Progress in Nucleic Acid Research and Molecular Biology, Volume 45

PROGRESS IN

Nucleic Acid Research and Molecular Biology Volume 45

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PROGRESS IN

Nucleic Acid Research and Molecular Biology edited by

WALDO E. COHN Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

KlVlE MOLDAVE Department of Molecular Biology and Biochemistry University of Calqornia, Zruine Iroine, California

Volume 45

ACADEMIC PRESS, INC. Harcourt Brace & Company

Son Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system,without permission in writing from the publisher.

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United Kingdom Edition pubfished by

Academic Press Limited 2 4 2 8 Oval Road, London NW17DX International Standard Serial Number: 0079-6603 International Standard Book Number: 0-12-540045-4

PFUNTEDW THE UNITED STATES OF AMERICA

939495969798

BB

9 8 1 6 5 4 3 2 1

Contents

ABBREVIATXONSAND SYMBOLS ........................................

ix

SOMEARTICLESPLANNED FOR F ~ U RVOLUMES E .......................

xi

Analysis of Rice Genes in Transgenic Plants Ray Wu, Xiaolan Duan and Deping Xu I. Analysis of Rice Genes ........................................ Methods for Gene Transfer in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of Transferred Genes in Transgenic Plants . . . . . . . . . . . . . . . . IV. Concluding Remarks and Future Prospects ....................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. 111.

2 9 16 22 24

Immunoglobulin Gene Diversification by Gene Conversion Wayne T. McCormack, Larry W. Tjoelker and

Craig B. Thompson Evidence for Somatic Gene Conversion . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Mechanism of Somatic Immunoglobulin Gene Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Enzymatic Activities Implicated in Immunoglobulin Gene Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

29

11.

32 38 43 44

ADP-ribosylation Factors: Protein Activators of Cholera Toxin Joel Moss and Martha Vaughan I. 11. 111.

Biochemistry of ADP-ribosylation Factors . . . . . . . . . . . . . . . . . . . . . . . . Structure of ADP-ribosylation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of ADP-ribosylation Factors in Animal Cells . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

49 55 60 63

vi

CONTENTS

Molecular Biology in the Eicosanoid Field Colin D. F u n k I. 11. 111. IV. V. VI. VII. VIII.

Lipoxygenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leukotriene A4 Hydrolase . . . . . . . . .. . Prostaglandin G / H Synthase ( ................... Prostaglandin-F Synthase . . . . . . . References

69 81

83

. . _ . . . . . . . . . . 89

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

93

Mamma Iian 6- Phosphofructo-2-kinase/fructose-2,6bisphosphatase: A Bifunctional Enzyme That Controls Glycolysis Guy G. Rousseau and Louis H u e I. PFK-2/ FBPase-2, A Bifunctional Enzyme That Synthesizes and Degrades Fructose 2,6-Bisphosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Molecular Characterization of PFK-2lFBPase-2 Isozymes . . . . . . . . . , 111. Characterization of PFK-2/ FBPase-2 Genes . . . . . . . . . . . . . . . . . . . . . . rV. Hormonal Control of PFK-Z/FBPase-Z Gene Expression . . . . . . . . . . . V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 106 116 119 124 124

tRNA Structure and Aminoacylation Efficiency Richard Gieg6, Joseph D. Puglisi and Catherine Florentz

.. .

I. Structural Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 11. Phenomenology and Early Structural Results . . . . . . . . . . . . . . . . . . . . . 111. Complexes between tRNAs and Aminoacyl-tRNA Synthetases . . . . . . IV. tRNA Identity for Aminoacyl-tRNA Synthetases . . . . . . . . . . . . . . . . . . V. General Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

. .

131 149 159 166 192 195

Evolution of Caz+-dependent Animal Lectins Kurt Drickamer 1. Classes of Animal Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 11. CaZ+-dependent Carbohydrate-Recognition Domains . . . . . . . . . . . . . . 208 111. Organization of C-type Animal Lectin Genes . . . . . . . . . . . . . . . . . . . . . 216

VII IV. Evolution of Saccharide-bindingSpecificity . . . . . . . . . . . . . . . . . . . . . . . 220 V. History of Carbohydrate-Recognition Processes . . . . . . . . . . . . . . . . . . . 229 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 INDEX

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

233

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Abbreviations and Symbols All contributors to this Series are asked to use the terminology (abbreviations and symbols) recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) and approved by IUPAC and IUB, and the Editors endeavor to assure conformity. These Recommendations have been published in many journals (I, 2) and compendia (3) and are available in reprint form from the Office of Biochemical Nomenclature (OBN); t h y are therefore considered to be generally known. Those used in nucleic acid work, originally set out in section 5 of the first Recommendations (I) and subsequently revised and expanded (2.3),are given in condensed form in the frontmatter of Volumes 9-33 of this series. A recent expansion of the one-letter system ( 5 ) follows. (5) SINGLE-LETTER CODERECOMMENDATIONS. Symbol

Origin of symbol

Meaning

G A TW) C

G A T(U) C

Guanosine Adenosine (ribo)Thym idine (Uridine) Cytidine

R

G or A T(U) or C A or C G or T(U) G or C A or T(U)

puRine pyrimidine aMino Keto Strong interaction (3 H-bonds) Weak interaction (2 H-bonds)

Y M K S

Wb

not not not not

or C or T(U) or T(U) or C or C or A or A or T(U)

G; H follows G in the alphabet A; B follows A T (not U); V follows U C; D follows C

D

A G G G

N

G or A or T(U) or C

aNy nucleoside ( i t . , unspecified)

Q

Queuosine (nucleoside of queuine)

H

B V

Q

‘Modified from h.Natl. Acad. Sci. U.S.A. 83, 4 (19E6). bW has been used for wyosine, the nucleoside of “base Y (wye). ‘D has been used for dihydrouridine (hU or H, Urd). Enzymes

In naming enzymes, the 1984 recommendations of the IUB Commission on Biochemical Nomenclature ( 4 ) are followed as far as possible. At first mention, each enzyme is described either by its systematic name or by the equation for the reaction catalyzed or by the recommended trivial name, followed by its EC number in parentheses. Thereafter, a trivial name may be used. Enzyme names are not to be abbreviated except when the substrate has an approved abbreviation (e.g.. ATPase. but not LDH, is acceptable).

ix

ABBREVIATIONS AND SYMBOLS

X

REFERENCES I . JBC241,527 (1966); &hem 5, 1445 (1966); /A/ 101. 1 (1966); ABB 115. 1 (1966). 129. 1 (1969); and e1smhere.t Gmeral. 2. EIB 15. 203 (1970); JBC 245. 5171 (1970); /ME 55, 299 (1971); and c1smhcn.t 3. "Handbook of Biochemistry" (G. Fasman, ed.), 3rd ed. Chemical Rubber Co., Cleveland, Ohio, 1970, 1975. Nuclcic Acids, Wls. I and 11, pp. 3-59. Nucleic acids. 4. "Enzyme Nomenclaturc" [Recommendations (1984) of the Nomenclature Committee of the IUB]. Academic Press, Ncw York, 1984. 5. EIB 150, 1 (1985). Nucleic Acids (One-letter system).? Abbreviotions of Journal Titles

Journals

Abbreviations used

Annu. Rev. Biochem. Annu. Rev. Genet. Arch. Biochem. Biophys. Biochem. Biophys. Res. Commun Biochemistry Biochem. J. Biochim. Biophys. Acta Cold Spring Harbor Cold Spring Harbor Lab Cold Spring Harbor Symp. Quant. Biol. Eur. J. Biochem. Fed. Proc. Hoppe-Seyler's Z. Physiol. Chem. J. h e r . Chem. Soc. J. Bacteriol. J. Biol. Chem. J. Chem. Soc. J. Mol. Biol. J. Nat. Cancer Inst. Mol. Cell. Biol. Mol. Cell. Biochem. Mol. Gm. Genet. Nature. New Biology Nucleic Acid Research Prcc Natl. h a d . M.U.S.A. Proc Soc Exp. Biol. Med. Progr. Nucl. Acid. Res. Mol. Biol.

ARB ARGen ABB BBRC Bchem BJ BBA CSH CSHLab CSHSQB EJB FP ZpChem JACS J. Bact. JBC JCS JMB JNCI MCBiol MCBchem MGG Nature NB NARes PNAS PSEBM This Series

tReprints available from the Office of Biochemical Nomenclature (W. E.Cohn, Director).

Some Articles Planned for Future Volumes Global Regulation of Mitochondrial Biogenesis in Saccharornyces cerevisioe

J. H. DE WINDEAND L. A. GRIVELL Adenoviral DNA Integration and Changes in DNA Methylation Patterns: A Different View of Insertional Mutogenesis

WALTERDOERFLER mRNA Binding Proteins in Eukaryotic Cells

TOMDONAHUE AND KEITH GULYAS Genomic Organization of T and W, A New Family of Double-Stranded RNAs from Sacchorornyces cerevisiae

ROSA ESTEBAN.NIEVES RODRIGUEZ-COUSIRO AND LUISM. ESTEBAN Enzymology of Homologous Recombination in Sacchorornyces cerevisiae

W. D. HEYERAND R. D. KOLODNER Regulation of mRNA Stability in Yeast

ALLAN JACOBSON AND

STUART h L T Z

Parallel-stranded DNA and RNA

THOMAS M. JOVIN,KARSTEN RIPPE, VITALY KURYAVYI AND ANGEL GARCIA Signal Transducing G Proteins: Basic and Clinical Considerations

MICHAEL A. LEVINEAND CHARLESEMALA Synthesis of Ribosomes

LASE LINDAHLAND

JANICE

M . ZENGEL

Drugs That Deplete Mitochondrial DNA in Vertebrates: Basic and Physiological Considerotions

REJEAN MORAIS DNA Polymerase II, the Epsilon Polymerase of Socchorornyces cerevisiae

ALAN MORRISONAND AKIO SUGINO Posttranscriptional Control of Lysogenic Pathway in Bacteriophage Lambda AMOS B. OPPENHEIM,DANIELKORNITZER, SHOSHY ALTUVIAAND

DONALDL. COURT Molecular Biology and Regulatory Aspects of Bacterial Glycogen and Plant Starch Synthesis

JACKPREISS xi

xii

SOME ARTICLES PLANNED FOR FUTURE VOLUMES

Mechanism of Action and Regulation of Protein Synthesis Initiation Factor 4E. Effects on mRNA Discrimination, Cellular Growth Rate, and Oncogenesis ROBERT E. RHOADS, SWATI JOSHI-BARVEAND CARRIERINKER-

SCHAEFFER Prosomes and Their Multi-catalytic Prateinase Activity: Relations to Messenger RNPs and the Cytoskeleton

KLAUS SCHERRER Regulation of Bacillus subtilis Gene Expression during the Transition from Exponential Growth to Stationary Phase

MARKA. STRAUCH The Egr Family of Immediate-Early Transcription Fodors

VIKASP. SUKHATME lntron Structure in Angiosperms VIRGINIAWALBOTAND KENNETH R.

LUEHRSEN

Analysis of Rice Genes in Transgenic Plants RAY Wu,' XIAOLANDUAN AND DEPING Xu Section of Biochemistry, Molecular and Cell Biology Cornell University fthaca, New York 14853

I. Analysis of Rice Genes ........................................... 11. Methods for Gene Transfer in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Analysis of Transferred Genes in Transgenic Plants . . . . . . . . . . . . . . . . . IV. Concluding Remarks and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . References .....................................................

2 9 16 22

24

Rice is the most important cereal crop in the world because, for over two and a half billion people, it is the major source of food (1). In 1990, over 510 million tons of rough rice were produced, and together with wheat, these two major crops feed most of the worlds population (2). Although much research has been carried out in rice genetics, breeding, and physiology, practically no work had been carried out prior to 1985 on the analysis of rice genes at the molecular level. As of 1984, there were only a handful of laboratories working on rice genes, and only three papers had been published on this subject. In contrast, dozens of papers were published on the molecular analysis of wheat and maize genes. In early 1985, The Rockefeller Foundation launched a major program to promote research on rice molecular biology and biotechnology. With a clear vision, adequate funding, and superb management by The Rockefeller Foundation, the number of first-rate laboratories working on rice molecular biology increased to 20 in 1986 and to over 70 in 1992. Largely as a result of this program, there has been an explosion of published studies: to date (April 1992) there are over 100 publications on the molecular analysis of rice genes. Some of these studies are described in Section I of this article. In addition to cloning and analyzing rice genes, it is essential to develop efficient methods for transformation and regeneration of rice plants. Transgenic plants are needed for analyzing the regulatory sequences of genes in vivo, and for transferring agronomically important genes into rice plants. As

To whom correspondence may be addressed. 1

Progress In Nucleic Acid Research and Molecular Blology, Vol 45

Copynght 8 1993 by Academic Press, Inc All rights of repmductmn in any form resewed

2

RAY WU ET AL.

of 1988, at least two methods had been developed for transforming rice protoplasts or intact cells that can lead to the regeneration of fertile transgenic plants; more details on transformation and regeneration of rice plants are given in Section 11. With these advances, rice has become the preferred model system for the molecular analysis of genes in cereal crops, for it is much easier to produce transgenic plants with rice (3-8) than with other cereals such as maize (9, 10), wheat ( I l ) , barley, or oat. Another advantage is that the genome size of rice is relatively small, only 4.2 x lo8 base-pairs per haploid genome, whereas the genome size of maize is six times larger and that of wheat is 30 times larger (12). In this article, we present information on molecular analyses of rice genes by in uiuo and in uitro methods (Section I) and in transgenic plants (Section 111).We have limited our discussion to nuclear genes and selected only a small number of representative genes for more detailed description. This treatment is not meant to be exhaustive: rather than including molecular analyses of all known rice genes, we give selected examples of several important types of rice genes, particularly those about which we have first-hand information. Another consideration in choosing specific genes for discussion involves selection of those genes that are unique to plants: for example, genes related to seed storage proteins in rice and other monocotyledenous plants such as wheat and barley. The response of rice phytochrome genes to light is another such example of gene regulation unique to plants. Finally, the development of specific applications, such as attempts to produce insectresistant transgenic rice plants, is a unique and important application of plant biotechnology.

1. Analysis of Rice Genes In general, molecular analyses of animal genes are far more advanced than those of plant genes. This is directly related to the fact that the level of funding for animal (including human) research has been more than ten times that for plant research. Moreover, in plant research, work on dicotyledenous plants such as tobacco and Arabidopsis is more advanced than that on monocotyledenous plants such as rice and wheat because these dicots are easier to transform and regenerate more plants than monocots. In this section, examples of the molecular analyses of several rice genes are given in order to show what types of experiments have been carried out, and how much is known about rice genes. It should be mentioned that, even though the genes described here are unique to plants, many of the methods used for analyzing plant genes are similar to those used in research on animals or bacteria.

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

3

A. Rice Actin Genes Actin is an essential component of the cytoskeleton of eukaryotic cells in a number of animal systems. Although the function of actin is less well understood in higher plants than it is in animals, actin is believed to play an important role in plant cell shape determination, cytoplasmic streaming, organelle movement, and extensive growth (13). In higher plants, actin proteins are encoded by multigene families. Four different actin genes have been isolated from a rice genomic library (14); however, these four do not represent all actin genes in rice, because genomic blot analysis shows the presence of 8-10 hybridizing bands, and at least seven different rice actin cDNAs have been isolated (15). Each of the four genomic actin genes contains three introns in the coding regions, and codes for a protein of 376-379 amino acids. In the actin 1gene there is also a 5' intron separating the 5' noncoding exon from the first coding exon.

1. In Vim ANALYSIS OF RICE ACTIN GENEEXPRESSION To determine whether the expression of the four isolated members of the rice actin gene family are differentially regulated, their respective transcript levels were determined. The results show that RNA transcribed from individual rice actin genes displays either a constitutive or developmentally differentiated pattern of accumulation in different vegetative tissues. Moreover, members of the actin gene family differ from each other in tissuespecific abundance of their respective transcripts. Gel blots of total RNA from rice were hybridized with radioactive actin gene-specific probes to determine the transcript size and the abundance of each transcript. Each of the four actin genes encodes a unique transcript 1.5 to 1.7 kb in length. The steady-state mRNA levels of each gene were assayed using RNAs isolated from different tissues at varying times during early vegetative growth. The results indicate that the transcript level of the four actin genes differ by approximately 20-fold in 4-day-old shoots. The relative mRNA level of act1 : act2 : act3 : act7 was 100 :35 : 6 : 5. The transcript levels of act1 and act7 were relatively constant in shoots 2, 4, 7, 13, and 35 days after germination. In contrast, the transcript levels of act2 and act3 35-day shoots dropped to approximately 20% of the level observed in 2-day-old shoots (1S). Abbreviations and special names: a d , rice actin 1 gene; bar, phosphinothricin acetyltransferase gene; bialaphos, a phosphinyl-substituteddialanine; CAT, chlorarnphenicol acetyltransferase; DAF, days after flowering; glufosinate, 2-amino-4-(hydroxymethylphosphiny1)butanoate; GUS, bacterial P-glucuronidase;gus, bacterial 0-glucuronidase gene; hpt, hygromycin phosphotransferase gene; nos, nopaline synthase gene; PEG, polyethylene glycol; Pfr, active form of phytochrome; phosphinothricin, the S-isomer of glufosinate; phyA, phytochrome A gene; p h y B , phytochrome B gene; Pr, inactive form of phytochrome.

4

RAY WU ET AL.

81

R1

-450

E l 1 2 61

81

MUM of Construct

O w Erprorrlon Efficiency (X)

PACn-F

100%

PActl-G

50%

PACtl-L

28%

pAcnJ

4%

Xbl

C l

R1

81

R1

Xbl

t

+i

-45n

81

61

81

Bl

81

Xbl

I

-45s

+1

R1

81

Xbl

2% -450

0

+1

&18EodlnO -ton

W 1 5 ’ roglon non-codlng nons

nos 3 iorrn~~tor sqwnso

AM1 5’ lntron

FIG. 1. Activity of GUS enzyme in rice protoplasts transformed with Actl-Gus constructs. The results represent the average of four independent experiments assayed 20 days after transformation of protoplasts (16). The lengths of the different regions of the constructs are not to scale. The indicated restriction enzyme sites are abbreviated as follows: B1, BomHI; 82, BglII, RI, EcoRI; S1, Sstl; Xbl, XbaI; Xhl, XhoI.

2. TRANSIENT EXPRESSION ANALYSIS

OF

RICE ACTIN-1 GENE

Analysis of the 5’ region of the rice actin-1 gene (actl)was carried out by constructing plasmids containing different lengths of the 5’ region joined to the coding sequence of a gene encoding a bacterial P-glucuronidase (GUS). Transient assays using rice protoplasts showed that a region of 1,400 base pairs upstream from the act1 translation-initiation codon contains all of the S‘-regulatory elements necessary for high-level gus expression (construct pActl-F, Fig. 1).Deletion of 384 bp from the 5’-end of pActl-F gives pActlG, which was 50% as active as pActl-F. Starting from pActl-G, by deleting 133 bp between the acceptor splice site and the putative mRNA branchpoint splice site from its 5’ intron (pActl-L), lowered the GUS activity to 28% of that observed for pActl-F. Deletion of the mRNA branch-point splice site (vertical arrows in Fig. 1) and 3’-donor splice site from its 5’ intron (pActl-J) lowered GUS activity to 4% of that for pActl-F. Deletion of the

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

5

entire act1 5’ intron (pActl -H) resulted in a drop of expression level to 2%of that for pActl-F. These and other data suggest that the intron-mediated stimulation of gus expression is associated, in part, with an in vivo requirement for efficient intron splicing (16). Additional deletion analysis of the 5’ region of act1 was carried out in the region upstream from the transcription start site (designated + 1 position). Transient assay identified two distinct cis-acting regulatory elements in the promoter region. The first regulatory element, a 40-bp poly(dA-dT) region between positions -186 and -146 (Fig. 1, pAct1-F; the location is marked by a large dot), was a positive regulator of the Act1 promoter activity. Deletion of this sequence lowers gus expression by at least 3-fold compared to pActl-F. By gel-retardation and footprinting analyses, we identified a ubiquitous rice protein that specifically recognizes this poly(dA-dT) element (17). Similarly, a yeast poly(dA-dT)-binding protein, named “T-rich binding factor,” has been characterized, and this factor stimulates transcription from templates containing poly(dA-dT) elements (18). The second regulatory element, which serves as a negative regulator of the act1 promoter, consists of a CCCAA pentamer-repeat (between positions -297 and -265). A CCCAA-binding protein was detected in rice root extract but not in rice leaf extract. Furthermore, there are several additional potential regulatory elements within the act1 promoter. Deletion between nucleotides -152 and -37 decreases GUS activity to about one-half. There is also a putative TATA box within this region (17). Thus, these findings suggest that the act1 5‘ region contains multiple cis-acting elements, each of which interacts with either ubiquitous and/or tissue-specific trans-acting factors to confer the observed constitutive pattern of act1 promoter activity.

B. Rice Phytochrome Genes Phytochrome is a regulatory protein that functions as a photoreceptor in response to light signals. The photoreceptor molecule is reversibly interconvertible between two forms: red light converts the inactive form of phytochrome (Pr) to the active form (Pfr). The active phytochrome controls the expression of selected responsive genes, and ultimately results in altered growth and development appropriate for the light environment. Phytochrome constitutes an ideal system for analyzing the molecular processes involved in the light-signal perception, transduction, and transcriptional response phases of phytochrome-regulated gene expression (19). The genes encoding phytochrome from half a dozen plant species have been cloned and characterized. In some plants, there are several different phytochrome genes (reviewed in 20). Kay et al. (21, 22) cloned a rice phytochrome cDNA, encoding a type I phytochrome (PI) (also known as phytochrome A, phyA), and the corresponding genomic sequence. Further,

6

RAY WU ET AL.

Dehesh et al. (23) cloned a second rice genomic sequence of the phytochrome gene, p h y B , and found that it shares only 50% amino-acid identity with the rice p h y A .

1. I n Vivo ANALYSISOF RICE PHYTOCHROME GENEE ~ R E S S I O N Levels of phytochrome mRNA, isolated from rice leaves after different light treatments, were determined by using a portion of the rice cDNA clone as the probe. Slot-blot hybridization analysis of mRNA isolated from etiolated rice leaves shows that a one-minute red-light pulse, followed by four hours of darkness, results in a substantial decrease in phytochrome mRNA abundance. The decline in the level of mRNA is due to phytochrome itself because the effect is partially reversed when plants are illuminated with red light followed by a far-red light. These results suggest that rice phytochrome decreases the expression of its own gene. Furthermore, nuclear “run-on” experiments show that this autoregulatory response is exerted on transcription of the phytochrome gene. Kinetic analysis of the phytochrome-induced decrease in mRNA abundance shows a very rapid decline within 15 minutes following a red-light or white-light treatment. After 30 minutes, the phytochrome mRNA was barely detectable (21). Expression of the rice p h y B has also been studied in parallel experiments. The results indicate that, whereas p h y A is negatively regulated by its gene product (phytochrome) in rice seedling shoots in a manner typical of monocots, p h y B is constitutively expressed irrespective of light treatment (23).

2. In VitrO ANALYSIS

OF A

RICE PHYTOCHROME GENE

Gel-retardation analysis was carried out using a fragment from the 5’ upstream region of a rice genomic phytochrome gene ( p h y l 8 , also known as phyA). The results show that protein extracts from etiolated rice-leaf nuclei contain a factor tHat binds to the p h y l 8 5’ region, and this factor is similar to GT-1, shown previously to bind light-responsive elements of photophilic genes such as the pea rbcS-3A. Indeed, an expected GT-motif sequence (Fig. 2) was found in the rice p h y 2 8 (between positions -242 and -220). The -220 ---------

-242

Rice

TAGGTTAATTA-TTGGCGGTAT

Oat

TAGGTTMTCAATTTCAGGTTAAT -1 76 -1 53

.**a*****

0 .

* * a

FIG.2. Nuclear protein binding sequences in the upstream conserved regions of the n c e and oaf phytochrome genes (21,24).-=, identical nucleotides between rice and oat phyA,- on top of the sequence, GT1 binding site, --- on top of the sequence, GT2 binding site.

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

7

binding of the rice nuclear factor to phyl8 was effectively competed for by the pea rbcS-3A box-I1 sequence, but not by a mutated box I1 fragment in which the conserved GG sequence was changed to a CC sequence (21). 3. TRANSIENT EXPRESSION ANALYSISOF A RICE PHYTOCHROME GENE

Functional analysis of a rice phyA gene promoter was carried out by transient expression assays using microprojectile-mediated gene transfer into etiolated rice seedlings. Results from deletional analysis and linker-scan mutation show that a GT motif, GCGGTAATT (see Fig. 2, sequence marked with dotted line on top), closely related to elements in the promoters of a number of other light-regulated genes, is important for phytochrome gene expression. An oligonucleotide containing the tandem GT motifs from rice phyA promoter was used to isolate a cDNA clone encoding a protein (GT-2) that bound to these motifs. GT-2 from the over-expressing clone specifically binds to the rice phyA promoter, and it has domains related to certain other transcription factors. RNA blot analysis indicates that GT-2 mRNA levels decline in white light, although red and far-red light pulses are ineffective (24). The data indicate that phytochrome exerts autoregulatory control over the transcription of its own phyA genes in monocots. Conversion of this photoreceptor to its active Pfr form initiates repression of phyA transcription within 5-15 minutes of light signal perception by a mechanism that does not require new protein synthesis (20).

C. Rice Storage Protein Genes The average protein content of milled rice is lo%, whereas prolamins and globulins are minor components and glutelin is the major component, constituting 80% of the total endosperm protein (25). The mature glutelin protein is composed of two subunits, one acidic (around 32 kDa) and one basic (around 22 kDa), that are produced by the post-translational cleavage of a precursor molecule (26-28). In several studies, cDNA (28,29)and genomic DNA (29-32) sequences of rice glutelin genes have been observed. The genes have been classified into three subfamilies, Gtl, Gt2, and Gt3, according to their DNA sequences. Each subfamily is composed of several copies (28, 29). The coding regions of G t l and Gt2 subfamilies are closely related and show 94-96% DNA sequence identity to each other, whereas the Gt3 subfamily shows only 77-82% DNA sequence identity to the other two subfamilies (29). These three subfamilies together have been named “subfamily A” (30),and several new glutelin genes that belong to a new subfamily, named “subfamily B,” have been described. Two members of the subfamily B glutelin genes show only 60% DNA sequence identity to those in subfamily A.

8

RAY W U ET AL.

1. I n Viuo ANALYSIS OF GLUTELINGENEEXPRESSION Very little is known about the regulation of glutelin gene expression, except that mRNA levels are regulated differentially during seed development. RNA hybridization analysis was carried out using cDNA probes specific for Gtl and Gt3. With both probes, glutelin mRNA transcripts were detected at five days after flowering (DAF), but the accumulation patterns for Gtl and Gt3 transcripts differ during further seed development. Gtl transcripts continue to increase in level throughout seed development, reaching a maximal level at 25 DAF. In contrast, Gt3 transcripts reach the maximal level at 10 DAF and then steadily decline between 10 and 25 DAF. Accumulation patterns for Gt2 appear to be similar to those obtained for Gtl (29). The mRNA accumulation pattern of a subfamily B glutelin gene indicates that the mRNA is first detectable at six DAF, and the level gradually increases reaching a maximum at 14 DAF, when it begins to decrease (30).This pattern of glutelin mRNA accumulation is similar to that observed for Gt3 (29).

2. I n Vitro ANALYSISOF RICE GLUTELINGENE5' REGION

In uitro analysis of a rice glutelin gene, pGL5-1 of the Gt2 subfamily, gave the following information. The DNA sequence at the 5' region of pGL5-1 and two other Gt2-related clones show a high degree of sequence identity between position -213 and the transcription start-site (+ 1). A DNA fragment (Fragment A, between positions -224 and -45) from pGL5-1 was incubated with partially purified nuclear extract prepared from immature rice seeds. Four major retarded bands were observed in a gel-retardation assay. To localize the protein-binding regions more precisely, DNase-I botprinting analysis of the same glutelin DNA fragment (Fragment A) was carried out. Several protected regions (boxes I, 11, 111, and IV)were observed (Fig. 3). Two regions that show strong protection are Box 111 (positions -164 and -146) and Box I (centered around position -90) (31). The protein-binding study was extended using DNA fragments further upstream from Fragment A of the rice glutelin gene pGL5-1. Fragment B spanned the sequence between -515 and -224, and Fragment C spanned the region between -677 and -515. Gel-retardation assay indicates two retarded bands using Fragment C. In footprinting analysis one region (Box V) centered around position -585 was protected from DNase-I digestion. To confirm the results of the protein-binding studies, two sets of synthetic oligonucleotides were made. GT1-14 includes 14 nucleotides of Box I, and GT3-13 includes 13 nucleotides of Box 111. Each oligonucleotide was selfligated to produce multimeric units. Using GT1-14 for gel-retardation analy-

b b b 5 9

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS -677 Nsll

Box V

-224

Box IV

Box I1 Box 111

BOX I (-103 to -86)

ATATCATGAGTCACTTCA a

BOX 111 (-164 to -148) BOXII (-122 to -108)

Box I

* a

a a a

maam

am

ACAAATGATGTGTCAATTA CTTCCGTGTACCACA a

aaa

aaa

a

BOX IV (-206 to -189)

ATCATCCATPTCATATT

Consensus (I to IV)

T---GT_GTCA--T

BOXV (-595 to -575)

-

AAGTCATAACTGA

--

.=.,

FIG.3. Protein-binding sequences in the promoter region of a rice glutelin gene, pGL5-1 (31). identical nucleotides between different protein-bindingsequences, dashed lines under the sequence GTCA represent the most conserved portion of the five sequences; a solid line on top of the Box-I sequence represents a sequence identical to the binding motif found in transcription factors jun and GCN4, + + below the Box V sequence, 5 bp inverted repeat.

sis, several protein-DNA coinplexes were observed; however, with GT1-13, a single complex was detected. It is worth noting that the TGAGTCA binding motif, to which transcription factorsjun and GCN4 are known to bind, is located in the middle of the Box I and Box I11 sequences (Fig. 3, the sequence marked with a solid line on top) (31).

II. Methods for Gene Transfer in Rice Gene transfer refers to a process that allows a specific fragment of DNA to enter protoplasts or intact cells. The gene transfer technology has served plant scientists in at least three ways. First, it serves as a rapid and simple method for assaying transient expression of foreign genes. Second, gene transfer, followed by regeneration of transgenic plants, serves as a way for assaying gene expression after stable integration of the foreign gene into the plant genome. Tissue-specific or developmentally regulated gene expression can be analyzed in transgenic plants. Third, gene transfer, followed by regeneration of fertile transgenic plants, serves as a new and powerful method for introducing agronomically useful traits into crop plants.

RAY WU ET AL.

A. Introduction of DNA into Rice Protoplasts 1. FORTRANSIENT ASSAYOF GENEACTIVITY Efficient methods for transfer of a foreign gene into rice protoplasts were established several years ago (reviewed in 33). For promoter analysis, different lengths of the upstream regulatory sequence to be analyzed are often ligated to the gus reporter gene, and these gene constructs are transferred into rice protoplasts by either electroporation or by incubation with polyethylene glycol (PEG). Several days after gene transfer, regenerated cells are assayed for GUS enzyme activity. In this method, as long as the same batch of protoplasts are divided into aliquots and are used for the analysis of different gene constructs at the same time, transformation efficiency is relatively constant and the values of the GUS activity can be compared.

2. FORPRODUCTION OF TRANSGENIC PLANTS For efficient selection of transformed cells and calli, it is desirable to transform protoplasts with a construct that contains a selectable marker gene. Selectable genes that are currently widely used for rice transformation include hygromycin phosphotransferase gene (hpt) and phosphinothricin acetyltransferasegene (bar) (9, 34). The selectable marker gene can be used in a ca-transformation experiment with a second construct to be analyzed, such as a d - g u s . However, it is preferable that the selectable marker gene be incorporated into the same construct that is carrying the foreign gene to be analyzed. One study reports the recovery of several fertile transgenic plants after transforming rice protoplasts with an hph-containing construct, using PEG treatment. These plants transmitted their integrated hph gene into the second generation (35). Similarly, we co-transformed rice protoplasts with two plasmids: pin2 promoterlactl intronlgus (50 pg DNA/ml), and CaMV 35-S promoterlbar (20 pg DNA/ml). After regeneration of cells and calli, resistant calli were selected using ammonium glufosinate as the selective agent. We found that 60-70% of the resistant calli show GUS activity. In a typical transformation experiment starting with 4 x lo6 protoplasts and 20 pg of plasmid DNA containing the bar gene, over 500 calli resistant to ammonium glufosinate can be obtained. Approximately 100-150 transgenic plants can be produced, out of which between 5-30 plants are fertile. An advantage of the protoplast method is that a number of investigators successhlly regenerated fertile rice plants from protoplasts; expensive equipment is not needed. The disadvantage of this method is that, at present, less than a dozen rice varieties (out of hundreds of varieties grown in the

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

11

field) efficiently regenerate fertile plants from protoplasts. Thus, the potential for the direct application of this method to produce agronomically superior plants for farmers is limited. However, regardless of its value as a practical application, the protoplast system serves the purpose successfully in studying gene regulation in both transient assays and in transgenic plants.

6. Introduction of DNA into Intact Rice Cells by the Biolistic Method The biolistic method, also known as the particle-gun-bombardment method, was developed by John Sanford and his collaborators at Cornell University (36). In this process, high-velocity microprojectiles coated with DNA are used to penetrate the walls of intact cells. The major advantage of this method is that any type of intact plant cell or tissue can be used as a target for receiving foreign DNA Thus, one can use the technique to transform rice varieties that may not be readily amenable to plant regeneration from protoplasts. Furthermore, one can study tissue-specific and developmentally regulated gene expression by introducing gene constructs directly into distinct plant parts at any developmental stage.

1. BASICDESIGNOF

THE

BIOLISTICDEVICE

There are three major types of the biolistic device, differing mainly in the process of accelerating the microprojectiles, which are made of either tungsten or gold particles. The original design used gunpowder charges to accelerate DNA-coated tungsten particles (36),but the gunpowder has now been replaced by compressed helium (37). The second design uses an electrical arc-discharge device to accelerate the DNA-coated gold particles (38).The third design uses compressed nitrogen to accelerate microprojectiles (39). THE BIOLISTICMETHOD GENE EXPRESSION

2. APPLICATIONOF FOR STUDYING

One major application of the biolistic method is in the study of gene expression in intact cells or specific tissues. For example, rice, maize, and wheat suspension cells have been bombarded by a plasmid containing either a gus reporter gene or a chloramphenicol acetyltransferase (CAT) reporter gene (40).Two days after bombardment, the GUS or CAT activity was measured. These experiments were carried out to optimize the biolistic method and to compare the expression of specific gene constructs in the major cereal crops (40).Using intact tissues, and bombarding them with plasmids containing different promoters of rice genes, we found that a glutelin gene is specifically expressed in rice immature seeds (34,and an a-amylase gene is specifically expressed in the aleurone cells of mature rice seeds (41). It would be

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RAY WU ET AL.

very diacult to isolate protoplasts from the above mentioned cells or tissues for transient assays of different constructs that contain tissue-specific promoters.

BIOLISTICMETHOD FOR PRODUCING AGRONOMICALLY USEFULTRANSGENIC PLANTS The second major application of the biolistic method is to produce transgenic rice plants without the need to regenerate plants from protoplasts. In one study, a foreign DNA was biolistically introduced into intact cells of embryo sections dissected from mature seeds of rice. The aim was to hit the embryogenic cells in the shoot apex region. Seven varieties of rice were tested, including five indica varieties, from which plants are not readily regenerated from protoplasts. After bombardment with a GUS gene construct the embryo sections were cultured. Between 40-70% of embryo sections developed directly into plantlets 2 to 3 weeks later. GUS assay and DNA blot hybridization showed that the GUS gene was integrated into the rice genome in 20 transgenic plants. However, the GUS gene was not found in plants of the next generation (42). In another study, two different plasmids were introduced separately into rice immature embryos by electric discharge particle acceleration. Six varieties of rice were tested, including two varieties currently grown commercially in the United States, and four indica varieties. One plasmid, containing both a visible reporter gene (gus) and a selectable marker gene (bar),was used. At 12 to 15 days after pollination, rice immature embryos were isolated and bombarded with DNA-coated gold particles. The target was the scutellar region of the immature embryos. After particle bombardment, embryos were plated in suitable culture media to allow the development of embryogenic calli, and then plantlets, under both selective (with bialaphos which contains phosphinothricin) and non-selective conditions. Transformed embryos and regenerated plants were obtained, which expressed GUS activity and contained the foreign gene as detected by DNA gel blot hybridization. Fertile transgenic plants (% generation) were recovered and Mendelian segregation of foreign genes was observed in the progeny (R,progeny). On average, out of 1OO immature embryos bombarded, three transformed plants were recovered. Thus, engineering of rice through transformation of organized tissue can be accomplished by particle bombardment (43).

3.

APPLICATION OF THE

4. ALTERNATIVEMETHODS FOR

PRODUCING TRANSGENIC PLANTS

As discussed in the previous section, bombardment of immature rice embryos with plasmids can lead to the creation of first and second generations of transgenic plants. However, the frequency of obtaining pure lines of

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

13

transgenic plants is relatively low, mainly because of the high frequency of chimeric plant formation, which, in turn, is probably due to cross-protection of nontransformed cells by transformed cells that results in inefficient selection of pure transformants from bombarded immature embryos. An alternative method for producing transgenic plants is to bombard embryogenic rice suspension cells with plasmids, followed by the efficient selection of resistant calli. To test the efficiency of this system, small clumps of cells from embryogenic suspension culture were bombarded with an uctllbur plasmid. Five to seven days after bombardment, cells were cultured for six weeks in a medium containing ammonium glufosinate to select resistant calli. In a typical experiment, several hundred resistant calli were produced, and almost all were pure transformants (not chimeric). This is probably due to the fact that, with small clumps of suspension culture cells, there is no cross-protection of nontransformed cells by transformed cells. Transformed calli readily regenerate into plants. Furthermore, transgenic plants were resistant to spraying with a 1% solution of the glufosinatebased herbicide BASTA, whereas nontransformed plants died of ammonia toxicity in 5-10 days (44). DNA gel-blot analysis of rice plants resistant to ammonium glufosinate showed the expected hybridization bands using digerent restriction enzymes to digest rice genomic DNA. The results demonstrate integration of the input foreign gene into the rice chromosome. A high percent of the transgenic plants were fertile, and the second generation plants (R,) also gave positive results in DNA gel blot analysis. This study clearly demonstrates the feasibility of introducing agronomically useful traits into rice by bombardment of suspension cell culture, followed by plant regeneration.

C. Introduction of DNA into Intact Cells via the Pollen-Tube-Pathway Method A method for transferring DNA into recently pollinated rice florets, the “pollen-tube-pathway” method, has been developed (45),based on the success of producing transgenic cotton using a similar method (46). In the original study (4.57, total DNA from a variety of rice with purple-tip glumes and purple leaves (donor DNA) was transferred to a variety with normal green pigmented tissues (recipient plant) after cutting off the stigma of the floret and applying a drop of DNA solution to the cut end of the style. It is assumed that the DNA reached the ovule by flowing along the pollen tube and entering the egg cells or the zygote in the ovary. The cells of young embryos are either without or with partial cell walls, so foreign genes can enter the cells more readily. Mature seeds were collected and planted. The results show that some progenies, including those of the D,, D,, and D,

14

RAY W U ET AL.

generations, possessed glumes with purple tips and awns, similar to the phenotype characteristics of the donor plants. In addition, some glumes became completely purple (45). In a second study (43, total DNA of two rice varieties with high levels of resistance to bacterial leaf blight (Xanthomonas campestris pv. Oryzae) was used to treat the florets of nine sensitive rice varieties using the same pollentube-pathway method. A total of 2,400 florets were treated and 233 viable seeds (Do) were produced. These seeds were planted, and the plants (D, generation) were inoculated with the bacterial pathogen. After two weeks, development of disease on leaves was scored. One D, plant was highly resistant to bacterial blight. All second generation plants that came from this D, plant were also resistant to bacterial blight. The results indicated that disease resistance was genetically transmitted to the next generation (47). Experiments involving the addition of purified plasmid DNA (instead of total DNA) to the cut end of the styles of recently pollinated rice florets have been carried out by at least four groups of investigators. Their results are summarized below. (a) Zhou and Gong (48) constructed kanamycin-resistant plasmids containing different rice repetitive DNA sequences. These plasmids were then introduced into rice, and D, and D, generation plants were obtained. Among the D, plants, one plant showed strong NPTII enzyme activity, and DNA gel-blot analysis showed a hybridized band of the expected size. (b) Luo and Wu (49) treated a total of 259 rice florets (variety Fujisaka-5) with DNA containing 3 5 3 promoter and NPTII coding sequences as well as histone-3 coding sequences; 54 seeds matured. After germination of the seeds, root tips from 6-day-old plantlets and leaf tissues from 8-week-old plants were analyzed. Using p35S/NPTII DNA as the probe, genomic DNA from seven plants out of 54 gave positive signals in DNA slot-blot hybridization. When genomic DNA from four transgenic plants was digested with an enzyme that does not cut within the plasmid, different hybridization patterns were observed, as expected. These results suggest that the foreign gene had integrated into the rice genome. Assay for NPTII activity demonstrated that several transgenic rice plants gave positive results (49). (c) Hensgens and Schilperoort (SO) used the rice variety Taipei 309 and a plasmid (HH271) that contains a Gus and a hpt gene in their experiment. By using the pollen-tube-pathway method, a number of F, and F, plants were produced (F, and F, are the same as D, and D,). Approximately 20%of the F, plants displayed GUS activity, and two of them (out of 12 F, plants) showed a signal in genomic blot analysis. Several individual F, plants, derived from a single F, plant, gave strong signals in DNA blot hybridization, thus indicating DNA integration into the rice chromosomes.

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

15

To further substantiate the integration of the plasmid DNA into rice chromosome, F, seeds were analyzed by in situ hybridization. A low but significant number of cells displaying a hybridization site on a rice chromosome were detected, thus indicating DNA integration (SO). (d) Xie and Fan (51)treated 993 florets using a plasmid containing a deltaendotoxin gene of Bacillus thuringiensis (B.t.) var. aizawai 7-29. Out of the 387 D, plants, 11 gave positive results in DNA dot-blot analysis. Genomic DNA blot analysis was also carried out, and one plant (D,-73) gave a positive signal. Also, the root of this plant showed GUS activity in the histochemical GUS assay. Thirty-three mature seeds obtained from the plant D,-73 were planted. Ten out of 30 plants examined gave hybridization signals in DNA dot-blot analysis. Southern blot analysis showed that one of them (D,-73-19) gave a positive hybridization signal. An additional 400 D, seeds were obtained but have not yet been analyzed (51). (e)X. Duan and R. Wu (unpublished data) carried out experiments using different varieties of rice (Taipei 309, Lemont, and Labelle), different plasmid constructs (eight different constructs), different time intervals between the beginning of flowering and the time of adding DNA (varying between 1, 2, and 3.5 hours), and the concentration of DNA (100, 200, 300, 400 pg/ml) applied to the florets. Eight experiments were carried out and 181 to 557 florets were treated in each experiment. Approximately 470 first-generation plants (D,) were obtained, and 240 plants fiom florets treated with the Guscontaining plasmids were analyzed for GUS activity. The results indicate that 12 plants (5%)gave a positive GUS signal in leaves or roots of young plants three weeks after germination. However, in older plants, the percentage of GUS positive plants progressively decreases. DNA hybridization results using either the slot blot or the wick blot (52) procedure show that approximately 1% (five plants of 470 plants analyzed) of D, generation plants (younger than 6 weeks) gave positive results. Southern blot analysis using DNA from mature plants gave suggestive evidence for the presence of the input plasmid in rice genomic DNA. In conclusion, we now believe that the pollen-tube-pathway method is not as simple and efficient as it appeared to us several years ago. The method can give transgenic plants at low frequencies. However, it is difficult to reproduce because important variables have not yet been well-defined. In 1987, when we started to use the pollen-tube-pathway method (49), other methods for producing fertile transgenic rice plants had not been established. Now, both the protoplast method and the biolistic method are relatively well established; thus, there seems to be no compelling reason to use less well-established and less efficient methods such as the pollen-tubepathway method.

16

RAY W U ET AL.

D. Introduction of DNA into Intact Rice Cells via the Agrobacterium System The Agrobacterium system has been widely used to transfom dicot plants by co-cultivation (53). However, monocot plants, especially cereal plants, are generally considered d s c u l t to transform by this method. In one experiment, mature rice (var. Nipponbare) embryos were inoculated with a wide-host range supervirulent strain of A. tumefaciens (54).The tumorigenic callus tissue grew on hormone-free medium. The transformation of this tissue was confirmed by DNA hybridization analysis that showed the presence of T-DNA in the rice genome. Additional experiments were carried out using Agrobacterium harboring a plasmid that included hpt and Gus. Hygromycin-resistant calli were selected and plants generated. Plants from independent transformants contained an intact gus fragment, and 50% of the seeds obtained from the selffertile plants germinated successfully. Seeds obtained from two plant lines were tested for their ability to germinate in the presence of 40 p.g/ml hygromycin. Both lines gave rise to healthy seedlings, whereas control seedlings turned black and died (55). The results from these and other experiments suggest that the hpt gene had been transferred, integrated, and then expressed in the rice cells (54). In another study (56), A. tumfaciens was treated with phenolic chemicals to increase its host range and then co-cultivated with rice suspension cells. The results indicate that both NPTZZ and nos were transferred and expressed in cultured rice cells. Integration of foreign genes into the rice genome was confirmed by Southern blot hybridization.

111. Analysis of Transferred Genes in Transgenic Plants Information on promoter analysis obtamed in vitro or by transient expression assays in uiuo must be confirmed and extended by analysis using transgenic plants. This includes transient expression analyses on tissue specificity and developmental-stage specificity after biolistic bombardment of different tissues. Regulation of gene expression is expected to be different when the gene of interest is on a plasmid, which exists as an extrachromosomal entity from when the gene is integrated into the rice chromosome. Therefore, analysis in transgenic plants is essential for understanding the regulation of gene expression.

A. Rice Actin-1 Gene (act?) To determine the pattern and level of activity of the act1 5' region, transgenic rice plants containing the 5' region of act1 ligated to the gus

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

17

coding sequence were generated. In a transformation experiment, the act1 (5'-region)lgus (coding) plasmid was incubated with rice protoplasts in the presence of PEG. Among the large number of regenerated plantlets, 44 exhibited gus expression in root and leaf tissues. From these plantlets, two clonal plant lines, named T8-1 and T8-2, were selected for detailed analysis

(57). One goal of this experiment was to provide proof that integration of the input plasmid into the rice genome had occurred. DNA gel blot analyses of leaf genomic DNA showed that each transgenic plant derived from T8-1 contained four copies of the input plasmid. Integration of the input plasmid, uctI(5')lgus, into the rice chromosome was shown definitively by three gel blot analyses. (a) In undigested rice genomic DNA, the hybridizing band is in the 25-50 kb size range, which is the average size of the isolated genomic DNA fragments. (b) In DNA digested with NcoI restriction enzyme, for which there are no restriction sites within the input plasmid, the T8-1 plants gave three hybridizing bands. Since each band differs in size compared to the input plasmid, this indicates that this plasmid has integrated into the rice chromosome at three independent integration sites. (c)When genomic DNA was digested with HindIII, which cuts once within the plasmid, the T8-1 line clearly showed four hybridizing bands (57). The second goal of this experiment was to analyze the GUS activity and the GUS protein level quantitatively in transgenic rice plants. GUS enzyme activity was measured and the specific activity calculated. From the known specific activity of the purified bacterial GUS enzyme, the amount of GUS enzyme produced in the transgenic plants was estimated. Quantitative immunoblot analysis was also carried out to determine the amount of GUS protein accumulated in the transgenic plants. The results of these analyses show that, in the T8-1 plants, the GUS enzyme in the leaves amounted to 3.2% of total soluble leaf protein (57).

B. Expression of Rice Phytochrome Genes in Transgenic Dicots In order to investigate the mechanisms of phytochrome action in vivo, rice phytochrome gene was fused to a suitable promoter and transferred to tobacco or Arubidopsis. The behavior of the transgenic plants was then studied. (1)A full-length rice phytochrome cDNA was ligated to the CaMV 35-S promoter and transferred to tobacco, and the progenies of the transgenic plants were analyzed. Some of the transgenic plants contained large amounts of rice phytochrome mRNA in green leaves. Indeed, extracts prepared from these plants contain up to fivefold more phytochrome than extracts from control plants. By using species-specific, anti-phytochrome monoclonal antibodies, it was found that the rice phytochrome assembles with the chro-

18

RAY WU ET AL.

mophore and is photoreversible. Analysis of the circadian pattern of cab mRNA levels in transgenic plants versus control plants showed that the overproduction of rice phytochrome extends the duration of the free-running rhythm of cub gene expression (58). Additional experiments showed that rice type-I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings. It was also shown that the hypocotyl elongation phenotype is dependent on both light quality and on light intensity. Furthermore, successive pulse irradiation with red light elicited short hypocotyls similar to those obtained under dim white light, and the effect was reversed by immediate far-red light treatment. This provides a direct indication that the phenotype is caused by biologically active rice phytochrome gene in transgenic tobacco (59). (2)The rice phytochrome B gene was ligated to CaMV 35-S promoter and introduced into Arubidopsis. Transgenic plants that overproduced phytochrome B were analyzed: the over-expressed phytochrome B is spectrally active, undergoes redlfar-red-light-dependent conformational changes, and the protein is stable in light. Overproduction of this protein is tightly correlated with a short hypocotyl phenotype in transgenic Arabidopsis seedlings. This phenotype is strictly light-dependent, showing that phytochrome B is a biologically functional photoreceptor. Based on similarities to phenotypes obtained by over-expression of phyA, it appears that phytochromes A and B can control similar responses in plants (60).

C. Expression of Rice Glutelin Gene in Transgenic Tobacco Whenever possible, it is desirable to analyze rice gene expression in transgenic rice plants because the results are more meaningful when obtained from a homologous system that matches the physiological conditions. The match between a gene and the plants into which the gene is transferred is even more important in the case of analyzing a rice seed storage-protein gene such as a glutelin gene, because of the complex packaging of rice seed storage proteins into protein bodies. On the other hand, since fertile transgenic rice plants were not easily produced until several years ago, results obtained in transgenic tobacco can provide some useful information. A chimeric gene construct, composed of the rice Gt3 promoter and the CAT gene, was introduced into tobacco. Analysis of the transgenic tobacco seeds indicate that 980 bp of the 5’ flanking sequence of Gt3 is sufficient for the full promoter activity and directs a seed-specific expression (61). The 5‘ upstream region of a type I1 glutelin gene joined to a gus reporter was introduced into tobacco. The chimeric genes were expressed specifically in developing seeds of the transgenic tobacco. Histochemical analysis demonstrated that the GUS activity wss restricted to the endosperm tissue. A deletion series of the 5’ flanking region was generated from positions -1329

ANALYSIS OF RICE GENES I N TRANSGENIC PLANTS

19

to -74 relative to the transcriptional initiation site. After transformation and regeneration of transgenic tobacco, the results show that GUS activity in the seeds reaches maximum when the region between positions -441 and -237 is included (62).

D. The Protease Inhibitor Genes Until now, the improvement of major crops has depended mainly on conventional breeding methods. However, in certain situations, introduction of a given agronomically desirable trait into crop plants has been either impossible or prohibitively time-consuming. For example, certain agronomically useful traits found in some crop species cannot be introduced into other crop species by conventional breeding due to a species barrier in sexual crosses. In other cases, introduction of a new genetic trait, such as disease resistance, is a slow process because it takes many years to accomplish the transfer of the desired genetic trait through repeated backcrosses. Recently, gene-transfer technologies have provided new solutions to some of these problems. We use insect-resistant transgenic plants as an example to demonstrate how gene transfer technology can engineer an agronomically useful trait into rice plants.

1. NEED FOR EXPRESSING INSECT-RESISTANT GENESIN PLANTS The losses of agricultural products caused by insect pests are enormous. For example, in rice alone, the worldwide loss of yield due to insect damage is at least several billion dollars a year (63).Furthermore, several devastating viral diseases in rice are transmitted by insects (64). Conventional control of insect pests in rice cultivation often depends upon the use of chemically synthesized insecticides; thus, environmental damage has long been one of the major concerns regarding the long-term, massive use of these synthetic chemicals. Increasing pressure to use nonhazardous, environmentally compatible (safe) pest control measures has spurred interest in natural insecticides such as plant-derived proteinase inhibitors and Bacillus thuringiensis crystal proteins. Plant-derived proteinase inhibitors are of particular interest because they are part of the natural plant defense system against insect attack. Studies on the effects of dietary proteinase inhibitors, either artificially introduced into defined diets or those already present in plant tissues, have shown that these native proteinase inhibitors can interfere with the growth and development of insects by inhibiting their digestive enzymes. These proteinase inhibitors are easily inactivated by cooking, and they show no harmful effect on either humans or animals who eat proteinase-inhibitorcontaining plant products (see 65 and 66 for review). Several recent reports demonstrate that, when either a potato proteinase

20

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inhibitor I1 (pi&) or a cowpea trypsin inhibitor (CpTi) gene is joined to a suitable promoter and introduced into tobacco plants by Agrobacterium-mediated transformation, those transgenic tobacco plants that produced relatively high Ievels of the protease inhibitors become resistant to tobacco insect pests (67, 68). Our first approach to achieving high level expression in rice was to use a constitutive and efficient promoter of rice, the actl promoter, which was isolated in our previous studies. Both actl lpin2 and actl lCpTi constructs were made and introduced into rice using our transformation and selection system (3).Over 300 transgenic rice plants have been regenerated and some of them have been confirmed by Southern blot analysis. The levels of proteinase inhibitors in different tissues of transgenic rice plants were analyzed. The protein extract from leaf tissues expressing PIN11 protein exhibited inhibitory activity against both purified trypsin and chymotrypsin, whereas the protein extract from leaf tissues expressing CpTi protein showed strong inhibitor activity only against trypsin. These results suggest that (a) these two proteinase inhibitorswere correctly synthesized and functionally active in transgenic rice plants; and (b) with the availability of an efficient transformation system for rice or other monocot plants, genetic engineering for applied purposes can be quickly extended to the major cereal crops by introducing agronomically important genes.

2. AN ADVANTAGEOF USINGA WOUND-INDUCIBLE PROMOTER TO EXPRESSUSEFULFOREIGNGENESIN TRANSGENICPLANTS For the production of insect-resistant transgenic crop plants, one desires to synthesize high levels of the insecticidal proteins and also to be able to regulate the synthesis of these proteins. High level expression of insecticidal protein genes in all tissues during the entire life cycle of the plant may have the following undesirable effect. The synthesis of high levels of insecticidal proteins in plants may compete for energy and building blocks for RNA and protein synthesis with the synthesis of other essential cellular components. This may lead to a decrease in crop yield. This undesirable effect can be largely overcome by using a wound-inducible gene construct to transform plants that will produce high levels of the insecticidal proteins only after insect attack. It has been reported that the potato proteinase inhibitor I1 gene ( p i d ) is not expressed in leaves, stems, or roots of potato plants until the plants are mechanically wounded; thus it mimics the attack of chewing insects. Upon wounding, the production of proteinase inhibitor is induced, not only in the wounded leaves, but also in non-wounded upper and lower leaves, as well as in the upper part of the stem; thus, this represents a systemic, woundinducible gene activation (69). When a p i d - g u s construct was introduced

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

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into potato or tobacco plants, the expression of the gus reporter gene followed the same pattern of expression as the endogenous pin2 gene in potato (70). The unique property of the pin2 gene promoter, the systemic wound inducibility, makes this gene promoter a potentially useful tool for genetic engineering of insect resistance.

3. TRANSFORMING RICE WITH A CONSTRUCT USING THE WOUND-INDUCIBLE mv2 PROMOTER As a first step towards evaluating the dicot pin2 promoter for use in rice, we constructed several pin2lgus plasmids. At the outset, we had reason to believe that the dicot pin2 promoter might not function efficiently in monocot plants such as rice. We therefore incorporated the 5’-intron from the rice act1 into the 5’-transcribed region of the pin2-gus fusion construct because the act1 5’ intron had previously been shown to greatly enhance gene expression of a dicot gene promoter in transformed monocots. The functionality and activity of two constructs, pin2lgus and pin2lactl intronlgus, were analyzed in transgenic rice plants. In transgenic rice plants, the pin2tgus construct showed a very weak wound response, as measured quantitatively by the GUS activity. However, in plants harboring the pin2lactl intronlgus construct, wounding of a leaf dramatically increased the production of GUS by 10-15 fold. Moreover, the wound response is systemic because high levels of GUS activity were also found in non-wounded leaves. We carried out parallel experiments by measuring the gus mRNA level using the gus coding region as the probe. Similar results were found, showing that wounding greatly increases the level of gus mRNA in transgenic rice plants. Next, histochemical analyses of the transgenic rice plants were carried out. The results show that the pin2 promoter is generally active in rice leaves, roots, and stems. However, the highest level of expression of the pin2lactl intronlgus construct was associated with the phloem part of the vascuIar tissue in both wounded leaves and roots (Xu and Wu, unpublished results). This result is consistent with the previous observation made in transgenic potato transformed with a similar pin2 promoterlgus construct, and this supports the conclusion that the signals mediating the wound response are transported via the vascular system of the tissues (70). We can conclude that systemic wound inducibility of a typical dicot gene promoter, pin2, works equally well in transgenic rice plants. This observation suggests that the wound signal(s)and its transduction pathway are highly conserved between dicot plants and monocot plants. As described above, a major application of gene transfer technology is the introduction of agronomically useful traits into crop plants. Although there is still much room for further improvement of several established transforma-

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tion systems, we believe that with extensive efforts of scientists in this field, the gene transfer technologies for rice and other major cereal crops will eventually become routine. Currently, the challenge for plant scientists is the identification and isolation of agronomically useful genes, including those responsible for insect resistance, viral, and/or fungal pathogen resistance. It can be predicted that, with the availability of efficient transformation systems for rice or other monocot plants, genetic engineering for introducing agronomically important genes can be quickly extended to the major cereal crops.

IV. Concluding Remarks and Future Prospects A. Analysis of Monocot Genes in Transformed Rice Protoplasts, Cells, and Transgenic Plants For analysis of gene expression directed by promoters from monocot genes, rice is now the preferred system because transgenic rice plants can be produced relatively easily, much more easily than other cereal plants such as maize, wheat, barley, sorghum, and oat. Since these monocots are all in the Grumineae family, rice is a close relative to them and thus can be used as a suitable system for the analysis of genes from these cereal plants. Transformation systems for rice protoplasts are well established for both transient expression assays of promoter activity and for production of transgenic plants to study tissue-specific and developmentally regulated gene expression. In the analysis of different rice genes, as well as genes of other monocots or dicots, we have identified several promoters with different tissue specificities and regulatory properties for expressing useful foreign genes in transgenic rice. For example, we have shown that rice act1 promoter is highly and constitutively expressed in rice plants. The rice act1 5' intron can substantially enhance the expression of a potato pin2 gene promoter in transgenic rice without affecting the favorable property of wound inducibility of this promoter. Glutelin gene promoter is immature-seedspecific, and a-amylase gene promoter is mature-seed-specific. Other tissue specific promoters, such as leaf-specific, root-specific, or pollen-specific, have been described by other investigators. These promoters will be useful for expression of foreign genes in transgenic plants to improve different desirable agronomic traits of major cereal crops.

B. Production of Agronomically Useful Transgenic Rice Plants Biological insect control agents are now an attractive alternative to chemical pesticides. Biological control will probably cost less and will have fewer adverse effects on the environment as compared to the use of chemical insecticides. Success in producing insect-resistant dicots by transferring

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

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genes that encode protease inhibitors (65-71) or B . t . toxins (72-75) into plants show that biological insect control is a feasible and practical approach. It can be predicted that within the next few years, transgenic rice plants transformed with genes encoding insecticidal proteins will be generated. Once insect-resistant transgenic rice plants are produced, the same approach can be used to transfer other agronomically important traits, such as disease resistance, into transgenic plants. Since each of the several known disease-resistance traits is coded for by a single gene based on genetic analysis, the chance of success will be high in generating disease-resistant rice plants. The large amounts of synthetic chemicals that have been used as herbicides have caused pollution of the environment. We believe that a superior alternative is to use insect-resistant crop plants to control weeds. For example, one can identdy the major weeds in a rice field and know which insects attack the weeds. Then, one can introduce one or two genes encoding insecticidal proteins into the rice plant so that these plants will be resistant to the insects and, thus, the insects will be forced to eat the weeds. Using a genetic engineering approach to produce transgenic rice plants that are tolerant to cold, drought, flood, or salt stresses is also possible as long as the genes encoding these useful traits are identified from different sources. Thus, identification and cloning of agronomically useful genes remains a challenge for plant scientists. Moreover, each of the above-mentioned traits is probably coded for by multiple genes instead of by a single gene, making the task of cloning genes more difficult. Nevertheless, with the rapid progress of plant molecular biology and plant biotechnology, the ultimate goals of identification and cloning a number of agronomically useful genes will be achieved. It is almost certain that many different varieties of transgenic rice plants will be grown in the fields around the world in the 21st century. Some transgenic rice plants will harbor several insect-resistant genes to protect them against insects, and other plants will carry several disease-resistant genes to protect plants against viral, fungal, and bacterial diseases. Some transgenic plants will include modified rice storage protein genes to increase the content of certain essential amino acids, and still other plants will include novel genes to make plants tolerant to low temperature, drought, flood, or salt stress. Thus, the future looks bright because the problem of food shortage can be largely solved by using superior transgenic plants to improve crop yield and quality.

ACKNOWLEDGMENTS This research was supported in part by a research grant RF 91001, #136, from the Rockefeller Foundation. D. Xu was supported by a predoctoral fellowship from the Rockefeller Foundation.

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40. Y. C. Wang, T. Klein, M. Fromm, J. Cao, J. C. Sanford and R. Wu, Plant Mol. Biol. 11,433 (1988). 41. J. K. Kim, J. Cao and R. Wu, MGG 232, 383 (1992). 42. J. Cao, Y. C. Wang, T. M. Klein, J. C. Sanford and R. Wu, in “Plant Gene Transfer” (C. J. Lamb and R. N. Beachy, eds.), pp, 21-33. Alan R. Liss, New York, 1990. 43. P. Christou, T. L. Ford and M. Kofron, BiolTechnology 9, 957 (1991). 44. J. Cao, X. Duan, D. McElroy and R. Wu, Plant Cell Rep. 11, 586 (1992). 45. X. Duan and S. Chen, China Agric. Sci. 3, 6 (1985). 46. G. Y.Zhou, J. Weng, Y. Zeng, J. Huang, S. Qian and G. Liu, in “Methods in Enzymology” (R. Wu, L. Grossman and K. Moldave, eds.), Vol. 101, p. 433. Academic Press, New York, 1983. 47. S. Chen and X. Duan, in “Rice Genetics 11: Proceedings of the Second International Rice Genetics Symposium.” Int. Rice Res. Inst., Manila, Philippines, p. 663,1991. 48. G. Y. Zhou and Z. Z. Gong, ”A Collection of Papers on Advances in Biochemistry for the Celebration of Professor Y. L. Wang’s Eightieth Birthday,” pp. 148-152. Chin. Publ. House Sci. Technol., Shanghai, China, 1987. 49. Z. X. Luo and R. Wu, Plant Mol. Biol. Rep. 6, 165 (1988). SO. L. A. M. Hensgens and R. A. Schilperoort, in “Gene Conservation and Exploitation” (R. Appels, P. Raven and J. P. Gustafson, eds.). Plenum, New York, in press, 1992. 51. X. Xie and X. L. Fan, Chin. Sci. Ser. B. 8, 830 (1991). 52. J. M. Irvine, J. V. Oakes, C. K. Shewmaker and A. Crossway Gene A d . Techn. Appl. 7,25 (1990). 53. S. G . Rogers, R. B. Horsch and R. T. Fraley, in “Methods in Enzymology” (A. Weissbach and H. Weissbach, eds.), Vol. 118, p. 627. Academic Press, Orlando, Florida, 1986. 54. D. M. Raineri, P. Bottino, M. P. Gordon and E. W. Nester, BiolTechnology 8, 33 (1990). 55. T. Ti, Y. Y. Bai, D. M. Raineri, M. P. Gordon and E. W. Nester, in “Fifth Annual Meeting of the International Program on Rice Biotechnology,”p. 46. Rockefeller Foundation, New York, 1991. 56. B. J. Li, Y. Xu, X. P. Xu, H. Shi, X. Ke and D. Yu, in “Fifth Annual Meeting of the International Program on Rice Biotechnology,” p. 61. Rockefeller Foundation, New York, 1991. 57. W. Zhang, D. McElroy and R. Wu, Pkmt Cell 3, 1155 (1991). 58. S. A. Kay, A. Nagatani, B. Keith, M. Deak, M. Furuya and N.-H. Chua, Plant Cell 1, 775 (1989). 59. A. Nagatani, S. A. Kay, M. Deak, N.-H. Chua and M. Furuya, PNAS 88, 5207 (1991). 60. D. Wagner, J. M. Tepperman and P. H. Quail, Plant Cell 3, 1275 (1991). 61. D. J. Leisy, Y. Hnilo, Y. Zhao and T. W. Okita, Plant Mol. Biol. 14, 41 (1990). 62. F. Takaiwa, K. Oono and A. Kato, Plant Mol. Biol. 16, 49 (1991). 63. E. A. Heinrichs, F. G. Medrano and H. R. Rapnsas, in “Genetic Evaluation for Insect Resistance in Rice,” p. 1. IRRI, Los Banos, Philippines, 1985. 64. K. C. Ling, in “Rice Virus Diseases,” pp. 11-33. IRRI, Los Banos, Philippines, 1972. 65. C. A. Ryan, Annu. Reu. Phytopathol. 28, 425 (1990). 66. C. A. Ryan, BioEssays 10, 20 (1989). 67. V. A. Hilder, A. M. R. Gatehouse, S. E. Sheerman, R. F. Barker and D. Boulter, Nature 330, 160 (1987). 68. R. Johnson, J. Narvaez, G. An and C. A. Ryan, PNAS 86, 9871 (1989). 69. H. Pena-Cortes, J. Sanches-Serrano, M. Rocha-Sosa and L. Willimitzer, Phnta 174, 84 (1989). 70. M. Keil, J. J. Sanches-Serrano and L. Willimitzer, EMBO J. 8, 1323 (1989).

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Immunoglobulin Gene Diversification by Gene Conversion WAYNET. MCCORMACK Department of Pathology and Laboratory Medicine Center for M a m d h n Genetics University of Florida College of Medicine Gainesville, Florida 32610

LARRYW. TJOELKER AND CRAIGB. THOMPSON' Howard Hughes Medical lnstitute Departments of lnternal Medicine and Microbiology lInimunalogy Uniuersity of Michigan Medical Center Ann Arbor, Michigan 48109 I. Evidence for Somatic Gene Conversion ........................ A. Somatic Diversity in Chicken Immunoglobulin Genes . . . . . . . . . . . . B. Gene Conversion in Chicken B Cell Tumors and Cell Lines C. Somatic Diversity in Rabbit V,, Genes .......................... 11. Molecular Mechanism of Somatic Immunoglobulin Gene Conversion . . . A. Gene Conversion vs. Double Homologous Recombination ......... B. Properties of Chicken IgL Gene Conversion ..... ..... C. Molecular Models for Immunoglobulin Gene 111. Enzymatic Activities Implicated in Immunoglobulin Gene Conversion . . A. Chromatin Structure and the Role of Transcription B. Initiation of Gene Conversion .................................. C. Bursa1 Expression of the Recombination-Activating Genes . . . . . . . . . D. Enzymes Involved in Homologous Recombination . . . . . . . . . . . . E. Heteroduplex DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29 29 31 32 32 33 34 36 38 38 39 41 42 43 43 44

Genetic recombination mechanisms have evolved in vertebrate species that allow their immune systems to generate an extremely diverse antibody repertoire. It is estimated that this repertoire can include at least loy antibodies, each with distinct antigen specificity. Antibody molecules are heterotetramers consisting of two Ig heavy (H) and two Ig light (L) chains; it is

To whom correspondence may be addressed.

27 Progress in Nucleic Acid Research and Molecular Biology, Vol 45

Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any fnmm reserved.

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WAYNE T. MCCORMACK ET AL.

6

*a I

-- -- --

t5Y I

I

*%/I-so)

u,

I

4(

-

JM

I

I0

119

-.c,-- I

01

FIG. 1. Representative genomic organization of (A) a mammalian heavy chain gene locus and (B) the avian heavy chain locus as defined by Reynaud et al. (15).

the N-terminal regions of these chains that encode the antigen binding sites. Instead of relying on individual germline genes to encode the required antibody repertoire, during lymphoid development each B cell creates a functional immunoglobulin heavy and light chain gene by a site-specific DNA recombination process referred to as V(D)J2joining (reviewed in 1-3). During V(D)J joining a functional antigen-binding site is created by the somatic assembly of variable (V), diversity (D, in the case of heavy chains), and joining 0) gene segments. In humans, these gene segments are found within large clusters in the Ig loci (for example, in the IgH locus there are hundreds of V genes, twelve D genes, and four J genes; Fig. 1A). It is the random assortment of the individual gene segments that are assembled into a complete antigen-binding exon and the junctional diversity inherent to the V(D)J joining process that generate most of the diversity required to generate a primary repertoire. Most higher vertebrates, including teleost fish (4), the amphibian Xenopus (5), the reptile Caiman (6), and most of the mammalian species studied, appear to rely on these combinatorial and junctional mechanisms to generate the primary antibody repertoire. However, some vertebrate species utilize V(D)J joining not to generate diversity, but only to activate Ig gene expression in a B cell-specific fashion. In these species, diversity is generated subsequent to V(D)Jjoining by a second type of DNA recombination event, called gene conversion. Somatic gene conversion has been documented as the molecular mechanism used by the chicken (reviewed in 7-10) and a variety of other avian species (11).More recently, gene conversion has Abbreviations used: Ig, immunoglobulin; H, heavy chain; L, light chain; V, variable region or gene segment, D, diversity region or gene segment, J joining region or gene segment; V(D)J joining, recombination of Ig V, D, and J segments. ALV, avian leukosis virus; CDR, complementaritydetermining region, RAG, recombinase activating gene.

IMMUNOGLOBULIN GENE DIVERSIFICATION

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also been demonstrated to generate somatic immunoglobulin diversity in the rabbit (12),a mammalian species well known for the diversity of its immune response. Although less is known about the mechanism of gene conversion than that of V(D)J joining, recent work has begun to shed some light on the molecular mechanisms involved in immunoglobulin gene conversion. In this review, we focus on recent advances in the study of gene conversion, primarily in the chicken, and on current models for the molecular mechanism of this form of somatic gene conversion.

1. Evidence for Somatic Gene Conversion A. Somatic Diversity in Chicken Immunoglobulin Genes The chicken IgH and IgL loci are novel in that they each contain only single V and J gene segments (Fig. 1B) that are capable of undergoing V(D)J joining in B cell progenitors (13-16). In addition, V(D)J recombination in the chicken is not an ongoing development process (17, 18). Chicken B-cell differentiation begins with a single wave of Ig gene rearrangement between days 10 and 15 of embryonic development, primarily in the splenic anlage (reviewed in 19-22). V(D)Jjoining is complete by day 18 of embryogenesis, and chicken progenitor B cells then migrate to a specialized lymphoid organ, the bursa of Fabricius, which is a posterior invagination of the cloaca of avian species. Within the bursa of Fabricius, an initial population of about 3-5 x 105 B cells expands by proliferation to approximately 1-2 x lo9 mature B cells. The diversity of the rearranged Ig genes in the progenitor B cells entering the bursa is relatively restricted, due to the limitations of the V(D)J joining process, which can utilize only single V and J gene segments in the IgL and IgH locus. However, during the expansion of the B-cell population within the bursal microenvironment, sequence diversification occurs in the rearranged VL and V, regions by intrachromosomal gene conversion, using sequence information copied from germline variable region pseudogenes (qV) located 5’ of the functional V genes (14,23).Each B cell accumulates at least 4-10 gene conversion events within the rearranged V regions before migrating from the bursa to the peripheral lymphoid organs (14, 24). Only those B cells that have completed this gene conversion process leave the bursa. Gene conversion in the chicken IgL locus has been shown to be restricted to the rearranged V gene segment, and does not occur in the leader region, the J gene segment, nor within a germline IgL allele that has not undergone V-J joining (23). Restriction-site analyses of other genes expressed at high levels in bursal lymphocytes have been performed to determine whether gene conversion is specific for the Ig loci. For example, the

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WAYNE T. MCCORMACK ET AL.

FIG. 2. Genomic organization of the chicken immunoglobulinlight-chain gene locus. The organization depicted is for the light-chain locus of the S3 inbred strain as defined by W. T. McCormack et al. (unpublished observations) and is similar to that previously described by Reynaud et 02. (14) for the CB strain.

lymphoid-specific Ca5-tubulin gene has a genomic organization resembling the IgL locus. This gene is actively transcribed in bursal lymphocytes and is located just 3' of a pseudogene segment with an extensive open reading frame (25). Despite these similarities in organization to the Ig loci, the Ca5tubulin gene does not appear to undergo gene conversion in bursal lymphocytes (26). Similarly, clusters of the chicken histone H2b genes do not appear to undergo gene conversion during B-cell development (26). Taken together, these data support the hypothesis that the gene conversion process is specifically targeted to the rearranged V gene segments of the Ig loci. Nucleotide sequence analyses (14) of chicken IgL cDNA clones confirm that all rearranged chicken IgL genes are encoded by the single functional V, and JL gene segments. When sequences of cDNA clones derived from the bursa at different developmental times were compared, it was observed that the number of nucfeotide substitutions increases with time (14). Most importantly, these substitutions are clustered in blocks of sequences that exactly match sequence blocks within a cluster of 25-27 WLsegments situated in the -20 kb region 5' of the functional V,, gene (Fig. 2). Although homologous to the functional VL element, each of these YVL segments is truncated at the 5' end and therefore lacks the leader exon and promoter elements required for transcription, and is mutated or truncated at the 3' end, resulting in loss of the recombination signal sequence (heptamer / 12 or 23 bp spacer / nonamer) required for V-J joining (1-3). Therefore, the WL segments cannot participate in the formation of a functional Ig gene by joining to the J, segment. The observation that nucleotide substitutions in the rearranged gene are found in the VVL segments suggests that the *V, segments act as templates for the diversification of V,, via a somatic recombination process, now known to be gene conversion. The chicken IgH locus is organized similarly to the IgL locus, in that only one functional V, and JH gene segment is available for V(D)J joining (15) (Fig. IS). The amount of combinatorial and junctional diversity in the IgH locus is somewhat greater than in the IgL locus, due to the presence of approximately fifteen D, gene segments. However, just as in the IgL locus, the primary mechanism for generating somatic diversity of the V, region is through gene conversion, using a family of 90 WHsegments located in a

-

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31

60-80 kb region just 5’ of the functional V, gene as donors. Interestingly, gene conversions in rearranged IgH genes often extend into the D, region.

B. Gene Conversion in Chicken B Cell Tumors and Cell Lines Just as the availability of murine B cell lines, such as the Abelson murine leukemia virus-transformed pre-B cell lines, has facilitated study of the V(D)Jjoining process (2), the availability of chicken B cell lines is contributing to our understanding of the gene conversion mechanism of Ig gene diversification. Infection of susceptible chicken strains with the avian leukosis virus (ALV) induces a preneoplastic proliferation of lymphocytes in single bursal follicles to form a structure called the transformed follicle (27, 28). In most cases, integration of ALV adjacent to the c-rnyc proto-oncogene causes deregulation of c-myc expression in these transformed follicle cells (29). After a latency period of up to several months, undefined secondary transforming events cause malignant progression of transformed follicle cells to produce a B-cell lymphoma, which may later give rise to metastatic tumors (27, 28). Transformed follicle cells are capable of homing to the bursa of Fabricius and continuing proliferation .(30),suggesting that they are arrested by ALVtransformation at the bursal stem cell stage of differentiation, and should also be capable of undergoing gene conversion in their rearranged Ig genes. Restriction-site analyses of sites within the rearranged V,, gene demonstrate that restriction sites are progressively lost in these cells (23, 30), suggesting that gene conversion does indeed occur in them. One of the cell lines derived from an ALV-induced bursal lymphoma and tested by this assay, the DT40 cell line (31), undergoes gene conversion constitutively in uitro (30, 32, 33). Therefore, the DT40 cell line provides an in uitro model for the study of gene conversion in chicken Ig genes. For example, surface IgM-negative variants of DT40 that arise spontaneously in culture are due to gene conversion events that result in frameshifts due to nucleotide insertion or deletion (33). Reversion of these variants back to surface IgM expression occurs through the superimposition of additional gene conversion events on the frameshift mutations. Nucleotide sequence analyses of the rearranged IgL locus of DT40 cells passaged over a period of one year (32)revealed that most (39151) subclones of DT40 represent novel variants of the parental tumor, and that gene conversions could account for 2031220 nucleotide substitutions observed in the variants. DT40 is the only ALV-induced chicken B-cell line that has been found to undergo gene conversion in uitro. Chicken B-cell lines generated by infection with the v-rel oncogene-containing retrovirus, reticuloendotheliosis virus (34),also appear not to undergo gene conversion. The assays that have been used to analyze these cell lines include nucleotide sequencing of the

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WAYNE T. MCCORMACK ET AL.

rearranged IgL genes of v-re2 cell lines (32), and restriction-site analysis of the rearranged IgL alleles (34).There has been one report of a v-reE cell line undergoing a low frequency of sequence diversification as detected by the loss of restriction sites (35),but this observation has not been confirmed by nucleotide sequencing data.

C. Somatic Diversity in Rabbit VH Genes The rabbit V, family, consisting of a few hundred V, genes, is similar in size to that of other mammalian species (36, 37). The rabbit V, genes all appear to belong to a single subgroup, and as many as 40-50% appear to be pseudogenes (37-39). The average spacing of rabbit V, genes is only 3-7 kb (36,40),resembling the close spacing of chicken *V segments more than the 8-25 kb spacing of other mammalian V, genes (41). Surprisingly, studies of V, gene rearrangement in rabbit B lymphocytes revealed that only a single V, gene, the most D,-proximal V, gene (V,,), is rearranged in nearly all rabbit B cells (42,43). Most of the potential diversity available through V(D)J joining of multiple functional germline V, genes is bypassed. Therefore, the rabbit IgH locus is functionally very similar to the chicken IgH locus, which only has one functional germline V, gene. The observation that a single V, gene is rearranged in most rabbit B cells explained the Mendelian inheritance of allotypic markers expressed on rabbit V, regions (V,al, V,a2, and V,a& reviewed in M), because the rabbit V,, gene encodes the V,a allotype expressed on 7040% of all serum Ig molecules. However, the question arises as to how diversity is generated in rabbit immune responses, which are used by biologists in many fields to generate antisera. Nucleotide sequencing of random rabbit V, cDNA and genomic clones revealed that blocks of nucleotide sequence are shared in different cDNA clones (44,suggesting the possibility that gene conversion might occur in rabbit V, genes. However, sequence analyses of rearranged V,, genes (12) demonstrated conclusively that blocks of nucleotide substitutions, insertions, and deletions appear in the V,,, gene after V(D)J joining. Furthermore, potential gene conversion donors for many of these diversified regions were identified within other genornic V, genes andlor pseudogenes. These results suggest that, as in the chicken IgL and IgH loci, somatic gene conversion is a major mechanism used by rabbit B cells to generate somatic diversity of the rearranged vH1 gene.

II. Molecular Mechanism of Somatic Immunoglobulin Gene Conversion Because of the relatively simple organization and small size of the chicken IgL locus, it is ideal for studies detailing the regulation of somatic gene conversion. The chicken IgL locus spans only about 25 kb, including a

IMMUNOGLOBULIN GENE DIVERSIFICATION

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cluster of 25-27 q V , segments and single V,, J,, and C, genes (14, 16). Transcription of the chicken IgL locus is regulated by a promoter site consisting of a conserved octamer element (46) and a TATA box located 5' of the functional V,, gene. The nucleotide sequences of all 25 *VL segments of three inbred chicken strains have been determined, allowing studies of gene conversion donor relationships in rearranged V,, genes. Using this information, several laboratories have undertaken detailed descriptions of the products of the gene conversion process. Based on these studies, an understanding of the molecular mechanism by which blocks of nucleotide sequence are transferred from the pseudogene pool into the functionally rearranged V genes is emerging.

A. Gene Conversion vs. Double Homologous Recombination The sequence exchange events that are observed in the chicken V,, gene are compatible with two types of recombination event between the V,, gene and TV, segments. One of these is gene conversion, defined as a nonreciprocal exchange of DNA sequence from a donor gene (W,) into a recipient gene (V,J. The second type of recombination event compatible with the observed sequence diversification in the rearranged V genes is double homologous recombination, that is, reciprocal exchange between the ?VL gene segments and the V,, gene. To distinguish between these possibilities, Carlson et ul. (47) examined the products of sequence exchange events in an F, cross between two inbred chicken strains that differed in their IgL loci by several restriction-site polymorphisms and nucleotidesequence polymorphisms in the TV, segments. On the basis of the sequence polymorphisms between the V,, and VV, segments of the parental alleles, the nucleotide-sequence exchanges between the rearranged V,, gene and the 'PV, segments were shown to occur exclusively within a single allele, that is, intrachromosomally. The reciprocality of sequence exchange events was determined by analyzing the 'PV, segments in a panel of chicken B-cell lines, derived from the same strain by direct transformation with a v-rel-containing retrovirus. These clonal B-cell lines had undergone many rounds of sequence exchange between the V, gene and q V L segments, as evidenced by restriction-site loss within the rearranged V,, gene (47). For three cell lines in which gene conversion events appeared to be specifically derived from the 9 V L 7 segment, the WV,, segment was isolated from both alleles of the cell lines and sequenced. In all cases, the sequences of the 9 V L , segments matched the germline sequence and was not modified. As an additional test for unidirectional sequence exchange, and to exclude the possibility that homologous recombination occurs between a V,, gene and *V, segment on sister chromatids, extensive restriction-site analyses were performed on a large panel of v-re1 B cell lines. Whereas this assay does detect sequence modification in

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WAYNE T. MCCORMACK ET AL.

the rearranged V,, gene, there was no evidence for sequence modification in the W,cluster (47).

B.

Properties of Chicken IgL Gene Conversion

1. PSEUDOGENE DONORUSAGE

The frequency at which VV, gene segments are used as gene conversion donors appears to depend on three variables. The first of these is proximity to the functional V,, gene. Reynaud et al. (14)pointed out that the 9 V L segments located proximal to V,, are used more frequently than those distal to V,, and the same pattern was observed in a comprehensive analysis of gene conversion events in rearranged V,, genes isolated from the bursa of Fabricius at day 18 of embryogenesis (24). The second factor influencing pseudogene usage is the overall sequence homology of the VVL segment with the V,, gene, which is determined by the length of the 9 V L segment and the percent of nucleotide-sequence identity to V, (24). For example, truncated "V, segments are used less frequently than full-length VVLsegments. The third factor determining the frequency of "VL usage as a gene conversion donor appears to be the relative orientation of the q V L segment (24).The W gene segments that are situated in the inverted or antisense orientation with respect to the V,, gene are used as donors more frequently than PV, segments in the same orientation as VLl. This bias for the usage of inverted VV, segments is independent of location within the W,cluster and overall homology to V,,. As gene conversions begin to accumulate within the rearranged V,, gene, the newly acquired sequence may influence further gene conversion events. For example, during sequence analysis of gene conversion events in a bursal lymphoma cell line, DT40, which undergoes gene conversion in oitro, Kim et al. (32) observed that an early gene conversion event during the passage of this cell line involved a 15-bp insertion derived from the "vL8 segment. In later sublines of the parental DT40 cell line, the later gene conversion events observed in this region were highly biased toward the use of the *VLl8 segment, which shares a 15-bp insertion similar to that found in ?VM. This observation suggests that sequence alterations of early gene conversion events may alter the ability of other pseudogenes to act as efficient sequence donors. 2. LENGTHAND LOCATIONS OF GENECONVERSION EVENTS

Diversification of the rearranged V,, gene segment occurs by multiple serial gene conversion events within the V region. The number of events per clone isolated horn the bursa of Fabricius at day 18 of embryogenesis is one to four (14,24),and the average number of events per clone from the bursa at

IMMUNOGLOBULIN GENE DIVERSIFICATION

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three weeks of age is three to seven (24).Rearranged V,, genes isolated from later stages of development accumulate so many gene conversion events that it is impossible to identify discrete events, due to overlaps among the gene conversion events (W. T. McCormack and C. B. Thompson, unpublished data). These observations support the hypothesis that gene conversion events accumulate within the rearranged V,, gene until the lymphocyte leaves the bursa1 microenvironment. The exact length of gene conversion events is impossible to determine, because of regions of sequence identity between the q V , donors and the target V,, gene at the boundaries of gene conversion events. However, the minimum lengths of gene conversion events may be determined by the length of sequence between the 5’-most and 3’-most substitution derived from an individual gene conversion event. The minimum length of gene conversion events in day 18 cDNA clones is usually between 8 and 120 bp, with an average of 27 bp (14, 24). The longest gene conversion event observed in day 18 clones was at least 249 bp (24). When the locations of gene conversion events within rearranged V,, genes isolated from the bursa at 18 days of embryogenesis were compared, using their minimum lengths, there appears to be a paucity of events in the most 5‘ portion of V,, gene segment (24), even though donor sequences homologous to the leader portion of the V,, exon and part of the leader intron are present in the pseudogene pool. There are sequence polymorphisms in this region of the pseudogene donors that would allow detection of gene conversion events in that region (14). Gene conversions occur throughout the remainder of the V,, exon to the V,-J, junction. In addition, gene conversion events occur more frequently within the complementarity-determining regions (CDR), which are the regions of the Ig molecule that form the antigen binding pocket of the antibody molecule. 3. POLARITYOF GENE CONVERSION EVENTS

At the 3’ end of the rearranged V,, region, gene-conversion events have been observed that extend past the region of homology between the donor lIrV segment and the rearranged V,, gene segment. The marked difference in the frequency of gene conversion at the 5‘ and 3’ ends of the V region suggests the possibility that gene conversion may have a 5’-to-3’ polarity. Analysis of the ends of gene conversion events also revealed other differences in the 5‘ and 3’ ends of individual events. The 5’ ends of gene conversion events always lie within a region of nucleotide sequence identity between V,, and the q V L donor. In contrast, the 3’ ends of gene conversion events often end in regions of nonhomology, and may result in an increase or decrease in the overall length of the rearranged V,, segment due to imprecision in the joining of sequences derived from the gene conversion donor and

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WAYNE T. MCCORMACK ET AL.

V,, (24). These observations support the hypothesis that the mechanism of gene conversion exhibits 5'-to-3' polarity within the target V gene. One explanation for these observations is that gene conversion is initiated at its 5' end by homologous pairing of the nontranscribed V,, strand with a WL segment, and is followed by strand extension in the 3' direction. Observations that gene conversions sometimes extend in the 3' direction into the JL region (14,24), and that gene conversions in the chicken V,, gene often extend beyond the region of W, homology with V,, into the D, region (15, 48) support this hypothesis.

C. Molecular Models for Immunoglobulin Gene Conversion 1. SINGLE-STRAND BREAK-REPAIR

The polarity that appears to be inherent to the gene conversion process may be explained by a variation of the Meselson and Radding single-strand break-repair model for gene conversion (49). As illustrated in Fig. 3, gene conversion may be initiated by single-strand breaks in the nontranscribed

e -

7

~V~donor rearranged VL, gene

DNA nick strand transfer

6

strand extension

6

resolution

6

DNA ligation

6

heteroduplex DNA repair

FIG.3. A single-strand break-repair model for sequence transfer associated with immunoglobulin gene conversion. ’PV gene donor sequence, thick lines. Rearranged functional V gene segment, thin lines.

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strand of the V,, gene, which are made accessible during Ig transcription in

the bursa1 lymphocyte. The unwound 3' end may then invade the DNA duplex of a homologous q V L segment to create a "D loop" by homologous pairing, perhaps mediated by a strand transfer activity analogous to the recA activity of Escherichia coli (50). The relative paucity of gene conversion events in the 5' region of the V,, gene and apparent 5'-to-3' polarity of gene conversion (24)would then be explained by the requirement for this homologous pairing with the donor gene segment. For example, homologous pairing mediated by the recA protein requires 35-50 bp of homology between the two DNA strands (50). A D N A polymerase could then extend the 3' end of the invading strand using the *VL sequence as a template for DNA synthesis. At this point, resolution of the D-loop structure without a cross-over event would produce an extended strand that may overlap with the pre-existing DNA strand, depending on the extent of DNA synthesis and the possible action of exonucleases on the free 3' and 5' ends. The newly extended strand could be preferentially rewound into the V,, gene by the rewinding action associated with the RNA polymerase-I1 enzyme complex during continued transcription (51). Imprecision in the religation of the extended and the nascent strand could result in the codon loss and duplication observed at the 3' ends of gene conversion events (24). The final product is a V,, gene with a region of heteroduplex DNA, which may or may not be repaired before the cell divides. If DNA repair does not occur, one daughter cell will inherit the gene conversion event, while the second daughter cell will inherit the unmodified DNA strand. If heteroduplex DNA repair does occur, the final product will depend on whether there is any strand bias during the repair process. A strand bias for the repair of the transcribed strand, for example, would result in a geneconversion event in all cases. Examples of such strand bias in heteroduplex DNA repair have been reported for some forms of DNA damage in mammalian cells (52-54).

2. DOUBLE-STRAND BREAK-REPAIR The products of chicken immunogIobulin gene conversion are also consistent with the double-strand break-repair model proposed by Szostak and co-workers for gene conversion in yeast (55, 56), if the observed polarity can be accounted for. According to this model (Fig. 4), gene conversion may be initiated by breaks on both strands of the rearranged V,, gene, followed by strand invasion of the *V, donor molecule. Strand extension would then occur on both strands of the V,, gene, and resolution of the recombination intermediate without crossovers would result in heteroduplex DNA formation. The lower frequency of gene conversion events in the 5' region of the

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WAYNE T. MCCORMACK ET AL.

yV~donor rearranged VL1 gene

7 7 9

DNA nicks I strand transfer

y p ,strand extensions

-

9

resolution / DNA ligation

F

9

heteroduplex DNA repair

7

FIG.4. A double-strand break-repair model for transfer of sequence information between a YV gene donor sequence (thick lines) and rearranged functional VL1 gene segments (thin lines).

V,, gene would suggest that extensions in the transcribed strand are not recovered as efficiently in the final product. This may be due to an inability of the transcription complex to wind this strand in as a result of a nick or gap. Alternatively, strand bias during heteroduplex DNA repair could remove the donated sequence on the transcribed strand.

111. Enzymatic Activities Implicated in Immunoglobulin Gene Conversion

A. Chromatin Structure and the Role of Transcription One of the distinguishing features of the rearranged IgL locus, as compared to an IgL locus that remains in the germline configuration, is the presence of clusters of DNase-I-hypersensitive sites, which are expressed in a lymphoid-specific fashion (23).These DNase-I-hypersensitive sites are not present in the unrearranged V,, gene segments in bursa1 lymphocytes, nor in T lymphocytes. This observation suggests that the chromatin configura-

IMMUNOGLOBULIN GENE DIVERSIFICATION

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tion of the rearranged IgL allele is more “open” than the germline allele, and is perhaps more accessible to the enzyme activities involved in the gene conversion process. This may result from localized changes in D N A topology associated with the transcriptional enzyme complex (57-59). In addition to the difference in DNase-I hypersensitivity, two features distinguish the rearranged V gene that is a target for gene conversion from the pseudogene donors and the unrearranged V gene. The first of these is simply the fact that the rearranged V gene has been joined to the J L gene and its flanking sequence by the V-J joining event; the second is that transcription is activated by this rearrangement event. There are, therefore, two potential molecular signals which may direct gene conversion to the rearranged V,, gene. One is the physical juxtaposition of the V, and JL gene segments and/or the deletion of the region between V, and JL. The second possible signal is the passage of the transcriptional enzyme complex. The possible roles of gene rearrangement vs. transcription may be separated by study of gene conversion in the Muscovy duck IgL locus. In contrast to all other avian species examined, the Muscovy duck IgL locus consists of more than one functional VL gene in addition to a large number of P V , segments (11).Interestingly, some of the Muscovy duck *VL segments have retained the recombination signal sequence in their 3‘ flanking region, which is required for V-J joining. However, these PV, segments are truncated at their 5’ ends and lack the promoter regions required for transcription. Tjoelker and co-workers (60 and unpublished data) have demonstrated that although such PV, segments cannot be transcribed they do undergo joining to the JL gene, and may be recovered from developing Muscovy duck B cells due to a functional V, gene rearrangement on the other IgL allele (Fig. 5). These rearranged P V , segments do not undergo gene conversion events in the bursa of Fabricius, suggesting that joining to the J L gene segment is not sufficient to target the rearranged PV, segment as a recipient of gene conversion events. Furthermore, nonfunctionally rearranged V, genes that can be transcribed but do not produce a functional protein product due to an out-of-frame V,-J, junction or stop codon do undergo gene conversion (33, 60). Taken together, these results suggest that V-J rearrangement is not sufficient to activate gene conversion, and that an active transcription process is required for the initiation of gene conversion.

6. Initiation of

Gene Conversion

As described above, the gene-conversion process appears to be intimately tied to the transcription of the rearranged VL, gene. One mechanism by which gene conversion may be initiated by transcription is via the introduction of random nicks in the rearranged V,, gene by nonspecific endonucleases during the localized unwinding and rewinding of the gene during

40

WAYNE T. MCCORMACK ET AL.

-w-}L

VL

qJVL

-1-1L

"L

JL

JL

JL

+

Gene Conversion

+

No Gene Conversion

> -,

Gene Conversion

FIG.5. Susceptibility of various VJ recombination products to gene conversion. (*), stop d o n . As described in the text, transcribed VJ recombination products, whether or not they encode a hnctional product are susceptible to gene conversion, while a recombinationproduct that lacks a promoter is not susceptible to gene conversion.

passage of the RNA polymerase. Alternatively, nicks may be left by the incomplete strand religation of topoisomerases associated with the transcriptional machinery (61). However, such nonspecific nicking would not explain the unique targeting of the gene conversion process to the rearranged V genes. Other genes that are transcribed at high levels in bursal lymphocytes, and that have potential donor genes, do not undergo gene conversion events in bursal lymphocytes (26),suggesting an Ig-specificcomponent to the initiation of gene conversion. One mechanism for the specific introduction of nicks into the rearranged V,, gene could be via an Ig-specificendonuclease activity, similar to the HO endonuclease that targets the MAT locus for gene conversion during yeast mating-type switching (62).Although no evidence for specific single-strand or double-strand breaks in the rearranged V,, has heen presented, chromatin structural studies reveal the presence of three dominant DNase-Ihypersensitive sites that are expressed in a bursal-specific manner and are localized within the rearranged V ,, gene (23).As discussed above, the increased DNase-I hypersensitivity suggests that the rearranged V,, gene in bursal lymphocytes is at least in a chromatin configuration that may be more accessible to enzymatic activities. Sequence analyses have suggested that gene conversion events occur more frequently near the complementarity-determiningregions of the rearranged V , gene, at sites corresponding to the locations of sequences that resemble the conserved heptamer element of the recombination signal sequence required for V(D)J joining (24). It has been suggested that these

IMMUNOGLOBULIN GENE DIVERSIFICATION

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heptamer-like elements may be targets for a site-specific endonuclease activity similar to the one involved in V(D)Jjoining (9, 10). Consistent with a role for these heptamer-like elements within the V,, gene, a bursal-derived chicken B-cell line that constitutively undergoes gene conversion in oitro that has acquired a point mutation in one of these heptamer elements appears to have ceased to accumulate gene conversion events in the vicinity of that heptamer element (32). These observations suggest a possible relationship between the V(D)Jjoining mechanism and gene conversion, such that the component(s) of V(D)Jjoining that recognize the heptamer elements may be involved in targeting gene conversion to the V,, gene via recognition of heptamer-like sequences. For example, endonucleolytic activities have been reported that cleave at or near the recombination signal sequences (63). However, it is unlikely that the complete V(D)J recombinase activity is involved in gene conversion because ongoing rearrangement of IgH and IgL genes is not detected during the bursa1 stage of chicken B-cell development (18).

C. Bursa1 Expression of the Recombination-Activating Genes To investigate the possibility that gene conversion and V(D)Jjoining are functionally related, Carlson et al. studied the expression of the recombination-activating genes RAG-1 and RAG-2 during chicken lymphoid cell differentiation (64). The recombination-activating genes were described in experiments designed to identify genes encoding V(D)Jrecombinase activity (65, 66). Random genomic fragments were transfected into a fibroblast cell line harboring a selectable V(D)J recombination substrate. Genes encoding the V(D)Jrecombinase activity could be positively selected by the expression of the selectable marker, which could be expressed only after a chromosomal inversion via a V(D)Jrecombination event. A pair of linked genes (RAG-1 and RAG-2) was discovered by this assay, and their patterns of expression in lymphoid cells were determined. The co-expression of RAG-1 and RAG-2 is observed in all mammalian lymphoid cells of the B and T cell lineages that can rearrange V(D)Jrecombination substrates. The function of RAG-1 and RAG-2 is under investigation. It is unknown whether RAG-1 and RAG-2 encode components of the a V(D)J recombinase enzyme complex, or whether their expression regulates the expression of recombinase genes. The chicken homologs of RAG-1 and RAG-2 have been identified and sequenced, and the expression of chicken RAG-1 and RAG-2 in various lymphoid tissues during development has been examined by Northern blot analysis (64).In contrast to the coexpression of RAG-1 and RAG-2 in mammalian cells undergoing V(D)Jjoining, chicken B cells undergoing gene conversion in the bursa of Fabricius express high levels of RAG-2 in the absence of RAG-1 expression. This selective expression of RAG-2 is unique

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WAYNE T. McCORMACK ET AL.

to the bursal lymphocyte population, as chicken T cells developing in the thymus [and undergoing rearrangement of their T-cell receptor genes via V(D)Jjoining] were found to coexpress RAG-1 and RAG-2. Mature lymphocytes in the chicken spleen express neither RAG-1 nor RAG-2. These results suggest that the expression of RAG-2 alone, or of genes regulated by RAG-2 in the absence of RAG-1, may be involved in the regulation of somatic Ig gene conversion in bursal lymphocytes. To test this hypothesis, Takeda et al. (67) deleted the RAG-2 gene by homologous recombination from the DT40 cell line, which expresses RAG-&and undergoes gene conversion in culture as described above. RAG-2.’- DT40 cells were found to retain the ability to undergo Ig gene conversion, demonstrating that RAG-2 expression is not absolutely required for Ig gene conversion in this cell line. Therefore, the exact role of selective RAG-2 expression in developing bursal lymphocytes remains speculative.

D. Enzymes Involved in Homologous Recombination Many of the enzymatic activities that appear to be involved in somatic Iggene conversion are also likely to be involved in normal DNA replication and repair processes, and may not be unique to the gene conversion event. For example, strand extension of an invading V,, gene strand using the VVL segment as a template requires the activity of DNA polymerase, and the extended strand may be re-ligated to the pre-existing strand by DNA ligase activity. According to the models for immunoglobulin gene conversion, for gene conversion to be an efficient mechanism for the generation of sequence diversity in developing chicken B cells, these cells must be capable of promoting homologous pairing of the donor and target sequences. These observations suggest that chicken B cells may also be proficient at other recombination events that involve homologous pairing. Chicken B-cell lines display a dramatic increase in the ratio of targeted to random integration events compared to other vertebrate and chicken cell lines (68).This process was shown to be cell-type specific, occurring in a variety of B-cell lines, but not in non-B-cell lines. However, this homologous recombination process differs from gene conversion in several ways. Homologous recombination in chicken B-cell lines is not specific for the immunoglobulin locus, since targeted integration into the P-actin locus was also observed. In addition, transcription of the target locus is not required, as targeted integration occurs efficiently at the nontranscribed vitellogenin locus in chicken B-cell lines. Finally, although targeted integration events occur in the bursal lymphoma line DT40, which also undergoes constitutive gene conversion, a number of B-cell lines that do

IMMUNOGLOBULIN GENE DIVERSIFICATION

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not undergo gene conversion in vitro did exhibit the homologous recombination activity (68).These results suggest that avian B cells are competent at homologous recombination activities, and may use some of the enzymes involved in homologous recombination in the gene conversion process. An enzyme activity that may be required for both of these types of recombination events is the strand transfer activity, which may resemble the E . coli recA protein (50).Similar strand-transfer activities have been partially purified from a number of eukaryotic cell types, including the rec-1 activity from the fungus Ustilago (69), and from a variety of human cell types, including bladder carcinoma, fibroblasts, HeLa cells, and B lymphoblasts (70-72).

E. Heteroduplex DNA Repair The single-strand break-repair and double-strand break-repair models for immunoglobulin gene conversion both involve the formation of a heteroduplex region within the V,, gene. As described above, the polarity of gene conversion events in the day 18 bursa1 IgL clones (24) may be explained in part by strand bias during heteroduplex DNA repair (52-54). In fact, if a strong directionality in heteroduplex repair exists, one might argue for an alternative model of gene conversion that involves heteroduplex DNA repair within a D loop formed between the target V gene and donor WV segment, in the absence of strand breakage. However, the nucleotide sequence data for gene conversion events do not support this model. The observation that the 3' ends of gene-conversion events can be imprecise, with nucleotide loss or duplication, suggests that strand breakage, extension, and religation must be involved.

IV. Conclusions The study of the molecular genetics of chicken Ig light chain locus has provided insights into several types of DNA recombination events. For example, studies of V(D)J type joining in this locus provided the first evidence for specific nucleotide addition to coding ends during Ig gene rearrangement (16).However, the expressed chicken V-gene repertoire is generated not by the combinatorial and junctional diversification of V(D)J joining, but by somatic gene Conversion, utilizing families of variable region pseudogenes as a pool of donor sequence information (8, 10).Chicken B cells have also been shown to be capable of targeted integration events via homologous recombination (68).Taken together, these results suggest that the chicken B cell provides a good experimental model system in which to study the mechanism of a variety of somatic DNA recombination events in higher vertebrate cells.

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37. S. J. Currier, J. L. Gallarda and K. L. Knight, J. Immunol. 140, 1651 (1988). 38. W. T.McCormack, S. M. Laster, W. F. Marzluff and K. H. Roux, NARes 13, 7041 (1985). 39. K. H. Roux, P. Dhanarajan, V. Gottschalk, W. T. McCornlack and R. W. Renshaw, J. Zmmunol. 146, 2027 (1991). 40. K. E. Bernstein, C. 8 . Alexander and R. G. Mage, J . Zmmunol. 134, 3480 (1985). 41. T. Honjo, Annu. Reu. Zmmunol. 1, 499 (1983). 42. R. S. Becker, M. Suter and K. L. Knight, Eur. J. Zmmunol. 20, 397 (1990). 43. K. L. Knight and R. S. Becker, Cell 60, 963 (1990). 44. R. G. Mage, K. E. Bernstein, N. McCartney-Francis, C. B. Alexander, G. 0. YoungCooper, E. A. Padlan and G. H. Cohen, Mol. Zmmunol. 21, 1067 (1984). 45. W. T. McCormack, P. Dhanarajan and K. H. Roux, J. Zmmunol. 141, 2063 (1988). 46. T. Parslow, D. Blair, W. Murphy and D. Granner, PNAS 81, 2650 (1984). 47. L. M. Carlson, W. T. McCormack, C. E. Postema, C. F. Barth, E. H. Humphries and C. B. Thompson, Genes Deu. 4, 536 (1990). 48. R. Parvari, A. Avivi, F. Lentner, E. Ziv, S. Tel-Or, Y. Burstein and I. Schechter, EMBO]. 7, 739 (1988). 49. M. S. Meselson and C. M. Radding, PNAS 72, 358 (1975). 50. C. M. Radding, BBA 1008, 131, (1989). 51. H. 8. Gamper and J. E . Hearst, Cell 29, 81 (1982). 52. I. Mellon, G. Spivak and P. C. Hanawalt, Cell 51, 241 (1987). 53. P. C. Hanawalt, Enuiron. Mol. Mutagen, 14 (Suppl. 16), 90 (1989). 54. H. Vrieling, M. L. Van Rooijen, N. A. Groen, M. Z. Zdzienicka, J. W. Simons, P. H. Lohman and A. A. van Zeeland, MCBiol9, 1277 (1989). 55. J. W. Szostak, J. L. Orr-Weaver and R. J. Rothstein, Cell 33, 26 (1983). 56. H. Sun, D. Treco, N. P. Schultes and J. W. Szostak, Nature 338, 87 (1989). 57. L. F. Liu and J. C. Wang, PNAS 84, 7024 (1987). 58. B. I. Osborne and L. Guarente, Genes Deu. 2, 766 (1988). 59. S. J. Brill and R. Sternglanz, Cell 54, 403 (1988). 60. L. W. Tjoelker, W. T. McCormack, L. M. Carlson and C. B. Thompson, in“Abstracts of the 7th International Congress of Immunology,” p. 13. Fischer, Stuttgart, 1989. 61. B. J. Thomas and R. Rothstein, Cell 56, 619 (1989). 62. A. J. S. Klar, in “Mobile DNA” (D. E. Berg and E. E. Howe, eds.), p. 671. Am. Soc. Microbiol., Washington, D.C. 1989. 63. T. J. Hope, R. J. Aguilera, M. E. Minie and H. Sakano, Science 231, 1141 (1986). 64. L. M. Carlson, M. A. Oettinger, D. G. Schatz, E. L. Masteller, W. McCormack, D. Baltimore and C. B. Thompson, Cell 64, 201 (1991). 65. D. G . Schatz, M. A. Oettinger and D. Baltimore, Cell 59, 1035 (1989). 66. M. A. Oettinger, D. G. Schatz, A. Gork and D. Baltimore, Science 248, 1517 (1990). 67. S. Takeda, E. L. Masteller, C. B. Thompson and J.-M. Buerstedde, PNAS 89, 4023 (1992). 68. J.-M. Buerstedde and S. Takeda, Cell 67, 179 (1991). 69. E. B. Kmiec and W. K. Holloman Cell 29, 367 (1982). 70. P. Hsieh, S. Meyn and R. D. Camerini-Otero, CeU 44, 885 (1986). 71. D. Ganea, P. Moore, L. Chekuri and R. Kucherlapati, MCBiol 7, 3124 (1987). 72. E. Cassuto, L.-A. Lightfoot and P. Howard-Flanders, MGG 208, 10 (1987).

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AD P- ribosy lat ion Factors: Protein Activators of Cholera Toxin’ JOEL MOSS AND

MARTHA VAUGHAN Laboratory of Cellular Metabolism National Heart, Lung, and Blood lnstitute National lnstitutes of Health Bethesrkr, Maryland 20892 I. Biochemistry of ADP-ribosylation Factors ........................... 11. Structure of ADP-ribosylation Factors .............................. 111. Functions of ADP-ribosylation Factors in Animal Cells . . . . . . . . . . . . . . .

References

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49 55

60 63

Some microorganisms affect their hosts by the action of secreted toxins that disrupt regulatory or metabolic pathways. Cholera toxin, a secretory product of Vibrio cholerae, closely related Escherichia coli secretory products (known as heat-labile enterotoxins, or LTs), and a Bordetella pertussis protein (with the dual names of pertussis toxin and islet-activating protein) all appear to affect cell metabolism through effects on proteins that require guanine nucleotides for activity and are responsible for transmembrane signalling (1).The toxins alter the activity of these guanine nucleotide-binding proteins (G proteins) by catalyzing the transfer of the ADP-ribose moiety of NAD to a critical amino acid in the a subunit. This ADP-ribosylation reaction results in either activation (e.g., cholera toxin, E . coli heat-labile enterotoxin) or inactivation (e.g., pertussis toxin) of the G protein. The G proteins are heterotrimeric signal-transducing proteins responsiAbbreviations: ARF, ADP-ribosylation factor; sARF, soluble ARF from bovine brain; hARF, human ARF; bARF, bovine ARF; yARF, yeast ARF; CPS, chicken pseudogene; G proteins, guanine-nucleotide-bindingproteins; G, and G,, the inhibitory and stimulatory G proteins, respectively, of the adenylyl-cyclase system; G , or transducin, the G protein coupled to rhodopsin and involved in visual excitation; Go, the G protein found in high concentration in brain; G,, the a subunit of the G proteins; DMPClcholate, dimyristoylphosphatidylcholine plus cholate; CTA, cholera toxin A subunit; CTA1, the A 1 protein of cholera toxin; LT, E. coli heat-labile enterotoxin; LT-Ih, LT serogroup type I that is encoded by an E. coli plasmid isolated From a human; LT-IIa, LT serogroup type 11, variant a; LT-IIb, LT serogroup type 11, variant b; GTPyS, guanosine 5’-[y-thio] triphosphate; GDPPS, guanosine 5’-[P-thio]diphosphate; GPP(NH)P, guanosine 5 -[P,y-imido]triphosphate; APP(NH)P, adenosine 5 ’ 4 3 , ~ imido]triphosphate . 47 Progress in Nucleic Acid Research and Molecular Biology, Vol. 45

48

JOEL MOSS AND MARTHA VAUGHAN

ble for coupling cell-surface receptors to their intracellular effectors (2-4). Activation of a receptor occurs through its interaction with a specific ligand or agonist. Activated receptor in turn activates the G protein. In its inactive state, the G protein consists of a, @, and y subunits, with the nucleotidebinding site of the a subunit containing GDP (4). Activation of a receptor by a ligand is associated with (i) release of GDP from the a subunit followed by binding of GTP, and (ii) dissociation of a(GTP) from Py. In most instances, a(GTP) is responsible for alteration of effector activity, although Py subunits may be involved in the control of phospholipases and certain other enzyme activities (4, 5). In this regulatory pathway, signal termination occurs, in part, as a result of the hydrolysis of bound GTP to GDP by an activity intrinsic to the 01 subunit (2-4). The a(GDP) is believed to reassociate with @y. Reactivation of the inactive a(GDP)Py complex results from its reassociation with agonist-activated receptor. Cholera toxin and E. coli heat-labile enterotoxins catalyze the ADPribosylation of the a subunit of G,, the stimulatory G protein of the adenylylcyclase system that is also believed to regulate Ca2+ channels (1, 6). There appear to be four forms of G,, generated by alternative splicingof transcripts of a single gene (7 ). Pertussis toxin can ADP-ribosylate several highly similar G proteins (8). These include: the Gai family (Gail, Gu,e, and Gai3), which is involved in the inhibition of adenylyl cyclase and control of ion flux; G,, which may also be involved in the regulation of ion flux; and Gat, or transducin, which plays a critical role in visual excitation in the retina (8).There are at least three different G,, genes and two G,, genes, and one G, gene that yields two proteins with different carboxy termini through alternative splicing of mRNA (9). ADP-ribosylation by pertussis toxin of the G,,, G,,, and G,, proteins on a cysteine residue near the carboxy terminus interferes with their ability to interact hnctionally with cell-surface receptors, thereby interrupting transmembrane signalling (8). In addition to these proteins, there is a family of G, subunits that appear not to be substrates for any of these bacterial toxins (10). Cholera toxin-catalyzed ADP-ribosylation of the Gas isoforms leads to activation of the protein, as a result of inhibition of its GTP hydrolytic activity and consequent persistent subunit dissociation into active a(GTP) and f3-y components (11,12).Inhibition of GTP hydrolysis thus prolongs the life-time of the activated species. In addition, ADP-ribosylation may promote release of GDP, thus clearing the nucleotide-binding site and permitting GTP binding (13, 14). ADP-ribosylation occurs on an arginine residue located near the center of the OL subunit (15). In the adenylyl-cyclasesystem, activation of G, by ADP-ribosylation results in a GTP-dependent enhancement of cyclase activity and increased formation of cyclic AMP (I). Early studies showed that cholera toxin-catalyzed ADP-ribosylation of

49

ADP-RIBOSYLATION FACTORS

G,, and/or activation of adenylyl cyclase is stimulated by GTP or an analog, and by membrane and soluble factors from a variety of species and tissues (16-28). Pigeon erythrocyte cytosol contains a factor of 15-20 kDa that, in the presence of GTP, enhances toxin activity; the cytosolic factor was sensitive to trypsin (16, 17). Enhancement of the capacity of membranes to respond to toxin has been achieved by incubation in the presence of GPP(NH)P and cytosolic factor. Adenine nucleotides are only weakly effective. A trypsin-sensitive cytosolic factor of -13 kDa that enhances cholera toxin-catalyzed activation of pigeon erythrocyte adenylyl cyclase and ADPribosylation of a 43-kDa protein has also been identified in horse erythrocytes (18).

-

1. Biochemistry of ADP-ribosylation Factors A. Purification and Characterization ARFs from a number of tissues and species including rabbit liver and bovine brain have been extensively purified and characterized (23-25, 27). The ARFs isolated from rabbit liver membranes were two proteins of -21 kDa (23), with Stokes radius (R,) of 2.38 nm, S20,w (S) of 2.10, partial specific volume (ml/g) of 0.74, M , of 21,500, and frictional ratio, fl/fO, of 1.19 (23).The protein appeared to be both activated and stabilized by detergents; it was stable to N-ethylmaleimide, but thermolabile. ARF was also isolated from bovine brain membranes and soluble fractions (24). The membrane ARF had a sedimentation coefficient similar to that of the rabbit liver protein in the presence of detergent. Mg2+ increases its stability and altered its chromatographic behavior. Two soluble forms of ARF (termed sAAF I and sARF 11) isolated from bovine brain had similar properties (25, 27).

B. Activation of Cholera Toxin by ARF To determine the mechanism of activation of cholera toxin by ARF, advantage was taken of the fact that cholera toxin, in addition to catalyzing the ADP-ribosylation of G,,, can use simple guanidino compounds (e.g., arginine, agmatine) and proteins unrelated to G,, as ADP-ribose acceptors (29, 30). Included among these protein acceptors is CTA1, the catalytic fragment of the toxin (31, 32). Cholera toxin-catalyzed ADP-ribosylation of simple guanidino compounds is stereospecific and proceeds by an SN2-like reaction with inversion of configuration (33).P-NAD is the substrate and the product is a-ADP-ribosyl-arginine (33). ADP-ribosylation of G,, by cholera toxin is stimulated by a purified bovine brain soluble ARF (sARF 11) or membrane ARF (mARF) in the

ADP-RIBOSYLATION FACTORS

51

presence of GTP or GTP analogs such as GTPyS or Gpp(NH)p; GDP, GDPPS or adenine nucleotides are inactive (Fig. 1)(25, 27). The reaction is further enhanced by DMPC/cholate. Under similar conditions, cholera toxin also catalyzes the auto-ADP-ribosylation of the CTAl catalytic unit (Fig. 1). Optimal modification also requires GTP or poorly hydrolyzable GTP analogs; GDP, GDPPS, and adenine nucleotides are ineffective (Fig. 1). DMPC and cholate are not necessary and, in fact, inhibit ARF- and GTPdependent auto-ADP-ribosylation, although they slightly enhance basal (in the absence of ARF and guanine nucleotide) auto-ADP-ribosylation. ARF also enhances ADP-ribosylation of simple guanidino compounds such as agmatine, which is stimulated by GTP and nonhydrolyzable GTP analogs, but not by GDP, GDPPS, and adenine nucleotides (25,27,34,35). The apparent EC,, for GTP was influenced by certain phospholipids and detergents (35). In the presence of SDS, the EC,, is -5 pM; with DMPC/cholate, it is -50 nM (35).Thus, ARF appears capable of activating cholera-toxin ADP-ribosyltransferase activities under conditions where it exhibits either relatively high or low S n i t y for guanine nucleotides. With SDS, activation is observed at relatively low, submicellar concentrations (-0.003%), whereas at higher concentrations, both ARF-stimulated and basal ADP-ribosyltransferase activity is inhibited (35).Activation by DMPC/ cholate is likewise dependent on both the concentrations and the ratio of the two agents. Maximal rates of reaction occur without a delay in SDS. However, in the presence of DMPC/cholate there is a lag before optimal activity is achieved. The delay is eliminated by incubation of sARF I1 with GTP before initiation of the reaction. The effects of phospholipids and detergents on the GTP-dependent activation of cholera toxin by ARF are also reflected in their effects on guanine nucleotide-binding. As expected from the activity data, high-affinity binding by soluble ARF from bovine brain is not observed in SDS (35). In DMPC/cholate, Mg2+-dependent, high-affinity GTP binding is not significantly affected by components of the transferase assay (cholera toxin, NAD, or agmatine). ARF purified from bovine brain membranes binds GTP, GDP, and GTPyS but not adenine nucleotides (24). Optimal binding of guanine nucleotides requires DMPC, high salt, and Mg2+ with a maximal stoichiomeFIG. 1. Effect of dimyristoylphosphatidylcholine,ARF, and nucleotides on ADP-ribosylation of G,, and auto-ADP-ribosylation of the cholera toxin A1 protein. Assays contained [32P]NAD, cholera toxin A subunit, G,, and, as indicated, dimyristoylphosphatidylcholine (DMPC), a soluble ARF purified from bovine brain (sARF 11). and guanine or adenine nucleotides. Shown are four autoradiograms of results of SDS-PAGE. Mobilities of CTAl and G,, are indicated on the left. Data are from Tsai et al. (27).

52

JOEL MOSS AND MARTHA VAUGHAN

try of almost 0.9 molGTP per molARF. Unlike many other GTP-binding proteins, ARF does not hydrolyze GTP. Binding of GTP or GTPyS increases the intrinsic fluorescence relative to that observed in the presence of GDP. Consistent with a lack of GTP hydrolytic activity is the finding that the effects of GTP are similar to those of GTPyS and are stable with time (24). Recombinant ARF 1, synthesized in E. coli, bound GTPyS in the presence of DMPC, cholate, and MgCl, with a K , of -0.1 p M (36).ARF had a significantly higher affinity for GDP than it did for GTPyS at low Mg2+ concentrations. At millimolar Mg2+, the apparent affinities for GTPyS and GDP are similar. Nucleotide binding is inhibited by 0.2 to 0.8 M NaC1. The recombinant ARF does not hydrolyze GTP (less than 0.0005/min).

C. Guanine-Nucleotide-Dependent Complex Formation with Cholera Toxin From kinetic studies, it has been concluded that ARF directly activates CTAl (Fig. 2) (34). It appears not to stimulate the toxin-catalyzed reaction by binding to G,,, as had been proposed (23).To determine whether stimulation by ARF is associated with formation of a stable ARFCTA complex, the proteins were incubated in the presence of guanine nucleotides and SDS, and the sizes of the products determined by gel permeation chromatography (37).In the presence of ARF and GTPyS, but not GDPPS, an active ARFtoxin complex was formed that eluted near the void volume of a Sephadex G-75 column. Only a minor fraction of ARF formed such complexes with toxin. CTAl in the aggregate was much more effective in catalyzing autoADP-ribosylation than was toxin that did not complex with ARF. The presence of both toxin and ARF in addition to SDS and GTPyS were required to obtain an active aggregate. ARF in the aggregate stimulated the ADPribosylation by added CTA of G,, and other proteins, but was itself a poor substrate for CTA. Monomeric ARF and CTA are more effective than the aggregated proteins in ADP-ribosylating model protein substrates such as albumin. The substrate specificities of the toxin in the monomeric form and in complex with ARF are clearly different. With DMPC/cholate (instead of SDS) and GTPyS, an ARF-toxincomplex was not detected. Instead, some of the ARF appeared to form a GTPyS-dependent aggregate, independent of toxin. Similar to its behavior with SDS, only a minor fraction of ARF formed aggregates in DMPC/cholate; the effects of the aggregated and monomeric ARF on the substrate specificity of cholera toxin were different (37)*

D. Immunological Characterization of ARFs Both anti-ARF peptide and anti-ARF protein polyclonal antibodies have been used to evaluate distribution and cross-species conservation (38, 39).

53

ADP-RIBOSYLATION FACTORS

IGTP 1 Thiol

ARF-GTP*

A,-SH

ARF*GTP*A,-SH"

ADP-RiboseGS,

"-J

ADP-RiboseG sa .GTP*

'Inactive

L

-

1

ADP-RiboseGS; GTP VActiive

b

ATP

Cyclic AMP

FIG. 2. Participation of a guanine nucleotide-binding protein cascade in the activation of adenylyl cyclase by cholera toxin. ARF is activated by GTP (or, in oitro, by a nonhydrolyzable GTP analog). ARF-GTP then complexes with and activates CTA1. Cholera toxin, composed of one A subunit consisting of A 1 and A2 fragments linked through a single disulfide bond, and five B subunits, is activated by thiol, generating an active A 1 protein. Activated CTAl catalyzes the transfer of ADP-ribose from NAD to G,,, which, in the presence of GTP, stimulates adenylyl cyclase catalytic unit, increasing the conversion of ATP to CAMP. Model is from Tsai et al. (27).

Rabbit anti-bovine brain sARF I1 antibodies react on immunoblots of bovine brain proteins with two soluble forms of ARF that behave electrophoretically like sARF I and sARF I1 and a membrane form of ARF, mARF (39). The antibodies do not react with a subunits of the heterotrimeric G proteins, G,, (transducin) from bovine retina, Go from bovine brain, or G,, and G,, from rabbit liver. Rabbit anti-sARF I1 antibodies react with -20-kDa ARF-like proteins in bovine, rat, and frog tissues (39). ARF appears to be present in highest concentrations in brain extracts. In rat brain, as in bovine brain, the predominant immunoreactive species is sARF 11. In peripheral tissues, the major immunoreactive species is sARF I. Anti-peptide antibodies detected ARF immunoreactivity in Saccharomyces cerevesiae and Dictyostehm discoidium, but not E . cob (38).Between 50 and 90% of ARF immunoreactivity was found in the soluble fractions of animal cells and tissues.

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JOEL MOSS AND MARTHA VAUGHAN

E. Developmental and Hormonal Regulation of ARF In rat brain, as in bovine brain, most of the ARF is cytosolic (39). Immunoreactivity showed two soluble ARF species, corresponding in electrophoretic mobility to bovine brain sARF I and sARF I1 (39). It should not be inferred that these are identical to sARF I and I1 purified from bovine brain cytosol. There is no increase with age in the protein corresponding to sARF I (39). On postnatal day two, the amounts of sARF I1 and sARF I in rat brain were equivalent. By the tenth postnatal day, sARF I1 was predominant. The relative increase in immunoreactive sARF I1 over sARF I was associated with an increase in ARF activity, quantified by its ability to stimulate cholera toxin ADP-ribosyltransferase activity, following partial purification by gel permeation chromatography from a soluble fraction (39). Some ARF mRNAs appear to be under hormonal control. Chronic corticosterone administration increases the amounts of mRNAs for ARF 1 and ARF 3 and increases A R F immunoreactivity in rat cerebral cortex (40). Consistent with possible physiological control of ARF mRNAs by corticosteroids, bilateral adrenalectomy decreases the amounts of mRNAs for ARF 1 and ARF 3.

F. Activation of E. coli Heat-Labile Enterotoxins by ARF Escherichia coZi heat-labile enterotoxins (LTs) are responsible in part for “travelers diarrhea” (41). The LTs include two serogroups, LT-I and LT-11, and two variants of the LT-I1 serogroup, LT-IIa and LT-IIb, have been analyzed in detail (1, 42-45). The LTs are similar to cholera toxin in that their effects on cells result from ADP-ribosylation of G,, (46-50). The LTs, however, do have some structural differences from CT. LT-I is most similar to cholera toxin in amino-acid sequence, enzymatic activity, and immunoreactivity, whereas LT-IIa and LT-IIb appear to represent a different branch of the toxin family tree (45, 51, 52). The A subunits of cholera toxin and all the LTs, however, share a significant degree of amino-acid homology (51, 52). Both purified bovine brain ARF and recombinant ARF synthesized in E . coli stimulate the ADP-ribosyltransferaseactivities of cholera toxin and the LTs (SO). In the presence of ARF, DMPC, and cholate, ADP-ribosylation of G,, by cholera toxin and LT-I is slightly greater than that observed with LT-IIa or LT-IIb. In all instances, the ARF effect is dependent on GTP (50). Like cholera toxin, the LTs catalyze the ADP-ribosylation of agmatine, a simple guanidino compound (SO). In the presence of ARF and GTP, but not with either alone, there is a significant increase in toxin-catalyzed ADPribosylagmatine formation (Fig. 3). SDS further enhances GTF- and ARFstimulated activity of all four toxins (Fig. 3).Significant differences in specific activities are observed when agmatine is used as the ADP-ribose acceptor.

55

ADP-RIBOSYLATION FACTORS

CT

LT-lh

LT-lla

LT-llb

FIG.3. Effects of ARF, GTP, and SDS on ADP-ribosylation of agmatine catalyzed by cholera toxin and E. coli heat-labile enterotoxins. Cholera toxin, and E . coli,heat-labile enterotoxins, LT-I, LT-Ila, and LT-IIb, were assayed for their ability to catalyze the ADP-ribosylation of agmatine in the presence of buffer (open bars), purified sARF I1 (a soluble ARF from bovine brain) (cross-hatched bars), GTP (double cross-hatched bars), or sARF I1 plus GTP (speckled bars). SDS was present as indicated. In the panels describing LT-IIa and LT-IIb, the presence of 103 on the ordinate indicates that the specific activities were multiplied by 1 8 to obtain numbers equivalent to those observed with cholera toxin. Data are from Lee et al. (50).

NAD:agmatine ADP-ribosyltransferase activities of cholera toxin and LT-I are over 100 times those of LT-IIa and LT-IIb. The differences in specific activities are evident both with and without ARF. It appears that the catalytic sites of LT-IIa and LT-IIb have a much more restricted substrate specificity and use “model” substrates with much lower efficiency than do cholera toxin and LT-I (50).

II. Structure of ADP-ribosylation Factors To identlfy the structural features of ARFs responsible for guanine nucleotide-binding, activation of cholera toxin, and reconstitution of yeast mutants, deduced amino-acid sequences of ARFs and ARF-like proteins were determined through the isolation of cDNA and genomic clones and the generation of cDNAs by reverse transcription and the polymerase chain reaction (53-61). At present, six mammalian, two yeast, and two Giardin ARF clones have been isolated (53-61). The mammalian ARF genes can be grouped into three classes based on deduced protein size and amino-acid sequence (Fig. 4) (58). Class-I ARFs, which include ARFs 1-3, contain 181 amino acids and differ from each other

56

JOEL MOSS AND MARTHA VAUGHAN

hARFl hARF3 bARF2 BARF4 hARF5 hARF6 y ARF hARFl hARF3 bARF2 hARF4 hARF5 hARF6 yARF hARFl BARF3 bARF2 hARF4 hARF5 hARF6 yARF

FIG. 4. Comparison of deduced amino-acid sequences of mammalian and yeast ARFs. All ARFs are compared to hARF 1 (55). Asterisks (*) indicate amino-acid identity with ARF 1, whereas hyphens (-) represent gaps introduced to optimize sequence alignment. Human ARFs 1, and 3-6 are abbreviated as hARF 1(55). hARF 3 (SS),hARF 4 (SS), hARF 5 (58),and hARF 6 (58), respectively. Bovine ARF 2 and yeast ARF 1 are indicated as bARF 2 and yARF I, respectively. Not shown in the figure are deduced amino-acid sequences for bovine (53) and rat ARF 1 and rat ARF 3 (Price et al., unpublished data), which are identical to those of the human counterparts. Data are, in part, from Tsuchiya et al. (58).

only near the amino and carboxy termini (Fig. 4) (58).Class-I1 ARFs (ARFs 4 and 5) contain 180 amino acids and differ from Class-I ARFs near the amino terminus and in the carboxy half of the proteins (Fig. 4).The single known member of Class I11 (ARF 6) has only 175 amino acids and differs from ClassI and -11ARFs most extensively near the amino terminus and in the carboxy half of the protein, but there are differences in amino-acid sequence throughout the protein. Separation of the mammalian ARFs into three classes based on similarities in size and amino-acid sequence is supported by phylogenetic analysis of the ARF coding region nucleotide sequences. In this scheme, yeast ARF is clearly differentiated from the six known mammalian proteins (58). The division of mammalian ARFs into three classes is also evident when coding region nucleotide and deduced amino-acid sequences are compared (Table I). Class-I ARFs are 95-96% and 80-84% identical in deduced aminoacid and nucleotide sequences, respectively (58). Class-I1 ARFs (human ARFs 4 and 5), are only 79-802 identical in amino acid sequence to Class-I

57

ADP-RIBOSYLATION FACTORS

6

5

4

3

2

1

ARF 2b

cDNA

ARF-Specific Probes

FIG. 5. Hybridization of rat brain poly(A)+ RNA with ARF-specific cDNA and oligonucleotide probes. Rat brain poly(A)+ RNA was hybridized with a coding region cDNA probe generated from a bovine retinal ARF 2 clone (ARF 2h) and oligonucleotide probes specific for ARFs 1 to 6. Data are from Moss et d.(69).

TABLE I COMPARISONS OF ARF-DEDUCED AMINO-ACID AND CODING-REGION NUCLEOTIDE SEQUENCES" hARFl hARFl bARFl hARF2 hARF3 hARF4 hARF5 hARF6 CPSl yARF

91 79 84 67 75

68 69 64

bARFl

hARF2

hARF3

hARF4

hARF5

hARF6

CPSl

yARF

100

96 96 -

80 80 80 79

80 80 80 79 90 65 65 64

68 68 69 68

67 67

80 84 68 73 69

96 96 95

-

77 77 77 76 72 69 65 65

59

-

68 66

80 68 71 64 63 66

-

71 73 66

68 65

77 60 60 67

64 64 89 60

68 68 64 64 99

0 Comparisons of the deduced amino-acid sequences are above the diagonal, and those of nucleotide sequences are compared below the diagonal. Comparisonsare expressed as percentage identity. Sources of the ARF sequences are noted in Fig. 4. Data are from Tsuchiya et 01. (58). Abbreviations are listed on page 47.

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JOEL MOSS AND MARTHA VAUGHAN

ARFs, and 64% identical to Class-111 ARF; however, they are 90% identical to each other. At the nucleotide level, the similarities are less obvious. ClassI1 ARFs are 67-75% identical to Class I and 60-65% identical to Class 111, but they are only 77% identical to each other. Human ARF 6 (Class 111)is 60-69 and 64-69% identical to ARFs 1-5 in coding region nucleotide and deduced amino-acid sequences, respectively. Despite the differences in deduced amino-acid sequences among Classes I, 11, and 111, all ARFs contain the conserved amino-acid signature sequences believed to participate in guanine-nucleotide binding and GTP hydrolysis (62). The phosphate-binding/GTP hydrolysis region (GX,X,X,X,GK; amino acids 24-30 in human A R F 1;Fig. 4) differs from other ras-like GTP-binding proteins in the presence of aspartate at position X, (63-65). In the ras proteins, this substitution, or the presence of almost any amino acid other than glycine at this site, is associated with a marked reduction in GTPase activity (62). One of the sequences involved in the coordination of Mg2+, believed by analogy to ras to be involved in the binding of the GTP p and y phosphates, bears more similarity to the 01 subunits of the heterotrimeric G proteins than to the cognate regions of other members of the ras superfamily of -20 kDa proteins (e.g., ras, rap, rho, rac, ral, rub) (62). This region, DVGGQ (amino acids 67-71 in human ARF 1; Fig. 4), is conserved among all of the ARFs. Similarly, the CAT sequence (amino acids 159-161 in human ARF 1; Fig. 4), which has been implicated in interaction with the guanine ring, is identical to a sequence in the 01 subunits of the heterotrimeric G proteins. It differs from those in the ras superfamily, in which only the alanine is present (62).The NKXD sequence (amino acids 126-129 in human ARF 1; Fig. 4) is believed to participate in binding of the guanine ring. In some members of the ras superfamily, such as rac, this sequence is TKXD (62). The ARFs are highly conserved among mammalian species, and between mammalian species and lower organisms, such as Saccharomyces cerevesiae and Giardia lamblia (53, 58, 59, 61, 62). In Saccharomyces cerevesiae, two ARF genes have been described, encoding two proteins that are 96% identical (61).There is an ARF-like gene (termed arl) in Drosophila melanogaster; the deduced amino-acid sequence i s -55% and -70% identical to the mammalian and yeast ARFs, respectively (66). The arl gene, encoded by a 1.05kb mRNA, was essential for viability. Use of one of two potential initiation codons gives rise to an open reading frame of 540 bp, encoding a 180 aminoacid protein of -20 kDa. The ARL protein reacted with an antibody made against a peptide representing amino acids 22 to 35 of ARF 1. It bound guanine nucleotides and, in contrast to ARFs, hydrolyzed GTP. It did not exhibit ARF activity in a cholera toxin ADP-ribosyltransferase assay and did not suppress ARF mutations in yeast (66). It thus appears that the ARF

ADP-RIBOSYLATION FACTORS

59

family includes related proteins similar in sequence but devoid of cholera toxin-stimulating activity. Among mammalian ARFs, human ARF 1 is 100% identical in deduced amino-acid sequence to rat and bovine ARF 1, and human ARF 3 is 100% identical to rat ARF 3 (53,55; S. R. Price, M . S. Nightingale, J. Moss and M. Vaughan, unpublished data). Human ARF 6 differs from a chicken ARF pseudogene only at position 158 where serine replaces threonine (58, 67). Conservation of amino-acid sequence is associated with conservation of coding region nucleotide sequence. Human ARF 1 nucleotide sequence is 91% identical to that of bovine ARF 1; those of human ARF 6 and the chicken pseudogene are 89% identical (58, 67) (Table I). This degree of identity is somewhat remarkable for cross-species comparison to a pseudogene. Yeast ARF 1is 65-77% and 60-67% identical to the mammalian ARFs in deduced amino-acid and coding region nucleotide sequences, respectively (53, 58) (Table I). ARF 2 mRNA, although present in bovine and rat tissues, has not been detected to a significant extent in human or monkey tissues by Northern analysis, by amplification of ARF-specific domains using the polymerase chain reaction, or by screening of human cDNA libraries (68; 1. M. Serventi, E. Cavanaugh, J. Moss and M. Vaughan, unpublished observations). Some ARFs, thus, may either diverge greatly in nucleotide sequence or be expressed in a tissue- or developmentally-specific manner. Expression of ARF 2 in rat brain, for example, appears to be developmentally regulated (39). There is considerable conservation of both mRNA size and sequence among the mammalian ARFs. A coding region probe excised from a bovine retinal ARF-2 cDNA with EcoRI and PvuII hybridized with multiple bands in poly(A)+-RNA from human, bovine, and rat tissues (68) (Fig. 5). ARFspecific hybridization was obtained by using specific oligonucleotide or cDNA probes under conditions of high stringency. In hybridization with rat brain poly(A)+-RNA, it was found that ARF 1corresponds to a 2.1-kb band, ARF 2 to one of2.6 kb, ARF 3 to bands at 3.8 and 1.3kb, ARF 4 to one at 1.8 kb, ARF 5 to a band at 1.3 kb, and ARF 6 to bands at 4.2 and 1.8 kb (Fig. 5) (69). Based on gene structure, it appears that the two mRNA species observed for ARF 3 result from the use of alternative polyadenylation sites (70). The human genes for ARFs 1and 3 have five exons (70, 71), four introns, and similar positions of exon-intron boundaries; a similar structure has been observed for the bovine ARF-2 gene (Fig. 6) (72). For all three Class I ARFs, exon 1 is not translated, exon 2 contains the GX,X,X,X,GK regions, exon 3, the DVGG, and exon 5, the CAT; the NKQD is divided, with NKQ in exon 4 and the aspartate in exon 5. For ARFs 1 and 3, the promoter regions are consistent with those of housekeeping genes and contain neither a TATA nor

60

JOEL MOSS AND MARTHA VAUGHAN GLDMQK

DVGG

NKQ

D CAT

FIG. 6. Structural comparison of human and bovine class I ARF genes. Exons and introns of the bovine ARF 2 (bARF 2), human ARF 1(hARF l),and human ARF 3 ( U R F 3) genes are indicated by boxes and solid (-) or broken ( I / )lines, respectively. All three genes are drawn to scale, with -//- indicating deletions in introns. Darkened boxes represent coding regions, with letters above the bARF 2 coding exons indicating, in single letter code, those amino acids believed to be involved in guanine nucleotide-binding and GTP hydrolysis (phosphate binding). Nucleotides shown below the non-coding exons in the 3'-untranslated regions indicate potential polyadenylation signals. Data are from Lee ct 01. (74, Serventi et al. (72), and Tsai et at. (70).

a CAAT box (70, 71).In both genes, there are potential Spl sites (GC boxes). Consistent with the promoter structure is the evidence from primer extension analysis and S1 and mung-bean nuclease mappings that there are multiple transcription initiation sites (70, 71).

111. Functions of ADP-ribosylation Factors in Animal Cells Although ARF proteins were initially identified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin in uitro, it is unclear what role they have in toxin action on cells and there is only very limited evidence that they influence endogenous ADP-ribosylation in animal cells (73). ARFs purified fiom soluble and membrane fractions of bovine brain have no effect on the activity of NAD:arginine transferases purified from turkey erythrocytes (J. Moss and S. J. Stanley, unpublished observations). Recently, however, four ADP-ribosyltransferasesthat modify non-muscle actin were purified fiom rat-brain extract and used to assess the effects of two soluble ARFs purified from rat brain by the procedure described earlier for bovine brain (73). Each of the enzymes incorporated a maximum of -1 mol of ADP-ribose per mole of G actin; F actin is apparently not a substrate. ADP-ribosylation of G actin prevented its polymerization. The enzymes had similar K,s for NAD (12-31 pM) and pH optima of -8, but different abilities to modify certain proteins, different responses to four phospholipids, and different responses to sARF I and sARF I1 (73). sARF I stimulated activity of transferase 11, inhibited transferase 111, and had no effect on the

ADP-RIBOSYLATION FACTORS

61

other transferases, whereas sARF I1 activated transferases I and IV, inhibited 111, and had no effect on transferase 11. These observations, albeit intriguing, are somewhat puzzling as there is no indication that GTP was present in the transferase assays, although ARF activation of toxin requires GTP (or an analog). ARF proteins have been identified as components of the non-clathrincoated vesicle system of the Golgi complex that transports newly synthesized proteins in eukaryotic cells (74).In NIH 3T3 cells, immunoreactive ARF is largely localized in the cytoplasmic surface of cis-Golgi membranes (60). Affinity-purified antibodies used in these studies were prepared against a peptide representing a sequence in bovine ARF 1 that is identical in mammalian ARFs 1 to 5 and has only one difference (leucine vs. valine) in ARF 6 (60).

Saccharomyces cerevisiae has two ARF genes encoding proteins of 181 amino acids that are 96%identical (61). ARF 1 represents -90% of the total ARF protein, ARF 2 only -10%. Disruption of the ARF 2 gene caused no detectable alteration in phenotype, whereas disruption of ARF 1 resulted in slowed growth, cold sensitivity, a defect in the secretion of invertase, and sensitivity to fluoride at concentrations that do not kill wild-type cells, perhaps due to the major reduction in ARF content. The effects of ARF-1 gene disruption were overcome by increasing the expression of ARF 2. They were also overcome in spontaneous revertants, some of which contained a functioning ARF-1 gene resulting from recombination with the ARF-2 gene. Overexpression of ARF also interfered with growth (61).It is possible that the inhibition of maturation of Xenopus oocytes produced by microinjection of ARF is related to this kind of effect (75).Deletion of both ARF genes was lethal (61). Such mutants could be rescued by the products of human ARF-1 and ARF-4 genes, from which it may be inferred that functions of human and yeast ARFs are, at least to some extent, conserved (76). In homogenates of RINm5F cells, a -20-kDa protein(s) that binds GTP on nitrocellulose blots was found almost entirely in the cytosol (13,000 x g, 30 min supernatant) (77). After incubation of permeabilized cells with GTPyS, the amount of the 20-kDa GTP-binding protein (and also others) in the particulate was markedly increased. GPP(NH)P (100 p,M) was somewhat less effective than GTPyS at the same concentration, and other nucleotides (GTP, GDP, GDPyS, ATP) were without effect. Further subcellular fractionation revealed that the increased binding was confined to plasma membrane- and Golgi-enriched fractions, whereas fractions enriched in lysosomes, mitochondria, and secretory granules contained no 20-kDa GTPbinding protein (77). Based on size, immunoreactivity, and ADP-ribosylation catalyzed by the C3 enzyme from Clostridium botulinum, which modifies certain rho pro-

62

JOEL MOSS AND MARTHA VAUGHAN

teins, rho, rub 3, and kreu 1were ruled out, and it was concluded that ARF is the GTP-binding protein that is translocated from cytosol to membranes in cells exposed to GTPyS (77). After electrophoretic separation, proteins in crude membrane and cytosol fractions from RINm5F cells were transferred to nitrocellulose and reacted with antibodies against ARF. Subsequently, the filter was stripped and used to assess GTP-binding. It is stated (77)that, after two-dimensional (as well as one-dimensional) electrophoresis, labeling by antibody and by GTP “coincided exactly”. In addition, distribution of ARF between the two fractions, and transfer of ARF from cytosol to membranes after exposure of permeabilized cells to GTPyS, was similar whether quantified by immunoreactivity or by GTP binding. Thus, there is clear circumstantial evidence of GTP binding, and that recombinant ARF immobilized on nitrocellulose also binds GTP (77). These observations may settle earlier disagreement about whether ARF immobilized on nitrocellulose does or does not bind GTP. Incubation of homogenates of PC12 cells with non-hydrolyzable GTP analogs resulted in accumulation of immunoreactive ARF from cytosol in membrane fractions; GTP, GDP, GMP and ATP were inactive (78).ATPyS was partially effective, presumably as a result of formation of GTPyS, catalyzed by nucleoside diphosphate kinase from endogenous GDP. Cytosolic ARF also associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in the presence of GTPyS (78). Similarly, incubation of purified recombinant ARF 1 with GTPyS or GTP, but not GDP, and dimyrisoylphosphatidylcholine resulted in association of ARF with the phospholipid vesicles (79).This system provides additional evidence for the view that ARF (like other “small” GTP-binding proteins) is not activated by AlFg, which does not support phospholipid binding by GDP-liganded ARF (79). Data relevant to a role for ARF in the structure and hnction of Golgiderived coated vesicles have been accumulating rapidly. Many of these observations are brought together in the recent report of Serafini et al. (74)that ARF is a major component of these so-called COP-coated vesicles, with an estimated approximately three molecules of ARF per molecule of a-COP. aCOP (-160 kDa) is one of four relatively large (61-160 kDa) proteins associated with three smaller proteins in a cytosolic complex referred to as a “coatomer”(80).In the model of Serafini et a2. (74),ARFaGDP in the cytosol would bind to an ARF-specific nucleotide exchange protein in the donor Golgi membrane. The resulting ARFvGTP could then itself interact with the adjacent phospholipid membrane to provide a site for assembly of coatomers and formation of a COP-coated vesicle that would be stable until interaction of the ARF with a specific GAP (GTPase-activatingprotein) in the acceptor membrane. There, GTP hydrolysis would produce ARF-GDP, which would

63

ADP-RIBOSYLATION FACTORS

dissociate, followed by disassembly of the coat structure and release of coatomers separately into the cytosol. This model accounts, of course, for the effect of GTPyS that would stabilize ARF in the GTP-liganded conformation, preventing disassembly of the coat and fusion of the uncoated vesicle with the target membrane, and thus interrupting vesicular transport. In principle, a specific nucleotide-exchange protein could be replaced by an ARF-specific nucleoside diphosphate kinase, which could catalyze the synthesis of GTP from GDP bound to the ARF molecule (81). The mechanism of generating ARF-GTP, whether by nucleotide exchange or by kinase action, would appear to be of secondary importance. The specificity is, however, critical, so that only when the ARF molecule is at the correct membrane is it activated by conversion to ARF*GTPand enabled to initiate vesicle budding. Likewise, the specificity of the ARF-GAP interaction ensures delivery of the Golgi-derived vesicle and its contents to the proper site. As pointed out by the authors (74), it was not proven that the 20-kDa protein in the Golgi-derived vesicles that bound GTP immobilized on nitrocellulose blots was, in fact, the immunoreactive ARF that was also on the blots, and there is still some lack of agreement concerning GTP binding by ARF on membranes. Nevertheless, it seeins probable that the model proposed is fundamentally correct. It has the added virtue of providing potential roles for the several ARFs that have now been identified, perhaps more than six in a single species. Whether each of these functions with its own specific nucleotide exchange protein (or nucleoside diphosphate kinase) and GAP, or whether at least some of the ARFs utilize the same exchange protein and GAP, i. e., operate between the same donor and acceptor compartments but are expressed only in specific cells or at specific times in development and/or differentiation, remains to be determined.

REFERENCES 1 . J. Moss and M. Vaughan, Ado. Entymol. R e h t . Areas Mol. B i d . 61, 303 (1988). 2. L. Birnbaumer, J. Codina, R. Mattera, A. Yatani, N. Scherer, M.-J. Tor0 and A. M. Brown, Kidney Znt. 32 (Suppl. B),514 (1987). 3. A. G. Gilman, ARB 56, 615 (1987). 4. L. Birnbaumer, R. Mattera, A. Yatani, J. Codina, A. M. J. VanDongen and A. M. Brown, in “ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 225. Am. SOC.Microbiol., Washington, D.C., 1990. 5. D. R. Manning, in “ADP-ribosylating Toxins and G. Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 349. Am. Soc. Microbial., Washington, D.C., 1990. 6. R. Mattera, M. P. Craziano, A. Yatani, Z. Zhou, R. Graf, J. Codina, L. Birnbaumer, A. G. Gilman and A. M. Brown, Science 243, 804 (1989). 7. P. Bray, A. Carter, C. Simons, V. Guo, C. Puckett, J. Kamholz, A. Spiegel and M. Nirenberg, PNAS 83, 8893 (1986).

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8. M. UI,in “ADP-ribosylating Toxins and G. Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 45. Am. SOC.Microbiol., Washington, D.C., 1990.

9. Y. Kaziro, in “ADP-ribosylating Toxins and G. Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 189. Am. Soc. Microbiol., Washington, D.C., 1990. 10. M. I. Simon, M. P. Strathmann and N. Gautam, Science 252,802 (1991). 11. D. Cassel and Z. Selinger, PNAS 74, 3307 (1977). 12. R. A. Kahn and A. G. Gilman, JBC 259, 6235 (1984). 13. D. L. Bums, J. Moss and M. Vaughan, JBC 258, 1116 (1983). 14 D. L. Bums, J. Moss and M. Vaughan, JBC 257, 32 (1982). 15. C. Van Dop, M. Tsubokawa, H. R. Bourne and J. Ramachandran, JBC 259, 696 (1984). 16. K. Enomoto and D. M. Gill, J. Supramol. Struct. 10, 51 (1979). 17 K. Enomoto and D. M. Gill, JBC 255, 1252 (1980). 18. H. Le Vine LII and P. Cuatrecasas, BBA 672, 248 (1981). 19. K. Enomoto and T. Asakawa, Biomed. Res. 3, 122 (1982). 20. M. 0. Pinkett and W. B. Anderson, BBA 714, 337 (1982). 21. L. S. Schleifer, R. A. Kahn, E. Hanski, J. K. Northup, P. C. Sternweis and A. G. Gilman, JBC 257, 20 (1982). 22. D. M. Gill and R. Meren, JBC 258, 11908 (1983). 23. R. A. Kahn and A. G. Gilman, JBC 259,6228 (1984). 24. R. A. Kahn and A. G. Gilman, JBC 261, 7906 (1986). 25. S.-C. Tsai, M. Noda, R. Adamik, J. Moss and M. Vaughan, PNAS 84, 5139 (1987). 26. D. M. Gill and J. Coburn, Bchem 26, 6364 (1987). 27 S.-C. Tsai, M. Noda, R. Adamik, P. P. Chang, H.-C. Chen, J. Moss and M. Vaughan, JBC 263, 1768 (1988). 28. D. A. Bobak, S.-C. Tsai, J. Moss and M. Vaughan, in “ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 439. Am. SOC. Microbiol., Washington, D.C., 1990. 29. J. Moss and M. Vaughan, JBC 252, ,2455 (1977). 30. J. Moss and M. Vaughan, PNAS 75, 3621 (1978). 31. J. B. Trepel, D -M. Chuang and N. H. Neff, PNAS 74, 5440 (1977). 32. J. Moss, S . J. Stanley, P. A. Watkins and M. Vaughan, JBC 255, 7835 (1980). 33. N. J. Oppenheimer, JBC, 253, 4907 (1978). 34. M. Noda, S.-C. Tsai, R. Adamik, J. Moss and M. Vaughan, BBA 1034, 195 (1990). 35. D. A. Bobak, M. M. Bliziotes, M. Noda, S.-C Tsai, R. Adamik and J. Moss, Bchem 29,855 (1990). 36. 0. Weiss, J. Holden, C. Rulka and R. A. Kahn,JBC 264, 21066 (1989). 37. S.-C. Tsai, R. Adamik, J. Moss and M. Vaughan, Bchem 30, 3697 (1991). 38. R. A. Kahn, C. Goddard and M. Newkirk, JBC 263, 8282 (1988). 39. S.-C. Tsai, R. Adamik, M. Tsuchiya, P. P. Chang, J. Moss and M. Vaughan, JBC 266,8213 (1991). 40. R. S . Duman, S. M. Winston, J. A. Clark and E. J. Nestler, J. Neurochem. 55, 1813 (1990). 4 1 . M. Field, Am. J. Clin. Nutr. 32, 189 (1979). 42. B. E. C . Guth, C. L. Pickett, E. M. Twiddy, R. K. Holmes, T. A. T. Gomes, A. A. M. Lima, R L. Guerrant, B. D. G. M. Franc0 and L. R. Trabulsi, Infect. Zmmun. 54, 587 (1986). 43. R. K. Holmes, E. M. Twiddy and C. L. Pickett, Infect. Immun. 53, 464 (1986). 44. B. E. C. Guth, E. M. Twiddy, L. R. Trabulsi and R. K. Holmes, Infect. Immun. 54, 529 (1986). 45. C. L. Pickett, E. M. Twiddy, B. W. Belisle and R. K. Holrnes, J. B u t . 165, 348 (1986). 46. J. Moss and S. H. Richardson, J. Clin. Inoest. 62, 281 (1978)

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47. D. M. Gill and S. H. Richardson, J. Infect. Dis. 141, 64 (1980). 48. J. Moss, J. C. Osborne, Jr., P. H. Fishman, S. Nakaya and D. C. Robertson, JBC 256, 12861 (1981). 49. P. P. Chang, J. Moss, E. M. Twiddy and R. K. Holmes, Infect. Zmmun. 55, 1854 (1987). 50. C.-M. Lee, P. P. Chang, S.-C. Tsai, R. Adamik, S. R. Price, B. C. Kunz, J. Moss, E. M. Twiddy and R. K. Holmes, J. Clin. Znoest. 87, 1780 (1991). 51. C. L. Pickett, D. L. Weinstein and R. K. Holmes, 1. Bact. 169, 5180 (1987). 52. C. L. Pickett and R. K. Holmes, in “Advances in Research in Cholera and Related Diarrheas” (R. B. Sack and Y. Zinnaka, eds.), Vol. 7, p. 165. KTK Sci. Publ., Tokyo, 1990. 53. J. L. Sewell and R. A. Kahn, PNAS 85, 4620 (1988). 54. S. R. Price, M. Nightingale, S.-C. Tsai, K. C. Williamson, R. Adamik, H.-C. Chen, J. Moss and M. Vaughan, PNAS 85, 5488 (1988). 55. D. A. Bobak, M. S. Nightingale, J. J. Murtagh, S. R. Price, J. Moss and M. Vaughan, PNAS 86, 6101 (1989). 56. L. Monaco, J. J. Murtagh, K. B. N-M, S.-C. Tsai, J. Moss and M.Vaughan, PNAS 87, 2206 (1990). 57. 2. Peng, I. Calvert, J. Clark, L. Helman, R. Kahn and H.-F. Kung, Biofactors 2, 45 (1989). 58. M. Tsuchiya, S. R. Price, S.-C. Tsai, J. Moss and M. Vaughan, JBC 266, 2772 (1991). 59. J. J. Murtagh, Jr., MI R. Mowatt, C.-M. Lee, F.-J. S. Lee, K. Mishima, T. E. Nash, J. Moss and M. Vaughan, JBC 267, 9654 (1992). 60. T. Stearns, M. C. Willingham, D. Botstein and R. A. Kahn, PNAS 87, 1238 (1990). 61. T. Stearns, R. A. Kahn, D. Botstein and M. A. Hoyt, MCBiol 10, 6690 (1990). 62. S. R. Price, A. Barber and J. Moss, in “ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 397. Am. SOC.Microbiol., Washington, D.C., 1990. 63. J. B. Gibbs, I. S. Sigal, M. Poe and E. M. Scolnick, PNAS 81, 5704 (1984). 64. J. P. McCrath, D. J. Capon, D. V. Coeddel and A. D. Levinson, Nature 310, 644 (1984). 65. R. W. Sweet, S. Yokoyama, T. Kamata, J. R. Feramisco, M. Rosenberg and M. Cross, Nature 311, 273 (1984). 66. J. W. Tamkun, R. A. Kahn, M. Kissinger, B. J. Brizuela, C. Rulka, M. P. Scott and J. A. Kennison, PNAS 88, 3120 (1991). 67. C. R. Alsip and D. A. Konkel, NARes 14, 2123 (1986). 68. M. Tsuchiya, S . R. Price, M. S. Nightingale, J. Moss and M. Vaughan, Bchem 28, 9668 (1989). 69. J. Moss, M. Tsuchiya, S. R. Price, S.-C. Tsai and M. Vaughan, 26th U.S.-Jpn. Cholera Con5 Kyoto, Japan in press (1990). 70. S.-C. Tsai, R. S. Haun, M. Tsuchiya, J. Moss and M. Vaughan, JBC 266, 23053 (1991). 71. C.-M. Lee, R. S. Haun, S.-C. Tsai, J. Moss and M. Vaughan, JBC 267, 9028 (1992). 72. I. M. Serventi, E. Cavanaugh, J. Moss and M. Vaughan, JBC in press (1993). 73. S. Matsuyama and S. Tsuyama, 1. Neurochem. 57, 1380 (1991). 74. T. Serafini, L. Orci, M. Amhedt, M. Brunner, R. A. Kahn and J. E. Rothman, Cell 67, 239 (1991). 75. T. D. Bahnson, S.-C. Tsai, R. Adamik, J. Moss and M. Vaughan, JBC 264, 14824 (1989). 76. R. A. Kahn, F. G. Kern, J. Clark, E. P. Gelmann and C. Rulka, JBC 266, 2606 (1991). 77. R. Regazzi, S . Ullrich, R. A. Kahn and C. B. Wollheim, BJ 275, 639 (1991). 78. M. W. Walker, D. A. Bobak, S.-C. Tsai, J. Moss and M. Vaughan, JBC 267, 3230 (1992). 79. R. A. Kahn, JBC 266, 15595 (1991). 80. M. G. Waters, T. Serafini and J. E. Rothman, Nature 349, 248 (1991). 81. P. A. Randazzo, J. K. Northup and R. A. Kahn, Science 254, 850 (1991).

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Molecular Biology in the Eicosanoid Field’ COLIND . FUNK Department of Pharmacology Division of Clinical Phannacology Vanderbilt University Nashuille, Tennessee 37232

I. Lipoxygenases . . . . . . . . . . . . . . A. 5-Lipoxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. 12-Lipoxygenase . . . . . . ...................... C. 15-Lipoxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... 11. Leukotriene & Hydrolase . . . . . 111. Prostaglandin G/H Synthase (Cy IV. Thromboxane-A Synth V. Prostaglandin-D Synthase .................................... VI. Prostaglandin-F Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Eicosanoid Receptors VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 69 74 78 81 83 86 87 89 89 93 93

Nearly 30 years have passed since the discovery of the biotransformation of arachidonic acid to prostaglandins ( I , 2). Since this original finding, the number of oxygenated arachidonic acid metabolites discovered has multiplied exponentially; collectively, these compounds are referred to as eicosanoids. For some of the eicosanoids, including thromboxane A,, prostacyclin, and leukotrienes, biological roles have been reasonably well delineated, whereas for other eicosanoids, like lipoxins and hydroxyeicosatetraenoic acids (HETEs), functions have not yet been elucidated (3-5). Eicosanoid involvement in the pathophysiology of inflammation and many other disorders has been the focus of research efforts worldwide. Since the biosynthetic pathways, biochemistry, and pathophysiological implications of prostaglandins and leukotrienes have been covered in detail in many excellent reviews (6-11) these topics are not addressed here to any great extent. Rather, I direct attention (see Fig. 1) to the most recent research on the molecular biology of the main enzymes and receptors involved in eicosanoid formation and signal transduction. Molecular biology made its main entrance to the eicosanoid field in 1987 when two groups simultaneously reported the cloning of the cDNA for leukotriene A, hydrolase (12,13).Subsequently, the field has expanded considerably to include many Abbreviations. PG, prostaglandin, LT, leukotriene, Tx, thromboxane, H(P)ETE, hydro(pero)xyeicosatetraenoicacid, FLAP, 5-lipoxygenase activating protein, COX, cyclooxygenase.

67 Progresa in Nucleic Acid Research and Molecular Biology, Val. 45

Copyright 0 1993 by Academic Press, Inc All rights of rrproductron In any form reserved

COOH

1

LEUKOTRIENE A,

mDRccAsE

PGGdH,

1

OH

\

OH COOH

FIG. 1. Metabolic transformation of arachidonic acid. Only those transformations discussed in the text are shown. Enzymes catalyzing the primary transformations are boxed.

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

69

interesting findings with the lipoxygenases, prostaglandin-synthesizing enzymes, and prostanoid receptors.

1. Lipoxygenases Lipoxygenases catalyze the incorporation of molecular oxygen into cis, cis-pentadiene-containinglipids to form the primary hydroperoxide reaction products (14). These enzymes contain a non-heme iron atom and they are widely distributed in the plant and animal kingdoms. Lipoxygenases differ in their specificity of molecular oxygen insertion. Within mammalian tissues, there are three major forms, the 5-, 12-, and 15-lipoxygenases, that catalyze the incorporation of dioxygen into positions 5, 12, and 15, of arachidonic acid, respectively.

A. 5-Lipoxygenase 5-Lipoxygenase (arachid0nate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes the first step in the formation of the biologically important leukotriene mediators. A hydrogen atom is abstracted &om position 7 of arachidonic acid and oxygen is inserted at C-5 to form 5-hydroperoxyeicosatetraenoic acid (15). A dehydrase activity of this enzyme catalyzes the subsequent rearrangement to the unstable allylic epoxide, Ieukotriene (LT) A, (16,lfl. The enzyme was first found in polymorphonuclear leukocytes (PMN) and was purified to homogeneity from human and porcine PMN and rat basophilic leukemia cells (18-21). To clone the human 5-lipoxygenase cDNA, polyclonal antibodies to the enzyme were used to immunoscreen a hgt-11 lung cDNA library (22).Purified human enzyme was also subjected to proteolytic cleavage with the lysine-specific protease from Achromobacter lyticus to generate peptide sequence information. Several clones were plaque-purified and one clone having a 0.4-kb insert, when sequenced, matched a 5-lipoxygenase peptide sequence. Using the 32P-labeled 0.4-kb insert, two clones were isolated from a human hgt-11 placenta cDNA library. From the sequence of clone pl5BS (excluding a 51-bp artifact), it was possible to deduce the complete primary sequence of 5-lipoxygenase (22).The cDNA sequence encodes a mature protein of 673 amino acids (674 including the initiator methionine) with a molecular weight of about 78,000. Although Ca2+ and -4TP are required for maximal activity, potential binding sites could not be predicted with high certainty from the primary sequence. The human 5-lipoxygenase cDNA was also cloned from a dimethyl sulfoxidedifferentiated HL-60 cell library using oligonucleotide probes based on peptide data from the purified human leukocyte enzyme (23). The placentaderived and HL-60 sequences were identical. Human 5-lipoxygenase is significantly related to other lipoxygenases

70

COLIN D. FUNK

(Table I). It is nearly identical to the rat 5-lipoxygenase (24) and it displays about 40% identity to other mammalian lipoxygenases (25-29) and 27% to the plant homologues (30-33). There are no significant homologies with other known proteins, with the exception of a short segment found in lipoprotein and hepatic lipases within the so-called interface binding domain. At present there are at least 10 cloned lipoxygenase sequences, and it has become increasingly evident that there is a core region in the central portion of the molecule that is highly conserved that probably cmtains the iron-binding site (Fig. 2). The motif His-X,-His-X,-His-XI,-His-X,-His is present in all sequences; included within this region are several interspersed conserved acidic and basic residues. There is a second region with another conserved histidine and glutamine residue, closer to the carboxy terminus and found in all 10 sequences, and a third region with a histidine, found in mammalian but not in plant lipoxygenases (Fig. 2). Mutagenesis studies have been carried out with 5-lipoxygenase to examine the role of many of these conserved residues with respect to enzyme activity (34-36). The results are summarized in Table 11. His-367, His-372 within the main core region and His550 in the second region are essential to enzyme activity and may be required for coordination of the iron atom. Studies with the soybean enzyme indicate that there are at least four imidazole ligands (37);however, similar experiments have not been performed TABLE I COMP~RISONS OF LIPOXYCENASES Lipoxygenase" h5LX r5LX hl2LX p12Lx hl5LX rbl5LX sbLXl sbLX2 sbLX3 PeLX

Amino acids

Molecular weight

mRNA length (kb)

Identity to h5LX

Identity to hl2LX

Identity to hl5LX

674 670 663 663 662 662 838 865 857 851

78,097 77,982 75,764 75,047 74,809 75,424 94,260 97,260 96,851 97,743

2.7 2.6 2.4 3.4 2.8 2.7b 3.0b 3.0b 3.0 3.0b

-

42 39

39 38 65 86 81 26 26 27 27

92 42 40 39 39 26 27 28 26

65 65 61 26 27 28 27

ah5LX, human 5-lipoxygenase (22, 23); r5LX. rat 5-lipoxygenase (24); hlBLX, human platelet 12-lipoxygenase (25, 26, 70); p12LX. porcine leukocyte 12-lipoxygenase (27); hl5LX, human 19lipoxygenase(28),rblBLX, rabbit 15lipoxygenase (29);sbLX, soybean lipoxygenase isozymes 1, 2, and 3 (3&32); peLX, pea seed lipoxygenase (33). Sequences retrieved from GenBank were analyzed by the software of Intelligenetics. bSizes of mRNA were not quoted in the original references and are estimates based on the cDNA clones obtained.

362

h5LX r5LX h12LX p12LX hl5LX rbl5LX sbLXl sbLX2 sbLX3 peLX

351 351 344 345 344 344 482 511 502 506

h5LX r5LX hl2LX pl2LX hl5LX rbl5LX sbLXl sbLX2 sbLX3 peLx

427 427 420 421 420 420

367

390

372

399

401 401 394 395 3 94 394 532 561 552 556 439 439 432 433 432 432

542 542 532 533 532 532 681 710 701 705

561 561 551 552 551 551 700 729 720 724

FIG. 2. Lipoxygenase sequence homology. The alignment of ten lipoxygenase sequences (one-letter amino-acid code) is shown in the putative iron-binding core region (top), and in two regions closer to the carboxy terminus (bottom). Conserved histidine, acidic and basic residues are boxed. The five histidines on top are numbered with respect to the 5-lipoxygenase (see Table 11). An arrow points to an essential glutamine residue. h5LX, human 5-lipoxygenase (22, 23); r5LX, rat 5-lipoxygenase (24);hllLX, human platelet 12lipoxygenase (25, 26, 70); p12LX, porcine leukocyte 12-lipoxygenase (27);hl5LX, human 15-lipoxygenase (28):rbl5LX, rabbit 15lipoxygenase (29);shLX, soybean lipoxygenase ixozymes 1, 2, and 3 (30-32);peLX, pea seed lipoxygenase (33).(Figure is adapted from Ref. 36.)

TABLE I1 SITE-DIRECTEDMUTACENESIS OF HUMAN~LIPOXYGENASE Enzyme activity"

A

Mutation

B

C

++

H362C H362K H362N H362Q H362S H367A H367C H367K H367N H367Q H367S H372N H372Q H372Sb H390A H390N H390Q H390S H399A H399N H399Q H399S H432A H432N H550A HSSOL H550N H5W D358N V374A v374s E376Q Y383F Y383S F393S F393W M435L Q557E COOH-6'

-

+ +++

++

+ +++ -

-

Lo

+

+++

+

++

-

++ +++

+++ ++ -

-

+ + +++ + + + +

+++

The results of in uitm mutagenesis studies from three research groups: (A) Refs. 35 and 38; (B) Ref. 34; and (C) Ref. 36. Enzyme activity is classified as: +, 67-1004 of native enzyme, , 34-66% of native enzyme; +, 1 4 3 % of native enzyme; -, no detectable activity; Lo, enzyme activity was detectable but expression was very low for accurate Q

++

++

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

73

with mammalian Iipoxygenases. It is also clear that Glu-358 and Gln-552, as well as the six residues at the carboxy-terminal are essential to activity and/or structural features of the molecule. It remains to be tested whether these mutant enzyme forms still bind iron. The 5-lipoxygenase enzyme has been expressed in several heterologous systems including baculovirus-infected insect cells, an osteosarcoma cell line, COS-M6 cells, Escherichia coli and yeast (38-43). The enzyme appears to retain all the essential characteristics of the native leukocyte enzyme in these systems, such as possession of both 5-lipoxygenase and leukotriene (LTA) synthase activities and dependence on Ca2+ and ATP for maximal activity. Studies performed &th the baculovirus/insect cells expressed enzyme have verified the presence of an iron atom (1.1 mol iron per mol enzyme) that is tightly bound and that is released after exposure of the enzyme to oxygen (44).No iron could be detected in the inactivated enzyme. Also, leukocyte microsomal and cytosolic proteins are not required for maximal enzyme activity as previously thought, but rather, they cause an enhanced stability of the enzyme in the in vitro assay (39). The 5-Lipoxygenase enzyme resides in the cytosol of unstimulated cells. Upon activation of leukocytes, H L 6 0 cells or rat basophilic leukemia cells with the calcium ionophore A23187 (calcimycin), the enzyme is translocated to a membrane fraction (45, 46). The activation is associated with a burst in leukotriene formation, and the enzyme found in the membrane apparently has undergone a “suicide-type’’ inactivation. The translocation step as well as the formation of leukotrienes in intact cells is blocked by the drug MK886 that binds to an 18-kDa membrane protein (47, 48). This small protein has been termed “5-lipoxygenase activating protein” (FLAP) (49). Experiments with osteosarcoma cells transfected with a 5-lipoxygenase expression vector revealed that these cells cannot make 5-lipoxygenase products from endogenous substrate when activated (40). However, when a vector expressing FLAP was co-transfected, activated cells synthesized leukotrienes; this synthesis was blocked by MK886 binding to FLAP (49).

TABLE I1 (Continued) assessment of activiv. Mutants are classifiedby the 1-letter code, e.g., H362C = histidine at position 362 mutated to cysteine. Expression studies were carried out in E. coli and in bacul-s-infected insect cells. H372S was originally reported to possess activityin the bacdovirus system (38). The explanation for this may have been an accidentalcontaminationof native5-Iipxygenasebaculwirus stockwith the mutant virus. c COOH-6 refers to deletion of the six carboxy-terminal residues.

74

COLIN D. FUNK

There is no solid evidence for a direct interaction of FLAP with 5lipoxygenase, so FLAP’S mechanism of action remains obscure (50). Interestingly, FLAP co-localizes with 5-lipoxygenase in leukocytes, HL60 cells, and several myeloid-derived cell lines, yielding credence to their mutual involvement in leukotriene formation (51). In the rat alveolar macrophage, the 5-lipoxygenase apparently resides in an active form within membrane fractions of unstimulated cells (52). It is possible, therefore, that some cell-type variability exists in the mechanism of formation of leukotrienes. Northern-blot data has revealed the presence of a predominant 2.7-kb mRNA transcript in human leukocytes, lung, placenta, and differentiated HL60 cells (22, 23). A band of similar size was observed in samples from rat cells and tissues, including basophilic leukemia cells, macrophages, lung, intestine, stomach, and epidermis (24). However, the presence of infiltrating white cells or resident mast cells could not be excluded as the source of 5lipoxygenase mRNA in some of these organs. A well-documented evidence for non-myeloid 5-lipoxygenase expression comes from immunohistochemical studies that located the enzyme in porcine pancreatic acinar cells (53). The human 5-lipoxygenase gene has been isolated from a series of genomic bacteriophage and cosmid clones (54).The gene spans at least 82 kb of DNA and is divided into 14 exons. There was one gap in the very large (>26 kb) third intron, so the actual size of the gene is unknown. The gene resides on human chromosome 10 distinctly separated from other lipoxygenase genes (55). All the members of the lipoxygenase gene family appear to be organized in the same exon/intron format (54-56). However, the 5-lipoxygenase gene appears to be much larger than other lipoxygenase genes. The site of transcription initiation was mapped to a thymidine residue 65 bp upstream of the start codon. The promoter region is rich in G-C boxes and lacks canonical TATA and CCAAT motifs. The sequence -179 to -56, which includes 5 tandemly arranged G-C boxes, is essential for transcription and from gel-shift analysis the Spl transcription factor can bind within this region (57). There was also evidence for two negative and two positive upstream transcription regulatory elements. Although there are many interesting features within the 5-lipoxygenase promoter region, very little is known about potential regulatory agents, and cis- and trans-acting factors that could control the expression of this gene and the resultant formation of leukotrienes. Obviously, there is still much to be learned in this area.

B.

12-Lipoxygenase

12-Lipoxygenase (arachid0nate:oxygen 12-0xidoreductase, EC 1.13. 11.31),found in platelets, was the first discovered mammalian lipoxygenase,

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

75

in 1974 (58).It catalyzes the stereospecific incorporation of molecular oxygen onto C-12 of arachidonic acid to yield 12(S)-hydroperoxyeicosatetraenoicacid (12-HPETE). 12-HPETE is reduced by cellular glutathione peroxidases to 12 (S)-hydroxyeicosatetraenoicacid (12-HETE). Although 12-HETE is synthesized in greater amounts than the cyclooxgenase-derived arachidonate metabolite, thromboxane A,, by various types of platelet stimuli, little is known about the biological relevance of this pathway. The enzyme is cytosoliclocalized, and it appears to undergo a translocation to a membrane fraction when (rat) platelets are activated by thrombin (59). 12-Lipoxygenase has been characterized from other sources, including porcine and bovine leukocytes, bovine tracheal epithelial cells, human uterine cervix, and human and mouse epidermal cells (60-66). The uterine cervix and epidermal cell enzymes are located in the microsomal fraction, and they may or may not be distinct from cytochrome P-450 enzymes (63-66). Much evidence has been obtained for the presence of 12-lipoxygenase isoforms (67), the two major forms being classified into “platelettype” and “leukocyte-type” 12-lipoxygenases. 12-Lipoxygenase from porcine leukocytes has been purified to apparent homogeneity, and several peptide sequences have been determined (27). The cDNA for this enzyme was cloned by hybridization screening with two sets of oligonucleotides based on one peptide sequence (27). On the other hand, human platelet 12-lipoxygenase cDNA was isolated by a polymerase chain reaction (PCR) homology approach (25). Oligonucleotide primers derived from conserved regions of 5- and 15-lipoxygenaseswere used to amplify a short 0.26-kb fragment from reverse-transcribed platelet RNA (25). This short fragment was radiolabeled and used to screen a human erythroleukemia (HEL) cell library to obtain the complete cDNA. Both the porcine leukocyte (662 amino acids) and human platelet/erythroleukemia cell (663 amino acids) 12-lipoxygenases encode = 75-kDa proteins. They share 65% amino-acid identity. The leukocyte-type enzyme exhibits more relatedness to 15-lipoxygenase than to the platelet 12-lipoxygenase. Additionally, the two enzymes have different substrate specificities and product profiles (68). The leukocyte 12-lipoxygenase will metabolize both arachidonic acid and linoleic acid whereas the platelet enzyme metabolizes virtually no C-18 fatty acids. The former enzyme forms small amounts of 15-lipoxygenase products, but the platelet enzyme forms exclusively lS-H(P)ETE (43). More recently, a bovine-trachea epithelial-cell 12-lipoxygenase cDNA was cloned by use of a 15-lipoxygenase probe from human reticulocytes (69). Its sequence is 89%identical to the porcine 12-lipoxygenaseand 86%identical to human 15-lipoxygenase. The relatedness of epithelial cell 12-lipoxygenase and human 15-lipoxygenase has been confirmed further by the fact that they share antigenic cross-determinants (69). Both the bovine trachea

76

COLIN D. FUNK

epithelial and porcine leukocyte 12-lipoxygenases,as well as 15-lipoxygenase have a Cys-X3-Cys-X3-His-X3-His zinc-finger motif (amino acids 532-544) which is not found identically in the platelet 12-lipoxygenase or 5-lipoxygenase sequences. The significanceof this motif is unknown. Two distinct 12lipoxygenase cDNAs have not yet been obtained from the same species, so the molecular nature of 12-lipoxygenase isoforms is still not clearly established. Even less clear is the biological significance of different 12-lipoxygenase isoforms. Northern-blot studies have revealed the presence of a single platelettype mRNA transcript in HEL cells and platelets (2.4, 3.0, or 3.1 kb sizes have been reported by three different groups) (25, 26, 70).Phorbol ester treatment of HEL cells increases this mRNA about 2-3 fold (25, 26). This mRNA is in the low abundance category being present at about five copies/cell(71). The mRNA has not been detected in other human tissues such as lung, placenta, adrenal gland, and liver by Northern-blot analysis. However, using reverse transcriptase (€IT)-PCR analysis, platelet-type 12-lipoxygenase mRNA was detected in small amounts in most tissues, probably due to platelets remaining within these tissues (55). Platelet-type 12-lipoxygenase was definitely present in cultured human umbilical vein endothelial cells (passage 4) since platelets would not represent a source of contamination in the PCR assay (55). The leukocyte-type 12-lipoxygenasemRNA (3.4 kb) was detected in several porcine tissues by Northem-blot analysis (27). The greatest amount was found in leukocytes followed by substantial amounts in pituitary gland and lung. Small amounts were detected in jejunum and spleen. No cross-hybridization on RNA blots, using the human platelet and porcine leukocyte 12lipoxygenase probes could be observed (70). Immunohistochemical studies have provided direct evidence for the presence of leukocyte-type 12-lipoxygenase in the parenchymal cells of porcine anterior pituitary and in porcine neutrophils and monocytes (72, 73). Other tissues positive by Northern blot appeared to contain 12-lipoxygenaseonly in resident mast cells or infiltrating granulocytes (73). Both forms of 12-lipoxygenasehave been expressed in heterologous systems (E. coli, baculovirus-infected insect cells, COS cells, and human embryonic kidney 293 cells) (25, 26, 43, 7 3 ~ )In. E . coli, the platelet-type 12lipoxygenase has been expressed as a large = 100-kDa fusion protein that retains activity, using the pGEX2T vector (74).The fusion moiety is a 26-kDa glutathione S-transferaseattached to the amino terminus of 12-lipoxygenase, which allows efficient affinity purification on a glutathione-agarose column. Sf9 insect cells infected with a recombinant platelet-type 12-lipoxygenase baculovirus can generate milligram amounts of enzyme from lo8 cells (73~).

77

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

A sequence coding for six histidines included after the initiator codon provides a simple one-step purification by Ni2 -NTA-agarose affinity chromatography. The native platelet enzyme has not previously been purified to homogeneity by conventional protein purification procedures. The expressed enzyme is highly active [up to 10 Fmol 12-H(P)ETE/mg p r o t e i d l 0 min incubation], is not dependent on any cofactors, and can be stored at -70°C in the presence of glycerol with retention of 7040% activity after 4 weeks. In oitro mutagenesis experiments show that the same histidine residues essential to catalytic activity for 5-lipoxygenase (His367, His372, and His550) are also essential to platelet-type 12-lipoxygenase activity (His360, His365, and His540) (73a).The nine N-terminal residues are also important to structure and/or function of the enzyme, as activity is abolished in their absence. The recombinant platelet-type 12-lipoxygenase synthesizes exclusively 12H(P)ETE from arachidonic acid. However, change of three adjacent amino acids Lys-416/Ala-417/Val-418 to Gln-416/11e-417/Met-418 (residues found in 15-lipoxygenases) enables the mutant recombinant enzyme to alter the specificity of 0, insertion to arachidonate substrate. 15-Lipoxygenase products [ lSH(P)ETE]account for = 25% of the total products synthesized from arachidonate by the mutant enzyme. These changes are discussed further in Section 1,C. The human platelet-type 12-lipoxygenase gene (12-lipoxygenase gene-1) has been isolated on several overlapping genomic cosmid and bacteriophage clones (55).The gene spans = 17 kb and exodintron junctions are analogous to the human 5-lipoxygenase gene. The putative promoter region has a “TATA-like” box (position -96 with respect to the ATG start codon), no CCAAT motif, and three G-C boxes in a 578-bp region from the ATG start. There were two sites that could account for the potential induction of transcription by phorbol esters at positions -531 and -511. However, this region requirw further characterization with transfection experiments using promoter-reporter gene constructs. Part of a second “12-lipoxygenase” gene (exons 7-8 and 10-11) was isolated on a series of overlapping bacteriophage clones from the same human genomic libraries (55). This gene (12-lipoxygenase gene-2) is most likely a pseudogene (Fig. 3) because: (i) it is not expressed in several tissues by RT-PCR analysis; (ii) it lacks two of the histidines in the translated exon-8 sequence, one of which is critical to lipoxygenase activity; (iii) there is no evidence for an exon-%like sequence; and (iv) there is an abnormal splice acceptor site before exon 8. The 12lipoxygenase gene-2 is = 85%identical to the 12-lipoxygenase gene-1 within exon sequences and both genes are located in human chromosome 17 (55).A leukocyte-type 12-lipoxygenase gene has yet to be found in humans. +

78

COLIN D. FUNK

394 hl ZLX-2

SWILKLRFQL

FIG.3. Alignment of the translated 12-lipoxygenase gene sequences within the putative iron-binding region coded by exons 8 and 9. hl2LX-1, human 12-lipoxygenase gene-1, corresponding to amino acids 344-394 of plateletlerythroleukemiacells enzyme (25, 55); h12LX-2, human 12-lipoxygenase gene-2. h12LX-2 is almost certainly a pseudogene (see Section I, B for details) and the amino-acid sequence, therefore, is hypothetical. h12LX-2 is lacking conserved residues (boxed) found in all lipoxygenases (see Fig. 2). The circled tyrosine residue would replace a conserved histidine that is essential to 5- and 12-lipoxygenaseactivity and would give rise to a nonfunctional enzyme (C. D. Funk, unpublished). An exon 9 sequence not present in h12LX-2 is denoted by dashes.

C. 15-Lipoxygenase 15-Lipoxygenase was first purified and characterized from rabbit reticulocyte-rich anemic blood cells (75). It was subsequently purified and characterized from rabbit and human polymorphonuclear leukocytes and human eosinophil-rich leukocytes (76-78). The enzyme is in the cytosol like other lipoxygenases, and there are no reports of translocation upon cellular stimulation as for the 5- and 12-lipoxygenases. The 15-lipoxygenasepathway is also present in lung, airway epithelial cells, and keratinocytes. Little is known about the function of t h i s pathway in these various cell types. The most plausible role for the reticulocyte enzyme lies with its ability to oxygenate lipid membranes directly (79, 80). Thus, it probably participates in the intracellular organelle degradation during maturation to the erythrocyte and possibly in the reconstruction and remodeling of the plasma membrane. The complete .15-lipoxygenase cDNA has been cloned from a human reticulocyte cDNA library using oligonucleotide probes based on the purified eosinophil sequence (28). The open reading frame encodes a 662aminoacid polypeptide with a molecular weight of 74,600. The cDNA from human tracheal epithelial cells encodes an identical protein (81).The rabbit reticulocyte 15-lipoxygenasecDNA has also been cloned and is 81% identical to the human homologue (29). Relationships to other lipoxygenases are shown in Table I. The rabbit cDNA contains a long 3’ untranslated region with ten copies of the tandemly arranged repeat sequence C,RC,TCTTC,AAG. This region has been proposed to be involved in the translational regulation of the mRNA. An issue yet to be resolved is whether there are isoforms of 15-lipoxygenase. One group has reported the purification of two 15-lipoxygenases from human leukocytes (82). Both forms have the same molecular mass (74 kDa), appear to be equally active with different substrates, and have the

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

79

same N-terminal sequence as the cloned human reticulocyte 15-lipoxygenase. The two forms were clearly separated by cation-exchange chromatography and their relative amounts dif€ered markedly between leukocytes of normal donors and from an individual with eosinophilia (82). Immunological evidence from another group has established the relatedness of 15-lipoxygenases from different cell-types (83). Specific localization experiments tend to indicate 15-lipoxygenase immunofluorescence only within the eosinophil population of leukocytes (83). In human trachea, 15-lipoxygenase is in both basal and ciliated epithelial cells. Mature reticulocytes, but not early or late stage reticulocytes, contain the enzyme (83). In another study, evidence for 15-lipoxygenase expression was found in both the cytosol and nuclei of human myometria (84). Southern-blot genomic DNA analysis apparently revealed the presence of a single copy gene both in rabbits and humans (56, 81). A major 2.7-kb mRNA for 15-lipoxygenase has been detected in human leukocytes, tracheal epithelium, and reticulocytes by Northern-blot analysis (81). Interestingly, the 15-lipoxygenase mRNA and protein have been detected in macrophage-rich human atherosclerotic lesions by in situ hybridization and immunofluorescence experiments (85). Moreover, these products co-localized with epitopes for oxidized low-density lipoprotein, and it has been proposed that the macrophage-induced modification of low-density lipoprotein may be mediated by 15-lipoxygenase, thus contributing to atherogenesis (86). In a more recent study, it was found that normal monocyte/macrophage populations do not contain the 15-lipoxygenase mRNA, but when induced with interleukin-4 they express 15-lipoxygenase mRNA and protein that is capable of oxygenating membrane lipids (87).Interferon y and hydrocortisone inhibit the interleukin-4 induction. These findings provide an interesting link between 15-lipoxygenase function and the immune/inflammatory response. The human 15-lipoxygenase has been expressed in E . coli, osteosarcoma cells, and COS-M6 cells (43, 88, 89). The recombinant enzyme retains the original characteristics of the protein isolated from eosinophil-rich leukocytes. Both 15-H(P)ETE and 12-H(P)ETE are synthesized in a ratio of 9 : 1. A comparison of three 12-lipoxygenase and two 15-lipoxygenase sequences showed that there are four conserved differences between the two lipoxygenases. A study to investigate the parameters controlling the specificity of molecular oxygen insertion at position 15 or 12 was carried out (go), based on mutagenesis of the conserved sequences. Change of a single methionine at position 418 of human 15-lipoxygenase to valine (found in 12-lipoxygenases) caused a shift in product profile with nearly equal amounts of 12-HETE and 15-HETE being formed. Further change of the two adjacent residues (Gln-416 to Lys-416, and lle-417 to Ala-417) to platelet 12-lipoxygenase resi-

80

COLIN D. FUNK

dues induced an even greater shift in the formation of 12-HETE accounting for over 90% of the products. Data from previous studies with the reticulocyte and soybean lipoxygenases (91)and these mutagenesis results provide the basis for a model that explains at least some of the parameters that control positional specificity of oxygen insertion (Fig. 4). Arachidonate fits into a substrate-binding cleft and the side-chain at position 418 of 15-lipoxygenase, and residues immediately adjacent control the deepness of penetration into the binding pocket. The different side chains in this region of 12-lipoxygenase apparently allow a deeper binding that consequently shifts the substrate relative to the site of hydrogen atom abstraction (C-10 for 12-lipoxygenation and C-13 for 15lipoxygenation). The complete rabbit 15-lipoxygenase gene and promoter sequence have been elucidated (92). Like the 5- and 12-lipoxygenase genes of humans, it is divided into 14 exons having the same exodintron format (Fig. 5). The gene is 8-kb long, the shortest of the mammalian lipoxygenase genes characterized so far. Transcription initiation starts 27-bp upstream from the ATG start codon. The 150-bp upstream segment of the start acts as a functional promoter in both erythroid and non-erythroid cell-lines. There is a “TATAlike” motif 30 bp upstream from the transcription initiation site, but no

,, Human enzyme

: 13

1,’

15-Lipoxygenase

‘...‘. 10

7

2

-;)‘Human

platelet

FIG.4. Determinants of positional specificity of molecular oxygen insertion in human 12and 15-lipoxygenases. The active site (&), presumably involving the non-heme iron atom, abstracts a hydrogen atom primarily at position C-13 in 15-lipoxygenase and exclusively at position C-10 in human platelet 12-lipoxygenase.The positioning of the arachidonate substrate relative to the active site could he controlled to some extent by residues 416-418 in 15lipoxygenase and 416-417 in platelet 12-lipoxygenase. R1 is CH3(CH2)4, Rz is CHzCH = CH(CHa),COOH. (Data are based on Refs. 73~2,90 and 91.)

81

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD I 3 kb

h5LX

I

I

h 12LX- 1

I

It

I

II

>82kb

> I

14

17 kb

14

1

h l2LX-2

. .

1

14

FIG. 5. Exonlintron structures of lipoxygenase genes. h5LX, human 5-lipoxygenase gene

(M), hl2LX-1, human 12-lipoxygenase gene-l which codes for platelet 12-lipoxygenase (55), h12LX-2, human 12-lipoxygenase gene-2, part of a probable pseudogene apparently lacking exon 9 (55),rblSLX, rabbit 15-lipoxygenase gene (56). Horizontal lines indicate gene loci and the vertical bars depict exons. Exons 1 and 14 are labeled for each gene, except for h12LX-2 where exons 7,8, 10, and 11 are shown. All genes are compared to the same scale except for five introns within the 5-lipoxygenase gene.

CCAAT-box is present in the correct position. The human 18lipoxygenase gene, like the 12-lipoxygenase genes-1 and -2, has been localized to chromosome 17 (55).It remains to be established if these three genes are linked to each other.

II. Leukotriene A, Hydrolase Leukotriene A, hydrolase (EC 3.3.2.6)catalyzes the conversion of leukotriene A, (LTA,) to the leukocyte chemotactic compound leukotriene B,. The hydrolysis reaction catalyzed by this enzyme is very specific for the LTA, epoxide substrate. The enzyme was originally found and purified from human PMN (93)but it has become increasingly evident that the enzyme is quite ubiquitous (94). cDNA clones encoding LTA, hydrolase have been isolated by immunoscreening with a polyclonal antibody, and by hybridization screening with a single 48-mer based on a purified PMN peptide sequence, from human lung and placenta, and spleen cDNA libraries, respectively (12, 13). The cDNAs from both sources were identical, covering an open reading fi-ame of 1833 bp encoding a mature 610 amino-acid protein of molecular weight = 69,000. More recently, cDNAs have also been isolated from mouse spleen and rat kidney cDNA libraries (95, 96). All three sequences are more than 93% identical. The 2.2-kb mRNA is widely distributed in virtually all tissues tested, although the highest relative amounts appear to be found in lung and leukocytes. From quantitative immunological

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COLIN D. FUNK

studies, small intestine epithelial cells are one of the richest sources of the enzyme (97). After the original cloning of the human sequences, data-base searches indicated that LTA, hydrolase is a unique epoxide hydrolase, unrelated to liver microsomal epoxide hydrolases and other proteins (12, 13).Two years later, however, a weak homology between LTA, hydrolase and aminopeptidase was reported (98). Independent observations revealed that LTA, hydrolase has a consensus motif found in zinc metalloproteinases (aminopeptidase N, thermolysin, endopeptidase, and collagenase are some of these enzymes) (99,100) (Fig. 6). After these observations, two groups experimentally verified the presence of a zinc atom in leukotriene A, hydrolase and found that the enzyme is bifunctional, displaying aminopeptidase activity, as well as LTA, hydrolytic activity (100-104). The endogenous peptide substrate, if one exists, has yet to be identified. The aminopeptidase homology lies within the central portion of the molecule (residues 233-340) adjacent to the most hydrophobic region (residues 190-205) of the protein. This hydrophobic region contains a cysteine at position 199, originally postulated to be a potential active-site residue within the LTA, substrate binding domain, LTA, hydrolase undergoes covalent inactivation during catalysis. However, from in uitro mutagenesis studies Cys-199 does not appear to be essential to activity. LTA, hydrolase with full activity is expressed in E . coli (95, 105).Mutagenesis studies with the expressed human and mouse enzymes have revealed that His-295, His-299, and Glu-318 constitute the three Zn atom coordination points (Fig. 6) since the mutated proteins were basically lacking zinc (106). Both LTA, hydrolase and aminopeptidase activities were reduced to nearly undetectable levels in the mutant proteins. The results verified the essential nature of the zinc atom to catalysis, but not to enzyme structural characteristics, as chromatographic and immunological properties were largely unaffected in the mutant proteins. LTA, hydrolase is inhibited by the aminopeptidase inhibitor bestatin and by the angiotensin-convertingenzyme inhibitor captopri1(104,107).Studies

human LTA 265 F P Y G G M E N P C L - T F V T P T L L A - G D - - - - - - K S - L S ~ I ~ I S ~ ~ G ~ 325 ~~T~H~~G mouse LTA 265 t t t * t * . f t * t _ t t * * * t t t * - * * - - - - - - * * * * * * * * * H * * * ~ ~ * * ~ * ~ * ~ * * * * ~ * * * *325 *~~~****~ rat LTR 264 t t * f * t t ~ t t t - t * f * t t t f * - * ~ - - - - - - * ~ * ~ * * ~ ~ ~ ~ * ~ ~ ~ * * * * ~ * * *324 * * * ~ * * * * M I N O P E P 349 ~NA*A*~*wG*v*YRENS**FDPLSSSSS"R"***B*FAS*V* 418

FIG. 6. Comparison of leukotriene A4 hydrolases (12, 13, 95, 96) and human aminopeptidase N (176) within the highly conserved zinc-ion binding region. Identity to the leukotriene A4 hydrolase sequence is indicated by *. Caps for alignment are shown by dashes. The two histidines and glutarnic acid residues that constitute the three zinc-binding ligands are boldfaced.

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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with the latter drug indicate that the sulfhydryl group is an important determinant for inhibitory activity and that there is a differential susceptibility of the two activities for captopril. The fact that LTA, hydrolase is so widely distributed, yet the first enzyme (5-lipoxygenase) in the synthesis of LTA, is restricted to only a few tissues suggests that the alternative functional role (aminopeptidase) could be very important. These inhibitors could help define more clearly the biological role of LTA hydrolase and the significance of LTA, transcellular metabolism.

111. Prostaglandin G/H Synthase (Cyclooxygenase) The release of arachidonic acid from membrane glycerophospholipids by various stimuli leads in many cell types to the formation of prostaglandins via the enzyme prostaglandin G/H synthase (also known as prostaglandin endoperoxide synthase and commonly referred to as cyclooxygenase; EC 1.14.99.1)(10). This enzyme has been characterized most extensively from the ram seminal vesicle, its richest source. The enzyme apparently functions as a homodimer (subunit molecular weight of = 72 kDa). It is a glycoprotein in the endoplasmic reticulum that contains one heme group/subunit. The enzyme carries out the bisoxygenation of arachidonic acid to form PGG, (cyclooxygenase activity), as well as the subsequent reduction of the 15hydroperoxy moiety of PGG, to yield PGH, (peroxidase activity). PGG/H synthase has received widespread attention over the past two decades since it was discovered that this enzyme is the main target site for aspirin and nonsteroidal anti-inflammatory drug action (11, 108, 109). The cDNA for the cyclooxygenase from ram seminal vesicle was cloned independently by three groups in 1988 (110-112). It encodes for a 600aminoacid protein with a 24-residue signal peptide and four potential Nlinked glycosylation sites, three of which contain high-mannose carbohydrate (113). Adjacent to the signal peptide sequence is an EGF-like domain, and the aspirin acetylation site (Ser-530) is found close to the carboxy terminus (114, 115). Partial and complete cDNAs from human sources (human umbilical vein endothelial cell, platelet/erythroleukemia cell, embryonic lung fibroblasts) have been reported as well as the complete mouse sequence (116-119). The cyclooxygenases from the different species are closely related (= 90% identity) and have the same basic features as the ram seminal vesicle cyclooxygenase. Collectively, these sequences are referred to as cyclooxygenase-1 (COX-1). Although there were earlier indications for the presence of cyclooxygenase isoforms (120), it was not until recently that this was realized at the molecular level. The serendipitous finding of a second form of cyclooxygenase (COX-2) arose independently from two groups not working in the

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COLIN D. FUNK

eicosanoid field who were investigating the nature of immediate-early response genes induced by the Rous sarcoma virus (src) in chick embryo fibroblasts and by phorbol esters in Swiss 3T3 mouse fibroblasts, respectively (121, 122). When the primary sequences were scanned with nucleotide data bases, it was found that these primary response genes were = 60% identical at the amino-acid level with COX-1 and around 80% similar when conservative substitutions were taken into account. The COX-2 cDNA encodes a 603 (chick)/604(mouse)amino-acid protein. The major differences between the COX-1 and COX-2 forms are at the two termini (Fig. 7). At the N-terminus, a 17-aminoacid stretch within the signal peptide of COX-1 is not present in COX-2. In contrast, COX-2 contains an 18-aminoacid region at the carboxy terminus not present in COX-1. All the other major features found in COX-1, like the aspirin-sensitive serine residue, EGF-like domain,

4++H++& 1 2 3 4 5 6-9

10

1

mRNA 64 n t

ATTTA

AmA

2 kb

124 n t

H1s2093 1 I 390(heme Interactlon)

His295 3 7 4 Asn53 130

eF5 16

protein

N signal peptide

OH peroxidase homology

602 amino acids

NH EGF-like

Tyr371 peroxidase homology

604 amino acids

FIG. 7. Comparison of mouse cyclooxygenase genes, mRNAs, and proteins. Top: cyclooxygenase gene loci. The two mouse cyclooxygenase genes are compared on the same scale. Vertical bars represent exons. Middle: cyclooxygenase mRNA forms. The start of transcription is indicated by the arrow. The coding region is represented by the open box and the sizes of the 5’ and 3 untranslated regions are noted by the thin lines. COX-2 mRNA contains 11 copies of the sequence ATTTA, a sequence found in mRNAs that turn over rapidly. Bottom: mouse cyclooxygenase isoforms. COX-1 and COX-2 differ primarily at the two ends (darkened boxes). Other salient features (EGF-like domain, N-linked glycosylation sites, potential heme interacting histidine residues, catalysis-essential tyrosine, and aspirin-sensitive serine residues) are shared between COX-I and COX-2. Numbering begins with the first rnethionine. Mouse COX-1 numbering differs from the sheep COX-1 (602 v5. 600 amino acids). The latter enzyme has been studied extensively and the features depicted here are based on the sheep sequence.

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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homology to peroxidases, and residues essential to catalytic activity, are present in COX-2. There are five potential sites for N-glycosylation and the three sites experimentally verified to have carbohydrate within COX-1 are conserved. COX-1 mRNA has been found in numerous tissues from various species, and is present as a = 2.8-kb transcript. The mRNA can be increased by many factors, such as phorbol ester, interleukin-1, progesterone, platelet-derived growth factor, EGF, CAMP, and serum, to mention a few (123-129). Glucocorticoids, aspirin, and heparin-binding (acidic fibroblast) growth factor-1 decrease COX-1 gene expression (130-132). The glucocorticoid effect is complex, probably mediated at several levels, and is cell-type specific (130, 133-135). The COX-1 3’-untranslated region (750-915 bp) is highly conserved among different species (= 50-70%) and this region is potentially involved in the regulation of expression of the gene (117, 118, 136). COX-2 mRNA transcripts are larger (= 4 kb); they appear to be expressed in very low amounts under basal or normal nonproliferating conditions, and are expressed in a tissue-type restricted fashion (121, 122). EGF, forskolin, phorbol ester, and serum induce a large and rapid increase in COX-2 mRNA levels in mouse fibroblasts. Glucocorticoids reduce the cycloheximide-induced increase in COX-2 mRNA levels (137). Luteinizing hormone/human chorionic gonadotropin (LH/hCG) induces a 40-fold induction of cyclooxygenase enzyme in rat granulosa cells of preovulatory follicles (138). The increase was not associated with a change in the 2.8-kb COX-1 mRNA. The specific cyclooxygenase induced by the treatment was partially purified from these cells and the N-terminal sequence was determined (139). The sequence is 96% identical to the mouse COX-2 indicating that LH/hCG specifically induced COX-2. Current data suggest, therefore, that there are two forms of COX mRNA and protein, each encoded by separate genes (see below), the one (COX-I) being more ubiquitously expressed and subject to low-level regulation (usually 2-4 fold changes in mRNA levels) and the other (COX-2), found in nearly undetectable levels by Northern-blot analysis, that is highly regulatable by various factors. COX-1 from ram seminal vesicles and human platelet/erythroleukemia cells, as well as Swiss 3T3 fibroblast-derived COX-2, have been expressed in COS cells (116, 117, 119, 140). So far, there have been no reports of expression of the heme-containing COX protein in E . coli, but recently it has been seen to be expressed in a baculovirus/insect cell system ( 1 4 0 ~ Exten). sive in uitro mutagenesis experiments have been performed with the expressed COX-1 enzyme (117, 119, 141-143). Replacement of Ser-530 with alanine does not markedly affect either cyclooxygenase or hydroperoxidase activity, indicating that the aspirin-sensitive residue is not essential to catalytic activity or substrate binding (119).

86

COLIN D. FUNK

The mutant enzyme, however, did not undergo irreversible inactivation by aspirin. Replacement by asparagine, though, abolished cyclooxygenase activity. Apparently, acetylation of Ser-530 causes an interference with substrate binding by introduction of a bulky side-chain. Mutagenesis of all 13 histidines in COX-1 to glutamine revealed the essential nature of His-207, His-309, and His-388 (141). It is likely that the imidazole group of His309 is an axial heme ligand and that His-388 is the distal ligand (143). His-207 may interact with a heme carboxyl side chain via ionic linkage. Site-directed mutagenesis has also shown the essential nature of Tyr-385 to cyclooxygenase, but not to peroxidase, activity (142). This residue probably lies at the active site and forms a tyrosyl radical during catalysis which is associated with the suicide-inactivation of the enzyme but not with the initial hydrogen abstraction step (143). Prostaglandin G/H synthase is a very complex enzyme and there is still much to be learned about its structurelfunction properties. The human and murine COX-1 genes have been cloned (144,145). They share the same exodintron boundaries and are divided into 11 exons spanning about 22 kb of genomic DNA. Characterization of the mouse COX-1 promoter has been performed (145). The murine COX-2 gene has been cloned and characterized (140). It contains 10 exons and spans 8 kb. The COX-2 gene is lacking an exon found in COX-1 (exon 2) that encodes the hydrophobic leader sequence. The eight internal exons are highly conserved between the COX-1 and COX-2 genes, with junctions located in analogous positions (Fig. 7). A 1.0 kb sequence upstream from the transcription initiation site conferred phorbol ester and serum inducibility in transfection studies. The human COX-1 gene is in chromosome 9 (117). The chromosomal position of the COX-2 gene is unknown at present.

IV. Thromboxane-A Synthase Thromboxane-A synthase (EC 5.3.99.5) catalyzes the conversion of PGH, to thromboxane A2 (TXA,). The enzyme is a ferrihemoprotein that undergoes inactivation during catalysis (146). TXA, is a potent platelet aggregator and a vasoconstrictor of vascular and respiratory smooth muscle. It has been implicated in the pathophysiologic expression of several disorders such as myocardial infarction, stroke, and atherosclerosis (147). Thromboxane-A synthase cDNAs have been isolated from human platelet and lung reverse-transcribed RNA by PCR using oligonucleotide primers based on direct peptide sequences (148, 149). Additional clones were isolated by hybridization screening of a human lung cDNA library. The platelet and lung enzymes appear to have identical 534 (or 533; there are two adjacent Met residues at the N-terminus) amino-acid sequences (TX synthase-I, with M, of = 60,684).However, the lung may have an alternative

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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form, as cDNA clones lacking 163bp near the 3' end of the translated region were obtained. The deduced sequence of the shorter enzyme, TX synthase11, would have 460 amino acids with an M, of 52,408. Two distinct thromboxane-A synthase species have been detected previously in platelets and WI-38 lung fibroblasts (150, 151). It is possible, therefore, that the two forms arise from alternative splicing and/or a variable extent of glycosylation. There are five potential N-linked sites and a highly hydrophobic segment at the Nterminus that could serve to anchor the protein in the endoplasmic reticulum. A single 2.1-2.2 kb mRNA can be detected by RNA blot analysis in human platelet, HEL, and THP-1 (a human monocytic leukemia cell line) cells (148, 149).Phorbol esters increase its expression in HEL cells. Thromboxane synthase has certain features characteristic of cytochrome P-450s (Fig. 8). When aligned with these enzymes, it is evident that thromboxane synthase is related especially to the P-450s of family IIIA (== 35%), with human nifedipine oxidase (cytochrome P-450IIIA4)showing the highest similarity. P-450s have conserved Cys residue in the carboxy-terminalregion that is thought to be the proximal ligand for the heme moiety, as well as a helix-heme-helix structure (152). Both the cysteine residue (Cys-480) and the helical structure are conserved in thromboxane synthase-I but not in thromboxane synthase-11. A more detailed characterization of the structurelfunction properties of this enzyme awaits expression and in vitro mutagenesis experiments.

V. Prostaglandin-D Synthase Prostaglandin-D synthase (prostaglandin-H, D-isomerase, EC 5.3.99.2) catalyzes the transformation of the cyclooxygenase-derived intermediate PGH, to PGD,. This prostaglandin is a major arachidonate metabolite in the

TxSyn-I 391 L P Y L D M V I A E T L R M Y P P A F R F T R E A A Q D C E V L G Q R I P A G A P S P E TxSyn-11 391 ________________________________________-----P450-IIIA4 353 ME-----VN----LF-I-M-LE-VCKK-V-IN-MF--K-V-~IPSY---R--KY-TE-TxSyn-I 451 TFNPERFTAEARQQHRPFTYLPFGAGPRS~LGVRLGLLNKLTLLHLHKFRFQACPETQ TxSyn-I1 451 ------YRCS* ~ 4 5 0 - I I I A413 ~ K-L----SKKNKDNID-YI-T---S---NCI-M-FA-MNM--A-IR--QN-S-KP-K--TxSyn-I TxSyn-I1 P450IIIA4

511 VPLQLESKSALGPKNGVYIKIVSR* 473 I--K-SLGGL-Q-EKP-VL-VE--*

FIG. 8. Alignment of carboxy-terminal regions of human thromboxane synthases and human cytochrome P450IIIA4. The cysteine believed to be the proximal heme ligand is boldfaced. Dashes indicate identity to the thromboxane synthase-I. The end of the coding sequence is shown by *. (Adapted from Refs. 148 and 149.)

88

COLIN D. FUNK

brain, where it exerts several central actions related to sleep, regulation of body temperature, and modulation of pain response (153-154). PGD, is also a major metabolite of mast cells and spleen (155, 156). PGD, shows several pharmacological activities such as inhibition of platelet aggregation, pulmonary vaso- and bronchoconstriction, and peripheral vasodilation. A polyclonal antibody against rat brain PGD synthase was used to isolate cDNA clones encoding the enzyme (157). An open frame of 564 bp codes for a 188 amino acid protein with a calculated molecular weight of 21,232. The purified protein has a molecular weight of approximately 26,000 and can be deglycosylated to two smaller protein bands of 23 and 20 kDa. Consequently, it is believed that there are two N-linked glycosylation sites at Asn-51 and Asn-78. The first 20 amino acids probably act as a signal peptide. The enzyme is apparently associated with the rough endoplasmic reticulum and outer nuclear membranes but is not integral to the membrane since there are no strongly predicted membrane spanning domains and detergents are not required to solubilize the enzyme (158). cDNAs of glutathione-independent PGD synthase have been isolated from human brain (259). The complete cDNA encodes a 190-residue protein and there appears to be significant genetic heterogeneity as 16 differences were observed between two different overlapping cDNA clones. Southernblot analysis of genomic DNA indicate a single gene. Thus, the many differences suggest the presence of allelic variants. The human and rat aminoacid sequences are approximately 80% identical and they share the same N-linked glycosylation sites. Homology searches reveal that glutathioneindependent PGD synthase is a member of the lipocalin “superfamily”(260). This group comprises various small (160-190 residues) secretory proteins that share a common feature for binding and transport of small hydrophobic molecules. There are at least 16 members of this superfamily. PGD synthase is most similar to a,-microglobulin (29% identity; 52% similarity) and, together with the y-chain of C8 complement component, these proteins form a distinct cluster in the phylogenetic organization of this superfamily. Based on analysis of other members of this superfamily, it is postulated that PGD synthase displays a similar tertiary structure consisting of an a-helix and an eight-stranded hydrophobic anti-parallel P-barrel, which probably binds the PGH, substrate. Cysteine at position 65, within this pocket, has been postulated to be an active-site residue. Upon Northern-blot analysis, a single 850-900 bp transcript is detected in human and rat brain and in rat spinal cord and epididymis, but not in rat spleen, liver, and kidney. These results tend to indicate that brain PGD synthase is different from “spleen-type” PGD synthase or g1utathione-Stransferases, which can also catalyze the formation of PGD,. Based on several immunological and biochemical observations mast cells contain the spleen-type PGD synthase (162). The relatedness of the brain and spleen/

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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mast cell types awaits the determination of the primary sequence of the latter.

VI. Prostaglandin-F Synthase Prostaglandin-F synthase (EC 1.1.1.188), an aldo-keto reductase, converts the endoperoxide intermediate PGH, to PGF,, and also converts PGD, to 901, IlP-PGF, on different active sites within the same molecule (162). This enzyme was initially purified to apparent homogeneity from bovine lung; it is a monomeric protein of M , 30,500 (163). Using two sets of short oligonucleotides of sequences based on aminoacid sequences, several cDNA clones were isolated from a bovine lung cDNA library (164). The clone with the longest insert (1.2 kb) had a 969 bp open reading frame coding for a 323 amino-acid polypeptide with a calculated molecular weight of 36,666. Sequence analysis revealed a strong similarity (65%) with human liver aldehyde reductase and rat lens aldose reductase and 77% similarity to frog lens p-crystallin, suggesting that these proteins form a gene family. The latter proteins share some of the enzymatic and cofactor features of PGF synthase but they do not share significant crossreacting antigenic determinants. Most striking is the exact homology of PGF synthase and p-crystallin in a 21-aminoacid stretch (residues 178- 198) with the exception of Pro-195. The dual functionality of PGF synthase is partially lost by mutagenesis of Pro-195 to Val-195 (found in crystallin) which suggests that this residue may be related to one of the active sites of the enzyme (165). By immunological studies and RNA blot analysis PGF synthase appears to be selectively distributed in lung and spleen (162). A 1.4-kb transcript has been observed in these tissues. The immunostained protein was localized primarily in alveolar interstitial cells, to a lesser extent in nonciliated epithelial cells of the bronchiole, and in both white and red pulp of the spleen (most likely in histiocytes and/or dendritic cells).

VII. Eicosanoid Receptors Prostaglandins and leukotrienes are known to transduce their signals via receptors linked to G-proteins. Distinct receptors for thromboxane A,, prostacyclin/PGE,, PGE (possibly three subtypes referred to as EP1, EP2, and EP3), PGF,, , LTB,, and LTD, present in various tissues have been described. Some of the receptors have been solubilized and partially purified (166-168) and the human platelet TXA, receptor has been purified to apparent homogeneity (169). The purified thromboxane receptor exhibited a very broad band on a SDS-polyacrylamide gel centered at = 57 kDa. Enough protein was obtained for partial sequence information. Oligonucleotide probes were used to screen a human megakaryocytic cell

90

COLIN D. FUNK

line (MEG-01)cDNA library (170).A partial length clone was obtained that, when sequenced, was found to encode the carboxy half of a putative Gprotein linked receptor. This clone was then labeled and used to screen a human placenta library. One full-length (= 2.9 kb) clone contained extensive 5’ and 3’ noncoding regions and a 1029 bp open reading frame coding for a 343 amino-acid protein of M , = 37000. The predicted sequence displays the characteristics of seven transmembrane G-linked receptors including two Nlinked glycosylation sites (Asn-4 and Asn-16) in the putative extracellular amino terminal tail (29 residues), conserved Cys residues in extracellular loops 1 and 2 (Cys-105 and Cys-183), and several other conserved residues within transmembrane regions, with the exception of the Asp residue found in transmembrane 3, known to be essential for receptors with small aminecontaining ligands (171). The sequence has a very short predicted third intracellular loop (27 residues). This portion of the molecule could possibly couple to the G-protein (Gs or larger G-protein) responsible for interacting with phospholipase C and causing subsequent changes in calcium ion flux (172, 173). The coding region for the thromboxane receptor is extremely G + C-rich (70%). It was nearly impossible to isolate this cDNA from placenta or platelet reverse-transcribed RNA under normal conditions of denaturation (94-95°C) with Taq polymerase (C. D. Funk, unpublished). However, a shifi of the denaturation temperature to 98°C and use of Vent polymerase (New England Biolabs, Beverly, MA) enabled amplification of the complete cDNA. The thromboxane receptor has been expressed in Xenopus oocytes. Apparently, it can couple with endogenous signal transduction components to elicit a calcium-activated C1- channel recorded by electrophysiological measurement (170).Binding studies have been performed with COS-1 cell membranes transfected by thromboxane receptor cDNA using the ligand S-145 (170). We have also shown high affinity binding of the thromboxane antagonist SQ-29548 in human embryonic kidney 293 cells and membranes transfected with thromboxane-receptor cDNA with maximal binding of 2-3 pmol/mg protein (C. D. Funk, unpublished). This level of expression is at least 5-10 times higher than in platelet membranes. On a per-cell basis assuming a 10% transfection efficiency, we estimate = 106 binding sites/ transfected cell as compared to = 1300 sites present on a platelet (174). Northern-blot analysis revealed the presence of a 2.8-kb band in the MEG-01 cell line, placenta, and lung. The mRNA is probably in the lowabundance category, based on the reported long exposure time (12 days) and amount of poly(A)+ RNA loaded (20 p,g) to see detectable signals. An approach to the isolation of other eicosanoid receptor genes by homology screening was taken, with the assumption that these receptors are related in primary structure (1 75).Primers were designed from the third and

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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sixth transmembrane domains of the thromboxane receptor and used to amplify a 418-bp clone from reverse-transcribed total RNA from mouse lung. This clone had 78% homology to the thromboxane receptor in the corresponding region. This clone was 32P-labeled and used to screen mouse lung, and subsequently, mouse mastocytoma cell cDNA libraries. Overlapping clones ML64 and MP660 were isolated; they showed 38% relatedness to the human thromboxane receptor (49% within transmembrane domains). The open reading frame encoded a 365-aminoacid polypeptide with an estimated M , of 40,077. There are two potential N-glycosylation sites, one at the amino terminus (Asn-16) and one in the second extracellular loop (Asn-193), and there are nine serine and threonine residues at the carboxy terminus that are potential phosphorylation sites. In order to identify the endogenous ligand of this receptor, clone MP660 was expressed in COS-1 cells and membranes of the transfected cells were prepared to perform binding studies. [3H]PGE, bound specifically and with high affinity (Kd = 2.9 nM) and binding was inhibited by unlabeled prostaglandins in the rank order E, = E, > iloprost (a PGI, analog} > F,, > D,. In Chinese hamster ovary cells stably expressing the receptor, PGE, and a subtype specific agonist induced a potent inhibition of forskolin-induced CAMP accumulation (175). Taken together, these results indicated that a mouse PGE (subtype 3; EP3) receptor had been isolated. Northern-blot analysis revealed the presence of 2.3-kb band in several tissues where PGE, exerts biological effects and where binding sites have been reported, with kidney showing the highest mRNA levels. A 7.0-kb band was also observed in kidney, uterus, brain, and P815 inastocytoma cells. We have also used the homology screening approach to isolate a related eicosanoid receptor (C. D. Funk, L. Furci and G. A. FitzGerald, unpublished). Using a 0.3-kb thromboxane receptor fragment that covers most of the transmembrane 5-7 region of this receptor, a 1.4-kb cDNA clone (PGQ) encoding a 402-aminoacid putative receptor was isolated from a human erythroleukemia cell cDNA library. PGQ has two potential N-linked glycosylation sites (Asn-8 and Asn-25) and is extremely rich in basic (mainly arginine) and serine residues in the predicted third intracellular loop and the carboxy-terminal tail. At present, the endogenous ligand of the PGQ receptor is unknown. It is clear from sequence alignments that the thromboxane, PGE, and PGQ receptors form a related subfamily within the superfamily of G-protein coupled receptors superfamily (Fig. 9). They are about 40% identical to each other, and the areas of highest homology are in transmembrane 7 and part of transmembrane 4 extending 10 residues into the second extracellular loop. The molecular biology of eicosanoid receptors is still in the infancy stage

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COLIN D. FUNK t

1

t

TXR

MSPCDPLNLSLAO~A’ITCAAPWVPNTSAVPPSGASPALPIFSMTLGAVSNLLALALLGR~RRRSATT 72 t t I I I I1 I M W P f f i S S ~ P C F R P T N I T L E E R R L I A S ~ F ~ S F C W G L A R - - S62 S

PGE

64

PGQ

t I I I II I MA~APEHSAEAHSNLSSTTDDCGSVSVAFPITMMVTGFVGNALAM-LLVSRSYRRRESKRKKS

2

PGQ

3

FLLFVASLLATDLAGHVIPGLVLRLrPAGRAPAGRAPAGG-----ACHFLGCMVFFGLCPLLLGCGMAVERCVGV 139 II I II I I I l l I Ill II I I I I FLTFLCGLVLTDFMLLVTGTIWSQHAALFEWHAVDPGCRLCR~~IFFGLSPLLLGAAMASERYLI134 II I It I I I l l I Ill II Ill1 F L L C I O W L A L T D L V G Q L L T S P W I L W L S Q R R W E Q L D P S G R ~ ~ ~ L ~ G L S S L L V A S136 ~~RA~I

TXR

PGE

-

4

PGQ TRPLLHAARVSVARARLALAAVAAVALAVALLP~VGRYE~YPGTWCFIGLGPPGGWR---QALLAGLFA208 t

I

II

I

II

Ill

Ill1

I I I I I I

I

I

TXR T R P F S R P A V A S Q R R L ~ ~ G L L P L L G V G R197G I I II I I1 I l l Ill1 I I I I I I 1 * I PGE -RAPHWYASHMRFRATPVLLGVWLSVLAFALLPVLGVGRYSVQh’PGTWCFISTGPAGNETDPAREPGSVAFA 207

PGQ TXR

PGE 6

PGQ GGSRSSGSARRARAHDVEMVGQLVGIMWSCI~SPULVLVALAV----------GGWSSTSLQRPLFLAVR 338 I

I I I I I I

I I I I

I

TxR ------AAQQRPRDSEVEMMAQLLGIMWASVCWLPLLVFIAQTVLRNPPAMSPAGQLSRTTE-XELLIYLR PGE

I

295 I I1 I l l 1 II 1 I __---QSSAQ~RITPETAIQLMGIMCVL~CWSPLLIMMLKMIFNQMSVEQCKTQMGKEKECNSFLIAVR 309

I

-7

PGQ LASWNQILDPWVYILLRPAVLRQLLRLLPP~G~GGPAGLGLTPSAWEASSLRSSRHSGLSHF

TxR

PGE

I IIIIIIIIIII II VATWNQILDPWWILFRRAVLRRLQPRLSTRPRSLSLQPQLMRSGLQ I IlIt1l111II II LASLNQILDPWVYLLLRKILLRKFCQIRDHTNYASSSTSLPCPGSSALMWSD2LER

402 343 3 65

FIG. 9. Comparison of prostanoid receptors contained within a subfamily of the seven transmembrane G-protein coupled receptor superfamily PGQ, a putative human prostanoid receptor (C. D. Funk, L. Furci and C . A. FitzCerald, unpublished); TxR, human thromboxane receptor (170); PGE, mouse prostaglandin E (subtype 3) receptor (175). The putative seven transmembrane regions are overlined. Vertical bars indicate identity between the three sequences. Potential glycosylation sites are indicated by *.

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and it is obvious that much remains to be learned about eicosanoid signalling transduction, mechanisms of receptor desensitization, and residues important for ligand binding. In addition, many members of this family including the leukotriene receptors remain to be cloned.

VIM. Concluding Remarks The molecular biology of the eicosanoids has come a long way since its inception in 1987. It is clear, however, that much remains to be learned in this area. For instance, a clear biological role for the 12- and 15-lipoxygenases needs to be defined. Availability of the genes for these enzymes should allow targeting strategies to discern potential functions. Clear information on the regulation and significance of leukotriene formation by 5lipoxygenase, 5-lipoxygenase activating protein, and leukotriene A, hydrolase is still lacking. The recent discovery of the second form of cyclooxygenase has reopened a wide interest in the academic and pharmaceutical communities in prostaglandin research. The differential regulation of the two cyclooxygenase forms and the development of selective isoform inhibitors will be areas of intensive research. Cloning of the different members of the eicosanoid receptor family should facilitate our unravelling of the mechanisms involved in the action of this interesting class of arachidonate metabolites known as eicosanoids. ACKNOWLEDGMENTS These studies were supported in part by NIH grants HL46017 and HL30400 and a Research Career Development Award HL02710.

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Mammalian 6Phosphofructo-2 kinase/fructose-2,6bisphosphatase: A Bifunctional Enzyme That Controls Glycolysis’ GUYG. ROUSSEAUAND

LOUISHUE HonnAne and Metabolic Research Unit Department of Biochemistry and Cell Biology University of Louvain Medical School and International Institute of Cellular and Mokcukzr Pathology Brussels, Belgium

I. PFK-S/FBPase-2, A Bifunctional Enzyme That Synthesizes and Degrades Fructose 2,6-Bisphosphate . . . . . . . . . . . . . . . A. Liver PFK-2lFBPase-2 .................................. B. PFK-2/FBPase-2 Isozymes . . . . . . . . . . . . . . . . . . C. Short-Term Control of PFK-2/FBPase-2 ......................... 11. Molecular Characterization of PFK-&/FBPase-2 Isozymes . . . . . . . . . A. Characterization of Several PFK-Z/FBPase-Z mRNAs . . . . . . .

A. Glucocorticoids . . . . . .

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

C. Insulin and Glucagon . D. Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V. Concluding Remarks . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 100 101 104 106 106 110 116 119 119 120 121 123 123 124

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Glycolysis is the process by which glucose is converted first to pyruvate and then either to lactate or to ethanol and CO,. It is an amphibolic pathway, as it can provide the cell with energy from glucose catabolism and can also

Abbreviations: FBPase-2, fructose-2,6-bisphosphatase(EC 3.1.3.46); Fru-6-P, fructose 6phosphate; Fru-2,6-P~,fructose 2,6-bisphosphate; GRE, glucocorticoid response element; IRE, insulin response element; ORF, open reading frame; PCR, polymerase chain reaction; PFK-1, 6-phosphofructo-1-kinase (EC 2.7.1.11); PFK-2, 6-phosphofructo-2-kinase (EC 2.7.1.105); PKA, CAMP-dependent protein kinase; PKC, protein kinase C. 99 Progress in Nucleic Acid Research

and Molecular Biology, Vol. 45

Capyngfit 5 1993 by Academic Press, Inc.

All r i s t s of reproduction in any form reserved.

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GUY G . ROUSSEAU AND LOUIS HUE

serve an anabolic hnction by yielding C , precursors for the synthesis of amino acids, fatty acids, and cholesterol. The first committed step of glycolysis is the conversion of fructose 6-phosphate (Fru-6-P)and ATP into fructose l,6-bisphosphate catalyzed by 6-phosphofructo-l-kinase(PFK-1). The most potent stimulator of PFK-1 is fructose 2,6-bisphosphate(Fru-2,6-P2),present in all eukaryotes, discovered in 1980 (I,2). Unlike other regulatory ligands of PFK-1, which act at millimolar concentrations, Fru-2,6-P, stimulates PFK-1 as an allosteric effector active in the micromolar (physiological)range. It does so by increasing the affinity of PFK-1 for Fru-6-P and by relieving the inhibition caused by ATP (3).Thus, Fru-2,6-P2exerts a strategic control over glucose utilization. Aside from PFK-1, present in all cells, three other enzymes are controlled by Fru-2,6-P2, but these are not ubiquitous. The gluconeogenic enzyme fructose-l,6-bisphosphatase,present mainly in liver and kidney, is inhibited by Fru-2,6-P2(4,5).In trypanosomatihe, pyruvate kinase is stimulated by Fru-2,6-P2 (6). Plants and a few microorganisms contain a pyrophosphate-dependent PFK-1, which is also stimulated by Fru-2,6-P2(7, 8). These effects of Fru-2,6-P2 have been reviewed elsewhere (9-11). The aim of this paper is to focus on the enzyme system that catalyzes the biosynthesis and biodegradation of Fru-2,6-P,. The characterization of this system and of its regulation is a prerequisite for understanding how fluctuations in Fru-2,6-P2 concentration integrate the hormonal and metabolic signals that control glycolysis. We limit ourselves to the mammalian enzymes and emphasize the recent input of molecular biology.

1. PFK-2/FBPase-2, A Bifunctional Enzyme That Synthesizes and Degrades Fructose 2,6-Bisphosphate The concentration of Fru-2,6-P2in a given tissue depends on the balance between the activity of 6-phosphofructo-2-kinase(PFK-2),which synthesizes Fru-&,6-P2,and of fructose-2,6-bisphosphatase(FBPase-2), which hydrolyzes it. The properties of this system were studied first in rat liver and are summarized below for comparison with those of other tissues. It has not been possible to separate PFK-2 &om FBPase-2 by any purification procedure; indeed, the two activities belong to a single homodimeric protein composed of SS,OOO-Al,. subunits. Evidence for distinct catalytic sites on each subunit of this bifunctional enzyme has been obtained from chemical modification and proteolytic treatment, and is reviewed elsewhere (10).

A. Liver PFK-2/FBPase-2 PFK-2 catalyzes the synthesis of Fru-2,6-P2 from Fru-6-P and MgATP. By contrast with PFK-1, PFK-2 is not inhibited by physiological concentra-

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101

tions of MgATP. In the presence of 5 mM Pi, a stimulator of PFK-2, the K , for Fru-6-P is about 50 FLM, a concentration also in the physiological range. PFK-2 is inhibited by citrate, phosphoenolpyruvate, and sn-glycerol3-phosphate at concentrations present in hepatocytes. Product-inhibition studies suggest that the reaction for Fru-2,6-P2 synthesis involves the formation of a ternary complex in an ordered sequential mechanism with a direct in-line transfer of the y-phosphate group of MgATP to the hydroxyl group of the C-2 of Fru-6-P. The hydrolysis of Fru-2,6-P2 to Fru-6-P and Pi is catalyzed by a specific phosphatase, FBPase-2, which is stimulated by Pi, sn-glycerol %phosphate, and nucleoside triphosphates. The K, for Fru-2,6-P2 is less than 0.1 p M , which is only & of the total concentration of Fru-2,6-P2 in livers of fed rats. Fru-6-P, one of the reaction products, inhibits FBPase-2 with an apparent & of 20 p M . As both the K , of PFK-2 and the K , of FBPase-2 are similar to the concentrations normally found in the liver, any change in Fru-6-P concentration will cause opposite changes in the two activities. The FBPase-2 reaction follows a “ping-pong” mechanism involving a phosphorylated enzyme intermediate. Incubation of the bifunctional enzyme with [2-32P]Fru-2,6-P2leads to the specific and transient phosphorylation of a histidine residue; the rate of phosphorylation is about 100 times the overall rate of hydrolysis, and thus is not rate-limiting. Vanadate, like Fru-6-P, inhibits FBPase-2 by binding to the phosphorylated intermediate (12). Interestingly, the phosphorothioate analogue of Fru-2,6-P2 is a better substrate than Fru-2,6-P2 itself (13). Liver PFK-2/FBPase-2 is a substrate of the CAMP-dependent protein kinase (PKA), which inactivates PFK-2 and activates FBPase-2 by phosphorylation. This results mainly from a change in their respective V,, values. The dephosphorylation of PFK-21FBPase-2 is catalyzed by phosphoprotein phosphatases [which correspond to phosphatases 2A and 2C in the classification of Cohen (13a)l.A more detailed account of the properties of liver PFK-2/FBPase-2 is given in Refs. 9 and 10.

B. PFK-2/FBPase-2 lsozymes Evidence for tissue-specific isozymes of PFK-2/FBPase-2 was first obtained in our laboratory when the properties of PFK-2IFBPase-2 purified from rat liver were compared with those of the bovine heart enzyme. The properties of the PFK-2/FBPase-2 purified from pigeon breast muscle and rat hindlimbs were also found to differ from those of the liver (reviewed in 14 and 15).The liver, heart, and skeletal muscle PFK-2IFBPase-2 have been termed the L, H, and M isozymes. They differ in molecular mass, kinetic properties, and response to protein kinases. All the mammalian PFK-2/ FBPase-2 isozymes can readily be purified by chromatography on anion-

102

GUY G . ROUSSEAU AND LOUIS HUE

exchange columns followed by affinity chromatography on either Blue Sepharose or phosphocellulose.

1. HEARTPFK-2/FBPASE-2 The H isozyme has relatively low FBPase-2 activity compared to the L and the M isozymes (Table I). The activity ratio of PFK-2 to FBPase-2 is about 100 for the H, 1 for the L, and 0.1 for the M isozyme. These ratios were obtained when FBPase-2 activity was measured at a physiological concentration of Fru-2,6-P,, i.e., in the micromolar range, which is & or less of the K, (40 pM) of the H isozyme for Fru-2,6-Pz. When the FBPase-2 activity is measured under V,, conditions, the PFK-2IFBPase-2 activity ratio decreases considerably. Another difference in the kinetic properties between the L and H isozymes is the poor PFK-2 sensitivity of the H isozyme to inhibition by sn-glycerol %phosphate, and its greater sensitivity to inhibition by citrate. These properties have been used as criteria to identify isozymes that differ from the L isozyme. Two forms of bovine heart PFK-2/FBPase-2 exist with subunit M,s of 54,000 and 58,000. Recent data (16)show that these forms result from alternative splicing of transcripts from the same gene (see Section II,A,3). Like the L isozyme, the H isozyme is a substrate for PKA. However, the phosphorylation sites differ, as does the response to phosphorylation. In the L isozyme, Ser-32 is phosphorylated and this inactivates PFK-2 and activates FBPase-2. In the H isozyme, the phosphorylation site has been identified in the Cterminal end at Ser-466 (164. Phosphorylation results in a slight stimulation of PFK-2 due to a decrease in K,,,for Fru-6-P. However, there is no effect on FBPase-2 activity. Unlike the L isozyme, the H isozyme is a substrate for protein kinase C (PKC) which phosphorylates Thr-475 without affecting the activity. The 54,000-M,form of the H isozyme does not contain these sites but is still phosphorylated by PKA and PKC, suggesting that other phosphorylation sites are present. The additional phosphorylation site for PKC has been TABLE I BIOCHEMICAL CHARhCrERISTICS OF PFK-2/FBPASE-2 ISOZYMES

PFK-2/FBPase-2 Phosphorylation by PKA PFK-2 FBPase-2 Phosphorylation by PKC Subunit M, ( x 10-3)

L

M

H

1 Yes

0.1 no

100

4

Yes

no

t

J.

-

t

no 55

T

no 53

Yes

Yes 55

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identified as Ser-84. No additional phosphorylation site for PKA has been identified so f