregation in the C, generation for either morphological characters or chromosome number. A certain degree of selection had been practiced in this material in earlier generations for improved fertility. The average number of quadrivalents was 7-8 per PMC. Matai in 1955 had determined that the C, generation showed 8-9 IV per PMC. There was thus only a slight reduction in quadrivalent frequency after 8 generations of selection. Bouharmont (1963a) obtained a similar result. Jones and Longley (1941) and Beachell and Jones (1945) felt that tetraploid sativas were unlikely to be of any practical utility (also see Siregar, 1953). But others have been more optimistic. Generally, fertility and vigor are improved when autotetraploids are intercrossed. Meiotic irregularities are also reduced. The fertilities of intervarietal autopolyploid hybrids are usually inversely correlated with those of their

248

N. M. NAYAR

diploid F, hybrids (Morinaga and Kuriyama, 1946, 1949; Cua, 1951b, 1952a,b; Hsieh, 1952; Oka, 1954b,c; Oka et al., 1954; Nagamatsu, 1954; Masima, 1952; Masima and Uchiyamada, 1955a,b; Masima e t a!., 1958) I n Cua’s (1952) studies, three intra-japonica hybrids which showed 91-94% diploid F, hybrid fertility gave only 3543% seed setting in their tetraploids. The seed fertility of the tetraploid F, generation varied from 7 to 64%. Of two japonica-indica hybrids, the first had 55% diploid F, fertility and 38% tetraploid F, see setting; their tetraploid F,s segregated 24-78% for seed fertility. In the second hybrid, the respective values were ll%,70%, and 12-91%. The mean number of quadrivalents in autotetraploids and japonica-indica tetraploid hybrids were nine and six, respectively. Cua (1952a) felt that “chromosome differentiation or heterozygosity was the cause of this increase in fertility” and thought that it would be possible to select superior tetraploid types from interracial hybrids. Masima and colleagues also obtained similar results. Their studies were more extensive. The causes of reduced fertility in a tetraploid were estimated to be due 15-20% to gametic sterility, 15% to envirionmental effects, and 50% to zygotic sterility (Masima, 1952). In a study of 16 autotetraploids and 41 tetraploid hybrids extending for five generations, fertility was found to be heritable and selection consequently effective. The highest fertility obtained was 79%. A few tetraploids were equal and even superior to their corresponding diploids in agronomic features. This improvement in fertility was attributed to reduction in quadrivalent frequency (Masima and Uchiyamada, 1955a,b). Based on these studies, they proposed that japonica and indica originated by structural changes of chromosomes such as small inversions and translocations. Since polyploidy reduced quadrivalent frequency, preferential pairing of chromosomes was assumed. Other workers have also observed low quadrivalent frequency in intervarietal autopolyploids (Yen and Pao, 1960; Thakur and Rao, 1966; Rangaswamy and Devasahayam, 1971). Oka (1955b3, on the other hand, advanced a genetic explanation for the reduction in quadrivalent frequency and increase in vigor obtained in tetraploid hybrids. In consonance with his theory of the nature of sterility in interracial hybrids, he attributed the reduction in quadrivalent frequency and increase in fertility also to complementary action of genes. Oka’s (1954a,b,c; also see Oka e t aZ., 1954) results are broadly similar to those obtained earlier by Cua and by Masima and colleagues. He found, however, no correlation between increased fertility and reduced quadrivalent number. Although he found that a great part of variation in fertility was heritable, and used plants with high fertilities to raise subsequent generations, it is not clear how mean fertility could

ORIGIN A N D CYTOGENETICS O F R I C E

249

go down in succeeding generations. Further, he proposed that no selective pairing of chromosomes was taking place during meiosis in these hybrids because four monogenically inherited characters showed segregational ratios between 35: 1 and 20:8: 1 and there was, besides, no deficiency of recessive genotypes. Although the studies of tetraploids in rice have not been done intensively and continuously as in other plants, such as barley, rye, and Dactylis, certain observations stand out. The average number of multivalent associations in rice autotetraploids (8-9 out of possi8ble 12 IV) is much higher than that obtained in other diploid species. For instance, the number of quadrivalents obtained by various workers is 1.9-3.8 in Hordeum vulgare, 1.1-3.9 in Secale cereale, and 3 . g 3 . 9 in Dactylis glomerata, in all cases out of a possible 7 IV (cf. Morrison and Rajhathy, 1960). Since the obscrvations of Kostoff (1940), it has been widely assumed that tetraploids of plants possessing small chromosomes form fewer quadrivalents than those with larger chromosomes, but Morrison and Rajhathy (1960) found that the opposite was probably more true in their studies of ten species. The rice data also support Morrison and Rajhathy’s findings. I n spite of the occurrence of large numbers of quadrivalents during M I of autotetraploids, the further course of meiosis is largely normal. Bouharmont (1963a,b) estimated that 15% of A1 showed irregular separation. The correlations for vigor and fertility observed by several workers also suggest that a proportion of the plants in early generations may be aneuploids. This aspect does not appear to have been studied in rice. Since fertility has been found to be heritable, it should be possible to carry out selection for economically useful types. Several methods for improving fertility of autotetraploids have been proposed (Morrison and Rajhathy, 1960; Ellerstrom and Sjodin, 1963; Reinbergs, 1964; Bender and Gaul, 1966; Sybenga, 1969). These methods need to be tested in rice also. The possibilities for the exploitation of autotetraploids do not appear to have been fully explored in rice. b. Tetraploids in Other Taxn. Ramiah et al. (1935) obtained a spontaneous tetraploid plant of perennial rufipogon. Its general features and cytogenetics were similar to those of sativa tetraploids. Hinata and Oka (1962~)produced a tetraploid of a glaberrima cultivar by colchicine treatment of seeds (also see Oka, 1968). Tetraploids have also been produced in australiensis and oficinalis (Gopalakrishnan and Shastry, 1964; Hu, 1967). As mentioned earlier, the autotetraploid officinalis showed a low number of quadrivalents and high seed set (p. 211). c. Allotetraploids. The usefulness of allopolyploids in determining species relationships has been amply proved in such genera as Gossypium and Nicotiana. They have been used only to a limited extent in Oryza.

250

N. M. NAYAR

The first allopolyploids to be obtained were those of glaberrima-sativa and sativa-breviligulata (Nayar, 1958; Gopalakrishnan, 1959; Gopalakrishnan et al., 1964). They were produced by treating buds of sterile F, diploid plants with colchicine. Both allotetraploids showed about 75% pollen stainability. The seed set was 47% in glaberrima-sativa and 13% in sativa-breviligulata. They showed about 6-8 IV during meiosis, The glaberrima-sativa amphidiploid was studied again in the Ca generation (Richharia and Govindaswamy, 196313). There was no change in quadrivalent frequency, but spikelet fertility was increased to 62%. Hinata and Oka (1962~)produced a sativa-glaberrima amphidiploid by crossing the autotetraploids of the two taxa (see Oka, 1968). The results have been reviewed already. Sampath (1969a) obtained allotetraploids of sativa-officinalis, puractata-eichingeri, and punctata-uustraliensis. Their cytology has not been reported.

4. Higher Ploidy Watanabe and Ono (1967, 1968) obtained three allohexaploids,

sa.tiva-latifo1ia (AA-CCDD) , sativa-m,inuta (AA-BBCC) , and minutaaustraliensis (BBCC-EE) . They were obtained by crossing-induced autopolyploids of the constituent taxa. All 9 combinations of sativa-latifolia and sativa-minuta polyploids were male-sterile. Interestingly, when hexaploid sativa-Zatifolia (AA-CCDD) was backcrossed to octoploid latifolia (CCCCDDDD) , an alloheptaploid was produced (ACD-CCDD)

that was 30% seed-fertile (Watanabe and Ono, 1968). Hybrids between A and C (D being a variant of the C genome) a t all lower levels of ploidy ( 2 ~ 6 s are ) sterile. The results of the sativa-australiensis amphidiploid have been presented already. Watanabe and Ono (1965, 1966) produced octoploids of minuta and latifolia by colchicine treatment of seeds. The expression of a gigas effect was manifest at this level only in the siee of spikelets, stomata, and pollen grains. The two octoploids differed in their meiotic behavior and fertility. These have been discussed already. Their allopolyploids were obtained by Morinaga et al. (1964). Ho and Li (1964) obtained an octoploid plant, but its identity was uncertain.

6. Aneuploidy

Most of the little work done has been directed toward developing trisomics in sativa. These have been isolated from progenies raised from selfed seeds of triploids, or from hybrid seeds obtained by crossing them with diploids. Nakamori (1932) raised a selfed progeny of twelve plants from a triploid plant. Their somatic chromosome number varied from 25 to 29. He was able to obtain eight different types of trisomics by 1949

251

ORIGIN A N D CYTOGENETICS OF RICE

(cf. T. Katayama, 1963; see also Yunoki and Masuyama, 1945). Ramanujam (193813) collected 150 seeds from a triploid plant. Of these, 60 seeds germinated and 50 seedlings grew to maturity. This population gave nine trisomics. Meiotic studies in trisomics showed 12 I1 1 I or 11 I1 1 111, as expected. If the univalent lay at or near the metaphase plate, it divided at A1 and the split halves migrated to either pole. Otherwise, it was included in one of the nuclei. If a trivalent was present, the three homologues separated normally. T. Katayama (1963) used two autotriploids, two completely asynaptic plants, and two partially asynaptic plants. Progenies were raised from open-pollinated seeds and from seeds obtained from triploid and diploid crosses. Open-pollinated seeds of partially asynaptic plants produced more trisomic plants. Progenies of triploid plants showed variations in chromosome number of 25 to 33; most progenies of asynaptics (both complete and partial) were diploids, and the rest had either 25 or 26 chromosomes only. Microsporogenesis of triploid plants was similar to that already described. The higher heteroploid plants showed variable numbers of trivalents and univalents. Katayama felt that asynaptic plants were superior to triploids for obtaining trisomics. Earlier, Jones and Longley (1941) also had obtained plants with 25 and 26 chromosomes in progenies of highly sterile diploid plants. Katayama (1966a) did not find a correlation between seed weight and somatic chromosome number. But Koga et al. (1967) found that perfect seeds produced plants that had relatively low chromosome numbers, while imperfect seeds produced plants with relatively high aneuploid numbers (also see Koga and Nagamatsu, 1967a,b,c). Jachuck (1963) interplanted stubbles of a triploid plant among diploid plants. I n the progeny of the triploid were plants with 25, 26, or more chromosomes, including a tetraploid (also see Karibasappa, 1961). The plants with 26 chromosomes were either tetrasomics (212 2 ) , or more frequently, double trisomics (2n 1 1). Incidentally, all the 26-chromosome plants obtained by T. Katayama (1963) were double trisomics. Sen (1965) studied the same material as used by Karibasappa and Jachuck. In the progeny, 11.6% of the plants were trisomics. He identified five trisomics on the basis of chromosome morphology. However, their morphological descriptions as given in two tables do not agree with each other for many characters. The pollen stainability of the five trisomics varied from 27% to 82%, and spikelet fertility from 23% to 72%. Watanabe et al. (1969) obtained 1% seed set in a triploid upon self-pollination, 5% on open pollination, and up to 14% upon pollination with a diploid. I n the progeny, 40% were trisomics and their pollen stainability ranged from 30 to 100%. It was less than 30% when the chromosome number was 27 or more. H u (1968) obtained 98 trisomic plants in the progeny of a triploid plant of a Taiwanese

+

+

+

+ +

TABLE 11

Frequencies of Aneuploid and Euploid Plants in Progenies of Triploid Plants

Rice Ramsnujam (1938b)

T. Katsyama (1963) Nakamore. in Wstanabe d. (1969) Koga et al. (1967)

el

H u (1968) Watansbe et d. (1969)

Total Expected Tomato Rick and Earton (1954)

No. of plants atudied ( %age)

Chromosome number Number of plants Percentage of plants

50 100.0 21 100.0 170 100.0 49 100.0 140 100.0 87 100.0

9 2n 10 2n 11 371 = 36 Other 2n = 24 2n 1 2n 2 2n 3 2n 4 2n 5 2n 6 2n 7 2n 8 2n 6 9 8 d 11 4 3 0 0 0 0 0 0 0 12.0 18.0 16.0 18.0 22.0 8.0 6.0 3 6 8 1 2 1 0 0 0 0 0 0 0 0 14.3 28.5 38.1 4.8 9.5 4.8 4 24 53 49 28 6 3 1 1 0 0 0 1 0 2.4 14.1 31.1 28.6 16.4 3.8 1.8 0.6 0.6 0.6 1 5 9 12 4 1 2 2 3 4 4 0 0 2 8.2 2.0 4.1 4.1 6.1 8.2 8.2 4.1 2.0 10.2 15.3 24.5 20 42 61 14 1 0 1 0 0 0 0 0 0 1 0.7 30.0 43.6 10.0 0.7 0 0.7 14.3 1 0 0 0 0 0 0 0 6 35 31 12 2 0 7.0 40.2 35.6 13.8 1.1 2.3

_

_

5 17 100.0

100.0 799 99.9

+

+

+

+

+

+

+

+

+

+

+

-

-

-

-

-

-

40 7.7

-

0.02

-

-

-

121 170 23.4 32.9

-

-

97 18.8

-

-

-

48 9.2

12 2.3

10 1.9

3 0.6

4 0.8

22.8

19.5

12.2

0.3

1.1

5.5

12.2

19.5

+

+

+

+

+

+

+

+8

2n = 24 2n 1 2n 2 2n 3 2n 4 2n 5 2% 6 2n 7 2n 0 0 303 342 13 7 0 0 0 37.9 42.7 16.4 0.9 -

-

4 0.8 5.5 2n + 9 0

-

-

4 0.8 1.1

0

-

-

3

0.2

0.6

0.3

0.02

0

0

+ 10 2n 4- 11 3n = 36

271

0

-

-

-

1

-

-

F

z 9

!d

Maize McClintock (1929) Punyaeingh (1947) Total Sugar beet Levan (1942b)

284 317 60 1 99.5

82 100.1

Snapdragon Sampson el 02. (1961)

582

Expected

582

100 Petunia Levan (1938) Rick (1971) Total Barley Tsuchiya (1960~) Spinach Janick et al. (1959)

2n=202n+12n+22n+32n+42n+5 2n+6 88 73 52 23 7 5 34 4 28 61 70 75 42 30 - - __ 9 1.5

62 10.0

149 24.8

143 23.8

127 21.1

65 10.8

2n=182n+12n+22n+32n+42n+5 5 4 22 14 8 19 6.1 4.9 26.8 17.4 9.8 23.2

+

2n+9 0 0 -

-

-

1

8

1.3

2n+6 4 4.9

2n+7 3 3.7

+ 3 2n + 4 2 n +

5 2n + 6

2n + 7 >

79 13.6

3n = 24 >

562

178 100

2n = 14 2n 1 2n + 2 2n + 3 2n + 4 2n + 5 2 n + 6 3n = 21 18 35 24 5 5 2 1 0 8 2 J 4 3 1 2 3 420 26 63 55 17 8 6 3 0 14.6 35.4 30.9 9.6 4.5 3.4 1.6 0

56 100.1

2n = 14 2n 1 2n 2 2n 3 2n 4 2n 5 In 6 3n = 21 Others 28 1 9 3 0 0 1 5 9 16.1 50.0 1.8 16.1 5.4 1.8 8.9

327 99.9

2n=122n+12n+22n+32n+42n+53n=18 90 118 23 3 9 41 43 2.8 12.5 13.1 27.5 36.1 7.0 0.9

90 88 -

+

+

+

+

+

+

0

2 n + 8 3n=27 2 1 1.2 2.4

--

2n = 16 2n 1 2 n + 2 2n 274 229 47.1 39.3 2 18

+

37 6.2

2n+7 2n+8 2 0 6 1 -

3n=30 0

0 0

-

254

N. M. NAYAR

indica variety, Kehtze, and classified them into twelve types. I n his study, 37% of the progeny survived to maturity and of these 30% were trisomics and 44% had 2n = 26 chromosomes. Nearly half the progeny of these 2 n + 2 plants also turned out to be trisomics. The twelve trisomic p!ants have been identified on the basis of their vigor, habit, stature, rolling of leaves, panicle and spikelet shape, and awning. They are generally shorter (81-110 cm vs 108 cm in diploid), later in maturity, and show variability in size of spikelets and panicles (16-20 cm long vs 18 cm in diploid and 100-165 grains per panicle vs 154 grains in diploid). The pollen stainability ranged from 68% to 95%. The transmission frequencies of the extra chromosomes upon selfing the twelve trisomics varied from 27% to 48%. Rice trisomics are similar to barley trisomics in these respects (cf. Tsuchiya, 1960b). A second set of twelve trisomic lines has been described in japonica rice by Iwata et al. (1970). They have been isolated in four cultivars, eleven from progenies of triploids and the twelfth from those of tertiary trisomics. Spikelet characters showed relatively less variation due to environment, and the authors felt that they were the most suitable characters for the identification of trisomics. The trisomics in rice have yet to be correlated with chromosome complement. The twelve possible linkage groups have been proposed for both japonica (Nagao and Takahashi, 1960) and indica (Misro et al., 1966) rices, but their identity with the haploid chromosome complement remains to be established. Thus, rice is way behind such crop plants as barley, maize, and tomato in this respect. There have been only a couple of reports of monosomics and nullisomics. Anandan and Krishnaswamy (1934) isolated a culture which segregated roughly in the ratio 3 : l for apparently normal plants and nonflowering plants. They termed the latter “barren sterile”. Some of the apparently normal-looking plants continued to segregate in similar fashion in successive generations. Somatic chromosome counts showed that the barren plants were nullisomic with 2n = 22 chromosomes (Sampath and Krishnaswamy, 1948). The authors suggested that a monosomic plant (2n = 23) would produce gametes with 11 and 12 chromosomes. Upon recombination, these gametes would produce plants with 24, 23, and 22 chromosomes in the ratio 1:2:1. The monosomic plant was morphologically similar to the normal diploid plant. The nullisomic plant, on the other hand, was stunted and produced no panicles. Chandrasekharan (1952) obtained a plant in a progeny of the above, in which some tillers appeared normal and the rest were nonflowering. The normal-looking tillers were found to be monosomic, and nonflowering tillers were nullisomic. During meiosis, the monosomic sectors showed 11 I1 1 I at diakinesis and mostly 12:ll chromosome separation a t

+

255

ORIGIN A N D CYTOGENETICS OF RICE

AI. The further course of meiosis was normal. The progeny of this monosomic segregated into normal-looking plants and nonflowering plants, and some plants were sectorial for monosomic and nullisomic condition, like the originally isolated plant. The second monosomic plant appeared as a sterile plant in the F, generation of an indica-japonica hybrid (Seshu and Venkataswamy, 1958). Upon vegetative propagation, it produced a few seeds. Six plants out of 45 raised from these seeds were all diploids, but in the PMCs they often showed 10 I1 1 IV indicating the presence of a reciprocal translocation. This suggested that only those gametes possessing the full haploid complement of 12 chromosomes were functional. Other kinds of aneuploid plants with variable numbers of chromosomes have been obtained by several workers in the progeny of triploid-diploid (2n = 36 X 2n = 24) crosses. The data are summarized in Table 11. For comparison, data from triploid-diploid crosses made in some other diploid species are also given. The results show that rice occupies an intermediate position between species like Petunia (2n = 14), on one hand, which is able to tolerate the complete range of aneuploidy from 2n to 3n, and those like Lycopersicon (2n = 24), on the other hand, which can tolerate only three extra chromosomes (up to 2n 3 ) . Rice is similar to Hordeum and Zea in this respect. The possible implications will be discussed in the following section. The characteristics of these aneuploids have not been studied in rice. There are also two reports of polysomaty or mixoploidy. The first case was observed in the roots of a sterile triploid interspecific hybrid (2n = 36), sativa-eichingeri (possibly tetraploid punctata). As the hybrid was sterile, it was being maintained vegetatively over the years. I n one year the root tips showed variation in chromosome number from 32 to 36 (Sampath, 1950). The second one was observed in microspore mother cells of a plant that was picked out from the field for its sterility and gigas character. Most PMCs showed 36 chromosomes, but one had 25 chromosomes and the remaining PMCs had 24 chromosomes (Sahay, 1963). Such mixoploid cells arise owing to irregularities in cell division, which can be caused by several factors. This has been reported from time to time in other organisms also (cf. Swanson, 1957).

+

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VII. Other Studies

A. SECONDARY ASSOCIATION This has been a topic of continuing interest to rice cytogeneticists.

It was in rice that secondary association of chromosomes was first clearly

256

N. 111. NAYAR

documented. Kuwada (1910) found that, in the second division of meiosis, 2 and sometimes even 3 chromosomes formed themselves into groups. He did not know their biological significance and termed them “pseudogemini” because the most frequent association was between 2 chromosomes. Darlington in 1928 called it “secondary pairing.” Lawrence (1931) then proposed that it indicated more remote affinities between chromosomes and suggested a polyploid origin of the species in which this was found. Based on Kuwada’s (1910) observation of secondary association and of Chao’s (1928) finding that certain characters in rice are controlled by duplicate genes, Lawrence (1931) advanced the theory that rice is a secondary polyploid derived from a form with seven pairs of chromosomes. Yamaura (1933) and Sethi (1937) supported the polyploid origin, but they felt that two species possessing 5 and 7 chromosomes were involved. But the strongest support for a polyploid origin of rice was given by Sakai (1935) and Nandi (1936). They proposed that rice was a secondary balanced allotetraploid which originated through hybridization between two species, both having 5 chromosomes, with 2 chromosomes duplicated. They observed a maximum association of 5 , comprising two groups of 3 chromosomes and three groups of 2 chromosomes [2(3) 3(2) 1. Nandi thought that the somatic chromosome morphology of rice also agreed with this proposal. The pairing observed by Ramiah et al. (1933b) in haploid rice and the occurrence of duplicate genes were mentioned as additional points of evidence. Parthasarathy (1938) and Okuno (1944) further supported this on the basis of their own findings. At the same time, Morinaga and Fukushima (1932, 1934) did not find any kind of affinity among chromosomes in the haploids that they examined. Hirayoshi’s (1957) study of Oryzeae and Zizanieae also showed that association occurred randomly. He even saw an association of all chromosomes. In recent years, Bouharmont (1962b3, Hu (1962), Katayama (1965a), and Chu (1967) have gone into this question in detail. Their results are conflicting. Bouharmont (1962b) considered the earlier work on secondary association, chromosome morphology and haploid chromosome pairing and supplemented them with his own observations on somatic and meiotic chromosomes of Oryza species. H e did not feel that there existed a case for considering rice as a polyploid species. Hu (1957, 1958b, 1960a, 1961, 1962) observed chromosomal associations in haploids of the two cultivated taxa and also in eight wild taxa. His observations supported the proposition of the basic chromosome number of rice being 5. I n haploid sativa, Hu (1958a,b) found a maximum pairing of 1 1 1 + 2 ( 3 ) + 2 ( 2 ) or 2(3) + 3 ( 2 ) . I n haploid glaberrima, it was 2 I11 1 I1 l ( 2 ) 2(1), with the 2 I attached to the nucleolus (Hu,

+

+

+

+

257

ORIGIN A N D CYTOGENETICS OF RICE

1960a). He took the two nucleolar univalents as one group, thus the maximum association observed in haploid rices was interpreted as five. Hu (1961, 1962) then studied the association of bivalents in PMCs of the two cultivated taxa and eight wild taxa (including subulata, which is now excluded from Oryza, and ridleyi, a tetraploid taxon). I n the diploid taxa, the highest association observed in 367 PMC was again 2(3) 3 ( 2 ) . The association data obtained in n’dleyi (2n = 48) differed significantly from the expected frequencies based on random distribution. Based on his own cytological observations and on the occurrence of several duplicate genes in rices (including those responsible for interracial hybrid sterility in sutiva, as suggested by Oka) , H u supported the hypothesis of the basic chromosome number of rice being 5. He felt that the secondary polyploid, nature of rice endowed the genus with a large potentiality for diversification. Hu’s (1962) data on bivalent association in 367 PMCs of seven diploid taxa show that they arranged themselves in 15 combinations. Of these, the association of 2(3) 3(2) was realized in only 8 PMCs. I n 5 cells, no association was found. In lO(1). A maximum of 76 cells showed another 13 PMCs, i t was l ( 2 ) l ( 3 ) 2(2) 5 ( 1). Katayama (1965a; also see Kudo and Katayama, 1965) and Chu (1967) made even more intensive observations. They saw that bivalents associated themselves in a large number of combinations in which several associations were higher than 2(3) 3 (2). They opposed the hypothesis that the basic chromosome number of rice is 5. Katayama (1965a) studied 2000 PMCs each at diakinesis and “diametaphase” or “diametastage” (possibly, prometaphase) in a japonica variety. Associations were more clearly seen a t prometaphase. He observed as many as 66 types of association out of a possible 77 types of association. Only 23 cells showed the association of 2(3) 3(2) a t prometaphase and 106 PMCs showed no association at all. An association of 2(2) 8(1) was seen in a maximum number of cells, viz., 211 PMCs, and another 194 PMCs showed l ( 3 ) 2(2) 5 ( 1 ) . When the association data were statistically analyzed on the basis of three types of randomness, general random association, linear random association and circular random association, the association was not found to be at random, but it did not support the hypothesis of a basic number of 5 either. Chu (1967) studied chromosome association in 680 PMCs of haploid japonica varieties. He observed 30 types of association. Only four PMCs showed 2(3) 3 ( 2 ) , while 36/680 cells showed higher associations than this and 79 cells had chromosomes randomly distributed. The results obtained by various authors show that while rice chromosomes do show some kind of association in most cells, there is no consistency in the associations. The evidence from secondary association

+

+

+

+

+

+

+

+

+

+

+

258

N. M. YAYAR

is inconclusive to support the hypothesis that rice originated by secondary polyploidy from one or two species that had 5 or 7 chromosomes.

B. NUCLEOLUS NUMBER In his study of several japonica rices, Kuwada (1910) found generally one nucleolus in the majority of PMCs. Selim (1930), who was a student of Gates, thought that japonica and indica rices showed specific differences in their nucleolar numbers. He studied five varieties and found two nucleoli in PMCs of indica varieties and one in japonica varieties. Gates believed that higher numbers of nucleoli were indicative of polyploidy, and his influence is apparent in the works of Nandi (19371, Parthasarathy (1938), and Pathak (1940). These authors described differences in number, shape, and size of nucleoli during the course of mitosis in several rice varieties. They found up to four nucleoli in a cell. Sakai (1938) proposed that japonica rices were binucleolar and indica rices quadrinucleolar. Oka (1944) and Oka and Kao (1956) studied nucleolar number in root tips of more than 100 sativa varieties. The average number of nucleoli varied from 2.0 to 3.6 per cell. Japonica and indica rices did not show any absolute differences in nucleolar number, but M i c a and bulu varieties possessed higher nucleolar numbers than japonica rices. The variation in nucleolar number was continuous. Shinohara (1962) extended the studies to rufipogon (annual and perennial) , glaberrima and barthii (as breviligulata). The African taxa were binucleolar (fewer than 2.1 nucleoli per cell) and rufipogon and indicn rices were predominantly quadrinucleolar. Shastry and colleagues observed the number and shape of nucleoli during pachytene stage in some wild and cultivated rices (Shastry et al., 1960a; Misra and Shastry, 1967). One, and sometimes two, nucleoli were seen, but, in addition, they found 2-6 small nucleolar bodies (sometimes up to 18) which stained similarly as the nucleoli. They compared them to the nuclear bodies (not nucleolar, as termed by the authors) observed by Walters (1963) in several Bromus species, but termed them instead “supernumerary nucleoli.” About a third of these were attached to chromosomes and the rest lay free. They proposed that evolutionarily advanced species might show more of these bodies consequent to increased competition in nucleolar activity in them as a result of chromosomal structural changes undergone by them. Walters (1963) found usually only one nucleolar body in a cell, and they were not present in somatic cells. It remains to be seen whether the bodies observed by Shastry and colleagues may be better comparable to the prenucleolar bodies observed in certain organisms.

ORIGIN A N D CYTOGENETICS O F RICE

259

C. CHIASMA FREQUENCY In Kuwada’s (1910) account of meiosis, the paired chromosomes were described as being ring- or X-shaped, later becoming square or dumbbell-shaped. These observations implied that they had either two or one chiasmata. Nandi (1936) estimated that the bivalents formed 1-3 chiasmata each. The mean number of chiasmata diminished from 1.7 per bivalent in early diakinesis to 1.5 at MI. The number of terminal chiasmata per bivalent was 0.9 in diakinesis and 1.2 in MI with terminalization coefficients of 0.55 and 0.83. Soriano (1961) also obtained about the same results, viz, 14-22 chiasmata per cell in M I of five indica varieties. Chu et al. (1969a) estimated chiasma frequency during diakinesis in two taxa, a sativa cultivar and a perennial rujipogon from India, and in twenty interspecific hybrids within the section Sativa. The mean number of chiasmata in the sativa variety was 1.98 z!z 0.65, in perennial rujipogon it was 1.91 & 0.61, and in the twenty hybrids it varied from 1.08 A 0.62 to 1.83 & 0.59. All hybrids showed normal pairing during meiosis, but they were mostly sterile. For instance, only three hybrids had more than 6% stainable pollen and as many as 11 hybrids were completely sterile. The data do not reveal any relation between pollen stainability and chiasma frequency.

D. ASYNAPSISAND DESYNAPSIS Plants that show reduced amounts of chromosome pairing during meiosis have been isolated in rice from time to time. They arise either spontaneously or, more frequently, in progenies of mutagen-treated material. Ramanujam and Parthasarathy (1935) found a sterile plant with erect habit, shorter stature and nonemergent panicles in a pure-line variety Co 4. It was both male- and female-sterile. It formed no bivalents a t diakinesis and MI. The spindles were of different sizes and shapes, and A1 separation was irregular. Consequently, dyads of unequal sizes and triads were formed. The second division was equally irregular, with the result that microspores of varying size and number were formed. Because of its complete sterility, its genetics could not be studied. Sakai (1940) observed asynaptic cells in three varieties. More of them were found at diakinesis than a t MI. H e attributed these to weather conditions. Chao et al. (1960) found seven sterile plants in a M, progeny of 34 plants after neutron irradiation. They set less than 1% seeds while their sib plants set about 80% seeds. Their chromosomes paired normally at pachytene, but they showed 10 I at diakinesis and 7 I a t M I . In

260

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addition, small chromosomal bodies, which were apparently fragments, were observed. The further course of meiosis was similar to that observed by Ramanujam and Parthasarathy (1935). They assumed that the sterile plant arose as a mutation for desynaptic behavior. Genetic studies showed that this character was monogenetically controlled (see also Chao and Hu, 1961). Chao and Hu (1961) studied the effect of three temperature regimes (20OC, 25OC, and 3OOC) on the expression of desynapsis in this mutant. The results were not consistent in two experiments, but, in general, high temperatures favored bivalent formation. I n agreement with similar work done earlier with asynaptic maize, RNA content in the florets of desynaptic rice was also found to be higher than that in florets of normal rice. Katayama (1960) obtained two completely asynaptic and two partially asynaptic plants in advanced generations of X-irradiated material. He used them to produce trisomics. This work has been reviewed already. Wang et al. (1965) also obtained a desynaptic mutant in M, generation after seed irradiation with X-rays. Chromosome pairing was complete a t pachytene, but most chromosomes were desynapsed by diakinesis and MI. The extent of desynapsis increased when plants were exposed to low temperature (15OC) for longer periods (2 days or more).

E. INTERCHANGE HETEF~OZYGOTES Reciprocal translocations are the most common form of mutagen-induced chromosome aberrations. Several workers have secured them also in rice (cf., Nayar, 1965; Gustafsson and Gadd, 1966). Because of their usefulness in genetic studies, a few attempts have been made in rice to develop translocation tester lines. Descriptions about their breeding behavior and fertility conform to the usual pattern (cf. Burnham, 1956). Because of the small size of rice chromosomes, the translocated ones show themselves as two bivalents in addition to their expected occurrence as rings and as chains of 4 chromosomes. Carpena and Ramirez (1960) found that stocks showing rings of 4 chromosomes gave about 55% pollen sterility and 65% ovule abortion, while those forming two rings showed only 8-20% pollen sterility and 6 2 3 % ovule sterility. Out of 27 stocks tested, 18 showed bivalents only. Hsieh and colleagues also isolated a number of translocation lines from X-irradiated populations (Hsieh, 1961; Hsieh et aZ., 1961a,b, 1962; also see Oka et al., 1953). They found that seed sterility was closely correlated with translocation frequency in PMCs. The agronomic features were not affected, or only slightly if at all, by the presence of translocations in homozygous form. Nishimura (1961) also studied meiotic behavior and fertility in a number of reciprocal translocation lines. H e was able to obtain a com-

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plete set of translocation tester lines in the variety Norin 8 (cf. Watanabe et al., 1969). The translocation heterozygotes showed 5 4 0 % seed fertility. The type and extent of quadrivalents formed showed wide differences. Li et al. (1964d) studied the relationships between the orientation of chromosomes in interchange heterozygotes and their fertility. The correlation coefficients between expected and actual pollen and seed fertilities were 0.6424 and 0.3512, respectively. These values are significant. By using translocation lines, Iwata and Omura (1971a,b) have now assigned ten linkage groups to respective chromosomes. Accordingly, linkage groups I to XI1 (of Nagao and Takahashi, 1963) have been assigned to chromosome numbers 6, 11, 4, 10, none, 2, 1, 9, 3, 8, 5 and none (of Nishimura, 1961).

F. NATURE OF RICEGENOME The idea that rice is a secondary polyploid has continued to haunt rice geneticists ever since the suggestion was put forward by Sakai (1935) and Nandi (1936), although it was Lawrence (1931) who proposed the hypothesis first. I n support of this, they pointed out the occurrence of secondary association of chromosomes, somatic chromosome morphology, and the observation of a duplicate mode of inheritance for five characters by Chao (1928). H u (1960a, 1962) strongly supported the hypothesis on the basis of his own observations on secondary association in several Oryza taxa, somatic chromosome morphology, formation of 1-2 I1 in certain frequencies in haploid microsporogenesis, and some additional instances of duplicate mode of inheritance. Sethi (1937), Parthasarathy (1938), and Okuno (1944) also supported the hypothesis on the basis of their findings on secondary association and chromosome morphology. Lawrence (1931), Yamaura (1933), and Ramanujam (1938a) pointed out that the proposed numbers of 5 or 7 are the most common basic chromosome numbers in Gramineae. Besides, the genus Zizania related to Oryza also has a basic number of 5 chromosomes. Karunakaran and Kiss (1971) obtained evidence from induced mutation studies in support of a polyploid origin of rice: higher chlorophyll mutation frequencies in M, generation than in M, and an increase in mutation frequencies after recurrent irradiation, both characteristics associated with polyploids. Thus, several lines of evidence have been advanced in support of a polyploid origin of rice. Bouharmont (1962b) dealt with some of the earlier arguments in respect to secondary association and chromosome morphology and felt that the evidence was not strong enough to support this contention. The extensive experimental data of Katayama (1965a) and Chu (1967)

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have shown that the association of rice chromosomes into five groups is only one of a large number of associations found during cell division and that chromosome associations do not show any regularity in rice. This was not even the most frequent type of association. Secondary pairing may provide evidence in support of a polyploid origin of a taxon or group (cf. Stebbins, 1950), but in rice it is apparently of limited reliability. The karyological studies in Gramineae show that the haploid chromosome number of all genera belonging to the tribes Oryzeae and the related Bambusae, except that of Zinania, is 12. In Zinania it is 15 (cf. Avdulow, 1931; Darlington and Wylie, 1955). Further, species with five pairs of chromosomes are uncommon in Gramineae. Also, the genus Ehrharta, which in many respects appears more like an ancestral prototype of the Oryzeae than does any other living genus, also has z = 12 (Stebbins, 1950). The possibility still exists that the tribe itself could have originated in some ancient past from forms with smaller chromosome numbers such as 5 or 7, but this event, if it has occurred a t all, could only have happened in such a remote p a s t s a y several millions of years ago-that it would be anyone’s guess if it would stil! leave any kind of residual attraction among chromosomes. It is also difficult to put much reliance on chromosome morphology to decide about the nature of the rice genome in view of their small size. Nandi (1936) saw in MI1 two groups of 3 univalents, two groups of 2 univalents, and 2 other univalents. The somatic chromosomes of rice are too small (3-4 p to 1 p long) to permit any kind of critical comparative morphological observations. The pachytene chromosomes should be more useful, but there are considerable differences in the observations made by various authors. The results obtained by Shastry and colleagues, who made the greatest number of studies, are themselves conflicting. In fact, two of their papers have been cited by different workers to support opposing viewpoints. Gustafsson and Gadd (1966) quoted Shastry et al.’s (1960a) study of pachytene morphology of variety Norin 6 to suggest that the haploid complement of rice consisted of 12 nonidentical fully differentiated chromosomes. Bouharmont (1962b3, however, cited Shastry and Rao’s (1961a) study of the three taxa, stapfii, glaberrima, and australiensis, in which they supported Nandi’s hypothesis that 5 is the basic chromosome number. Before Karunakaran and Kiss (1971) presented their results from mutational studies supporting a polyploid origin, Siddiq and Swaminathan (1968~)obtained exactly opposite results: “an extremely low frequency of mutations . . . in M, in nonsegregating lines” and reduced mutation frequency in recurrently irradiated material. They suggested

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that the mutational response of rice was like that of diploids. Karunakaran and Kiss’s (1971) data are more extensive, and they calculated mutation frequencies on the basis of M, and M, populations. However, the data also show some lack of dosage effect in some cases. It appears, therefore, that more extensive and critical studies are needed on this aspect. The appearance of 1-2 I1 in certain frequencies in PMCs of haploid rices has been advanced as another argument in support of its polyploid origin. But bivalents are found in similar frequencies in haploids of such true diploid species as rye (Levan, 1942a), snapdragon (Rieger, 1957), barley (Sadasivaiah and Kasha, 1971), and several others. Kimber and Riley (1963) estimated that half of 34 diploid species for which published data were available showed occasional bivalents during haploid meiosis. Archaic polyploidy and aneuploid origin of one or more chromosomes may be responsible for such behavior, but these aspects have been discussed already in rice. The occurrence of duplicated segments is another possibility. If duplicated segments exist, it may show preferential pairing leading to nonrandom association of nonhomologous chromosomes. Chu (1967) attempted to study this in rice without much success because of the difficulty in distinguishing all chromosomes. He found that chromosome 1 associated with three other chromosomes (3, 5 or 6, and 11 or 12) mainly a t three sites, viz., distal part of the long arm, and distal and proximal parts of the short arm. Chromosome 1 is considerably longer than the remaining 11 chromosomes. Several workers beginning with Chao (1928) have noted the presence of duplicate genes in rice. I n his summation of genetic studies in rice, Jodon (1964) pointed out that “possibly an exceptional number of characters are controlled by duplicated genes” in rices and also that duplicate and complementary gene action appeared to be involved in the inheritance of certain characters for which only single gene segregations were previously known. Complementary modes of inheritance have also been observed in the few genetic studies conducted on interspecific hybrids (Nayar, 1958; Nayar et al., 1966). Complementary genes may represent intermediate steps between duplicate genes and independent genes (Stephens, 1950). The role of duplications in evolution has been stressed from time to time, more especially in recent years (Metz, 1947; Stephens, 1951; Spofford, 1969; Mayo, 1970; Ohno, 1970). They have played important roles in the evolution of several groups. Duplication may occur by one of several ways : translocation, unequal crossing-over, regional redundant duplication, and polyploidy (cf. Ohno, 1970). Even a single-locus heterosis may afford a sufficient drive for the incorporation of a newly arisen

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duplication into a species gene pool (Spofford, 1969). After duplications arise by unequal crossing-over, approximate reversions can readily occur for tandem repeats (Metz, 1947; Mayo, 1970). Further evidence for genetic duplication in rice may be deduced from its tolerance of aneuploidy (cf. Table 11 in Rick, 1971). Reviewing the work on duplicate genes in maize, Rhoades (1951) stated: “that the architecture of the germplasm of maize contains duplicated regions can hardly be doubted, but whether or not they represent vestiges reflecting an amphidiploid origin, or represent later occurring duplications cannot be decided a t this time.” I n rice, the available evidence indicates that the rice genome is most probably monoploid (or monohaploid) in nature in which chromosomal and genetic duplications have taken place. The confirmation for this and its extent can probably be better obtained by comprehensive genetic and linkage studies because of the difficulty in identifying individual rice chromosomes with certainty. ACKNOWLEDGMENTS This work was prepared during the tenure of an Alexander von Humboldt Foundation Fellowship while in residence at the Institute of Plant Breeding, University of GGttingen, West Germany. I thank the Foundation for the award and Professor G. Robbelen, Director of the Institute, for hospitality. Quite a few articles on rice continue to appear in periodicals of limited circulation and these have been published in at least ten languages. I am grateful to Mr. P. P. Khanna, Mr. C. Gangadharan, Dr. H. Morishima, Dr. T. Tateoka, and the International Rice Research Institute for sending me copies of some publications on short notice, and to Dr. A. Azael and the International Rice Research Institute for translating some of the articles. I thank also Dr. W. D. Clayton and Dr. H. Jacques-FBlix for clarifying certain taxonomic problems. The constant encouragement of Profeessor E. W. Caspari helped me considerably in completing this manuscript.

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on desynapsis in rice. Bot. Bull. Acad. Sin. 6, 197-207. Watanabe, Y., and Ono, S. 1965. Cytogenetical studies on the artificial polyploids in the genus Oryza. I. Colchicine-induced octoploid plants of 0. latijolia Desv. Jap. J . Breed. 15, 149-157. Watanabe, Y., and Ono, S. 1966. Cytogenetical studies on the artificial polyploids in the genus Oryza. 11. Colchicine-induced octoploid plant of Oryza minuta Presl. Jap. J . Breed. 16, 220-230. Watanabe, Y., and Ono, S. 1967. Cytogenetical studies on the artificial polyploids in the genus Oryza. IV. Fertile allohexaploid rice minuta-australiensis. Nippon Idengaku Zasshi 42, 203-212. Watanabe, Y., and Ono, S. 1968. Cytogenetical studies on the artificial polyploids in the genus Oryza. 111. Two kinds of allohexaploid rice sativa-latifolia (AACCDD) and sativa-minuta (AABBCC). Jap. J . Breed. 18, 15-21. Watanabe, Y., Ono, S., Mukai, Y., and Koga, Y. 1969. Genetic and cytogenetic studies on the trisomic plants of rice Oryza sativa L. I. On the autotriploid plant and its progenies. Jap. J . Breed. 19, 12-18. Watson, W. 1969. Early cereal cultivation in China. In ‘‘The Domestication and Exploitation of Plants and Animals” (P. J. Ucko and G. W. Dimbleby, eds.), pp. 397402. Duckworth, London. Watt, G. 1892. Rice. I n “Dictionary of Economic Products of India,” Vol. 5, pp. 498-653. Superintendent, Gov. Printing, Calcutta. Wilson, E. 0. 1965. The challenge from related species. In “The Genetics of Colonizing Species” (H. G. Baker and G. L. Stebbins, eds.), pp. 7-27. Academic Press, New York. Wrigley, C. 1960. Speculations on the economic prehistory of Africa. J. Ajr. Hkt. 1, 189-203.

Wu, H. K., and Li, S. S. Y. 1964. Chromosome morphology of 0. sativa and 0. australiensis and their pairing in the F, hybrid a t earlier meiosis. Bot. Bull. Acad. Sin. 5, 162-169. Wu, H. K., Kwan, S. C., and Li, H. W. 1964. A preliminary note on the pachytene analysis of japonica x indica hybrids. In “Rice Genetics and Cytogenetics,” Proc. Symp., Los Banos, Philippines, 1963, pp. 187-188. Elsevier, Amsterdam. Wu, L., Tsai, K. S., and Li, H. W. 1967. Cytogenetical studies of Oryza sativa L. and its related species. 12. An alien additional line second backcross generations of 0. sativa.X 0. australiensis. Bot. Bull. Acad. Sin. 8, 165-168. Wu, S. H., and Tsai, C. K. 1963. Cytological observations on the F, hybrid rice (Oryza sativa L. x Pennisetum sp.). Nung Yeh Hsueh Pa0 11, 293-307 (in Chin.). [Plant Breed. Abstr. 34, 5866 (19641.1 Wuu, K. D., Jui, Y., Lu, K. C. L., Chou, C., and Li, H. W. 1963. Cytogenetical studie of Oryza sativa L. and its related species. 3. Two intersectional hybrids 0. sativa Linn. x 0. brachyantha A. Chev. et Roehr. and 0. minuta Presl. x 0. brachyantha A. Chev. et Roehr. Bot. Bull. Acad. Sin. 4, 51-59. Yamaura, A. 1933. Karyologische und embryologische Studien uber einige BambusArten. Shokubutsugaku Zasshi 47, 551-555. Yang, K. K. S., Ho, K. C., and Li, H. W. 1965. Cytogenetical studies of 0. sativa L. and its related species. 8. Studies on meiotic division of F, hybrid of 0. sativa L x 0. brachyantha A. Cheval. e t Roehr. Bot. Bull. Acad. Sin. 6, 32-38.

Yao, 5. Y., Henderson, M. T., and Jodon, N. E. 1958. Cryptic structural hybridity as a probable cause of sterility in intervarietal hybrids of cultivated rice 0.

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sativa L. Cytologia 23,4555. Yasui, K. 1941. Diploid bud formation in a haploid Oryza with some remarks on the behaviour of nucleolus in mitosis. Cytologia 11,515425. Yeh, B. P., and Henderson, M. T. 1961a. Cytological studies of rice. Proc. Meet. Rice Tech. Working Group, 9th, Lafayetta, La., 1960 pp. 9-10. Yeh, B. P., and Henderson, M. T. 1961b. Cytogenetic relationship between cultivated rice Oryza sativa L. and five wild diploid forms of Oryza. Crop Sci. 1, 445-450. Yeh, B. P., and Henderson, M. T. 1962. Cytogenetic relationships between African annual diploid species of Oryza and cultivated rice 0. sativa L. Crop Sci. 2, 463467.

Yen, D. E. 1970. Sweet potato. I n “Genetic Resources in Plants: their Exploitation and Conservation” (0. H. Frankel and E. Bennet, eds.), pp. 341-350. Blackwell, Oxford. Yen, Y. J. and Pao, W. K. 1960. Studies on the technique of breeding cereal crops utilizing polyploidy. I. Tetraploid rice. Nung Y e h Hsueh Pa0 11, 1-9 (in Chin.). [Plant Breed. Abstr. 30, 3976 (1960).1 Yunoki, T., and Masuyama, Y. 1945. Investigations on the later generations of autotriploid rice plants. Kyushu Daigaku Nogakubu Gakugei Zasshi 11, 182-216 (in Jap.). Cited in International Rice Research Institute (1964). Zohary, D. 1965. Colonizer species in the wheat group. I n “The Genetics of Colonizing Species” (H. G. Baker and G. L. Stebbins, eds.), pp. 403423. Academic Press, New York. Zohary, D. 1969. The progenitors of wheat and barley in relation to domestication and agricultural dispersal in the Old World. I n “The Domestication and Exploitation of Plants and Animals” (P. J. Ucko and G. W. Dimbleby, eds.), pp. 47-66. Duckworth, London. Zohary, D. 1970. Centres of diversity and centres of origin. I n “Genetic Resources in Plants: their Exploration and Conservation” (0. H. Frankel and E. Bennett, eds.), pp. 3342. Blackwell, Oxford. Zukovsky, P. M. 1950. “Cultivated Plants and their Wild Relatives,” 107 pp. Commonw. Bur. Plant Breed. Genet., Cambridge, England. (An abridged translation from the Russian.)

SOME ASPECTS OF THE REPRODUCTIVE BIOLOGY OF Drosophila: SPERM TRANSFER, SPERM STORAGE, AND SPERM UTILIZATION G. 1. Fowler* Deportment of Biology, University of Oregon, Eugene, Oregon

I. Introduction . . . . . . . . . 11. Anatomy of the Reproductive Systems . A. Female System . . . . . . . B. Malesystem. . . . . . . . 111. Sexual Behavior of the Male and Female A. Acquisition of Sexual Maturity . . B. Courtship and Copulation . . . . C. The Duration of Copulation . . . IV. Sperm Transfer . . . . . . . . A. The Time of Ejaculation . . . . B. Number of Sperm Ejaculated . . . V. Post-Mating Responses in the Female . A. State of Receptivity. . . . . . B. Post-Copulation Changes in the Female VI. Sperm in the Female: Sperm Storage. . VII. Sperm in the Female: Sperm Utilization . VIII. Conclusions . . . . . . . . . References . . . . . . , . .

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1. Introduction

Although the normal anatomy, histology, and development of Drosophila has been fully described by various authors (see, e.g., Demerec, 1950; Fristrom, 1970), the reproductive biology of this organism is still not well known. Nonidez (1920) was one of the first to describe the internal phenomena of reproduction. Subsequent experiments designed to elucidate the mechanism by which the female utilizes sperm were carried out by Kaufmann and Demerec (1942). Since that time, however, the study of sperm transfer, sperm storage, and sperm utilization in D. melanogaster has been the subject of few investigations, and the majority

* Present address : Institut fur Allgemeine Biologie, Universitat Dusseldorf, Dusseldorf, Germany. 293

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of these have been carried out in the last decade. This fact is not surprising since the entire area of reproduction, particularly in the male Drosophila, has become of considerable interest recently in attempts to shed some light on the mechanism or mechanisms responsible for the very interesting phenomenon of “meiotic drive” in Drosophila, a term first coined by Sandler and Novitski (1957) to describe the condition in which heterozygotes for two given alleles fail to produce the two kinds of expected gametes with equal frequency. In most of the work on “meiotic drive,” the phenomenon has been concluded to be a function of events occurring during meiosis (e.g., Novitski and Sandler, 1957; Lindsley and Sandler, 1958; Peacock and Erickson, 1965; Hartl et al., 1967). At the present time there is no evidence to refute this idea. It has been shown, however, that postmeiotic events, such as sperm transfer, sperm storage, and sperm utilization, may also be playing more than a passive role in the ultimate recovery of progeny from a particular mating (e.g., Zimmering and Fowler, 1968; Mange, 1970; Childress and Hartl, 1972; Olivieri et al., 1970). For this reason, then, it seems worthwhile to review these particular aspects of the reproductive physiology of D. melanogaster (with reference to other species of Drosophila where appropriate). Even though it is the aim of this review paper to deal primarily with only certain areas of the reproductive biology of Drosophila, some detailed attention is also given to descriptions of the male and female reproductive systems, spermatogenesis, sperm morphology, sexual behavior of the male and female, and the postcopulatory physiology in the female, in the hope that the reader will be better able to see the specific areas of sperm transfer, sperm storage, and sperm utilization in the overall perspective of the physiology of sex in Drosophila, in general. It is hoped that the material presented ‘here will succeed in demonstrating the paucity of our understanding and the confusion which still exists with regard to some of the aspects of the reproductive physiology of Drosophila as well as to draw attention to those areas where future research might be most fruitful. II. Anatomy of the Reproductive Systems

The first full account of the morphology and function of the male and female reproductive systems of D. melanogaster was published by Nonidez (1920). More recently, these systems have been described in detail by Miller (1950) and modified in some respects by Lefevre and Jonsson (1962a). Furthermore, a complete morphological analysis of the testis, genital canal, and accessory glands of the male D. melanogas-

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ter, has recently been carried out by Bairati (1967, 1968). The internal reproductive system of the adult female is described in some detail by King (1970). The following descriptions of the morphology and functions of the various components of the reproductive tracts of D. melanogaster are based mainly on the publications of these workers. A. FEMALE SYSTEM The reproductive system of the adult female of D. melanogaster (Fig. 1) occupies the posterior two-thirds of the abdomen and is composed

FIO.1. Diagram of the female reproductive system in Drosophila melanogaster.

E, egg; Ft, fat tissue; G, accessory gland (parovarium); Odc, common oviduct; Ov, ovary; SmRcp, seminal receptacle; Spt, spermatheca; Utrs, uterus; Vag, vagina. (Reprinted from Patterson and Stone, 1952.)

of (1) the paired ovaries, (2) the oviducts and the vagina-uterus, (3) three sperm-storing organs, and (4) the paired accessory glands, the parovaria. The pair of ovaries are located a t the anterior end of the abdomen and are joined by two lateral oviducts, which in turn unite to form the common median oviduct. The posterior end of the common oviduct is connected with the anterior end of the genital chamber, an elongated

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FIG.2. Portions of the female reproductive system in Drosophila melanogaster. Stained with 2% acetic orcein. (A) Nomanki. Ft, fat tissue; G, accessory gland (parovarium) ; SmRcp, seminal receptacle: px, proximal portion; ds, distal portion;

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muscular pouch. The larger anterior portion of this pouch is termed the vagina. The sperm-storing organs, which in the majority of Drosophila are three in number, are: the single ventral receptacle (seminal receptacle) and the two spermathecae (Fig. 2A). The seminal receptacle is a long, often coiled tube, whose overall size in Drosophila varies considerably from species to species. I n D . melanogaster, for example, it is approximately 2 mm in length with an outside diameter of approximately 15 pm increasing to about 30 pm a t the distal end and finally narrowing again to form a closed pouch. The seminal receptacle, which opens into the common oviduct a t the top of the uterus, is divided into two distinct regions, the proximal and distal, each of which comprises about one-half of the total length of the tube. The lumen in the proximal half of the seminal receptacle is quite narrow, comprising approximately one-third of the overall diameter. Proceeding distally, however, the lumen gradually widens until, in the distal half of the receptacle, the diameter of the tube and the diameter of the lumen are one and the same in overall dimension (Fig. 3 ) . Consequently the bulk of the sperm storage in the seminal receptacle is in the distal half (Fig. 2B). The spermathecae are a pair of mushroom-shaped organs, each of which consists of a dark brown sclerotinized capsule and a slender trachealike duct. The ducts arise by small openings in the dorsal wall near the anterior end of the uterus (Fig. 2A). At the distal end of each duct is the capsule, which is approximately 70 pm in diameter and resembles an inverted double-walled bowl. Each capsule is covered externally by a single layer of cuboidal epithelial cells and is surrounded by a small mass of fat (Fig. 4 ) . As sperm storing organs, the spermathecae are considered to be “minor” when compared to the seminal receptacle which routinely accommodates 80-90% of a medium sized ejaculate of 300-500 sperm. Cytological examinations of the spermathecae by Fowler e t nl. (1968), however, have shown that the description of the spermathecae as “minor” storage organs is considerably less accurate than originally thought. On the basis of sperm counts in Oregon-R females following a single insemination by males 5-24 hours old, there is, in fact, evidence that the distribution of sperm in the seminal receptacle and the spermathecae may be directly related to the size of the ejaculate (Table 1 ) . Spt, spermatheca; Sptd, spermathecal ducts; Utrs, uterus. (B) Phase microscopy. Note (1) the distention of the distal portion of the seminal receptacle (at arrows) due to the presence of large numbers of sperm and (2) detail topography of the uterus with particular attention directed to ‘‘channels” (at arrows) through which sperm move to the storage organs.

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It can be seen in Table 1, that when the mean number of sperm in the seminal receptacle is less than 50, there is an approximately equal amount of storage in the spermathecae. When 50-100 sperm are found in the receptacle, the proportion in the seminal receptacle and the spermathecae is approximately 60: 40, respectively. When greater than 100 sperm are found in the receptacle, the distribution in the seminal receptacle and the spermathecae resembles that found in medium to

FIO. 3. Proximal (px) and distal (ds) coils of the seminal receptacle (brackets indicate lumens of each coil) containing sperm (head of sperm at arrow) which hm been stained with 2% acetic orcein. Phase microscopy.

large-sized ejaculates (i.e., approximately 80:20). At the present time, there is no explanation for the relatively high proportion of sperm stored in the spermathecae when the total number of sperm stored is small. It is possible that the observation is peculiar to the type of males used in these experimenh (the description of which follows in a later section of this paper), but there is no a priori reason to suspect that this is so. Even though in the D . mulleri subgroups of the repleta species group, sperm is never observed in the spermathecae, which in these Drosophila

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FIG.4. Spermatheca (Spt) of female Diosophila melanogaster. Ft, fat tissue; Sptd, spermathecal duct. Phase microscopy of material stained with 2% acetic orcein.

have become modified into glandlike structures (Wheeler, 1954), the major function of the spermathecae in Drosophila seems to be as spermstoring organs. In D. melanogaster, however, they are apparently essential to the survival of sperm in the seminal receptacle, as well. For example, Anderson (1945), from studies of mutants of D . melanogaster

a. L.

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in which the spermathecae are partly or entirely absent, concludes that the spermathecae, specifically the capsular portions, are necessary for high fertility. In this regard, it has been suggested (Davey, 1965) that the secretory epithelium of the spermathecae may provide nutriment for sperm, a hypothesis that might explain the fact that Drosophila sperm are still partially viable in the female even after 3 months of storage (Muller, 1940). The paraovaria represent a pair of accessory glands lying just behind the spermathecae and connected with the uterus by fine ducts (Fig. 2A). The specific function of these organs is not clear, but they certainly TABLE 1 Mean Number and Mean Percent of Sperm Stored in the Spermathecae and Ventral Receptacle of Wild-Type (Oregon-R) Females Mated Singly to Heterozygous SD Males Raised at 26°C and Aged upon Eclosion from 5 to 24 Hoursasb Range of sperm stored

Mean No. of sperm stored

1-50 51-100 101-200 201-300 301-400 401-500 501-600

21.0 77.4 150.8 253.6 331.5 413.0 509.6

a

Mean No. of sperm in Mean No. of sperm in Spermatheca

Ventral receptacle

12.2 26.6 32.1 42.6 46.8 45.5 136.3

8.8 80.8 118.7 211.0 284.7 367.5 373.3

Sperma- Ventral No. of theca receptacle females 58.1 34.4 21.3 16.8 14.1 11.1 26.8

41.9 65.5 78.7 83.2 85.9 88.9 73.2

27 22 60 43 12 2 3

Data of Fowler el al. (1968).

* Cases where no sperm were found in either the spermathecae or the ventral recep-

tacle are omitted.

are not sperm receptacles. On dissection of the female soon after insemination, sperm is often seen in the vicinity of the parovaria, but never inside what appears to be a lumen. Nonidez (1920) has suggested that the parovaria, which contain “small refractive granules,” may be essential to the process of sperm storage by ( 1 ) reactivation of the sperm immediately after ejaculation, (2) dilution of the thick fluid portion of the ejaculate, thus removing one of the obstacles to free motion of the sperm, or (3) both. Concerning these possible functions, the present evidence is that sperm are ejaculated in a highly active state (see Section IV, B ) , therefore, there is no support for the idea that the parovaria are necessary for sperm “reactivation.” On the other hand, the parovaria may somehow be involved in producing the copious secretion found in

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the vagina of the Drosophila female during insemination (Patterson and Stone, 1952), but this is not yet unequivocally established. According to Riley and Forgash (1967) the function of the secretions produced by the accessory glands of the female Drosophila is t o produce a mucoprotein adhesive which causes eggs to adhere to the surface upon which they are laid.

B.

h/IALE

SYSTEM

In contrast to the female reproductive system, there is an impressive amount of information on the anatomy and physiology of the reproductive system of the male D . melanogaster, much of it relatively recent (e.g., Bairati, 1967, 1968). The internal organs of the reproductive system of the male Drosophila (Figs. 5A and 5B) consist of ( 1 ) the paired testes, (2) the paired vasa deferentia (vas deferens), dilated in part to form the (3) seminal vesicles, (4) the paired accessory glands (paragonia), and (5) an unpaired ejaculatory duct with (6) an appended ejaculatory bulb, the sperm pump. The testes are elongated tubes about 2 mm long with a diameter of approximately 100 pm. There are three major portions: the apical, the intermediate, and the terminal. Each of these zones is responsible for a different functional activity of the testes of the X Y male, the normal spermatogenesis of which has been extensively reviewed by a number of investigators (e.g., Bairati, 1967, 1968; Hess and Meyer, 1968; Meyer, 1968). For example, in the apical zone of the testes, cysts of sixteen primary spermatocytes are produced from a single spermatogonial cell by four synchronous mitotic divisions; the sixteen spermatocytes so formed are connected by intercellular bridges (Meyer, 1961, 1968; Abro, 1964; Bairati, 1967; Hess and Meyer, 1968; Kiefer, 1966). I n the intermediate zone, all the spermatocytes in a cyst undergo the meiotic divisions in unison, producing 64 spermatids which are also interconnected by cytoplasmic bridges and which, in the terminal or spermatic zone of the testes, undergo synchronous maturation into spermatozoa (Cooper, 1950; Baccetti and Bairati, 1964; Bairati, 1967, 1968). Bundles containing more than 64 spermatids have not been observed in D. melanogaster (Fig. 6 ) . On the other hand, fewer than 64 spermatids per bundle is not unusual. Recent work by Kiefer (1966) and Anderson (1967) indicates, in fact, that only 20% of the mature sperm bundles contain the expected number of spermatids in D. metanogaster (the mean number per bundle being 61). I n other species of Drosophila, 64 sperm per bundle is not necessarily the rule. For example, in the standard strain of D . pseudoobscura, spermatozoa come to maturity in bundles of 128

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FIQ.5. See opposite page for legend.

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RQ. 6. Transverse sections of several testicular cysts of a Drosophila melanogaster male (wild-type) containing tails of late spermatids. The mean number of spermatids per cyst is 58. Nine microtubules surround each pair of central tubules (short arrows) in the flagellum (F) which, itself, is partially surrounded by the dense mitochondria1 crystalloid (MI. Glutaraldehyde-osmium fixation, Epon-Araldite embedding. x 25,100. spermatids each (Dobzhansky, 1934; Policansky and Ellison, 1970) , and in D. hydei no more than 32 spermatozoa per cyst have ever been found (Hess and Meyer, 1968). When the testes are gently broken open, an association of sperm bunFIG.5. (A) Diagram of the male reproductive system in Drosophih melanoguster. Aed, anterior ejaculatory duct ; Amp, ampullary portion of the anterior ejaculatory duct; c, common unpaired deferent duct; D, vas deferent; Eb, ejaculatory bulb; G, accessory glands; Og, outer genitals; P, anterior ejaculatory duct papilla; Ped, posterior ejaculatory duct; T to v, testis; Tub, tubular portion of anterior ejaculatory duct, v, testiculodeferential valve; Sv, seminal vesicles. (B) Portions of the male reproductive system in Drosophila melanogaster. Note (arrows) masses of granules (“filamentous bodies”) in the lumen of the accessory glands (G). Phase contrast microscopy of unstained material’. (Fig. 5. (A) reprinted with permission, from Bairati, 1968.)

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dles with large cells of the terminal epithelium is observed (Kaplan and Gugler, 1969). The earlier work of Guyenot and Naville (1929) and Geigy (1931) suggested that the cells are nutritive. Ultrastructural analysis of these cells by Bairati (1967) revealed that they contain large numbers of mitochondria and lysosomes, which are mostly concentrated in the extensions of cytoplasm that surround the ends of each sperm bundle. The physiological significance of this finding is not known but recent findings of Tokuyasu et al. (1972) suggest that these “nutritive cells” in D . melanogaster may, in fact, be the “cystic bulges” formed during a normal morphogenetic process which “transforms spermatids from a syncytial state to a state in which each spermatid is invested in its own membrane” and in which one finds excess cytoplasm, ribosomes, and organelles of the spermatids which have become eliminated during this “individualization process.’’ I n D. melanogaster, the testes are fully formed and contain mature sperm a t the time of eclosion, however, spermatogenesis (including spermiogenesis) may still occur after the male has reached senile infertility more than 32 days after eclosion (Philip, 1942). The opinions of authors differ with respect to when meiosis first begins in the immature testis of Drosophila. In melanogaster, Khishin (1957) found no evidence of the beginning of meiosis in third-instar larvae (approximately 112 hours old). On the other hand, labeling experiments of Kaplan and Sisken (1960) showed that meiotic prophase is typical of larvae that are 20-39 hours of age. Variable results have also been obtained by a number of other investigators attempting to determine the duration of the spermatogenic process. For example, Demerec and Kaufmann (1941) determined the minimum time necessary for 6-day-old ixradiated males to produce spermatozoa carrying chromosomes which had been damaged by the irradiation. These workers found that it was usually day 19 after the treatment, even though some sperm production occurred as early as day 12 or 13. On the other hand, Mossige (1955) determined that depressions of fecundity in brooding patterns of males that had been X-irradiated at eclosion first appeared 6-9 days after treatment. These results were confirmed by Chandley and Bateman (1962) using autoradiography. On the basis of their experiments, these authors concluded that the period which lasts from the synthesis of DNA in the spermatocyte until fecundation is 10 days in males that are continuously brooded. In this interval, 4 days are required for the maturation of the spermatocyte, 5 days for spermiogenesis, and 1 day for the maturation of the spermatozoon. Using both X-irradiation and autoradiography, Martin (1965) confirmed the work of Chandley and Batemen (1962) and, in addition, concluded that the duration of spermatogenesis in the

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immature testes of larvae (approximately 9 days) is not the same as that of mature testes (adult males) ; there is a prolongation of 24 hours in the former with respect to the latter. It is suggested, however, that this finding may be a reflection of the particular stocks used in the experiments, and not applicable to Drosophila generally. Recent work by Bairati (1967), in which morphological observations were made using the light microscope, generally supports the experimental evidence of Martin (1965) and also suggests that there is a regular cycle of maturation of the male germinal cells in Drosophila, even though no direct observations of movements of the germinal cells from the apical to the terminal zone of the testis were possible. Cytologically, it can be seen that the first motile sperm in D. melanogaster males are found 7 1 hours after eclosion (Khishin, 1955). On the other hand, dissections made by Lefevre and Jonsson (1964) a t short intervals after emergence demonstrate that, in some males a t least, sperm motility begins to occur within less than an hour. Recent cytological observations on the testes removed from males a t various stages of development (Bairati, 1967) suggest that sperm in the testes of pupae also show motility. On the basis of these conflicting reports, it is difficult to make any definitive statements concerning the time of appearance of sperm motility in Drosophila. It is possible that the various results are merely reflections of differences in particular stocks and different physiological conditions during development. Whatever the specific time of its occurrence, it is generally agreed that motility is first seen in the terminal zone of the testes, often as movement involving entire bundles, the sperm soon becoming disengaged from the bundles and moving about individually. Passage of the mature sperm through the testiculodeferential valve (Fig. 5A) and into the seminal vesicles (as described by Bairati, 1968) occurs in greatest numbers 6-10 hours after eclosion, even though a few motile sperm can be found in this portion of the vas deferens earlier. The movement of the mature spermatozoa into the seminal vesicles is a continuous process. According to Lefevre and Jonsson (1962a) the vesicles are endowed with a certain degree of elasticity and continue to enlarge in volume as increased numbers of sperm enter and accumulate. At dissection, if the seminal vesicles are ruptured so that the sperm are liberated, intense motility is observed. Under the same conditions, sperm bundles liberated from the testes are immotile. The length of the mature spermatozoon in the vas deferens of the male and in the seminal receptacle of the female Drosophila has been the subject of several investigations (e.g., Yanders and Perras, 1960; Sidhu, 1963; Hess and Meyer, 1963; 1968; Beatty and Sidhu, 1969;

*

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Policansky, 1970). As first observed by Sidhu (1963), subsequently published with Beatty in 1969 (Beatty and Sidhu, 1969) and independently observed by Policansky (1970), there are three classes of sperm in some members of the D. obscura species group: Short (30-90 pm), Medium (100-170 pm), and Long (250300 pm) (Fig. 7). With the exception of D. obscura, in which there is a virtual disappearance of the short

FIQ.7. Sperm of three different sizes in Drosophita pseudoobscura. Phase contrast copy of unstained material. (A) sperm 0.05 mm in length; (B) sperm 0.10 mm in length; ( C ) sperm 0.30 mm in length. Note sperm heads at arrows. (Reprinted, with permission, from Policansky, 1970.) class in the storage organs of the female, this trimorphic condition remains evident a t all stages of the passage of the spermatozoa through the male and female tracts. During this passage, the measurements o f the length of the spermatozoa remain unaltered, thus indicating that spermatozoa neither shrink nor elongate in their journey from the seminal vesicles of the male to the storage receptacles of the female. Furthermore, they remain in the same proportions as they left the seminal vesicle of the male. It is not yet known whether all three types are

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equally functional in fertilization. Most recently, size classes of the head and flagellum of Drosophila spermatozoa have been reported in some detail by Beatty and Burgoyne (1971). While sperm populations that are trimorphic in composition have not been discovered in any other species group of Drosophila, sperm possessing wide variation in length from species to species and from genotype to genotype is characteristic of Drosophila in general. For example, the average mature spermatozoan in the vas deferens of the male or in the seminal receptacles of the female in D. melanogaster has a total length of approximately 1.76 mm with a tail that is more than 200 times the length of the head (Cooper, 1950). On the other hand, single sperm in both D. funebris and D. hydei have been measured to be as much as 6.6 mm in length (Yanders and Perras, 1960; Hess and Meyer, 1963; respectively). Hess and Meyer (1963, 1968) have demonstrated a direct correlation between sperm length and the amount of Y chromosome present. For example, in D. melanogaster, a male that is X/O produces sperm with an average length of 1.1 mm while X/Y and X/X/Y sperm measure 1.8 mm and 3.7 mm, respectively. The genetic basis of this observation is not yet clear since there is very little information on the specific effect of the morphogenetic genes of the Y chromosome on spermatogenesis and spermiogenesis even though developmental aberrations appearing during spermiogenesis in partially Y -deficient males of Drosophila have been extensively studied by a number of investigators (e.g., Meyer et al., 1961; Kiefer, 1966; 1968; Hess, 1967; Meyer, 1968; 1969). Generally speaking, the experimental results seem to support the hypothesis, as stated by Meyer (1968), that “the Y-chromosomal factors control the coordination of the various synthetic and morphogenetic processes during critical phases of development leading to the formation of functional sperm” . . . but . . . “without contributing structural information on the molecular level.” Unfortunateiy, it is not entirely clear by what mechanism(s) this is accomplished. The overall length of the spermatozoan is also closely correlated with the length of the testes and the ventral receptacle (Yanders and Perras, 1960; Sidhu, 1963; Beatty and Sidhu, 1969) in most species of Drosophila. The significance of this observation relative to, say, the function of the Y chromosome, is undetermined. Concerning the fine structure of sperm and the biochemistry of sperm motility, there is a voluminous amount of literature. For example, in vertebrates (see, e.g., review by Fawcett, 1958), sperm is morphologically divided into head, mid-piece, and tail, corresponding to the genetic, metabolic, and locomotor functions, respectively. The sperm head consists

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of acrosome and nucleoprotein, and the mid-piece is composed primarily of mitochondria which arc closely associated with the flagellar apparatus of the tail. The mid-piece and tail combined contain all the necessary elements for motility, i.e., those elements necessary for energy production and energy translation. The sperm of invertebrates (e.g., Insects, notably Drosophila, Yasuzumi et al., 1958; Kiefer, 1966, 1970; Anderson, 1967; Meyer, 1968; Tates, 1971; Tokuyasu, et al., 1972) is essentially the same as that of vertebrates except that there is no separately discernible midpiece. In D. melanogaster the sperm is very long and does not appear to be structurally differentiated along its length (Meyer, 1968; Bairati, 1967; Kiefer, 1966) except for the head region and the very tip of the tail. The “neck region” in the head of mature sperm of D. melanogaster contains a short centriole derivative encased within a deep nuclear “implantation fossa” and is accompanied for most of its length by the principal mitochondrial derivative (Perotti, 1969a,b). The tail consists of two longitudinally oriented portions, one of which is homogeneous in appearance, the other with radially arranged substructure containing the nebenkern and the axial region (Blaney, 1970). These two regions will split apart by treatment with acid (Oster et al., 1966) and osmotic stress (Blaney, 1970). Because of the structure of its axial filament and the paracrystalline hexagonal texture of its mitochondrial derivative, the sperm of D . melanogaster appears to be one of the most complex ever studied (Kiefer, 1970). At the proximal end of each of the seminal vesicles, the paired vas deferens connect with the anterior (ampullary) portion of the ejaculatory duct (Fig. 5A) which, according to Bairati (1968), is that portion of the male genital system which produces the “non-cellular part of the sperm.” At the same level the two accessory glands (paragonia) also empty into the ejaculatory duct (Figs. 5A and 5B). The accessory glands of the male are a pair of elongate sacs approximately 400 pm wide and 130 pin long, the walls of which consist of large binucleate cells. In the apical portion of each gland and interspersed between the binucleate cells are ovoid cells which contain large vacuoles and project into the lumen. The number, structure, and distribution of these glandular cells varies in different species of Drosophila, but 58 is the average number in D. melanogaster (Gill, 1964). These vacuolate cells are in an active secretory phase and produce the viscous fluid which contains “refractive granules of unequal size” (Nonidez, 1920), the needlelike “crystals” of Gill (1964), and the “filamentous bodies” of Bairati (1968) (Figs. 5B and 8 ) . Gottschewski (1937) first suggested that the secretion of the accessory glands is necessary for

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FIG.8. Portion of an accessory gland wall secondary cell (adult virgin wild-type Drosophila melanogaster male). Filamentous body (Fb)which encloses a portion of homogeneous electron-opaque material (horn) which, itself, is surrounded by bundles (t) of tubular elements (short arrows). Osmium tetroxide fixation. Vestopal embedding. x 18,200. (Printed, with permission, from unpublished work by Bairati.)

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effective fertilization of the female in Drosophila, since sperm taken from the seminal vesicles and injected into the uterus produce offspring much more rarely than does normal ejaculate that is artifically transferred. However, repeats of the experiments of Gottschewski (1937) have not been successful, and to date no other case of artificial insemination in Drosophila has been reported. Lefevre and Jonssoii (1962a, 1964) have more recently suggested that successful sperm transfer depends upon the presence of the accessory

FIG.9. Transverse section of a portion of the ventral receptacle of a Drosophila melanogaster female after mating. Note the presence of numerous paragonial tubular elements (t) tightly intermingled with sperms. Osmium tetroxide fixation. Vestopal embedding. x 24,000. (Printed, with permission, from unpublished work by Bairati.)

gland secretion. These workers found that after completing 5 successive matings during a period of 3-4 hours, males, on subsequent matings, no longer produced any offspring. Dissection of such males, however, clearly showed that spermatozoa were still to be found in the seminal vesicles. It was, therefore, concluded that the physiological cause of such sterility was the temporary exhaustion of the accessory glands. Since, according to Stromnaes and Kvelland (1962), a newly eclosed male requires 12 hours to synthesize accessory gland secretion, it is clear that in males which are successively mated there is not adequate time for the continuously depleted accessory glands to be replenished and sterility would result. Similar conclusions were also reached by Garcia-

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Bellido (1964), suggesting, then, that there may indeed be a close correlation between the quantity of accessory gland secretion available at the time of mating and the number of sperm successfully transferred. The granules, i.e., ‘Lfilamentousbodies,” which are typical of the contents of the accessory glands of Drosophila have recently been the object of particular interest. Acton (1966) and Bairati (1967, 1968) have not

FIQ.10. Portion of a filamentous body. In cross section, the wall of the filamentous structure appears to consist of a regular arrangement of globular subunits. Osmium tetroxide fixation. Vestopal embedding, x 160,000. (Reprinted, with permission, from Bairati, 1968.)

only shown that presence of these bodies in the lumen of the male accessory glands of D . melanogaster, but can also demonstrate their tubular components along the male genital duct and in the female receptacles after copulation (Fig, 9 ) . At the electron microscope level these structures are similar t o contractile elements (Fig. 10) and derive from the epithelial cells of the accessory glands, and it was suggested by Bairati (1968) that the accessory glands of the male Drosophila secrete these filamentous structures as a component of the spermatic fluid which may

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(1) aid in the transfer of sperm along the female reproductive tract, (2) serve as reserve material used in some way by the spermatozoa

during their long storage in the female receptacles, or (3) contain some substance that would activate and support the process during which the spermatozoan penetrates the ovum. Similar inclusions have been found in the paragonial fluid in male D . paulistoruin (Tandler et al., 1968) and D. virilis, D. sintulans, D. pseudoobscura, and D . hydei (Perotti, 1970). A number of investigators have been interested in characterizing the accessory gland secretion of Drosophila males, and attempts to determine its specific role in the reproductive process have been extensive. For example, Chen and Diem (1961) located a peptide in the paragonia of adult males of D. melanogaster. Judging from its mobility on paper chromatography and amino acid composition, it corresponded very well to the “sex-peptide” found earlier by Fox (1956a,b). From a study of the relationship of reproduction to the life-span in Drosophila, Kummer (1960) suggested that the secretion of the accessory glands probably has a stimulating effect on oviposition in the female. Since then, this view has been confirmed both by transplantation of these glands (Garcia-Bellido, 1964; Leahy, 1966; Merle, 1969) or by the injection of their extracts (Leahy and Lowe, 1967) into virgin females. Further evidence to support this view has been recently obtained by Chen and Buhler (1970a,b), who have injected virgin females with a single purified peptide of the acidic type. As clear as these findings are, there is, as yet, no information about the mechanism of stimulation of fecundation by the secretion of the male paragonia. It has been suggested that the paragonial peptide may either (1) act as a trigger to initiate and maintain the synthesis of yolk proteins or (2) supply a chemical stimulus to activate the neuromuscular system involved in egg deposition (David and Merle, 1966). At the present time there is no evidence to either support or refute the possibility of any kind of relationship between the filamentous bodies in the secretion of the paragonia described by Acton (1966) and Bairati (1967, 1968) and the sex-peptides of Fox (1956a,b) and Chen and Diem (1961). Peristaltic movements exhibited by the seminal vesicles and the accessory glands aid in the transfer of spermatozoa from the male to the female during copulation, even though it is the contraction of the ejaculatory duct, particularly the tubular portion (Fig. 5A) which is primarily responsible for propelling the sperm across the ejaculatory bulb at the time of ejaculation. The release of spermatozoa from the seminal vesicles into the anterior ejaculatory duct occurs during mating, the process being

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regulated by a sphincter which, itself, is under nervous control (Bairati, 1968). The ejaculatory bulb (sperm pump) is usually considered to be the organ in charge of ejaculating the sperm (Nonidez, 1920; Miller, 1950). However, some new functional interpretations have been suggested by the work of Bairati (1968), all of which lead him to conclude that “the ejaculatory bulb should be regarded primarily as a gland in charge of producing a highly viscous secretion that is subsequently released into the female genital canal.” The functional significance of this “waxy plug” produced by the ejaculatory bulb is, a t present, unknown. The fact that it appears in the female before sperm are introduced suggests that it might serve as a factor favoring the travel of sperm from the uterus to the seminal receptacles (Bairati, 1968; Bairati and Perotti, 1970). Brieger and Butterworth (1970) have recently biochemically characterized a lipid which is also found in the ejaculatory bulb of adult male D. melanogaster and is transferred t o the female during mating (Butterworth, 1969). The lipid (cis-vaccenyl acetate) may be the same as, or similar to, the “waxy plug” described by Bairati (1968), but this is not definitely known. I n addition to the viscous secretions, the ejaculatory bulb also produces a number of esterases. Johnson and Bealle (1968) have studied these esterases in 93 different species of Drosophila. A correlation of the presence or absence of the bulb esterases with results from interspecific hybridization tests, leads these authors to suggest that there is a greater chance for a successful interspecific cross when males possess the esterase in their ejaculatory bulb, than when they do not. The specific function of the enzyme in Drosophila sexual physiology is, however, not yet clear. It has been noted by a number of different investigators that there is a significant decrease in the fecundity (i.e., the number of descendants produced) of male Drosophila with increasing age, which leads, ultimately, to complete sterility immediately after the average fertile period of 32 days. This age-related reduction in fecundity, which, itself, is variable from male to male and from strain to strain, has been attributed to a number of different conditions: (1) a lack (or complete absence of) accessory gland secretion (Lefevre and Jonsson, 1962a; PerrinWaldemer, 1965; Garcia-Bellido, 1964), (2) a possible inactivity of the ejaculatory bulb due to enfeeblement (Duncan, 1930) and/or too little or no bulb secretion being available for the formation of the viscous “plug” (Bairati, 1968), or (3) the progressive inactivation or loss of the stem cell spermatogonia (Hannah-Alava, 1965). I n regard to this last point, Hannah-Alava (unpublished) has recently shown that a t 32 days of age the testes of Drosophila melanogaster males begin to atrophy

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(Figs. 11A and 11B). At the same time there are large numbers of sperm stored in the seminal vesicles, presumably unable to be ejaculated for some of the reasons stated previously. Accordingly, Hannah-Alava (1968) reported that, just prior to death, a brooded male will frequently produce a large brood, following a long period of decreasing ones, indicative of the fact that, with age, some of the normal mechanisms for ejaculation are beginning to fail.

FIO.11. Portions of the male reproductive system of Drosophila melanogaster. Light microscopy of Feulgen-stained material. (A) 5-day-old unmated male. Sv, seminal vesicle containing sperm; T, testis. (B) 32day-old unmated male. Testes (at arrows) showing atrophy. (Printed, with permission, from unpublished work by Hannah-Alava.)

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FIQ.11. (Continued).

In light of all the observations cited in this section it seems reasonable, then, t o assume that every portion of the genital canal plays an important role in ensuring a normal performance of the male reproductive function.

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Ill. Sexual Behavior of the Male and Female

A. ACQUISITION OF SEXUAL MATURITY Most male Drosophila are sexually active within a few hours of their emergence from the pupal case even though in D. melanogaster this characteristic varies greatly from strain to strain and from individual to individual within different strains. On the average, however, newly emerged males do not ordinarily mate before they have attained sexual maturity, which is usually a t about 12 hours of age (Stromnaes and Kvelland, 1962; Lefevre and Jonsson, 1964). While this does not necessarily mean that significant proportions of a population of D. melanogaster males will mate a t 12 hours after eclosion, the assumption used repeatedly for the process of virgin collecting is that in cultures kept a t 25OC this is sufficient time for some copulation to take place. While the majority of wild-type Drosophila males do not mate and transfer sperm until 18-24 hours after eclosion, some strains will exhibit sexual activity as early as 7-8 hours after eclosion, even though Stromnaes and Kvelland (1962) reported that in 75% of such matings, no sperm is transferred. This is, however, not the case in some mutant strains of D. melanogaster. For example, preliminary experiments by Fowler and Levine (unpublished) show that approximately 50% of a population of males heterozygous for the second chromosome markers, SegregationDistorter (SD) (for description, see Sandler et al., 1959), and cinnabar (cn) and brown ( b w ) , not only exhibit heightened sexual activity, but will even copulate and transfer sperm as early as 7-12 hours after eclosion (Table 2a). It can be seen in Table 2a, that the same degree of sexual activity is not observed in the control (wild-type) population until 18-24 hours after eclosion. Subsequent experiments carried out in an attempt to determine the genetic basis of such early mating in the SD/cn bw flies clearly demonstrated that early mating in these strains is not a function of the presence of the SD locus. The chromosome(s) on which the factor(s) responsible for such precocious sexual behavior resides, has not been unequivocally determined, but inferences suggest that either the second chromosome or the X chromosome may somehow be involved. De Wilde (1964) has stated that the reason for the high percentage of sterile matings in males 7-8 hours of age is due to the “state of the testis.” This is taken to mean that there has simply not been sufficient time for the entire processes of spermatogenesis and spermiogenesis to be completed and, therefore, mature spematozoa are not yet present

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in the seminal vesicles. The fact that males do show sexual activity and will even copulate a t this time, however, suggests that the development of the testis, i.e., “genital function,” and the acquisition of sexual behavior may be independent developmental events. While it is generally true that fewer males exhibit sexual activity 7-8 hours after eclosion TABLE 2a Comparison of the Sexual Activity (i.e., Time of Mating) of SD/cn bw and Wild-Type (Oregon-R) Drosophila melanogaster Males Mated to 5-Day-Old Wild-Type Females at Various Times after Eclosiona

Male genotype SD/m bw Oregon-R

Percentage of males that mated in the following hour intervals after eclosionb 7-12

12-18

18-24

24-30

58. 3 (186) 6.8 (190)

77. 6 (149) 37.3 (144)

68.7 (145) 55.3 (168)

81.2 (170) 78.8 (151)

Data of Fowler and Levine (unpublished).

* The numbers in parentheses represent total number of cultures of

one female.

one male and

TABLE 2b Comparison of the Numbers of Progeny Produced by SD/cn bw and Wild-Type (Oregon-R) Drosophila melanogaster Males Mated to &Day-Old Wild-Type Females a t Various Times after Eclosiona Average number of progeny/female in the following hour intervalsb Male genotype SD/m bw Oregon-R

7-12

12-18

18-24

24-30

82.9 (86) 118.1 (12)

114.8 (113) 101.7 (53)

119.8

136.7 (128) 160.1 (115)

(97)

121.2 (113)

Data of Fowler and Levine (unpublished). The numbers in parentheses represent the total number of females transferred.

than do older males, it can be seen in Table 2b that those males that do mate in this time interval also transfer sperm and produce progeny a t levels comparable to their older brothers. Comparing the data with those in Table 2a, it can be seen that SD/ cn bw males produce sizable numbers of progeny a t the same time (i.e.,

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7-12 hours after eclosion) that they also demonstrate heightened sexual activity, a characteristic also typical of those few (6.8%) wild-type males who also copulate and produce progeny in this time interval. Dissection of the testes of all males (SD/cn bw and Oregon-R) following mating, shows large masses of motile spermatozoa in the seminal vesicles. At the same time, no mature sperm were observed in the testes of those males that did not mate in this interval. These findings, then, taken together, would tend to suggest that, a t least in some strains of D . rnelanogaster, genital function and sexual activity may not be independent events. However, this does not necessarily mean that the presence of mature sperm in the male Drosophila is a prerequisite for sex drive. Lefevre and Jonsson (196213) have, in fact, shown that sperm in the seminal vesicles of males subjected to temperatures of -1OOC for 10 minutes are no longer motile and, furthermore, seem to be irreversibly inactivated, i.e., “dead.” Even though such sperm is not transferred, the treated males continue to mate repeatedly over a period of 24 hours following treatment and show no indication of a reduction in sex drive. In addition to males who can mate and can produce progeny a t times considerably earlier than 12 hours, there are also populations of males whose acquisition of sexual vigor is slower than normal. For example, tests on males from two wild-type strains, different only in the fact that they have been maintained for a number of years in different laboratories, produce very different results with respect to the acquisition of sexual activity (Fowler, unpublished). For example, in one strain, maximal sexual activity of a large population of males is exhibited 18-24 hours after eclosion (thereby supporting the observations reported above) but the same degree of sexual activity is not observed in a comparable population of males in another strain until 48-66 hours after eclosion. The reason(s) for this finding is not clear. Bosiger (1960, 1962, 1963) has established that there is a relationship between sexual vigor and the degree of genetic heterozygosity such that in strains which are made more and more homogeneous by many generations of sib matings, one notes a significant diminution in both the onset and the overall sexual vigor of the males. This, however, is not a completely adequate explanation to account for the findings mentioned above since, presumably, both wild-type stocks have been maintained by sib-matings. Such findings do, however, point to the fact that the genes (polygenes?) involved in the mating behavior in Drosophila are probably complex systems which do not yield easily to experimentation (see, e.g., “mating speed” experiments by Spiess and Langer, 1964, for further details). As a reflection of his sexual vigor, a male may copulate more than

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once during a certain period of time, but this is also a variable behavioral characteristic in Drosophila and, furthermore, may be modified by a number of different factors. For example, Stromnaes and Kvelland (1962) report that the frequency of mating is a function of the age of the male such that in a 12-hour period, wild-type males 72-96 hours old will mate with twice the number of females as do the same type of males 24-48 hours old. On the other hand, McSheehy (1963) finds that it is the number of females available to the male which determines his frequency of mating. Specifically, 50% more copulations occur when one male is placed with 8 females than when 1 male is placed with only 2 females, a result which is probably a reflection of some kind of competition between females during courtship by the male. There is a point, however, a t which additional females available to a male do not increase the number of progeny produced, i.e., the male does not copulate indefinitely (Lefevre and Parker, 1963). This is probably due t o a “crowding effect” which results in an interference with normal courtship patterns and ultimately leads to an unsuccessful copulation. Indeed, that the frequency of copulation in Drosophila males is a volume-related phenomenon was first suggested by the work of Del Solar (1964), who showed that when the number of pairs of flies varies from 1 to 5, there are fewer matings in a small volume than in a larger one. Female D. melanogaster are much less variable in their sexual activity than are males. Usually a newly emerged female actively repels a courting male and does not reach her maximum receptivity until 48 hours after eclosion. However, some mating prior to that time is not uncommon, but it is generally a function of the species and is also correlated with the maturation of the ovaries and an increase in juvenile hormone (Manning, 1967). The receptive period of a virgin female continues until approximately 8-10 days after eclosion, a t which time she gradually becomes more unreceptive to the male.

B. COURTSHIPAND COPULATION The study of Drosophila courtship dates back to 1915 when Sturtevant first described the detailed behavior of D. melanogaster and some of its mutants (Sturtevant, 1915). Since then, there have been a number of studies and descriptions of courtship behavior in the genus, notably those of Spieth (1952), Bastock and Manning (1955), Bastock (1956), and Manning (1959, 1967). Any of these are recommended t o the reader who wishes to have more detailed information on the subject of courtship in Drosophila than is presented here.

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Drosophila males never attempt to mount females without performing some preliminary courtship, although only a few minutes of such will usually suffice. This characteristic, however, is a species-dependent variable as well as being a function of the volume available to the flies (Del Solar, 1964). The male approaches the female and performs various courtship patterns in a rather rigid sequence and then attempts to copulate, If the male is unsuccessful, he repeats the courtship. One of the most important stimuli given by the male comes from his wing vibration display which drives a current of air over the female’s antennae. Indeed, Ewing (1964) has shown that there is, apparently, a linear relationship between a male’s wing area and his courtship success; wingless males were least successful (Bastock, 1956; Ewing, 1964). For the details of the male sexual behavior including the nuptial dance, orientation of the male to the female, vibrations of wings and licking, see, for example, the publications of Bastock and Manning (1955), Hoenigsberg (1960), and Hoenigsberg and Santibaiiez (1960). All the various stimuli from the male have a cumulative effect upon the female such that ultimately the sexual response threshold in the female is sufficiently lowered so that the male is accepted. The acceptance response from the female stimulates the male to mount the female and achieve intromission of the phallus (aedeagus) . Physiologically, the onset of copulation primes the sphincter-opening reflex of the anterior ej aculatory duct in the male, thereby releasing the spermatozoa from the seminal vesicles and into the anterior ejaculatory duct (Bairati, 1968). Subsequent contractions of the ampullary and tubular portions of this duct (Fig. 5) propel the sperm across the ejaculatory bulb.

C. THEDURATION OF COPULATION The length of the copulatory period is fairly constant for each species (Spieth, 1952) but varies enormously within the genus e.g., from about 30 seconds ( D . polychaeta, 25 seconds; D . mulleri, 29 seconds; D. victoria, 33 seconds) to over 1.5 hours in D . acanthoptera. I n D . melanogaster, there is a range from about 10 minutes (Wheeler, 1947) to 24 minutes, even though matings allowed to go to completion seem to last, on the average, about 20 minutes (Duncan, 1930; Manning, 1962b). Macbean and Parsons (1967) have suggested that copulation time in D. melanogaster is male-determined and may be proportional to the amount of sperm that is transferred. However, studies of the mating behavior of four wild-type strains of D . melanogaster by Yanders (unpublished) show no such correlation. In addition, it is commonly observed that males which lack a Y chromosome and, therefore, transfer

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no sperm, copulate for periods of time that are similar in duration to that of normal XY individuals, who do transfer large numbers of spermatozoa in a single copulation and ejaculation. This observation is evidence of an indirect nature, but, nevertheless, further suggests that there may be no relationship between the duration of copulation and the amount of sperm which is transferred a t the time of ejaculation. I n D . melanogaster, there is considerable evidence for the fact that the duration of copulation is under genetic control (see, e.g., a summary of the various laboratory experiments on the mating behavior of Drosophila by Parsons, 1967). Merrell ( 1949), Hildreth (1962), and Parsons (1964) found that mating takes place a t different rates in a number of pure and hybrid strains of D. melanogaster and Hosgood and Parsons (1965) reported differences between strains from single inseminated females taken from natural populations. Furthermore, Manning (1961) has shown the effects of artificial selection on the duration of mating from his studies in which crosses of a “slow” strain (80 minutes) with a “fast” strain (3 minutes) of D. melanogaster produce hybrids for which the duration of mating is intermediate between the two. IV. Sperm Transfer

A. THETIMEOF EJACULATION Nonidez (1920) first reported that sperm ejaculation takes place (in wild-type matings of D. melanogaster) 9-10 minutes after the beginning of copulation. This observation was later supported by Manning (1962b). Since this work, a number of experiments of the “interrupted-mating” type have been carried out and have yielded similar results. For example, Garcia-Bellido (1964) interrupted matings a t 4 and 8 minutes after the beginning of copulation and found that after 4 minutes of copulation not one spermatozoan was transferred, but a t 8 minutes masses of sperm were observed in the female genital tract. By means of phase contrast microscopy, Bairati (1968) has also studied the sequence of events during the transfer of sperm. His findings show that a t 7-8 minutes after the beginning of copulation, some transfer of sperm has taken place. However, masses of sperm are not visible throughout the uterus and vagina of the female until 10-12 minutes after copulation has begun. Bairati’s observations, furthermore, indicate that prior to the ejaculation of spermatozoa, a (‘waxy plug” (see Section 11, B for discussion) is transferred to the female. The plug is visible in the uterus of the female from 5-7 minutes after the beginning of copulation until approximately 6 hours after the end of copulation, a t

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which time i t either shrinks and finally disappears or is removed from the genital canal by the passage of the first egg. While all these data seem to suggest that ejaculation of spermatozoa does not generally occur until approximately 8 minutes after the beginning of copulation, none of it, on the other hand, completely eliminates the possibility of ejaculation a t an earlier time. Indeed, this is undoubtedly a characteristic of Drosophila males which is variable from strain to strain and, perhaps, also from individual to individual. For example, a preliminary study in which wild-type flies were separated a t various times during copulation indicates that the time a t which 24-hour-old males first begin to ejaculate sperm ranges anywhere from 1 minute after the beginning of copulation to just before the end of copulation (Fowler, unpublished). In addition to individual differences between males, the physiological basis for which is not understood, experiments by Lefevre and Moore (1967) seem to suggest that the age of the male may also play a role in the determination of the time at which sperm is first ejaculated, a t least in D . melanogaster. For example, using males and females of wild-type as well as certain mutant strains, these authors find that after the initiation of mating, sperm transfer occurs earlier in older (e.g., 3-day-old) males than it does in younger (e.g., 24-hour-old) males. Whether this finding is typical of Drosophila males, in general, is presently not known.

B. NUMBEROF SPERMEJACULATED At the time of ejaculation the sperm are deposited in the female genital tract enclosed in a membranous sac (the “spermatophore”), which is formed by the secretion of the male accessory glands (Hinton, 1963). According to Nonidez (1920), sperm in the uterus immediately after ejaculation appear to be completely immotile. This, however, is not confirmed by the microscopic examinations of the genital tract of females, dissected immediately after copulation, by D e Vries (1964), among others, who reports that the sperm a t this time are in vigorous motion. Even though the spermatozoa actively move it is not yet known how they disengage themselves from the spermatophore in the uterus of the female and subsequently move into the storage organs since in vitro studies of sperm movement in Drosophila do not show that undulating sperm can exhibit any forward movement (De Vries, 1964). Concerning the actual number of sperm transferred a t ejaculation, it seems probable, a t least a t first approximation, that this is a function of the number of sperm which have accumulated in the seminal vesicles

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of the male a t the time of copulation (Lefevre and Jonsson, 1962a; Mossige, 1955). This is, of course, an entirely reasonable assumption, particularly if spermatozoa are not reabsorbed and/or spontaneously ejaculated, as is suggested by the work of Liining (1952) and Muller (1951). But, as already mentioned (see Section 11, B ) , the quantity of sperm transferred by the male is closely correlated with the quantity of accessory gland secretion available a t the time of mating (Lefevre and Jonsson, 1962a; Garcia-Bellido, 1964). I n this regard, then, the age of the male is, apparently, an important factor in the ultimate determination of the number of sperm transferred since the older the male the more accessory gland fluid would, presumably, be present. According to Lefevre and Jonsson (1962a) the accessory glands possess very little secretion a t eclosion but enlarge with time. Therefore, the quantity of sperm that can be transferred a t copulation increases as the male ages. For example, the very first mating of wild-type males less than 18 hours old results in the transfer of relatively small numbers of sperm (e.g., 200 or less) and frequently none a t all (Lefevre and Jonsson, 1962a). Judging from the relatively few progeny they produce, even wild-type males a t 24 hours of age (Table 2b) probably also transfer very little sperm. With increasing age, however, motile sperm continue to accumulate in the seminal vesicles of virgin wild-type males, so much, in fact, that Lefevre and Jonsson (1962a) estimate that 7-day-old males have as many as twice the number of vesicular sperm as do males aged for 3 days. Such age affects in wild-type strains are, however, not characteristic of Drosophila males, in general. For example, as has been previously discussed, SD/cn bw D . melanogaster males exhibit precocious sexual behavior and show a t least equal fecundity with comparably aged wildtype males (Tables 2a and 2b). I n preliminary experiments (Fowler, unpublished), in which sperm from such males was actually counted in the storage organs of wild-type females after copulation, there is evidence that as early as 13-14 hours after eclosion, 60% of a population of virgin males of this particular genotype transfer, on the average, approximately 250 sperm. While there is considerable variability in the individual counts, it is clear, a t least in this strain of Drosophila, that less than 18 hours of age does not necessarily severely limit the number of sperm that can be transferred in the first mating. Indeed, the number of sperm counted in the seminal receptacles of females mated to males aged an additional 6 hours, e.g., 18 hours old, is approximately the same as that from the matings to the younger males. Data of this kind, then, tend t o suggest that while there may be a basic positive correlation between the age of the male and the numbers of sperm transferred a t any one ejaculation, such a relationship may

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reflect (1) strain differences and/or (2) may have a limiting point beyond which additional sperm are not transferred regardless of the number present in the seminal vesicles and the amount of accessory gland secretion available a t the time of copulation. These possible modifying factors are, of course, in addition to the well documented observation that increased age of the Drosophila male leads to the malfunctioning of a number of the components of the reproductive system (see Section 111, B for discussion), any one of which could lead to a reduction in the number of sperm which are transferred at copulation. Considerable modification in the numbers of sperm transferred by an adult virgin Drosophila male a t copulation can be related to factors other than differences in strains, age, and the amount of available accessory gland fluid. For example, the observation of a 30% increase in progeny when males are subjected to high (e.g., 36OC) temperature shocks, suggested to Iyengar and Baker (1962), as only one of a number of other possible explanations, that the number of sperm ejaculated during copulation might be a function of temperature. Even though an increase in male fecundity and, presumably, also in the numbers of sperm transferred, can be accomplished by exposures to high temperature for short periods of time, it is known that temperatures above 31OC for a long period of time induces sterility in approximately 95% of a male Drosophila population so treated (Young and Plough, 1926). With regard to cold temperature shocks, Lefevre and Jonsson (196213) exposed males to -1OOC for 10 minutes and noted that all the spermatozoa in the seminal vesicles were rendered immotile, a condition leading to almost complete sterility in subsequent matings. Similar results have also been obtained in studies of this type by Iyengar and Baker (1962) and Wedvik (1962). In addition to temperature, the transfer of sperm in D . melanogaster can also be affected by the treatment of males with X-rays prior to mating. For example, on the basis of the microscopic examination of the relative fullness of excised ventral receptacles of inseminated females, Yanders (1959, 1964) first reported that the number of sperm delivered to the D . melanogaster female is inversely proportional to the dose of X-irradiation. Zimmering and Fowler (1966) confirmed this result and provided additional information on the actual number of sperm transferred a t the various doses (Table 3 ) . Judged on the basis of sperm counts in the ventral receptacle, it appears from the data in Table 3 that relatively high doses of X-rays (at 1000 r/min) of 5,000 r, 25,000 r, and 50,000 r delivered to the male prior to mating results in reductions of some 20-25%, 60-65%, and 85-90%, respectively, in the number of sperm transferred to the female.

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What specific effect(s) such acute doses of X-ray may have on the various components of the male reproductive system remains t o be determined. On the basis of the previous discussion of the numerous ways in which the number of sperm transferred at copulation might be affected, it is obviously very difficult to make generalizations about what fraction of the sperm population contained in the seminal vesicles is actually ejaculated by the male a t a single copulation. This is particularly true since (1) sperm cannot be counted in the male or (2) in the female immediately after copulation since the sperm a t that time are still parTABLE 3 Effects of Irradiation of Drosophila Males (Wild Type) on the Number of Sperm Transferred to (I) Yellow (y) Females and (11) Wild-Type (+) Females" Mean No. of sperm in Expt. No. I

I1

Dose of X-rays (r) 0 5,000 25 ,000 50 ,000 0 5,000 25 ,000 50 ,000 ~~~~~

a

Ventral recep- Spermatacle thecae 236.9 183.7 90.1 31.5 300.0 219.6 112.6 22.5

10.4 14.9 11.1 21.3 35.5 35.9 18.8 5.0

Mean percent sperm in

Percent Ventral decrease, recep- Sperma- ventral No. of tacle thecae receptacle females 95.8 92.5 89.0 59.7 89.4 85.0 85.7 81.8

4.2 7.5 11.0 40.3 10.6 14.1 14.3 18.2

22.5 62.0 86.7 26.8 62.5 92.5

46 36 40 30 57 89 22 17

~

Data of Zimmering and Fowler (1966).

tially enclosed in the spermatophore. In both the male and the female, then, the sperm heads (that part of the mature spermatoeoan which is differentially stained with acetic orcein and, therefore, used for purposes of counting) are very difficult to discern one from the other. However, it is possible to make estimates of the number of sperm transferred a t copulation (with no particular reference to the number in the male before ejaculation) and these have been done by a number of investigators. For example, the number of sperm initially deposited by 6-dayold wild-type virgin males in the female reproductive tract was first approximated by Kaufmann and Demerec (1942). They reported that nearly 4000 sperm are transferred in the first mating of such males. Similar results with 24-hour-old wild-type males have also recently been obtained by Zimmering (personal communication). The results from ex-

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periments with wild-type males, however, cannot (or should not) be extrapolated to other strains of Drosophila. For example, counts made on the sperm mass in the vagina-uterus of yellow (y) females mated to 24-hour-old SD/SD+ males and dissected immediately after insemination indicate that the average transfer in such matings is on the order of 300400 sperm (Peacock and Erickson, 1965). The considerable difference observed between the number of sperm transferred by the wild-type males and those ejaculated by SD males is, perhaps, not as surprising as it might otherwise be in the light of the recent observations by Tokuyasu et al. (1972), and some independent findings by the present author (unpublished), which seem to indicate that in the testes of SD/SD+ males there is some alteration in the normal processes of sperm development. This alteration in the normal development of the spermatozoa may manifest itself in different ways. For example, as seen in Fig. 12, there may be reduced numbers of spermatids in each bundle, an observation which confirms the earlier findings of Nicoletti (1968) and Bertolini and Nicoletti (1968), who found the same thing in the males. I n addition to reduced numbers of sperm testes of SDRoma/SD+ in the spermatid bundle, there is also evidence (Nicoletti, 1968) that in some bundles of 64 spermatids about 30 exhibit “abnormal” tail structure. This observation has recently been confirmed by Tokuyasu et al. (1972), who, in addition, have observed that in heterozygous SD stocks, particularly those with high distorting (i.e., k ) values, approximately half of the spermatid nuclei are seen to contain “incompletely condensed chromatin,” a failure of normal spermatogenesis which, according to the authors, may be largely responsible for the fact that such sperm are usually not “individualized” and, therefore, do not complete normal development. All of these variations in the normal pattern of sperm development can lead to sperm which can be described, generally, as “degenerate.” If such “degenerated sperm” are, in fact, not ejaculated, as has been suggested by Zimmering et al. (1970a), among others, the observation of reduced numbers of sperm in the female immediately after insemination might be expected. It should be stressed at this point, however, that sperm development in the testes of SD males is, itself, often a variable trait ranging from strains of SD in which sperm development appears to be completely normal to those in which there is evidence of considerable aberrancies in the normal developmental processes and some degeneration. Mutants that lead to aberrancies in the normal development of sperm have been previously reported in Drosophila. For example, in D . melanogaster, Shoup (1967) reported that in a male heterozygous for a translocation between the X chromosome and the second chromosome, there

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is a failure in the differentiation of the sperm head, a condition which ultimately leads to complete sterility. Normal spermatogenesis and/or spermiogenesis is, apparently, also affected in sex-ratio (SR) mutations occurring in the X-chromosomes of some populations of D. simulans

FIQ. 12. Transverse sections of several test,icular cysts of an SD-72 heter-

ozygous Drosophila melunogaster male containing tails of late spermatids. The mean

number of spermatids per cyst is 32. See Fig. 6 for description of normal spermatid structure and technique. x 25,100.

(Faulhaber, 1967) and D.pseudoobscura (Policansky and Ellison, 1970). Because of the difficulties involved in making reliable counts of sperm in the female genital tract immediately after copulation, most of the reports of “sperm transfer” have, in reality, been extrapolations from actual counts of sperm in the storage organs of the female, which in

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heavily inseminated females ranges from 500 to 700 sperm (Lefevre and Jonsson, 1962a; Kaplan et al., 1962). According to these authors, the figure of 500-700 sperm represents the “storage capacity” of the female, a number somewhat larger than that in average-sized ejaculates which, from males aged 2 4 6 6 hours, is on the order of 300-500 sperm (Peacock and Erickson, 1965; Zimmering and Fowler, 1966, 1968). Peacock and Erickson (1965) report that a t least in the upper part of this range, ejaculates from, for example, SD/SD+ males, there is an approximate 1: 1 correspondence between the number of sperm transferred and the number of sperm stored. T o make a generalization from this observation to include other strains of D . melanogaster is, however, probably not valid. If, for example, the counts of sperm in the ejaculates of wild-type males by Kaufmann and Demerec (1942) and Zimmering (personal communication) are correct, one must somehow account for a reduction by about 80-90% of the sperm population to that which finally appears in the storage organs of the female. Kaufmann and Demerec (1942) and Lefevre and Jonsson (1962a) suggest that there is considerable “wastage” of sperm in Drosophila, such that, in fact, only about 10-20% of the sperm transferred is actually stored. If this is so, the “excess” from an ejaculation of several thousand must be expelled from the genital tract of the female within 2-3 hours after the end of copulation, since when dissections of the genital tract of the female are made approximately 3 hours after the end of copulation, virtually all the sperm visible in the female are seen in either the ventral receptacle or the spermathecae. Since the dissection of the female is almost always done prior to the laying of the first egg after insemination, an expelling of such a mass of sperm by the passage of an egg through the genital tract (Kaufmann and Demerec, 1942) would seem to be an unlikely explanation to account for this absence of sperm in the vagina-uterus of the female. The proportion of sperm transferred relative to the number of sperm stored is, as yet, an unsolved problem in studies of Drosophila reproductive biology, but it seems almost certain that there is rarely a 1:l relationship between sperm transfer and sperm storage. More on this particular point is presented in Section VI. V. Post-Mating Responses in the Female

A. STATEOF RECEPTIVITY Mating produces a number of rapid and marked changes in both the behavior and the reproductive physiology of Drosophila females. For example, even though the male Drosophila commonly courts and mates

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with other females almost immediately after one mating, this is not typical behavior of the female, who generally exhibits unreceptivity to any male for a t least some time after copulation (Chapman, 1969; Lefevre and Jonsson, 1962c; Meyer and Meyer, 1961; Dobzhansky and Pavlovsky, 1967; among others). This period of unreceptivity, which can vary from 4 hours (Smith, 1956) to 10 days in length (Manning, 1962a,b, 1967), may be largely due to two different, but related, factors: (1) a “sperm effect” in which receptivity remains switched off so long as there are sperm in the storage organs of the female, and/or (2) a “copulation effect” in which the act of copulation, itself, has induced the lack of receptivity (Manning, 1962a,b, 1967). Experiments by Manning (1962a) suggest that the “sperm effect” may be due to the presence of some chemical substance(s) contained in the ejaculate which acts as an inhibitor of receptivity. It is difficult to say, however, what specific component of the ejaculate is actually responsible for the effect. A logical first choice would be the sperm themselves. Even though there is virtually no evidence on this point in D . melanogaster, in a t least one other species of Drosophila it seems unlikely that the reason for the lack of female receptivity to further mating is due to the presence of sperm. Specifically, Smith (1956) has shown that normally inseminated females of D.subobscura rarely mate again even if their sperm supply has been depleted by continued oviposition. There is a similar paucity of evidence in Drosophila to implicate the supporting medium for the sperm (i.e., the paragonial fluid) as being responsible for the lack of receptivity of the mated female even though chemical agents which eliminate receptivity in other Diptera are found predominantly in the seminal fluid. Indirect support for this possibility in Drosophila, however, may come from the fact that Drosophila females usually require about 24 hours to become receptive after their seminal receptacles are empty of sperm, an observation that might suggest the waning of a chemical influence from seminal fluid still present in the female genital tract. Furthermore, Merle (1969) has reported that virgin females with grafted paragonia tend to refuse copulation and this might implicate the paragonial secretion with this aspect of female sexual behavior. Even with the lack of strong evidence of their origin, the presence of “receptivity-inhibiting substances” in the female Drosophila may be advantageous t o the male possessing them since according to Manning (1967) “males whose sperm tends to cause the switch-off of a female’s receptivity will be a t a considerable advantage, because their sperm will be used to the full. Both males and females whose sons have sperm with this effect will be favored.” I n addition to (or, perhaps, in concert with) the “sperm effect,” Man-

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ning (1962a,b, 1967) also suggests that the act of copulation, itself, may play a role in reducing receptivity to additional mating in Drosophila females. Evidence to support the hypothesis is not very extensive. As one example, however, Smith (1956) reported that females mated to males without testes exhibit unreceptivity for periods of time ranging from 4 to 24 hours, but regain their receptivity sometime later. Since these males transfer no sperm a t ejaculation, it is possible that the mere act of copulation is producing the effect in the female. Whatever the general effects of factors that lower the receptivity of the female to additional matings, it is a well known fact that the female rejection reactions displayed during this period of unreceptivity can be overcome by a particularly persistent male. This is true of all species of Drosophila, but it is very common in D . melanogaster. Such instances, however, are not the rule and are probably mainly cases of rape.

B. POST-COPULATION CHANGES IN THE FEMALE REPRODUCTIVE PHYSIOLOGY With no particular reference to the effect on female receptivity, Di Pasquale (1959) has described the effect of copulation on the reproductive physiology of certain mutant strains of D. melanogaster. I n the pleurae of the females of these strains, superficially pigmented areas appear soon after copulation. D i Pasquale and Zambruni (1965) report that these “brown spots” (mutant, bsp) are, apparently, induced by the copulatory act, itself, Furthermore, since such spotting will also appear in the female following the introduction of a thin glass needle into the vagina (Di Pasquale and Zambruni, 1967), it is suggested that some mechanical stimulus during copulation is the causative agent. Probably the most extensively studied post-mating change in the reproductive physiology of the female Drosophila occurs in the genital tract (vaginal pouch). Patterson (1946) was one of the first investigators to study these changes which collectively comprise the phenomenon known as the “insemination reaction.” For a full account of this reaction, see Patterson (1947) and Patterson and Stone (1952). Briefly, the insemination reaction occurs both in intraspecific (homogamic) matings and in interspecific (heterogamic) matings. It usually follows immediately after coitus, but in some matings it may begin before copulation is even completed. The reaction is revealed in the vagina through a rapid secretion of fluid into its cavity, resulting in an increase in the vaginal pouch to three or four times its normal size. In females from homogamic matings the vagina returns to a normal

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condition usually within a period of 8-9 hours, but in those from heterogamic matings it may remain swollen for several days and sometimes as long as a week. I n the latter type of mating this reaction may (1) reduce the number of sperm which enter the storage organs of the female, (2) interfere with fertilization, and (3) cause cytolysis of the eggs, all of which act to reduce the number of hybrids which might be produced, and in some crosses may eliminate them altogether. The term “reaction mass” has been used to designate the contents of the swollen vagina. In heterogamic matings the mass persists for a long time as a discrete crystalline structure. On the other hand, in homogamic matings, the reaction mass remains soft and is expelled by the female usually within 6-12 hours. In a large number of homogamic matings there is no visible reaction mass a t all. Nonetheless, there is some evidence that changes in the genital tract of the female do take place. For example, in D . melanogaster, there is no visible reaction mass in the reproductive tract of the female following a homogamic mating, however, within 30 minutes after the end of copulation, sperm which have not already been stored in the seminal receptacles are largely nonmotile. Furthermore, 1 hour after the end of mating, the vagina is seen to be free of sperm and a mass of nonmotile sperm is found on the surface of the food (Wheeler, 1947). According to Wheeler (1947), who studied homogamic matings in several species of Drosophila, this finding affords direct evidence that sperm are expelled by the female in some homogamic crosses and supports the suggestion that some kind of insemination reaction is taking place. If, in fact, insemination reactions of this type were the rule in homogamic matings in Drosophila, it might explain the large discrepancy between counts of sperm in the vaginauterus of the female immediately after copulation and the numbers in the storage organs sometime later (see Section IV, B for discussion). At this point it is important to note that there does not seem to be any obvious correlation between the “waxy plug” observed in the female genital tract of D.melanogaster described by Bairati (1968) (see Section 11, B) and the fluid secretion which becomes the reaction mass in homogamic and heterogamic matings. One of the most important and interesting questions concerning the insemination reaction is the agent or agents which might be responsible for triggering the response. It seems clear from the experimental results of Patterson (1947) and Wheeler (1947) that the reaction mass in both homogamic and heterogamic matings might be induced by either (1) the living spermatozoa or (2) the seminal fluid. I n studying the early stages of the formation of the reaction mass, it has been observed that in a few cases the reproductive tract of the female contains no detectable

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sperm and yet the typical insemination reaction takes place immediately after copulation. From this evidence and the observation that an insemination reaction can occur after insemination of a female by sterile hybrid males, it may be assumed that motile sperm are not the active agents in producing the reaction. Not much specific information is known about the chemical composition of the seminal fluid in Drosophila even though the liquid part of the ejaculate is composed primarily of the secretions of the paragonia (which have been described in some detail in Section 11, B). On the basis of the striking similarity between these secretions and the material observed in the vaginas of many species of Drosophila immediately after copulation (Wheeler, 1947), it seems reasonable to suggest that a direct relationship exists between the male paragonial fluid and the insemination reaction. This possibility, however, is not experimentally supported and, in fact, Lee (1950), who injected various substances into the vagina, suggests that the insemination reaction is induced by a secretion from the testis (possibly a protein), not by the paragonial secretion. The presence of an insemination reaction in intraspecific matings forces one to consider the possibility that such a reaction may be a normal consequence of insemination and may have useful functions in insemination, fertilization, or oviposition. Indeed, according to Patterson (1946), “It (the insemination reaction) may have the effect . . . of preparing the reproductive tract for the fertilization mechanism which is to follow.” The strong reaction noted in large numbers of interspecific matings, on the other hand, does not allow the same conclusion. I n this case, Patterson and Stone (1952) suggested that the prolonged reaction in heterogamic matings reduces hybridization and is, therefore, an important species isolating mechanism. As Smith (1956) pointed out, however, a female which shows such an extended insemination reaction suffers a great reduction in fertility, and the general reaction cannot, therefore, be due to the direct result of natural selection for species isolation. It seems, then, that neither the function of the insemination reaction nor the way in which it has become established in populations of Drosophila is yet completely clear. Much more clearly understood than the “insemination reaction” is the effect of the presence of sperm on other facets of the reproductive physiology of the female Drosophila. In this regard, David (1963) has shown that eggs laid by inseminated females are about twice the size and the number of those laid by virgin females who, a t least in D. melanogaster, lay 40% fewer eggs than do mated females (Wilson et al., 1955).

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The females of D. melanogaster begin egg laying on days 2-3 posteclosion and maximally ovipost from day 6 to 10, even though this is, apparently, a strain-dependent characteristic (McMillan et al., 1970a). After this maximum period, egg laying geometrically declines, e.g., about 2.7% per day (Narain, 1962), until the death of the female, which occurs approximately 30 days after eclosion (Robertson, 1957, among others). I n the inseminated female, the number of eggs produced during the egg laying lifetime of the animal varies tremendously and is a function of any number of different factors, e.g., age of the female, genotype, humidity, degree of adult crowding, nutrition of the adult (see King, 1970, p. 50, for additional factors and specific references). Determination of the actual numbers of eggs produced by D. melanogaster females for periods less than lifetime have been measured by a number of investigators (see McMillan et al., 1970b, for specific references), but as a rule these figures are inconsistent from strain to strain and from experiment to experiment. This is particularly true for estimates of egg production over the entire lifetime of the female, an observation which is hardly surprising given the fact that oviposition is a function of so many different factors, all of which must be similarly controlled from experiment to experiment in order to provide consistency. Several workers, however, have addressed themselves, either directly or indirectly, to the problem of a t least estimating the total numbers of eggs produced by a D. melanogaster female over a lifetime. For example, David and Merle (1966) concluded from their studies that a female can daily lay twice as many eggs as its total number of ovarioles. Since, in an inbred strain of average fecundity the ovarioles/ovary number about 16 1, this means that a single female can produce approximately 64 eggs per day. On the assumption, then, that a female will live for about 30 days, it is expected that under the most favorable conditions a female can produce approximately 2000 eggs during her lifetime. It should be stressed at this point, however, that this figure is only a reproductive physiological potential which is, to the author’s knowledge, not yet experimentally supported. Because of the difficulties of obtaining an actual reliable figure of lifetime egg production of D . melanogaster females by experimentation, McMillan et al. (1970a,b) have placed the factors which influence female fecundity into a theoretical framework and have derived a mathematical model which characterizes, among other parameters, the daily egg production of females over their lifetime. The conclusion reached by these workers is that the total potential lifetime egg production is equivalent to the number of primordial egg cells a t the time of initiation of

*

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egg laying, a conclusion, in fact based upon the growth/decay features of typical lifetime egg production curves. As derived, the model appears to be sound and, in fact, has been used to determine chromosomal effects upon egg production in D . melanogaster (Fitz-Earle, 1971 ; 1972) as well as to examine lifetime fecundity in other organisms (G. W. Friars, personal communication). As already mentioned, mating, itself, exerts some influence on the production of eggs, possibly as a trigger to stimulate oviposition. Further, if such a mating stimulus is not supplied, oviposition is delayed. After a once inseminated female has been laying eggs for several days, that is, after the maximum egg laying period has been passed, the rate of oviposition decreases to the point where the female is laying eggs a t a level similar to that of virgins (David and Croissant, 1956; David, 1963). With a second copulation, however, fecundity again rises quickly. This pattern is repeated throughout the reproductive period of the female and suggests that the presence of spermatozoa in the female plays an essential role. in the maintenance of what David (1963) has termed, the “activated” state of the female. Extensive work by a number of investigators has led to the suggestion that the effect of spermatozoa on female fecundity may, in reality, be more a function of the medium supporting the sperm than of the sperm themselves. Evidence to support this hypothesis has already been discussed in some detail (see Section 11,B) . The effect of mating on oviposition in Drosophila females is, apparently, not a function of the type (genotype) of the male with which the female mates. For example, copulation of females with sterile males carrying deficient Y chromosomes lay as many eggs as do females inseminated by normal XY males (Muller, 1944). Similarly, Cook (1970) finds that females mated to sterile males who lack the Y chromosome completely (i.e., XO) also lay eggs at a level comparable to that of normally fertilized females, even though the increase in fecundation in this case is transient returning rapidly to the virgin level. Results from such sterile matings, taken together, might support the “activation-byparagonial-fluid” hypothesis, on the assumption, of course, that the secretions of the paragonia are being ejaculated in the absence of spermatozoa. However, there is no evidence that this occurs. If this is not the case, increased fecundation of the Drosophila female following mating may be a reflection, according to Muller (1944), of “some nervous reaction attendant upon the act of mating of the female that leads to ovulation (and oviposition) ”. Further experimentation will, hopefully, provide a resolution to the problem as well as to the many other confusing aspects of the post-mating responses in the Drosophila female.

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VI. Sperm in the Female: Sperm Storage

Sperm storage is one of the most important factors in the reproductive physiology of the Drosophila female since it is unlikely that a significant number of eggs (if, indeed, any) are fertilized by sperm that are not first resident in the ventral receptacle or the spermathecae. It is a well known fact that, during ejaculation, sperm are deposited in the vagina of the female in the form of the “spermatophore” (see Section IV, B for discussion), but from this point on, the literature on the behavior of sperm in the female genital tract is sufficiently variable and/or unclear so as to make generalizations impossible. For example, there is no evidence to suggest the manner in which the sperm travel from the spermatophore to the seminal receptacles for storage. On the basis of what is known in other organisms, e.g., mammals, it seems reasonable to suggest that muscular contractions of the walls of the genital tract might play an important role in the transport of sperm in the Drosophila female. Even though there is no evidence to the contrary, the fact that in Drosophila, the length of the genital chamber is so short (e.g., less than the length of the tail of the sperm), it hardly seems necessary, to use muscular contraction as a sperm-transporting mechanism in this organism. In this regard, it has been suggested by Bairati (1968) that the “tubular elements” in the male paragonial fluid (i.e., the supporting medium for the sperm) may, a t least be partly responsible for transporting the sperm to the seminal receptacles (see Section 11, B for discussion). Indeed, the fact that such elements are observed interspersed among spermatozoa stored in the ventral receptacles (Fig. 9) could be taken as evidence for such a hypothesis. Regardless of the particular methods by which sperm are moved to the storage organs, i.e., by muscular contraction or by contractile components in the seminal fluid, or both, or neither, it is interesting to note that in this movement to the seminal receptacles, the sperm mass does not fill the entire lumen of the genital chamber. Specifically, a number of dissections made immediately after the end of copulation and before the mass of sperm has been completely stored, indicate that the sperm which has become free of the spermatophore are generally contained and moving in what could, perhaps, be most accurately described as “channels” (Fig. 2B), all of which seem to be physical extensions of the seminal receptacles (Fowler, unpublished). If, indeed, sperm do move in such a contained fashion from the spermatophore to the storage organs and if there are direct connections between the vagina

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(i.e., the spermatophore) and the seminal receptacles, it is possible that such channeling would increase the efficiency and rapidity with which a large number of sperm are stored. For example, Nonidez (1920) observed that spermatozoa do not enter the seminal receptacles immediately after their deposition in the vagina, there being a 2-3 minute pause during which the sperm become active (or activated). However, within 2-5 minutes after this “activation,” the entire lumen of the ventral receptacle was observed to be filled. Even though the activation of sperm in the female immediately after ejaculation has not been substantiated, to the present author’s knowledge, by any other published investigation, the time of the appearance of the sperm in the storage organs reported by Nonidez (1920) is generally in agreement with most subsequent experimental observations. For example, after dissecting females at various times during copulation, Yanders (unpublished) established that the first spermatozoon reaches the ventral receptacle “only a few minutes after ejaculation,” i.e., about 20 minutes after the beginning of copulation, the seminal receptacles reaching their maximum fullness within 1 hour. Similar results have been obtained by Gugler et al. (1965), who observed that sperm can enter the storage organs within 20 minutes after the end of copulation; by De Vries (1964), who reported the appearance of sperm in the storage organs less than 1 minute after the interruption of copulation; and by Lefevre and Jonsson (1962a), who observed sperm being stored during copulation. Generally speaking, the majority of these conclusions have been based on saline squashes of the dissected storage organs of the female either during or immediately after copulation. Even though this technique is entirely satisfactory to determine the first appearance of sperm in the seminal receptacles, it has recently been of some interest to determine the actual number of sperm being stored relative to a certain length of time, and in this regard, a cytological technique has been developed for counting sperm. The technique of actually counting sperm in the seminal receptacles of D . melanogaster females was first outlined by Lefevre and Jonsson (1962a) and then modified in some details by Peacock and Erickson (1965). Generally speaking, the technique involves dissecting out the female storage organs approximately 2-3 hours after the end of copulation, staining the entire preparation with a 2% solution of natural orcein in 60% acetic acid for a minute or so, and then applying a cover slip and observing under oil with phase microscopy. The head is that part of the spermatozoon that can be stained in this fashion (Fig. 3) and it is a relatively easy, albeit tedious, procedure to merely count the stained heads from the proximal to the distal end of the ventral recep-

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tacle in which they are more or less oriented in a linear fashion. Accurately counting sperm in the spermathecae is, however, more difficult because of the chitinous walls of the capsule through which the individual sperm head is difficult to see (Fig. 4). However, this difficulty can generally be overcome if the spermathecae are broken open by gentle pressure so that the chitinous walls are ruptured (Fowler et al., 1968) with the result that the sperm contained therein are spread out, making the deeply stained heads more discernible. Using this technique, Fowler (unpublished) has carried out experiments in which wild-type D. melanogaster females were dissected a t &minute intervals after the end of copulation with 24-hour-old SD/ cn bw males. The results of these experiments are generally consistent with the findings cited earlier, in that as early as 15 minutes after the end of mating well over a hundred sperm are observed in the seminal receptacles. Furthermore, after 1 hour no sperm are found anywhere in the female reproductive tract except in the ventral receptacle and the spermathecae, which, combined, contain about 350 sperm. Even though the experimental evidence cited here seems to suggest that sperm of D. melanogaster is stored relatively rapidly after copulation and, indeed, in some instances, even during copulation, this is not typical of all species of Drosophila. As a case in point, in D. unispina and D . brachynephros no sperm appear in the storage organs of the female until 1 hour after the end of copulation (Shima et al., 1967). The rapidity with which sperm storage takes place in D. rnelanogaster has been experimentally shown to be dependent on a number of different factors. For example, observations by Lefevre and Moore (1967) suggest that the speed of sperm storage may be an age-related phenomenon. These authors reported that a t 8.5 minutes after the completion of mating, sperm from 3-day-old males appear in the ventral receptacle and the spermathecae. On the other hand, comparable storage occurs in only 1 minute when the same type of male is mated a t 1 day of age. In both cases, maximum storage occurs within 15-20 minutes. There is also some evidence that the rapidity with which sperm storage takes place may be a reflection of the genotype of the female. Specifically, Yanders (1963) carried out inter- and intrastrain crosses between four different wild-type strains of D. melanogaster and determined the relative number of sperm in the storage organs sometime after the completion of copulation. The results of these experiments indicate that the migration of sperm (as determined by estimates of numbers of sperm in the storage organs by the criterion of “relative fullness”) is proportionately slower in certain combinations than in others, a finding which Yanders (1963) attributes to some kind of inhibiting action of the female

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genital tract on the motility of certain classes of sperm. De Vries (1964) carries this hypothesis a step further by making the suggestion that the quantity (and presumably the type) of sperm stored in a particular female may be a function of a particular male-female combination. Such a hypothesis suggests that the entry of sperm into the storage organs of the female may involve some processes which are dependent on the nature of the individual sperm themselves. What this ‘‘nature of the individual sperm” may be is not entirely clear, but is probably not a function of the genotype, per se, of the sperm since it is generally conceded that the ATP of the spermatozoan is completely inert and that the functional aptitude of sperm is completely independent of its genetic content (Muller and Settles, 1927). This point, however, is discussed in more detail in Section VII. Without specifiying the biological bases for the findings, a number of observations have been made in D . inelanogaster which might be interpreted as examples of differential sperm storage and which might also be reflections of the kind of “interaction” suggested by De Vries (1964). For example, Zimmering et al. (1970a) have shown that while an SD/cn bw male mated to a yellow (9)female shows a 1.1 relationship bettween sperm transferred and sperm stored, the ratio is 2:1, respectively, when the same SD/cn bw male is mated with a wild-type (+) female (Zimmering, personal communication). I n addition, males that carry a Y chromosome with a deficiency in the proximal region of the YL fertility complex (i.e., KL-1-; see Brosseau, 1960, for description) produce motile sperm but are sterile. Kiefer (1969) reported that females examined immediately (and a t 24 hours) after copulation with these males show sperm filling the uterus and moving up to the seminal receptacles but not entering them. I n line with these observations and consistent with the hypothesis of some kind of sperm-female genital tract “interaction” is the report by Sidhu (1963) and Beatty and Sidhu (1969) that in the trimorphic populations of sperm from males of D . obscura, there is a virtual disappearance of the Short class in the combined contents of the storage organs of the female, even though they are observed in the uterus following copulation. Even though not all these cases of “differential sperm storage’’ necessarily need to be interpreted as examples of sperm-female “interactions”-for example, the inability of KL-1- sperm to enter the storage organs has been suggested by Kiefer (1969) to result from an inefficient motile apparatus (i.e., lowered ATP production) -it is still possible, and indeed likely, that there may be subtle biochemical differences between populations of sperm from different males which may be recognized by some female genital tracts as being immunologically “foreign”

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and, accordingly, selected against (i.e., not stored or utilized). Such a suggestion has, in fact, already been made by De Vries (1964) and, recently, by Hart1 and Childress (1973), who observed that even though some males which are homozygous for the locus, Segregation-Distorter (SD1/SD*) produce copious quantities of sperm which are both motile and stored in the female, the males are nonetheless, sterile by virtue of the fact that the sperm are not used for fertilization. The majority of the observations of sperm in the female following multiple inseminations seem to further imply, if not strongly suggest, the possibility of differential responses of sperm to the genital tract environment of the female. For example, considering the phenomenon of %perm displacement,” Lefevre and Jonsson (1962a,c) have found that upon a second mating to a male carrying different markers it can be shown genetically that the mutant sperm (e.g., “vermilion,” “forked,” “carnation”) are easier to displace in the storage organs of females than those derived from wild-type (+) males. Such displacement has been interpreted in terms of the observed circulation of sperm within and between the storage organs, and the data seem to suggest that sperm of different genetic constitutions may have different abilities in this respect, presumably as a function of the female environment. This conclusion is supported by the earlier work of Dubinin (1928), who suggested some kind of “biological antagonism” between sperm from different males and the genital tract of the female, and more recently, the experiments of Gugler et al. (1965), who demonstrated that within 20 minutes sperm from a second mating can replace first sperm. On the other hand, interpretations from double mating experiments by Nonidez (1920), Nachtsheim (1928), Lobashov (1939), Kaufmann and Demerec (1942), Bateman (1948), Ehrlich (1959), Meyer and Meyer (1961), Manning (1967), and Dobzhansky and Pavlovsky (1967) range from complete mixing of the two kinds of sperm to the suggestion that the sperm are stored in layers. None of the hypotheses, however, are necessarily supportative of some kind of sperm-female interaction. On the basis of such conflicting observations, it is clear th a t sperm storage, a t least in multiple inseminations in Drosophila, is not a consistent phenomenon. The numbers of sperm actually stored in the receptacles of the female can be modified by prior exposure of the male to certain agents, notable among which is X-irradiation. It has been previously mentioned (see Section IV, B for discussion) that Yanders (1959,1964) found a reduction in the numbers of sperm stored in the ventral receptacle of the female D.melanogaster if the male had been irradiated before mating. Further-

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more, there is a negative correlation between the dose of X-irradiation delivered to the male and the number of sperm transferred, an observation confirmed by Zimmering and Fowler (1966) by the direct counting of sperm in the storage organs (Table 3 ) . According to Yanders, the basis for such an observation may be that some aspect of sperm behavior (e.g., motility) may be the physiological (i.e., nongenetic) damage sustained by X-irradiated sperm. Furthermore, on the basis of experiments carried out by Lefevre and Jonsson (1962a), Yanders suggested that the reduction in storage may be due to the limited ability of sperm with a defective motile apparatus to compete with the more vigorous unaffected sperm of the population for available storage space. While there is no clear-cut specific evidence either to support or refute this idea, preliminary experiments carried out by the present author (unpublished) demonstrate that loss of motility cannot be solely responsible for the reduction in the numbers of sperm transferred, and presumably stored, after X-irradiation, a t least with respect to the higher (i.e., 100,000 r) doses. Because of their relative anatomical positions in the female, it has been suggested that during storage sperm first fill the ventral receptacle and then the spermathecae (Nonidez, 1920). This observation has, apparently, not been confirmed or refuted by any other published investigation, but on the basis of the possible organization of the female genital tract into “channels” through which the sperm may move to the receptacles, it is conceivable that the majority of sperm in the ejaculate might be preferentially directed to one receptacle (e.g., the ventral receptacle) at the time of storage. There is no direct evidence to support this possibility but on the basis of numerous dissections of the female storage organs made at selected intervals both during and immediately after the end of copulation, one has the impression that there is no preferential storage of the seminal receptacles, sperm appearing in both storage organs in a completely randomized fashion. Without hypothesizing on the possible priorities of the seminal receptacles for sperm storage, Lefevre and Jonsson (1962a) suggest that once sperm storage has occurred, there is a continuous circulation of sperm within and between the seminal receptacles. In terms of the actual numbers of sperm which can be stored by the seminal receptacles, on the basis of direct counts of sperm, it appears that under normal circumstances (i.e., a moderate-large sized ejaculate of about 300-700 sperm), approximately 80% of the storage takes place in the ventral receptacle, a completely reasonable phenomenon given the larger size of this storage organ (see Section 11, A for description). However, as discussed previously (see Table 1) this 80:20 relationship

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34 1

between sperm storage in the ventral receptacle and the spermathecae, respectively, varies considerably with the size of the ejaculate (Fowler et al., 1968). In addition to considerations of where the sperm are stored, there is, in some strains of D . melanogaster and with ejaculate sizes in the upper part of a range of 300-700 sperm, an apparent 1:l relationship between the number of sperm transferred and the number of sperm stored. As has been previously mentioned, however, this is not typical of Drosphila males, in general, nor is it typical of ejaculates of, say, older males, where the number of sperm transferred may be in the thousands, at which time all but about 20% of the ejaculate is lost (Kaufmann and Demerec, 1942). I n addition to the possibility that a t least part of this large discrepancy between the numbers of sperm transferred and those which are subsequently utilized for fertilization is a function of some kind of “interaction” between the sperm and the female genital tract, about which there is no direct evidence, it is interesting to note that in terms of actual numbers of sperm ultimately stored, approximately the same number of sperm are found in the seminal receptacles whether 80% or 20% of the ejaculate is being stored. Such a finding suggests that there may be a physical limitation of the storage organs, themselves, such that, generally no more than 700 sperm can be stored by the ventral receptacle and the spermathecae, combined. That this may, indeed, be the case has been suggested by Lefevre and Jonsson (1962a) and Gugler et al. (1965)) who described the sperm storage organs of D . melanogaster females as %on-elastic and non-contractile.” More on this particular point, however, is discussed in the next section. It is clear, then, from the observations reported in this section that there is considerable variability in the amount of time required for sperm to be stored, as well as variability in the number of sperm stored relative to the number of sperm transferred, I n addition, there is also the underlying suggestion that the entry of sperm into the female storage organs at all involves some process (es) which may be dependent upon the nature of the individual sperm, themselves. VII. Sperm in the Female: Sperm Utilization

Once storage has taken place, sperm in both the ventral receptacle and the spermathecae are used for fertilization. According to Nonidez (1920)) sperm stored in the ventral receptacle of D . melanogaster is used first in fertilization and those in the spermathecae are used later after the receptacle is exhausted of sperm. This sequence of sperm utiliza-

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tion is, apparently, also characteristic of a number of other species of Drosophila. For example, in four strains of D . virilk, together with D. americani, D . novamezicana, D . funebris, and D . hydei, the sperm disappear from the ventral receptacle before those in the spermathecae are exhausted (Patterson, 1947). Similarly, in homogamic matings of D. nigromaculata, D. brachynephros, and D. unispina, Shima (1966; Shima et al., 1967) reports that sperm present in the seminal receptacle disappear prior to the disappearance of those in the spermathecae. Even though it is reasonable, primarily because of the relative positions of the storage organs in the female genital tract (e.g., the proximal portion of the ventral receptacle opens directly into the common oviduct a t the top of the uterus) to expect that sperm in the ventral receptacle would be used first in fertilization, there are some species of Drosophila where this is not true. Specifically, in the classes of four different subgenera including D . duncani, D. victoria, D . bwcki, and D . pseudoobscura, and D. immigrans of the subgenus Drosophila, sperm are drained from the ventral receptacle and the spermathecae a t about the same rate (Patterson, 1947). Regardless of the differences between the various species of Drosophila with regard to which stored sperm are used first in fertilization, when females of all species are dissected a t intervals after mating, the sperm store is observed to diminish progressively so that by the time the females no longer lay fertile eggs, the ventral receptacle and the spermathecae are completely exhausted of sperm (Nonidez, 1920; Patterson, 1954). It was originally thought that the utilization (i.e., fertilization) of sperm in Drosophila was as inefficient a process as was the storage of sperm (see Section IV, B) i.e., a great number of sperm were “wasted” since only one of a number of sperm entering the egg would fuse with the egg nucleus. This view was largely supported by the work of Huettner (1924), who occasionally found more than 30 sperm in a single ovum, and by Kaufmann and Demerec (1942) and Counce (1959), all of whom agreed on the basis of their findings that polyspermy was the rule in Drosophila, as i t is in the majority of other insects. By using somewhat different cytological techniques from those of their predecessors, however, Hildreth and Luchessi (1963) failed to find any significant degree of polyspermy in either D. melanogaster or D. virilis (i.e., less than 1% of fertilized ova contained more than one sperm), and from their work comes the presently accepted view that sperm recovery in Drosophila can be explained only with an inference of monospermy. This view was subsequently confirmed by the work of Lefevre and Jonsson (1962a), who carried out experiments to recover all the progeny from single-mated females and obtained results which indicate that there

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is 8 1: 1 relationship between the progeny recovered and the number of sperm counted in the seminal receptacles. Nothing is known of the specific manner in which the female controls the utilization of stored sperm whereby only one sperm is used at a time. Lefevre and Jonsson (1962a) suggested that stored sperm are in a continual state of circulation, such that at any time “some sperm will be approaching the basal end of the receptacle and, perhaps, even partially emerging from it into the uterus. Should no egg be present, the sperm could reverse direction and travel back through the receptacle.” This idea is consistent with the observations by Peacock and Erickson (1965) that, in many dissections of storage organs, a single sperm is seen near the opening of the ventral receptacle even though a large number are present more distally. Occasionally, the discharge of sperm from the opening of the spermathecae and the orifice of the ventral receptacle was observed by Wheeler (1954). In these cases, the discharge of the sperm from the receptacles seemed to be synchronized with the movement of an egg down the oviduct, and it is possible that this movement is the stimulus that brings about the release of a single sperm from the receptacles. It has been previously thought that fecundation is a phenomenon subject to the chance presence of an egg reaching the uterus a t the moment when a spermatozoon comes out of the receptacle. In this regard, the storage organs play virtually no role other than to act as passive repositories of the sperm. For example, Demerec (1950) concluded that the ventral receptacle lacked any musculature whatsoever, and Lefevre and Jonsson (1962a) described the sperm storage organs as “non-elastic and non-contractile.” The possibility, however, that the structure of the ventral receptacle, itself, may play less than a passive, and, perhaps, an active role in the utilization of sperm has recently been suggested by the work of Blaney (1970). Electron microscopy of individual coils of the ventral receptacle show, for example, the presence of an outer muscle coat which, in transverse section, is seen to be composed of thick and thin filaments of myosin and actin, respectively (Figs. 13A and 13B). Such findings suggest that contractions of the ventral receptacle may assist the active movement of the spermatozoa in the emission of stored sperm. Postulating some sort of regulation by the musculature of the ventral receptacle whereby one sperm a t a time is emitted for fertilization may, indeed, be the basis for the previously discussed finding that once sperm is stored in Drosophila, it is used in a very efficient manner. According to Garcia-Bellido (1964) , however, the indispensable mechanism necessary to assure both the admission and emission of spermatozoa

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FIG.13A. Transverse section of the seminal receptacle of the female Drosophila melanogaster. Note sperm (sp) in the lumen of the receptacle, the cuticular lining (cu), the cells of the receptacle wall (wc), the basement membrane (bm) and external to this a layer of visceral muscle (vm). Electron microscopy. Scale line = 1.0 pm. (Reprinted with permission from Blaney, 1970.)

FIQ. 13B. Transverse section of part of the wall of the seminal receptacle of the female Drosophila melanogaster. Note visceral muscle (vm). Each thick filament is surrounded by 12 thin filaments (see arrow). Electron microscopy. Scale line = 0.5 p. (Reprinted with permission from Blaney, 1970.)

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to and from the ventral receptacle in order for fecundation of the oocytes to occur, is the secretion of the male paragonia which, as can be seen in Fig. 9, is the medium which surrounds and presumably provides support to the sperm a t storage. In this particular interpretation, the accessory gland fluid apparently also acts as the stimulus for the opening (and closing?) of the seminal receptacles. If this possibility is taken together with the findings of Blaney (1970), one might imagine that the “target” of the paragonial fluid is the musculature of the ventral receptacle. Furthermore, if, as originally suggested by Meyer (1956), the paragonial fluid is important in normal fecundation, and if some minimal amount of it is necessary before stimulation (contraction?) of the ventral receptacle leading to an expulsion of a spermatozoan and normal fecundation can occur, this might explain why a male that has been continually mated to a number of females, thereby severely reducing the fluid in its accessory glands (Lefevre and Jonsson, 1962a), is essentially sterile even though large numbers of sperm are observed in the female storage organs at the end of copulation. As has been previously discussed, the observations of Lefevre and Jonsson (1962a) lead to the suggestion that the female Drosopkila uses sperm with a high degree of efficiency, meaning that virtually 100% of the sperm which is stored is used in fertilization. It is obvious, however, that the degree of efficiency with which the sperm are used by any particular female in fertilization is directly related to the physiological state of the female a t the time of oviposition and fertilization. It is, for example, a well known fact that the food on which the female is placed can, to a large degree, determine the number of progeny recovered. If, for example, the female is not well fed she will invariably produce fewer offspring, presumably because fewer eggs are produced. Along these lines, Olivieri et al. (1970) reported that if D . melanogaster females are prevented from laying eggs immediately after fertilization (by keeping them on glucose agar medium), fewer progeny are produced than from controls. On the other hand, if the food is relatively rich, particularly in protein, the maximum number of offspring (as a function of the number of sperm stored) can often be recovered. Several investigators have reduced the fertility of inseminated females by submitting them to cold treatment (e.g., Novitski and Rush, 1948; Scossiroli, 1954; Frydenberg and Sick, 1960; Myszewski and Yanders, 1963; Barnett and MunGz, 1970). In most cases there appears to be a direct correlation between the length of exposure and the number of progeny produced, however, this seems to vary from strain to strain. Even though all these treatments affect (i.e., reduce) the numbers of progeny recovered from a particular female, it is impossible to say

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where the specific effect is being manifested. It is likely that the reduction in progeny which is characteristic of all such treatments is a compounding of many different effects. For example, in all such cases it is almost certain that a certain percentage of the reduction in the recovery of progeny is contributed by an adverse effect on the egg-producing capacity of the female. As has been previously discussed, any number of different factors can alter the physiology of the female relative to egg development and laying (see Section V, B for details). In addition, the sperm themselves may undergo alterations of some kind in the storage organs such that the eggs they fertilize are “abortive.” Such a specific possibility has been suggested by Guyenot (1913), among others, to explain the observation that females who were kept undernourished on potato medium (to prevent egg laying) produced significantly fewer progeny than did controls. It is equally possible that the recovery of reduced numbers of progeny as a result of a specific treatment may be reflecting some kind of induced sperm loss. Along these lines, Yanders (unpublished) has irradiated females 7 days before mating with X-ray doses which cause degeneration of the ovaries, thus completely eliminating egg laying and the possibility that any observed reduction in the numbers of sperm in the storage organs might be due to the fertilization of eggs. Even with no sperm being used in fertilization, such treated females still exhibited a significant loss of sperm from the ventral receptacle with time. The reason(s) for such sperm loss is unclear. On the basis of work previously described (see Section V I ) , it is tempting to suggest the existence of an incompatibility (i.e., an “interaction”) of some kind between some sperm and the reproductive tract of a female, such that sperm which have been rendered inactive by some specific treatment or are generally less “fit” (e.g., have reduced motility) are ejected or “leak out” from the seminal receptacles. There is, finally, the possibility that the treatments previously described, when applied to a particular female Drosophila, may bring about a change in the efficiency of sperm utilization with the result that fewer progeny are recovered than are expected. Even though there is no direct evidence that undernourishment or treatment with X-rays or cold, for example, are responsible for a lowering of sperm utilization in the Drosophila female, it is not difficult to imagine that any of these treatments might bring about physiological changes in the storage organs capable of upsetting the normally high efficiency with which sperm are used for fertilization. The efficiency of sperm utilization in D . melanogaster, however, may also vary in the absence of any specific treatment applied directly to

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the inseminated female. This was first suggested by De Vries (1964), who studied the rate of loss of sperm from the ventral receptacle of D. melanogaster females over a period of time. The results of these experiments showed that there are differences in the rates of loss when one strain of females was inseminated by different strains of males. Furthermore, the differences may be correlated with the chromosomal constitution of the male. More specific information on the subject was obtained by the work of Zimmering and Fowler (1968), who observed that the ratio of sperm counted in the seminal receptacles to the number of progeny recovered from similarly inseminated single females ie., the TABLE 4 Progeny and Sperm Counts Resulting from Matings of Oregon-R (+) Males with Oregon-R (+) Females, and Oregon-R (+) Males with Yellow (y) Femalesa Expt. No.

l3

0

I IIa b IIIa b IVa b

+ + + + + + +

+ + Y + Y +

0

2/

Mean No. of sperm

No. of observations

Mean No. of progeny

No. of observations

Progeny: sperm ratio

413.3 399.2 435.1 484.9 542.7 566.7 572.8

54 18 15 14 17 25 13

329.0 302.7 166.9 372.6 261.5 350.2 242.7

54 20 16 17 16 23 13

0.79 0.75 0.38 0.77 0.48 0.62 0.42

Data of Zimmering and Fowler (1968).

P:S ratio, was different when two different strains of females were inseminated by males of the same strain. Specifically, as can be seen in Table 4, under conditions yielding a P : S ratio of 0.5 from matings of Oregon-R (+) males with yellow (y) females, in excellent agreement with results from similar experiments reported by Peacock and Erickson (1965), significant departures toward a ratio of 1.0 were observed from matings of Oregon-R males (brothers of the above) with Oregon-R females. Progeny:sperm ratios that vary with the genotype of the female have also been reported in D. melanogaster that carry the SD (SegregationDistorter) locus. From matings of SD males (k = 1.0) with females of eight different genotypes, Zimmering et al. (1970a) have found ratios of 0.3-0.5 from five of these types and 0.7-0.9 from three. Even though there is some recent statistical evidence (Fowler and

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Siervogel, unpublished) which seems to cast some doubt on the validity of any relationship between counts of sperm and counts of progeny as expressed as a ratio between the two, one is still tempted to interpret the experimental findings discussed above as reflections of the relative efficiencieswith which different female, Drosophila utilize similar populations of sperm. One cannot, of course, rule out yet another possibility, that of an “interaction” between the genotype of the sperm and the genotype of the female (as previoulsy discussed) which would in these cases determine the types of sperm and the proportions thereof utilized in fertilization (Zimmering and Fowler, 1968). There is an impressive amount of evidence, most of it fairly recent, to support the hypothesis of such nonrandom utilization of sperm in D. melanogaster. For example, observations by Denell and Judd (1969) indicate that segregation ratios from SD males may be modified as a function of the genotype of the female. I n addition, Olivieri et al. (1964) have shown that in populations of sperm which are composed of both nullosomic and disomic sperm (i.e., X.Y vs 0) there is a shift in the recovery ratio with storage time in the female. For example, in mating to Oregon-R females, Olivieri et al. (1970) find that the X.Y-bearing sperm from YSXEN(l).YL/Omales are recovered less frequently in the later subcultures than in the early ones. These results have been confirmed by Johnsen and Zarrow (1971), who independent of Olivieri et al. (1970) also carried out experiments in which YsXEN(*).YL/Omales were mated to yellow (y) females. However, in contrast to these early findings, Johnsen (1971) has subsequently observed that another X.Y chromosome (i.e., XpYL.Ys from Bs males) is recovered from a different strain of females more frequently in the later transfers than in the early ones. The difference in the recovery patterns of the two X.Y chromosomes was initially interpreted by Johnsen and Zarrow (1971) to be a possible reflection of the differences in the strains of females used in the two experiments. If this is so, it is yet another likely case of the differential utilization of sperm whereby different females use the same population of sperm (i.e., X.Y) in different ways. There is some recent evidence, however, that the specific structure of the chromosomes, i.e., YsXEN(l).YLor XpYL.Ys, may be a determining factor in the individual pattern of recovery of these chromosomes completely independent of either the female strain or the genetic (i.e., autosomal) background (Johnsen, personal communication). While Johnsen and Zarrow (1971) and Olivieri et a,?. (1970) interpreted their results to be a case of “sperm competition’’ (see, e.g., review of Parker, 1970, for discussion of the word), the nonrandom utilization

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of sperm observed by Childress and Hartl (1972) in attached-X strains of D . melanogaster females mated to males carrying the translocation T(1,4)Bscar (i.e., Bs = Bar of Stone; car = carnation), is considered to be a case of “sperm preference.” In this particular case, several different attached-X strains of females were examined and each was found to have a characteristic and different ratio of recovery of the two types of sperm possible from the translocation male. From these results, Childress and Hartl (1972) concluded that females of different strains of D . melanogaster have different degrees of “sperm preference” and an “interaction of the sperm with the genital tract of the female, leading either to preferential sperm storage or to preferential fertilization” is suggested as the basis of the phenomenon. Finally, Mange (1970), studying the adult sex-ratio in D.melanogaster from matings of Oregon-R males to Oregon-R females, observed that among roughly the first 2070 of the eggs laid by the females there was a marked decrease in the proportion of sons, a finding which is directly related to the age of the fathers (from 1 day to 13 days). This decrease, however, was absent, or possibly reversed, among later eggs. Mange (1970) interprets these results as reflecting the possibility that Y-bearing sperm from younger males, but X-bearing sperm from older males, is preferentially (i.e., “nonrandomly”) utilized even though the overall sex ratio a t the time of the exhaustion of sperm by a female is approximately 50%.

Regardless of the terminology used, i.e., “sperm preference,” “sperm competition,’’ or %on-random utilization,” the physiological mechanism affecting the differential utilization of sperm of different types, as seen in all the experiments cited in this section, is completely unknown. However, a t least one presumption underlying all the observations of sperm utilization in Drosophila is that the ultimate recovery of a particular sperm may be a reflection of a functioning of some X- or Y-linked genes contained in its genetic complement. This conclusion is very difficult to reconcile with the observations by Muller and Settles (1927), which were subsequently confirmed by McCloskey (1966), that sperm lacking virtually all of its genetic material can function perfectly well. Muller and Settles (1927) were the first to address themselves specifically to this question of the functioning of genes in the mature sperm, reasoning that if spermatozoa were deficient for certain parts of their genome they should be incapable of carrying out normal sperm function. Therefore, if such sperm were mixed with normal spermatozoa in a single population of spermatozoa, a shift should be observed in the ratio of progenies developing from eggs laid a t various times following insemination, a shift which would be a reflection of which sperma-

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tozoa (i.e., normal or deficient) were being used in fertilization. Critically testing their prediction utilizing sperm from D. melanogaster males which were heterozygous for a translocation between chromosomes 2 and 3 (from which deficiency gametes could be regularly produced) Muller and Settles observed that the relative frequencies of the deficient sperm remained constant after different storage periods in the female; that is, no shift in the recovery ratio was observed. On the basis of this finding and the fact that the particular deficiency which was utilized comprised more than 37%of the haploid autosomal complement, Muller and Settles concluded that the entire sutosomal complement must be devoid of “functioning” genes and that genes, in general, do not function in the mature spermatozoa. Attesting to the correctness of this hypothesis is the work done some forty years later, also in D. melanogaster, by McCloskey (19661, who arrived at essentially the same conclusion but with a different genetic system. McCloskey utilized a number of compound autosome-bearing lines and measured the relative survival rates of the reciprocal dp-dfbearing gametes (e.g., nullo-aL, diplo-2R vs diplo-2L, nullo-2R, etc.) . Noting no shift in the relative proportions of the different sperm types as a function of increasing time of storage in the seminal receptacles of the female also suggested to McCloskey, in support of the work of Muller and Settles, that “active” genes are not required for the maintenance of viability and function in the mature spermatozoan. The conclusions of Muller and Settles (1927) and McCloskey (1966) are well supported by subsequent work carried out by a number of different investigators. For example, Holm et al. (1967) tested the recovery ratios of spermatozoa in populations containing mixtures of nullosomic and disomic sperm (e.g., C(3L)RM; C(3R)RM vs 0) and found that the recovery of the two sperm types was essentially equal when summed over all storage times. In addition, Lindsley and Grell (1969), by testing similar populations of nullosomic and disomic gametes, have shown that normal spermiogenesis can occur in the virtual absence of any chromosomes a t all and suggest, as one possibility that synthesis, but not the formation of genetic information, occurs throughout spermiogenesis and may be mediated through “stable messenger RNA” which, when distributed to the haploid products, directs a normal spermiogenic process. I n general, then, it is clear that together these findings provide little basis in fact for the possibility stated earlier that differential sperm utilization (or differential sperm storage, for that matter) might be a reflection of the active functioning of genes in the mature spermatozoan. A more plausible possibility, then [and one suggested by the conclusions

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of the work of Lindsley and Grell (1969)], is that there might be certain products of earlier gene expression-e.g., premeiotic or from the diploid (i.e., primary spermatocyte) constitution-which are ultimately distributed as “immunological labels,” as it were, to the mature spermatozoan. I n a given female environment a mixed population of such “labeled” sperm might be differentially utilized if, for example, a particular spermatozoan was not compatible with its genetic surroundings (in this case, the storage organs of the female). Such incompatibility could, then, manifest, itself as a shift in the recovery of certain sperm genotypes with increased length of storage in the female receptacles. For a discussion of other models by which females could differentially utilize populations of sperm in t,he absence of active genes in the spermatozoa, see a recent excellent review of the phenomenon of “meiotic drive” by Zimmering et al. (1970b). VIII. Conclusions

It is clear from the evidence presented here that from the beginning of the development of sperm in the testis of the male up to and including the ultimate recovery of that sperm in the form of progeny, variability is the only consistent feature in all aspects of the reproductive biology of Drosophila. This variability exists a t virtually all levels of the reproductive systems in both the male and the female and, not surprisingly, is a reflection of a multitude of complex and, seemingly, hopelessly intertwined interactions between the genetic constitution of the organism and its particular environment. On the basis of the observations reported in this paper, then, one can say with some certainty that the ultimate recovery of progeny in Drosophila probably represents the aggregation of a great number of largely uncontrollable physiological events. Even though, as mentioned in the Introduction, it seems unlikely that a phenomenon such as “meiotic drive” in Drosophila is solely the result of such postmeiotic events, it seems almost certain that variation in any of the aspects of reproduction discussed here could substantially alter the ultimate recovery of two alternative alleles from a heterozygote from that expected on the basis of Mendelian considerations alone. Unhappily, it is not entirely clear how the variation in the reproductive physiology of Drosophila might be experimentally controlled. It is clear, however, that this must be done before interpretation of the data can be completely meaningful. It is hoped, however, that in designing future experiments in Drosophila, particularly those whose interpretation depend on the control of this variability in reproduction, this review will

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help to determine a better experimental approach to the problem at hand than was previously available.

ACKNOWLEDGMENTS I would like to acknowledge my wife, Janet, for her understanding and, seemingly, unlimited patience with me during the trying months of writing this manuscript. Special thanks are also due to Professor E. Novitski for providing me with a stimulating laboratory environment in which to work and write. The many critical discussions and the numerous suggestions he made, as well as those offered by Professor E. Caspari for improvement of the manuscript, are greatly appreciated. Finally, I wish to thank the secretarial staff of the Department of Biology, University of Oregon, and particularly Pat Nickerson, for the typing of the rough drafts and final copy, respectively, of the manuscript. Without their technical abilities, not to mention their tolerance of “last minute” alterations, this work would not have been possible. REFERENCES

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Drosophila melanogaster und D . hydei. Z . Zellforsch. Mikrosk. Anat. 84, 141-175. Meyer, G. F. 1969. Experimental studies on spermiogenesis in Drosophila. Genetics 81, Suppl., 79-92. Meyer, G. F., Hew, O., and Beermann, W. 1961. Phasenspezifische Funktionsstrukturen in Spermatocytenkernen von Drosophila mebnogaster und ihre Abhiingigkeit vom Y-Chromosom. Chromosoma 12,676-716. Meyer, H. U. 1956. Failure of inseminated females to produce fertilized eggs unless additional copulation takes place. Drosophila Inform. Serv. 30, 135. Meyer, H. U., and Meyer, E. R. 1961. Sperm utilization from successive copulations in females of Drosophila. Drosophila Inform. Sew. 35, 90-92. Miller, A. 1950. The internal anatomy and histology of the image of Drosophila melanogaster. In “Biology of Drosophila” (M. Demerec, ed.), pp. 420-534. Wiley, New York. Mossige, J. C. 1955. Sperm utilization and brood patterns in Drosophila melanogao. ter. Amer. Natur. 89, 123-127. Muller, H. J. 1940. An analysis of the process of structural change in chromosomes of Drosophila. J. Genet. 40, 1-66. Muller, H. J. 1944. Use of males with defective Y’s to promote the laying of unfertilized eggs. Drosophila Inform. Serv. 18, 58. Muller, H. J. 1951. Homosexual copulation in the male of Drosophila and the problem of the fate of sperm of males isolated from females. Drosophila Inform. Serv. 25, 118. Muller, H. J., and Settles, F. 1927. The non-functioning of the genes in spermatozoa. Z . Indukt. Abstamm.-Vererbungsl. 43, 285-312. Myszewski, M. E., and Yanders, A. F. 1963. The effect of storage upon the differential survival among sperm. Drosophila Inform. Sew. 38,35-36. Nachtsheim, H. 1928. Eine Methode zur Priifung der Lebensdauer genotypisch verschiedener Spermien bei Drosophila. Z . Indukt. Abstamm.-Vererbungs1., Suppl. 11. Narain, P. 1962. Effect of age of female on the rate of egg production in D . melanogaster. Drosophila Inform.Serv. 38, 96-97. Nicoletti, B. 1968. I1 controllo genetic0 della meiosi. Atti Ass. Genet. Ital. 13, 1-71. Nonidez, J. F. 1920. The internal phenomenon of reproduction in Drosophila. Biol. Bull. 39, 207-230. Novitski, E., and Rush, G. 1948. Desemination by low-temperature shocks. Drosophila Inform. Serv. 22, 75. Novitski, E., and Sandler, I. 1967. Are all products of spermatogenesis regularly functional? Proc. Nut. Acad. Sci. U.S.43,318-324. Olivieri, G., Pica, L., and Olivieri, A. 1964. Sulla possibiliti che la presenza del chromosoma Y condizioni in Drosophila melanogaster lattivita degli spermatozi nei processi di fecondazione. Atti Ass. Ital. Genet. 9, 169-171. Olivieri, G., Avallone, G., and Pica, L. 1970. Sperm competition and sperm loss in Drosophila melanogaster females fertilized by YsX.YL/O males. Genetics 64, 323-335.

Oster, I. I., Duffy, J., and Binnard, R. 1966. Observations on a piece of tail. Drosophila Inform. Sew. 41, 136. Parker, G. A. 1970. Sperm competition and its evolutionary consequences in the insects. Biol.Rev. Cambridge Phil. SOC.45,525676.

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Parsons, P. A. 1964. Genotypic control of mating times in Drosophila melanogaster. Ezperientia 20, 569-570. Parsons, P. A. 1967. “The Genetical Analyeis of Behaviour,” 174 pp. Methuen, London. Patterson, J. T. 1946. A new type of isolating mechanism. Proc. Nat. Acad. Sci. U.S. 32, 202-208. Patterson, J. T. 1947. The insemination reaction and its bearing on the problem of speciation in the mulleri subgroups. Tex., Univ., Publ. 4720, 3741. Patterson, J. T. 1954. Fate of the sperm in the reproductive tract of the Drosophila female in homogamic matings. Tex., Univ., Publ. 5422, 19-37. Patterson, J. T., and Stone, W. S. 1952. “Evolution in the Genus Drosophikz,” 610 pp. Macmillan, New York. Peacock, W. J., and Erickson, J. 1965. Segregation-distortion and regularly non-functional products of spermatogenesis in Drosophila melanogaster. Genetics 51, 313-328.

Perotti, M. E. 1969a. The neck region in mature sperm of Drosophila melanogaster. Comp. Spermatol., Proc. Int. Symp., lst, Accad. Naz. Lincei Quademo No. 137.

Perotti, M. E. 1969b. Ultrastructure of the mature sperm of Drosophila melanogaster. J . Submicrosc. Cytol. 1, 171-196. Perotti, M. E. 1970. Filamentous structures in the male accessory glands of Drosophila. Congr. Znt. Microsc. Electron. 7th, Grenoble. Perrin-Waldemer, C . 1965. Biologie de la reproduction du male et des spermatozoides chez Drosophikz melanogaster. Ann. Biol. Anim., Bwchinz., Bwphys. 6, 553-585. Philip, U. 1942. Meiosis in Drosophila. Nature (London) 149,527428. Policansky, D. 1970. Three sperm sizes in Drosophila pseudoobscura and Drosophila persimilk. Drosophila Inform. Serv. 45, 119. Policansky, D., and Ellison, J. 1970. ‘(Sex ratio” in Drosophila pseudoobscura: Spermiogenic failure. Science 148, 516-517. Riley, R. C., and Forgash, A. J. 1967. Drosophik melanogaster egg shell adhesive. J . Insect Physiol. 13, 509-518. Robertson, F. W. 1957. Studies in quantitative inheritance. XI. Genetic and environmental correlation between body size and egg production in D . melanogaster. J. Genet. 55, 428443. Sandler, L., and Novitski, E. 1957. Meiotic drive as an evolutionary force, Amer. Natur. 91, 105-110. Sandler, L., Hirakumi, Y., and Sandler, I. 1959. An instance of meiotic drive in a natural population of Drosophila melanogaster. I. The cytogenetic basis of segregationdistortion. Genetics 44, 233-250. Scossiroli, R. E. 1954. Desemination of fertilized Drosophila melanogaster females. Physiol. Z001.27, 157-162. Shima, T. 1966. Notes on the copulation, insemination reactions and sperm storage of Drosophila nigromaculata in homoganic matings. Drosophila Inform. Serv. 41, 170.

Shima, T., Kaneko, A., and Momma, E. 1967. On some aspects of the copulation, insemination reaction and sperm storage in two species of the quinariu group. Drosophila Inform. Serv. 42, 100. Shoup, J. R. 1967. Spermiogenesis in wild type and in a male sterility mutant of Drosophila melanogaater. J . Cell Biol. 32, 663-675. Sidhu, N. S. 1963. Genetic effects on the spermatozoa of Drosophikz. Ph.D Thesis, Univ. of Edinburgh, Edinburgh.

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Smith, J. M. 1956. Fertility, mating behavior and sexual selection in Drosophila subobscura. J. Genet. 54, 261-279. Spiess, E. B., and Langer, B. 1964. Mating speed control by gene arrangements in Drosophila pseudoobscura homokaryotypes. Proc. Nut. Acad. Sci. U.S. 51, 1015-1019. Spieth, H. T. 1952. Mating behavior within the genus Drosophila (Diptera). Bull. Amer. Mus. Natur. Hist. 99, 399474. Stromnaes, O., and Kvelland, I. 1962. Sexual activity of Drosophila melanogaster males. Hereditas 48, 44M70. Sturtevant, A. H.1915. Experiments in sex recognition and the problem of sexual selection in Drosophila. J. Anim. Behav. 5, 351-366. Tandler, B., Williamson, D. L., and Ehrman, L. 1968. Unusual filamentous structures in paragonia of male Drosophila. J. Cell Biol. 38, 329-336. Tates, A. D. 1971. Cytodifferentiation during spermatogenesis in Drosophila melanogaster. Ph.D Thesis, Transitorium voor Geneeskunde, Leiden, The Netherlands. Tokuyasu, K. T., Peacock, W. J., and Hardy, R. W. 1972.Dynamics of spermiogenesis in Drosophila melanogaster. I. Individualization process. Z. Zellforsch. Milcrosk. Anat. 124, 479-506. Wedvik, M.1962. The effect of low temperature on fertility of Drosophila melanogaster males. Drosophila Inform. Serv. 36, 127. Wheeler, M. R. 1947. The insemination reaction in intraspecific matings of Drosophila. Tex., Univ., Publ. 4720, 78-115. Wheeler, M. R. 1954. Taxonomic studies on American Drosophiliae. Tex., Univ., Publ. 5422, 47-64. Wilson, L. P., King, R. C., and Lowry, J. L. 1955. Studies on the tuw strain of Drosophila melanogaster. I. Phenotype and genotypic characterization. Growth 19, 215-244. Yanders, A. F. 1959. The effect of X-rays on sperm activity in Drosophila. Genetics 44, 545-546. Yanders, A. F. 1963. The rate of Drosophila melanogaster sperm migration in inter- and intra-strain matings. Drosophila Inform. Serv. 38, 33. Yanders, A. F. 1964. The effect of X-rays on insemination and sperm retention in Drosophila. Genetics 49, 309-317. Yanders, A. F., and Perras, J. F. 1960. Sperm length in four Drosophila species. Drosophila Inform. Serv. 34, 112 [correction, 38, 145 (1963)l. Yamzumi, G., Fujimura, W., and Ishida, H. 1958. Spermatogenesis in animals as revealed by electron microscopy. V. Spermatid differentiation of Drosophila and grasshopper. Exp. Cell Res. 14,268-285. Young, W. C., and Plough, H. H. 1926. On the sterilization of Drosophila by high temperature. Biol. Bull 51, 189-198. Zimmering, S., and Fowler, G. L. 1966. X-irradiation of the Drosophila male and its effect on the numzber of sperm transferred to the female. 2. Vererbungsl. 98, 150. Zimmering, S.,and Fowler, G. L. 1968. Progeny: sperm ratios and nonfunctional sperm in Drosophila melanogaster. Genet. Res. 12, 359-383. Zimmering, S., Barnabo, J. M., Femino, J., and Fowler, G. L. 1970a. Progeny: sperm ratios and segregation-distorter in Drosophila melanogaster. Genetica 41, 61-64. Zimmering, S., Sandler, L., and Nicoletti, B. 1970b. Mechanisms of meiotic drive. Annu. Rev. Genet. 4, 409-436.

MEIOTIC DRIVE IN Drosophila: NEW INTERPRETATIONS OF THE SEGREGATION DISTORTER AND SEX CHROMOSOME SYSTEMS W. J. Peacock and George 1. Gabor Miklos Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra, A.C.T., Australia, and Research School at Biological Sciences, Australian National University, Canberra, A.C.T., Australia

I. Introduction . . . . . . . . . . . . 11. Segregation Distorter . . . . . . . . . . A. The Mechanism of SD Action . . . . . . B. The Genetics of SD . . . . . . . . . C. Discussion . . . . . . . . . . . . 111. Sex Chromosome Meiotic Drive Systems . . . . A. Description of the sc4sc8 System. . . . . . B. Interpretation of the s c 4 s c 8 System . . . . . C. Reexamination of the S C ~ S C *System. . . . . D. A New Hypothesis . . . . . . . . . E. Tests of the Model . . . . . . . . . F. Modification of Meiotic Drive by Temperature . G. Discussion . . . . . . . . . . . . . IV. General Conclusions . . . . . . . . . . References . . . . . . . . . . . . .

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1. Introduction

In many plants and animals, heterozygotes for a genetic marker (gene, chromosome segment or chromosome) do not yield the expected equality of the two alleles in t,heir progeny. This departure from Mendelian expectation is in some cases shown to result from a meiotic event rather than by simple gametic or zygotic lethality; these have been termed cases of meiotic drive (Sandler and Novitski, 1957), the implication being that particular alleles or chromosomes can be “driven” to a higher population frequency as a consequence of the meiotic event. A recent review of Zimmering et al. (1970a) has considered many of these systems; prominent among the systems they discussed were several which have been described in male Drosophila melanogaster. I n this review 36 1

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we shall be concerned with recent developments in the understanding of these Drosophila systems. I n particular we will focus on the Segregation Distorter ( S D ) system where the understanding of the genetic and morphological basis of drive has drastically altered in the last two years. I n addition we will examine a number of sex chromosome meiotic drive systems and present a new hypothesis as to their basis. II. Segregation Distorter

Sandler et al. (1959) described the segregation distorter ( S D ) locus, located near the centromere region of chromosome 2, which when heterozygous in a male (SD/SD+)results in the transmission of the S D chromosome to the virtual exclusion of the SD+ chromosome. Thus in a cross to SD+/SD+females there is a large excess of SD/SD+ progeny compared to SD+/SD+progeny. Distortion was measured in terms of k value, where k is the proportion of SD progeny among total progeny ( S D / ( S D SD+)). Females heterozygous for the locus showed no evidence of distortion in the recovery ratio of the two alleles.

+

A. THEMECHANISM OF SD ACTION 1. Time of Effect

Sandler et al. (1959) demonstrated by egg-hatch tests that zygotic mortality did not contribute significantly to the segregation distortion, and concluded that either (i) SD gametes were produced in excess of SD+ gametes, or (ii) the two kinds of gametes were produced in equal frequency, the majority of the SD+ gametes being nonfunctional. The second alternative was shown to be the case by Peacock and Erickson (1965). They constructed an SD heterozygote in which the SD+ chromosome was cytologically distinguishable from the SD chromosome. The SD+ chromosome occurred in half of the secondary spermatocytes ; furthermore these heterozygotes contained the full complement of 64 heads in each cyst of developing spermatids. Although the SD+ gametes thus appear to fail sometime between the initiation of sperm development and the time of fertilization, the primary SD effect appears to occur a t an earlier stage-at the first meiotic division. Mange (1968) showed that some SD lines were temperature sensitive and that the most sensitive period occurred 8-9 days prior to sperm maturity. This corresponds to the time taken for spermatocytes to develop from early meiosis I through to mature sperm (Chandley and Bateman, 1962). Hihara (1971) confirmed Mange's finding of temperature sensitivity of the SD system and cytologically verified that the

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early primary spermatocyte was the sensitive stage. The importance of this stage in the SD effect has also been demonstrated by Murnik (1971), who found that irradiation of primary spermatocytes led to depression in the magnitude of the SD effect. She suggested that prophase I might be the most sensitive period since the maximum depression of SD activity corresponded to the time of maximum induction of homologous nonsister chromatid breakage and reunion. The implication, from Murnik's analysis, that the time of synapsis of homologs in prophase I i,s the time of SD action, is in line with a conclusion made by Sandler et al. (1959) that the S D phenomenon showed a dependence on synapsis. SD+ chromosomes having rearrangements with breakpoints in the vicinity of the S D locus reduced or eliminated the effect, and on this basis Sandler et al. suggested that pairing in the region of the SD locus was a prerequisite for SD action. However, a translocation involving a distal second chromosome breakpoint (T(2,3)bwV4)reduced SD activity as much as some of the rearrangements involving proximal breakpoints. Another difficulty in making conclusions about the importance of synapsis is that most of the SD+ rearrangements suppress both SD chromosomes which have normal gene sequence and SD chromosomes in which paracentric and pericentric inversions occur. Thus we may conclude that there is strong evidence to indicate that the initial SD effect occurs in the primary spermatocyte, but further experimentation is needed before a more precise statement can be made. 9. Breakage-Reunion Hypothesis

In their account of the SD system, Sandler et al. (1959) proposed a cytogenetic explanation for the nonfunctionality of the SD+ sperm and presented some cytological observations in support of the model. They proposed that when the SD chromosome paired with its homolog in prophase I, an isochromatid break was induced in the SD+ chromosome. They further supposed that sister-union then regularly occurred so that the SD+ chromosome would produce a chromatid bridge a t anaphase 11. The presence of the bridge, or a breakage product from the bridge, was postulated to be the causative agent in preventing the SD+ cells from proceeding normally through spermiogenesis. Cytological analysis of SD heterozygotes showed sister-union loop chromatids a t metaphase I1 and dicentric bridges a t anaphase 11, but failed to disclose any acentric fragments at anaphase I. However, a chromosome element which was interpreted as the acentric fragment was regularly seen a t metaphase 11. These observations appeared to provide confirmation of the breakage-reunion hypothesis but have not been repeatable in subse-

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quent analyses. Peacock and Erickson (1965) found the meiotic cytology of SD heterozygotes to be normal; they found no evidence of either dicentric chromatids or of acentric fragments. Furthermore, as mentioned earlier in this review, by using an SD+ chromosome which could be distinguished cytologically from the SD chromosome, they were able to show that each primary spermatocyte did in fact produce the expected four meiotic products. Crow et al. (1962) presented some evidence that X-ray-induced recombination events in chromosome d occurred in higher frequency in SD heterozygotes than in controls, and they concluded that this may have been due to an interaction of X-rayinduced and SD-induced breaks. However, in the light of the cytological normality of meiosis in SD heterozygotes, it must be concluded that the breakage-reunion hypothesis is untenable. 3. Functional Pole Hypothesis On finding no aberrations a t meiosis, Peacock and Erickson (1965) extended their observations to the developing spermatid bundles. They found that the mean number of spermatid heads per cyst was similar in S D heterozygotes and controls, approaching the expected 64. They then examined the number of sperm transferred to females and again found a correspondence between SD heterozygotes and controls. All sperm transferred in a mating, by males stored for up to 66 hours, were stared in the spermathecae and ventral receptacle of the female, but only 40-50% of these sperm were recoverable as progeny. This discrepancy between stored and recoverable sperm in both SD heterozygotes and controls led Peacock and Erickson to propose that the mechanism behind the SD effect does not involve the production of extra nonfunctional sperm. Instead they suggested that the sperm recovery data could be interpreted in terms of a regular production of functional and nonfunctional sperm, and that in SD heterozygotes the SD chromosome is nonrandomly included in the functional class of sperm. Thus the SD phenomenon was seen to result from a specific chromosome orientation a t metaphase I. Peacock and Erickson proposed that the primary spermatocyte is differentiated so that one pole of the anaphase I spindle will subsequently give rise to two functional sperm, the other pole giving rise to two sperm which would not effect fertilization. The concept of a regular production of functional and nonfunctional sperm had earlier been introduced by Novitski and Sandler (1957) as a logical necessity to explain recoveries of gametic classes in a case of meiotic drive involving a translocation between the X and chromosome 4. Although the functional pole hypothesis was attractive because it could be applied to other cases of meiotic drive (Novitski et al., 1965; Peacock,

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1965), the only direct evidence in its support was that only 50% of stored sperm were recovered as progeny. The hypothesis should now be discarded because it has been shown that the proportion of stored sperm which is recoverable as progeny is dependent upon the genotype of the female. For example, Zimmering and Fowler (1968) confirmed the recovery ratios obtained by Peacock and Erickson (1965) when yellow (y) females were used in mating to Oregon-R males, but found that from similar matings of Oregon-R males to Oregon-R females up to 85% of sperm were recovered. Zimmering et al. (1970b) showed this to be also true for heterozygous SD males and concluded that virtually all sperm transferred by SD heterozygous males could be recovered as progeny. This means that the majority of sperm transferred by an SD heterozygote, having a k value of 1.0, are in fact SD-bearing sperm. An emphatic demonstration that this is so is given by Peacock et al. (1972a), where in some matings of S D heterozygotes all stored sperm were recovered and all were SD sperm. This contradicts the conclusion originally made by Peacock and Erickson (1965) that both SD and SD+ sperm were transferred in equal frequencies and must mean, a t least in the SD line used by Peacock et al., that the SD+ sperm are rendered nonfunctional and are presumably discarded in the male.

4. Sperm Dysfunction Hypothesis The initial suggestion of Sandler et al. (1959) that the SD+ sperm failed to function, or were eliminated, was supported by Hartl et al. (1967). These authors found a high correlation between the total number of progeny (lifetime productivity) and l/k. A similar finding was reported by Nicoletti et al. (1967). The correlation was almost totally ascribable to length of the fertile period, the daily production of all different SD lines (and the controls) being similar. Peacock and Erickson (1965) had earlier found that SD males became sterile sooner than their controls, but the average daily productions were similar for heterozygous S D and controls, and concordantly the controls produced about double the number of progeny than did the SD heterozygotes in their lifetime productivities. Hartl et al. (1967) found an exception to the equal daily production in the first 24-hour period, where it was noted that there was a proportionality in progeny production to l/k. The conclusions of these authors were (i) in the first 24-hour period the number of sperm available in the seminal vesicle is limiting with respect to sperm transfer during mating, and therefore differences in numbers of functional sperm per bundle will be obvious, (ii) that after 24 hours the number of sperm is not limiting and therefore there will be a lack of correlation between k and number of sperm transferred, and (iii) that “the total number

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of sperms is already present in the fairly young flies, and that the supply is exhausted sooner” in the S D stocks. Although these observations suggested dysfunction of SD+ sperm, there are difficulties with the interpretation of the data. First, many lines of evidence show that the adult male continues to produce and transfer sperm on a regular basis such that daily brood experiments yield satisfactory samples of daily sperm production. An example is given by the temperature and X-ray experiments discussed in Section 11, A, 1, and additional, more extensive, data are given in Section 111, C. Hence a 48-hour male clearly does not contain his total sperm supply. Second, although Hartl et al. found a proportionality in the first 24 hours of sperm production in young males, Peacock and Erickson (1965) found equal production of progeny by S D heterozygotes and controls in the same period. Another factor known to be operative is that the ratio of sperm recovered to sperm transferred can be dependent upon the size of the insemination (Peacock et al., 1972a). An extensive discussion of the large number of components which may be involved in the sampling of sperm is to be found in the review by Fowler in this volume. The conclusion can be drawn that progeny numbers do not provide a reliable estimate of the total numbers of functional sperm. Thus although the experiments of Hartl et al. are suggestive of dysfunction of SD+ gametes in S D heterozygotes, and thus agree with the conclusion drawn in the last section that the majority of sperm transferred by an S D heterozygote are S D sperm, direct evidence for sperm dysfunction would be desirable. Such evidence has now been made available through electron microscope analyses. 8. Spermiogenesis and Sperm Dysfunction

The first morphological indication that not all sperm develop normally in an S D heterozygote was given in a brief report by Nicoletti (1968). Nicoletti found by electron microscope analysis that in transverse sections of the testis some cysts of developing spermatids contained both normal and abnormal tails, these occurring in approximately equal numbers; in some cysts he found only 32 sperm tails. His report led to a study of spermiogenesis in Drosophila by Tokuyasu et al. (1972a,b) which has provided knowledge of a number of processes of basic importance in sperm development. Theses authors were then able to clearly document that, in SD heterozygotes, SD+ sperm frequently failed to follow the normal sequence of development (Peacock et al., 1972a). A summary of normal spermiogenesis and the departures from normality in various S D stocks may be found in Peacock et al. (1972b). With respect to normal spermiogenesis some of the pertinent features are as

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follows. All 64 postmeiotic nuclei are synchronously transformed into spermatids, then finally into mature sperm. These 64 developing spermatids are contained within a common cytoplasm-a syncytium. The syncytium is surrounded by a t least two cyst cells, the head cyst cell and tail cyst cell, and possibly there are more. The head cyst cell surrounds the spermatid syncytium in the region of the heads, and, after the transformation of the postmeiotic nuclei into sperm heads and the elongation and development of sperm tails, the process of individualization begins and encloses each spermatid in a membrane, thus separating each from the other members of the cyst. During this process the bulk of cytoplasm and nucleoplasm is removed and discarded. After individualization the 64 sperm are coiled into the basal region of the testis. The heads and surrounding head cyst cell (and some small part of the tail cyst cell) have a t this time formed an intimate association with one or more cells of the terminal epithelium. During coiling, the enveloping cyst cells and the contained “waste bags” of discarded cytoplasm, nucleoplasm, and organelles are drawn down with the coiled sperm into the basal region of the testis. After completion of coiling the sperm are released into the lumen of the testis and pass through the testicular duct into the seminal vesicle. The remaining cyst cells and waste bags are broken down and finally phagocytosed by the terminal epithelial cells. Electron microscopy of SD heterozygotes confirmed the light microscope determinations that there were 64 postmeiotic products and has shown that all 64 do not proceed synchronously through spermatid development. Quite early in sperm head development, two classes of sperm are detectable in each cyst. SD heterozygotes which show complete SD action (k = 1.0) invariably have 32 heads with chromatin condensation patterns similar to that of controls, and 32 heads in which the chromatin condensation is abnormally delayed. This pattern of 32 normal-32 abnormal in head development becomes increasingly apparent in subsequent stages (Fig. l ) ,and is often accompanied by aberrations in tail development (Fig. 2 ) . The two stocks which have been examined extensively by electron microscopy, SD-72 Madison and SD-72 Canberra, differ in the morphological degree of aberration in head development but always show the 32-32 segregation in every cyst. They do not show a regular 32-32 segregation in tail development, and even SD-72 Canberra, which is the more extreme, frequently shows fewer than 32 abnormal tails in a cyst. Serial sectioning has shown that every abnormal tail is associated with an abnormal head. At the time of individualization the existence of two classes of spermatids becomes very obvious: frequently in SD-72 Canberra heterozygotes only 32 of the 64 spermatids are individualized, 32 remaining in the syncytium. The spermatids re-

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maining in the syncytium are invariably those with abnormal heads (and tails). When coiling takes place, those sperm in the syncytium are separated from the individualized sperm (Fig. 3 ) , presumably because those sperm in the syncytium are not coiled with the geometrical precision of the other sperm. In sections across the cyst which is in the midst of coiling, the profiles nearest the heads will often show only a group of 32 normal, individualized tails whereas the more caudal profiles, as yet uncoiled, will show both the 32 individualized tails together with 32 tails within a syncytium. The group of abnormal syncytial tails

FIG.1. Transverse section through the head region of a cyst of spermatids in the testis of an SD heterozygote. The dense chromatin of the 32 normally developed SD spermatids contrasts with the irregular appearance of the chromatin in the SD' spermatids. Not all 32 SD' spermatids are visible in this particular section. X 10,500.

undergoes breakdown and, together with the waste bag with which it has a direct cytoplasmic connection, is finally phagocytosed by the terminal epithelial cells in the basal region of the testis (Fig. 4). The abnormal spermatids in the cyst have been identified as the SD+ spermatids by the following criteria: (i) the sperm transferred by SD-72 Canberra have been shown to be SD sperm (see Section 11, A, 4), therefore the sperm destroyed in the testis are in all likelihood the SD+ sperm; (ii) when SD-72 Canberra is heterozygous to an SD+

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chromosome which is insensitive to SD action, there are no abnormal spermatids in the cyst, and equal numbers of SD and SD+ chromosomes are recovered; (iii) when SD-72 Canberra is heterozygous to an SD+ chromosome which is partially sensitive to SD action, there are fewer than 32 abnormal spermatids in the cyst, the proportion of abnormal spermatids per cyst being consistent with the recovery ratio of S D and SD+chromosomes. Although SD+sperm are rarely recovered from SD-72 Canberra, there are not always 32 spermatids left in the syncytium. Both intra- and

Fro. 2. Transverse section through the tail region of two spermatid cysts of an SD heterozygote. In the right-hand cyst 32 (SD)tails have been individualized, and 32 (SD') tails have remained in the syncytium. The syncytium is present as 8 areas in this section but is actually continuous. In the left-hand cyst only 4 tails have been left in the syncytium, 59 being individualized. x6,500.

intermale variability exists with respect to the number of nonindividualized spermatids, but 32-32 segregation is always clearly detectable in the appearance of the heads. Where SD+ sperm are individualized, it seems likely that they are still broken down within the testis. Sperm breakdown has been detected in the base of the testis, in the testicular duct, and even in the seminal vesicle. In SD-72 Madison where the developmental lesions seem less extreme, some XD+ sperm may be transferred to the female, but even in this case the lesion is apparently sufficient to prevent sperm from effecting fertilization. These electron microscope studies have left little doubt that SD+ gametes are recovered in less than expected numbers because SD induces an effect on the AD+chromosome which leads to improper development of the SD+gamete. The abnormalities of the SD+ gametes are generally such that some processes of importance in sperm maturation fail to operate cor-

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FIQ.3. See opposite page for legend.

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37 1

rectly and lead to the breakdown of these gametes. The same processes have been shown (Tokuyasu et al., 1972a,b) to discriminate against the occasional abnormal spermatids which occur in control stocks : this emphasizes that the electron microscope studies of Tokuyasu et al. and Peacock et al. have elucidated the consequence of SD action and have not provided information on the primary effect of the SD locus. Whatever SD action may be in molecular terms, it apparently causes the SD+ chromosome to be a gametic lethal. A genetic demonstration of this point has been provided by Sandler and Carpenter (1972), who have shown that when an SD' chromosome is included in the same nucleus as the SD chromosome by nondisjunction, dysfunction of that gamete occurs.

B. THEGENETICSOF SD Investigations of the basic genetics of the SD system have revealed an array of genetic components as well as many unusual associated phenomena. Some of the unusual properties of the SD system which have been described are as follows: (i) genetic instability-certain populations of SD heterozygous males have far greater between-male variance in the amount of distortion than do others according to Sandler and Hiraizumi (1960a), who postulated the existence of many SD states each of which mutated with a high frequency per generation, the degree of mutability being controlled by a Stabiliser gene; (ii) the Activator gene-the SD gene apparently cannot function without an initiator or Activator gene, which is closely linked and which switches on SD activity (Sandler and Hiraizumi, 1960b) ; (iii) conditional distortion-the SD gene can induce a sex chromosomal Suppressor of itself even when SD is not operative (Sandler and Hiraizumi, 1961a) ; (iv) translocal modification-alleles of SD and SD+ were reported to translocally modify, FIG.3. (a) A part of the transverse section of the basal testicular zone in an SD heterozygote. In the A, B, C, or D cyst cells, the bundle profiles with the hexagonal close-packing of normal tails far outnumber those of loosely packed abnormal tails (small arrows in the A and D cells). In some profiles (long arrows) in the E and F cyst cells, both normal and abnormal tails are seen together. In the G cyst cell, almost all profiles show abnormal tails. TE, terminal epithelial cells. (b) An enlarged portion of the profile of an abnormal tail bundle (small arrow in the C cyst cell in a). (c) An enlarged portion of the G cyst in a.

Various degeneration stages of sperm tails are recognized. (d) An enlarged field continuous to the left of (a) in a consecutive section (turned clockwise by roughly 90'). The difference between the normal and abnormal bundles (abnormal ones indicated by arrows) in the A and B cyst cells is clearer in this enlargement than in (a). Refer to explanation of (a). (a) x 2,100; (b) and (c) x 11,500; (d) X 3,450. (From Tokuyasu et al., 1972b.)

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W. J. PEACOCK A N D GEORGE L. GABOR MIKLOS

FIO.4 (a) Longitudinal section of the most basal region of the terminal testicular zone. (b) Demagnified tracing of (a). Spherical or ellipsoidal bodies which are

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a phenomenon in which the behavior of an allele could be altered by the presence of another allele even in the absence of crossing-over (Sandler and Hiraizumi, 1959). These properties have suggested to Sandler and Carpenter (1972) that “it may . . . be that the elements involved are not conventional.” However, a mode of analysis introduced by Miklos and Smith-White (1971) permits an evaluation of the SD system in standard genetic terms. I n this section we shall briefly outline the approach and consider its application to some of the phenomena listed above. 1. The Measurement of SD Activity The strength of distortion has been estimated using k value (Sandler et al., 1959) or using standard deviations of a normal curve (Miklos and Smith-White, 1971). The latter measurement simplifies the interpretation of SD data. The rationale behind this system of measurement is that an SD+sperm can have only one of two fates-it can be capable of fertilization or not. This determination must be made a t the individual spermatocyte level since SD heterozygotes may produce both classes of SD+sperm. Miklos and Smith-White concluded that this implied the existence of an underlying variability among spermatocytes such that those which have a level of the variable greater than a given threshold value, will give rise to dysfunctional SD’ gametes. If there were no variability between spermatocytes all SD+ gametes would be above or below the threshold and hence all would be either dysfunctional or functional. Such males would have k values of either 1.0 or 0.5, respectively. Miklos and Smith-White found that an assumption of a normal distribution of the underlying variable yielded good fits to the published data, and furthermore, the magnitude of between male variance over the range of SD distortion levels fitted the predicted curve. This mode of analysis is shown in Fig. 5 where the probability of SD+ dysfunction is given by the area of the normal curve above the threshold. Distortion is measured in standard deviation units by the distance of the mean from full of myeloid whorls, degenerating sperm tails, waste materials, and vacuoles are seen in (a) a t the locations indicated by solid line-hatched areas in (b). A cyst which shows profiles of a coiled bundle in (a) is indicated by a dotted line in (b). The large space of the cyst indicated by the broken line-hatched area in (b) resembles the interior of the spherical bodies. Mitochondria of apparently normal morphology (M) are seen in the inset, an enlarged part of the spherical body indicated by an asterisk in (b). Liberated sperm are recognized in the spaces outlined by broken lines. An arrow in (b) indicates the direction toward the testicular duct. MW, myeloid whorls. (a) x 1,350; inset, x 14,400. (From Tokuyasu et al., 1972b.)

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W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

the threshold. I n contrast to this measurement of SD effect in terms of probability of dysfunction of SD+gametes, k measures this probability indirectly. The expression of SD activity in terms of k value or in terms of standard deviation units can lead to quite different interpretations. For example, in considering temperature-induced changes in SD activity, Hihara (1971) found that the SD heteroaygote, R(cn)-l4/SD+,changed in k from 0.94 at 25OC to 0.83 a t 17OC, a reduction of 0.11 in k ; the SD heteroaygote, SD-72/SD+ changed only from 0.997 to 0.969, a reduction of 0.028 in k . Hihara commented on the relative temperature stability of the SD-72 allele as compared to the recombinant SD allele, R(cn)-l4, but when these results are expressed in standard deviation units, R (cn)-14 altered from 6 . 5 ~to 5 . 8 ~ and SD-72 from 7 . 7 ~to 6 . 9 ~changes ~ THRESHOLD

-

2

-

1

0

1

2

3

4

STANDARD DEVIATIONS

FIQ.5. This figure represents the distribution in an SD heterozygote of the variable determining dysfunction of SD' sperm. The population of dysfunctional SD' sperm is given by the unshaded area of the normal curve. Distortion is measured in standard deviations as the distance of the mean of the curve from the threshold. (After Miklos and Smith-White, 1971.)

of 0.7 and 0.8 standard deviation units, respectively. The conclusion now would be that the two alleles have similar temperature responses. In Fig. 6 the magnitude of the difference between these two systems of evaluation of SD activity is readily seen for males with means 0, 1, 2, and 3 standard deviation units above the threshold; the k values for these males are 0.67, 0.86, 0.98, and 0.999, respectively, an apparently irregular progression. 8. Genetic Instability One of the unusual properties of SD is that of genetic instability. Sandler and Hiraiaumi (1960a) found that a number of SD chromosomes which had recombined with the SD+ homolog in heterozygous females showed much increased variability in k values as compared to the original unrecombined SD chromosome. They classified these recombinant lines as being either semistable or unstable and suggested that

Drosophila

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the difference between the original SD lines and the recombinants was the loss of a Stabiliser gene from the tip of the SD chromosome. They found that recombinant males of a particular k value generated progeny males having a wide range of k values, and this effect was maintained in subsequent generations. They concluded that the SD locus was highly mutable and that the action of the Stabiliser gene was “either that (8)

k$O.SSS

THRESHOLD

I

k

R

Ibl

,=o.sn

kp0.Sn

c ,

10

-

0

1u

1

2

3

STANDARD D EVIAT10NS

FIQ.6. This figure demonstrates the apparent increase in variance of k as mean levels of distortion are reduced. In each of (a), (b), and (c), the distributions of the dysfunction variable are shown for two males (A and B). In each case the means of the two males remain a constant distance of lo apart, but the differences in k values vary markedly.

of increasing the Ic value associated with any state of SD or . . . to decrease mutation rate to the lower Ic states.” In analyzing the same data Miklos and Smith-White (1971) have shown that it is possible to envisage the system without recourse to interpretations of high mutability or to genes which control mutation rate. When the data were expressed in standard deviation units the recombinants were seen to differ in their mean levels of distortion but with the variability between males being approximately the same in all recombinant lines. Thus there is no evidence for a Stabiliser gene whose function is to maintain low

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W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

variability between males. The effect of recombination can be simply interpreted as the loss of linked modifiers affecting SD activity. A demonstration of apparent increase in between-male variability in k value as mean distortion is reduced is given in Fig. 6. If the distortion values of two males A and B are kept the same distance apart (one standard deviation) as the mean distortion of the population is lowered, the distance which separates them in k value increases from 0.02 to 0.11 to 0.20; there thus appears to be more variability between males as distortion is reduced.

3. The Activator Gene The normal distribution analysis also leads to different conclusions from the k analysis with regard to the genetic complexity of the SD region itself. Sandler and Hiraizumi (1960b) proposed the existence of an Activator gene ( A c ) , which was responsible for initiation of SD activity, this gene being closely linked to the SD locus. Some doubt as to the validity of this conclusion was introduced when it was discovered that the SD lines used in this recombination analysis contained a small pericentric inversion in the region of the SD locus (Lewis, 1962). However, Hiraizumi and Nakazima ( 1967) repeated the recombination analysis with a chromosome free of the pericentric inversion and again concluded that Activator was a component of the SD system. Miklos (1972) has noted that in the analysis of Hiraizumi and Nakazima, Activator was identified with different distortion levels than those originally proposed by Sandler and Hiraizumi. He showed that when the distortions of the various recombinant chromosomes were expressed in standard deviations, Activator could be regarded as a modifier comparable t o others which increase SD activity, There is no evidence which implies that in the absence of Activator the SD locus is unable to function. On the contrary, an SD chromosome lacking the Activator shows a level of distortion consistent with the remaining modifiers.

4. Sensitivity in the SD System Tests of different chromosome 2’s have shown that these chromosomes have characteristic sensitivities, i.e., the susceptibility of the chromosome to distortion by an SD allele. Sandler and Hiraizumi (1959) termed an insensitive SD+ chromosome one which in combination with an SD chromosome yielded a k value of 0.5, and a sensitive chromosome as one which was actively distorted by an SD chromosome. By examining recombinants between SD+ chromosomes having different sensitivity levels, they concluded that the region of sensitivity was close to the

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SD locus. From further recombination studies Sandler and Hiraizumi (1960b) concluded that in fact the SD+ locus and the locus of Sensitivity were coincident. Their analysis appears to have been in error, perhaps because of the presence of the pericentric inversion. Crow et al. (1962) and Hiraizumi and Nakazima (1967) have used chromosomes free of the pericentric inversion and have obtained clear evidence that the locus of Sensitivity is separable from the SD locus. Both the X-ray-induced recombinants of Crow et al. and the spontaneous crossovers of Hiraizumi and Nakazima placed the Sensitivity locus to the left of cn and to the right of the SD locus. All the recombinants displayed the level of Sensitivity of either the SD or the SD+chromosome. Sensitivity then is attributable to a specific locus on the second chromosome, and different alleles have different phenotypes. This is one complexity that must be kept in mind in further analyses of the S D system. For example, the recognition of the Sensitivity locus leads to a problem in the analysis of homozygous SD males (cf. Section 11,C). Additional information on the position of the Sensitivity locus is given by Sandler and Carpenter (1972) in their reanalysis of the Sandler and Hiraizumi ( 1960b) recombinant data. Sandler and Carpenter have recognized evidence for a Sensitivity locus distinct from the S D locus; they have termed the Sensitivity locus “sensitive-receptor.” Their additional information on the position of the Sensitivity locus results from the demonstration that the removal of the region 41-43A by an insertional translocation into the Y chromosome did not remove the Sensitivity locus from the SD+ chromosome. Thus the Sensitivity locus lies between SD and cn and may be either to the left or right of the region defined by’ the insertional translocation. 6. Translocal Modification

Translocal modification was listed earlier in our mention of some of the unusual properties of the SD system. I n fact this process was held to be a characteristic of the SD+ chromosome rather than of the SD chromosome. It was attributed to chromosomes obtained from normal laboratory stocks. I n the original description, Sandler and Hiraizumi (1959) took two second chromosomes: (i) bwD, a n insensitive chromosome, and (ii) cn bw having a sensitivity of 1.0. These chromosomes were combined in a male, bwD/cnbw, where crossing-over does not occur, and mated to an SD-5/cn bw female. The two classes of S D sons, SD-5/bwD and S D - 6 / m bw, were then tested for distortion; it was found that the SD-5/cn bw males did not show the characteristic mean k value of 0.99, but in fact demonstrated reduced S D action with a mean

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W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

k value of 0.84. Sandler and Hiraizumi concluded that there had been a directed partial genetic change of state of the SD+ allele on the cn bw chromosome, so that its sensitivity was decreased. Furthermore, the SD+ bwD chromosome induced a change of state in the SD-5 allele, reducing its capacity to distort. These “translocal modifications” were characteristic not only of the bwD chromosome but were found with numerous other second chromosomes. Although the changes mentioned above were directed from sensitivity toward insensitivity, and from high SD action toward lower SD action, translocal modification has also been detected from insensitivity toward sensitivity. This latter change was achieved in the case of the bwD chromosome only with repeated backcrosses to the cn bw stock. It is unnecessary to invoke any unusual explanations for these phenomena since all are consistent with the changes that can be expected to occur during standard outcrossing of an inbred stock, changes which involve reassociation of modifier genes. A single outcross of the cn bw stock could obviously radically alter the existing associated system of modifiers and result in a significant decrease in sensitivity. This decrease is not due to any change in the major sensitivity locus per se, but only in the segregating modifiers. Repeated backcrossing of the “translocally modified” SD-5/cn bur male to the cn bw stock would be expected to restore sensitivity as the inbred genotype is regained. 6. Conditional Distortion

We have seen so far that all the unusual properties of the SD system we have considered are consistent with standard quantitative genetic interpretations when the available data are analyzed by parameters of a normal distribution. There is no need to imagine that the genes of the SD system are highly mutable nor that they are highly mutagenic. A possible exception is the phenomenon of conditional distortion. Sandler and Hiraizumi (1961a) found that “for certain SD lines, if the SD bearing chromosome is inherited from the female parent, then, in a fraction of F, male sibships, only one half of the heterozygous SD sons exhibit the phenomenon of segregation distortion; in the other half of the sons segregation is normal.’, They concluded that in males carrying the SD gene the X chromosome may become changed into a specific inhibitor of segregation distortion. The X chromosome did not have to pass through a female in order that the induced suppressor be expressed since the changed X could be kept in males by using crosses to attached X females and again the sons would not distort. Sandler (1962) has mapped one such induced suppressor on the X chromosome. However the data and pedigrees in the major report (Sandler and

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Hiraizumi, 1961a) are insufficient for a detailed evaluation of the phenomenon. Some difficulties in precisely defining the events arise from the facts (i) that the phenomenon may not occur for several successive generations in the same stock which at other times demonstrates the effect, (ii) that, even when it occurs, the effect in that sibship is only found in approximately 20% of all possible X chromosomes, (iii) that there is a probability of approximately 0.2 that a conditioned X will appear among the progeny of an unconditioned female, (iv) that the phenomenon is dependent only on the presence of S D , not on the existence of S D activity. Miklos (1971) has noted that the histograms of lc values of sibships from conditioned females are similar to histograms from situations in which it is known that there is large between-male variance. Thus the S D locus may have the capacity to induce a suppressor on the X chromosome, but further analysis is needed to define the phenomenon accurately. One attempt to reproduce this phenomenon has failed to detect the effect (Miklos, 1972).

7. Sex Ratio Hiraizumi and Nakazima (1967) first noted that the sex ratio in the SD+ class differed significantly from the 1:l expectation; they found a correlation between lc and the magnitude of sex ratio. At the time, they interpreted this effect in terms of the functional pole hypothesis, and they concluded that the data indicated a region on the X chromosome having partial homology to the S D region on chromosome 9. However, Denell et al. (1969), although corroborating the effect and extending the observation of the depression of the Y ;SD+class with increasing k value found no suggestion of dependence of sex ratio on lc value in the S D gametic class. They concluded that the sex ratio effect was unlikely to be explicable on the functional pole hypothesis and instead provided evidence in favor of the sperm dysfunction model. Denell and Miklos (1971) extended the relationships found between the probability of dysfunction of the SD+ gametes and their sex chromosome content. In their examinations of gametes which contained either the X , Y , both X and Y , or no sex chromosomes at all, they found that the highest probability of dysfunction occurred in the nullo;SD+ gametes, th a t the Y;SD+ dysfunction was greater than that of the X;SD+, and th a t attached X Y ; S D + was least susceptible to dysfunction. Curiously, in situations with a free X and a free Y , the nondisjunctional X / Y gametes showed much greater dysfunction than attached X Y gametes. The most significant aspect of the Denell and Miklos analysis was

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W. J. PEACOCK A N D GEORGE L. GABOR MIKLOS

their demonstration that the seemingly complex relationship between k value and sex ratio in the SD+class did in fact conform to predictions made with the normal distribution mode of analysis. If it is assumed that the presence (or absence) of particular sex chromosomes modifies the probability of dysfunction by a given amount, then particular threshold values can be ascribed to each of the gametic types. If these 0.6

!

o'4

+

0.3

0

3

>

5

0.2

0.1

0

0.6

0.0

0.8

0.7

0.0

1.0

k

FIG.7. (a) The observed rates of change of sex ratio and k for SD' gametes of SD heterozygotes having differing sex chromosome constitution. (A, X / Y ; B, attached X Y / Y ; C , attached X Y / O ) . (b) Predicted rates of change generated by the normal distribution method of analysis. (After Denell and Miklos, 1971.)

thresholds are considered with increasing mean distortion levels, a remarkable agreement is found between predicted and observed relationships (Fig. 7 ) . This provides a further example of the usefulness of approaching the SD phenomenon with two basic assumptions (i) that there exists a normally distributed underlying variable, and (ii) that certain thresholds of this variable exist with respect to the probability of gametic dysfunction.

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C. DISCUSSION

It seems clear from all the available evidence that the segregation distorter effect is the consequence of the dysfunction of SD+ sperm, this dysfunction being induced by the SD allele. The electron microscope analysis has given clear evidence that the sperm dysfunction is generally manifested as a failure in the development of the SD+ sperm. Although this analysis has given us important insights into the nature of the SD system and the fate of the SD+ sperm, it has not provided any clues as to the primary effect of the S D allele, an effect which apparently occurs early in the primary spermatocyte. It has also been seen that the SZI system conforms to the expectations of a quantitative genetic system, including both major and minor gene effects. However, the normal distribution mode of analysis cannot specify the mechanism involved. Apart from the SD locus itself, a sensitivity locus has been identified by recombination analysis. This locus maps a short distance to the left of cn which is in the salivary region 43A-43EF (Lindsley et al., 1972). The SD locus maps to the left of the sensitivity locus. Sandler and Hiraizumi (1960b) originally postulated that the SD locus was to the right of the centromere, but the existence of a pericentric inversion in their S D stocks led to confusion in the recombination analysis (Miklos, 1972). An examination of the data of Hiraizumi and Nakazima (1967), using an SD chromosome free of the pericentric, shows that the SD locus may even map to the left of pr, a locus on the left arm of chromosome 2 (38B2-38F7) (Roberts, 1968). Particular S D chromosomes have characteristic distortion potentials, and particular sensitivity alleles have characteristic susceptibilities to distortion. I n S D heterozygotes, when different SD chromosomes are tested against various SD+ chromosomes, it is evident that the extent of distortion is limited either by the SD chromosome or the sensitivity locus on the SD+ chromosome, depending on their respective values. Thus the observed level of dysfunction of SD' alleles is dependent on these two components of the SD system. One limit to the amount of dysfunction is the inherent distorting potential of the S D chromosome, An SD chromosome which when heterozygous to an SD' chromosome causes dysfunction of all SD+ spermatids is defined as having a distorting potential of 1.0. Similarly, the SD+ chromosome in this situation is defined as having a sensitivity of 1.0. If the SD chromosome of the above example is made heterozygous to another SD+ chromosome and causes only 50% of the SD+ spermatids to become dysfunctional, then this second SD+ chromosome has a sensitivity of 0.5. I n this case the limit to distortion has been

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W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

set by the SD' sensitivity allele. Conversely, an SD chromosome which, when heterozygous to an SDtchromosome carrying a sensitivity allele of 1.0, results in 50% of SD+ chromatid dysfunction, is defined as having a distorting potential of 0.5. Here the limit of SD+spermatid dysfunction is set by the SD chromosome. We have been able to deduce these rules principally from the data of Hiraizumi and Nakazima. Sensitivity has previously been expressed in terms of k, but should be expressed in terms TABLE 1 Dysfunction Levels in Males Having SD and SD+ Chromosomes of Varying Sensitivities and Distortion Potentials@Vb SD Chromosome

Chromosome Original SD R(SD-%)-l R(SD-36)-1 SD(Wm)) SD(R(m)) R(SD-%)-l

Distortion potential 0.997 0 .85 0.85

0.36 0.36 0 .85 -

Homolog

Sensitivity

Chromosome

Distortion potential

0 0 0 0.80 0.80 0

SD+(m bw) SD+(cn bw) SD+(R(pr)) S D + ( m bw) SD+(R(pr)) SD(R(m))

0 0 0.18 0 0.18 0.36

Sensitivity 1.0 1.0 0

.o

1

0 0.80

Observed resultant dysfunction 0.997 0.85 0.04 0.36 -0.15' 0.80

Data from Hiraizumi and Nakaaima (1967). The definitions of Sensitivity and Distortion potentials are given in the text (Section 11, B, 4). The underlined entries indicate in each cross which component of the SD system is limiting and determines the observed resultant dysfunction. This negative value indicates a possible slight reverse distortion. b

of the probability of dysfunction of SD+ gametes and in fact ranges from 0 to 1 and not from 0.5 to 1 (Table 1). The constraint to mechanism implied by these data is simply that the two loci concerned interact in a direct fashion. They do not interact as two, independent loci which would determine the final resultant level of distortion as a product of their respective probabilities of action. An S D chromosome which has the potential of rendering SD+ gametes dysfunctional with a probability of 0.8 will only achieve this level of dysfunction when heterozygous with an SD' chromosome of sensitivity of sufficient magnitude ; against an SD+ chromosome of lower sensitivity this SD chromosome will distort only to the limit set by the SD+chromo-

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some. The data in Table 1 also show that, if the “SD+”chromosome actually carries an S D allele, all of the conclusions reached above are still valid. I n summary, we conclude that (i) the action of an SD allele is a trans event, i.e., SD affects its homolog, (ii) the trans event is operative even if the homolog has an S D allele, (iii) the effect of the SD chromosome is independent of the magnitude of its own sensitivity locus. One possible exception of the trans limitation of the SD effect is also found in Hiraizumi and Nakazima. In a particular cross (their Table 7B), an S D heterozygote was constructed such that the SD+ chromosome had a sensitivity of 0 (i.e., k = 0.5), and the SD chromosome had a sensitivity locus which had been characterized at 0.80 (k = 0.83) ; this heterozygote showed a k value of 0.46-a slight reverse distortion. If this really is a cis action it is of a much lower magnitude than would be expected on the basis of the trans determined sensitivity value (0.15 rather than 0.80). The only other data which demonstrate cis distortion are those of Sandler and Hiraizumi (1960b). These data unfortunately involve recombinant chromosomes from an SD chromosome with the pericentric inversion and characterization of the recombinants is difficult. Further evaluation of the possibility of cis distortion awaits additional analysis. Although we have seen that an SD allele will cause dysfunction of gametes carrying its homolog provided that homolog has a sensitivity greater than 0, a problem arises in consideration of certain homozygous SD data. Hartl (1969) found that many SD/SD combinations result in almost complete sterility of the male. This sterility is unexpected in combinations where the sensitivity of each of the chromosomes is 0 (k = 0.5): for example R(SD-SG)/R(SD-S6) (Hartl, Table 2, entry 5 ) would be expected to have normal fertility since it is homozygous for a sensitivity allele of 0 value-neither chromosome should distort its partner. Similarly Peacock et al. (1972a) found an SD-72 Canberra homozygote to be almost sterile and furthermore found abnormal development of virtually all spermatids in each cyst. It is possible in these situations that the sterility is not brought about by each chromosome causing dysfunction of its homolog but is a result of homozygosity per se. This point will be further considered in the general conclusions (Section IV). The electron microscope analyses showed that there was substantial breakdown of SD+ sperm in the testes of SD heterozygotes, but Peacock et al. (1972a) found that the extent of breakdown differed in different SD stocks even where the distortion potentials were equivalent. They pointed out that it was likely that some SD+ sperm could be transferred

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to the female. The viability of these sperm would probably depend to some extent upon an interaction with the microenvironment of the female storage organs as also pointed out by Hart1 and Childress (1972) : this may well be the basis of the female effect reported by Denell and Judd (1969). These authors reported that different lc values were obtained when S D heterozygous males were mated to a number of genetically different females. When the female effect is expressed in standard deviations, it reflects only slight alterations of the recovery ratio. A major role for the female was proposed by Zimmering et al. (1970a) in their suggestion that “segregation distortion is the consequence, in some way, of the difference between X-bearing and Y-bearing sperm.” This was part of a general proposition that “primary sex ratio is controlled by genes in the female that distinguish between, and control the relative frequency of, fertilization by X-bearing and Y-bearing sperm, and that meiotic drive in males results from an aberration in the system by which sperm of the two types are distinguished.” The electron microscope analysis has shown that this suggestion is highly unlikely to be operative in the case of SD. 111. Sex Chromosome Meiotic Drive Systems

We shall now examine other meiotic drive systems in the Drosophila melanogaster male and ask whether in any or all of them the unequal EUCHROMATIN

-

HETEROCHROMATIN D

C

B

A

7 k~.-.Ad

NORMAL X

Nucleolus organizer IN (1) sc4

IN (1) sc8

IN (1) sc4Ls~*R

.mn

-

Cantromere

FIG.8. Diagrammatic representation of the distribution of heterochromatic bloch (A, B, C, and D) in various X chromosomes (based on Cooper, 1959). Z ~ ( I ) ~ C “ . S C ~ ~ is derived by recombination in an Zn(l)sc‘/Zn(l)sc* female.

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gametic recovery ratios are brought about by improper sperm development similar to the spermiogenic lesions described for SD. The system which has been most extensively characterized genetically and cytologically is that involving the In(1 )scZtsc"X chromosome. This chromosome is deficient for a large portion of the basal heterochromatin. Its exact origin and structure are diagrammed in Fig. 8 together with the main morphological divisions of a normal X chromosome as described by Cooper (1959). Further details of this and other chromosomes mentioned in this section can be found in Lindsley and Grell (19688).

A. DESCRIPTION OF THE sc4scs SYSTEM Gershenson's (1933) pioneering study revealed that males carrying the sc4scs X chromosome together with a normal Y produced a considerable number of nullo (no sex chromosome) and X / Y gametes. The high TABLE 2 Gametic Frequencies Produced by sc4~c8X/ Y Males Mated Both to Free X and Attached X Females Type of X chromosomes of female Free Attached Free A ttached Free Attached Attached

X

Y

nullo

Y chromosome X/Y used

0.47 0.52 0.44 0.43 0.64 0.61 0.50

0.27 0.22 0.18 0.16 0.32 0.33 0.38

0.23 0.23 0.37 0.39 0.04 0.06 0.08

0.03 0.03 0.01 0.02 0.01 0.01 0.03

Gametic types

y+Y y+Y B5Y BSY BSYy+ BSYy+ y+Y

References Sandler and Braver (1954) Sandler and Braver (1954) Peacock (1965) Peacock (1965) Peacock (1965) Peacock (1965) Ramel (1968)

nondisjunction level led him to suggest and cytologically verify that the X chromosome was deficient for much of the region homologous to a normal Y . Gershenson also noted the unequal recovery of complementary gametic types, especially the excess of nullo over X / Y , and he inferred that the univalents were frequently lost in the meiotic divisions. High nondisjunction levels and unequal gametic recoveries have been subsequently confirmed in similar studies where use of marked Y chromosomes has permitted direct identification of gametic classes (Sandler and Braver, 1954; Zimmering, 1963; Peacock, 1965; Ramel, 1968). The sc4sc8 X is generally recovered more often than the Y and the nullo is usually found in great excess of the X / Y gametes; some results typical of the system are shown in Table 2.

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W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

It may be that nondisjunction values depend on the Y chromosome used as well as other genetic components, e.g., the data of Sandler and Braver vary quite markedly from those of Ramel, even though the .y+Y has been used in both studies. Differential viability problems are insignificant since crosses to free X and attached X females yield similar recoveries. Culture temperature also influenced the behavior of the sc4sc8 system TABLE 3 Effects of Temperature on Gametic Frequencies Produced by sc4sc*X/Y Males Mated Both to Free X and Attached X Females Type of X chromosomes of female

Free Attached Attached Attached Attached

Gametic types

X

Y

nullo X / Y

0.50 0.50 0.53 0.50 0.51

0.24 0.42 0.31 0.38 0.41

0.25 0.02 0.05 0.03 0.14 0.02 0 . 0 8 0.03 0.06 0.02

Y Temperchromosome ature used (“C) y+Y y+Y y+Y y+Y y+Y

25 18 30 25 19

References Zimmering (1963) Zimmering (1963) Ramel (1968) Ramel (1968) Ramel (1968)

(Zimmering, 1963; Ramel, 1968). At 18OC the frequency of nondisjunction as well as the inequalities in gametic recoveries were reduced, whereas a t 3OoC they were increased (Table 3 ) . B. INTERPRETATION OF THE sc4scs SYSTEM 1 . Meiotic Loss Hypothesis

Gershenson postulated that the deficient sc4sc8 X frequently failed to pair with the Y and that the univalents were distributed randomly, producing X , Y , nullo, and X / Y gametes. He argued that the univalents were sometimes lost at the meiotic divisions and formulated algebraic expectations for the gamete classes. Sandler and Braver (1954) added to Gershenson’s hypothesis the assumption that the Y was lost in higher frequency than the sc4scs X , since the recovery of the Y was depleted relative to that of sC1scR. Zimmering (1963) also interpreted the unequal recovery ratios as resulting from meiotic loss of unpaired chromosomes and further suggested that the marked alterations in gametic frequencies at 18OC were due to “regularization of the transmission of the Y.” Both Cooper (1964) and Peacock (1965) cytologically confirmed Gershenson’s original supposition that the sc4sc8 X and a Y frequently failed to synapse, but no chromosome loss was found during meiosis even though in the latter study several marked Y chromosomes were

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used and the frequency of nondisjunction differed in the various stocks.

It was further discovered (Peacock, 1965) that unpaired chromosomes did not move a t random, but always proceeded together to a pole a t anaphase 1. Thus disjunctional anaphase 1 separations yielded equal numbers of X and Y containing secondary spermatocytes, whereas nondisjunctional separations led to the formation of equal numbers of nu210 and X / Y secondary spermatocytes (Table 4). Gershenson had also argued that in scJscR X / Y / Y males the X would remain as a univalent since it had reduced synaptic activity. Genetically, he found mostly X / Y and Y gametes, and he argued that the unsynapsed X was often lost, leading to an increase in the relative number of Y sperm. Cytological analyses of meiosis in sc4sc8/Y/Y males corroborated TABLE 4 Cytological Data from sc4scsX/YMales with Various Marked Y Chromosomes4 Metaphase I

Y chromosome used Paired Y+Y BSY BSYy+ w+Y

Unpaired

42 87 89 85

19 39 5 44

Metaphase I1 and Anaphase I1

Anaphase I DisNondisjunctional junctional 44 54 24 38

9 20

14

X

Y

71 72 43 37 106 107 161 159

nullo X / Y 26 13 10 76

21 15 9 81

From Peacock (1965).

Gershenson’s inference that the sciscs X generally remained as a univalent and that the two Y’s formed a bivalent (Cooper, 1964; Peacock, 1965), but once again chromosome loss was not observed. 2. Functional Pole Hypothesis

Peacock (1965) found in cytological observations that there were equal frequencies of reciprocal meiotic products. This observation contrasted with the disparate genetic recoveries. He further found a close agreement between the frequency of synapsis failure a t metaphase 1, the frequency of nondisjunctional second division meiocytes, and the genetic frequency of nondisjunction (Table 5 ) . The close agreement between the cytological and genetical estimates of nondisjunction, in spite of gametic depletions, led Peacock to suggest that this system may have been explicable on the functional pole hypothesis elaborated earlier (Section 11, A, 3 ) . He assumed that when the sc4scs X and Y were paired, the X oriented toward the functional pole more frequently than the Y , and that in

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W. J. PEACOCK A N D QEORGE L. GABOR MIKLOS

nondisjunctional cases both univalents proceeded to the nonfunctional pole with a high probability. Yanders et al. (1968) did find evidence for a differentiation between the meiotic poles of primary spermatocytes in their scores of codistribution of chromosomes and certain intracellular symbionts. However, this did not provide direct evidence for the functional pole hypothesis, which as we have already seen is not applicable in the case of SD. We have therefore reexamined the sc4scs system using TABLE 5 Comparison of Cytological and Genetic Measures of Nondisjunction Proportion of nondisjunctional Y chromosome secondary spermatocytes used (nullo XY)/Total

+

Proportion of genetic nondisjunction as measured by nullo/(nullo X )

+

~~

BSY BSY BSYy+ Y+Y

0.44 0.18 0.08 0.39

0.46 0.18 0.07 0.46

References ~

~~~~

Peacock (1965) Peacock (1965) Peacock (1965) Yanders et al. (1968)

both published and previously unpublished information (Peacock, unpublished; Miklos, 1970) and as a result propose a new pairing-dysfunction hypothesis. OF THE sc4sc8 SYSTEM C. REEXAMINATION

In both the meiotic loss and functional pole hypotheses it was assumed that the events leading to unequal gametic recovery occurred a t meiosis. We have determined the critical period by temperature shock experiments. Males (sc4sc* X / Y ) were raised a t 1 8 O , 25O, and 27OC and subsequently serially mated and maintained at 25OC. The results are shown in Fig. 9. The 27O and 1 8 O nondisjunction values returned to the 25OC level after a period of culture at this temperature, demonstrating the following points: (i) the critical period occurs in the primary spermatocyte; the lag period of 7-9 days corresponds to the time taken for primary spermatocytes to develop into mature sperm, a timing determined autoradiographically by Chandley and Bateman (1962) ; (ii) these data emphasim an earlier point (Section 11, A, 1) that adult males continue to produce and sequentially transfer mature sperm if a continuing supply of virgin partners is available. Thus although we have reason for discarding both the meiotic loss and functional pole hypotheses, each of which relates directly to the

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389

meiotic divisions, these temperature shift experiments demonstrate that the sc4scs dehavior is determined a t meiosis. Further analysis of individual male data is in agreement with this conclusion in that we have found a relation between the probability of synapsis and the probability of gametic recovery (Fig. 10).

..............o............. o.l~o,, / ,

.'U.

w

, , ,

U

Lu

E z

1

o

I(b),

,

, , ,

I

,

,

0

2

3

4

8

7

8

1

6

I

0

,

1011

,

,

I I

121314

TIME IN DAYS

FIG.9. (a) Results of temperature shift experiments showing the change in nondisjunction frequency over time for sc'scsX/BSY males raised at 27", 25" and 18"C, then mated and maintained at 25°C. (b) Results of similar experiments using sc'sc*X/~+Ymales. A, males raised a t 27°C; 0, males raised a t 25°C; 0 , males raised at 18°C.

This figure demonstrates that as the frequency of disjunction is reduced, the discrepancy between the complementary classes of both disjunctional and nondisjunctional gametes becomes more extreme. From these data we infer that chromosome pairing a t meiosis has a direct bearing on gametic recovery. Before discussing the significance of these relationships in more detail, we will present further data pertaining to the unrecovered gametes. The sc'sc" system is similar to SD in that all meiotic products are present as secondary spermatocytes, in having 64 spermatids per bundle, and in lacking any significant zygotic lethality

390

W. J. PEACOCK A N D GEORGE L. GABOR MIKLOS

(Peacock, 1965). Another point of similarity between the two systems lies in the inverse correlation between progeny number and the extent of meiotic drive-males with high levels of nondisjunction yield fewer progeny than those with lower values (Table 6). We regard these data

FIG. 10. The observed relationship between nondisjunction frequency and the extent of departure from equality in recovery of reciprocal gamete classes from sc4scsX/Y (data from Peacock, Miklos, and Goodchild, 1973). TABLE 6 Comparisons of the Productivities of sc4scaX/y+Y and sc4scsX/BSY Males Period of sperm sampling (days) 3 3 15 15 15 15 15 15

Y chromosome used

Progeny/male

No. of males

Frequency of nondisjunction

Y+Y

209 113 699 635 576 446 328 235

176 71 20 17 4 17 13 6

0.10 0.54 0.12 0.17 0.22 0.17 0.22 0.30

B5Y Y+Y

only as suggestive evidence for sperm dysfunction and have already discussed the limitations of progeny number as a parameter. The most convincing evidence for a sperm dysfunction mechanism should come from electron microscope analysis of developing spermatids.

MEIOTIC DRIVE IN

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391

This is as yet unavailable for the sc4scu system, but light microscope observations show considerable sperm breakdown in the testes of sc'sc8 males (Peacock et al., 1973). Peacock e t al. (1972a) showed that in SD/SD+ males the sperm breakdown visualized in the electron micro-

FIG.11. Photomicrograph of the basal region of a testis dissected from an In(l)sc'LscSR/g+Ymale. SV, Seminal vesicle containing large numbers of sperm ; CB, coiled bundle of sperm in base of testis; WB, waste bags predominantly of degenerating sperm. The smallest waste bags are similar to those seen in control males and probably represent discarded portions of the germinal cyst cell. scope could also be detected at the level of the light microscope. For example, the "waste bags" of discarded organelles and abnormal sperm were more obvious and of larger dimensions in SD males than in controls. Comparison of sc4scRmales with full sib controls has shown evidence of massive sperm breakdown in the sc4scRsystem (Fig. 11).

392

W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

The light microscope observations together with the progeny number analyses are thus consistent with a sperm dysfunction explanation for the unequal recoveries observed between reciprocal gamete classes in the sc'scs system. Actual loss of gametes is also indicated by the genetic data (Table 2), in which the X class exceeds 50%.

D. A NEW HYPOTHESIS Some characteristics of the scJscR system which have emerged from the previous considerations are as follows: (i) Cooper (1964) has demonstrated that there are several sites in the basal X heterochromatin which can participate in pairing of the X and the Y , that there are also multiple sites on the Y chromosome, and that the sc4sc8X chromosome is deficient for some of the sites involved in pairing with a normal Y chromosome. (ii) At 25OC the sc4sc8 gamete is recovered in excess of Y , and nullo gametes are recovered far more frequently than X / Y gametes. (iii) These discrepancies in gametic recoveries are correlated with the frequency of nondisjunction. (iv) Disjunctional and nondisjunctional gametes are produced by separate populations of meiocytes-nullo and X / Y gametes are the sole products of synaptic failure. These properties, when considered with the observation that the absence of sex chromosomes from a spermatid nucleus does not interfere with the formation and operation of sperm, suggest to us a direct relationship between chromosome pairing and sperm development. We propose that the presence rather than the absence of a particular chromosome results in abnormal sperm development. This is comparable to the situation which has been shown to hold for SD. Our basic premise is that pairing of a normal X and a normal Y is a prerequisite f o r normal sperm development. Perturbations in pairing lead to abnormal sperm development. Our pairing-dysfunction model generates predictions which are satisfied both within the sc4sc8 system and in other sex chromosome meiotic drive situations. It permits analysis of the sc4sc" system in terms of normally distributed variables, a characteristic of biological systems. 1. Development of the Pairing-Dysfunction Model

For any given metaphase 1 cell, it is clear that the sex chromosomes will be present as a bivalent or two univalents. Since these two alternatives occur within the population of spermatocytes of a single male, and since there are many pairing sites on both the X and Y , there must be cell-to-cell variability in the number of pairing sites that inter-

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act. We postulate that if a sufficient number of pairing sites interact, disjunction is assured. When the number of interacted sites is less than this threshold value, univalents result a t metaphase 1 and nondisjunction ensues. We also know (see preceding section) that there is a positive correlation between the magnitude of the recovery ratios and the frequency of nondisjunction. This suggests that noninteracted pairing sites contribute to irregular spermatid development. Hence, the lower recovery of the Y relative to the S C ' S C ~ X is explicable in terms of the inequality of pairing sites-the S C ' S C ~ X is deficient in some pairing sites, so that even in cells in which this S is completely paired with a Y there will be noninteracted Y sites remaining. The noninteracted Y sites confer a certain probability of developmental failure upon a gamete carrying the Y . I n a similar way, in nondisjunctional gametes, both the sc4sc8 X and Y contribute to the probability of a developmental lesion in that they will both have noninteracted sites. Nullo gametes, being devoid of any sex chromosomes that could upset sperm maturation, should not suffer developmental failure. When a comparison is made of the frequencies of nullo and sc4sc8X gametes, the genetic and cytological frequencies of nondisjunction are very close (Table 5 ) . This indicates that we can discuss sperm dysfunction in terms of the developmental failure of Y and X / Y gametes and that our best genetical estimates of nondisjunction should be obtained from only the sc4sc8 X and nullo classes. The slight discrepancy in estimates a t high nondisjunction suggests that a t these high levels the X may also undergo some dysfunction because of the increased number of noninteracted sites.

3. Quantitative Aspects of the Model Although it is obvious in Fig. 10 that a correlation exists between recovery ratios and the frequency of nondisj unction, analysis shows that this relationship in the nondisjunctional class is best described by a second-order regression curve. However, if the data for gametic recoveries and for nondisjunction frequency are plotted in standard deviation units, a straight-line relationship is found for both the disjunctional and nondisjunctional classes (Fig. 12). The slope for each line approaches unity, indicating a direct correlation between the pairing and dysfunction variables. The assumption that normally distributed variables underlie the observed range of recovery and disjunction values is consistent with the basic tenet of our pairiny-dysfunction model-that of the existence of cell-to-cell variability in the number of interacted pairing sites on the X and Y chromosomes. Implicit in the assumption is that a certain threshold number of interactions has to occur to ensure normal disjunc-

394

W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

tion of the sex chromosomes and the subsequent normal development of the gametes. The difference in intercept between the disjunctional and nondisjucof the dysfunction variable) provides a measure in tional plots (1.2~ the nondisjunctional class of the added probability of dysfunction which can be attributed to the existence of noninteracted sites on the X as well as the Y . At 2% nondisjunction (-2.00) there is approximately a 10% dysfunction of Y and approximately a 50% dysfunction of X / Y . Dysfunction of X gametes may also occur a t higher levels of nondisjunction and this may account for the slight departure of the disjunction

NONDISJUNCTION (STANDARD DEVIATIONS)

FIO.12. Plot of the extent of dysfunction of Y gametes (A), and X / Y gametes (B) against the frequency of nondisjunction InuZZo/(nuZZo X / Y ) I . Both dysfunction and nondisjunction are plotted in standard deviation units.

+

plot from a slope of 1. The measurement of nondisjunction and dysfunction in standard deviations avoids the complication of apparently different orders of change in different experiments, e.g., a change from 1% to 476 nondisjunction is equivalent to a change from 31% to 50% nondisjunction when measured in standard deviations. Hence if a modifier in one stock were to move nondisjunction from 1% to 4% and in another stock from 31% to 50%, it actually would be having the same effect in both cases. 3. Biological Aspects of the Model Muller and Settles (1927) originally made the observation that sperm function was not impaired by the absence of portions of the Drosophila

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395

genome. More recently this observation has been expanded by Lindsley and Grell (1969), who in an extreme case demonstrated that a sperm carrying only a chromosome 4 could fertilize an egg. The implication from these observations is that information for sperm development is dependent on the diploid genotype. I n the case of segregation distorter the SD locus determines a change on the SD+ homolog which dictates that the gamete carrying the SD+ chromosome will exhibit a developmental lesion. Chromosome 2 can be omitted from the nucleus, and normal development is not impaired. Therefore SD+ gamete dysfunction is dependent upon the presence of the SD+ chromosome. We feel that the same argument applies to the sc4sc8 system. The pairing-dysfunction model suggests that bivalent association of the X and Y a t meiosis is important for normal development. This may be because pairing effects a physical modification of chromatin, where such a modification is an essential prerequisite to normal sperm development. For example, a pairing-induced conformational change in chromatin may be necessary for the packaging of chromatin which occurs in the conversion of secondary spermatocytes to mature sperm. Alternatively, pairing may mediate repression of specific groups of genes; if repression fails, then continued transcription results in gametic dysfunction. Since we lack molecular data on the mechanisms involved, we cannot distinguish between these or other alternatives and will therefore discuss the postulated change in nonspecific terms. We will refer to the prepairing condition as being active and the postpairing condition as inactive.

4. Summary of the Model We envisage that in scPscRX / Y males, pairing between the X and

Y chromosomes depends first on many sites present on both chromosomes. These sites, or the loci controlled by them, are normally genetically active and need to be switched off during meiosis in order that normal sperm development may ensue. The active loci are inactivated by pairing between the X and Y . When pairing is efficient, all pairing sites on the X will have interacted with sites on the Y , but because the sc4scs X is a deficient element, the Y will still possess noninteracted sites and will continue to be active and thus contribute to Y gamete dysfunction. As the efficiency of pairing decreases, so also the frequency of nondisjunction increases and more and more sites are left noninteracted on both the X and Y chromosomes. One can imagine this as a zipper being unfastened although noninteracted sites may not necessarily be contiguous. As more sites are exposed, a higher level of Y gamete dysfunction follows, and some sc4scxX gamete dysfunction may also occur. In the nondisjunctional gametes the null0 class is not susceptible to

396

W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

gametic dysfunction because it is devoid of sex chromosomes. The X / Y class on the other hand contains two chromosomes which have previously not paired sufficiently to allow a disjunctional separation a t anaphase 1, so both contain many active sites and interact to produce a high level of gametic dysfunction. Furthermore, as nondisjunction increases, the mean number of interacted pairing sites in the nondisjunctional class decrease and dysfunction of the X / Y gametes will increase.

E. TESTSOF

THE

MODEL

1. The sc4scaX / Y / Y Genotype

Males having the sc4scuchromosome together with two Y chromosomes have been studied by Gershenson (1933), Sandler and Braver (1954), and Zimmering and Green (1965). The behavior of this genotype has been puzzling. Virtually all the gametes produced by a sc4scs/Y/Y male are X / Y or Y , the latter class being predominant. This is seemingly complete reversal of meiotic drive, since in sc4sc8/Y males it is the Y that suffers dysfunction; in sc4scs/Y/Y males it is the X which undergoes dysfunction. Gershenson’s deduction that in a sc4scR/Y/Ymale the two Y chromosomes would form a bivalent and the X would remain as a univalent has been cytologically verified (Cooper, 1964; Peacock, 1965). This observation of the univalent nature of the sc4sc8 X is important in that it demonstrates that the genetic behavior of the sc4sc8/Y/Y is in direct accord with the model we have proposed for the sc4sca/Y system. On our model we would predict the two Y chromosomes are rendered inactive through pairing, disjoin normally, and do not contribute to gametic dysfunction. The univalent X , being unpaired, is not switched off and contributes to gametic dysfunction. Thus those gametes which include an X will suffer some dysfunction whereas those which do not will develop quite normally. In contrast to the sc4scs X / Y / Y genotype, males having a normal X and two Y chromosomes show no evidence of inequality of recovery of reciprocal classes. Cytologically the metaphase 1 configuration in such males is that of a trivalent involving all these sex chromosomes (Cooper, 1964). Apparently all chromosomes achieve inactivation through pairing, and little or no dysfunction results. 2. The bblX Chromosome

If our model is correct, we would expect any X chromosome having a partial deletion of the pairing region in the basal heterochromatin to exhibit behavior similar to that of the sc4scs X chromosome. Lindsley

MEIOTIC DRIVE IN

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Drosophila

et al. (1960) produced a number of X chromosomes deficient for the locus of bobbed ( b b ). These deficient X chromosomes segregated irregularly from the y+Y in spermatogenesis producing nondisjunctional gametes in significant frequencies. The detailed analysis of one of these, D f (1) bb2481is given below (Moore and Peacock, unpublished data). The genetic data (Table 7) show the same trend as the sc4scUsystem-a high level of nondisjunction and grossly unequal recovery of reciprocal products. As in the sc4sc8 system, crosses to females having free x' or attached X chromosomes TABLE 7 Gametic Frequencies Produced by bblX/y+Y Males Mated Both to Free X and Attached X FEMALES at 25OC Type of X chromosomes of female Free Attached

Gametic types

X

Y

nullo

X/Y

Total progeny

0.28 0.20

0.08 0.07

0.63 0.71

0.02 0.06

3507 787

TABLE 8 Cytological Data From bblX/y+Y Males Metaphase I

Y chromosome Unused Paired paired YfY

Y

21 71

39 105

Anaphase I

Metaphase I1

Disjunctional

Nondisjunctional

X

Y

60 65

116 110

38 60

34 71

Anaphase I1 nullo X / Y 74 98

78 107

demonstrate that relative viabilities are insignificant in the results. Since the frequency of nondisjunction exceeds 50%, the maximum frequency expected on the basis of random movement of the two chromosomes, these genetic data indicate that as in the case of the sc4scs system there is nonrandom movement of univalents a t anaphase 1. This is confirmed in cytological analysis (Table 8). The correspondence between the frequency of univalents a t metaphase I on the one hand and the genetic and cytological measures of nondisjunction on the other is striking. I n bbl/y+Y males the proportion of metaphase I cells having univalent X and Y chromosomes is 0.65, the proportion of nondisjunctional secondary spermatocytes is 0.68, and the genetically measured frequency of nondisj unction is 0.69. This cor-

398

W. J. PEACOCK AND GEORGE L. GABOR MIKLOB

respondence indicates as in the sc4sc8 system, that the inequality in recovery of reciprocal gametic types can be ascribed to the dysfunction of the Y and X / Y gametes. The magnitude of the recovery ratios for the bbl X are similar to those obtained for the sc4sc8 X a t comparable levels of nondisjunction. Thus, although in the bbz X the remaining pairing sites are proximal rather than distal as in the sc4sc8 X chromosome, the properties of the system are compatible with the pairing-dysfunction model. 3. Attached X Y Chromosomes Situations in which the interaction of the pairing sites of the X and Y has been altered are also found in various rearrangements between the X and Y chromosomes. Zimmering (1963) and Johnsen (1971) have investigated the meiotic behavior of a chromosome in which a portion of the TABLE 9 Recovery Ratios From Various Attached X Y Chromosomes Arrangement of X and Y elements

Probability of dysfunction of the attached X Y 0.70 0.39 0.15 0.14 0.12 0.01-0.19 0.21-0.37

Authors Johnsen (1968) Johnsen and Zarrow (1971) Lindsley and Sandler (1958) Sandler and Braver (1954) Olivieri et al. (1970)

basal X heterochromatin is appended to the long arm of the Y chromo* so that there is no free Y chromosome. These authors some ( X p Y LY") have shown that the recovery of the X p Y L Y . x is reduced relative to that of the null0 gamete, the depression of the X p Y L *Y" class being approximately 75%. Johnsen (1971) has further shown that the X p Y L * Y 8is regularly univalent at metaphase I and that there is negligible meiotic loss. We suggest that sperm dysfunction may be the operative mechanism here, the dysfunction resulting from the lack of a pairing partner for the X p Y L* Y" and the subsequent resulting presence of noninteracted pairing sites. If this reasoning is correct we would expect that in other chromosomes in which all the essential elements of the X and Y are on the one centromere that we would find abnormal recovery ratios. This appears to be the case but to differing extents in various experiments (Table 9). These attached XY's are in general not as deviant as the XpYL*Y".

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Certain observations made by Cooper (1964) reveal why this may be so. Cooper showed that a univalent Y s X . Y L loops back and pairs with itself. Intrachromosomal pairing of this type may be equivalent to interchromosomal pairing with respect to interaction of chromosomal pairing sites. Thus the expected depletion of the X Y class may be lessened depending on the extent of intrachromosomal pairing. I n the attached X Y chromosomes in the table, the X and Y elements are arranged differently and they may thus differ substantially in their potential for intrachromosomal pairing. I n addition, in none of these chromosomes can there be a complete definition as to the content of X and Y heterochromatic pairing sites. The significant point is that there is consistently a depletion of the attached X Y class, but when a pairing partner is provided this depression is reduced; for example, Sandler and Braver (1954) found instead of a 14% depression, only a 3% depression when fragment 2, a modified Y L chromosome, was included with the Y s X *Y L chromosome. A further example of the behavior of an attached X Y with and without a free Y chromosome can be found in Denell and Miklos (1971) ; the chromosome they used was again the Y s X -Y L ,which showed a 13% depression without a Y versus a 2% depression when a Y was present. I n both the examples given above, we may suppose that the regularization of the recovery ratio resulted from more complete saturation of pairing sites involving, perhaps, loop pairing as well as homologous pairing. The absence of any significant frequency of primary nondisjunction in these examples is consistent with the assumption of regular bivalent formation. Attached X Y recovery has also been modified by the addition of X heterochromatin duplications. Lindsley and Sandler (1958) repeated and extended some earlier work of Gershenson (1940), who investigated in some outstanding studies the nature of the so-called inert heterochromatin of the X chromosome. Lindsley and Sandler showed that of 8 duplications free of viability complications, 7 showed regular segregation from an attached X Y chromosome. In all 7 cases the attached XY was recovered less frequently than the duplication but in only 2 cases was there a significantly lower recovery than the 14% depression of the attached XY class observed in the control. I n the remaining case the attached X Y and the duplication segregated independently and reciprocal products were recovered in approximately equal frequencies. Since Cooper has shown that the most proximal region of the X heterochromatin does not contain any pairing sites, we would predict on our model, that a cytological investigation would show the following results: (i) The duplication which showed independent segregation would be small and would not be paired with the attached X Y a t metaphase 1; the depression of the attached XY gametes and of the attached

400

W. J. PEACOCK AND GEORGE L. GABOR MIKLOS

XY/duplication gametes would then be ascribed solely to the attached X Y chromosome as in controls. (ii) The two duplications which segregated regularly but were recovered in considerable excess of the attached X Y would be of a larger size class, would be regularly paired with the attached X Y a t metaphase 1, and must interact in such a way as to prevent loop pairing to a considerable extent. (iii) The five duplications which showed regular disjunction but did not significantly alter the recovery of the attached X Y would also be large and would form a metaphase 1 bivalent, but pairing must be of such a nature as to permit some noninteracted pairing sites. This relationship of size and meiotic behavior has been observed by Cooper (1964), who noted that of 40 X chromosome duplications he tested, 10 segregated randomly from an attached X Y chromosome and all were small or smaller than chromosome 4. The remaining 30 deletions which segregated regularly from the Y 9 X .Y L chromosome were all greater in length than chromosome 4. A similar correlation was observed by Gershenson (1940) in a series of tests of X duplications in X / Y / d u plication males. Lindsley and Sandler also tested some of the duplications with X . YLYs (Parker, 1954), an attached X Y in which the distribution of the heterochromatin differed from that in Y s X . Y L . They concluded that there was no apparent effect of the distribution of the heterochromatin on the meiotic behavior of an attached XY/duplication combination, but in fact their data do show significant alterations in the recovery ratios. Hence, the recovery of reciprocal gametic types probably depends to some degree on the pairing relationships within the bivalents; this in turn depends on the arrangement of the heterochromatic blocks.

F. MODIFICATION OF MEIOTIC DRIVEBY TEMPERATURE We briefly introduced data earlier (Table 3) showing that temperature had a sizable effect on recovery ratios. Zimmering (1963) first showed that a t 1 8 O C in sc4sc8/Y males, there was (i) a lowered level of nondisjunction and (ii) a reduction in inequality of the two gametic ratios. He also found that the X p Y L * Y 8 / 0system exhibited a reduction in the deficiency of the X p Y L * Y 8class at 1 8 O . Zimmering argued by analogy from the X p Y L * Y s / O to the sc4sscS/Y system that, since temperature effects were independent of pairing in the X p Y L .Y s case, they were likewise independent of pairing in the sc4scS case. However, temperature effects have a number of components. Peacock (1965) demonstrated that a t 18OC there was no longer nonrandom movement of univalents (Table 10). It can be seen that for all the 2 5 O ex-

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periments the frequencies of nondisjunction measured a t the second division of meiosis are very close to the frequencies of unpaired chromosomes a t metaphase 1. At 1S0C, however, the frequency of nondisjunction is approximately halved, indicating that the movement of univalents is now effectively randomized. Table 10 also shows that there is an effect on pairing at metaphase 1. This effect varies from zero to approximately 0 . 7 ~of the pairing variable in the different experiments. If we allow for this change in univalent behavior, then by reference to our previous curves we can determine (i) the effect of temperature on the pairing variable and (ii) the effect of temperature on the dysfunction variable. For both disjunctional and nondisjunctional gametes dysTABLE 10 Cytological Data From sc4scSX/Y Males Raised at 25' and 18OC

Y chromosome used

Temperature ("C)

BSY

25 18 25 18 25 18

Y+Y Y+Y

a

Metaphase I (% unpaired) 0.37 0.28 0.33 0.32 0.43 0.15

(276)" (169) (323) (234) (126) (96)

Anaphase I (% nondisjunctional) 0 . 3 9 (101) 0 . 2 3 (71) 0 . 2 5 (109) 0.15 (60) 0.42 (154) 0 . 1 7 (71)

Metaphase I1 and anaphase I1 ( % nondisjunctional) 0.40 0.15 0.31 0.21 0.40 0.06

(257) (316) (422) (235) (471) (223)

Figures in parentheses indicate number of cells scored.

function is reduced. The reduction for the Y varies from 0 . 2 to ~ 1 . 2 and ~~ to 2 . 1 ~ . for the X / Y varies from 0 . 5 ~ Thus the temperature effect on drive is compounded of effects on (i) the probability of pairing, (ii) univalent movement, (iii) probability of dysfunction. Ramel (1968) had commented that the existing data demanded a temperature effect on both univalent movement and pairing. I n the case of the s c 4 s c U / Y / Y system, no cyto1,ogical estimates of the frequency of the univalent X condition exist for 18°C cultures. Such cytological studies are necessary before it is possible to determine the relative contributions of the pairing and dysfunction variables in this particular system. Cytological data are available, however, for the b b z / Y and indicate that there is no temperature effect on univalent movement, leaving only pairing and dysfunction as components of the temperature effect (Table 11).

402

W. J. PEACOCK A N D GEORGE L. GABOR MIKLOS

TABLE 11 Cytological Data From bb' X / Y Males Raised at 25" and 18°C

Y chromosome used

Temperature ("C)

?/+Y

25 18 25 18

Y

Metaphase I (% unpaired) 0.65 0.61 0.60 0.55

(60)~ (242) (176) (64)

Anaphase I (% nondisjunctional) 0.66 0.59 0.63 0.70

,. The numbers in parentheses indicate the number of

(176) (138) (175) (60)

Metaphase I1 and anaphase 11 (% nondisjunctional) 0.68 0.59 0.61 0.52

(224) (628) (336) (209)

cells scored.

G. DISCUSSION The thesis developed for the sc4sc8 system, that pairing of the sex chromosomes is of importance in preestablishing conditions for normal sperm development, has been strengthened by several independently derived situations in which pairing was disrupted to some degree. I n these cases the reduction in frequency of pairing is associated with physical loss of sites in the pairing regions. Another chromosome, sc4scs1, in which the proximal heterochromatic break is suspected of being even closer to the centromere than in sc4scs (Cooper, 1959), has been found by Ramel (1968) to exhibit similar meiotic behavior to that of the sc4scs chromosome. There are some data available in which nondisjuction has been reported where the chromosomes involved have had no actual loss of chromosome material. For example, Tokunaga (1971) has extensive data on both spontaneous and temperature-induced nondisjunction a t the first meiotic division in the Drosophila male. I n all her data the nullo class is in a 3-fold excess of the X / Y gametic class. She took the precaution of using a marked Y chromosome and has established that the nullo gametes are in fact lacking both sex chromosomes rather than merely representing a loss of the Y marker by chromosome rearrangement. This point was overlooked in some previous analyses where nondisjunction was induced by irradiation (Strangio, 1962) ; however, Zimmering and Wu (1964) have some well controlled irradiation experiments which again show an excess of nullo over X / Y gametes. We can place little emphasis on these results because we have no knowledge of the stage a t which the perturbation of normal meiotic events occurred. For example, if the temperature- and irradiation-induced nondisjunction events resulted from spindle aberrations after completely normal pairing had

MEIOTIC DRIVE IN

D'rOSOphdCl

403

taken place, then the excess of nzillo to X / Y would not supply any support for our model. We can do no more a t present than note that the results are consistent with the expectations on the basis of our model. A better opportunity to test the model is provided in the mutants isolated by Baker and Carpenter (1972). These authors produced mutants in the X chromosome which were characterized by significant frequencies of sex chromosome nondisjunction. They have placed two of the mutants in the euchromatin, possibly a t different loci, and have suggested that these are loci controlling the heterochromatic pairing sites. Again the nullo class exceeded the X / Y class and, furthermore, among disjunctional progeny, X gametes were in excess of Y gametes. I n their stocks it should be possible to determine cytologically whether nondisjunction resulted because of a failure of X / Y bivalent formation. Baker and Carpenter noted that there was a negative correlation between the frequency of males among disjunctional progeny and the frequency of nondisjunction. However, if their data are plotted in terms of a recovery ratio of X / ( X Y ) ,then there is no correlation evident; the nondisjunctional data are too small to determine if there is any significant correlation of the nullo/(nullo X / Y ) ratio to nondisjunction frequency. These mutants will be of much interest in further analysis since they represent a homogeneous group which has a cis action on the pairing region of the X chromosome such that Y gamete dysfunction occurs, but in a manner which is independent of the frequency of nondisjunction. It will be important to further test our suggestions by examining a number of attached X Y chromosomes, in the absence of a pairing partner, both cytologically and genetically. The predictions of our model concerning unequal recovery and intrachromosomal pairing are straightforward and directly amenable to analysis. Similarly, cytological behavior of X heterochromatic duplications with attached X Y chromosomes will provide a further evaluation. It would be desirable to test the hypothesis by producing Y chromosomes deficient in pairing sites and in this way complement the X chromosome studies, but this is likely to prove difficult because of the distribution of the essential male fertility factors throughout both the long and short arms of the Y chromosome (see Lindsley and Grell, 1968). One well documented meiotic drive system in male Drosophila is the T(1;4)Bssystem. This translocation between the X chromosome and chromosome 4 is characterized by unequal recovery of reciprocal gametic classes (Novitski and Sandler, 1957), by low fertility (Novitski, 1970), and shows a temperature response in the extent of meiotic drive (Zimmering and Perlman, 1962). There may well be sperm dysfunction in this system, but we see no immediate application of our model since the X chromosome breakpoint is euchromatic and does not automatically

+

+

404

W. J. PEACOCK A N D GEORGE L. GABOR MIKLOS

lead to any expected difficulties in pairing. However, the translocation per se may cause a disturbance to normal pairing, and unequal recovery may result from developmental lesions which are a consequence of these pairing difficulties. On the other hand, the basis of meiotic drive in this system could be quite distinct from the mechanism we have discussed. At least one other cause of gametic dysfunction has been documented, this being the Y chromosome breakage which Erickson (1965) has described in the case of recovery disruptor. Another case is the sex ratio system of Drosophila pseudoobscura. In their cytological analysis of the system Novitski et al. (1965) noted regular X and Y bivalent formation, thus it seems unlikely in this case that the primary developmental failure is attributable to any failure of pairing. Policansky and Ellison (1970) have reported that sex ratio males have only half the numbers of sperm per cyst as controls. However Novitski et al. (1965) had shown that these males had 128 sperm heads per cyst, and since the sections shown by Policansky and Ellison were of closely packed, individualized sperm tails, it seems likely that they examined cysts in the post coiling stage when the defective sperm tails had already been separated (Peacock et al., 1972b). IV. General Conclusions

Some of the recent developments which have given us new insights into meiotic drive in male Drosophila have been made primarily in the Segregation Distorter system. Electron microscope analyses have given clear evidence of spermatid dysfunction, this sperm dysfunction being limited, in S D heterozygotes, to the SD' class. The breakdown of these particular spermatids generally within the testis, results in the observed meiotic drive; in the extreme situation only S D gametes are recovered. The cause of the developmental abnormalities in the SD+ spermatids is not known, but the following points have been established : (i) The S D allele is responsible for an event which is limited to its homolog, this event subsequently resulting in the dysfunction of the gamete carrying this chromosome. (ii) The S D homolog can be completely susceptible to S D action, completely insensitive, or have some intermediate level of susceptibility. A discrete region of the chromosome in the centromere region, the sensitivity locus, is principally responsible for the degree of susceptibility. (iii) The time of the initial SD-mediated event has been localized to the early primary spermatocyte and may possibly occur during a stage in which homologs are paired. Although we have no evidence as to the actual nature of the SD effect, some of the more unconventional genetic processes which have been attributed to the S D system appear to be unnecessary assumptions.

MEIOTIC DRIVE IN

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405

When the extensive genetic data that are available are analyzed in the light of two basic assumptions (viz. that SD action is a process which can be analyzed in terms of a normally distributed variable, and that the probability of gametic dysfunction is associated with threshold values of this variable) , they reflect a standard quantitative genetic situation. There is evidence of two major loci, the SD locus and the sensitivity locus, and of many modifying genes located on the second chromosome and on the other chromosomes of the complement. Some of these modifiers, or a t least some chromosome regions, have greater effects than others, but we have not found any data which argue for a controlling switch mechanism (Activator) for the SD gene; nor have we found any evidence for a gene (Stabiliser) which controls mutability of the XD locus. The demonstration of sperm dysfunction in the S D system led us to reconsider the sc4scxsex chromosome meiotic drive system. This case had been interpreted in terms of a proposed regular production of nonwhen this hyfunctional sperm-the functional pole hypothesis-but pothesis was excluded as a possibility for the SD system, we searched for an alternative explanation. Examination of sc4scRmales has shown that a large amount of sperm dysfunction occurs; it is likely that this is associated with the unequal recoveries of reciprocal classes of gametes. On the basis of a striking correlation between the extent of meiotic drive and the frequency of nondisjunctional separations of the sex chromosomes, we have proposed that sperm dysfunction in this case has its origin in the failure of normal meiotic pairing of the sex chromosomes (the pairing-dysfunction hypothesis). Temperature shift experiments are consistent with this suggestion in that they have localized the effect to the primary spermatocyte. Other sex chromosome meiotic drive systems lend considerable support to the hypothesis, in that a number of separately derived chromosomal situations which interfere with pairing result in genetic data consistent with the predictions of the pairing-dysfunction model. Thus for the sex chromosomes a consistent picture has emerged implicating meiotic pairing as having a role in controlling postmeiotic developmental sequence. The model is open to further experimental tests, some of these being outlined in earlier discussion. The suggestion that pairing is responsible in the sex chromosomes for controlling steps of importance in spermiogenesis may well be extended to the autosomes. For example, the SD system is interpretable in terms of deficiencies, comparable to the arguments used for deficient X situations. There are no compelling data for this suggestion, but we had noted earlier that the sperm dysfunction seen in S D homozygotes may not be correlated directly with the SD effect and may be the result of homozygosity per se. It could be supposed that a homozygous defi-

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W. J. PEACOCK A N D GEORGE L. GABOR MIKLOS

ciency in this case led to a failure of normal sperm development, this failure not being due to SD action. The different SD alleles could be deficiencies of varying extents. We wish to emphasize that there is no direct evidence that SD action does have its basis on an interruption to normal pairing, and although we have shown an interaction between the SD and sc4sc8 systems (Miklos e t a!., 1972), this could occur even if the basic mechanisms were unrelated. Nevertheless, we feel that it may be fruitful to examine meiotic drive systems in the light of the possible importance of the disruption of pairing. We have already made the point that the T ( 1 ; 4 ) B Bsystem, although not involving X heterochromatin displacement or deletion, is an effective driving system and have suggested that the translocation of chromosome segments may in itself lead to difficulties in pairing and subsequent gamete dysfunction. Examination of other translocation heterozygotes may reveal evidence of sperm dysfunction.

ACKNOWLEDGMENTS We would like to thank Dr. C. W. Hinton and Dr. D. L. Lindsley for their critical reading of this manuscript. We are also grateful to Dr. K. Tokuyasu and Mr. R. Hardy for valuable discussions and help with the illustrations. Both of us are grateful to Dan Lindsley for providing a stimulating atmosphere at the University of California, San Diego; much of the original discussion took place in his laboratory.

REFERENCES Baker, B. S., and Carpenter, A. T. C. 1972. Genetic analysis of sex chromosomal meiotic mutants in Drosophila mehnogaster. Genetics 71, 255-286. Chandley, A. C., and Bateman, A. J. 1962. Timing of spermatogenesis in Drosophila melanogaster using tritiated thymidine. Nature (London) 193, 299-300. Cooper, K. W. 1959. Cytogenetic analysis of major heterochromatic elements (especially X h and Y ) in Drosophila melanogaster, and the theory of “heterochromatin”. Chromoaoma 10, 535-588. Cooper, K. W. 1964. Meiotic conjunctive elements not involving chiasmata. Proc. Nut. Acad. Sci. U.S. 52, 1248-1255. Crow, J. F., Thomas, C., and Sandler, L. 1962. Evidence that the segregationdistortion phenomenon in Drosophila involves chromosome breakage. Proc. Nut. Acad. Sci. U.S. 48, 1307-1314. Denell, R. E., and Judd, B. H. 1969. Segregation distorter in Drosophih melanogaster males: An effect of female genotype on recovery. Mol. Gen. Genet. 105, 262-274. Denell, R. E., and Miklos, G. L. G. 1971. The relationship between first and second chromosome segregation ratios from Drosophila melanogaster males bearing segregation distorter. Mol. Gen. Genet. 110, 167-177. Denell, R. E., Judd, B. H., and Richardson, R. H. 1969. Distorted sex ratios due to segregation distorter in Drosophila melanogaster. Genetics 61, 129-130. Erickson, J. 1965. Meiotic drive in Drosophila involving chromosome breakage. Genetics 51, 555-571.

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Gershenson, S. 1933. Studies on the genetically inert region of the X-chromosome of Drosophila. J . Genet. 28, 297-312. Gershenson, S. 1940. The nature of so-called genetically inert parts of chromosomes. Vid. Akad. Nauk URSR, Kiev 116 pp. (in Russ.). (Engl. transl. by Eugenia Krivshenko.) Hartl, D. L. 1969. Dysfunctional sperm production in Drosophila melanogaster males homozygous for the segregation distorter elements. Proc. Nut. Acad. Sci. U.S. 63, 782-789. Hartl, D. L., and Childress, D. 1972. Genetic studies of sperm formation and utilization in Drosophila melanogaster. Proc. Int. Symp. Genet. Spermatozoon, 269-288.

Hartl, D. L., Hiraizumi, Y . , and Crow, J. F. 1967. Evidence for sperm dysfunction as the mechanism of segregation distortion in Drosophila melanogaster. Proc. Nut. Acad. Sci. U.S. 58, 2240-2245. Hihara, Y. K. 1971. Genetic analysis of modifying system of segregation distortion in Drosophila melanogaster. Nippon Idengaku Zasshi 46, 75-82. Hiraizumi, Y., and Nakazima, K. 1967. Deviant sex ratio associated with segregation distortion in Drosophila melanogaster. Genetics 55, 681-697. Johnsen, R. C. 1968. An X * Y " Y L chromosome. Drosophila Inform. Serv. 43, 158. Johnsen, R. C. 1971. Cytogenetics of a univalent chromosome in Bs Drosophila melanogaster males. Can. J . Genet. Cytol. 13, 8149. Johnsen, R. C., and Zarrow, S. 1971. Sperm competition in the Drosophila female. Mol. Gen. Genet. 110, 36-39. Lewis, E. B. 1962. Balivary gland chromosome analysis of segregation distorter lines. Drosophila Inform. Serv. 36, 87. Lindsley, D. L., and Grell, E. H. 1968. Genetic variations of Drosophila melanogaster. Carnegie Inst. Wash., Publ. 627. Lindsley, D. L., and Grell, E. H. 1969. Spermiogenesis without chromosomes in Drosophila melanogaster. Genetics, Suppl. 61, 69-77. Lindsley, D. L., and Sandler, L. 1958. The meiotic behaviour of grossly deleted X chromosomes in Drosophila melanogaster. Genetics 43, 547463. Lindsley, D. L., Edington, C. W., and Von Halle, E. S. 1960. Sex-linked recessive lethals in Drosophila whose expression is suppressed by the Y chromosome. Genetics 45, 1649-1670. Lindsley, D. L., Sandler, L., Baker, B. S., Carpenter, A. T. C., Denell, R. E., Hall, J. C., Jacobs, P. A., Miklos, G. L. G., Davis, B. K., Gethmann, R. C., Hardy, R. W., Hessler, A., Miller, S. M., Nozawa, H., Parry, D. M., and Gould-Somero, M. 1972. Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 71, 157-184. Mange, E. J. 1968. Temperature sensitivity of segregation-distortion in Drosophila melanogaster. Genetics 58, 399413. Miklos, G. L. G. 1970. Segregation distortion in Drosophila melanogaster. Ph.D. Thesis, University of Sydney, Sydney. Miklos, G. L. G. 1971. S D distributions and the measurement of distortion. Drosophila Inform. Serv. 47, 67. Miklos, G. L. G. 1972. An investigation of the components of segregation-distorter systems in Drosophila melanogaster. Genetics 70, 405-418. Miklos, G. L. G., and Smith-White, S. 1971. An analysis of the instability of segregation-distorter in Drosophila melanogaster. Genetics 67, 305-317.

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Miklos, G. L. G., Yanders, A. F., and Peacock, W. J. 1972. Multiple meiotic drive systems in the Drosophila melanogaster male. Genetics 72, 105-115. Muller, H. J., and Settles, F. 1927. The non-functioning of the genes in spermatozoa. Z. Indukt. Abstamm.-Vererbungsl. 43, 285-312. Murnik, M. R. 1971. Environmental effects on segregation-distorter in Drosophila melanogaster: irradiation of SD-72 a t the onset of spermiogenesis. Genetica 42, 457-465.

Nicoletti, B. 1968. I1 controllo genetic0 della meiosi. Atti Ass. Genet. Ital. 13, 1-71. Nicoletti, B., Trippa, G., and De Marco, A. 1967. Reduced fertility in SD males and its bearing on segregation distortion in Drosophila melanogaster. Atti Accad. Naz. Lincei, C1. Sci. Fis., Mat. Natur., Rend. 43, 383-392. Novitski, E. 1970. The concept of gamete dysfunction. Drosophila Inform. Serv. 45, 87-88.

Novitiski, E., and Sandler, I. 1957. Are all products of spermatogenesis regularly functional? Proc. Nut. Acad. Sci. U.S. 43, 318-324. Novitski, E., Peacock, W. J., and Engel, J. 1965. Cytological basis of “sex ratio” in Drosophila pseudoobscura. Science 148, 516417. Olivieri, G., Avallone, G., and Pica, L. 1970. Sperm competition and sperm loss in Drosophila melanogaster females fertilized by Y’X. Y L / O males. Genetics 64, 323335.

Parker, D. R. 1954. Radiation induced exchanges in Drosophila females. Proc. Nat. Acad. Sci. U.S. 40, 795-800. Peacock, W. J. 1965. Nonrandom segregation of chromosomes in Drosophila males. Genetics 51, 573-583. Peacock, W. J., and Erickson, J. 1965. Segregation-distortion and regularly nonfunctional products of spermatogenesis in Drosophila melanogaster. Genetics 51, 313328.

Peacock, W. J., Tokuyasu, K. T., and Hardy, R. W. 1972a. Spermiogenesis in Segregation Distorter ( S D ) males of Drosophila melanogaster. In preparation. Peacock, W. J., Tokuyasu, K. T., and Hardy, R. W. 1972b. Spermiogenesis and meiotic drive in Drosophila. Proc. Int. Symp. Genet. Spermatozoon., 347-268. Peacock, W. J., Miklos, G. L. G., and Goodchild, D. J. 1973. Sex chromosome meiotic drive systems in Drosophila. I. Sperm dysfunction in males carrying a heterochromatin deficient X chromosome. To be published. Policansky, D., and Ellison, J. 1970. “Sex Ratio” in Drosophila pseudoobscura: Spermiogenic failure. Science 169, 888-889. Ramel, C. 1968. The effect of the Curly inversions on meiosis in Drosophila melanogaster. 11. Interchromosomal effects on males, carrying heterochromatin deficient X chromosome. Hereditas 60, 211-222. Roberts, P. A. 1968. Large size of recovered p r deficiencies. Genetics 60, 216. Sandler, L. 1962. A directed, permanent, genetic change involving the segregation-distortion system in Drosophila melanogaster. Amer. Natur. 96, 161-165. Sandler, L., and Braver, G. 1954. The meiotic loss of unpaired chromosomes in Drosophila melanogaster. Genetics 39, 365-377. Sandler, L., and Carpenter, A. T. C. 1972. A note on the chromosomal site of action of SD in Drosophila melanogaster. Proc. Int. Symp. Genet. Spermatozoon, 233-246.

Sandler, L., and Hiraizumi, Y. 1959. Meiotic drive in natural populations of Drosophila melanogaster. 11. Genetic variation a t the segregation-distorter locus. Proc. Nut. Acad. Sci. US.45, 1412-1422.

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Sandler, L., and Hiraizumi, Y. 1960a. Meiotic- drive in natural populations of Drosophila melanogaster. IV. Instability a t the segregation-distorter locus. Genetics 45, 1269-1284. Sandler, L., and Hiraizumi, Y. 1960b. Meiotic drive in natural populations of Drosophila melanogaster. V. On the nature of the SD region. Genetics 45, 1671-1689. Sandler, L., and Hiraizumi, Y. 1961a. Meiotic drive in natural populations of Drosophila melanogmter. VII. Conditional segregation-distortion : a possible nonallelic conversion. Genetics 46, 585-604. Sandler, L., and Hiraizumi, Y. 1961b. Meiotic drive in natural populations of Drosophila melanogaster. VIII. A heritable aging effect on the phenomenon of segregation-distortion. Can. J. Genet. Cytol. 3, 33-46. Sandler, L., and Novitski, E. 1957. Meiotic drive as an evolutionary force. Amer. Natur. 91, 105-110. Sandler, L., Hiraizumi, Y., and Sandler, I. 1959. Meiotic drive in natural populations of Drosophila melanogaster. I. The cytogenetic basis of segregation-distortion. Genetics 44, 232-250. Strangio, V. A. 1962. Radiosensitivity during spermatogenesis in Drosophila melanogaster. Amer. Natur. 96, 145-149. Tokunaga, C. 1971. The effects of temperature and aging of Drosophila males on the frequencies of X X Y and X O progeny. Mutat. Res. 13, 155-161. Tokuyasu, K. T., Peacock, W. J., and Hardy, R. W. 1972a. Dynamics of spermiogenesis in Drosophila melanogaster. I. Individualization process. Z. Zelljorsch. Mikrosk. Anat. 124, 479-506. Tokuyasu, K. T., Peacock, W. J., and Hardy, R. W. 1972b. Dynamics of spermiogenesis in Drosophila melanogaster. 11. Coiling process. Z . Zelljorsch. Mikrosk. Anat. 127, 492-525. Yanders, A. F., Brewen, J. G., Peacock, W. J., and Goodchild, D. J. 1968. Meiotic drive and visible polarity in Drosophila spermatocytes. Genetics 59, 245-253. Zimmering, S. 1963. The effect of temperature on meiotic loss of the Y chromosome in the male Drosophila. Genetics 48, 133-138. Zimmering, S., and Fowler, G. L. 1968. Progeny: sperm ratios and nonfunctional sperm in Drosophila melanogaster. Genet. Res. 12, 359-363. Zimmering, S., and Green, R. E. 1965. Temperature-dependent transmission rate of a univalent X chromosome in the male Drosophila melanogaster. Can. J . Genet. Cytol7, 453-456. Zimmering, S., and Perlman, M. 1962. Modification of abnormal gametic ratios in Drosophila. 111. Probable time of the A-type effect in BarStone translocation males. Can. J . Genet. Cytol. 4, 333-336. Zimmering, S., and Wu, C . K. 1964. X - Y nondisjunction and exchange induced by X-rays in primary spermatocytes of the adult Drosophila. Genetics 49, 499-504. Zimmering, S., Sandler, L., and Nicoletti, B. 1970a. Mechanisms of meiotic drive. Annu. Rev. Genet. 4, 409436. Zimmering, S., Barnabo, J. M., Femino, J., and Fowler, G. L. 1970b. Progeny: sperm ratios and segregation-distorter in Drosophila melanogaster. Genetica 41, 61-64.

SYMMETRY IN PROTEIN-NUCLElC ACID INTERACTION AND ITS GENETIC IMPLICATIONS Henry M. Sobell Deportment of Chemistry, The University of Rochester, Rochester, New York; Department of Radiation Biology and Biophysics, The University of Rochester School of Medicine and Dentistry, Rochester, New York

I. Introduction . . . . . . . . . . . 11. Symmetry in Protein-Nucleic Acid Interaction . A. The Principle for Dimer Recognition. . . B. The Principle for Tetramer Recognition . . 111. Regulation Mechanism for RNA Transcription . IV. Mechanism for Genetic Recombination . . . A. Eukaryotic Systems. . . . . . . . B. Prokaryotic Systems . . . . . . . C. Viruses . . . . . . . . . . . V. Mechanism for DNA Replication . . . . . A. I n Vitro Replication Mechanism . . . . B. I n Viuo Replication Mechanism . . . . VI. DNA Restriction and Modification Mechanisms VII. Chromosome Structure . . . . . . . . VIII. Summary. . . . . . . . . . . . References . . . . . . . . . . .

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. 411 .412 . 412 . 418 . 420 , 424 . 425 . 441 . 451 . 464 . 464 . 466 . 470 ,474 . 476 . 476

1. Introduction

Symmetry principles are known to play a fundamental role in biological organization, governing the assembly of macromolecular subunits into viruses, membranes, oligomeric globular and fibrous proteins, and cellular organelles (Crick and Watson, 1956; Caspar and Klug, 1962; Monod et al., 1965; for an excellent review, see Engstrom and Strandberg, 1968). Thus, for example, small spherical viruses utilize icosahedral symmetry in their construction, identical protein subunits being used to form large protein shells in which each subunit has, as nearly as possible, 411

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HENRY M. SOBELL

the same local environment (Caspar and Klug, 1962; Finch and Klug, 1966; Klug and Finch, 1968; Finch et al., 1970; for a review, see Klug et al., 1966), Multisubunit enzymes utilize symmetry in their construction, endowing them with allosteric properties important in their regulation (Monod et al., 1963; Monod et al., 1965; for an excellent overview, see Structure and Function of Proteins at the Three Dimensional Level, Cold Spring Harbor Symp. Quant. Biol., 1971). The cooperative properties of biological membranes have also been ascribed to an underlying structural symmetry, which, in addition, may promote their self-assembly (Changeux et al., 1967; Changeux, 1968). The possible role which symmetry plays in protein-nucleic acid interaction is discussed in this chapter. These considerations suggest the existence of an alternate branched configuration for DNA induced by binding specific structural proteins to symmetrically arranged polynucleotide base sequences. The concept that such sequences exist at ends of genes or operons leads to a molecular theory interrelating genetic recombination, DNA replication, and RNA transcription. Several preliminary communications describing elements of this theory have already appeared (Sobell, 1972,1973).

11. Symmetry in Protein-Nucleic Acid Interaction

A. THEPRINCIPLE FOR DIMERRECOGNITION 1 . Stereochemistry of Actinomycin-DNA Binding

The ideas presented here stem from the stereochemical model which has been advanced for actinomycin-DNA binding (Sobell et al., 1971a,b; Jain and Sobell, 1972; Sobell and Jain, 1972; for a review, see Sobell, 1973) . Actinomycin is a cyclic polypeptide containing antibiotic which binds to deoxyguanosine residues in double-helical DNA and specifically inhibits RNA synthesis (for a review, see Reich and Goldberg, 1964) (see Fig. 1 ) . We have successfully cocrystallized actinomycin with its DNA substrate, deoxyguanosine, and have solved the three-dimensional structure of the complex by X-ray crystallography. The configuration observed in the crystalline complex explains in a natural way the stereochemistry of actinomycin binding to DNA (refer to Figs. 2-5). The phenoxazone ring system on actinomycin intercalates between the basepaired dinucleotide sequence, dG-dC, while the peptide subunits (related by 2-fold symmetry) lie in the narrow groove of the DNA helix and

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

413

L mew1 so1

L pro D vol CH,-

CH3

&jyJ

C-;H HN,

HC’HC-, ‘NH CH3

CH3

0

1 thr

2

CH3

FIG. 1. Structure of actinomycin C, (D). Abbreviations: meval, methyl valine; sar, sarcosine; pro, proline ; val, valine; thr, threonine.

FIG.2. The actinomycin-deoxyguanosine complex viewed down the approximate 2-fold axis of symmetry. Cyclic pentapeptide chains lie behind deoxyguanosine molecules and are drawn with lighter lines. One deoxyguanosine molecule is stacked above, the other below, the actinomycin phenoxazone chromophore (these are drawn with heavier lines). Dotted lines indicate hydrogen bonds between deoxyguanosine residues and pentapeptide chains.

414

HENRY M. SOBELL

FIQ.3. The actinomycin-DNA complex, viewed as in Fig. 2. The phenoxazone ring system on actinomycin intercalates between the base-paired dinucleotide sequence, d G d C , while the peptide subunits (related by %fold symmetry) lie in the narrow groove of the DNA helix and interact with deoxyguanosine residues on opposite chains through specific hydrogen bonds.

interact with deoxyguanosine residues on opposite chains through specific hydrogen bonds. The binding of actinomycin to DNA demonstrates a general principle governing protein-nucleic acid interaction (i.e., the principle for dimer

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

415

FIQ.4. Corey-Pauling-Koltun (CPK) space-filling model representation of the actinomycin-DNA complex, viewed as in Fig. 3. For clarity, the DNA molecule has been shaded to distinguish it from the actinomycin molecule.

recognition). This is shown schematically in Fig. 6A. If a protein molecule has identical subunits related b y 2-fold symmetry when it binds to DNA-the 2-fold axis coinciding with the dyad axis on DNA-then a necessary consequence is that the base sequence in the recognition site have 2-fold symmetry.

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HENRY M. SOBELL

Fro. 5. Corey-Pauling-Koltun (CPK) space-filling model representation of the actinomycin-DNA complex, viewed from a direction opposite to that shown in Fig. 4. The light area delineates the actinomycin pentapeptide chains, which make numerous van der Waals contacts with methylene protons on sugar residues on both chains in the narrow groove of the DNA helix.

I.>

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

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NUCLEASE SPECIFICITY

ACTINOMYCIN SPECIFICITY

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TETRAMER RECOGNITION GIERER (1966)

FIG.6. A schematic diagram illustrating the general principle governing protein-nucleic acid interaction, as exemplified by actinomycin-DNA binding specificity (A) and endonuclease specificity (B). If a protein molecule has identical subunits related by 2-fold symmetry when it binds to DNA-the 2-fold axis coinciding with the dyad axis on DNA-then a necessary consequence is that the base sequence in the recognition site has 2-fold symmetry. (C) Extension of this general principle for dimer recognition to include tetramer recognition. One postulates a tandem genetic duplication of the DNA sequence involved in dimer recognition, followed by a hydrogen-bonding rearrangement. This nucleic acid structure can then be recognized by a tetrameric protein having identical subunits related by 4-fold symmetry. Patterns of recognition such as these may exist between operators and repressors. See text for discussion.

2. Nuclease Specificity

Although the binding of actinomycin to DNA represents the only direct structural information we currently have concerning symmetry in protein-nucleic acid interaction, chemical evidence indicates that several nuclease enzymes cleave between symmetrically arranged nucleotide sequences, forming double-strand scissions in DNA (Bernardi, 1968 ;

418

HENRY M. SOBELL

Kelly and Smith, 1970) (see Fig. GB). The first of these, a splenic acid deoxyribonuclease, is a dimer containing identical subunits. The enzyme is strongly inhibited by actinomycin, and was therefore thought to attack guanine rich sequences. Bernardi postulated that if the subunits of this enzyme were related by 2-fold symmetry, this would allow the enzyme to recognize the dyad axis on DNA, and, if each subunit had an active site, permit simultaneous double-strand scissions of the sugar-phosphate backbone. A necessary consequence of this type of recognition would involve symmetrically arranged sequences, such as dC-dG or dG-dC, the latter being the sequence which actinomycin binds preferentially. The second nuclease enzyme is a restriction enzyme isolated from Hemophilus influenzae; it recognizes the symmetrically arranged hexanucleotide sequence shown in Fig. 6B and introduces a double-strand scission at its central singularity. Although the subunit structure of the enzyme has not yet been established, Kelly and Smith postulate a similar protein-nucleic acid recognition pattern for this restriction enzyme. It is of interest in this connection that Meselson and Yuan have demonstrated that two classes of heteroduplex h(0:K) DNA produced by annealing isolated strands, one modified, the other not modified, are resistant to Escherichia coli endonuclease attack (Meselson and Yuan, 1968). Thus, modification (presumably by methylation) of one strand imparts immunity from the restriction enzyme to the other strand, suggesting some type of 2-fold symmetry in the recognition site. For further discussion of symmetry in restriction and modification, see Section VI. B. THEPRINCIPLE FOR TIWRAMER RECOGNI~ON 1 . Identical Subunits

The binding of actinomycin to DNA (in particular, to dG-dC sequences) and its specificity in inhibiting the RNA polymerase reaction suggest a primitive repressor-operator character for this complex, which may prove to have more general meaning with regard the recognition of naturally occurring operators by repressors. While no structural evidence is yet available concerning the arrangement of subunits in the lac or the h repressors, the lac repressor is known to bind DNA as a tetramer, whereas the A repressor initially binds DNA in dimeric form, and then subsequently, in oligomeric form (Gilbert and Muller-Hill, 1967; Riggs et al., 1970; Pirrotta et al., 1970; Chadwick et al., 1970). The general principle for dimer recognition can be extended to include tetramer recognition in the following way (see Fig. 6C). One postulates

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

419

a tandem genetic duplication of the D N A sequence involved in dimer recognition, followed by a hydrogen-bonding rearrangement of the D N A (Gierer, 1966). This then generates a cloverleaflike structure that can be recognized by a tetrameric protein having identical subunits arranged in one of three ways. If the subunits of the protein are related by 4-fold symmetry, then this means that the operator must be a structure which itself possesses 4-fold symmetry, as indicated in Fig. 6C. Alternatively, if the subunits of the protein are related by only 2-fold symmetry (in particular, by one 2-fold axis of symmetry), then the operator structure must also have this symmetry. Such a structure can be obtained from the cloverleaf structure by bending the horizontal leaves forward and

A

FIG. 7. Alternate symmetries possible for protein-nucleic acid interaction. (A) Two-fold symmetry relating protein subunits with cloverleaf nucleic acid structure. This gives rise to two distinct chemical environments in protein-nucleic acid recognition and seems less likely when the tetrameric protein has identical subunits. This symmetry, however, can be used by a protein possessing two nonidentical pairs of subunits, i.e., two a and two p subunits. An example may be the interaction between the Escheiichia coli RNA polymerase and certain classes of promotors. See text for additional discussion. (B) 222 symmetry relating protein subunits with nucleic acid cloverleaf structure. Exact 222 symmetry is not possible without interrupting the continuity of the polynucleotide chain; however, pseudo-222 symmetry remains an alternative possibility to 4-fold symmetry.

the vertical leaves backward, in a roughly tetrahedral arrangement (see Fig. 7A). This gives rise to two distinct chemical environments involved in protein-nucleic acid recognition, however, and seems less likely when the tetrameric protein has identical subunits (see, however, the next section, which describes the situation for a tetrameric protein having two different subunits). Higher symmetry, i.e., 222 symmetry, is not possible for the operator structure shown ; however, pseudo-222 symmetry remains an alternative possibility to 4-fold symmetry (see Fig. 7B).

420

HENRY M. SOBELL

The precise symmetry relating subunits of the lac and the A repressors will eventually be revealed by X-ray crystallography ; however, strong genetic evidence already points to the existence of 2-fold symmetry in the lac operator genetic map, with higher order subdivision possible (Smith and Sadler, 1971; Sadler and Smith, 1971). These findings, along with the observation that lac repressor binds d (A-T) polymer selectively with high affinity (Lin and Riggs, 1970), directly support the Gierer-like operator structure shown in Fig. 6C. Definitive evidence, of course, must await the nucleotide sequence data of the lac and A operators, and these studies are in progress (Gilbert and Ptashne, personal communication) . 2. NonidenticaE Subunits

If a tetrameric protein contains two different subunits (say, two a! and two p subunits), then the most likely symmetry for the DNA complex is 2-fold symmetry, the protein subunits lying in a roughly tetrahedral arrangement. This would be similar to that shown in Fig. 7A, except that the base sequences in the horizontal and vertical leaves need not be the same. The interaction between the RNA polymerase and certain promotors may utilize this type of interaction, and this will now be discussed.

Ill. Regulation Mechanism for RNA Transcription

This section describes a structural model for positive and negative control a t the level of RNA transcription for those operons of the Monod-Jacob type (Jacob and Monod, 1961). The model may also be applicable to those genes, or clusters of genes, possessing promotor elements capable of binding the RNA polymerase and initiating RNA transcription in the absence of additional structural proteins, such as the cyclic AMP receptor protein or the arabinose C gene product. The theory does not address itself to those operons whose control may be more complex, involving, perhaps, translational control mechanisms. An example of one such operon is the histidine operon (Silbert et al., 1966; Roth et al., 1966; for a review of mechanisms known to regulate amino acid metabolism, see Umbarger, 1969). Basic ideas are summarized in Fig. 8. Negative control is envisioned to involve a cloverleaflike operator structure possessing only small loops of denatured DNA (Fig. 8A). The repressor may act to block movement of the RNA polymerase past the operator site, it having initiated binding and messenger RNA synthesis a t a promotor site “downstream”

42 1

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

(Reznikoff e t al., 1969). Positive control, on the other hand, involves the formation of larger loops of denatured DNA, these acting as sites of attachment for the RNA polymerase and initiation of messenger RNA synthesis (Fig. 8B). Both mechanisms postulate the existence of symmetrically arranged polynucleotide base sequences a t the ends of genes A 5'

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FIG. 8. A model for positive and negative transcriptional control. (A) Operator-repressor negative control is envisioned to involve a cloverleaflike operator structure possessing only small loops of denatured DNA. The repressor may act to block movement of the RNA polymerase past the operator site, it having initiated binding and messenger RNA synthesis a t a promotor site "downstream." (B) Promotor-activator positive control is envisioned to involve the formation of larger loops of denatured DNA, these acting as sites of attachment for the RNA polymerase and initiation of messenger RNA synthesis.

or operons capable of existing either as Watson-Crick structures, or, in the presence of specific structural proteins, Gierer-like cloverleaf structures. The model makes several predictions: 1. Negative control elements (operator regions) should demonstrate 2-fold (or mirror) symmetry in their genetic maps. For example, operator constitutive point mutants in the lac operon possessing similar levels

422

HENRY M. SOBELL

of constitutive p-galactosidase activity should occur pairwise and map in a symmetric fashion. Mutations that occur in the middle of the operator region (therefore giving rise to noncomplementary base pairs in both Gierer loops) would be expected to show higher levels of constitutive synthesis than those occurring a t either end of the operator region. Mutants of the latter type, in fact, may be only marginally constitutive since one would not expect an altered complementary base pair (particularly, of a transition type) to perturb the protein-nucleic acid interaction to any great extent. Evidence pointing to the existence of 2-fold symmetry in the lac operator genetic map has recently appeared (Smith and Sadler, 1971; Sadler and Smith, 1971). A similar prediction can be made concerning the fine structure genetic map for positive control elements (promotor regions), with the exception that asymmetry would be expected to occur in its central region. 2. Positive control elements should have the capability of demonstrating bidirectional control of messenger RNA synthesis in certain operons, since, potentially, there are two sites of attachment for the RNA polymerase (an example of one such operon may be the ma1 B region of the maltose operon ; Hofnung and Schwartz, personal communication ; Schwartz, 1967). Normally, however, one would expect to have unidirectional control, mediated, perhaps, through specific protein factors, such as the sigma factor. These would dictate the site of attachment for the RNA polymerase and initiate strand selection in transcription (Burgess et al., 1969; Travers and Burgess, 1969; Travers, 1969, 1970; Rautz and Bautz, 1970). 3. A given structural protein may behave either as a repressor or an activator, depending on the specific nucleotide sequence i t interacts with (as shown in Fig. 8 ) . Evidence suggesting that the arabinose C gene product can demonstrate both repressor and activator properties a t different controlling sites in the arabinose operon has been presented by Englesberg et al. (1969). A classic example of an operon demonstrating both positive and negative control is the lac operon (see Beckwith, 1967; Perlman et al., 1970; Arditti et al., 1970; decrombrugghe et al., 1971a,b,c; for an overview, see Beckwith and Zipser, 1970). In addition to the i-o repressor-operator system important for negative control, there exists a positive control system which involves the promotor element, cyclic adenosine 3’,5’monophosphate (cyclic AMP), and a cyclic AMP receptor protein (also called a catabolite gene activator protein). The latter control system underlies the phenomenon of catabolite repression, a phenomenon whereby elevated concentrations of glucose (or catabolite end products from glucose metabolism) repress the maximal expression of cer-

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

423

tain operons, even when fully induced. It is now known that this glucose effect is due to the lowering of intracellular levels of cyclic AMP, and that cyclic AMP plays a central role in the activation of catabolitesensitive operons a t the level of transcription (Makman and Sutherland, 1965; Perlman and Pastan, 1969; Ullman and Monod, 1968; Zubay et al., 1970; Perlman et al., 1969; decrombrugghe et al., 1969). I n the presence of increased concentrations of cyclic AMP, lac mRNA synthesis is stimulated in vivo and in vitro. This effect requires the presence of a cyclic AMP receptor protein and a functional promotor region (Perlman et al., 1970; Arditti et al., 1970; Silverstone et al., 1969). Recent experiments suggest that the cyclic AMP receptor protein-cyclic AMP complex acts on lac DNA (presumably a t the promotor site) to stimulate an early step in the initiation of lac transcription by the RNA polymerase holoenzyme (decrombrugghe et al., 1971a,b,c). A possible structural mechanism underlying cyclic AMP activation is shown in Fig. 8B. A similar control mechanism may regulate ribosomal RNA synthesis (Travers et al., 1970a,b; see, however, Haseltine, 1972; Pettijohn, 1972). Normally, an abrupt cessation of RNA synthesis occurs when E . coli cells are deprived of required amino acids. This stringent response to amino acid starvation is relaxed in certain mutants (RC- or rel-) which continue to accumulate RNA in the absence of protein synthesis (Stent and Brenner, 1961). The nucleotide, guanosine 5’-diphosphate, 3’(or 2’) diphosphate (ppGpp) has been demonstrated to accumulate during the stringent response to amino acid starvation (Cashel and Kalbacher, 1970), and its presence appears to interfere with RNA synthesis (Cashel, 1970; Travers et al., 1970a). Possible explanations are that ppGpp interferes with guanosine triphosphate (GTP) biosynthesis and (or) selectively inhibits the initiation or elongation of RNA chains. An alternative explanation is that ppGpp inactivates the psi factor, a protein factor, which, in addition to the RNA polymerase holoenzyme, is necessary for ribosomal RNA synthesis in vitro (Travers et al., 1970a,b). Although the latter explanation is an attractive one (the psi factor may activate promotor regions to initiate ribosomal RNA synthesis, analogous to the cyclic AMP receptor protein), conflicting evidence has appeared (Haseltine, 1972; Pettijohn, 1972), and further work is necessary to resolve this question. Burgess (1971) has recently reviewed the chemistry and structure of the E . coli RNA polymerase enzyme. The enzyme consists of two identical cr subunits, a p and p’ subunit (approximate molecular weights are 40,000, 150,000, and 155,000) and two additional factors, the u and o factors (molecular weights 90,000 and 10,000, respectively). The a&?’

424

HENRY M. SOBELL

aggregate is called the core enzyme and is capable of binding DNA and ’ U U J is called the holoenzyme catalyzing RNA synthesis. The C Y ~ ~ ~ complex and demonstrates enhanced ability to transcribe phage DNA with correct strand selection. The u factor appears to stimulate binding and initiation a t specific sites on DNA, these presumably being the promotor regions. The function of UJ is unknown. Although there is no information as yet concerning the spatial arrangement of subunits within the RNA polymerase, one can imagine two distinct mechanisms for its attachment to DNA. The first of these has been discussed previously and involves activation of the promotor region through the binding of a specific structural protein important in positive control (i.e., the cyclic AMP receptor protein, the arabinose C gene product, etc.) . This denatures the DNA and exposes single-stranded loops, which can then serve as specific binding sites (mediated through U ) for the RNA polymerase. The second mechanism may involve a different class of promotors capable of direct activation through interaction with the RNA polymerase alone. For this to happen, the subunits of the enzyme must be related by approximate 2-fold symmetry (relating the two a subunits and the p and p’ subunits). The enzyme could then interact with a cloverleaflike promotor structure possessing approximate 2-fold symmetry, as discussed in Section 11, B, 2. RNA polymerase molecules with less complexity, such as the T7-induced RNA polymerase, may utilize additional factors or exist in multimeric form when activating promotors that control the expression of late genes during phage infection (Chamberlin et al., 1970; Summers and Siegel, 1970; Gelfand and Hayashi, 1970; for a review, see Studier, 1972).

IV. Mechanism for Genetic Recombination

The concept that regions of DNA possessing symmetrically arranged polynucleotide base sequences can exist either as Watson-Crick structures or cloverleaf Gierer-like structures (the latter being induced by a specific structural protein or proteins), leads to a simple but powerful molecular model t o explain genetic recombination. The model involves the following features: 1. Chromosomes pair, owing to the formation of Gierer-like structures, which are induced by a specific recombination structural protein (8). Synapsis occurs, therefore, not randomly along the genome, but at specific pairing regions (identified as promotor regions). 2. A Holliday (1964) heteroduplex structure is formed which can mi-

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

425

grate into the structural gene area in either direction along parental DNA molecules. 3. Reciprocal recombination results from breakage and reunion of parental DNA molecules, this reflecting the action of an endonuclease possessing 2-fold symmetry. Breakage and reunion involves either singleor double-strand exchange. 4. Nonreciprocal recombination results from exonucleolytic activity by this same enzyme, which eliminates one of two hybrid DNA regions. Subsequent events (i.e., excision and repair) give rise to a phenomenon termed, in eukaryotes, gene conversion, and in prokaryotes and viruses, asymmetric (or nonreciprocal) genetic exchange. The theory will first be presented to explain eukaryotic recombination. It will then be expanded to include general and site specific recombination mechanisms in viruses and bacteria. A. EUKARYOTIC SYSTEMS Eukaryote genetics, particularly the area of fungal genetics, has provided a wealth of data on genetic recombination which places rigid constraints on possible underlying molecular mechanisms. For outstanding recent reviews in this area, see Emerson (1969) and Fincham (1970). For general reference, see Fincham and Day (1971). 1 . Chromosome Synapsis and the Holliday Structure

Although the mechanism of pairing homologous chromosomes during meiosis is poorly understood and little is known about the physicochemical basis underlying chromosomal recognition (for a review in this area, see Grell, 1969), it is generally agreed that chromosomal pairing must ultimately reflect pairing a t the nucleotide level to account for precise interchange of genetic information. Electron microscopy has revealed the presence of a complex structure, the synaptinemal complex which is believed to be associated with some aspect of meiosis, perhaps chromosomal synapsis and (or) crossing-over (Fawcett, 1956; Moses and Coleman, 1964; Westergaard and von Wettstein, 1966). This section describes a mechanism for pairing homologous chromatids a t the nucleotide level which results in intimate synapsis. It docs not attempt to deal with the multitude of presynaptic events that precede, or the complex structures (i.e.J the synaptinemal complex) that accompany, genetic recombination during meiosis. Details of the model are as follows (see Figs. 9 and 10). Symmetrically arranged polynucleotide base sequences on homologous chromatids (promotor regions, spaced every cistron or operon length or so along

426

HENRY M. SOBELL

FIG.9. A model for genetic recombination. Homologous chromatids, A-C and B-D, possess specific regions (promotor regions, placed every cistron or operon length along the chromosome) capable of forming Gierer-like structures in the presence of a specific recombination structural protein. Regions such as E-G and H-F form single-stranded denatured loops outside the immediate environment of the protein (which senses only the symmetry related nucleic acid structure shown) and are therefore susceptible to nuclease attack (shown by the arrows). When complementary loops, G, H are nicked and opened, Watson-Crick base pairing occurs, this followed by extensive propagation of the heteroduplex (shown in the lower two figures). The final structural intermediate is shown in the center of Fig. 10.

the chromosome) are first converted to their Gierer structures in the presence of a specific structural protein, which, for simplicity, we shall call a recombination protein. A protein such as this may recognize spe-

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

427

cific structural features common to many different promotor regions, and, in this way differ from more specific activator proteins utilized for positive tzanscriptional control. Typically, a symmetrically arranged sequence contains central sequences [E,G] and [H,F] which do not possess -symmetry. Therefore, the Gierer structure contains loops of sinA

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FIO.10. A model for genetic recombination (continued). The central structural intermediate can give rise to the Broker-Lehman (Broker and Lehman, 1971) hybrid DNA structure in the absence of polynucleotide ligase, or to the Holliday (1964) hybrid DNA structure in the presence of polynucleotide ligase. The Holliday structure possesses 2-fold symmetry. It can therefore be recognized by a nuclease possessing 2-fold symmetry, which would be able to simultaneously nick strands of the same polarity a t homologous sites (independent of base sequence). This would give rise to reciprocal recombination involving either single- or double-strand exchange, depending on which strands are cut and joined. Another feature of this heteroduplex structure which results from its 2-fold symmetry is its ability to migrate with a zipperlike action along parental DNA molecules without unwinding difficulties. This would allow recombination to occur randomly throughout the genome and could give rise to polarity effects observed in gene conversion. gle-stranded DNA outside the immediate environment of the protein (which senses only the symmetry related nucleic acid structure shown) ; these are susceptible (either randomly or with specificity) to nuclease attack. When complementary loops are nicked (i.e., [G,H] ), homologous Gierer structures can then come together through base pairing, this followed by extensive propagation of the hybrid DNA through branch mi-

428

HENRY M. SOBELL

gration (the details of which are shown in the lower diagrams of Fig. 9) (Lee et al., 1970; Kim et al., 1972). One then arrives a t the central intermediate shown in Fig. 10. Since this process is basically a polynucleotide renaturation reaction, one would expect it to occur spontaneously, and no additional structure or energy source need be postulated. I n the absence of polynucleotide ligase, this intermediate can become the Broker-Lehman structure, a structure observed by electron microscopy after abortive T4 polymerase- ligase- phage infection (Broker and Lehman, 1971). In the presence of ligase, one can form the Holliday hybrid DNA structure (Holliday, 1964). This results from base pairing of sticky ends [H,G], followed by sealing of nicks in the polynucleotide chain by ligase. The Holliday structure is a particularly interesting structure in that it has 2-fold symmetry. It can therefore be recognized by an endonuclease possessing 2-fold symmetry, which would simultaneously act to nick strands of the same polarity a t homologous sites (independent of base sequence). This would give rise to breakage and reunion events involving either single- or double-strand exchange, depending on which strands are cut and joined (see next section). Another feature of the heteroduplex structure (which results from its 2-fold symmetry) is its ability to migrate (by rotary diffusion) with a zipperlike action in either direction along parental DNA molecules without unwinding difficulties (see Fig. 11). This would allow genetic recombination to occur throughout the genome and, in addition, may explain polarity effects observed in gene conversion (see Section IV, A, 3). 3. Single-Strand and Double-Strand Exchange An important feature of the Holliday heteroduplex is its ability to give rise to two distinct types of breakage and reunion events, depending on which strands are cut and joined (Holliday, 1964). This is shown schematically in Fig. 12. The first type, denoted double-strand exchange, results in the reciprocal exchange of flanking markers. The second type, denoted single strand exchange, does not. Both types of events are associated with the formation of hybrid DNA regions which, if heterozygous, may result in either postmeiotic segregation or gene conversion (see Section IV, A, 3 ) . Recently, Sigal and Alberts (1972) have investigated the detailed stereochemistry of the half-chiasma portion of the Holliday structure using molecular models. These studies have shown that the transfer of strands from one helix to another can be achieved within one length of sugar-phosphate backbone. Thus, strand exchange can occur with all bases remaining paired without giving rise to bond strain or unfavor-

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

429

FIG. 11. A schematic diagram illustrating how the Holliday synaptic structure can migrate into the structural genome without unwinding difficulties. Striped arrows denote direction of propagation. Smaller arrows with ellipses convey sense of rotation experienced by parental DNA duplexes. Long central bidirectional arrow represents axis of 2-fold symmetry.

able contacts. This cross connection can readily diffuse along the joined helices (resulting in continued strand exchange) by a zipperlike action in which the two identical bases above or below the cross connection exchange places. On a gross scale, this process results in rotation of the two helices in the same sense about their helical axes, this being driven by rotary diffusion (Meselson, 1972). An important additional observation concerns the phenomenon of crossed strand interchange. If each of the rods supporting the two space-filling helices is cut at the

430

HENRY M. SOBELL

level of the cross connection (these being artifacts anyway), the top sections of both double helices can be swiveled around each other without bond breakage to yield an identical crossed strand exchange in which the “outside strands” in the original model become “bridging strands,” and the original “bridging strands” become “outside strands.” Since both forms of strand exchange must be in rapid equilibrium at physiological

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FIQ.12 A schematic diagram illustrating how the Holliday synaptic structure can give rise to two distinct types of breakage and reunion events, depending on which strands are cut and joined. The first type (shown on the left), denoted double-strand exchange, gives rise to the reciprocal exchange of flanking markers. The second type (shown on the right), denoted single-strand exchange, does not. Both types of events give rise to hybrid DNA regions, which, if heterozygous, can give rise to either postmeiotic segregation or gene conversion. temperatures, only one nuclease need be postulated to effect either singleor double-strand breakage and reunion events. 3. Polarity in Gene Conversion Implicit in this model for genetic recombination is the idea that synapsis between homologous chromatids occurs between genes, not within genes (Whitehouse, 1966). This results in the formation of hybrid DNA, which can then migrate into the structural gene area. It will now be shown that such a migratory heteroduplex structure can give rise to hybrid DNA of different classes in recombinant molecules, this leading

431

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

to a n explanation of polarity effects in single-site conversion and coconversion as documented by extensive studies of unselected tetrads in yeast by Hurst, Fogel, and Mortimer (1972). Refer to Fig. 13A and B: We envision a fixed-length migratory hybrid +

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Fh. 13. An explanation for polarity in gene conversion. (A) Schematic figure showing the Holliday hybrid DNA structure migrating from right to left encountering mutant allele 17 in the arg, locus, this triggering endonuclease attack. In addition, the nuclease possesses exonucleolytic activity ( 0 )and jumps onto either of the two nicked strands. Subsequent migration to the left causes the right half of the migratory duplex to fall apart, this fixing the starting point a t which hybrid DNA begins in the recombinant molecules. Further migration, followed by nicking of homologous strands, results in reciprocal exchange, either of the single- or double-strand type. The latter is associated with exchange of flanking markers and is shown in this figure. ---, DNA polymerase repair. Diploid cross is (1 2 +I/(+ 16 17). (B) Polarity in coconversion is interpreted as arising from heteroduplex regions of variable size in one recombinant molecule with subsequent excision and repair of the complete heteroallelic area. Coconversion events of the type, 17-16-2-1are more frequent than 17-16-2,which, in turn, are more frequent than 17-16. This may reflect the diminishing probability of forming hybrid DNA regions with progressively smaller size. Coconversion events of the type, 16-2-1, 16-2, and 2-1 are infrequent or absent in the diploid cross (1 2 +)/(+ 16 17),and single site conversions a t 17 are far more prevalent than at 16, 2, or 1. These observations are readily interpretable in terms of the model presented. Although this model is specifically addressed to explaining gene conversion in eukaryotes, a similar model can also be used to explain prokaryotic recombination (in particular, transformation) and nonreciprocal genetic recombination among viruses. See text for discussion.

+

+

+

+

432

HENRY M. SOBELL

DNA structure (containing, perhaps, fifty to one hundred nucleotide pairs) moving (by rotary diffusion) from right to left and encountering the first mutant allele 17 in the arg, locus. This triggers endonuclease attack, nicking homologous strands (Fincham and Holliday, 1970) [the possibility that nicking need not be triggered by a mismatched base pair, but can, instead, occur randomly to the right of 17 with high probability is also possible ; however, this would not as readily explain asymmetry in gene conversion (discussed below), and marker effects that influence integration efficiencies in transformation (see Section IV, B, l ) ] . In addition, we postulate this nuclease to have exonucleolytic activity (analogous to the nuclease coded by the rec B and C cistrons in E . coli (Goldmark and Linn, 1972)), degrading either of the two nicked strands. Subsequent migration of the hybrid DNA structure to the left [driven, perhaps, by the ATP-dependent exonuclease (Cassuto and Radding, 1971) J causes the right half of the migratory duplex to fall apart, this fixing the starting point at which hybrid DNA begins in the final recombinant molecules. Further migration, followed by nicking of homologous strands, results in reciprocal exchange, either of the singleor double-strand type. Owing to exonucleolytic action, only one of two recombinant molecules contains hybrid DNA. DNA polymerase activity repairs the other duplex either before or immediately after the final recombination event, this giving rise to homoallelic and heteroallelic duplexes. Polarity in coconversion is readily interpreted as arising from heteroduplex regions of variable size (containing, for example, alleles 17-16-2-1, 17-16-2, 17-16, or 17) in one recombinant molecule with sub. sequent excision and repair of the complete heteroallelic area (as shown in Fig. 14). The model readily explains why coconversion events of the type, 16-2-1, 16-2, and 2-1 are infrequent or absent in the diploid cross (1 2 +)/(+ 16 17) and why single-site conversions a t 17 are far more prevalent than a t 16, 2, or 1. Implicit in the model is the prediction that the exchange of flanking markers should accompany gene conversion events approximately 50% of the time, and this is in agreement with the yeast gene conversion data (see Table 1 ) . Although the yeast data indicate that conversion events of the type 1+:3m and 3+: l m occur with equal frequency, this does not seem to be the case with Ascobolus immersus (Rossignol, 1969). When point mutants in gene 75 were classified by the ratio of frequencies of conversion from wild type to mutant and mutant to wild type (i.e., 1+:3m/3+:lm), they were found to fall into three classes (as defined by coefficients of asymmetry), a, p, and y , with ratios 14:1, 1:1, and 0.73 :1, respectively. Within each class, polarity was evident, with singlesite conversion frequencies increasing from left to right according to

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+

+

FIQ. 14. An explanation for polarity in gene conversion (continued). (A) Fourchromatid stage during meiosis before heteroduplex repair. The central DNA duplexes have undergone synapsis and genetic recombination as described in Figs. 9, 10, and 13A, with the exchange of flanking markers. One recombinant molecule is homoallelic in the argr locus, owing to exonucleolytic activity and DNA polymerase repair associated with events accompanying the recombination process. The other recombinant molecule is heteroallelic in the arg4 locus. If this area is not excised and repaired, postmeiotic segregation will result, giving 3:5 or 5:3 ascospore types in octad analysis. (B) Four-chromatid stage during meiosis after heteroduplex repair. The central DNA duplexes have undergone synapsis and genetic recombinaton as described above, with the exchange of flanking markers. Although originally only one recombinant molecule was homoallelic, now both recombinant molecules are homoallelic owing to excision and repair of the heteroallelic recombinant molecule. This can either result in normal 2:2 or 4:4 segregation, or in gene conversion, i.e., 1:3 or 3:1 segregation in tetrads, 2:6 or 6:2 segregation in octads. Dashed lines indicate single DNA stand which has been excised and resynthesized. Each continuous line represents a single DNA strand.

the position of mutant alleles on the genetic map. In addition, there were large differences in the total conversion frequencies for different classes of mutants which mapped near one another (the relative conversion frequencies were approximately 1.0, 4.2, and 2.8, for a, p, and y , respectively), so that without this classification scheme the relationship between conversion frequency and map position could not be detected.

434

HENRY M. SOBELL

TABLE 1 Association of Recombination with Conversion in argda C

petl

arg4

4

19 1 2

/

aw4

Diploid

genotype

Number of asci

517

petl-17 4-thrl petl -thrl c-2 4-thrl c-thrl

8 42 5 1 18 2

7 19 3 0 10 2

313

17

petl-thrl

22

10

243

2

petl-thrl

22

13

petl-17 2-thrl petl-thrl petl-2 1-thrl petl-thrl c-2 1-thrl c-thrl petl -16 19-17 16-thrl petl -thrl petl-17 19-thrl petl -thrl c-2 2-17 lbthrl 1-17 C-17 1-thrl c-thrl

1 7 28 3 14 19 2 6 5 19 16 41 10 2 82 42 4 2 15 4 2 31 74

1 3 14 1 8 8 2 3 4 12 9 21

690

24049 23932

4 2

+

BZ28

17

23956 23957 23958

17

+ +

17 2 17

BZ140

2 + 17

+

544

X841

1 + + 2

367

x901

1 + t 2

116

X2961

19

X2988 X2976

+ 1

+

16

2

+ +

17

+

+ +

a

2566

1505

16 17

Total From Hurst et al. (1972).

Interval

Number of conNumber versions of conwith versions recom3 :1 plus bination 1:3 in interval

4

4 + -

+

\

16 17

17 4-17 4 2 4-2

BZ34

+

Allele(s) converted

thrl

6861

2 17 2-17 1 2 1-2 1 2 1-2 19 16 17 19-17 19-16 16-17 19-16-17 1 16 17 2-16 1-2-16 2-16-17 1-2-16-17

549

6

2 38 21 1 1 3 4

1 11 31 268

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

435

A possible explanation is shown schematically in Fig. 15. One can imagine in the migratory hybrid DNA structure three types of mismatched base pairs, which may act with varying efficiencies to trigger endonucleolytic attack on homologous strands. The first arises from

WILD TYPE

GC-AT a 1.0 [14:i]

GC-cTA y 2.0 [1:0.73]

p 4.2 [Id]

GC-cCG

FIO. 15. Diagram illustrating a possible explanation for Rossignol's coefficients of asymmetry in gene conversion. (A) Hypothetical wild-type allele in migratory Holliday heteroduplex structure. Owing to crossed strand interchange (see text for discussion) it is equally likely that GG or OC are in bridging strands. (B) GC -+ AT transition mutation, resulting in two different mismatched base pairs in the migratory Holliday heteroduplex, i.e., GT and CA. These cause only minimal distortion, triggering endonuclease activity with relatively low efficiency (relative conversion frequency = 1.0). An absolute specificity for exonucleolytic activity to begin with a pyrimidine following endonuclease attack and a high relative affinity (14: 1) for cytosine compared with thymine, can explain marked asymmetry in gene conversion for this class of mutations. (C) GC + TA transversion mutation, resulting in CT and GA mismatched base pairs. These cause more severe helix distortion and trigger endonuclease activity with relatively higher efficiency (relative conversion frequency = 2.8). Due to crossed strand interchange, exonuclease activity can begin with either cytosine or thymine, resulting in less asymmetry in gene conversion (1:0.73). The departure from unity may reflect the preference which the exonuclease has for cytosine compared to thymine. (D) GC + CG transversion mutation, resulting in OC and GG mismatched base pairs. These cause severe helix distortion, and trigger endonuclease activity with high efficiency (relative conversion frequency = 4.2). Owing to crossed strand interchange, exonuclease activity has an equal probability of effecting either direction in gene conversion, resulting in perfect symmetry in gene conversion (1:l). See text for additional discussion.

436

HENRY M. SOBELL

the transition mutation [G-C + A-TI , and causes minimal helix distortion (see Fig. 15B). The second and third, however, arise from transversion mutations [G-C +T-A; G-C + C-GI, and these give rise to more severe helix distortion (see Fig. 15C and D). If the efficiency triggering endonuclease attack is directly related to the degree of helix distortion, the latter types of mismatched base pairs would be expected to give rise to hybrid DNA more frequently. This would be manifested by their relative single-site conversion frequencies (i.e., a, 1.0; y, 2.8; p, 4.2). Asymmetry in frequencies of conversion from wild type to mutant and mutant to wild type may reflect an absolute requirement that exonucleolytic activity begin with, say, a pyrimidine, immediately following endonuclease attack. Both y and p classes contain bridging strands which contain a purine and a pyrimidine. Owing to crossed strand interchange (see Section IV, A, 2), it is equally likely that either heteroduplex is eroded, and this would give rise to approximately equal numbers of 1+:3m and 3+:1m gene conversion events. The a class, on the other hand, possesses either two purines or two pyrimidines in the bridging strands. If the affinity of the enzyme for, say, cytosine is fourteen times that for thymine, then one can explain the 14:l bias observed for this class of mutants. This explanation carries with it the interesting prediction that double mutants containing closely linked alleles belonging to different classes should exhibit a coconversion frequency and asymmetry characteristic of the leftmost marker. It also predicts that transition mutations should give rise to gene conversion events possessing large coefficients of asymmetry, whereas the reverse is true for transversion mutations. Polarity in gene conversion need not be unidirectional throughout a gene. A good example of bidirectional polarity has been provided by Mousseau (1967) in series “19” of Ascobolus. Here, there appears to be a group of centrally placed sites of rather low conversion frequency flanked by sites of high conversion frequency at either end of the gene. Similar findings exist for three genes in Neurospora crassa, me-$ me-6, and me-7. Using random spore analysis of crosses between allelic mutants, Murray (1969) assessed the relative frequencies of conversion a t two me mutants in a particular cross from different frequencies of two parental flanking marker combinations among me+ recombinants. I n each case, higher conversion frequencies were noted for those sites located at the ends, compared with the middle, of genes. This can be explained by assuming that synapsis occurs a t promotor elements located on either side of a gene in these cases, irrespective of their involvement with transcriptional control. Although postmeiotic segregation is rare in Saccharomyces cerevisiae,

SYMMERTY IN PROTEIN-NUCLEIC ACID INTERACTION

437

Neurospora crmsa, Aspergillus nidulans, and the European strain of Ascobolus immersus, it is rather frequent in Sordaria fimicola, Sordaria brevicollis, and the American strain of Ascobolus immersus. Postmeiotic segregation is manifested as 5+:3m or 3+:5m wild-type to mutant octad segregation, as well as 4+:4mP aberrant segregation. The 5:3 and 3:s segregation pattern is most readily explained as reflecting the failure to excise and repair heterozygous DNA (usually present in only one of two recombinant molecules) before mitotic division (otherwise, correction mechanisms would result in gene conversion, i.e., 6+ :2m or 2+ :6m segregation ; or no gene conversion, i.e., 4+ :4m segregation). 4+:4mp segregation can be explained as arising from hybrid DNA in both recombinant molecules, this reflecting the occasional absence of exonucleolytic activity earlier in the recombination process. A certain fraction of potential 4f :4mp segregants could undergo excision and repair of one of two heterozygous recombinant molecules to give rise to 5 :3 or 3 :5 segregation. However, this would predict that gene conversion (as manifested by an apparent double crossover of intragenic alleles without flanking marker exchange) should accompany 5:3 or 3:5 segregation approximately half the time, and this is not observed. A prediction which the model makes is that 4+:4mm segregation events should be rare or absent, since this would necessitate the excision and repair of two hybrid DNA recombinant molecules to give the reciprocal gene conversion event. Unfortunately, events such as these are not readily separable from 4+:4mP events in the usual octad analysis, and no data are available to assess their frequency. Table 2 tabulates the observed frequencies of different conversion patterns among spore color mutants in a variety of eight-spored ascomycetes. I t is seen that whereas 4+:4mP aberrant segregation is common in Sordam’a fimicola (Kitani and Olive, 1967), it is uncommon in Ascobolus immersus (Emerson and Yu-Sun, 1967, 1968). In examples 1-7 (mutant alleles in Sordaria fimicola), there appears to be a rough correlation between the 6+ :2m/2+ :6m and the 5+ :3m/3+ :5m ratios, as one would predict from the model assuming that either strand in the heteroduplex recombinant molecule has equal probability of undergoing excision and repair. However, this is not the case in examples 14-16 (mutant alleles in Ascobolus immersus), and this may reflect unequal probabilities for repair of specific strands in heterozygous DNA in these organisms.

4. Xntragenic versus Intergenic Recombination Although intragenic recombination in eukaryotes is usually nonreciprocal (i.e., involves gene conversion), reciprocal recombination between

TABLE 2 Variations in Frequencies of Different Conversion Patterns among Spore-Color Mutants in EightrSpored Ascomycetesa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a

99 96 97 100 49 53 47 6 162 644 169 74 139 57 1 99 1 2661 439 693

00

Relative frequencies (%) of different conversion patternse

Converted asci Example* No.

I+

w

%

6+:2m

0.20 0.23 0.23 0.22 0.20 0.23 0.22 0.08 0.73 2.11 0.50 0.26 4.0 8.4 12.3 17.7 18.6 18.7

31.3 (21.744.3) 7.3 (2.9-15.0) 6.2 (2.3-13.5) 3.0 (0.6-8.8) 0.0 (0.0-7.5) 1.9 (0.05-10.5) 4.3 (0.5-15.4) 0.0 (0.0-61.7) 3.1 (0.1-6.4) 51.2 (46.8-56.6) 75.4 (68.0-82.8) 58.1 (40.5-70.3) 15.8 (10.1-23.7) 45.0 (39.8-50.3) 22.1 (19.3-24.9) 71.8 (68.7-74.4) 72.0 (64.7-79.3) 46.8 (42.2-51.1)

5+:3m 35.4 27.0 28.8 9.0 42.8 7.6 23.4

(24.8-49.3) (18.0-39.5) (19.5-41.4) (4.1-17.1) (27.1-65.6) (1.3-19.5) (11.9-41.5) -

-

-

-

64.0 (51.8-78.4) 10.2 (7.7-13.1) 17.5 (15.8-20.3) 3.2 (2.6-3.9) 1.1 (0.5-2.9) 16.3 (13.6-19.5)

4+:4mP

3+:5m

2+:6m

13.1 (7.1-22.5) 36.4 (25.7-50.8) 48.4 (35.8-64.3) 35.0 (24.7-48.8) 38.8 (24.1-60.2) 30.2 (17.449.1) 23.4 (11.9-45.5) Undetected Undetected Undetected Undetected Undetected Undetected Undetected 1.2 (0.6-2.l)d Undetected 0.9 (0.2-2.3)d 0.9 (0.3-1.8)d

14.1 (7.9-23.6) 23.9 (15.4-36.6) 12.4 (6.5-21.6) 40.0 (29.9-54.3) 18.4 (8.6-34.7) 52.9 (35.7-76.0) 42.6 (26.8-65.6)

6 . 1 (2.2-13.1) 5.2 (1.7-12.1) 4 . 1 (1.1-10.5) 13.0 (7.0-22.0) 0.0 (0.0-7.5) 7.6 (1.3-19.5) 6.4 (1.3-18.7) 100 (38.3-100) 96.9 (93.6-99.9) 48.8 (43.4-54.2) 24.6 (17.2-32.0) 41.9 (29.7-59.5) 2.9 (0.8-7.2) 21.0 (17.5-25.0) 17.9 (15.2-20.4) 20.2 (18.5-21.9) 25.1 (20.8-30.1) 19.3 (16.2-22.8)

-

-

17.3 (11.5-25.9) 23.8 (20.3-25.0) 41.4 (37.4-54.2) 4.8 (4.1-5.9) 0.7 (0.1-2.0) 16.7 (13.9-20.1)

From Emerson (1969).

* Examples 1to 7, respectively, alleles g,,

h,, h2=,ha, h,,, h4 and ha, at the gray-spored locus in SorduriujimieOZu (Kitani and Olive, 1967); 8 to 10, respectively, alleles 1604, 63, and 137, of series 46 of Ascobolus immersus (Rossignol, 1964); 11 and 12, respectively, 14, mutant w-62 X alleles 77 and 775 of series Y of Aswbolus immersus (Kruszewska and Gajewski, 1967); 13, mutant w-6 X 15, double mutant w-62 gr-1 X 16, w-lO(P) X 17, w-1O(P) X gr-1; and 18, w-78(p) X gr-1; among these w-10 and w-78 are presumptive allelea and gr-1 an independently segregating marker (Emerson and Yu-Sun, 1967, 1968). figures in parentheses are 95% confidence limits based on frequencies among total aaci, method of W. L. Stevens (Fisher and Yates, 1957, Table VIII1). Figures in boldface indicate the most frequent pattern. Postmeiotic 4+:4mp segregation in unordered asci was detected by the use of a second, unlinked, ascospore character.

++;

+;

+;

+;

3

2 5

8

F

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

439

mutant alleles in the same gene can occur as well, particularly if these are well spaced (Fogel and Hurst, 1967; Lissouba et al., 1962; Rossignol, 1967). This is in agreement with the fixed-length migratory hybrid DNA structure proposed earlier, which allows for the exchange of flanking markers (in this case, these being well spaced mutant alleles in the same gene) via a double-strand breakage and reunion event. Gene conversion can still occur between these intragenic flanking markers (Rizet and Rossignol, 1966), utilizing the mechanisms described in the previous section. It is of interest that in higher organisms, such as Drosophila melanogaster, intergenic recombination, even among the most closely spaced genes, is about ten times the maximum intragenic recombination values (e.g., see Pontecorvo, 1958; Thomas, 1970). This agrees with the concept that chromosomal synapsis occurs between genes, not within genes. I n higher organisms, specific regions may have evolved (i.e., long genetically silent regions containing symmetrically arranged nucleotide base sequences) that are expressly reserved for synapsis. Such regions, for example, could dictate that genetic recombination occur between specific genes, such as master genes, but not between slave genes (Callan and Lloyd, 1960; Callan, 1967). Regions such as these may be capable of forming branched DNA structures extending several thousand angstroms to synapse in the synaptinemal complex. These regions would be distinct from promotor regions, envisioned to be smaller regions of symmetrically arranged nucleotide sequences existing on the ends of master and slave genes, important for their transcriptional control. 6. High Negative Interference and M a p Expansion

Before closing this section, it seems worthwhile to include a brief discussion of high negative interference and map expansion, two effects that come up in fine structure genetic mapping, and to discuss possible underlying molecular mechanisms. The phenomenon called high negative interference, originally described by Pritchard (1955) in Aspergillus and Chase and Doermann (1958) in bacteriophage T4, refers to the observation that recombination between two closely linked alleles increases the likelihood that further genetic exchange occurs in the immediate neighborhood. Although the effect is primarily encountered in fine structure intragenic mapping, it also exists between allelic sites in neighboring genes (Calef, 1957; Putrament, 1967; Morpurgo and Volterra, 1968; Murray, 1970). Amati and Meselson (1965) studied negative interference in A using mutants containing three or more closely linked alleles. They found that, exchanges tended to occur within short clusters (two on the average,

440

HENRY M. SOBELL

but occasionally as many as four) and estimated the mean length of DNA involved in the cluster of exchanges to be about 1500 nucleotides. Two possible explanations for this localized negative interference have been discussed by Meselson (1967a). The first involves a single primary event and involves the formation of a hybrid DNA segment with a mean length of about 1500 nucleotides joining both parental DNA mole-

M a p Distance

FIG.16. Examples of fine structure map expansion. The map distance, or expected recombination frequency, is the sum of the map intervals between mutants two or more intervals apart. Recombination is the actual frequency of recombinants observed when such mutants are crossed. The solid lines show the relationship between the map distance and recombination which would be observed if the map was strictly additive. Frequencies are as follows: (A) Schizosaccharomyces su-3, prototrophs per 10' ascospores; (B) Ascobolus ser-19, wild-type spores per loa tetrads; ( C ) Neurospora t r y p d , prototrophs per 1V ascospores. Fine structure map expansion may reflect a marker-induced excision-repair mechanism, triggered by a mismatched base pair. Redrawn from Fincham and Holliday (1970).

cules. Clustering of exchanges could arise as a consequence of excision of mismatched base pairs by endonucleolytic cleavage and limited exonucleolytic action, followed by DNA repair. If each site were corrected independently, the final duplex would have alternating segments with either parental genotype, appearing to have undergone multiple genetic exchanges. The other po.ssibility is that the formation of hybrid DNA accompanying recombination occurs many times during the course of

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

44 1

infection (i.e., multiple primary events), and that 1500 nucleotides represents the mean length of a region containing several short hybrid DNA segments, each having undergone excision and repair independently. Although it is not possible to discriminate between these two alternatives, the concept that mismatched base pairs can trigger localized excision and repair events a t numerous heterozygous sites in a single stretch of hybrid DNA (i.e., formed from a single primary event) is the simpler, and therefore the more attractive, explanation of high negative interference. The phenomenon of map expansion is seen in many linear fine structure maps in fungi and refers to the tendency for recombination frequencies between widely spaced mutant alleles within a gene to greatly exceed estimates of the same intervals obtained by adding the recombination frequencies given by more closely spaced markers. Fincham and Holliday ( 1970) have explored the consequences of a marker-induced excisionrepair explanation of map expansion. Their analysis (the details of which cannot be presented here) predicts three phases in the mapping curve: an initial additive phase when the recombining sites are closely linked, a phase of increased slope corresponding to map expansion, and a final additive phase of reduced slope beyond the expansion region. Several sets of experimental data show a clear transition from the initial additive region to that involving map expansion (see Fig. 16), but as yet there is little evidence that pertains to the predicted final phase. The concept that endonuclease attack is triggered by a mismatched base pair is an attractive one, since, in addition, it explains polarity and asymmetry in gene conversion (Section IV, A, 3) and marker effects that influence integration efficiencies in transformation (Section IV, B, 1 ) .

B. PROKARYOTIC SYSTEMS Genetic exchange in prokaryotes is mediated by several different types of transfer mechanisms. These include transformation, transduction, conjugation, and episomal transfer (for a general reference, see Hayes, 1968). Each differs in the size of DNA transferred and in the mechanism of transfer. It will be shown that most prokaryotic genetic recombination can be accounted for by single-strand exchange, involving a mechanism similar to that discussed for gene conversion in eukaryotes. Doublestrand exchange may be used by the F sex factor for integration into and excision from the recipient chromosome. This will be taken up in Section IV, C, 2, which discusses site-specific recombination in temperate bacteriophages. The discovery of recombination-deficient mutants of E . coli and their

442

HENRY M. SOBELL

mapping into three distinct loci (recA, recB, and recC) has been an important recent advance toward understanding recombination in prokaryotic systems (Clark and Margulies, 1965; Clark et al., 1966; Van de Putte et al., 1966; Howard-Flanders and Theriot, 1966; HowardFlanders, 1968; for a review, see Clark, 1971). Although all are sensitive to ultraviolet irradiation, the recA mutants are particularly sensitive, breaking down their DNA about thirty times more extensively than normal strains (Clark et aZ., 1966). The recB and recC mutants, on the other hand, show less breakdown of DNA than normal strains following ultraviolet irradiation, either individually, or as the double mutant. When combined with recA, both recB and recC strains show the same ‘(cautious” ultraviolet-induced breakdown of DNA (Willetts and Clark, 1969)- These strains, however, possess the high ultraviolet sensitivity and absence of recombination characteristic of recA alone (although recA strains show little or no recombination, low, but significant, levels of recombination remain in recB and in recC strains). The recB and recC cistrons are now known to code for an ATP-dependent exonuclease which also possesses endonuclease activity (Goldmark and Linn, 1972). The function of the recA cistron is not known. This cistron could perhaps code for the structural protein necessary to activate promotor regions to effect synapsis in general bacterial recombination.

I . Transformation Bacterial transformation offers an excellent opportunity to relate the physical and chemical properties of DNA with events that accompany genetic recombination (for a recent review, see Hotchkiss and Gabor, 1970). Although first described in Pneumococcus (Griffith, 1928; Avery et aZ., 1944), transformation has since been found to occur in other bacterial species, notably, Hemophilus influenme (Alexander and Leidy, 1951) and Bacillus subtilis (Spizizen, 1958). Each system shows variability with respect to the proportion of cells transformed (this reflecting a special physiological state called competence) , the size and nature of DNA molecules effective in transformation, and the fate of the transforming DNA immediately prior to its integration into the host genome (the eclipse period). Competence in transformation may reflect the presence of lytic enzymes necessary for passage of DNA through the cell wall (Young and Spizizen, 1963 ; Akrigg et d.,1967). Alternatively, noncompetent cells may destroy transforming activity of donor DNA by endonucleolytic cleavage (Haseltine and Fox, 1971). Almost nothing is known about the nature of cellular receptor sites for DNA attachment, or the mechanism by which DNA is transported into the cell.

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

443

Native DNA is the most active form for usual transforming systems. Denatured DNA, fractionated into single strands, shows only marginal activity in Pneumococcus (Peterson and Guild, 1968 ; Guild and Robison, 1963; Roger, Beckmann and Hotchkiss, 1966), Hemophilus (Mulder and Doty, 1968), and B. subtilis (Rudner et al., 1968) ; however, see Goodgal and Postel (1967) and Tevethia and Mandel (1970) concerning this point. Cat0 and Guild (1968) have conducted a broad study of cellular affinity, competition, and transformation rate of physically sheared DNA, carefully fractionated according to size. They have demonstrated that DNA fragments having molecular weights between 0.3 and 0.8 million bind pneumococcal cell sites tightly and are extremely active in transformation. DNA fragments of molecular weights under 300,000 (corresponding to 450 base pairs or less) are taken up by the cell; however, the transforming activity of these fragments abruptly diminishes as their size decreases. The eclipse period in transformation refers to an interval of time during which donor marker activity decreases following DNA uptake by competent cells (Fox, 1960; Venema et al., 1965). Thus, for example, pneumococci, exposed to transforming DNA for a period of 10 minutes contain intact donor marker integrated into the host chromosome (Fox and Hotchkiss, 1960). However, prior to this time, donor marker activity in the recipient cells falls to very low values, reappearing by about the sixth minute (Fox, 1960). Similar results were reported by Venema et al. (1965), who found virtually complete loss of marker activity shortly after exposure of B. subtilis cells to transforming DNA. This eclipse period disappears rapidly (in about 15 minutes), in a time slightly shorter than that required for the completion of integration. A donor-recipient complex has been detected during the eclipse period (Bodmer and Ganesan, 1964; Pene and Romig, 1964; Fox and Allen, 1964; Harris and Barr, 1969; Notani and Goodgal, 1966; Dubnau and Davidoff-Abelson, 1971). This is a heteroduplex structure in which the donor moiety has an approximate molecular weight of 750,000. Available evidence indicates that this complex exists transiently in a form with low recombinant-type transforming activity, and that the reappearance of recombinant activity accompanies the integration process (Dubnau and Davidoff-Abelson, 1971). Integration is known to proceed by a process resulting in single-strand insertion into the recipient genome (Fox and Allen, 1964; Bodmer and Ganesan, 1964; Notani and Goodgal, 1966). The size of the integrated region has been estimated to be between 2000 and 3000 nucleotides in Pneumococcus (Fox and Allen, 1964), and considerable DNA degradation may accompany the integration process (Lacks, 1962; Stuy, 1965;

444

HENRY M. SOBELL

Fox and Allen, 1964). The recombinant DNA region exists as a physical and genetic heterozygote in Pneumococcus. Guerrini and Fox (1968a) have shown that transformants give almost exclusively mixed clones carrying recipient and a single transformant type a t the earliest stages. Excision-repair processes are induced by mitomycin or ultraviolet irradiation, and this converts heterozygous regions into homozygous ones (Guerrini and Fox, 1968b). I n B . subtilis, however, it appears that heterozygosis occurs only transiently, being readily corrected by excision-repair mechanisms to give gene conversion (Bresler et al., 1968; Spatz and Trautner, 1970). These and other data are interpretable in terms of the following molecular mechanisms for transformation: a. Synapsis is mediated through interaction between specific pairing regions (promotor regions) on the donor and recipient DNA, using the mechanism shown in Figs. 9 and 10. This requires that donor DNA be (on the average) greater than a certain critical size (roughly cistron or operon length) to effect transformation, in agreement with the physical shear studies. b. Double-stranded donor DNA is obligatory immediately prior to synapsis, since, for promotor regions t o be activated to their Gierer state, they must interact with a recombination structural protein (this may be the recA gene product, or its equivalent) using symmetry in protein-nucleic acid interaction (see Section 11, B, 1). Single-stranded DNA may effect transformation ; however, it must first be transformed to its double helical form (by replication) to undergo synapsis. c. The Holliday heteroduplex structure may correspond to the donor-recipient synaptic complex which occurs during the eclipse period in transformation. d. Genetic recombination then proceeds in a manner entirely analogous to intragenic recombination in eukaryotes (i.e., nonreciprocal recombination leading to gene conversion) as shown in Fig. 17. Integration of donor DNA into the recipient genome is accomplished by nicking the Holliday migratory DNA structure a t equivalent sites on homologous strands, and then degrading one of two hybrid DNA regions by exonucleolytic activity (ie., this nuclease may be analogous to that coded by the recB and recC cistrons, which has both endonucleolytic and exonucleolytic activities). Subsequent migration of the hybrid DNA structure (now only a half-chiasma) driven, perhaps, by this ATP-dependent exonuclease, continues until the complete donor fragment is incorporated into the recipient genome. This gives rise t o the formation of an insertion heteroduplex, with the accompanying degradation of an equivalent amount of recipient DNA (see Steinberg and Herriott, 1968).

SYMMETRY I N PROTEIN-NUCLEIC e

d

C

b

a

e

d

C

b

0

t

t

t

t

t

b

a

.

b

0-

+

+

C

b

a

+

,

f

e

d

,

445

-

C C

-1

ACID INTERACTION

-

-4

FIQ.17. A molecular mechanism for transformation. Donor and recipient chromosomes possess specific regions (promotor regions) capable of forming Gierer-like structures in the presence of a specific recombination structural protein. Synapsis occurs through the mechanism shown in Figs. 9 and 10. Integration of donor DNA into the recipient genome is accomplished by nicking the Holliday migratory DNA structure a t equivalent sites on homologous strands (this perhaps triggered by a mismatched base pair), and then degrading one of two hybrid DNA regions by exonucleolytic activity (i.e., a single strand of the recipient chromosome is degraded). Subsequent migration of the hybrid DNA structure (now only a halfchiasma) driven perhaps by this (iecBC ATP dependent) exonuclease continues until the complete donor fragment is incorporated into the recipient genome. This gives rise to the formation of an insertion heteroduplex, which may either undergo excision and repair to give pure transformed clones, or give mixed clones by segregadonor DNA; -, tion. Exonuclease activity; ---,DNA polymerase repair; -, recipient DNA. See text for additional details.

e. The heterozygous region can then either undergo excision and repair to give pure transformed clones (gene conversion) or give rise to mixed clones by segregation. Marker efficiency in transformation may reflect varying efficiencies which different mismatched base pairs have in triggering the excision-repair process, as suggested by Ephrussi-Taylor and Gray (1966). Alternatively, marker efficiency may reflect a mechanism similar to that proposed to explain asymmetry in gene conversion (see Section IV, A, 3) ; however, this would predict the occasional exclusion

446

HENRY M. SOBELL

of low efficiency marker effects by high efficiency markers, and this apparently has not been observed (Ephrussi-Taylor and 'Gray, 1966; Lacks, 1966).

b. Transduction Transduction has generally been classified into two categories, generalized and specialized transduction, according to the range of bacterial markers capable of being transferred by the transducing phage. Typical examples of generalized transduction are P22-mediated transduction in S. typhimurium (Zinder and Lederberg, 1952) and P1 transduction in E. coli (Lennox, 1955). I n this type of transduction almost any genetic marker in the donor strain can be transferred to a recipient strain. On the other hand, phage x in E . coli K12 (Morse et al., 1956) is capable of transducing specialized markers only (for example, markers in the gal or bio loci), these being located within a very limited region of the bacterial chromosome and lying on either side of the attachment site specific for the x prophage. This section will discuss briefly the phenomenon of generalized transduction. Specialized transduction will be discussed in Section IV, C, 3. For a recent review of generalized transduction, see Ozeki and Ikeda (1968). Two general models have been advanced to explain the mechanism of generalized transduction. The first (sometimes referred to as the unified model) equates the mechanism of generalized transduction with that of specialized transduction to the extent that, in both cases, fragments of bacterial chromosome are incorporated into the phage genome to yield defective phages. According to this concept, generalized transduction differs from specialized transduction in the ability of the phage genome to interact with many regions along the host chromosome with which it undergoes recombination during vegetative multiplication. The discovery by Luria, Adams, and Ting (1960) that the generalized transducing phage P1 can form a defective variant carrying lac genes ( P l d l ) which is, in many respects, similar to hdg, initially supported the unified model for the origin of generalized transduction. Later, however, Ikeda and Tomizawa (1965a) conclusively demonstrated that most P1 transducing particles incorporate fragments of bacterial DNA which contain no detectable phage DNA. It appears a t the present time that P1 can mediate two types of transduction, generalized and specialized, and that the former is overwhelmingly predominant in transduction. A similar situation has been inferred for the P22 Salmonella system. Smith-Keary (1966) has succeeded in isolating defective transducing phages of P22 which carry the pro (proline) gene, this being located near the P22 attachment site on the Salmonella chromosome. This type

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

447

of transductant, however, is the minority class; the majority of transductants contain markers randomly distributed along the bacterial genome and contain no viral genome. It appears, therefore, that lysogenic phages (such as P1 and P22) can give rise to both types of transduction, each involving different mechanisms. A simple explanation for the origin of transducing particles in generalized transduction is that a phage-sized piece of bacterial DNA is incorporated into the head protein envelope in place of a phage chromosome to give a “head-full” of DNA, analogous to the situation in T 4 (Streisinger et al., 1964, 1967). Supporting evidence for this comes from the observation that, in normal populations of phage P1, about onefourth are of small size, having heads 650 A instead of 900 A in diameter (Ikeda and Tomizawa, 1965b). These small phage particles (their DNA is about 40% the length of normal phage DNA) are defective; however, their genomes are circularly permuted, since, in multiple infections, they can complement one another to yield normally infectious particles. A proportion of these small particles are transducing and carry a diminished length of bacterial DNA, as evidenced by the reduced probability of cotransduction of neighboring bacterial genes. This implicates the role of the head structure as the primary determinant specifying the length of DNA (whether it be phage or bacterial) to be wrapped up into the phage particle. Other examples of phages known to perform generalized transduction which have circularly permuted genomes (these presumably arising from the packaging of high molecular weight concatenated DNA structures into phage-sized lengths by the “head-full” mechanism) are P22, ~ 1 5 ,and €34 (Thomas and MacHattie, 1967; Toyama and Uetake, personal communication cited in Ozeki and Ikeda, 1968). Virtually nothing is known about the mechanism for the incorporation of the generalized transducing bacterial genome into the recipient chromosome. Breakage and reunion which involves double-strand integration necessarily involves two separate synaptic events, whereas a mechanism similar to transformation (Section IV, B, 1) necessitates only one synaptic event. This latter mechanism is a particularly attractive one since the integrity of the bacterial chromosome remains uninterrupted throughout the integration process. However, further data are necessary before a specific mechanism can be postulated to explain genetic recombination mediated by generalized transduction. 3. Conjugation

Conjugation in E . coli requires the presence of a conjugal fertility factor (the F factor) integrated into the chromosome of the male donor

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HENRY M. SOBELL

strain, this regulating chromosomal transfer into the female recipient strain (for excellent recent reviews in this area, see Curtiss, 1969; Susman, 1970). The F factor determines the formation of surface structures known as pili, which act as attachment organs to couple mating bacteria (Brinton, 1965). These serve as conduits through which chromosomal material is transferred from male to female, and also act, perhaps as sensory organs through which the male bacterium receives the signal to begin chromosomal transfer (Ippen and Valentine, 1967). The F factor is known to exist in two distinct states, the autonomous state and the integrated state (see next section for further discussion of this), the latter allowing the transfer of genetic markers with high frequency from the male (Hfr) to the female (F-) (Lederberg et al., 1952; Hayes, 1953; Jacob and Wollman, 1961). Typically, transfer begins a t a specific origin (this depends on the particular Hfr strain used) and proceeds in a given direction in an orderly sequence (Wollman e t al., 1956). Since transfer of the whole chromosome is rare, those markers that are close to the origin are transferred with much higher frequency than those located far away. A portion of the integrated F factor seems to be transferred a t the leading end of the Hfr chromosome; however, the rest of the F factor remains a t the distal terminus (Jacob and Wollman, 1961). For this reason, the transfer of donor capacity (which depends on transfer of the whole F factor) is a rare event. I n their model to explain the mechanism of chromosomal transfer during conjugation, Jacob, Brenner, and Cuzin (1963) proposed that transfer required the concomitant replication of the donor genome, one of two replica duplexes being transferred to the recipient as synthesis proceeded. It is known that the exogenote DNA fragment consists of one donor strand and one newly synthesized strand (Gross and Caro, 1966), the latter possibly reflecting DNA synthesis in the recipient cell (Bonhoeffer and Vielmetter, 1968; Freifelder, 1967). Ihler and Rupp (1969) have demonstrated that only one of two x strands can be recovered from recipients after conjugation with labeled lysogenic donors. Opposite strands of the phage could be detected in the recipient by using donors which inject their chromosome in opposite directions. It appears that a specific strand of the sex factor is broken and that transfer is initiated a t this site, transfer commencing with a free 5’ terminus on the donor strand (Ihler and Rupp, 1969). Little is known about the mechanism for integration of the donor chromosome into the recipient genome. Breakage and reunion appears to be the most likely mechanism (Siddiqi, 1963; Bresler and Lanzov, 1967; Oppenheim and Riley, 1966; Bresler et al., 1967; Piekarowicz and Kunicki-Goldfinger, 1968); however, it has not yet been clearly

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

449

established whether integration involves single- or double-strand transfer (for a discussion, see Curtiss, 1969). The mechanism which has been proposed for transformation necessitates only one synaptic event (in contrast to double-strand transfer) and is an attractive one for both generalized transduction and conjugation in that the continuity of the bacterial chromosome is not interrupted by the integration process (see Section IV, B, 1 ) . However, further data are required before a specific mechanism can be postulated to account for genetic recombination mediated through conjugation.

4. Episomal Transfer For an excellent discussion of sex factors and other plasmids, see Hayes (1968). The mating system in E . coli is comprised of two types of donor strains, F+ and Hfr, which have different properties with regard to the mechanism of genetic transfer and their bacterial phenotype. Both strains produce filamentous appendages called sex fimbriae (or pili) , which enable them to make intimate contact with the recipient (F-) cell. These appendages, in addition, confer susceptibility to a group of RNA-containing spherical viruses (Loeb, 1960; Loeb and Zinder, 1961; Dettori et al., 1961) and certain filamentous DNA viruses (Marvin and Hoffman-Berling, 1963; Zinder et al., 1963), to which F- cells are resistant. The F+ character is readily transmissible to an F- recipient strain independently of the bacterial chromosome (Lederberg et al., 1952; Hayes, 1953; Cavalli-Sforza et al., 1953), this, in many ways, resembling a viral infection. Treatment of F+ donor strains with low concentrations of acridine orange leads to a loss of the sex factor, and a conversion of the population to F- genotype, a process known as “curing” (Hirota, 1960). “Cured” strains can be reconverted to F+genotype by conjugation with F+ strains, simultaneously regaining the ability to produce sex fimbriae and becoming susceptible to infection by donor-specific phages (Crawford and Gesteland, 1964; Brinton et al., 1964; Brinton, 1965). These and other data suggest that, in F+ cells, the sex factor is in an autonomous state, existing as a separate cytoplasmic element distinct from the bacterial chromosome. Hfr bacteria, on the other hand, differ in several fundamental ways from F+ bacteria. These strains arise a t low frequency from F+ strains and promote conjugation resulting in a high frequency of genetic exchange between donor and recipient. I n contrast with F+-mediated conjugation, the recipient usually remains F-, the sex factor being transferred last as a chromosomal marker (Jacob and Wollman, 1958). Different Hfr strains demonstrate different origins and directions of transfer (Jacob and Wollman, 1961), and, in contrast with F+ strains, the H f r

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HENRY M. SOBELL

character is not “cured” by acridine orange treatment (Hirota, 1960). It is now known that the transition from the F+ state to the Hfr state is a consequence of a single reciprocal recombination event, which acts to insert, the F sex factor a t one of many possible sites on the E . coli chromosome. Since both the F+ and Hfr chromosomes are circular structures (Cairns, 1963; Taylor and Adelberg, 1961), it has been inferred that the F factor is also circular, undergoing integration (and excision) by the same basic mechanism proposed for phage h (Campbell, 1962). The phenomenon of sexduction was discovered by Adelberg and Burns (1960), who discovered intermediate donor strains (denoted primary donors, I) which arose as variants of different Hfr strains. The sex factor could be readily transferred to recipient F- cells (to form secondary donors, 1’), these then demonstrating the same oriented sequence of transfer as the original Hfr strain, but a t about one-tenth the efficiency. When the intermediate primary donor strain was treated with acridine orange, it was converted to an F- strain through loss of its sex factor; however, on reinfection of this same strain with a normal sex factor (from an F+ strain) it regained the I’ secondary donor character, suggesting that the original integration site for the F factor on the bacterial chromosome had been modified so as to retain a high affinity for the sex factor. A logical interpretation of these observations is that both intermediate donor strains (I and 1’) possess altered F-prime (F’) sex factors that carry a segment of the bacterial chromosome, this due to a rare recombination event in the parental Hfr strains between regions of the integrated sex factor and neighboring regions of the bacterial chromosome (analogous to the formation of gal-transducing particles of phage A, i.e., Xdg). Because the segment of bacterial chromosome carried by the sex factor has virtually perfect homology for the allelic region of a recipient chromosome, one would expect the frequency of insertion and release of the F’ sex factor to be much greater than the wild-type F factor, giving rise to Hfr and F+ states which alternate rapidly within a given cell. This results in a population of bacteria (I’ secondary donors) which can transfer both the autonomous sex factor and the chromosome at high frequency, and this is observed in the intermediate I’ donor strains. It is of interest that heterogenotes which contain, say, a Z- allele on the chromosome and a Z+ allele on the episome (Z-/F-Z+) are unstable for the Z+ character, segregating Z- progeny with a probability of about per cell per generation. These heterogenotes also give rise to small numbers of homogenotes (for example, Z-/F-Z-) and larger numbers of inverse heterogenotes (Z+/F-Z-) , this reflecting recombination between regions of homology in the episome and chromosome (Jacob

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

45 1

et al., 1960; Scaife and Gross, 1963; see Hayes, 1968). Herman (1965)

has shown that recombination between an F’ factor and the chromosome is often (although, not always) reciprocal. He constructed the merodiploid E. coli strain (z+y-/F-zy+), z- being a strongly polar mutation in the p-galactosidase gene in the lac operon. Lac+ recombinants, having z+y+ in either the episome or the chromosome were selected. Of these, eleven were found which had the z+y+genotype on the episome; these were then treated with acridine orange to remove the episome. Seven of the eleven recombinant strains had the reciprocal genotype (zy-) on the chromosome, the remainder had nonreciprocal genotypes. Evidence for predominantly reciprocal recombination between well spaced mutant alleles in prophage A (where the A prophage is located both on the chromosome and on the episome) has been provided by Meselson (1967b). He studied recombinants arising from the parental merodiploid strain ( c m i h/F +I, where c, mi, and h are markers on the prophage genetic map, and observed that the majority were due to reciprocal crossing over (i.e.) 36 were c +JF - +mi h, and 68 were c mi +/F h ) , while a minority were nonreciprocal (i.e., 8 were c + +/F h, and 8 were c m i +/F - mi h ) . A possible molecular mechanism explaining these as well as other aspects of episomal genetics will be presented in Sections IV, C, 2 and 3.

++

++ ++

+

+

C. VIRUSES Bacteriophages have provided a wealth of information on varied aspects of genetic recombination. Formal genetic analysis has shown that (general) recombination between viruses is usually nonreciprocal, yielding one parental and one recombinant genotype from a single mating event. Recombinant molecules often possess a hybrid DNA region which is heterozygous, this giving rise to segregation upon subsequent replication. Studies with isotopically labeled phages have demonstrated a breakage and reunion mechanism which is often accompanied by extensive DNA synthesis. Classical techniques of bacterial genetics have identified specific genes in bacteria and viruses which govern alternate pathways in recombination. These pathways differ in their ability to promote either reciprocal or nonreciprocal recombination in different regions of the chromosome. This section will discuss the evidence for general and site-specific recombination in temperate viruses (leaning heavily on evidence available from A genetics), and will include a brief discussion of specialized transducing phages and their origin.

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HENRY M. SOBELL

1. General Recombination

For a recent review, see Signer (1971). Genetic recombination was first demonstrated in bacteriophages with phage T2 of E. coli by Delbruck and Bailey (1946) and Hershey (1946). From single-burst experiments in which parental phages had closely spaced markers, Hershey and Rotman (1949) concluded that recombination was primarily nonreciprocal, a finding subsequently confirmed in further studies by Bresch (1955) for phage T1 and by Boon and Zinder (1969) for phage f l . Strong reciprocity, however, has been observed when recombination occurs between prophages containing well spaced mutant alleles in partially diploid bacteria (see discussion above and Meselson, 1967b). It is now known that there are three distinct recombination pathways during (temperate) bacteriophage infection and that each has different properties with regard the site and nature of the recombination event. The rec genes in E. coli are defined by recombination deficient (rec-) mutants which are incapable of undergoing recombination by generalized transduction, conjugation, or episomal transfer. These map into three cistrons, recA, recB, and recC, and possess characteristic phenotypes (see introduction in Section IV, B). The pathway on which the rec gene products promote recombination has been called the rec pathway. Since phage A was found to recombine a t approximately normal frequency in rec- strains (Brooks and Clark, 1967), it was inferred that h had its own recombination systems (s) . These have been subsequently discovered to be of two kinds, the red system, which promotes general (non-site specific) recombination (Franklin, 1967; Gottesman and Yarmolinsky, 1968; Echols and Gingery, 1968; Signer and Weil, 1968a; Shulman et aZ., 1970) and the int system, important for site-specific recombination and the establishment of lysogeny (Zissler, 1967; Gingery and Echols, 1967; Gottesman and Yarmolinsky, 1968; Signer and Weil, 196813). Red- mutants define two genes, called reda and reap, these coding for a 5' specific exonuclease and a ,8 protein whose function is unknown (Shulman et al., 1970; Radding, 1970; Signer et al., 1968). The p protein appears to complex with the exonuclease, increasing its affinity for DNA (Radding and Carter, 1971). A third gene, y , may also be part of the red pathway, although this is not yet completely certain (Zissler et al., 1971). The int system is composed of two genes; the int gene, for integration of A into the bacterial chromosome, and the xis gene, necessary (in addition to int) for excision (see next section). Table 3 compares the rec, red, and int pathways in their ability t o promote recombination between different intervals of the A chromosome. The interval J-cI includes the prophage attachment region, while the interval cI-R does not. It is seen that recombination is mediated by

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

453

all three pathways in the interval J - c I ; however, in the interval, cI-R, only the rec and red pathways promote recombination. This reflects the int site-specific recombination pathway, which is restricted to the prophage attachment region (Signer and Weil, 196813).Along similar lines, red- phage integrate efficiently into the host chromosome in rec+ or recbacteria ; conversely, integration defective int- mutants show normal general recombination in red+ or rec- strains. Furthermore, in a rechost, the red system promotes almost as much A recombination as in a rec+ host; however, the rec system alone is less efficient, red- mutants TABLE 3 General and Site-Specific Recombinationa ~~

Phage

~~

~

Recombinationb (%)

red

int

Host recA

+ +-

+

+

-

+

-

+-

+-

+

2.0 1.3

-

<0.05

-

-

J-c

C-R

7.5

3.6

4.1

3.0 <0.05 1.3

7.8

3.1

<0.05

Adapted from Signer (1971), after Signer and Weil (196813). The interval J-c includes the prophage attachment region att, hence recombination within it may be general or site specific; the interval c-R does not, and recombination within it is only general. The red- mutation inactivates both exonuclease and 0 protein, and the rec- mutation is recA-. a

showing about one-tenth the wild-type recombination frequency (Echols et al., 1968; Signer and Weil, 1968a; Weil and Signer, 1968; Gottesman and Yarmolinsky, 1968). Weil (1969) has presented evidence that i n t promoted recombination is reciprocal whereas red promoted recombination is generally nonreciprocal. He studied progeny from single cells in a mixed infection of a rec- host by two strains of h carrying three mutations, SUSA,b,, and cIII. In the absence of rec function, recombination in the susA-b, interval was exclusively red promoted; however, in the b,-cIII interval (which includes the A attachment site) it was found that 75% of the recombinants were int promoted. For the s a d - b , interval, Weil found little correlation between reciprocal recombinants, but for the b,-cIII interval the correlation was significant (correlation

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HENRY M. SOBELL

coefficients 0.16 and 0.64, respectively). The nonreciprocality of red-promoted recombination could explain why red function, unlike rec function, rarely allows a second A (int-) genome to be integrated into an (int-) prophage (Gottesman and Yarmolinsky, 1968). The nature of the molecular events accompanying recombination in A has been studied recently in the presence of different recombination pathways (rec, red, or int) by Kellenberger-Gujer and Weisberg (1971), Kellenberger-Gujer (1971), F. W. Stahl and Stahl (1971), and M. M. Stahl and Stahl (1971). All systems appear to effect the physical exchange of DNA during recombination, the int system being particularly efficient in this regard (Kellenberger-Gujer and Weisberg, 1971). Evidence for a “ring to ring” insertion between h and hdv [i.e., hdv is an autonomously replicating DNA fragment of h which possesses all the genes necessary for DNA replication, but none of the late genes that would lead to lysis and destruction of the cell (Matsubara and Kaiser, 1968) ] has been provided by Kellenberger-Gujer (1971). An entire hdv monomer can be inserted into the h chromosome, implying a double-stranded reciprocal crossover event in crosses such as these (Campbell, 1962). General h recombination may involve a similar mechanism, a “terminase” cleaving between genes A and R with sufficient nucleotide overlap to result in single h chromosomes which possess cohesive ends (Mousset and Thomas, 1969). It should be noted that a double-stranded reciprocal crossover event between two circular chromosomes can still result in nonreciprocal recombination for markers which are closely spaced and in the hybrid DNA region (see below). Singlestrand insertions between circular duplexes have not been ruled out by these studies ; these would involve monomer “ring to ring” single-strand exchanges which, again, may or may not give reciprocal recombination (see below). Extensive DNA synthesis accompanies red (and possibly, rec) mediated recombination, this varying according to the chromosomal interval examined (F. W. Stahl and Stahl, 1971 ; M. M. Stahl and Stahl, 1971). Red promoted recombination, therefore, appears to be more involved than simple breakage and reunion. These and other data are interpretable in terms of the following mechanism for general recombination: a. Synapsis between homologous chromosomes (these being either linear or circular) is mediated by interaction through specific pairing regions (promotor regions), these first being “activated” to their Gierer state by a specific recombination structural protein (see Figs. 9 and 10). We speculate these recombination proteins to be the p protein in the h red pathway, and the recA protein in the bacterial rec pathway. Proteins such as these may be capable of recognizing specific nucleotide

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

455

sequences common to many different promotors, and one need not postulate all promotors to have identical base sequences (this point will be discussed again more completely in the next section, which discusses int and xis promoted recombination). The p protein would be expected to recognize common elements of A promotors more efficiently than the recA protein, which has evolved independently for bacterial recombination. The model predicts homology, therefore, between h and E . coli promotor sequences to account for recA promoted h recombination. b. The Holliday heteroduplex structure (mediating synapsis) can then migrate (by rotary diffusion) in either direction along parental DNA molecules (see Fig. 11). Recombination occurs through the mechanism shown in Fig. 18. Since the Holliday structure has 2-fold symmetry, it can act as a substrate for an endonuclease possessing 2-fold symmetry, which could simultaneously act to nick strands of the same polarity a t homologous sites (independent of base sequence) , this, perhaps, being triggered by mismatched base pairs in the hybrid DNA region. We postulate this nuclease to have, in addition, exonucleolytic activity, degrading either of the two nicked strands (in the rec pathway, this may correspond to the ATP-dependent nuclease coded by the recB and recC cistrons, which possesses both endonucleolytic and exonucleolytic activity ; in the red pathway, this may correspond to the reda coded 5’ exonuclease which (we postulate) has endonucleolytic activity not yet detected) . This drives the hybrid DNA region to the left and causes the right half of the migratory duplex to fall apart, fixing the starting point a t which hybrid DNA begins in (one of two) recombinant molecules. Exonucleolytic activity may be extensive, resulting in significant DNA repair synthesis. This may be especially prevalent in red-promoted recombination. c. Further migration of the hybrid DNA region, followed by nicking of homologous strands, results in reciprocal exchange, either of the singleor double-strand type. The hybrid DNA region, present in only one of two duplexes, may either remain heterozygous or, more probably, can undergo extensive excision and repair to give the recombinant or parental genotype. Markers that lie within the hybrid DNA region (this may correspond to more than half the chromosome in f l , for instance) would give rise to nonreciprocal recombination (this being the equivalent of gene coconversion in eukaryotes) . Markers that lie outside the hybrid DNA region could either remain in the parental configuration (singlestrand exchange) , or undergo reciprocal flanking marker exchange (double-strand exchange). Both types of events can take place between circular DNA molecules ; double-strand exchange would generate a new circle with double the circumference, whereas single-strand exchange would not.

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HENRY M. SOBELL

FIG. 18. A molecular mechanism for recombination in viruses. (A) Recombination event between two deletion mutants to give a parental genotype ( + a ) and a wild-type recombinant genotype (+ +). (B) Recombination event between two deletion mutants to give a double mutant genotype (ba) and a wild-type +). (C) Recombination event between a deletion mutant recombinant genotype (a) and a point mutant (b) to give a double mutant genotype (ha) and a wild-type recombinant genotype (++). (D) Recombination event between a deletion mutant (a) and a point mutant (b) to give a double mutant genotype (ha) and a wild-type DNA polymerase repair. recombinant genotype (+ +).0 ,Exonuclease activity; ---,

(+

The model is attractive in its simplicity and logical design. Moreover, it leads to a molecular mechanism for the Campbell (1962) model to explain site-specific recombination, and this will now be described.

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

457

2. Site-Specific Recombination

Lysogeny by temperate bacteriophages, such as A, involves the integration of a circular viral genome into the host chromosome, this involving a reciprocal recombination event between specific attachment sites on the viral and bacterial chromosomes (Campbell, 1962; for an outstanding review of lysogeny and site-specific recombination, see Signer, 1968 ; see other excellent recent reviews by Campbell, 1969; Gottesman and Weisberg, 1971; Echols, 1971). For A, this is accomplished through the int recombination pathway, which uses int function for integration (Zissler, 1967; Gingery and Echols, 1967; Gottesman and Yarmolinsky, 1968) , and int and zis function for excision (Guarneros and Echols, 1970; Kaiser and Masuda, 1970). Both int and xis function depend on the presence of attachment regions on the bacteria and virus, now known to be complex loci which, for purposes of discussion, shall be defined as follows: the bacterial attachment site, B (attB/att,) B’; the viral attachment site, P(attp/attp)P’. As will be seen later, the att, and att, regions are considered to be the sites for intimate synapsis between the bacterial and viral chromosomes and therefore would be expected to have exact homology. B, B’, P, and P’, however, need not have exact homology (only related homology) since (we postulate) these regions to be involved in protein-nucleic acid interaction, not in intimate synapsis. During prophage integration, two new attachment sites are generated : B (att,/attp) P’ and P (att,/att,) B’. One of the clearest demonstrations that B (attB/attB)B’, P(attp/attp)P’, B (attB/attp)P’, and P (att/att,)B’ are not identical comes from crosses between the transducing phages hgal and hbio under conditions in which general recombination is eliminated by mutations in red and rec (Echols, 1970). These transducing phages carry the prophage attachment sites, B (attB/attp)P’ and P (attp/attB)B’, respectively, and recombine with each other a t these sites to generate nontransducing h carrying P ( attp/attp)P’, and a hgal-bio transducing phage carrying B (att$attn) B’. This recombination mimics prophage excision and has been found to depend on xis as well as int function. Site-specific recombination in the reverse cross, A X hgal-bio, requires int but not xis (Echols, 1970). These findings can be summarized as follows: X[P(attp/attp)P’] X Xgal-bio[B(att~/att~)B’] int,zis 11int

Xgal[B(attB/attp)P’] X Xbio[P ( a t t ~ / a t t aB’l ) The role of helper phage in facilitating hgal transduction has been studied by Echols and Court ( 1971) and Weisberg and Gottesman (1971).

458

HENRY M. SOBELL

The integration defect of hgal may arise because of the combination hgal[B (attB/attp)P’] and host [B (ath/attB)B’] (the “structural defect” hypothesis of Guerrini, 1969). A helper phage may integrate first to provide two new potential integrative recombination structures: [B (attB/attp)P’] (prophage left end), and [P(attp/attB)B’] (prophage right end). These may then provide the proper steric environment for hgal integration. Experimental support for the general features of the structural defect hypothesis has ,been provided by a study of site-specific TABLE 4 Site-Specific Recombination by XgalaJ % Recombination for Xgal(BP’) with

int and xis genotype

Xgal(BP’)

Abio(PB’)

hgalbio(BB‘)

int+xis+ &-xis+ int+xis-

4.8 0.02 5.6

10 0.05 0.03

0.07 0.03 0.05

Adapted from Echols and Court (1971). Parental phages carried the susA32 or susP80 mutation (Campbell, 1961). The int- and xis- mutations employed were int2 (Gingery and Echols, 1967) and sisl (Guarneros and Echols, 1970). The Xgal phage used was the gal8 plaque-forming phage isolated by Court and Adhya and described previously (Echols, 1970). General recombination was eliminated by the use of a rec- host (strain 152) and red- phage carrying the red114 mutation (Echols and Gingery, 1968; Signer et al., 1968). Media (supplemented T-broth) and cross conditions have been described previously (Echols, 1970). Total progeny phage were scored by plating on C600 su+,and sus+ recombinants were scored by plating on MSO su-. The numbers in the table are (plaques on MSO/plaques on (3600) X 100. The data in column 1 represent the average of two separate experiments. The data in columns 2 and 3 are taken from Echols (1970) and are included for comparison. The red+ recombination frequency was 5.6 for gal X gal, 9.3 for gal X bio, and 5.3 for gal X gal-bio.

recombination between hgal and hgal, hbio and hgal-bio (Table 4). It is seen that in the presence of int and xis function, Agal[B(att,/att,)P’] can recombine with both hgal [B (attB/uttp)P’] and h’bio [ P (attp/attB)B’] , but poorly with hgal-bio [B (attB/attB)B’]. I n the presence of only int function, however, Agai [B (attB/attp)P’] recombines with only hgal [B (att,/att,)P’], this corresponding to the prophage left end (Echols and Court, 1971). The specificity which the int system has in promoting site-specific recombination had been inferred earlier by studies with the related lambdoid phage ~$80(Signer and Beckwith, 1966). Although the $80 attachment site (designated attsoB) is located close to the trp region, it

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

459

is possible to transpose the lac region to a position close to attRoBand to form $80dlac transducing particles. These transducing particles carry affinity for both the lac and the att,oB regions, and, potentially, can be inserted at either. Mixed infection with $80 and $8Odlac in a rec- host results in the attachment of $80dlac at attsoB,but not a t lac. Although +80dlac cannot attach a t either site by itself in a rec- host, it can in a re@ host, attachment occurring at the lac region. These experiments were interpreted to mean that $80 (but not $80dlac) directs the synthesis of a special site-specific recombination protein (s), which allows the defective transducing particle to attach at the attsoBsite. I n single infection, $80dlac lacks this site-specific integration pathway ; however, it can use the bacterial recombination system to integrate into the lac region. These and other data are consistent with the following mechanism for site-specific recombination: a. Synapsis between bacterial and viral chromosomes (these both circular) is mediated by interaction through specific pairing regions (the attachment regions designated attB and attp), these first being “activated” to their Gierer state by the int recombination structural protein. The int protein is capable of recognizing common homology in B, B’, P, and P’, this homology also appearing in portions of the att, and attp regions (see Fig. 19). After nicking complementary Gierer loops (we leave the specific endonuclease unspecified), synapsis occurs (through the structural intermediates shown in Figs. 9 and 10) to give the Holliday heteroduplex structure. We estimate the region for exact homology (attBand attp) to be between 20 and 30 base pairs (however, see Davis and Parkinson, 1971). The int protein differs from the p protein or the recA protein in its absolute specificity for the attachment region, being analogous to activator proteins which have absolute specificity for specific promotors (see Section 111). b. Owing to its small compact structure (in this case, 20-30 base pairs in either hybrid DNA region) and the nonidentity of B, B’, P, and P’, the Holliday heteroduplex does not migrate, but remains fixed a t the region of exact homology. It can undergo two fates (shown in Fig. 20) : single-strand exchange, resulting in excision ; double-strand exchange, resulting in integration. These two events may not be exactly equivalent, this, perhaps reflecting the steric influence of asymmetric combinations of B, B’, P, and P’, as discussed below. c. Starting from the prophage, this process can be reversed as follows: The attachment region, prophage left end [B (attB/attp)P’], and prophage right end [ P (attp/attB)B’] , are activated to their Gierer state by the int protein (see Fig. 19). Synapsis then occurs, as described previously. The synaptic structure can then undergo two fates (shown

460

HENRY M. SOBELL

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FIO.19. A hypothetical mechanism for the activation of the attachment region by the int protein using symmetry in protein-nucleic acid interaction. The bacterial attachment site is denoted IB(att~/att~)B’l.The viral attachment site is denoted [P(attp/attp)P’]. Subsequent events (shown in Figs. 9 and 10) lead to formation of a nonmigratory synaptic structure which leads to sitespecific recombination. Area shaded by slashed lines indicates base sequences which are exactly symmetric. Area shaded by stippled pattern indicates approximately symmetric base sequences (i.e., symmetry relating purines with purines, pyrimidines with pyrimidines). Both types of symmetric sequences can be utilized for intimate contact in protein-nucleic acid interaction.

in Fig. 21) : double-strand exchange, resulting in excision; single-strand exchange, resulting in integration (the model is very similar to that proposed by Cross and Lieb, 1967). The double-strand exchange event is associated with crossed strand interchange (Section IV, A, 2 ) . The configuration on either side of the synaptic structure is, therefore, [P,P’]/ [B,B’], immediately before excision. This differs from the configuration immediately prior to double-strand integration (which also requires a crossed strand-interchange event), [P,B’]/ [B,P’]. This asym-

461

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

metry may somehow code for excision (which requires both int and

xis function) or integration (which requires only int function). F-lac or (in the absence of site-specific recombination) +80dlac episomes can integrate into the lac region of the bacterial chromosome

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FIQ. 20. A molecular mechanism for site-specific recombination. Activation of the bacterial attachment site [ B ( a t t ~ / a t t ~ ) B ‘and l the viral attachment site [P(attp/attp)P’l is accomplished by the int protein, which uses symmetry in protein-nucleic acid interaction (see Fig. 19). This then allows synapsis to occur through the mechanism shown in Figs. 9 and 10. Because of the nonhomology between B, B’, P and P’ (as well as between the rest of the bacterial and phage chromosome), the synaptic structure does not migrate. It can undergo two fates: excision, due to single-strand exchange ; integration, due to double-strand exchange. See text for additional discussion.

by means of the rec pathway. Here, recA protein could “activate” promotor regions in the lac region (both on the chromosome and on the episome) to promote synapsis. I n contrast with synapsis in site-specific recombination, however, the Holliday heteroduplex can migrate throughout the region of homology. Recombination (leading to either excision or integration) can occur a t any time, this, perhaps accompanied by ex-

462

HENRY M. SOBELL

tensive excision and repair in regions of hybrid DNA. Double-strand exchange would result in integration, and, depending on the position of the synaptic structure with regard to marker alleles a t the moment of exchange, this may result in inversion of the transfer sequence of markers during conjugation by a given ,donor strain (Scaife and Gross,

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FI~. 21. A molecular mechanism for sitespecific recombination (continued), Activation of the prophage left end attachment site [B(att~/attp)P'l and the prophage right end attachment site CP(att~/atts)B'l is accomplished by the int protein. Synapsis occurs through the mechanism shown in Figs. 9 and 10. The synaptic structure can undergo two fates : excision, due to double-strand exchange ; integration, due to single-strand exchange. In addition to int, xis function is required for excision. See text for discussion. 1963; consult Fig. 139, p. 792, in Hayes, 1968). Subsequent synapsis followed by excision can lead to the interchange of marker alleles on the chromosome and on the episome. This can effect either the reciprocal exchange of alleles between the sex factor and the chromosome (i-e., z+y-/F - z y + becoming zy-/I? - z+y+) or (if the mutant allele happens to fall within the region of hybrid DNA) nonreciprocal exchange (i.e.,

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

463

z+y-/F - z y + becoming z+y-/F - zgT’) (Herman, 1965). A similar mechanism can account for Meselson’s data (Section IV, B, 4) (Meselson, 1967b). 3. Origin of Transducing Phage One of the attractive features of the Campbell (1962) model is its ability to explain a t least partially the origin of specialized transducing phages (see Signer, 1968; Echols, 1971). Rare “illegitimate” crossover events between neighboring circularized portions of the prophage and bacterial chromosome could conceivably occur, these leaving a piece of viral genome behind and adding a piece of bacterial genome. Events such as these would produce either defective or functional viruses depending on the extent of the viral genome deleted and its importance in viral function (Matsushiro, 1963; Manly et al., 1969; Shapiro et al., 1969). Although it is conceivable that illegitimate crossover events can arise as a result of weak homology between bacterial and viral DNA (present in promoter elements, for example), several lines of evidence point otherwise. First, the amount of bacterial and phage DNA in transducing particles is highly variable, and there is no obvious correspondence between breaks (primarily intragenic) that occur a t different regions in the prophage and bacterial genome (see Campbell, 1971). Second, mutations in int, red, or rec, either individually or in combination, do not interfere with the formation of gal transducing particles; in fact, increased numbers of transducing particles have been detected (see Franklin, 1971). This suggests that the excision of specialized transducing particles involves a mechanism that may differ from general or site-specific recombination, being perhaps similar to that responsible for the formation of bacterial and viral deletions (Franklin, 1971). Two different mechanisms can be considered to explain illegitimate recombination. The first involves a Campbell-like crossover between regions of DNA possessing limited homology or even nonhomology. This would involve circularization of the region involved, followed by doublestrand scissions, these, perhaps, mediated by a protein serving both a structural and catalytic role. Staggered cleavage could result in short single-stranded regions with cohesive ends, provided short sequences (even, perhaps, as small as dinucleotide sequences) exist which are complementary. Otherwise, one would have to postulate that sealing of polynucleotide chains (resulting in the circularization of episomes or in the formation of a continuous bacterial chromosome possessing a deletion) can take place in the absence of nucleotide pairing, the process being catalyzed by a protein able to transfer and join double helical ends

464

HENRY M. SOBELL

immediately after cleavage. The second mechanism depends on errors during DNA replication. Single-strand segments of DNA which possess weak secondary structure could be bypassed during replication, giving rise to deletions. The initial product of such an event would be a heterozygote, which could either segregate out during subsequent replication or undergo excision and repair. Both mechanisms unfortunately are highly speculative and difficult to test or distinguish experimentally. The origin of illegitimate recombination remains, therefore, a major unsolved problem. V. Mechanism for DNA Replication

This section examines current models of DNA replication in light of the theory which has been presented to explain structural aspects of transcriptional control and genetic recombination. I n particular, the presence of symmetrically arranged nucleotide sequences a t the ends of genes or operons (i,e., promotors) may serve the important additional role of priming the formation of Okazaki fragments with RNA oligonucleotides during DNA replication. The initiation of bidirectional chromosomal replication a t a specific origin may also reflect the activation of 8 replicator locus by an initiator protein using symmetry in protein-nucleic acid interaction, analogous to the mechanism postulated for positive control of RNA transcription. Abnormal branched DNA structures observed after extensive DNA synthesis in vitro could (in part) reflect the phenomenon of branch migration which can occur in regions possessing symmetrically arranged polynucleotide base sequences. Various aspects of DNA replication have been reviewed extensively in recent years (Gross, 1972; Klein and Bonhoeffer, 1972; Goulian, 1971; Pato, 1972; Lark, 1969; Helmstetter, 1969). A great deal of additional information in this area has appeared in recent months.

A. In Vitro REPLICATION MECHANISM Pioneering work by Kornberg and his associates led to the discovery of

DNA polymerizing enzymatic activity (DNA polymerase I) which is able to synthesize high molecular weight DNA using all four nucleoside triphosphates, Mg2+and double helical DNA as a template (for a classic review, see Kornberg, 1961). Synthesis proceeds by chain elongation in the 5‘ to 3’ direction, involving reaction of the 3‘-hydroxyl group on the growing chain with 5’-deoxyribonucleoside triphosphates. I n many respects, the enzymatically synthesized DNA in vitro appears to be a faithful copy of the input template DNA, having the same overall base

465

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

composition and nearest-neighbor frequency. However, the product demonstrates marked branching in electron micrographs and renatures readily after heat or alkaline denaturation. This, combined with the additional observation that extensively replicated DNA lacks biological activity (as measured by transformation) , early pointed to additional unknown factors important for in vivo DNA replication. A great deal of progress has taken place in recent years since these early studies;

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FIG.22. A mechanism for in vitro DNA replication. DNA polymerase first attaches to nicked regions of double-helical DNA possessing free 3’-OH termini. Synthesis then proceeds unidirectionally (5’ to 3’), displacing one of two DNA strands. When the growing point reaches a symmetrically arranged nucleotide sequence (postulated to be a portion of a promotor sequence), branch migration occurs allowing synthesis to continue “backward” from the replication fork. This gives rise to extensively branched DNA structures which rapidly renature after heat or alkaline denaturation. Although the fidelity of replication on the nucleotide level may be highly accurate during the in vitro synthesis, one would still expect the DNA to be inactive in transformation (even after extensive shear) since i t lacks the pairing region important for synapsis a t the ends of genes (promotors). The model predicts that synthetic DNA-like molecules which lack symmetrically arranged nucleotide sequences should remain unbranched, even after extensive in vitro replication.

however, it still seems meaningful to understand the possible origin of these branched structures. I n particular, the presence of symmetrically arranged nucleotide sequences a t the ends of genes could give rise to the phenomenon of branch migration during in vitro replication, and this (in part) may explain the origin of these branched structures. Refer to Fig. 22. DNA polymerase first attaches to nicked regions of DNA possessing free 3’-OH termini. Synthesis then proceeds unidirectionally (5’ to 3’) , displacing one of two DNA strands. When the growing point reaches a symmetrically arranged nucleotide sequence (postulated

466

HENRY M. SOBELL

to be a portion of a promotor sequence), branch migration occurs (Lee et al., 1970), allowing synthesis t o continue “backward” from the replication fork (Guild, 1968; Kornberg, 1969; Inman and Schnos, 1971). This would give rise to extensively branched DNA structures, which rapidly renature after heat or alkaline denaturation. Although the fidelity of replication on the nucleotide level may be highly accurate during the in vitro synthesis (Goulian and Kornberg, 1967), one would still expect the DNA to be inactive in transformation since it lacks the pairing region important for synapsis a t the ends of genes (promotors). A prediction which this model makes is that DNA molecules lacking ,symmetrically arranged nucleotide sequences should not demonstrate branching, even after extensive in vitro replication. Synthetic DNA-like polymers of the type, poly (dA) * poly (dT) , poly (dI) poly (dC) and poly [dG-dA-dA) J -poIy[dT-dT-dC) J should therefore remain unbranched, whereas polymers of the type, poly [ d (A-T) ] and poly [d (I-C) ] should exhibit marked branching. Although no information is available concerning poly [d (I-C) 1, poly [d (A-T) ] is, in fact, a branched structure as seen by electron microscopy (see Kornberg, 1961). Branching during ’replicationof homopolymer structures such as poly (dA) .poly (dT) would result in the formation of random-block copolymers, containing stretches of A’s and T’s covalently joined. This has not been observed.

-

B. In Vivo REPLICATION MECHANISM Advances in our understanding of the in vivo mechanism for DNA replication have taken place in three major areas in recent years. The discovery by De Lucia and Cairns (1969) of a mutant of E . coli (polAl) which lacks the Kornberg enzyme (DNA polymerase I) has stimulated the search for other DNA polymerase activities. Two other distinct polymerase activities have since been detected (DNA polymerases I1 and 111) (Knippers, 1970; Kornberg and Gefter, 1970, 1971; Moses and Richardson, 1970a,b; ,Gefter et al., 1971). Although their role in DNA replication is not clear, both enzymes catalyze 5‘ to 3t polymerization in the presence of the four nucleoside triphosphates in the same manner as DNA polymerase I. The isolation of polymerase-defective strains has led to characterization of membrane fractions actively engaged in semiconservative DNA synthesis (Smith et at., 1970; Knippers and Stratling, 1970; Okazaki et al., 1970; Ganesan, 1968). These systems are greatly stimulated by the addition of adenosine triphosphate (ATP). For reasons that are still unclear, however, the membrane activity is

SYMMETRY I N PROTEIN-NUCLEIC ACID INTERACTION

467

short lived; synthesis begins to level off after 1 or 2 minutes a t temperatures between 2OoC and 37OC (Smith et aZ., 1970). Moses and Richardson (1970~)have made the important additional observation that cells exposed to low concentrations of toluene become permeable to deoxynucleoside triphosphates and can continue to synthesize DNA in the presence of ATP. Replication has been shown to be semiconservative, and proceeds for about 1 hour a t a linear rate comparable to that observed in whole cells. This same rate of synthesis is observed in pol’ and polAl cells, implicating either polymerase I1 or polymerase 111 (or both) as the true replication enzymes. In addition, a type of DNA synthesis was detected that was not semiconservative (after exposure of toluenetreated cells to low concentrations of pancreatic DNase or to EDTA). This “repair” synthesis depends on DNA polymerase I activity; it is reduced a t least 20-fold in pol- cells, shows no ATP dependence, and is inhibited by DNA polymerase I specific antiserum. The discovery by Okaeaki et al. (1968) that a significant fraction of newly replicated DNA exists in the form of single-stranded fragments of low molecular weight (having sedimentation coefficients between 7 and 11 S) has provided strong evidence that favors a discontinuous DNA replication mechanism. Iyer and Lark (1970) and Werner (1971) have found that approximately half the pulse-labeled E . coli DNA is recoverable as small fragments, the rest remaining in larger-sized material. Through analysis of pulse-labeled DNA fragments using specific exonucleases, Okaeaki and Okazaki (1969) have demonstrated that phage T4 DNA synthesis proceeds in vivo in a 5‘ to 3’ direction. Direct electron microscopic visualization of replicating phage A DNA has shown that one of two branches at each replicating fork is frequently connected with the unreplicated parental double helix by a segment of singlestranded DNA, as might occur during discontinuous DNA synthesis (Inman and Schnos, 1971). These data, taken together, are compatible with models (C) and (D), shown in Fig. 23; both models involve the continuous replication of one DNA strand and the discontinuous replication of the other. Considerable excitement has been generated by the recent demonstration that RNA synthesis primes the initiation of DNA synthesis (Brutlag et al., 1971; Wickner et aZ., 1972; Sugino et al., 1972; Clewell et al., 1972). It had been known for many years that DNA polymerases from E. coli and phage-infected cells could extend preexisting chains, but were incapable of initiating new ones (e.g., see Goulian and Kornberg, 1967; Goulian et al., 1968). Thus, although single-stranded circles of M13 or 4x174 phage DNA act as excellent templates for E . coli DNA polymerase, small oligonucleotides must first act as primers to initiate

468

HENRY M. SOBELL

synthesis (Mitra et al., 1967; Goulian and Kornberg, 1967; Goulian, 1968). RNA polymerase, on the other hand, can initiate new chains (Maitra and Hurwitz, 1965), and a transcriptional event could initiate DNA synthesis. Brutlag et al. (1971) has discovered that the conversion of single-stranded DNA of bacteriophage M13 to the double-stranded

(C)

( D)

FIQ.23. Four models for DNA replication at the growing fork: (A) 5’ to 3’ and 3’ to 5‘ synthesis for both strands. Synthesis can be continuous (as shown) or discontinuous; (B) 5’ to 3’ discontinuous synthesis for both strands; (C) 5’ to 3’ continuous synthesis for one strand, discontinuous for the other; (D) 5’ to 3’ continuous synthesis for one strand, looping back periodically to the other parental template strand. Periodically, the apex of the loop is nicked, giving rise to discontinuous synthesis of the other strand.

replicative form in E . coli is blocked by rifampicin, an antibiotic that specifically inhibits the host RNA polymerase. Rifampicin also blocks the multiplication of the double-stranded replicative form. The conversion of single-stranded M13 to the double-stranded replicative form is also inhibited by actinomycin (Schekman et aE., 1972). This implies a transcriptional priming role for RNA polymerase and the presence of a double-helical hairpin structure a t or near its site of attachment. Sugino, Hirose, and Okazaki (1972) have demonstrated the existence

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

(A)

469

(C)

FIG.24. A mechanism for in vivo DNA replication. (A) Initiation of replication begins with a bidirectional transcriptional event at the origin of replication. The origin contains the replicator locus, a DNA segment consisting of a symmetrically arranged nucleotide sequence which must first be activated to its Gierer state by a specific structural protein (the initiator protein) to enable attachment by RNA polymerase. (B) Short RNA oligonucleotides can then prime for bidirectional DNA chain growth. (C) At each replicating fork, one DNA strand is synthesized continuously, the other discontinuously (the model can readily be modified to allow discontinuous synthesis of both DNA strands). The latter type of synthesis reflects the presence of promotor sequences which (when single stranded) can assume hairpin structures that bind RNA polymerase tightly. (D) This then allows RNA-primed DNA synthesis to proceed backward from the replication fork. (E) Subsequent removal of RNA oligonucleotides by nuclease action, followed by the repair of gaps by DNA polymerase activity and the sealing of nicks by polynucleotide ligase completes the mechanism for semiconservative DNA replication. -, RNA oligonucleotide primer; - DNA single strand; --- excision of RNA/ DNA and polymerase repair; 0, origin; P, promotor.

of short RNA chains covalently attached to newly replicated DNA fragments in E . coli. DNA extracted from E. coli (previously labeled with a brief pulse of radioactive thymidine) and then denatured with either heat, formamide or formaldehyde bands in a cesium sulfate equilibrium

470

HENRY M. SOBELL

density gradient a t a position with a density higher than that expected for single-stranded DNA. If, however, first treated with alkali or RNase, it exhibits the density expected for single-stranded DNA. Further studies of these nascent DNA fragments after pulse-labeling cells with tritiated uridine suggest the RNA segment to be between 50 and 100 nucleotides. The DNA segment is between 1000 and 2000 nucleotides, a length corresponding to the size of a typical cistron (Okazaki et al., 1968). These and other data are consistent with the following model for in vivo DNA replication (see Fig. 24): Initiation of replication begins with a bidirectional transcriptional event a t the origin or replication. The origin contains the replicator locus, a DNA segment consisting of a symmetrically arranged nucleotide sequence (analogous to a promotor sequence), which must first be activated to its Gierer state by a specific structural protein (i.e., the initiator protein) to enable attachment by RNA polymerase (Fig. 24A). Short RNA oligonucleotides can then prime for bidirectional DNA chain growth (Fig. 24B). At each replicating fork, one DNA strand is synthesized continuously, the other discontinuously (the model can readily be modified to allow discontinuous synthesis of both DNA strands). The latter type of synthesis reflects the presence of promotor sequences which (when single stranded) can assume hairpin structures that bind RNA polymerase tightly (Fig. 2 4 0 . This then allows RNA-primed DNA synthesis to proceed backward from the replication fork (Fig. 24D). Subsequent removal of RNA oligonucleotides by nuclease action, followed by the repair of gaps by DNA polymerase activity and the sealing of nicks by polynucleotide ligase completes the mechanism for semiconservative DNA replication (Fig. 24E). VI. DNA Restriction and Modification Mechanisms

For excellent recent reviews in this area see Arber and Linn (1969), Boyer (1971), and Meselson, Yuan, and Heywood (1972). The phenomenon of host-controlled modification and restriction refers to the ability of a bacterial strain to allow or to restrict growth by an infecting bacteriophage, this depending on the modification properties of the previous strain on which the phages were grown. Thus, for example, phage A prepared on E . coli strain K (A.K) plates efficiently on strain K; however, when plated on E . coli strain B, only meager growth is observed. On the other hand, phage A prepared on E. coli strain B (LB) plates efficiently on strain B; however, i t now plates with poor efficiency on E. coli strain K. The restriction and modification of phage A by several host specificities found in E . coli has been extensively studied by genetic and biochemical methods, and this has led to the

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

47 1

general concept that two enzymes, an endonuclease and a methylase, are responsible for the restriction and modification of DNA. The restriction endonuclease makes a double-strand scission a t a specific sequence of base pairs if it is unmodified, and the modification methylase modifies this sequence (by forming, for example, 6-methylaminoadenine) to render it insensitive to the restriction enzyme. Most of our understanding of the biochemistry of restriction and modification comes from studies of five different systems of E. coli, called B, K, P, RI and RII. In addition, the properties of a restriction endonuclease from Hemophilus influenzae have been studied, and, as mentioned previously (Section 11, A, 2 ) ) these have had interesting implications for the mechanism of restriction. The B and K systems are named after the E . coli strains in which they are found. They are closely related, being specified by alleles of the same genetic locus, the hs (host specificity) locus. The P system is carried by phage P1 and is expressed in cells lysogenic for P1, while the Rr and RII systems are carried by plasmids called drug-resistance transfer factors. The restriction endonucleases can be categorized into two general classes (see Boyer, 1971; Meselson et al., 1972). The first class (type I) includes the B, K, and P endonucleases. These are all high molecular weight enzymes (the estimated molecular weights for both the B and K endonuclease is about 400,000; for the P endonuclease, about 200,000), and all possess subunit structure. Each requires adenosine triphosphate (ATP), S-adenosylmethionine (SAM) and Mgz+ for activity. Highly purified enzyme preparations are active on native unmodified DNA and produce a limited number of double-strand breaks using a two-step mechanism (Meselson and Yuan, 1968; Roulland-Dussoix and Boyer, 1969). Unmodified single-stranded DNA is not attacked by the B restriction endonuclease (Linn and Arber, 1968) and h DNA consisting of one modified and one unmodified polynucleotide chain is completely resistant to attack by the K restriction endonuclease (Meselson and Yuan, 1968). Massive hydrolysis of ATP accompanies type I endonuclease activity. This could reflect a conformational change accompanying enzyme-substrate recognition (which, for some reason, occurs repetitively in the in vitro reaction), and it has been speculated that this involves the formation of a Gierer-like DNA structure a t the recognition site (see Meselson et al., 1972). The second class of restriction endonucleases (type 11) include the Hemophilus restriction enzyme and the RI and RII enzymes. These enzymes have relatively low molecular weights (the Hemophilus enzyme is estimated to be about 67,000; the R, enzyme about 80,000; the RII enzyme about lOO,OOO), and all are probably dimers with identical sub-

472

HENRY M. SOBELL

units. I n contrast with the type I endonucleases, neither ATP or SAM is required for activity; only Mgz+ and unmodified double-helical DNA is required. Another difference concerns the number of double-strand breaks per unit length of unmodified DNA mediated by these enzymes. Both the Hemophilus and R-factor restriction endonucleases make approximately one break per 1000 base pairs, whereas the B restriction enzyme makes 1-2 breaks per 5000 base pairs. This suggests that the recognition site on DNA for the Hemophilus and R factor restriction endonucleases may involve fewer nucleotides (therefore coming up more frequently on a random basis) than the K or B recognition sites, which may be slightly longer. Figure 25 shows sequences of recognition sites for three restriction endonucleases: (A) Hemophilus influenme endonuclease R (Kelly and Smith, 1970), (B) E. coli RI endonuclease (Hedgpeth e t al., 1972), (C) E . coli RII endonuclease (Bigger, Murray, and Murray, 1973; Boyer, Dugaicyzk, Chou, Hedgepeth, and ,Goodman, 1973). Symmetric sequences possessing exact symmetry have been shaded with slashed lines, while those related by “approximate” symmetry (i.e., symmetry which only relates purines with purines, pyrimidines with pyrimidines) have been shaded by B stippled area. It is evident that both types of symmetric sequences play a role in specificity; this depends on the detailed stereochemistry of protein-nucleic acid interaction (see discussion in Section IV, C, 2 and examine Fig. 19). Figure 25(D) demonstrates that approximate 2-fold symmetry exists in (and around) the sequence for the cohesive ends of A (Wu and Taylor, 1971; Weigel, Englund, Murray, and Old, 1973). It may therefore be a substrate for a nuclease enzyme possessing 2-fold symmetry, akin to a restriction endonuclease. An interesting finding observed with several type I restriction endonuclease enzymes (in particular, the K and P endonucleases) is their possession of modification (methylase) activity. Recent purification of the modification methylase of E . coli B has shown that the enzyme contains two nonidentical polypeptides, p and 7, molecular weights 60,000 and 50,000, respectively (Linn et al., 1972). The structure appears to be plyl (it is not yet certain, however, whether the active form is p 2 y 2 ) . The enzyme has an absolute requirement for S-adenosylmethionine and, although it can methylate a wide variety of DNAs, it cannot methylate E . coli B DNA. These workers have also purified the restriction endonuclease of E. coli B. The enzyme consists of three polypeptides, (Y, p, and y, molecular weights 135,000, 60,000, and 55,000. The p and y subunits are indistinguishable from the p and y subunits found in the modification methylase. These observations are consistent with a previous

SYMMETRY IN PROTEIN-NUCLEIC ACID INTERACTION

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prediction by Boyer (1971) that type I restriction endonucleases and modification methylases have similar subunit structure: (a) the methylase enzyme may consist of two recognition subunits and two

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methylating (SAM-requiring) subunits ; (b) the endonuclease enzyme may have this same core structure, but in addition possess two ATPrequiring nuclease subunits, the complete structure retaining 2-fold symmetry, SAM may play a regulatory role, inhibiting methylase activity and promoting nuclease activity by an allosteric mechanism. Type I1 endonucleases and methy lases are distinct from one another, and possibly consist of dimers containing identical subunits. Since both enzymes (presumably) recognize the same restriction site, they probably have regions of exact homology in amino acid sequence. This may reflect their common ancestral origin during evolution billions of years ago. VII. Chromosome Structure

It is clear that any theory for genetic recombination (and, for that matter, DNA replication and RNA transcription) must take into account some of the known structural features of eukaryotic chromosomes. This section is included to present a speculation concerning the structure of the lampbrush chromosome during the diplotene phase of meiosis. Because of space (and time) limitations, no attempt will be made to review the extensive literature in this area. One can imagine two states possible for a long DNA molecule: an extended linear state, shown in Fig. 26A (the dark line represents either B

FIG.26. A model for DNA folding. (A) Extended linear state for a long DNA molecule. The dark line represents either the DNA double-helix or, more probably in eukaryotic organisms, a coiled-coil structure composed of DNA-histone. (B) More compact folded state formed by interactions between specific structural protein(s) which bind at ends of genes or clusters of genes.

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FIG.27. Possible origin of the morphology of the lampbrush chromosome observed during the diplotene phase of meiosis. Chromosomal replication initiates a t promotor regions and progresses to give a paired bivalent structure stabilized by protein-protein interactions. RNA synthesis initiates at promotor regions (which lie along the axis of the chromosome) to give a gradient of increasing RNA chain lengths as one traverses the loops. Synapsis between homologous chromatids would be expected to occur near the axis of the chromosome and not in the peripheral loops. Line denotes DNA double helix, or more probably, a coiled-coil structure composed of DNA-histone.

the DNA double helix or, more probably in eukaryotic organisms, a coiled-coil structure composed of DNA-histone) and a more compact folded state, shown in Fig. 26B, formed by interactions between specific structural protein (s) (these being similar, perhaps, to the recombination structural protein (s) postulated to “activate” promotors for synapsis during viral, bacterial and fungal genetic recombination) which bind at the ends of genes or clusters of genes. A possible origin of the morphology of the lampbrush chromosome is shown in Fig. 27. Chromosomal replication first initiates a t promotor regions, and this progresses to give a paired bivalent structure stabilized

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by (meiotic) protein-protein interactions. RNA synthesis then initiates at promotor regions (which now lie along the axis of the chromosome) to give a gradient of increasing RNA chain lengths as one traverses the loops (for evidence concerning this, see Gall, 1955; Callan, 1963). Synapsis between homologous bivalent chromatids would be expected to occur near the axis of the chromosome and not in the peripheral loops. For a discussion of the morphology of lampbrush chromosomes and their possible etiology, see Gall (1963). VIII. Summary

Symmetry considerations of protein-nucleic acid interaction suggest the existence of an alternate branched configuration for DNA induced by binding specific structural proteins to symmetrically arranged polynucleotide base sequences. The concept that such sequences exist at the ends of genes or operons leads to a molecular theory interrelating genetic recombination, DNA replication, and RNA transcription. The theory accounts for a large amount of data in these areas and makes a wide range of testable predictions. Its most fundamental prediction (i.e., the presence of symmetrically arranged nucleotide base sequences a t the ends of genes or operons) can be tested in the near future. ACKNOWLEDGMENTS This work has been supported in part by granta from the National Institutes of Health, the American Cancer Society, and the Atomic Energy Commission. This paper has been assigned report No. UR-3490-180 at the Atomic Energy Project, the University of Rochester.

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AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed.

A Aase, H. C., 241, 268 Abd-El-Al, A., 48, 69 Abelson, J. N., 11, 12, 13, 21, 69, 82, 88, 97 Abelson, P. H., 42, 43, 69 Bbro, A., 301, 362 Acton, A,. B., 311, 312,352 Adams, D. H., 116, 132, 146 Adams, J. N., 446, 484 Adelberg, E. A., 2, 77, 450, 476, 489 Agrawal, B. B. L., 6, 14, 24, 98 Akaboshi, E., 18,86 Akrigg, A., 442,47& Alberts, B., 428,487 Albrecht, A. M., 44, 69 Alexander, H. E., 442,476 Alexander, J., 184, 264 Alikhanian, S. I., 57, 69 Allchin, F. R., 161,264 Allen, M. K., 6, 69, 443, 444, 480 Allen, S. L., 37,71 Allison, A., 143, 146 Altman, S., 11, 21, 69 Amati, P., 439, 476 Ames, B. N., 15, 17, 23, 26, 27, 30, 34, 63, 64, 65, 69, 79, 83, 96, 102, 420, 488 Amos, B., 142,146 Anandan, M., 254, 264 Anderson, E., 164, 165, 168, 176, 196, 232, 264, 265 Anderson, K. W., 13, 69 Anderson, N. A., 116, 131, 148 Anderson, P. M., 48, 69 Anderson, R. C., 299, 362 Anderson, R. L., 58, 78 Anderson, W. A., 301, 308, 362 Anderson, W. B., 422, 423, 479

Andersson-Kotto, I., 43, 69 Ando, S., 200, 290 Andoh, T., 11, 69 Angladette, A., 154, 179, 186, 193, 194, 196, 197, 266 Anton, D. N., 420, 487 Antonovics, J., 117, 137, 1.45 Apirion, D., 36, 77, 97 Appella, E., 24, 74 Arasu, N. T., 108, 146 Arber, W., 470, 471,476, 484 Arditti, R. R., 16, 17, 51, 69, 98, 422, 423, 463, 476, 487 Arnold, C. G., 117, 137, 146 Arnott, S., 13, 70 Ashwcll, M., 56, 70 Atkins, J. F., 16, 17, 27, 28, 70, 96 Atkinson, D. E., 29, 70 Atwood, K. C., 20, 61, 70, 96 Aubertin, M., 117, 137, 148 Avallone, G., 294,345,348,354 398, 408 Avdulow, N. P., 262,266 Avery, 0. T., 442, 477 Ayad, S. R., 442, 4Y6

B Baccetti, B., 301, 362 Backer, C. A., 162, 168, 207, 211, 220, 221, 225, 266 Bacon, D. F., 44, 45, 70, 86, 101 Baglioni, C., 30, 31, 70 Bahn, E., 38, 42, 70 Baich, A., 44, 45, 70,101 Bailey, W. T., 452, 479 Baille, D. L., 32, 70 Bairati, A., 295, 301, 303, 304, 305, 308, 311, 312, 313, 320, 321, 331, 335, 362 Baker, B. S., 381, 403, 406, 407

491

492

AUTHOR INDEX

Baker, C. M. A.,46,90 Baker, H.G.,165, 183,266 Baker, R. M., 324,366 Balbinder, E.,16,70, 74 Banerjee, S.,59,80 Barben, H., 20,26,70 Barbour, S. D.,4, 46,47,70 Bardenas, E.A., 180,186,187, 188,266 Barigozzi, C.,67,70 Barish, N.,30, 70 Barker, G. R., 442, 476 Barnabo, J. M.,326, 338, 360, 365, 409 Barner, H.D.,57,76 Barnett, B., 345, 362 Barnett, H.L., 130, 146 Barnett, L.,6, 11, 12, 13, 17, 18, 22, 27,69,70, 73,76, 98 Barnoux, C.,466, 481 Baron, L. S.,59,78 Barr, D.,27,10.4 Barr, G.C.,443, 481 Barratt, R. W., 42, 53, 71 Barrau, J., 161, 178,266 Barrett, D.K.,116,132,146 Barry, E.G.,42,71 Barth, P.T., 57,58,71 Barton, D.W.,252, 286 Basak, S. L., 165, 172, 201, 266 Bastock, M.,319, 320,362 Bateman, A. J., 304, 339, 362, 363, 362, 388, 406 Bauch, B., 116, 129,145 Bauerle, R. H.,10, 15, 16, 17, 63, 64, 71, 83, 90, 98, 103 Baumberg, S.,44,45,85,101 Bautz, E. K.F., 422,477, 478 Bautz, F.A,, 422,477 Baylor, M.B., 37,71 Beacham, I. R.,57,58,71 Beachell, H. M., 241, 244, 247, 266 Beadle, G.W., 30,31,71,100,122,145 Beale, R.A.,162,866 Bealle, S.,313, 366 Beatty, R. A., 305, 306, 307, 338, 362 Becker, E.,30, 7 3 Beckmann, O.,443,486 Beckwith, J. R., 2, 15, 16, 17, 20, 35, 36,51,58,68, 69,71, 82, 86, 93, 97, 421, 4B, 423, 458, 463,476, 486, 487,488. 490

Beermann, W.,307, 368 BeissonSchechroun, J., 109, 146 Belcour, L.,131, 146 Bender, K.,249,266 Benson, C.E.,54,71 Benzer, S.,20, 71 Berg, P.,22, 61, 62, 71, 74, 79, 80, 84, 98 Berger, H., 6, 18, 47, 71, 72, 88, lo4 Berman, M.,52, 72 Berman-Kurtz, M., 52, 72 Bernardi, G.,417,477 Bernet, J., 109, 131, 146 Bernhardt, S. A., 39, 40, 42, 72, 103 Berry, E. A., 128,145 Bertani, E. T.,37, 71 Bertolini, B., 326,363 Bertrand, H., 55, 56, 72, 83 Bertsch, L. L.,468,486, 487 Bevan, E. A., 116, 127, 128, 145, 162 Bhalerao, S. G., 162,266 Bhanduri, P.N.,235, 266 Bhide, R.K.,162, 265 Bianchi, A., 154, 266 Bigger, C.A., 472, 477 Biggs, R.,130,145 Binnard, R.,308, 368 Birge, E.A., 36,72 Birnstiel, M.L.,61,72 Bjare, U.,36,72 Blaich, R.,109, 111, 146 Blaney, W.M., 308,343,344,345,363 Blank, J., 15,58,96 Blinkova, A. A.,448,477 Blume, A. J., 16,70 Bodenstein, D.,20, 87 Bodmer, W.F.,443,477 Bosiger, E.,318, 363 Boidin, J., 130, 146 Bojalil, L.F.,43,91 Bolle, A.,11, 78 Boncinelli, E.,61,96 Bonhoeffer, F. J., 448, 464, 466, 467, 477, 483, 488 Bonner, D. M., 23, 24, 42, 43, 72, 78, 99, 101 Bonner, M., 10,16,lO4 Bonney, R.J., 58,72 Bonnier, G.,2,72

493

AUTHOR INDEX

Boon, T., 452,grr Bor, N. L., 168, 207, 208, 211, 266 Bouharmont, J., 168, 172, 190, 191, 213, 214, 216, 227, 228, 235, 237, 247, 249, 256, 261, 262, 266, 266 Boulter, A. B., 42, Y9 Boulter, M. E., 130,146 Bourgeois, S., 17, 63, Y2, 418, 486 Boyce, R. P., 442, 478 Boy de la Tour, E., 11,78 Boyer, H., 58, Y8, 471,487 Boyer, H. W., 470, 471, 473, 477, 482, 487 Boyer, S. H., 46, 72 Bradford, R. M., 57, 73 Bradley, A. C., 253, 286 Brambl, R. M., 56,72 Brammar, W. J., 6, 18, 71, Y2, 104 Braver, G., 385,386, 396, 398,399,408 Brawner, T. G., 123,161 Breitman, T. R., 57, 73 Brenneman, J. A., 116, 121, 161 Brenner, S., 6, 11, 13, 15, 16, 17, 18, 21, 22, 27, 69, YO, 73, 76, 82, 87, 93, gy, 98, 423, 448, 483, 488 Bresch, C., 452, 47Y Bresler, S. E., 444, 448, 477 Brewen, J. G., 388,409 Bridges, C. B., 2, 30, 31, 32, 45, 62, 63, 66, 73, 92, 97, 99 Brieger, F., 108, 146 Brieger, G., 313, 363 Brinton, C. C., 448, 449,4Y7 Brinton, C. C., Jr., 449, 47Y Brito da Cunha, A., 59,90 Brockman, H. E., 8,YS Brodie, H. J., 117, 133, 146 Brody, S., 62, 73 Broker, T. R., 427, 428, 477 Bromberg, F. G., 51, 97 Brooks, G. T., 66, 67, 73 Brooks, K., 452,477 Brosseau, G. E., Jr., 338,363 Brown, D. D., 61, 73 Brown, L. R., 154, 266 Brown, S. W., 224, 266 Brownstein, B. L., 36,88 Bruni, C. B., 16, 73, 96 Brutlag, D., 467, 468, 477, 487, 489 Bryant, P. J., 67,73

Erzozowski, T. H., 48, 73 Bucher, T., 56, 97 Buchi, H., 22, 87 Buhler, R., 312, 363 Burdette, W. J., 61, 67, 68, Y3 Burgess, R. R., 422, 423, 478, 489 Burgoyne, P. S., 307, 362 Burkill, I. H., 158, 161, 266 Burnet, B., 66, 67, 73 Burnett, J. H., 129, 130, 143, 146 Burnham, C. R., 260, 266 Burns, R. O., 32, 33, 34, 48, 49, 73, 83 Burns, S. N., 450,4Y6 Burt, M. E., 48,49,101 Burton, E. G., 53, 54,73, 98 Butcher, A. C., 116, 124, 125, 146 Butenandt, A., 30, 73 Butler, L., 333, 367 Butterworth, F. M., 313, 363 Buttin, G., 46, 103 C

Cabet, D., 42, Y4 Cairns, J., 450, 466, 478, 4Y9 Calef, E., 439, 478 Callahan, R., 111, 16, 74 Callan, H. G., 439,476, 478 Calvo, J. M., 32, 33, 34, 35, 48, 73, 74 Campbell, A., 2, 74 Campbell, A. M., 450, 454, 456, 457, 458, 463, 478 Campbell, J. H., 22, 23, 88 Campbell, W., 24, 98 Capes, N., 34, 74 Capinpin, J. M., 162, 207, 209, 210, 244, 266 Carbon, J., 21, 27, 62, Y4, 84, 96, 98 Carlile, M. J., 116, 119, 146 Carlton, B. C., 5, 6, 8, 74, 104 Carmichael, J. W., 116, 127, 161 Carnahan, J., 449, 4Y7 Caro, L., 448, 481 Caroline, D. F., 41, 42, 74 Carpena, A. L., 260, 266 Carpenter, A. T. C., 371, 373, 377, 381, 403, 406, 407, 408 Carr, A. J. H., 128, I46 Carsiotis, M., 24,74 Carter, D. M., 452, 486

494

AUTHOR INDEX

Carter, T., 15, 74 Case, M. E., 23, 24, 57, 76 Cashel, M., 423, 478, 489 Caspar, D. L. D., 411, 412, 478 Caspari, E., 32, 76 Cassuto, E., 432, 478 Castaiieda, M., 42, 76 Castiglioni, M. C., 67, 70 Caten, C. E., 108, 116, 124, 125, 146, 148 Cato, A., Jr., 443,478 CavalliSforza, L. L., 448, 449, 478, 484 Chadwick, P., 418, 478, 486 Chalmers, J. H., Jr., 23, 76 Chamberlin, M., 424, 442, 478 Champe, S. P., 20, 71 Chan, T.-S., 21, 23, 76 Chandler, R. F., Jr., 154,266 Chandley, A. C., 304, 363, 362, 388, 406 Chandraratna, M. F., 154, 196, 199, 203, 266 Chandrasekharan, P., 209, 254, 266, 276 Chang, C. C., 168, 208, 210, 211, 214, 215, 231, 272 Chang, F. N., 36,76 Chang, K. C., 161, 162, 221, 266 Chang, T. D., 260, 283 Chang, T. M., 260,271 Chang, T. T., 154, 180, 186, 187, 188, 200, 205, 214, 265, 266, 268, 290 Chang, W. T., 163, 166, 170, 174, 175, 178, 179, 180, 181, 184, 185, 186, 188, 193, 194, 196, 202, 203, 213, 241, 242, 247,260, 266, 271, 279,286 Changeux, J. P., 411, 412, 478, 486 Chao, C. Y., 259, 260, 266 Chao, L. F., 256, 261, 263, 266 Chapman, R. F., 329, 353 Chargaff, E., 443, 487 Charles, H. P., 41, 76 Chase, M., 439, 478 Chater, K. F., 64, 65, 76 Chatterjee, D., 155, 156, 157, 158, 159, 162, 166, 168, 170, 185, 186, 207, 211, 213, 217, 218, 220, 266 Chen, B., 422, 423, 479, 486 Chen, C. C., 168, 169, 172, 186, 191, 206, 210, 214, 217, 222, 223, 226, 227, 228, 2-30,231, 87'6,277

Chen, P. S., 312, 363 Chen, R., 422,423,479 Cherest, H., 76 Chevalier, A., 155, 156, 162, 163, 180, 181, 204, 205, 207, 211, 213, 217, 218, 220, 221, 222, 266, 267 Chevalley, R., 11, 78 Childress, D., 294, 339, 349, 363, 366, 384, 407 Chipchase, M., 61, 72 Chou, C., 222, 229, 2991 Chou, S. L., 202, 2b7 Chovnick, A,, 32, 70, 350, 365 Chowdhury, K. A., 161,267 Chu, Y. E., 135, 146, 163, 166, 172, 181, 185, 186, 187, 190, 191, 195, 204, 206, 234, 235, 239, 240, 241, 242, 244, 256, 257, 259, 261, 263, 2b7 Church, R. J. H., 179,267 Clark, A. J., 2, 4, 46, 47, 70, 76, 77, 88, 103, 442, 452,477, 478,489 Clark, J., 116, 119, 120, I46 Clark, J. D., 179, 182, 183, 267 Clavilier, L., 54, 55,76, 94 Clayton, C. W., 116, 121, 162 Clayton, W. D., 156, 167, 180, 206, 207, 213, 216, 267 Clewell, D. B., 467, 479 Coen, D., 57, 76 Cohen, S. S., 57, 76 Colin, M., 17, 63, 72, 103 Coleman, J. R., 425, 486 Collins, 0. R., 116, 117, 119, 120, 146, i49 Connaway, S., 422, 423,476 Contesse, G., 15, 76 Cook, R. M., 334, 363 Coonradt, V. L., 122, i46 Cooper, K. W., 301, 307, 363, 384, 385, 386, 387, 392, 396, 399, 400, 402, 406 Cordaro, C., 15, 76 Coun:ne, S. J., 342, 363 Coursey, D. G., 184,264 Court, D., 457, 458, 479 Cox, B. S., 26, 76 Cox, E. C., 10, 86 Cramer, F., 13, 76 Cranston, J. W., 467, 479 Crasemann, J. M., 15,99

AUTHOR INDEX

Cravens, M., 14, 99 Crawford, E. M., 449,4Y9 Crawford, I. P., 15, 86 Crick, F. H. C., 6, 17, 18, 21, 22, 25, 27, YO, 73, Y6, 411, 479 Croft, J. H., 116, 124, 125, 146, 148 Croissant, J., 334,363 Crosby, E. F., 46, 72 Cross, R. A., 460,479 Crow, J. F., 293, 366, 364, 365, 377, 406, 407 Crowe, L. K., 117, 136, 149 Csuk&s-Szatl6czky,I., 67,94 Cua, L. D., 201, 244, 247, 248, 267 Cuellar, H. S., 117, 146 Cunin, R., 16, 17, 63, 64, 76, 78 Cunningham, S., 57, 78 Curry, J. B., 21, 62, 74 Curtiss, R., 448, 4Y9 Cuzin, F., 448,483

D Dana, S., 165, 172, 266 Darby, L. A,, 117, 136,148 Darlington, C. D., 158, 262,268 Das, D. C., 234, 235, 237, 268 Datta, P., 20,46, Y6 Davey, K. G., 300,363 David, J., 312, 332, 333, 334, 363 Davidoff-Abelson, R., 443, 479 Davidson, N., 428, 466, 483, 484 Davies, G. E., 36, 76 Davies, J., 2, 36, 76 Davis, B. K., 381,407 Davis, P. H., 212, 268 Davis, R., 459, 4Y9 Davis, R. H., 38, 39, 40, 41, 42, 43, 72, Y4, Y6, YY, Y9, 102, 103 Davis, R. W., 428, 466, 484 Dawid, I. B., 61, 73 Dawson, G. W. P., 9, 10, 33, 34, 77, 96, 98, 99 Day, P. R., 425, 480 De Busk, A. G., 40,96 de Candolle, A,, 162, 171, 177, 268 de Crombrugghe, B., 422,423,479,486 De Duve, C., 143, l4Y Dee, J., 116, 119, 120, 146, 147, 161

495

de Issaly, I. M., 38, 39, 41, 42, 96 Deland, M., 350, 366 DeLange, R. J., 55, 57,77 Delbruck, M., 452, 479 Del Solar, E., 319, 320, 363 DeLuca, M., 29, Y7 De Lucia, P., 466, 479 De Marco, A., 365,408 Demerec, M., 2, 44, 48, 49, 50, 60, 77, 81, 86, 91, 103, 293, 304, 325, 328, 339, 341, 342, 343, 364, 366 DeMoss, J. A., 23,72 Denburg, J., 29, 77 Denell, R. E., 18, 77, 348, 364, 379, 380 381, 384, 399, 406, 407 Denhardt, G. H., 11, 78, 447, 488 Dennert, G., 15, 84 Deppe, G., 15, 84 deRobichon-Szulmajster, B., 76 De Serres, F. J., 123,147 de Souza, H. L., 59,90 de Toledo, J. S., 59,90 Dettori, R., 449, 479 Deusser, E., 36, 77 Deutseh, J., 57, 76 Devasahayam, P., 247, 248, 684 De Vries, J. K., 322, 336, 338, 339, 347, 364 de Wet, J. M. J., 165, 212,232,268,870 De Wilde, J., 316, 364 Dhillon, T. S., 144,147 Diacumakos, E. G., 55,7Y Diem, C., 312,363 Dimbleby, G. W., 193, $90 Di Pasquale, A. P., 67, 70, 330, 364 Dixon, D. M., 182, 193,268 Dobzhansky, T., 194, 268, 303, 329, 339, 354 Dodds, K. S., 212, 268 Doermann, A. H., 439, 478 Doggett, H., 177, 668 Doida, Y., 201, 282 Donachie, W. D., 41, 77 Donalson, L. J., 46, 76 Doty, P., 443, 486 Doy, C. H., 57,Y6 Drake, J. W., 10, 87 Drapeau, G. R., 5,104 Dreyfuss, J., 49, 50, 77, 78

496

AUTHOR INDEX

Dubinin, N. P., 339,364 Dubnau, D., 443, 479 Dubnau, E., 33, 78 Duffy, J., 308, 368 Duncan, F. N., 313, 320,364 Dunn, J. J., 422, 478 Duntze, W., 24, 78, $0 Duphil, M., 42, 87

E Eakin, R. T., 56, 57, 78 East, E. M., 108, 135, 147 Echols, H., 452, 453, 457, 458, 463, 479, 481, 487, 488 Eddleman, H., 11, 102 Edgar, R. S., 11, 78, 103, 447, 488 Edidin, M., 67, 93 Edington, C. W., 396, 407 Edwards, D. L., 56,57, 78 Ehrensvard, G., 43, 69 Ehrlich, E., 45, 93, 339, 364 Ehrman, L., 312, 360 Eichler, F., 76 Eisenstark, A., 57, 58, 71, 78 Eisenstark, R., 57, 78 Ellerstrom, S., 249, 268 Ellingboe, A. H., 8,9,91 Ellison, J., 303, 327, 369, 404, 408 Elseviers, D., 16, 17, 28, 63, 64, 70, 76, 78 Emerson, S., 30, 60, 78, 425, 437, 438, 479, 480 Emmer, M., 422, 423, 486 Emrich, J., 18, 27, 93, 99, 100, 447, 488 Engel, J., 364, 404,408 Engelhardt, D. L., 78,93 Engle, L. M., 200, 268 Englesberg, E., 58, 59, 63, 78, 81, 422, 480 England, P. T., 489 Engstrom, A., 411, 480 Ensign, S., 24, 78 Ephrussi, B., 30, 31, 32, 71, 78 Ephrussi-Taylor, H., 445, 440, 480 Epstein, R. H., 11, 78 Erickson, J., B3, 326, 328, 336, 343, 347, 369, 362, 364, 365, 366, 404, 406, 408 Eron, L., 422, 423, 463, 476, 487

Eroshevich, K. E., 297, 300, 337, 341, 364 Eskin, B., 472,484 Esposito, M. S., 30, 79 Esser, K., 108, 109, 111, 112, 113, 115, 146, 146, 147, 161 Evenchik, B., 467, 479 Ewing, A. W., 320,364 Exner, B., 200, 870

F Fairley, J. L., 41,7Q Falk, R., 18, 35, 59, 79, 89 Fan, D. P., 15,21,22,97 Fankhauser, D. B., 16, 34, 79 Faulhaber, S. H., 327, 364 Fawcett, D. W., 307, 364, 425, 480 Fellenberg, G., 117, 137,146 Femino, J., 326, 338, 360, 365, 409 Fennell, D. L., 124,161 Ferretti, J. J., 22, 79 Finch, J. T., 412, 480, 483 Fincham, J. R. S., 8, 9, 42, 43, 46, 79, 99, 425, 432, 440, 441, 480 Finck, D., 38,39,79 Fink, G. R., 15, 23, 24, 34, 35, 74, 79, 98, 420, 488 Fischer, G. A,, 54,79 Fisher, R. A., 438, 480 Fitz-Earle, M., 333, 334, 364, 367 Flaks, J. G., 36, 76 Flavin, M., 52, 53, 54, 79, 87, 97 Flentje, N. T., 116, 131, 149, 162 Floor, J., 66, 79 Fogel, S., 431, 434, 439, 480, 482 Foley, J. M., 8, 79 Folk, W. R., 61, 79, 80 Forbes, E. C., 43, 80 Forgash, A. J., 310,559 Forrest, H. S., 31,65, 66,81 Foster, J. W., 30, $9 Foulds, J., 62, 84 Fowler, G. L., 294, 297, 300, 324, 325, 326, 328, 337, 338, 340, 341, 347, 348, 364, 360, 365, 409 Fox, A. S., 30, 66, 70, 80, 312, 364 Fox, M. S., 442,443,444,480, @l, 482 Fradkin, J. E., 59,80

AUTHOR INDDX

Fraenkel, D. G., 59, 80 Franklin, N. C., 15, 80, 452, 463, 480 Fredga, K., 234, 237,276 Frcese, E., 52, 80 Freifelder, D., 37, 80, 448, 480 Freundlich, G., 17, YG Freundlich, M., 34, Y4 Frieden, C., 35, S O Friedman, S. B., 17, 33,80 Friedman, T. B., 31, 96 Friis, J., 30,84 Fristrom, J. W., 293,354 Frydenberg, O., 3,45, 355 Fujimura, W., 308, 360 Fukasawa, T., 58, 80 Fukuhara, H., 55, 75 Fukushima, E., 231, 232, 240, 241, 242, 244, 245,246, 256, 278, 279 Fuller, G. F., 46, 72 Fulton, I. W., 117, 133, l4Y Fuscaldo, K. E., 66, 80, 89, 90

G Gahor, M., 442, 482 Gadd, I., 260, 262, 269 Gaines, E. F., 241, 268 Gajewski, W., 54, SO, 438, 483 Gall, J. G., 61,80, 476, 480 Gallegly, M. E., 116, 121, 152 Galliers, E., 52, 80 Gallucci, E., 6, 26, 80, 102 Galsworthy, P. R., 49, 50, 93 Ganem, D., 16,37,94 Ganesan, A. T., 443, 466, 477, 480 Ganguly, P. M., 164, 173, BY8 Cans, M., 42, 65, Y4, 80 Garber, E. D., 144, l4Y Garcia-Bellido, A,, 311, 312, 313, 321, 323, 343, 356 Gardner, E. J., 66, 81, 101 Garen, A., 2, 6, 11, 20, 21, 23, 26, 76, 80, 81, 82, 102 Garen, S., 11,81 Garner, H. R., 53, 54, 102, 103 Garnjobst, L., 53, 55, 71, 77, 81, 116, 123, 124, 147, 152 Garvin, R. T., 36,106 Garza-Chapa, R., 116, 131, 148

497

Gatlin, L., 25, 86 Gaul, H., 249, 265 Gefter, M. L., 11, 12, 13, 69, 97, 466, 481, 483 Geider, K., 468, 48Y Geigy, R., 304, 355 Gelfand, D. H., 424, 481 Gemski, P., Jr., 449, 4YY Genson, M., 42, 88 Germershausen, J., 24, Y4 Gersh, E. S., 65, 81 Gershenson, S., 385, 396, 399, 400, 407 Gesteland, R. F., 449, 4Y9 Gethmann, R. C., 381, 407 Ghatge, M. B., 154, 172, 186, 204, 205, 268

Chose, R. L. M., 154, 157, 159, 163, 168, 170, 172, 174, 177, 178, 186, 188, 193, 204, 205, 268, 284 Ghosh, A. K., 162, 163, 166, 170, 269, 285 Ghosh, B. N., 247, 268, 270 Ghosh, D., 31, 32, 66, 81 Ghosh, H., 22, 87 Ghosh, M., 172, 201, 234, 237, 265, 268 Ghosh, S. S., 161, 267, 268 Gibson, J. B., 177, 290 Gielow, L., 63, 81 Gierer, A., 419, 481 Gilbert, W., 418, 481 Gilbertson, R. L., 117, 133, 160 Giles, N. H., 8, 23, 24, 52, 53, 57, 76, 79, 81 Gill, K. S., 308, 366 Gillespie, D. H., 49, 50, 60, 77, 81, 91 Gillie, 0. J., 9, 81 Gilmore, R. A., 23, 24, 25, 26, 81, Q,t? Gingery, R., 452, 453, 457, 458, 479, 481 Ginsburg, A., 49,81 Glansdorff, N., 16, 17, 63, 64, YG, Y8, 81 Glass, B., 66, 67, 81, 94 Glassman, E., 30, 31, 67, 82, 84 Glazer, A. N., 55, 57,77 Gold, L., 21, 23, 84 Goldberg, I. H., 412, 486 Goldmark, P. J., 46, 82, 432, 442, 481 Gollub, E. G., 54, 82 Goodchild, D. J., 388, 390. 391, 408, 409 Goodgal, S. H., 443,481,486

498

AUTHOR INDEX

Goodman, H. M., 11, 12, 21, 82, 88, 472, 482 Goodman, M., 34, 74 Gopalakrishnan, R., 164, 168, 170, 171, 173, 185, 186, 191, 208, 210, 213, 214, 218, 219, 220, 222, 223, 224, 226, 227, 229 230, 231, 234, 235, 249, 250, 263, 268, 269, 281 Gopinathan, K. P., 21, 23, 82 Gorini, L., 2, 20, 25, 28, 36, 45, 68, 70, Y2, 88, 91, 98, 106 Gossop, G. H., 124,148 Gotoh, K., 230,269 Gots, J. S., 42, 48, 54, 71, 82 Gottesman, M. E., 422, 423, 452, 453, 454, 457, 479, 481, 486, 489 Gottschewski, G., 308, 310, 366 Gould-Somero, M., 381, 407 Goulian, M., 464, 466, 467, 468, 481 Govindaswamy, S., 162, 163, 166, 170, 171, 174, 175, 188, 193, 202, 203, 241, 247,250,269, 284, 286, 286 Gowans, C. S., 30,92 Gowdridge, B., 56, 82 Granger, G. A., 142,148 Grant, V., 117, 137, 148, 176, 195, 196: 211, 215, 225, 269 Grant, W. F., 165, 269 Grasso, V., 129, 148 Gray, C. W., 46, 72 Gray, R., 183, 194, 269 Gray, T., 445, 446, 480 Graziani, F., 61, 96 Green, M. M., 20, 30, 31, 32, 65, 68, 82, 83, 96, 233, 286 Green, R. E., 396, 409 Greenberg, G. R., 58,89 Gregg, T., 32,90 Grell, E. H., 2, 20, 30, 31, 32, 35, 86, 89, 101, 350, 351, 367, 385, 395, 403, 407 Grell, K. G., 138,148 Grell, R. F., 425, 481 Grieshaber, M., 10, 83 Griffith, F., 442,481 Griffiths, A. J., 55,56,83 Grindle, M., 116, 124, 148 Grist, D. H., 154, 163, 669 Grodzicker, T., 16, 83, 106

Gros, F., 15,76 Gross, J. D., 448, 451, 462, 464, 481, 487 Gross, S. R., 34, 48, 49, 73, 83, 86, 123, 148 Gross, T. S., 64, 65, 76,83 Grun, P., 117, 137, 148 Guarneros, G., 457,458,481 Giinther, E., 117, 136, 148 Guerrini, F., 444, 458, 481 Guertin, M., 15, 92 Guespin-Michel, J. F., 52, 83 Guest, J. R., 5, 10.4 Gugler, H. D., 304, 328, 339, 341, 366, 366 Guild, W. R., 443, 466, 478, 481, 486 Guirard, B. M., 30, 83 Gupta, N., 22, 87 Gustafsson, A., 260, 262,269 Gustchin, G. G., 158, 197, 198, 269 Guyknot, E., 304, 346, 366

H Haagen-Smit, A. J., 30,100 Haddox, C. H., 60,102 Hakim, K. L., 163, 181, 185, 190, 191, 195, 224, 269, 273 Hall, J. C., 381, 407 Hallick, L. M., 452, 487 Halvorson, H. O., 26,97 Hamada, H., 158, 170, 178, 202, 203, 207, 269, 270 Hamlett, V., 47, 88 Hankin, L., 117, 134,148 Hannah-Alava, A., 313, 314, 366 Hara, S., 197, 200, 274 Hardesty, B. A., 55, 83 Hardman, J. K., 10,92 Hardon, J. J., 117, 137, 148 Hardy, R. W., 304, 308, 326, 360, 365, 366, 371, 373, 381, 383, 391, 404, 407, 408, 409 Harlan, J. R., 160, 165, 176, 177, 270 Harland, S. C., 173, 270 Harmsen, R., 32, 106 Harris, H., 46, 83 Harris, W. J., 443, 481 Harrison, A. P., Jr., 57, 83 Harrison, B. J., 117, 136,148

AUTHOR INDEX

Hartl, D. L., 293,294,339,349,353,355, 365,383,384,407 Hartman, P. E., 2,6, 15,17,23, 24,26, 27, 34, 63, 64, 65, 69, 77, 83, 92, 93, 96, 420,487 Hartman, Z., 23, 26, 27, 65, 83 Haschemeyer, R. H., 48, 49, 101 Haseltine, F. P.,442,482 Haseltine, W.A,, 423,482 Haskins, E. F., 116,120,146 Haskins, F. A., 55,56,84,101 Haslam, J. M., 57,89 Hawkes, J. G., 219,270 Hawthorne, D. C., 23, 24, 26, 30, 46, 84,92 Hayashi, M., 424,481 Hayes, W., 441, 448,449,451, 462,482, 489 Healy, S. K., 31,96 Heath, S.,18,19,27,104 Hedayatullah, S., 247,270 Hedgpeth, J.,472,482 Heiser, C. B., Jr., 164,165,176,196,270 Helbaek, H., 160,270 Helinski, D. R.,5,7,84 Helmstetter, C. E., 464,482 Henderson, A,, 61,84 Henderson, A. S., 67,84 Henderson, E.J.,10,84,92 Henderson, M. T.,170, 171, 172, 186, 188, 190, 191, 199, 200, 206, 226, 228, 240,241,269,270,291,292 Henney, H. R.,116,120,148 Henney, M.R.,116,120,148 Henning, U., 5,6,15,84 Henninger, M., 11,103 Herman, R.K., 451,463,482 Herndon, C. N., 46,72 Herold, J. L.,32,78 Herriott, R.M., 444,488 Hershey, A. D., 452,482 Hess, O., 301, 303, 305,307, 355, 358 Hessler, A., 381,407 Heyn, A. N.J., 247,270 Heywood, J., 470,471,484 Heywood, V. H., 212,268 Hihara, Y.K., 362,374,407 Hildreth, P. E., 67,84, 321, 342, 355 21, 27, 62, 74, 84 Hill, C. W.,

499

Hill, J. M., 39,84 Hinata, K., 170, 172, 174, 180, 181,185, 186, 187, 188, 190, 191, 192, 194, 195, 196, 204, 249, 250, 270, 279, 280 Hinton, C. W., 322,355 Hiraieumi, Y., 293, 316, 356, 359, 362, 363, 365, 371, 373, 374, 376, 377, 378, 379, 381, 382, 383, 407, 408, 409 Hirayoshi, I., 230,256,270 Hirose, S.,467,468,489 Hirota, Y., 449,450,466, 481,482 Hirsch, M.-L., 39,42,74,84 Hirsh, D., 21,23,84 Hirvonen, A. P.,44,102 Hitchcock, A. S.,163,271 Ho, K.C., 168, 169, 222, 223, 232, 241, 242, 243, 250, 261, 271, 277, 291 Ho, K. M., 207, 244, 245, 272 Hoenigsberg, H. F., 320,355 Hoffee, P.A., 15,58,78,96 Hoffman, E.P., 26, 84 Hoffmann, J. A,, 130,148 Hoffman-Berling, H., 449,484 Hofnung, M., 51,63,85 Holliday, R.,424,427,428,432,440,441, 480, 482 Holloway, B.W., 123,148 Holm, D.G., 350,355 Hong, M.S.,260,283 Hooker, J. D.,162, 207, 208, 271 Hopkins, N., 418,478 Horiuchi, K., 20,85 Horn, V., 6,8,10,16,lo4 Hosgood, S.M.W., 321,356 Hotclikiss, R.D., 442,443,480,482,486 Houlahan, M. B., 38, 39, 40, 85 Howard-Flanders, P., 442,478,482 Howarth, S.,50,85 Hsich, S.C., 192,200, 201,241,242,247, 248,260,266,271,283 Hsu, K. J., 198,200,201,271 Hu, C. H., 168, 169, 190, 191, 207, 208, 210, 211, 214, 215, 216, 219, 222, 227, 228, 230, 231, 232, 234, 235, 236, 239, 240, 241, 242, 244, 245, 249, 251, 252, 256, 257, 261,271, 272, 877, 289 Hu, S. Y., 207,272 Hu, W. L., 259,260,266 Huang, T.S.,192, 201, 247, 248, 283

500

AUTHOR INDEX

Huettner, A. F.,342,366 Hunter, A. W.S., 253,286 Hunter, J. H., 116, 121, 162 Hurst, D. D.,37, Yl, 431, 434, 439, 480, 482 Hurwitz, J., 46,103,468,484 Hutchinson, J. B., 173, 177, 279 Huttenhauer, G.,58, 78 Hwang, L. H., 10,84,86

Jacobson, K. B., 30, 31, 32, 86, 101 Jacques-FClix, H., 221, 222, 2Y3 Jain, S.C.,412,483, 488 Janick, J., 253, 273 Janosko, N.,24, 90 Jargiello, P.,15,58,96 Jennings, P. R.,199,f l 3 Jeon, K. W., 117, 139, 148, 1.69 Jinks, J. L., 10, 86, 108, 116, 124, 146,

I

Jobbitgy, A. J., 40,86,96 Jodon, N. E., 200, 240, 263, BY3, 291 Johnsen, R. C.,348, 366, 398, 4OY Johnson, F.M., 313,356 Johnston, B. F., 179, 182, 184, 2YS Jokura, K., 58, 80 Jones, A. M., 194, 273 Jones, D.A,, 116,125,148 Jones, E. E.,44, 86, 101 Jones, E.W., 30,86 Jones, J. W., 198, 202, 241, 242, 244, 247, 251, 266, 2YS Jones, K., 233, 273 Jones, M.E.,42,86 Jonsson, U. B., 294, 305, 310, 313, 316, 318, 323, 324, 328, 329, 336, 339, 340, 341, 342,343,345,366,367 Jordan, E., 15, 17, 18, 29, 58, 86, 99, 104 Joseph, C.A.,190, 195,2Y3 Judd, B. H., 65,86, 348,364,379,384,406 Jiittenonke B., 117, 136, 148 Jui, Y.,222, 229, 291 Juill, J. L.,124, 148 Jukes, T.H., 25,86 Jungwirth, C.,34, 48, 83, 86

Ichijima, K., 244, 245, 246, 8'2 Ihler, G.,448, 463, 482, 48Y Ikeda, H., 446,447,482,486 Ikediugwu, F.E.O., 143,148 Iljina, T.S.,57, 69 Imae, Y.,466,485 Imamoto, F.,15,86 Inge-Vectomov, S.G., 22,86 Ingraham, J. L.,48, 69 Inman, R. B.,466, 467, 468, 482, 486 Inoue, Y.,244,251, 252,276 Inouye, M.,18, 27, 86, 93, 99, 100 Ippen, K., 18,86,463,487 Ippen, K. A.,448, 482 Ishida, H., 308, 360 Ishidsu, J . 4 , 48, 101 Ishii, K., 234, 236, BY2 Issaly, A. S., 38, 39, 40, 41, 42, 96 Issar, S. C.,218,288 Itikawa, H., 44, 45, 60, 81, 86 Ito, J., 6, 10, 15, 86, lo4 Ito, K., 37, 86 Iwata, N.,254, 261,,273 Iyengar, S. V., 324,366 Iyer, V. N.,467, 482

J Jabbur, G., 8,93 Jachuck, P. J., 163, 166, 169, 181, 185, 190, 191, 195, 273 Jackson, E. N.,17, 86 Jacob, F.,15, 17, 63, 64, 69, 86,.103, 412, 420, 448, 449, 450, 482, 483, 486, 489 Jacob, T.M.,22,87 Jacobs, K. F.,247, I 7 3 Jacobs, P. A.,381, 4OY

14s

K Kaback, H. R.,51,86 Kafer, E.,43,86 Kainuma, R.,467, 470, 486 Kaiser, A. D.,454, 457, 483, 484 Kaji, H., 36, 87 Kalbacher, B.,423,478 Kalckar, H. M., 58, 99, 104 Kaliaeva, E.S., 57, 69 Kalman, S. M.,48, 73 Kalyanpur, M.G., 48,Y4

AUTHOR I N D a

Karnen, R. I., 423,489 Kameneva, S. V., 57,69 Kanazir, D. T., 44, 87 Kanehisa, T., 66, 87 Kaneko, A., 337, 342, 369 Kano, Y., 15, 85 Kao, C. H., 258, 282 Kaplan, J. G., 42, 87, 89 Kaplan, S., 11, 24, 78, 87 Kaplan, W. D., 304, 328, 339, 341, 366, 366 Karam, J. D., 47, 87 Karibasappa, B. K., 205, 208, 221, 222, 244, 245, 251, 273, 274, 287 Kariya, B., 18, 51, 73 Karkas, J. D., 443, 487 Karunakaran, K., 234, 235, 239, 261, 262, 263, 274 Kasha, K. J., 263,286 Katayama, T., 168, 210, 214, 227, 228, 229, 230, 231, 244, 245, 251, 252, 256, 257, 260, 261, 274, 276 Katayama, T. C., 154, 162, 168, 169, 172, 186, 190, 191, 202, 207, 208, 209, 210, 212, 214, 215, 217, 218, 220, 221, 223, 225, 226, 227, 228, 230, 232, 274, 275, 281 Katayarna, Y., 241,274 Kato, S., 197, 200, 274 Katsuo, K., 199, 274 Katz, E. R., 6, 22, 7 3 Kaudewitz, F., 56,97 Kaufrnan, S., 30, 31,87 Kaufmann, B. P., 293, 304, 325, 328, 339, 341, 342, 364, 366 Keep, E., 117, 136,148 Kellenberger, E., 11, 78 Kellenberger-Gujer, G., 454, 483 Kelly, T. J., 418, 472, 483 Kernper, J., 17, 48, 49, 87 Kendrick, E. L., 130,148 Kerr, D. S., 52, 53, 87 Khishin, A. F., 304, 305,36G Khorana, H. G., 22,87 Kidd, W. D., 339, 341, 366 Kiefer, B. I., 301, 307, 308, 338, 356 Kihara, H., 154, 168, 170, 172, 175, 186, 190, 191, 202, 208, 210, 214, 215, 217,

501

218, 220, 223, 226, 227, 228, 230, 231, 232, 176, 281 Kikkawa, H., 30, 87 Kikkawa, S., 166, 171,275 Kim, J.S., 428, 483 Kimber, G., 241, 242, 243, 263, 276 Kirnura, K., 130, 148 King, J., 11,87, 103 King, M. E., 57, 87 King, R. C., 20, 87, 295, 332, 333, 356, 3GO Kirschner, I., 37, 80 Kiss, I. S., 261, 262, 263, 274 Kitarnura, E., 200, 276 Kitani, Y., 437, 438, 483 Kittel, C., 412, 478 Klein, A., 464, 483 Kline, D. M., 126, 150 Kling, D., 10, lo4 Klofat, W., 52, 80 Klopotowski, T., 34, 79 Klug, A., 411, 412, 4Y8, 480, 483 Klug, W. S., 20, ST Knauert, F. K., 41,100 Knippers, R., 466, 483 Kobel, H. R., 67, 87 Koch, R. E., 10, 87 Kossel, H., 22, 87 Koga, Y., 244, 245, 251, 252, 261, 276, 280, 291 Kohno, T., 18, 19, 27, 87, lo4 Kolh, W. P., 142, 148 Konigsberg, W., 93 Kopac, M. J., 42, 43, 101 Korah, M., 222, 234, 235, 237, 276 Kornberg, A,, 464, 466, 467, 468, 477, 481, 483, 486, 487, 489 Kornberg, H. L., 29, 87 Kornberg, T., 466, 481, 483 Kosaka, H., 197, 200,274 Koshland, D. E., Jr., 29, 88 Kostoff, D., 249, 276 Kozinski, A. W., 47,71 Krauss, M., 46, 101 Kreider, G., 36, 88 Kreneva, R. A., 444,477 Krisch, H. M., 47, 88 Krishnamurthy, A,, 170, 175, 193, 202,203, 269

502

AUTHOR INDEX

Krishnaswamy, N., 209,276 Krishnaswamy, V., 254, 264, 286 Kroh, M., 108, 1.69 Kruszewska, A., 438, 483 Ku, Y., 223, 276 Kuang, H. H., 162, 198, 199, 200, 201, 207, 241, 276 Kudo, A., 257, 276 Kuhner, R., 130,149 Kuenen, R., 108, 109, 115, 147 Kummer, H., 312, 366 Kung, T. S., 199, 200, 247, 286 Kunicki-Goldfinger, W. J. H., 448, 486 44, 88 KUO,T.-T., Kurahashi, K., 58,80 Kurek, L. I., 29,99,100 Kuriyama, H., 186, 190, 191, 195, 199, 202, 206, 214, 215, 218, 223, 226, 228, 230, 244, 247, 248, 250, 276, 279, 289 Kurland, C. G., 36, YC Kushev, V. V., 444,4Y7 Kushner, S. R., 47, 88 Kuwada, Y., 172, 233, 256, 258, 259,276 Kuwana, H., 30, 88 Kuwano, M., 15,88 Kvelland, I., 310, 316, 319, 360 Kwan, S. C., 200, 291 Kwon, K. J., 116, 125,149 L

Lacks, S., 443,446,483, 484 Lacroute, F., 42, 87, 88 Lacy, A. M., 23, 30, 88 Ladizinsky, G., 165, 276 Landy, A., 11, 12, 13, 21, 69, 82, 88, 9Y Lange, M., 130,149 Langer, B., 318, 360 Langridge, J., 22, 23, 88 Lanka, E., 6, 11, 102 Lanzov, V. A., 448,477 Largen, M., 63, 81 Lark, K. G., 464, 467, 482, 484 Launert, E., 156, 222, 2Y6 Lautenberger, J. A,, 472,484 Laven, H., 117, 139, 140, 144, 1.49 Laviola, C., 116, 121, 162 Lawless, M. B., 43, Y7 Lawrence, D. A., 64, 65, 76, 89

Lawrence, W. J. C., 256, 261, BY6 Lazdunski, C., 52, 89 Leahy, S. M. G., 312, 366 Leberman, R., 412, 480,483 Lederberg, E. M., 446, 448, 449, 4Y8, 484, @6 Lederberg, J., 446, 448, 449, 478, 484, 484 490 Lee, C. S., 428,466,484 Lee, H. T. Y., 332,366 Lee, L.-W., 65, 89 Lee, N., 58, 78 Lee, S. S. Y., 260,290 Lefevre, G., Jr., 65, 89, 294, 305, 310, 313, 316, 318, 319, 322, 323,324, 328, 329,336, 337, 339, 340, 341, 342, 343, 345, 366, 36Y Lehman, I. R., 427,428,4YY Leidy, G., 442, 476 Lein, J., 59, 89 Lein, P. S., 59, 89 Lemke, P. A., 116, 130,149 Lennox, E. S., 446,484 Lerner, S. A., 46, 51,100 Leslie, S. E., 46, 7 2 Levan, A., 234, 237, 242, 253, 263, 276 Levin, D. A., 195, 196,276 Levinthal, M., 15, 64,89 Lewis, D., 54, 89, 108, 117, 136, 1.49 Lewis, E. B., 376, 407 Lewis, H., 195, 212, 225, 232, 233, 276 Lewis, H. S., 67, 89 Lewis, H. W., 67, 89 L’Hhritier, P., 140, 1.49 Li, D., 259,266 Li, H. W., 168, 169, 172, 186, 191, 200: 206, 210, 214, 217, 222, 223, 226, 227, 228, 229, 230, 231, 232, 241, 242, 243, 250, 260, 261, 2Y1, 276, 276, 277, 290, a91 Li, S. S. Y., 223, 261, 277, ,291 Lieb, M., 460, 479 Lielausis, A., 11, Y8 Lielausis, I., 11, 103 Lifschytz, E., 18, 59, 89 Lilly, L. J., 54, 89 Lin, E. C. C., 46, 51, 52, 72, 89, 100 Lin, S. Y., 420, 484 Lindegren, C. C., 122,1.@ Lindsley, D. L., 2, 20, 32, 35, 61, 89,

AUTHOR INDEX

96, 294, 350, 351, 367, 381, 385, 396, 398, 399, 403, 407 Ling,, H., 117,119,146,149 Linn, S.,46, 82, 432, 442, 470, 471, 476, 481, 484 Linnane, A. W., 57, 89 Linskens, H. F., 108,149 Lissouba, P., 439, 484 Litwihska, J., 54, 80 Lloyd, D. G., 117, 136,149 Lloyd, L., 439,478 Lobashov, M. E., 339,367 Loeb, T., 449, 484 Love, A., 212,277 Lohrman, S., 141, 161 Lomax, M. S., 58, 89 Longley, A. F., 202, 241, 242, 247, 213 Longley, W., 412,483 Loo, W. S., 247,289 Loper, J. C., 16, 17, 70 Lorch, I. J., 117, 139, 148, 149 Low, B., 46, 10s Lowe, M. L., 312,556 Lowenstein, R., 57, 89 Lowry, J. L., 332,360 Lu, K. C. L., 168, 169, 222, 223, 229, 230, 231, 276, 277, 291 Lu, P., 20, 26,89 Lucas, Z. J., 467, 481 Lucchesi, J. C., 342, 366 Luck, D. J. L., 56, 96 Lue, P. F., 42, 89 Luning, K. G., 323, 367 Luftig, R. B., 11, 102 Luig, N. H., 9, 22,89 Luria, S. E., 15, 80, 446, 484 Lyle, H., 11, 102

M Maas, W. K., 45, 51, 89, 97, 102 Mabuchi, T., 226, 276 Macbean, L. T., 320,367 Maccacaro, G. A,, 449, 479 McCarron, M. Y., 66, 80, 89, 90 McCarron, S. M. J., 66,80 McCarty, M., 442, 477 McClintock, B., 252,277

395, 472,

251,

227,

503

McCloskey, J. K., 349, 350, 367 McDougall, K. J., 40, 41, 90 McGrath, J., 424,478 McGuire, E. J., 67, 96 MacHattie, L. A., 447, 463, 487, 489 McIntire, S. A,, 32, 90 MacKechnie, C., 26,97 McKeen, C. G., 117, 134, 149 McKenzie, A. R., 116, 131, 149, 162 McLellan, W. L., 44,102 MacLeod, C. M., 442,477 McMillan, M., 333, 367 McNeilly, T., 117, 137, 149 Macrae, R., 116, 131, 133, 149, 160 McSheehy, T. W., 319, 367 Magalhles, L. E., 59, 90 Magasanik, B., 17, 98, 423, 488 Magnaye, A. B., 210,266 Magni, G. E., 22, 26, 90 Mahdihassan, S., 159, 277 Mahoney, D. L., 253, 273 Maitra, U., 468, 484 Majisu, B. N., 177,268 Makhlin, E. E., 117,139,160 Makman, R. S., 423,484 Malamy, M. H., 15, 18,90 Malva, C., 61, 96 Mandel, M., 443, 489 Mange, A. P., 294, 349, 367 Mange, E. J., 362, 407 Mangelsdorf, P. C., 174, RY Manly, K. F., 452, 458, 468, 484, 4888 Manney, T. R., 23, 24, 78, 90 Manning, A,, 319, 320, 321, 329, 330, 339, 362, 367 Manwell, C., 46, 90 Marais, W., 180, 287 Marcou, D., 143, 160 Margoliash, E., 6, 14, 24, 98 Margolin, P., 10, 15, 16, 17, 32, 33, 34, 48, 49, 63, 64, 71, 73, 74, 78, 80, 86, 87, 90, 99 Margulies, A. D., 442, 478 Markert, C. L., 46, 90 Marsho, N. J., 42, 71 Martin, A. O., 304, 305, 367 Martin, F. W., 117, 137,150 Martin, R. G., 15, 16, 23, 26, 27, 73, 79, 90, 96, 102

504

AUTHOR INDEX

Martuscelli, J., 42,76 Maruyama, Y.,197,200,274 Marvin, D.A.,449,484 Marzluf, G.A.,30, 31, 90 Masima, I., 201, 248,277 Masson, M., 42, 80 Masuda, T.,457, 483 Mather, K.,117, 136,160,177,203,277 Matile, P.,143, 160 Matsubara, K.,454, 484 Matsumura, S.,226, 276 Matsuo, T.,197, 198, 199, 277 Matsushiro, A.,463, 484 Matsuyama, Y.,251,292 Mattoon, J. R.,55,90 Matusima, K.,246,277 Maynard Smith, J., 176, 203, 206, 277 Mayo, M. J., 116,131,149 Mayo, O.,263,264,277 Mayr, E.,176, 177, 194, 196, 203, 206, 211, 216, 225,277 Meister, A.,48,49,69,101 MelloSampayo, T.,200,277 Melville, R.,219,278 Merle, J., 312, 329, 333, 363, 367 Meronk, F.,Jr., 63,78, 422,480 Merrell, D.J., 321,367 Merrill, E.D.,211,278 Meselson, M., 418, 429, 439, 440, 451, 452,463,470,471,476,484 Messenguy, F.,43,91 Messmer, I., 42, 87 Mete, C.W.,263,264,278 Metzenberg, R. L., 30, 54, 73, 91 Meyer, E.R.,329,339,368 Meyer, G. F., 301, 303, 305, 307, 308, 366, 367, 368 Meyer, H. U., 329, 339, 345, 368 Michaelis, A., 233, 286 Michaelis, G.,18,91 Michels, C.A.,15,16,91 Miklos, G.L. G., 373, 374, 375, 376, 379, 380, 381,388,390,391, 399,406, 407, 408 Miller, A.,294, 313, 368 Miller, C.G., 22,91 Miller, J. D., 117,160 Miller, J. H.,16, 37, 94, 421, 423, 486, 488 Miller, S. M., 381,407

Mills, D. I.,8,9,91 Mills, S.E.,23,72 Misra, R. N., 191, 200, 206, 235, 239, 240,243,258,262,278,288 Misro, B.,199,254,278, 286 Mitchell, H. K., 2, 38, 39, 40, 41, 42, 43, 45, 46, 55, 56, 57, 60, 78, 83, 84, 86, 91, 97, 101, 102 Mitchell, M. B., 41, 42, 43, 55, 56, 60, 84, 91 Mitra, S., 468,486 Mitra, S.K.,164, 173,278 Mitsukuri, Y.,234, 236, 240, 272, 278 Miyake, T.,44,91 Miaobuchi, K.,49,50,77,91 Mizushima, U.,197, 198, 199, 274, 290 Model, P.,22, 91 Mohanty, H.K.,200, 286 Mohanty, N. R., 235, 266 Momma, E.,337,342,369 Momose, H.,36,91 Monod, J., 15, 17, 63, 86, 103, 411, 412, 420, 423, 482, 486, 489 Monty, K. J., 49, 77 Moore, D.M., 233,278 Moore, L., 322, 337, 357, 452, 453, 458, 479, 488 Mora, J., 42,43,76,77, 91 Moreau, F.,122, 160 Morgan, D.H., 9,43,91 Morgan, R.,22, 87 Morgan, T.H., 2,45, 63,92 Morinaga, T.,168, 186, 190, 191, 195, 196, 198, 199, 202, 206, 210, 214, 215, 217, 218, 223, 226, 227, 228, 230, 231, 232, 240, 241, 242, 244, 245, 246, 248, 250, 256, 278, 279 Morishima, H., 135, 146, 163, 166, 170, 171, 172, 174, 180, 181, 185, 186, 187, 188, 190, 191, 192, 194, 195, 196, 204, 205, 206, 210, 211, 221, 222, 224, 244, 259, 267, 279, 280, 283 Morita, T., 32, 92 Morpurgo, G.,439, 486 Morrison, J. W.,245, 249, 280 Morrison, R. M., 116, 128, 160 Morse, D.E.,15, 16, 17, 88, 92 Morse, M. L.,446,486

AUTHOR INDEX

Mortimer, R. K., 23, 24, 25, 26, 46, 84, 92, 431, 434, 482 Moruzi, C., 122,160 Moses, M. J., 425, 486 Moses, R. E., 466,467,486 Mossige, J. C., 304, 323, 368 Mosteller, R. D., 15, 92 Mounce, I., 133, 160 Mount, D. W., 46,103 Mousseau, J., 436, 439, 484, 486 Mousset, S., 454, 486 Moyed, H. S., 29,101 Muller-Hill, B., 418, 481 Mukai, F. H., 23,33,92 Mukai, Y., 244, 245, 251, 252, 261, 291 Mukherjee, K. L., 116, 119, 160 Mukhopadhyay, S., 240, 287 Mulder, C., 443, 486 Muller, H. J., 45, 92, 93, 300, 323, 334, 338, 349, 350, 368, 394,408 Munkres, K. D., 56, 103 MunBz, E. R., 345, 362 Murdock, G. P., 179, 182, 183, 184, 193, 194, 280 Murnik, M. R., 363, 408 Murray, K., 472, 4YY, 489 Murray, M. L., 34, 92 Murray, N. E., 15, 52, 53, 92, 99, 436, 439, 477, 486 Myers, J. S., 10, 92 Myszewski, M. E., 345, 368

N Nachtsheim, H., 339, 368 Nagai, I., 163, 196, 280 Nagaishi, H., 4, 47, 70, 88 Nagamatsu, T., 199, 200, 244, 248, 251, 252, 276, 280 Nagano, H., 10, 84, 96 Nagao, S., 254, 261, 280 Nair, N. R., 162, 171, 176, 177, 280 Nakagahra, M., 254, 273 Nakamori, E., 244, 246, 250, 280 Nakamura, K., 30,922 Nakamura, S., 241, 242, 280 Nakao, S., 175, 202, 203, 276, 280 Nakata, A., 15, 99 Nakatorni, S., 233, 280

505

Nakazima, K., 376, 377, 379, 381, 382, 4 0 ~ Nandi, H. K., 168, 172, 210, 227, 229, 234, 256, 258, 259, 261, 262, 280 Naono, S., 15, YO Narain, P., 333, 368 Narang, S. A., 22, 87 Narita, K., 14, 9.2 Nash, D., 35, 92, 93 Nashed, N., 8,93 Natarajan, A. T., 235, 266 Naville, A., 304, 366 Nayar, N. M., 162, 163, 164, 165, 166, 168, 170, 171, 172, 173, 174, 176, 177, 181, 185, 186, 188, 190, 191, 193, 194, 195, 206, 250, 260, 263, 269, 280, 281 Nazario, M., 40, 41, 93, 96 Neet, K. E., 29, 88 Nelson, 0. E., 117,160 Nelson, R. R., 116, 126, 160, 162 Netter, P., 57, 76 Neuhard, J., 38, 42, 48,93 Neuhauser, K. S., 117, 133, 160 Newmeyer, D., 53, 68, Y l , 93, 116, 122, 160, 161, 162 Newton, A., 15, Y4 Newton, D., 16, 93 Newton, J., 18, 27,93,99 Newton, W. A,, 15, 16, 74, 93, 106 Nezu, M., 168, 172, 186, 190, 191, 210, 214, 215, 217, 218, 223, 226, 227, 228, 230 232, 2Y6, 281 Nicoletti, B., 326, 351, 363, 368, 360, 361, 365, 366, 384, 408, 409 Nikaido, H., 15, 58, 80, 89, 93 Nishimura, Y., 260, 261,281 Nissley, P., 422, 423, 479 Nobrega, F. G., 47,93, 100 Nolte, D. J., 32, 59, 93 Nomura, M., 36, 93 Nonides, J. F., 293, 294, 300, 308, 313, 321, 322, 336, 339, 340, 341, 368 Norby, S., 38, 42, 70 Nordman, C. E., 412, 488 Notani, G. W., 93 Notani, N., 443,486 Novitski, E., 294, 345, 368, 369, 361, 364, 403, 404, 408, 409 Novitski, M., 16, 106 Noyes, A. N., 46,Y2

AUTHOR INDEX

Nozawa, H., 381,407 Nyst, P., 247, 281 0

O’Donovan, G. A., 38, 42, 48, 93 Oeschger, N.S.,27,93 Ogur, M.,54, 101 Ohno, S.,46,93,263,281 Ohta, N., 49, 50, 93 Ohtsuka, E., 22, 87 Oishi, M., 46,47,93, 100 Oka, H. I., 135, 146, 162, 163, 164, 166, 170, 171, 172, 174, 175, 178, 179, 180, 181, 184, 185, 186, 187, 188, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 203, 204, 205, 206, 210, 211, 213, 217, 218, 219, 220, 221, 222, 224, 243, 244, 247, 248, 249, 250, 266, 258, 259, 260, 267, 270, 271, 279, 880, 281, ,982, 283 Okada, T., 57, 93 Okada, Y.,18, 27, 86, 93, 99, 100 Okazaki, R.,466,467,468,470,486,489 Okazaki, T.,466, 467, 470, 486 Okuno, S.,256, 261, 283 Okura, E.,210, 229, 230, 244, 269, 283 Olbrich, B.,56,97 Old, R. W.,489 Olive, L. S., 116, 128, 146, 161, 437, 438, 483 Olivieri, A., 348, 368 Olivieri, G.,294, 345, 348, 368, 398, 408 Omura, T., 199,244,254,261,278,280 Ono, S.,223, 229, 230, 231, 232, 244, 245, 250 251,252,261,279, 291 Oppenheim, A. B.,448,486 Oppenheimer, S. B.,67,93 Orgel, L.,17,63,72 Orr, C.W., 67,93 Ortigoza-Ferado, J. A.,44, 86 Oster, I. I., 45, 92, 93, 308, 368 Ostman, R.,40, 90 Ozeki, H.,11, 69, 446, 447, 486

P Padhye, A. A,, 116,127,161 Pancho, J. V., 162, 168, 207, 209, 211, 282, $83

Pandey, K.K.,117,136,161 Pao, W.K.,248, 292 ParLdi, E.,67, 94 Pardee, A. B.,49, 50, 77, 78, 93, 94 Parker, D. M., 319,367 Parker, D.R.,400,408 Parker, G.A.,348,368 Parker, J. H.,6, 14, 24, 55, 94, 98 Parkinson, J., 459, 4Y9 Parmeter, J. R.,Jr., 116, 131, 162 Parnell, D. R., 117, 137,161 Parry, D. M.,381,407 Parsons, P. A., 320, 321, 366, 367, 369 Parthasarathy, N., 241, 244,245, 246, 249, 256, 258, 259, 260, 261, 283, 284 Pasetto-Nobrega, M.,47,93 Pastan, I., 423, 479, 486 Paterniani, E.,117, 137, 161 Pathak, G. N., 214, 231, 258, 283 Pato, M.L., 464, 486 Patterson, J. T.,295, 301, 330, 331, 332, 342, 369 Pavan, C., 59, 90 Pavlovsky, O., 329, 339,364 Peacock, W.J., 293, 304, 308, 326, 328, 336, 343, 347, 369, 360, 362, 364, 365, 366, 371, 373, 383, 385, 386, 387, 388, 390, 391, 396, 400, 404, 406, 408, 409 Peltier, M., 190, 191, 283 Pendyala, L.,40,100 Pene, J. J.,443, 486 Pkrk, G.,54, 55, 76, 94 Perkins, D.D., 53,71,122,123,161, 16i? Perlman, M., 403, 409 Perlman, R. L.,422, 423, 479, 486 Perotti, M. E.,308, 312, 313, 362, 369 Perras, J. F.,305,307,360 Perrin, D., 17,63,103 Perrin-Waldemer, C., 313,369 Perry, R. P., 20, 100 Pestka, S., 36, 94 Peterson, J. M.,443, 486 Petrochilo, E.,57, 76 Pettijohn, D.E.,423,486 Pfahl, M.,37, 94 Pfahler, P. L.,253,273 Philip, U.,304, 369 Pica, L.,294, 345, 348, 368, 398, 408

AUTHOR INDEX

Piccinin, G. L., 449, 479 Piekarowicz, A,, 448, 486 PiBrard, A., 42, 88 Pierog, S., 59, 99 Pilger, R., 156,220,221, 283 Piotrowska, M., 44, 94 Pirrotta, V., 418, 478, 486 Pittenger, T. H., 55, 56, 72, 83, 94, 116, 123, 161 Plaine, H. L., 66, 67, 81, 94 Platt, T., 16, 37, 94 Plough, H. H., 2, 94, 324, 360 Policansky, D., 303, 306, 327, 369, 404, 408 Polito, L., 61, 96 Pontecorvo, G., 439, 486 Ponticello, G., 412, 488 Port, L. A., 43, 77 Portiires, R., 157, 162, 163, 170, 178, 179, 180, 181, 183, 185, 186, 187, 188, 193, 194 196, 197, 203, 204, 208, 217, 283, 284 Postel, E. H., 443, 481 Poulter, R. T. M., 116, 120, 161 Pradines, M., 55,76 Prasad, I., 126, 161 Preer, J. R., Jr., 67, 94 Prestidge, L. S., 49, 50,94 PrBvost, G., 42, 74 Primakoff, P., 15, 92 Pritchard, R. H., 22, 57, 58, 71, 94, 439, 443, 486, 489 Prodoehl, A., 155, 156, 213, 218, 284 Provost, P., 24,74 Ptashne, M., 418, 478, 486 Puglisi, P. P., 26, 90 Puhalla, J. E., 117, 134, 148, 161 Punyasingh, K., 252,284 Putrament, A., 439, 486 Putterman, G. J., 6, 14, 24, 98

R Radding, C. M., 432, 452, 458, 463, 478, 484, 486, 487, 488 Radford, A,, 39, 42, 53, 71, 94, 96 Raj, A. Y., 199,284 Rajhathy, T., 245, 249, 280 Raman, V. S., 190, 195, 273

507

Ramanujam, S., 168, 169, 188, 190, 227, 232, 241, 243, 244, 245, 246, 249, 251, 252, 256, 259, 260, 261,284 Ramel, C., 385, 386, 401, 402, 408 Ramiah, K., 154, 157, 159, 162, 163, 168, 170, 174, 177, 178, 188, 193, 196, 241, 244 245, 246, 249, 256, 284 Ramirez, D. A., 200, 260, 266, 268, 290 Rangaswamy, S. R. S., 247, 248, 284 Rao, D. R. R., 168, 169, 210, 222, 223, 224, 227, 231, 235, 239, 258, 262, 288 Rao, G. M., 199, 284 Rao, H. K. S., 214, 226, 229, 284 Rao, J. R., 248, 290 Rao, M. B. V. N., 154, 157, 159, 162, 163, 170, 174, 185, 187, 196, 205, 214, 220, 284, 286 Rao, P. K. M., 234, 235, 237, 239, 262, 288 Rao, R. K., 199, 286 Rapaport, F. T., 141, 151 Raper, J. R., 108,147,161 Raper, K. B., 116, 124, 125, 14, 161 Rapin, A. M. C., 58,99 Rasmuson, B., 65, 96 Rau, N. S., 172, 233, 234, 284 Ravel, J. M., 65, 89 Rayle, R. E., 65,96 Rechler, M. M., 16,73, 96 Reeves, R., 21, 96 Reich, E., 412, 486 Reichard, P., 468,486 Reiff, I., 117, 139,161 Reinbergs, E., 249, 284 Reissig, J. L., 38, 39, 40, 41, 42, 93, 96 Reznikoff, W. S., 2, 15, 17, 35, 36, 63, 91, 96, 421, 423, 463, 486, 487, 488 Rhoades, M. M., 264,284 Rich, A,, 20, 26, 89 Richardson, C. C., 466, 467, 486 Richardson, R . H., 379, 406 Richey, D. P., 52, 72 Richharia, A. K., 247, 250, 284 Richharia, R. H., 162, 163, 168, 170, 171, 185, 190, 191, 192, 199, 200, 204, 218, 220, 224, 226, 228, 229, 231, 254, 87'8, 284 286 Rick, C. M., 240, 252, 253, 264, 286 Riddle, D. L., 4, 27, 28, 96

508

AUTHOR INDEX

Rieger, R., 233,263,286 Rifkin, M. R., 56, 96 Riggs, A. D., 418, 420, 484, 486 Riley, M.,448,486 Riley, R., 241, 242, 243, 263, 676 Riley, R. C.,301,369 Ritossa, F. M.,20, 61, 84, 96, 96 Riyasaty, S.,9,27,96 Rizet, G.,109, 161, 439, 484, 486 Rizki, M. T.M., 31, 32, 67,96 Rizki, R. M.,31,32,96 Roberts, C.F.,8, 79 Roberts, P. A.,381, 408 Robertson, B. C.,15, 58,96 Robertson, F. W.,333, 369 Robison, M.,443,481 Robson, D.S.,333,367 Robyns, W.,216, 286 Roehrich, O.,180, 181, 213, 221, 667 Rorsch, A.,442,489 Roess, W.B.,40,96 Roger, M., 443,449,486,,490 Rola, F. H., 47, 93 Romagnesi, H.,130, 149 Romig, W.R., 443,486 Roschevicz, R. J., 155, 156, 157, 158, 159, 163, 166, 167, 168, 170, 177, 180, 185, 188, 189, 193, 204, 205, 207, 208, 209, 211, 213, 216, 217, 219, 220, 222, 286 Roseman, S., 15,51,67,76,93,96,97 Rosenthal, G.A.,48, 69 Rosset, R., 36, 106 Rossignol, J. L., 432, 438,439,484, 486 Roth, J. R., 4, 21, 22, 27, 28, 34, 65, 91, 96, 96, 420, 487 Roth, L. F., 116, 132,146 Roth, S., 67, 96 Rothman, J., 16, 106 Rotman, R., 452,482 Roulland-Dussoix, D., 471, 487 Rowbury, R. J., 64, 65, 76, 83, 89 Roy, S. C.,162, 163, 164, 174, 286 Roy, S.K., 199,289 Ru, S.K.,199,200,247,286 Rudner, R., 443,&'7 Runemark, H.,195, 286 Rupp, W.D., 448, 48.9 Rush, G.,345,368 Russell, E.S.,67,96

Russell, R. L., 11, 12, 13, 69, 97, 98 Russo, S.,154,286 Ryan, S. L.,141, 161 Rytka, J., 24,98 S

Sadasivaiah, R. S., 263, 286 Sadler, J. R., 420, 422, 487, 488 Saedler, H., 15,17,18,86,91 Sahadevan, P. C.,162, 171,176, 177, 280 Sahay, B. N.,255, 286 Saier, M.H., Jr., 51,97 St. Lawrence, P.,2, 20, 104 St. Pierre, M.L., 16,97 Sakabe, K.,467,470,486 Sakai, K.I., 172, 247, 256, 258, 259, 261, 286, 686 Sakami, W., 53, 98 Sakore, T.D., 412,488 Salazar, J., 24, 90 Salgo, M.,34,74 Sambrook, J. F., 15, 21, 22, 97 Sampath, S., 155, 156, 157, 159, 162, 163, 164, 166, 169, 170, 171, 173, 174, 180, 181, 185, 186, 187, 191, 193, 199, 200, 202, 204, 205, 208, 210, 211, 213, 214, 216, 217, 219, 220, 221, 222, 224, 226, 228, 229, 231, 250, 254, 255, 263, 669, 273, 281, 286, 286 Sampson, D. R., 253,286 Sand, G., 17,63,7'6, 81 Sandberg, A. A., 234, 237, 276 Sanderson, K. E., 2, 34, 35, 49, 97 Sandler, I., 316, 369, 362, 363, 364, 365, 373, 403, 408, 409 Sandler, L., 294, 316, 351, 367, 368, 369, 360,361,362,363,364,365, 371,373,374, 376, 377, 378, 381, 383, 384, 385, 386, 396,398, 399, 406, 407, 408, 409 Sang, J. H., 66, 67, 73 Sansome, E.,121,161 Santibaiiez, S. K.,320, 366 Sarabhai, A.,6, 16,97 Sasaki, T.,156, 286 Sastry, N.S., 170,286 Sato, H., 248, 277 Sauer, C.O.,161,d87 Saul, G.B.,141,161

AUTHOR INDEX

Savage, E. J., 116, 121, 162 Savic, D., 34, 97 Savin, M. A., 53, $7 Sawicki, M., 44, 94 Scaife, J. G., 16, 17, 35, 51, 69, 97, 421, 451, 462, 486, 487 Scala, G., 61, 06 Gchaefer, E. W., Jr., 46,72 Schaefer, F., 452, 490 Schaeffer, P., 450, 483 Schaefler, S., 51, 97 Schaller, H. E., 466, 467, 488 Scharloo, W., 177, 287 Schekman, R., 467, 468, 477, 487, 489 Schleif, R. F., 423, 489 Schlessinger, D., 15, 36, 88, 97 Schmiedt, I., 8, 106 Schnos, M., 466,467,482 Schultz, J., 2, 30, 31, 45, 63, 97 Schultz, M. E., 117, 137, 162 Schwartz, D., 423, 490 Schwartz, M., 51, 63, 86, 422, 487 Schwarz, P., 40, 103 Schweickerdt, H. G., 180, 287 Schweizer, E., 26, 97 Scossiroli, R. E., 345, 369 Scott, w. A., 56,57,9r Seale, T. W., 23, 26,76, 97 Sebald, W., 56,97 Seetharaman, R., 185, 190, 191, 192, 195, 202, 214, 226, 229, 284, 286, 286, 287 Selhub, J., 53, 98 Selim, A. G., 172, 233, 258, 287 Sen, S. K., 234, 235, 236, 237, 239, 240, 245, 251, 287 Senaratna, J. E., 162, 208, 287 Sercarz, E. E., 45, 98 Seshu, D. V., 205, 218, 221,255,286,287 Sethi, B. L., 234, 256, 261, 287 Settles, F., 338, 349,350, 368, 394,408 Setzer, V., 59,90 Shaffer, B., 24, 98 Shah, D. B., 47, 88 Shapard, P. B., 31, 98 Shapiro, B. M., 89 Shapiro, J. A., 18, 58, 86, 98 Shapiro, J. L., 463, 487 Sharma, A. K., 240, 287 Sharma, S. D., 162, 163, 164, 166, 168, 169,

509

204, 205, 208, 209, 210, 226, 227, 269, 287, 288 Sharp, P. A., 428,485 Shastry, S. V. S., 162, 163, 164, 166, 168, 169, 170, 172, 191, 199, 200, 204, 205, 206, 208, 209, 210, 218, 222, 223, 224, 226, 227, 229, 230, 231, 234, 235, 237, 239, 240, 243, 249, 258, 262, 268, 269, s78, 287, 288 Shaw, S. F., 43,91 Shepard, M. K., 46, 72 Sheppard, D., 422, 480 Sheppard, D. E., 63, 65, 78, 98 Sherman, F., 6, 14, 23, 24, 25, 26, 54, 55, 57,81,90, 94, 98, 99 Shima, T., 337, 342, 369 Shinohara, M., 258, 288 Shipman, N., 14,99 Shive, W., 65,89 Shoup, J. R., 32,98, 326,369 Shulman, M. J., 452, 458, 487, 488 Shulman, R. G., 17, 18, 27, 70 Shumas, S. R., 54,71 Sick, K., 38, 42, 70, 345, 366 Siddiq, E. A., 201, 202, 262, 288 Siddiqi, 0. H., 448,487 Sidhu, N. S., 305, 306,307, 338,362, 669 Siegel, R. B., 424,489 Siegel, R. W., 117, 139, 162 Sigal, N., 428, 487 Signer, E. R., 452, 453, 457, 458, 463, 484,487,488,489, 490 Silagi, S., 55, 56, 98 Silbcrt, D. F., 15, 90, 420, 488 Silver, S. D., 37, 71 Silverstone, A. E., 17, 98, 423, 488 Simchen, G., 116, 124, I48 Siregar, H., 197, 199, 247, 288, 290 Sisken, J. E., 304,366 Sjodin, J., 249, 268 Slaughter, C., 54, 79 Slonimski, P. P., 54, 55, 57, 76, 94, 98 Smith, D., 10,98 Smith, D. A,, 64,65,89, 98 Smith, D. W., 466, 467, 488 Smith, D. W. E., 15,90 Smith, E. L., 55,57,77 Smith, H. O., 418,472,483

510

AUTHOR INDEX

Stern, C., 61,99 Stevens, C. M.,48, 74 Stewart, J. W., 6, 14, 23, 24, 25, 26, 55, 57,81, 98, 99 Stocker, B. A. D., 44,88 Stoeckenius, W., 449, 490 Stoffler, G.,36, 77 Stokes, J. L.,30,99 488 Stone, W. S., 295, 301, 330, 332, 369 Smith-White, S.,373,374,375,407 Stratling, W., 466, 483 Snell, E.E.,30,83 Sobell, H.M.,412,483, 488 Strangio, V. A., 402,409 SOU, D.,21,98 Strandberg, B.,411, 480 Strauss, B. S., 52, 59, 99, 101 Soliman, A.,31, 96 Streisinger, G., 18, 27, 86, 93, 99, 100, Soll, L.,22,62,84,98 Somers, J. M.,116, 127, 146, 162 447, 488 Stretton, A. 0. W., 11, 87 Somlo, M.,55, 76 Stretton, H. M.,116, 131, 149, 162 Sonneborn, T. M.,117, 139, 140, 162 Stromnaes, O.,310, 316, 319, 360 Soriano, J. D., 259,288 Spata, H.C.,444, 488 Studier, F. W., 424, 489 Speirs, J., 61,72 Sturtevant, A. I+., 2, 45, 59, 63, 92, 99, 319, 360 Spencer, J. E.,161,288 Stuttard, C., 10, 99 Spiegelman, S.,20,61,96,96 Spiess, E.B.,318,360 Stuy, J. H., 443,489 Spieth, H.T.,319, 320, 360 Subrahmanyam, V., 154, 172, 186, 204, 205, 268 Spirin, A. S.,36,98 Spizizen, J., 442,488,490 Subramaniam, N. K., 199, 289 Spofford, J. B.,263,264,288 Subramanyam, M. D.,208, 211,217, 286 Squires, C., 21, 27, 62, 63, 74, 78, 84, Sugimoto, K.,466, 467, 470, 486 98, 422,480 Sugino, A,, 466,467,468,470,486,489 Srb, A. M.,43, 56, 98, 99 Sukhodolec, V. V.,57,69 Srinivasan, P. H., 10, 100 Summers, W. C.,424,489 Stadler, D.R., 18,51,73, 99 Sundarajan, T. A., 58, 99 Stadtman, E.R.,49,81 Suskin, S.R.,6,24,29,74 83,99,100 Stahl, F. W., 15, 99, 454, 488 Susman, M., 11, 78, 448,489 Stahl, M.M., 447,454, 488 Sutherland, E.W., 423,484 Stahl, R. C.,23, 26, 27, 65, 83 Suyama, Y., 38, 39, 79 Stark, G. R.,36, 76 Suauki, H., 418, 486 Stark, M.B.,66,99 Swallen, J. R.,163, 217, 218,220, 289 Starlinger, P.,17,18,86,91 Swaminathan, M.S.,201,202,262,288 Stasiowski, S., 10, 16, 104 Swanson, C.P.,224,255,289 Stebbins, G. L., 165, 172, 176, 192, 196, Sybenga, J., 249, 289 201, 216, 233, 237, 262, 266, 288, 289 Szolyvoy, K.,15,84 Steffensen, D.M.,20,99 T Steinberg, C. M.,11, 22, 26, 78, 90 Tabata, H., 247, 289 Steinberg, R.,418, 478 Steinberg, W. L.,444, 488 Tabor, C.W., 45,100 Steiner, E.,117, 137,162 Tabor, H., 45,100 Takahashi, H.,241, 289 Stent, G. S., 423, 488 Takahashi, M.,247, 254, 261, 280, 289 Stephens, S. G., 173, 263, 289

Smith, J. D., 11, 12, 13, 21, 69, 8.9, 88, 97, 98 Smith, J. M.,329, 330, 332, 360 Smith, P.,219, 270 Smith, S.I.,41,100 Smith, T. F.,420, 422, 487, 488 Smith-Keary, P. F., 33, 34, 77, 98, 446,

511

AUTHOR INDEX

Takahashi, R., 174,289 Takata, R., 11, 100 Takenaka, Y., 241, 189 Takigushi, Y., 197, 200, 874 Tamaki, H., 16, 70 Tamir, H., 10,100 Tanka, S., 46, 51, 100 Tandler, B., 312, 360 Tanemura, S., 18, 19, 27, 87, 100, lo4 Tang, P. S., 247, 989 Tanner, D., 47, 100 Targa, H. J., 59, 90 Tarrab, R., 43, 91 Tartof, K. D., 20, 30, 31, 61, 100 Tateoka, T., 154, 155, 156, 162, 163, 166 168, 180, 193, 204, 205, 207, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 226, 229, 232, 241, 289, 290 Tates, A. D., 308,360 Tatum, E. L., 30, 31, 55, Y7, 81, 100, 123, 162 Taylor, A. L., 2, 100, 450, 489 Taylor, C. W., 68,93, 116,122,161,169 Taylor, E., 472, 489 Templin, A., 4,47, 70, 88 Ten Berge, A. M. A., 8 , 105 Terao, H., 197, 198, 290 Terry, W., 16, 95 Tersaghi, E., 18, 27, 93, 99, 100 Tevethia, M. J., 443,489 Thakur, R., 248, 254, 278, 290 Theodor, J. L., 117, 142,169 Theriot, L., 442, 482 Thiery, J., 412, 478 Thoday, J. M., 177, 290 Thomas, C., 364, 377, 406 Thomas, C. A,, Jr., 439, 447, 489 Thomas, F. L. X., 14, 99 Thomas, R., 454, 485 Thorpe, D., 8,104 Thurmon, T. F., 46,72 Thwaites, W. M., 40, 41, 77, 100 Tindeholt, V. E., 328, 366 Ting, R. C., 446, 484 Ting, Y., 159,160, 177,290 Tissieres, A., 55, 56, 84, 91, 101 Titani, K., 14, 92 Tocchini-Valentini, G., 422, 423, 4Y6

Tokunaga, C., 402, 409 Tokuno, S., 52, 101 Tokuyama, T., 32,92 Tokuyasu, K. T., 304, 308, 326, 360, 365, 366, 371, 373, 383, 391, 404, 408, 409 Toledo, F., S. A., 59, 90 Tomizawa, J., 37, 101, 446, 447, 488 Toshima, H., 48, 101 Tourney, R., 216,986 Townsend, C. E., 108, 135, 169 Trautner, T. A., 444, 488 Travers, A. A., 422, 423, 478, 489 Trippa, G., 365,408 Trotta, P. P., 48,49,101 Tsai, C. K., 207, 291 Tsai, K. S., 223, 991 Tsuchiya, T., 253, 254,290 Tsuda, Y., 52,101 Tsugita, A., 11, 18, 27, 86, 93, 99, 100 Tu, D. S., 198,201,%?76 Tuli, V., 29, 101 Tung, Y., 412,478 Turner, B. C., 122,162 Turner, J. H., 66,101 Twardzik, D. R., 30, 32,101 Tyagi, 0. P., 231,283

U Uchiyamada, H., 201, 247, 248, U7, 986 Ucko, P. J., 193, 290 Ulane, R., 54, 101 Ullmann, A., 17, 63, 86, 423, 489 Umbarger, H. E., 29, 32, 33, 34, 46, 48, 49, Y3, 74, 83, 86, 101, 420, 489 Unger, L., 44, 101 Uscuplic, M., 116, 132,146

V Valentine, R. C., 448, 449, 489, 490 van Breugel, F. M. A., 67, 87 Vandendries, R., 116, 129,169 Van de Putte, P., 442,489 van Schouwenberg, J. C., 197, 199, 990 Varmus, H. E., 422, 423, 4Y9, 486 Vasconcellos, J. de C., 154, 990 Vavilov, N. I., 158, 160, 165, 990

512

AUTHOR INDEX

Velasco-Demeterio, E.,200, 290 Venema, G.,443,489 VenemaSchroder, T.,443, 489 Venkataswamy, T.,200, 255, 287, 290 Verkov, P.,18,91 Verrall, A. F.,133,152 Vesell, E. S.,46, 101 Vielmetter, W., 448,477 Vogel, H. J., 41, 42, 43, 44, 45, 69, 70, 85, 86, 101, 102, 104 Vogel, R. H.,41, 42, 43, 44, 101, 102 Voll, M.J., 34,102 Volterra, L.,439, 486 Von Borstel, R. C., 22,26,90, 102 Von Halle, E.S., 396,407 von Wettstein, D., 425,489 Vrablik, G.R.,46,72 Vyas, S.,45, 102 W

Wagenaar, G.A. W., 197, 199, 290 Wagner, R. P.,2, 45, 46, 60, 102 Wagoner, D. E.,46,102 Walters, M. S.,258,290 Wampler, D.E.,41,79 Wang, S.,260,290 Wang, W. H.,172, 186, 191, 206, 210, 214, 217, 222, 223, 226, 228, 230, 276 Ward, S.,11, 102 Warner, H.R.,47,102 Waskell, L.,424, 478 Watanabe, K.,50,94 Watanabe, Y.,223, 229, 230, 231, 232, 244, 245, 247, 250, 251, 252, 261, f l 6 , 291 Watson, J. D., 411,479 Watson, W., 161,291 Watt, G.,157, 158, 159, 162, 163, 166, 167, 168, 171, 174, 177, 211, 232, 291 Wattiaux. J. M.,20,W Watts-Tobin, R. J., 17, 18, 27, 70, 76 Weber, A., 16, 70 Weber, K.,16,37,94 Webster, J., 143, 148 Webster, R. E., 21, 22, 23, 48, 76, 78, 83, 91 Webster, R.K., 126,162

Wechsler, J. A.,466, 481 Wedvik, M., 324,360 Wegelenski, P.,43, 44, 94, 102 Weidel,W., 30, 73 Weigel, P. H.,489 Weigert, M.G.,6,11, 102 Weil, J., 452, 453, 458, 487, 488, 489 Weinberg, R.,58, 78 Weinfeld, H., 58,72 Weisberg, R. A., 454, 457, 481, 483, 489 Weiss, R. L.,43, 102 Well, R. D.,22, 87 Wellner, V. P., 48,69 Weng, T.S.,172, 186, 191, 206,210, 214, 217, 222, 223, 226, 228, 230, 276 Wenk, M., 16,106 Werner, R.,467, 489 Westergaard, M., 426,489 Westergaard, O.,468, 487 Weyer, E. M.,46, 101 Wheals, A. E.,116, 120, 162 Wheeler, M. R., 299, 320, 331, 332, 343, 360

Whitehead, E., 29,102 Whitehouse, H. L. K.,430, 489 Whitfield, H. J., Jr., 15, 23, 26, 69, 90, 10.2 Whitney, H.S., 116, 131, 168 Wiame, J. M.,42, 88 Wiame, J.-M., 42, 43, 88, 91 Wickner, W., 467, 468, 487, 489 Wiebers, J. L.,53, 54, 102, 103 Wiesmeyer, H., 58,104 Wilhelm, R. C.,11, 26, 78, 81, 84 Wilkins, M.D.,65,89 Willetts, N. S., 46, 103, 442, 489 Williams, C. A., 111, 116, 123, 162 Williams, L.G.,38, 39, 40,41,42, 103 Williamson, D.L.,312,360 Willson, C.,17,63,103 Wilson, E.O.,225,291 Wilson, J. F.,55, 81, 111, 116, 123, 147, 162 Wilson, J. H., 11,102 Wilson, L.P.,332,360 Wimber, D.E.,20, 99 Wittmann, H.G.,36,77 Wollman, E. L.,448, 449, 450, 482, 483, 489

513

AUTHOR INDEX

Wood, S.B., 11, 103 Wood, W. B., 11,87,102 Woodward, C. R., Jr., 30,99 Woodward, D. O., 56, 57, 72, 78, 103, 106 Woodward, V. W., 39, 40, 41, 7Y, 84, 90, 103 Work, T. S., 56, YO Wright, M., 46, 103 Wrigley, C., 183, 184, 291 Wu, C. K., 402,409 Wu, H. K., 168, 169, 200, 223, 227, 229, 230, 231, 2Y6, 27Y, 291 w u , L., 223, 291 Wu, R., 472,489 Wu, S. H., 207, 291 Wuesthoff, 0. G., 16,103 Wun, K. D., 222, 223, 226, 228, 276 Wuu, K. D., 222,229,291 Wyche, J. H., 34,65,103 Wylie, A. P., 262,268 Wyman, J., 411, 412,486 Wyttenbach, E. G., 144,147

Y Yamaura, A., 256, 261,291 Yamazaki, H. I., 67, 103 Yan, Y., 48, 103 Yanders, A. F., 305, 307, 324, 337, 339, 345, 358, 360, 388, 406, 408, 409 Yang, K. K. S., 168, 222, 261,277, 291 Yanofsky, C., 2, 5, 6, 7, 8, 10, 15, 16, 17, 18, 20, 29, 61, 62, 69, Y2, Y3, Y4, 84, 85, 86, 92, 100, 104 Yao, S. Y., 200, 240, 291 Yarmolinsky, M. B., 58, 104, 452, 453, 454, 457, 481 Yasui, K., 234, 236, 241, 292 Yasuzumi, G., 308,360 Yates, F., 438, 480 Yeh, B. P., 170, 171, 172, 186, 190, 191, 200, 206,226,228,270,298 Yeh, J., 64, 89 Yeh, P. Z., 260, 290

Yen, D. E., 160,292 Yen, M. C., 130,149 Yen, Y. J., 248, 292 Young, F. E., 442, 490 Young, H. C., 260,271 Young, W. C., 324,360 Young, W7.S., 111,51,9Y Yourno, J., 18, 19, 27, 87, 100, lo4 Yuan, C. H., 223, 2Y5 Yuan, R., 418, 470, 471, 484 Yudin, A. L., 117, 139,150 Yuill, E., 124, 148 Yunoki, T., 251, 292 Yura, T., 42, lo4 Yu-Sun, C. C. C., 437, 438, 480 2

Zabell, S., 16, 105 Zabka, G. G., 116, 119, 160 Zadrazil, S., 11, 12, 21, 82 Zalkin, H., 10, 84, 86, 92, lo4 Zalokar, M., 54, 60, 105 Zambruni, L., 330, 354 Zarro, S., 348, 356, 398, 407 Ziegler, I., 32, 106 Zimmering, S., 294, 297, 300, 324, 325, 328, 337, 340, 341, 347, 348, 354, 360, 361, 365, 384, 385, 386, 396, 398, 400, 402, 404, 409 Zimmermann, F. K., 8, 93, 105 Zimmermann, R. A., 8,105 Zinder, N. D., 20, 21, 22, 23, 75, 78, 85, 91, 93, 446, 449, 452, 477, 484, 490 Zipser, D., 15, 16, 22, 83, 91, 93, 105, 422, 477 Zissler, J., 452, 457, 490 Zohary, D., 160, 165, 176, 178, 270, 2Y6, 292 Zollinger, W. D., 56,105 Zubay, G., 422, 423.4Y6, 490 Zukovsky, P. M., 170, 292 Zwaig, N., 52, 72 Zwenk, H., 442, 489

SUBJECT INDEX A

female, 295-301 male, 301-315 female post-mating response, post-copulation reproductive physiology, 330-334 state of receptivity, 328-330 segregation distorter, discussion, 381-384 genetics of, 371-380 mechanism of action, 362-371 sex chromosome meiotic drive systems, 384-385 description of sc'sc' system, 385-386 discussion, 402-404 interpretation of sc4sc8system, 386-388 modification by temperature, 400402 new hypothesis, 392-396 reexamination of sc4sc8 system, 388-392 tests of model, 396-400 sexual behavior, acquisition of sexual maturity, 316-319 courtship and copulation, 319-320 duration of copulation, 320-321 sperm in the female, storage, 335-341 utilization, 341-351 sperm transfer, number ejaculated, 322328 time of ejaculation, 321-322

Africa, rice, origin, 178-196 subspeciation, 203-204 Animals, heterogenic incompatibility in, 138-141 Asia, rice, origin, 157-178 subspeciation, 196-203 Asynapsis, rice, 259-260 C

Chiasma, frequency in rice, 259 Chromosomes, rice, morphology, 233-240 number, 240-255 structure, 474-476 Codon(s), different letters, supression and, 5-6 Copulation, Drosophila, 319-320 duration, 320-321 Courtship, Drosophila, 319-320

D Deoxyribonucleic acid, replication mechanism, in vitro, 464-466 in vivo, 466-470 restriction and modification mechanisms, 470474 Desynapsis, rice, 259460 Drosophila, anatomy of reproductive systems, 294-295

E Enzyme(s), active conformation, reconstitution, 28-37 Eukaryotes, genetic recombination mechanism, 425441 514

515

SUBJECT INDEX

F Frameshif ts, double, suppression and, 18-19 Fungi, heterogenic incompatibility in,

0 Oryza see rice

115-135

P G

Gene product, limiting, effective dosage, 6 0 4 6 Genetic recombination, mechanism, 424-425 eukaryotic systems, 425-441 prokaryotic systems, 441-451 viruses, 451-464 Genetics, heterogenic incompatibility in Podospora, 109-113 suppressors, 1-2 Genome rice, nature of, 261-264 relationships, 225-233

H Heterogenic incompatibility, animals, 138-141 conclusions, 142-145 fungi, 115-135 higher plants, 135-138 histoincompatibility and, 141-142 Podosgora, genetic basis, 109-113 Heterozygotes, interchange, rice, 26&261 Histoincompatibility, heterogenic incompatibility and, 141-142 1

Initiators, new, suppression and, 14-17

M Meiosis, irregularities, cross sterility and, 114

N Nucleoli, rice, number, 258

Parsexual processes, genetic control, basic control of propagation, 113-114 cross sterility due to irregular meiosis, 114 species concept, 115 sterility genes, 114 Peptide (s) , toxic suppression, 17-18 Plants, heterogenic incompatibility in, 135-138

Podospora, heterogenic incompatibility, genetic basis, 109-113 Polarity, elimination, suppression and, 14-17 Post-mating response, Drosophila female, post-copulation reproductive physiology, 330-334 state of receptivity, 328-330 Prokaryotes, genetic recombination mechanism, 441-451 Protein(s), active conformation, suppression and, 6-13

substitute activity, suppression and, 37-58

Protein-nucleic acid interaction, symmetry, dimer recognition, 412-418 tetramer recognition, 418420

R Reproductive systems, Drosophila, female, 295-301 male, 301-315 Ribonucleic acid, transcription, regulation mechanism, 420424

516

SUBJECT INDEX

Rice(s), asynapsis and desynapsis, 259-260 chiasma frequency, 259 chromosome complement, early studies, 233 morphology, 233-240 number, 240-255 classification of, 154-157 interchange heterozygotes, 260-261 nature of genome, 261-264 nucleolus number, 258 origin, African, 178-196 Asian, 157-178 secondary association, 255-258 species relationships, genome relationships, 225-233 interrelationships, 206-225 subgeneric classification, 204-205 subspeciation, African, 203-204 Asian. 196-203

S Segregation distorter, Drosophila, discussion, 381-384 genetics of, 371-380 mechanism of action, 362-371 Sex chromosome, meiotic drive systems, Drosophila, 384-385 description of sc4scssystem, 385-386 discussion, 402404 interpretation of sc’sc* system, 386-388

modification by temperature, 400-402

new hypbthesis, 392-396 reexamination of sc4scs system, 388-392

tests of model, 396-400

Sexual maturity, acquisition in Drosophila, 316-319 Sexual processes genetic control, basic control of propagation, 113-114 cross sterility due to irregular meiosis, 114 species concept, 115 sterility genes, 114 Species, concept, 115 Sperm Drosophila, number ejaculated, 322-328 storage in female, 335-341 time of ejaculation, 321-322 utilization by female, 344-351 Sterility, genes and, 115 Suppression, intergenic, 14-20 effective dosage of limiting gene product, 60-66 elimination of deleterious accumulation, 58-60 informational, 20-28 reconstitution of active enzyme conformation, 28-37 substitute protein activity, 37-58 intragenic, 4-5 active conformation, 6-13 different letters of a codon, 5-6 double frameshifts, 18-19 elimination of a toxic peptide, 17-18 new “initiators” and elimination of polarity, 14-17 nomenclature, 24 other interesting cases, 66-68 Suppressors, genetics and, 1-2

V Viruses, genetic recombination mechanism, 451464