Advances in Genetics Volume 38

Volume 38

Advances in Genetics

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Advances in Genetics Edited by Jeffery C. Hall

Jay C. Dunlap

Department of Biology Brandeis University Waltham, Massachusetts

Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire

Theodore Friedmann

Francesco Giannelli

Department of Pediatrics Center for Molecular Genetics School of Medicine University of California, San Diego La Jolla, California

Division of Medical and Molecular Genetics United Medical and Dental Schools of Guy’s and St. Thomas’ Hospital London Bridge, London, United Kingdom

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Copyright 0 1998 by ACADEMIC PRESS 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. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2660198 $25.00

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Contents Contributors

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Body Plan Genes and Human Malformation

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Edoardo Boncinelli, Antonio Mallamaci, and Vania Broccoli I. Introduction 1 11. Establishing the Body Axes 3 111. Patterning the Limb 16 IV. Patterning the Ocular Anlage 21 V. Patterning the Tooth Anlage 22 References 24

Molecular Genetics of the Hereditary Ataxias

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Massimo Pandolfo and Laura Montermini I. Introduction 32 11. The Autosomal Dominant Progressive Ataxias 111. Friedreich Ataxia 49 References 60

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The Minute Genes in Drosophila and Their 69 Molecular Functions Andrew Lambertsson I. Introduction

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11. Historical Review 76 111. Minutes as Genetic Tools in Studies of Growth and Development 84 88 IV. Minutes and the Protein Synthesis Theory 122 V. Conclusions and Prospects References 124 V

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4 Genetics of Biological Rhythms in Drosophria Jeffrey C. Hall I. Introduction 135 11. Chronogenetics 136 111. Chronogenetic Biology 163 IV. A Final Chronogenetic Thought References

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5 DNA Breakage and Repair

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P. A. Jeggo I. Introduction 186 11. Formation of DNA Strand Breaks 186 111. Mechanisms for the Repair of DNA DSBs 188 191 IV. Identification of Genes Required for NHEJ V. Characterization of Proteins Involved in NHEJ by Biochemical, Molecular, and Genetic Analysis 196 VI. DNA-PK as a Protein Kinase 200 VII. The Mechanism of NHEJ 201 VIII. The Contribution of NHEJ and HR to DSB Rejoining in Yeast versus Mammalian Cells 205 206 IX. DNA-PK-Defective Mice 207 X. Radiosensitivity and Immune Deficiency XI. A Model for NHEJ 208 XII. Summary 210 References 2 11 Index

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Edoardo Boncinelli DIBIT, Instituto Scientifico H San Raffaele; and Center for Cellular and Molecular Pharmacology, CNR, 20132 Milan, Italy (1) Vania Broccoli DIBIT, Instituto Scientifico H San Raffaele, 20132 Milan, Italy (1)

Jeffrey C. Hall Department of Biology and NSF Center for Biological Xming, Brandeis University, Waltham, Massachusetts 02254 ( 135) Penny A. Jeggo MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, United Kingdom (185) Andrew Lambertsson Department of Biology, Division of General Genetics, University of Oslo, N-0315 Oslo, Norway (69) Antonio Mallamaci DIBIT, Instituto Scientifico H San Raffaele, 20132 Milan, Italy (1) Laura Montermlni Centre de Recherche Louis-Charles Simard, McGill University, Montreal, Quebec, H2L 4M1 Canada (31) Massimo Pandolfo Centre de Recherche Louis-Charles Simard; Departement de Medecine, Universite de Montreal; and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H2L 4M1 Canada

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Malformation Edoardo Boncinelli,*lt Antonio Mallamaci,* and Vania Broccoli* *DIBIT, Istituto Scientific0 H San Raffaele, 20132 Milan, Italy; C e n t e r for Cellular and Molecular Pharmacology, CNR, 20132 Milan, Italy

1. Introduction 1 3 11. Establishing the body axes A. Rostrocaudal patterning 3 B. Dorsoventral patterning 14 111. Patterning the limb 16 A. Limb development 16 16 B. Genes encoding secreted molecules 18 C. Homeobox genes and limb development 21 IV. Patterning the ocular anlage A. Eye formation 21 21 B. Homeobox genes and the patterning of the eye anlage 22 V. Patterning the tooth anlage A. Tooth development 22 23 B. Homeobox genes and patterning the tooth anlage References 24

I. INTRODUCTION Development initiates with a series of symmetry-breaking events leading to the establishment of fundamental polarities along the rostrocaudal and dorsoventral body axes and, subsequently, along the proximodistal axes of limbs and appenAdvances in Genetics, Vol. 38 Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved. 0065-2660/98 $25.00

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dages. These polarities have to be maintained for some time and subsequently translated into positional values and regional specification, ultimately leading to cell differentiation and maturation. All of these events are genetically controlled by developmental genes and the identification of these genes is a major task of current biology. Many of these genes act in circuits and there is a plenty of redundancy or better, overdetermination, in this genetic control. A key role is played by the so-called regulatory genes, genes acting through the control of the expression of other genes laying hierarchically downstream from them and sometimes termed target genes. Regulatory genes generally code for transcription factors; that is, nuclear proteins able to recognize specific DNA sequences, bind to them, and modulate, through this specific binding, the level of expression of the corresponding target genes. A relatively large proportion of regulatory genes are the homeobox genes (McGinnis and Krumlauf, 1992). Homeobox genes are regulatory genes, originally discovered in Drosophila, characterized by the presence of a specific, evolutionarily conserved DNA sequence termed homeobox and able to code for a protein domain of some 60 amino acid residues, termed homeodomain. It is through the action of their homeodomain that the protein products of the homeobox genes, the homeoproteins, bind to the regulatory regions of specific genes and control their expression. Vertebrate homeobox genes occur in families (Stein et al., 1996). Most of these gene families have counterparts in Drosophila and are often termed according to this similarity. Thus, for example, vertebrate homeobox genes similar to the fruit fly homeotic genes are termed Hox genes; those related to the Drosophila paired ( p d ) gene are termed Pax; those related to the Drosophila engrailed (en) gene are termed En, and so on. A relevant role is also played in development by genes coding for growth factors and, in general, secreted proteins. Also these genes occur in families; they play a major role the Wnt gene family, related to the Drosophila wingless (wg) developmental gene and genes coding for various FGFs (fibroblast growth factors) and BMPs (bone morphogenetic proteins). Recently, the expression patterns of all these genes have been extensively studied in the mouse and have provided useful suggestions for the function they exert during mammalian development. Moreover, their function has been often assayed in vivo by so-called reverse genetics. A large subset of these genes have been knocked-out via homologous recombination in embryonic stem (ES) cells and animals bearing homozygous null mutations have been generated (recently reviewed in St. Jacques and McMahon, 1996). The analysis of these animals enabled us to understand some major mechanisms controlling vertebrate development, with special emphasis on the identification of genes involved in managing positional information and establishing the body plan. But, despite the recent incredible expansion of our knowledge in molecular biology of development, there has been a relative lack of reports about involvement of body plan

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genes in inherited human disease, but the situation is now changing (reviewed in Engelkamp and van Heyningen, 1996 and in Boncinelli, 1997). O n one hand, the increasing number of gene disruption experiments in transgenic mice (St. Jacques and McMahon, 1996) provides a number of suggestions for possible spontaneous inherited pathologies; on the other, the two converging strategies of candidate disease-genes and positional cloning lead to new discoveries. The field has been recently reviewed (Engelkamp and van Heyningen, 1996; Boncinelli, 1997). In this chapter we focus on recent progress and analyze the problem of patterning the embryo along the major body axes and in well-studied morphogenetic fields, namely the developing limb, eye, and tooth anlagen.

II. ESTABLISHING THE BODY AXES A. Rostrocaudal patterning 1. Three genetic domains O n the sole basis of the regional expression of different families of regulatory genes, the central nervous system, if not the entire body, of vertebrates can be subdivided into at least three broad domains along its rostrocaudal axis (Figure 1.1). Each domain also appears to follow a specific developmental pathway for its specification and regionalization (Bally-Cuif and Boncinelli, 1997 for a recent review). Domain 1, including the fore- and midbrain, represents the domain of action of the homeobox genes of the Otx and Emx families. Domain 3 , corresponding to the rhombencephalic regions posterior to rhombomere 1 ( r l ) and spinal cord, is the domain of the homeobox genes of the Hox family. Domain 2, essentially r l and the so-called met-mesencephalic boundary region, remains outside the domains of action of both the OtxlEmx gene families and the Hox gene family: Pax and En genes play a key role in its developmental regulation and extend their influence also into the posterior region of the adjacent mesencephalon. Domain 2 appears to represent something different from all the rest of the neuraxis and to follow unique developmental criteria, possibly owing to its relative evolutionary novelty (Bally and Wassef, 1995; Joyner, 1996; Bally-Cuif and Boncinelli, 1997; Boncinelli, 1997); the met-mesencephalic boundary included in it could be a source of long-range active morphogen(s) involved in patterning the adjacent fields.

2. The fore- and midbrain region Two couples of homeobox genes, Emxl and Emx2 (Simeone et al., 1992a,b), and Otxl and Otx2 (Simeone e t al., 1992a, 1993; Finkelstein and Boncinelli, 1994), respectively, are related to Drosophila head gap genes empty spiracles (ems) and

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Hox

Figure 1.1. Schematic subdivision of the vertebrate CNS on the basis of the expression of specific families of regulatory genes. The vertebrate CNS can be subdivided along its anterior-posterior axis into at least three domains. Domain 1, comprisedof fore- and midbrain, represents the domain of action of Otx and Emx genes families (Fig. 1.2). Domain 2 includes essentiallyrhombomere 1 ( r l ) and the so-called met-mesencephalicboundary region: Pax, Gbx, and En genes play a key role in its development. Domain 3, corresponding to the rhombencephalon posterior to rhombomere 1 ( r l ) and spinal cord, is the realm of Hox genes (Fig. 1.3). In particular, 3' Hox genes belonging to groups 1-4 control the development of the rhombencephalon and its regionalization into a number of neural segments,termed rhombomeres.Central Hox genes of groups 5-8 and 5' Hox genes of groups 9-13 control the cervicothoracicand the lumbosacralregionsof the spinal cord, respectively (see also Fig. 1.4).

orthodentick (otd), and seem to play a relevant role in patterning the vertebrate fore- and midbrain. In the developing central nervous system of mouse embryos at day 10.5 of development (E10.5) most of the specific differentiative events have not yet occurred and at this stage all four Emx and Otx genes are expressed. Their expression domains (Simeone et al., 1992a) are continuous regions of the developing brain contained within each other in the sequence Emxl < Emx2 < Otxl < Otx2 (Figure 1.2). The Emxf expression domain includes the dorsal telencephalon. Emx2 is expressed in the dorsal and ventral forebrain with an anterior boundary slightly anterior to that of Emxl and a posterior boundary within the roof of presumptive diencephalon. The Otxl expression domain contains the Emx2 domain: it covers a continuous region including part of the telencephalon,

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Figure 1.2. Nested expression domains of Otx and Emx homeobox genes in the developing brain of a El0 mouse embryo. T h e Emxl expression domain includes the dorsal telencephalon. EmxZ is expressed in dorsal and ventral forebrain with a posterior boundary within the roof of presumptive diencephalon. T h e Otxl expression domain contains the Emx2 domain: it covers a continuous region including part of the telencephalon, diencephalon, and mesencephalon. Finally, the O t x Z expression domain contains the Otxl domain, both dorsally and ventrally, and practically covers the entire fore- and midbrain, to the exclusion of the early optic area. Te, telencephalon; Di, diencephalon; Mes, mesencephalon; Met, metencephalon; Mye, myelencephalon; os, optic stalk.

the diencephalon, and the mesencephalon. Finally, the Otx2 expression domain contains the Otxl domain, both dorsally and ventrally, and practically covers the entire fore- and midbrain, to the exclusion of the early optic area. Expression of Emx and Otx genes identifies several regions in the forebrain. Some of these regions seem to correspond to presumptive anatomical subdivisions, whereas the significance of others remains to be assessed. The first appearance of the products of the four genes is also sequential during development: Otx2 is already expressed at least as early as at E5.5 (Simeone et al., 1993), followed by Otxl and Emx2 at E8-8.5 and finally by Emxl at E9.5 (Simeone et al., 1992a). Thus, it seems reasonable to postulate a role for the four homeobox genes in establishing the identity of the various embryonic brain regions. In this line, the regionalization of the early rostral brain seems to be a centripetal process progressing through discrete steps and ultimately leading to the specification of dorsal telencephalon. Studies on mouse, frog, chick, zebrafish, and sea urchin Otx2 (reviewed in Boncinelli and Mallamaci, 1995) imply a role of this gene in the early establishment of the head and rostral brain. An Otx2-like gene is also present in planaria with a comparable role in head development (our unpublished data). Transgenic mice bearing null mutations for all four genes have been produced and analyzed. Otx2 is the first of these genes to be expressed during development and

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it was expected to play an essential role in anterior head formation and probably axis determination (Boncinelli and Mallamaci, 1995). Analysis of mice bearing null mutations in Otx2 produced in three different laboratories confirms these expectations (Acampora et al., 1995; Matsuo et al., 1995; Ang et al., 1996). These mice fail to gastrulate and stop developing at early midgestation. The most conspicuous phenotype of these midgestation embryos is the deletion of rostra1 brain regions, including forebrain and midbrain, anterior to rhombomere 3 (1-3).It is highly likely that the deletion of anterior neural structures is a consequence of the defective formation and migration of anterior axial mesendoderm cells. A similar early phenotype is exhibited by embryos lacking L i d , another homeobox gene expressed almost as early in development (Shawlot and Behringer, 1995). This could implicate these genes in cases of mouse or human anencephaly, but so far no evidence for this connection is available. Otxl null mutants also showed some interesting features that may suggest Otxl as a candidate locus for certain congenital malformations in man. In fact, Otxl null mutants show multiple abnormalities affecting several areas of the cerebral cortex, hippocampus, mesencephalon, and cerebellum, as well as special sense organs (Acampora et al., 1996; Suda et al., 1996). All of these localizations are in agreement with the expression of this gene during embryogenesis. Conversely, it was probably not easy to anticipate that homozygous Otxl null mice are affected by epileptic seizures. Epilepsy is a phenotypically and genotypically heterogeneous disorder; many different forms of epilepsy are known and for some of them a genetic component has been suggested. A number of transgenic mouse models are known for this disease (Noebles, 1996) and Otxl-deficient mice represent a promising new addition to this catalog. Emx2 null mutants have also been analyzed (Pellegrini e t al., 1996; Yoshida et al., 1997). O n the basis of its expression domain, this gene was proposed to play a role in the control of proliferation and migration of cortical neurons (Gulisano et al., 1996). Emx2 null mice are born but do not survive for long since they lack kidneys. Inspection of the brain of late-gestation embryos or newborn animals shows a generalized reduction of the cerebral cortex, both in extension and in thickness, severe malformations of the hippocampus and medial limbic cortex, and complete absence of dentate gyrus. It is not clear why the latter anatomical structures are particularly affected in these mutant mice. It is conceivable that the products of the gene Emx2 play specific roles in defining the identity of these structures. On the other hand, it is known that the hippocampus and dentate gyrus formation requires a prolonged cell proliferative period: so it is reasonable that these structures could suffer in a more intense way detrimental effects deriving from the missing EMX2 homeoprotein. Emxl null mutants do not show any striking cerebral phenotype other than occasional absence of corpus callosum (Yoshida et al., 1997;Qiu e t al., 1996). Although developmental and phylogenetic data suggest a very important role for

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Emxl , we have at present no hints for its function. Perhaps, Emxl may be more relevant in later developmental stages and the analysis of phenotypes deriving from the absence of its products may require refined experiments. Mutations in the human homolog of Emx2, that is EMXZ, have also been associated with a human congenital malformation termed schizencephaly (Brunelli et al., 1996; Granata et al., 1997). Heterozygous de n o w mutations in this gene have been found in sporadic cases of this extremely rare developmental defect of the cerebral cortex associated with full-thickness clefts of the cerebral hemispheres with consequent communication between the ventricle and pericerebral subarachnoid spaces. Based on the space separating the walls of the fissure, an open-lip form and a closed-lip form can be distinguished (Guerrini et al., 1996). The molecular nature of the various mutations detected in these patients is fairly heterogeneous. Even if the number of cases studied is still very restricted, a case may be made for a correlation between the molecular nature of these mutations and the observed clinical severity. In fact, predictably deleterious molecular defects, like frameshift mutations or mutations affecting the splicing pattern are invariably associated with severe, open-lip, bilateral schizencephaly, whereas subtle or leaky mutations are associated with mild, closed-lip, seemingly unilateral schizencephaly (Granata et al. , 1997). This may be ultimately useful for prognosis, given the difficulties associated with the differential diagnosis for this particular pathology and its extreme clinical heterogeneity.

3. The isthmic region As proven by heterotopic homochronic transplants in chickenkquail embryos, the boundary between rhombencephalon and mesencephalon acts as an organizing center. If the midbrain-hindbrain junction region is included in inverted reimplants or transplanted to the diencephalon or rhombencephalon, the tissue maintains its developmental fate and also induces the surrounding cells to form mesencephalic or metencephalic (mes-met) structures (reviewed in Puelles et al., 1996). The growth factor FGF8 is expressed at the mes-met boundary and its application to caudal diencephalon can mimic the effects of a mes-met graft (Crossley et al., 1996). Genes belonging to the Wnt, Pax, En, and Gbx families, namely W n t l , Pax2, P a d , Pax8, En1 , En2, and Gbx2, are specificallyexpressed around this boundary in an highly dynamic way (reviewed in Bally-Cuif and Wassef, 1995; and in Joyner, 1996); it is also noteworthy that the Drosophila homologs, namely wingless, paired, and engrailed, are together involved in estabilishing embryonic intersegmental boundaries in the fruit fly. W n t l , En1 , En2, Pax2, and Pax5 have been knocked-out in the mouse and mutant embryos display various anomalies and deletions in the area of cerebellum and/or midbrain (reviewed in Joyner, 1996). Recently, the phenotype shown by mice homozygous for a naturally occurring mutation in the gene Pax2

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has been analyzed (Favor e t al., 1996). The mutated allele Pax21Neudiffers from the wild-type by a I-nucleotide insertion, which is identical to a previously described mutation in a human family with renal-coloboma syndrome (Sanyanusin et al., 1995). In homozygous mutant embryos, the development of the optic nerve as well as of the metanephric kidney is severely affected, as already described for other Pax2-1- embryos (Torres et al., 1995; Keller et al., 1994). In addition, Pax21Neuhomozygous animals display a deletion of cerebellum and posterior mesencephalon, which was not described for either Pax2-1- embryos (Torres et al., 1995;Keller et al., 1994) or reported in mutant humans (Sanyanusin et al., 1995). These mice should provide an ideal animal model for future studies on the congenital abnormalities associated with human PAX2 mutations.

4. The rhombospinal region a. The Hox genes clusters: Structure and expression A key role in patterning the anatomical regions along the body axis, from the branchial area through the tail, is played by Hox genes. They are homeobox-containing genes which encode transcription factors involved in modeling the definitive embryo shape and represent the true vertebrate homologs of the Drosophila homeotic genes. In the fruit fly embryo, homeotic genes provide biological information for specifying the identity of the various body segments; their mutations result in bizarre phenotypes wherein one body segment gives rise to structures appropriate for another one. Eight of these genes are located in two contiguous chromosomal loci called Antennupedia (ANT-C) and Bithorax (BX-C) complexes, col-

Figure 1.3. Alignment of the four vertebrate Hox loci. Paralogy groups are shown as well as a comparison with Drosophila homeotic (HOM) genes. The grey boxes represent Drosophila homeotic genes: labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), and Antennapedia (Antp), all belonging to the Antennapedia complex ( A N T C ) , and Ulmabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B), forming the Bithorax complex (BX-C). The black boxes represent the 39 vertebrate Hox genes; above each of them is the current nomenclature (Scott, 1992). They are ordered in 13 paralogy groups, labeled at the bottom; at the top, their homology relationships with Drosophila HOM genes are represented. The horizontal arrow indicates direction of transcription of Hox genes. Under it, HOMIHox modulators are listed, which seem to act by regulating the chromatin structure of the loci. Th e Drosophih gene nithorax and its vertebrate homolog MLL should allow an open chromatin conformation; conversely, Drosophila Polycomb genes and their vertebrate relatives h i - 1 , mel-18, and eed would promote heterochromatin formation. Hox genes belonging to groups 1 4 are expressed first, are necessary for hindbrain patterning and display the most prompt and efficient response to retinoic acid in vim0 (reviewed in Boncinelli et al., 1991). Hox genes of groups 5-8 are expressed later, control the patterning of the thoracic region and display an intermediate response to retinoic acid. Hox genes belonging to groups 9-13 are expressed last, control the lumbosacral region, and are the most refractary to retinoic acid stimulation.

DrosoDhila ANlr-C

Vertebrates Hox-a

HOX-c Hox-d Paralogygroups

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Anterior Early 3'

High RA response

4 ' 5 hindbrain I

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8 ' 9 10 11 12 13 thoracic I lumbo-sacral regron I regron 6

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~~ ~ ~ p a~e a n 0 bmtl mel-I8 eed

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lectively called HOM-C, for Homeotic complex. They have arisen from a single gene array that was split during insect radiation. Conversely, along the phyletic line leading to vertebrates, this common ancestral cluster has undergone two duplications giving rise to Hox gene clusters (reviewed in Kenyon, 1994). In mouse and man these complexes, approximately 120 kb in length, were analyzed in detail. Their 39 genes are oriented in the same 5’40-3’ direction of transcription (Figure 1.3). The four loci are highly homologous and can be easily aligned. Corresponding genes in different Hox clusters share the highest sequence similarity; they belong to 13 different sets of genes, which are termed paralogy groups, and are numbered from 1 to 13, starting from the 3‘ end of the various clusters. Most of the Hox genes can be correlated with specific Drosophila homeotic genes. Moreover, a unique feature of both Hox and HOM clusters is the correlation between the physical order of genes along the chromosomes and their anterior boundaries of expression along the rostrocaudal axis of the embryo (reviewed in McGinnis and Krumlauf, 1992). In vertebrates, 3’ Hox genes are expressed early in development and control anterior regions, whereas progressively more 5’ genes are activated later and control more posterior regions; members of the same paralogy group often show coincident expression boundaries. This spatial-temporal colinearity is followed by all 39 Hox genes. Such a characteristic pattern is detectable in several developing embryonic structures, namely the central nervous system, paraxial mesoderm, neural crest, limbs, and genitalia, providing a coordinate system of axial signals involved in generating different regional identities (reviewed in Krumlauf, 1994). Figure 1.4 show an updated map of the expression domains of several developmental genes along the developing central nervous system.

b. Mutations in Hox genes So far, only three inheritable deseases, namely mouse hypodactyly (Hd), human synpolydactyly, and hand-foot-genitalia (HFG) syndrome, affecting limb development, have been associated to naturally occuring mutations in Hox genes (see Section 111,C).In addition, it can be hypothesized that HOX genes, or some of their target, could be good candidates for mutations causing segmental defects of hindbrain or spinal cord development. In humans, this might include Moebius syndrome (OMIM 157900), Arthrogryposis multiplex congenita (OMIM 208100), Axenfeld-Rieger anomaly (OMIM 10920), or segmental spinal muscular atrophy (OMIM 183020).l Here, we will summarize some relevant results obtained by gene targeting strategies in mice. Such artificial mutations help us to circumvent the lack of naturally occurring mutants and provide useful cues for possible spontaneous human inherited pathologies. A large body of evidence have been accumulated on ‘OMIM (&Line Mendelian Inheritance in Man) can be contacted at the URL: http://www3.ncbi.nlm.nih.gov/ornim/

Telencephalon

Diencephalon

Mesencephalon Isthmus Rhomboncephalon

Spinal cord I

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Hoxl Figure 1.4. Expression domains of some major developmental genes in the developing central nervous system (Shimamura et al., 1995 and references therein). Telencephalon and diencephalon are shown as subdivided into six prosomeres according to the model of Puelles and Rubenstein (Shimamura et al., 1995).

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the functions played by Hox genes in controlling the regional identity of axial skeleton. Generally speaking, phenotypes derived from loss-of-function experiments show repeated structures like vertebrae or ribs to undergo anterior transformations; that is, for example, the case of Hoxc8 gene inactivation, which leads to the homeotic transformation of the first lumbar vertebra into the seventh toracic one, which bears an extra pair of ribs (LeMouellic et al., 1992).These phenotypes resemble the changes in body segments displayed by homeotic mutant flies, even though they never show a full transformation as it happens in Drosophila. Conversely, mice bearing complementary gain-of-function mutations display posterior transformations, as is the case for gain-of-function mutant Drosophila embryos. For example, Hoxd4 and Hoxa7 ectopic expression cause posterior transformations of skull occipital bones and cervical vertebrae, respectively (Lufkin et al., 1992; Kessel et al., 1990). This data support the concept of functional homology between Hox and HOM genes (reviewed in Boncinelli et d., 1994).However, this is not an invariant rule, because gain-of-function mutations in Hoxc6 and Hoxc8 lead to anterior transformations (Jegalian and De Robertis, 1992; Pollock et al., 1992). Hox genes are involved not only in patterning the axial skeleton. They also provide molecular cues for specifying the regional identity of other embryonic structures, like hindbrain, branchial arches, genitalia, and the various regions of the digestive tract. In the hindbrain, Hoxbf and Hoxb2 mutant mice fail to form the somatic motor nucleus of the VIIth nerve, which controls the muscles responsible for facial expression, leading to a facial paralysis (Studer et al., 1996; Barrow and Capecchi, 1996; Goddard et al., 1996). Features of this phenotype closely resemble the clinical signs associated with Bell's Palsy (OMIM 134100) and Moebius syndrome in humans. In the branchial area of Hoxa2-1mutant embryos, the identity of the mesenchymal neural crest of the second arch is changed into that of the first arch, resulting in homeotic transformations of second- to first-arch skeletal elements (Rijili et al., 1993). In female genitalia, the Hoxaf 0 mutation causes the homeotic transformation of the proximal part of uterus into oviducts; a parallel transformation is observed in mutant males at the juction between the epididimus and ductus deferens (Benson et d., 1996). Finally, the organization of the smooth layers of the rectum, the most caudal part of the digestive tube, is severely perturbed in Hoxdf 2 and Hoxdl3 mutant embryos (Kondo et al., 1996). In general, single Hox mutant mice display phenotypes which are much less severe than expected on the basis of their expression patterns. These defects correlate with the sites where a single Hox gene is expressed. This can be partially explained by compensatory mechanisms among different Hox genes. A n outstanding example is the axial skeletal abnormalities found in Hoxa9/Hoxd9 double mutants. Here, a homeotic transformation more extended than just the sum of the phenotypes of the individual mutations was observed and this finding may indicate a functional redundancy between the two genes in regions where

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they are coexpressed (Fromental-Ramain et al., 1996). This kind of interaction probably takes place not only between members of the same paralogy group, but also between the various Hox genes of the same cluster. Hoxb5 and Hoxb6 compound homozygous mutants display an anterior homeotic transformation of some cervicothoracic vertebrae, which is absent in mice lacking either one or the other of these genes (Rancourt et al., 1995). In all double mutant combinations and in the triple mutant generated though the knockout of Hoxa4, Hoxb4, and Hoxd4, progressively more dramatic homeotic transformations of the axial skeleton were observed and their severity increased with the total number of mutated alleles (Horan et al., 1994). This also suggests a dose-dependent requirement for functionally interchangeable gene products and that these proteins may interact on a quantitative basis. The malformations derived from targeted Hox mutations cannot always be explained as simple homeotic transformation. In particular, the complete absence of the atlas in Hoxa3/Hoxd3 compound mutant mice supports the hypothesis that Hox genes can be also necessary for the very existence of some embryonal structures and not only for their later specification (Condie and Capecchi, 1994). In keeping with that, suppression of the Hoxal function results in the deletion of rhombomere r5, reduction of r4, and loss of specific rhombencephalic nuclei (Chisaka et al., 1992).

c. Regulators of the Hox genes In Drosophila, members of the Polycomb group gene family (Pc-G) act as negative regulators of the homeotic genes by binding to sites in the HOM-C complex and inducing the formation of heterochromatin (reviewed in Lawrence and Morata, 1994). Three vertebrate homologs of these genes have been discovered: h i - 1 , mel-18, and eed. The first two show high homology and their targeted mutations offer similar results. Changes of vertebral identities occur that lead to a general posterior transformation of the axial skeleton (van der Lugt et al., 1994;Akasaka et al., 1996).That is attributable to an anterior shift of the expression domains of some Hox genes, so altering positional values in the sclerotome precursors (van der Lugt et al., 1996;Akasaka et al., 1996). In humans, h i - 1 was found to act as a proto-oncogene, whereas mel-18 appears to display tumor suppressor activity (Kanno et al., 1995). The third vertebrate homolog of Pc-G Drosophila genes, namely eed (for embryonic ectoderm development), has been identified via positional cloning of the corresponding classical mouse mutation (Shumacher et al., 1996). Mice carrying a hypomorphic eed allele exhibit posterior transformations along the axial skeleton. Moreover, mice homozygous for an eed null mutation show disruption of the primitive streak during gastrulation, which might reflect very early eed involvement in regulating anteroposterior patterning (Shumacher et al., 1996).

The Drosophila gene crithorax ( t r x ) and its vertebrate homolog MLL can

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activate HOM and Hox gene transcription, respectively, by acting on their chromatin genomic environment. Mll null mice are embryonic lethal and fail to express several Hox genes, whereas MU+/- individuals have aberrations of segment identity and corresponding caudal shifts in the anterior Hox expression boundaries (Yu et al., 1995). MLL is disrupted by chromosomal translocation in some patients affected by acute leukemias, often displaying a mixed lymphoid-myeloid phenotype. In the developing nervous system, kreiskr and krox20 genes seem to act as Hox regulators. Spontaneous kreisler mutant mice are deaf and lack presumptive r5 and r6 territories. The early absence of krox20 and group 4 Hox gene expressions in these areas suggest a possible involvement of Kreisler in their functional regulation, even if a direct interaction has yet to be shown (Frohman et al., 1993). In contrast, Krox20 is a direct modulator of the r3/r5 expression of both Hoxu2 and Hoxb2 (Nonchev et al., 1996). A key role in regulating the overall Hox gene expression is played by retinoic acid (RA), a molecule belonging to the group of retinoids that are vitamin A (retinol) derivatives. RA has been known for long time to produce teratogenic effects in humans (Sporn et al., 1994). The first hint about the functional relationship between RA and Hox genes came from the sequential induction of their expression observed in embryonal carcinoma cell lines upon treatment with exogenous RA (reviewed in Boncinelli et al., 1991). Hox genes located at the 3' end of the four loci are activated first and respond to relatively low concentrations, whereas progressively more 5' Hox genes are activated later and require higher concentrations of RA (Figure 1.3).In vivo experiments subsequently confirmed the importance of its role in normal development of several embryonic structures (reviewed in Conlon, 1995; Kastner et al., 1995). Exposure of murine embryos to teratogenic doses of different retinoids can induce anterior shifts of Hox gene expression domains in paraxial mesoderm that elicits, later in development, posterior transformations of several vertebrae along the axial skeleton (Kessel and Gruss, 1991). Similar manipulations also lead to complex malformations in the central nervous system. In the hindbrain, exogenous RA causes transformation of rhombomere r2 into an r4 identity, a phenomenon associated with changes in Hoxbl expression (Marshall et al., 1992). This findings provide cues about the mechanisms by which RA might induce teratogenesis in the human embryo.

B. Dorsoventral patterning

1. Role of notochord and floor plate The fate of cells located a t different dorsoventral positions within the neural tube and the paraxial mesoderm depends upon signals that derive initially from adja-

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cent axial mesodermal cells forming a notochord. These signals induce the differentiation of ventral cell types within the neural tube and the paraxial mesoderm and suppress the differentiation of dorsal fates. Floor plate cells differentiate in the neuroepithelium immediately adjacent to the notochord, motor neurons in the ventral lateral neuroepithelium, and sclerotome cells in the ventralmost paraxial mesoderm. Induced floor plate cells now share with the notochord its patterning activities, so that a source of ventralizing signals is available in close proximity to the neuroepithelium and paraxial mesoderm even after the ventral displacement of the notochord, which occurs as development proceeds (reviewed in Placzek, 1995).

2. Sonic hedgehog A secreted protein encoded by a gene termed Sonic hedgehog (Shh) is expressed specifically in the notochord and in the floor plate as well as at the posterior edge of limb buds (reviewed in Ingham, 1995, and Roelink, 1996): this suggests it can mediate dorsoventral patterning properties exhibited by these structures. Support for this suggestion comes from experimental expression of the Shh gene or placement of SHH protein-releasing beads at ectopic locations in developing embryos, as well as from treatment of explanted embryonic target tissue with the purified protein. In the first case, SHH mimics notochord and floor plate by promoting expression of ventral markers in neural tube, brain, eye and somites (reviewed in Ingham, 1995; and Roelink, 1996; Ericson et al., 1995; McDonald et al., 1995). In the second, purified SHH protein induces ventral cell-specific genes, with floor plate markers induced at higher concentration and motor neurons and sclerotome markers at lower (Roelink et al. , 1995; Fan et al., 1995). Knock-out mice confirm Shh involvement in dorsoventral patterning of vertebrate embryonic tissues, including brain, spinal cord, and axial skeleton. Early defects are detectable in the establishment or maintenance of midline structures, like notochord and floor plate; later defects include the absence of spinal column and most of the ribs, the absence of ventral cell types in the neural tube, cyclopia, and defects in distal limbs (Chiang et al., 1996). Heterozygous alterations in the Shh locus are specifically associated with the occurrence of some cases of holoprosencephaly in our species. Holoprosencephaly (HPE) is a common developmental defect of the forebrain and the midface in humans, involving incomplete development and septation of midline structures in the central nervous system with a broad range of clinical severity. Alobar HPE, the most severe form, involves complete lack of division of the forebrain into two hemispheres and is associated with facial anomalies including cyclopia, a primitive nasal structure (the so-called proboscis) and/or midface clefting. A t the mild end of the spectrum there are microcephaly, mild hypertelorism, single maxillary central incisor, and other defects (Cohen, 1989a,b; Cohen and Sulik, 1992).The phe-

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notypic variability is common also between affected members of the same family. A t least four types of familial forms have been described with autosomal dominant or recessive inheritance. Cases of autosomal dominant holoprosencephaly have been reported to be associated to different types of mutations at the HPE3 locus (Roessler et al., 1996, Belloni et d., 1996).In humans, loss of one SHH allele is sufficient to cause HPE, whereas both alleles need to be lost in Shh-/- mice to produce a similar phenotype (Chiang et d., 1996). The presence of only one copy of SHH in humans, though able to disturb ventral midline neurogenesis, is not sufficient to cause defects stemming from somite ventralization or limb abnormalities characteristic of Shh-1- mice (Belloni et al., 1996; Roessler et al., 1996).

111. PATTERNING THE LIMB A. Limb development The limb is one of the best characterized morphogenetic fields in the developing mammalian embryo, thanks to its accessibility to experimental manipulations as well as to the possibility of extrapolating to mammals the large body of evidence accumulated in chicken. In the limb bud, the apical ectodermal ridge (AER), namely a thickening of the distal ectoderm of the bud, stimulates the proliferation of mesenchyme present in the underlying progress zone (PZ). The interaction between ridge and mesenchyme is reciprocal and a signal from the latter maintains the former. During limb bud elongation, as cells leave the PZ, they lay down structures along the proximodistal axis of the limb in sequence, starting with proximal structures and progressing distally. A region of the posterior bud mesenchyme, called the zone of polarizing activity (ZPA), has the ability to direct the formation of mirror-image duplications when transplanted to the anterior margin of a host limb bud. This has been proposed to be a source of a morphogen, the concentration of which could provide anteroposterior positional information to limb bud cells. Finally, ectodermal signals control the dorsoventral patterning of mesenchyme, which determines the disposition of tendons and muscles (reviewed in Johnson et al., 1994; Xckle, 1996).

B. Genes encoding secreted molecules Candidate molecules have been identified which could mediate the patterning function played by the AER, ZPA, and dorsal ectoderm (DE) (see Figure 1.5A). Shh is expressed in the ZPA and mimics some properties of the ZPA when applied to the anterior edge of the limb bud (reviewed in Tickle, 1996). Retinoic acid also mimics the ZPA and is present in the limb bud where it has been suggested to act

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Figure 1.5. Genes involved in patterning the vertebrate developing limb. In A the expression of genes encoding secreted molecules in the limb bud and their cross-talk are represented. Sonic hedgehog (SM) is expressed at the posterior edge, while various FGFs (including FGFZ, FGF4, and FGF8) are present along the distal margin of the bud; the dorsal ectoderm is marked by Wnt7a. A positive feedback loop takes place between SM and Fgf4, whereas Wnt7a stimulates Shh expression (bent arrows). In B the expression domains of some homeobox genes in mid-late developing limb are summarized. Lmxl transcripts are in the dorsal mesenchyme, whereas En1 products are in the ectoderm lining the ventral half of the limb. Genes belonging to the 5' end of the Horn cluster are restricted in a proximal4istal way: Howl 3 transcripts are in the autopod anlage, Horn11 in the developing zeugopod, HoxulO is expressed throughout the field. Conversely, genes of the 5' region of Hoxd (d13, d12, dl I , dIO, and d9) are restricted along the anterior-posterior axis. Their Russian doll-shaped domains share the posterior margin, at the caudal edge of the limb, and have anterior boundaries colinear with the position of the gene within the cluster: the rostralmost being d9 and the caudalmost being d13.

upstream of Shh (Helms et al., 1996), but its function in this field remains to be clarified. Molecules belonging to the FGF family, namely FGFZ, FGF4, and FGF8, are variously expressed in the AER: the application of them to a limb bud, from

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which the AER has been previously removed, leads to limb outgrowth. Wnt7a is specifically expressed in the DE and it has been suggested to mediate DE action. It has been shown that the various sources of patterning signals cross-talk between each other: positive feedback loops take place between Shh and Fgf4 and Wnt7a sustains Shh expression (reviewed in Tickle, 1996; Cohn and Tickle, 1996). Mice lacking Wnt7a activity have been generated and their limb mesoderm displays dorsal to ventral transformations in cell fate. Many mutant animals also lack posterior digits (Parr and McMahon, 1995). This may be attributed to the interruption of the positive feedback loop normally occurring between Wnt7u and Shh and to the physiological role played by the latter in promoting the development of posterior digits. In Shh-1- mice the distal structures are most affected, with a complete absence or fusion of the zeugopod bones: in the hindlimb, the most severely affected, the femur, is formed, but tibia and fibula are completely absent; in the forelimb, a bony extension of the humerus may represent either a fused ulna/radius or a long, bent humerus. A reasonable link between these distal truncations and loss of Shh function could be the apparent role of FGFs in promoting limb outgrowth and the requirement for Shh in the maintenance of Fgf4 expression in the posterior AER (Chiang et al., 1996). Patients bearing one SHH mutated allele and affected by holoprosencephaly, like Shh+/- mice, don’t display any limb phenotype (Roessler et al., 1996; Belloni et al., 1996). Finally, retinoids have been described to cause teratogenic effects in limbs, like digit truncations and reductions in long bones (Sucov et al., 1995).

C. Homeobox genes and limb development Positional information within this field is probably first conveyed as a graded concentration of diffusible molecules along the three main limb axes and is subsequently translated into the expression levels of some homeobox genes. Lmxl is expressed in dorsal mesenchyme in response to Wnt7a signaling and appears to mediate the dorsalizing effects acted by it (Riddle et al., 1995; Vogel et al., 1995). En2 is expressed in ventral limb ectoderm (see Figure 1.5B): it is necessary for the proper ventral limb patterning and acts in part by repressing dorsal differentiation induced by Wnt7a. Enl-1- mice display dorsal transformations of ventral paw structures and dorsal duplications of ventrodistal structures (Loomis et al., 1996). The Horn and Hoxd expression patterns are highly dynamic: in the midlate developing limb, genes belonging to the 5’ end of the Hoxa cluster (a23, al2, and a20) are restricted in a proximodistal way; genes at the 5‘ of Hoxd (d23 , d2 2 , d2 2 , d20, and d9) are restricted along the anteroposterior axis (see Figure 1.5B for more details) (reviewed in Johnson et al., 1994). It has been proposed that Horn and Hoxd genes can simply pattern the limb along these two axes, respectively, by giving rise to combinatorial code units able to specify the identity of all major morphogenetic subfields within the limb itself. The mirror-image duplication of the H o d expression pattern, which can be obtained together with the analogous

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duplication of the digital pattern by applying RA-releasing beads to the anterior border of the limb (Izpisua-Belmonte et al., 1991), seems to be a good support for that. However, this model is an oversimplification and must be reconsidered more carefully. All Hox genes involved in limb patterning have been knocked out one by one and, via genetic crosses, in different combinations; in addition, among these genes are the only two Hox genes that have been demonstrated to be affected in naturally occurring mutants, namely Hornl3, involved in murine hypodactyly and human HFG syndrome, and HOXD13, involved in human s p polydactyly (see below). The phenotype displayed by some of these mutants casts some doubts on the simple patterning model described above. Functional redundancy and extensive expression overlapping between genes belonging to the same paralogy group could explain some discrepancies between our expectations and the actual situation. That is the case for Hoxa genes: their knock-outs yield roughly the expected phenotypes, but only if associated to the ablation of the corresponding Hoxd paralogs. For example, the zeugopod derivatives, radius and ulna, still present in Hoxal J -1- mice, even if abnormal (Small and Potter, 1993), and are completely absent in the double-mutants Hoxal 1 -/-/Hoxdll-/- (Davis et al., 1995). In a similar way, Hoxal3-/-/Hoxdl3-/- double knock-outs lack almost any chondrified condensation in the autopods (Fromental-Ramain et al., 1996), at variance with hypodactyly Hd/Hd mice (Mortlock et al., 1996) and artificially generated Hoxal3-l- mice (Fromental-Ramain e t al., 1996), which display one or more fingers. However, relevant difficulties remain about the function postulated for Hoxd genes. In chicken tulpid mutants, for example, all 5' Hoxd genes are expressed uniformly along the anteroposterior axis of the limb and a large number of morphologically similar digits are present. However, they look like third digits and not fourth, as predicted by the model. In addition, Hoxdl3-1mice (Doll6 et al., 1993) don't display any digit V-to-digit IV homeotic transformation, as expected, but simply a reduction in size of the digits and delay in their development. It has been proposed (Doll6 e t al.,1993) that the main role of Hoxd genes in the developing limb is not in specifying positional identities of already formed blastemas, but rather in molding their shape by regulating their proliferation, growth, and differentiation. The model of Shubin and Alberch (1986) can possibly help in resolving these difficulties. In this model, the autopod is a skewing of the topological proximodistal axis of the limb, which runs from the ulna through the ulnare and then bends anteriorly through the distal carpals. According to Shubin and Alberch, the five digits share all the same anteroposterior positional value, which is in good agreement with the late expression of Hoxdl3, spreading up to the digit I anlage. For this reason, Hoxd genes, acting in limb as selectors of anteroposterior identity, cannot pattern the digital anlagen as identity selectors. They might do it only through different mechanisms: for example, by modulating cell proliferation and growth, as suggested by Doll6 et al. (1993). Hypodactyly (Hd) is a semidominant mutation in mice that maps in a genetic interval overlapping the Horn cluster. Hd/+ animals have a shortened dig-

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it I on all four limbs but are otherwise normal and fertile; they also display alterations in timing of ossification of specific cartilagineous elements of the autopod. Limbs of Hd/Hd animals usually have a single digit (possibly IV) and are affected by loss of some carpal and tarsal bones; the few retained carpal and tarsal elements are small. In Hd mutants a 50-bp deletion has been specifically detected in the first exon of the Horn13 gene, and it probably originated through heterologous recombination between triplet repeats. The mutation could lead to the production of aberrant proteins able to act in a dominant-negative manner by interfering with the function of other homeoproteins and/or by altering directly the expression of target genes (Mortlock et al., 1996). Remarkably, the phenotype displayed by Hd/Hd mutants is similar but not identical to the one shown by artificially generated Hoxa13-/- mice, which lack only one digit, the first (Fromental-Ramain et al., 1996). The human HOXA13 gene quite recently had been found to be mutated in a family with hand-foot-genital syndrome, an autosomal dominant, fully penetrant disorder implying hand and foot anomalies remarkably similar to those of hypodactyly in the mouse (Mortlock and Innis, 1997). These include short first metacarpals, small distal phalanges of the thumbs, short middle phalanges of the fifth fingers, and fusion or delayed ossification of wrist bones. Similarly, in the feet, the great toe is shorter due to a short first metatarsal and a small, pointed distal phalanx. Associated to these limb anomalies are also uterine anomalies typically involving a partially divided (bicomate) or completely divided (didelphic) uterus, represeting defects of Mullerian duct fusion. The nonsense mutation found in this family causes a truncation of the corresponding HOXA13 protein before the end of the homeodomain. Mutations in HOXD13 have been found in five families affected by synpolydactyly, an autosomal, dominantly inherited human abnormality of the hands and feet (Muragaky et al., 1996; Akarsu et al., 1996). Heterozygotes display proper synpolydactyly, namely a malformation involving both webbing between fingers and duplication of them. The homozygous phenotype includes the transformation of metacarpal and metatarsal bones to short carpal- and tarsal-like bones. Affected people of these families were found to contain a HOXD13 gene mutated in a novel way. Many HOX homeoproteins are known to contain in their amino-terminal portion a number of short sequences of repeated amino acid residues, mostly alanine and serine, but sometimes also glutamine and proline, that are believed to participate in the function of gene repression, or activation, exerted by this region. In particular, in the HOXD13 homeoprotein there are two serine stretches and one alanine stretch consisting of 15 residues in a row. Affected individuals were found to be heterozygous or homozygous for an expansion of this alanine stretch. In the various families analyzed, this expansion ranges between 7 and 10 additional alanine residues as compared to the wild-type situation (Mugaraky et d., 1996; Akarsu et d., 1996). All mutated chromosomes within a given family exhibit the same type of expansion, 7, 8, 9, or 10 additional residues,

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showing that the situation is relatively stable through several generations. These mutations are not likely to act through a mere loss of function mechanism. In fact, mice lacking the corresponding Hoxdl3 gene show a similar but distinct phenotype (Doll6 et al., 1993). Conversely, the authors propose that the observed expansions of the alanine tract may alter the function of the protein. For example, the mutated protein may still be able to interact with DNA but may not be able to interact with other proteins. The hypothesis of gain of abnormal function is potentially supported by the observation that mice simultaneously lacking Hoxdl3, Hoxdl2, and Hoxdl 1 genes show a synpolydactyly phenotype (Zakany and Duboule, 1996).

IV. PATTERNING THE OCULAR ANLAGE A. Eye formation The vertebrate eye originates through the reciprocal interaction between two tissues early in the development. At the late headfold stage, the optic sulcus is formed by an evagination of the presumptive forebrain neuroectoderm: this grows out to form the optic vesicle, which approaches the surface ectoderm and remains connected to forebrain through the so-called optic stalk. When the optic vescicle reaches the surface ectoderm, a thickening of the surface ectoderm, also called the lens placode, becomes apparent; later on during development, this placode invaginates forming the lens vesicle and finally the lens. The optic vesicle invaginates to form the two-layered optic cup surrounding the developing lens: the outer layer of the cup forms the pigmented epithelium, the inner layer differentiates into the neuroretina. Finally, the ectoderm overlying the lens and some associated mesenchyme gives rise to the cornea. Two key points in this process are the refinement of the positional information along the proximodistal axis of the developing optic vesicle, which preceeds its subdivision into optic stalk, pigmented retina and neuroretina, and the restriction of lens forming properties to the overlying placodal ectoderm. Plenty of genes have been demonstrated to be expressed specifically in the developing eye: among them at least three homeobox genes, namely P a d , Pax6, and Chxl 0, are likely to play a key role in these patterning processes.

B. Homeobox genes and the patterning of the eye anlage As reported elsewhere, Pax2 and Pax6 display a wide and complex expression pattern (reviewed in Chalepakis and Gruss, 1996).Among the optic vesicle derivatives, Pax2 expression is confined to cells within the optic vesicle that contribute to the optic stalk and parts of the ventral retina around the choroid fissure, which is the domain of retina where ventral nasal and ventral temporal retina fuse to create the closed optic cup. Conversely, Pax6 transcripts are restricted to the op-

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tic cup, being absent in the optic stalk (reviewed in McDonald and Wilson, 1996). In zebrafish cyclops mutants, where SM expression is reduced, Pax6 expression expands toward the medial portion of the optic recess and these cells acquire some properties characteristic of retina. Conversely, in wild-type embryos microinjected with Shh synthetic mRNA complementary alterations occur. The Pax2 expression domain expands and there are more cells with features typical of optic stalk derivatives. These findings are in keeping with the idea that Pax2 and Pax6 could promote the differentiative programs specific for derivatives of optic stalk and optic cup, respectively (McDonald et al. 1995).Chxf 0 RNA is detectable in neuroblasts of the inner layer of the optic cup and in part of their derivatives (Burmeister et al., 1996). In addition, Pax6 products, originally detectable throughout the anterior ectoderm, become subsequently confined to the lens and corneal anlage (reviewed in McDonald and Wilson, 1996). A mutational analysis of PAX2 has been conducted in a human family with optic nerve colobomas, renal hypoplasia, mild proteinuria, and vesicouretral reflux, and a heterozygous point mutation has been found, causing a frame-shift of the PAX2 coding region in the octapeptide domain (Sanyanusin et d., 1995). Noticeably, optic nerve colobomas, in which the choroid fissure fails to close, occur also in mice lacking Pax2 (Torres et al., 1995; Keller et al., 1994). PAX6 has been shown to be involved in semidominant developmental disorders implying eye defects, like Aniridia in man and S d eye (Sey) in mice and rats. Heterozygous Sey-’+ mice show defects in both optic cup and placodal derivatives, like reduced eye, iris hypoplasia, and lens vacuolization and dislocation; humans affected by heterozygous Aniridia mutation display iris hypoplasia, cataract formation, and corneal vascularization (reviewed in Hanson and Van Heynigen 1995).Homozygous Sey mice lack eyes and nasal cavities and exhibit brain abnormalities (Schmal et d., 1993);in a similar way, humans compound heterozygous for mutant PAX6 alleles display bilateral anophthalmia with fused eyelids, a small malformed nose, and severe forebrain abnormalities (Glaser et d.l 1994). Blocking the expression of the homeobox gene ChxlO in zebrafish embryos results in delayed development and reduction of neuroretina and in microphthalmia (Barabino et d., 1997). Recently, an involvement of ChxlO in ocular retardation in the mouse has been reported: nonsense mutations within its homeodomain affect proliferation of retinal progenitors and differentiation of bipolar cells in the retina (Burmeister et al., 1996).

V. PATTERNING THE TOOTH ANLAGE A. Tooth development Odontogenesis takes place on both maxillary and mandibular processes of the first branchial arch. It requires the correct positioning of the odontogenetic field along

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the lingual-buccal axis of the developing branchial arch processes as well as a finer specification of medial-lateral positional values inside this field, related to the capability to give rise to different dental types (incisors, canines, premolars, and molars). The epithelium derived from the oral ectoderm forms the enamel organ, while the remainder of the tooth and its supporting tissues are derived from the mesenchyme of the first branchial arch (Lumsden, 1988). The process encompasses a complex series of epithelial-mesenchymal interactions by which the hystogenetic properties of the two forming tissues are refined and information concerning positional identities is reciprocally transferred.

B. Homeobox genes and patterning the tooth anlage It is reasonable to hypothesize that some of the homeobox genes expressed in the first branchial arch could bear positional information necessary to set up the correct onset and progression of the odontogenetic process: Otlx2/RIEG and Msxl are among them. Otlx2IRIEG expression, originally distinguishing stomodeum from other ectoderm, becomes subsequently restricted to the teeth forming areas (Mitsiadis,personal communication). The distal ectomesenchymeof first branchial arch processes is patterned by Msxl and Msx2: at the lingual border of their domains, a stripe of Msxl +/Msx2- mesenchymal cells corresponds exactly to the overlying primary epithelial thickening, namely the area from which tooth buds will develop (reviewed in Sharpe, 1995). It has been suggested that lesions in any of these genes could interfere with the onset and/or the progression of tooth formation. RIEG, the human homolog of murine RieglOtlx2, has been identified and shown to be mutated in cases of Rieger syndrome, an autosomal dominant human complex disorder that essentially includes three cardinal features, namely hypodontia (both oligo- and microdontia), developmental problems in the anterior chamber of the eye, and umbilical anomalies (Semina et d., 1996).In a humanfamily with autosomal dominant tooth agenesis mapped to chromosome 4~16.1,a missense mutation within the MSX 1 homeodomain was found to segregate at high penetrance with the defect: all affected individuals lacked both maxillary and mandibular second premolars and third molars; some of them lacked other teeth too (Vastardis et d., 1996). These findings are in keeping with data from phenotypic analysis of MsxI-/- mice (Satokata and Maas, 1994). Finally, it was suggested that Dlxl and Gsc,by patterning the mandibular ectomesenchyme along the medial-latera1 direction, could specify the type of tooth which will develop in each area (molar in Dlxl +/Gsc-,canine in Dlxl +/Gsc+,and incisor in Dlxl-/Gsc+ areas, respectively) (Sharpe, 1995). This prediction has not yet been confirmed.

Acknowledgments We thank Thimios Mitsiadis, who provided us data before their publication; we are also indebted to Wolfgang Wurst for interesting and helpful discussions. Work of our group is supported by grants from

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Telethon-Italia Programme, the EU BIOMED and BIOTECH Programmeand the Italian Association for Cancer Research, AIRC.

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Hereditary Ataxias Massimo Pandolfo*st**and Laura Montermini* *Centre de Recherche Louis-Charles Simard tDepartement de Medecine, Universite de Montreal $Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H2L 4M1 Canada

I. Introduction 32 II. The autosomal dominant progressive ataxias 34 A. Genetic classification of the autosomal dominant ataxias 35 35 B. Triplet-repeat expansions in the dominant ataxias C. Polyglutamine proteins in the dominant ataxias 39 D. Cloned dominant ataxia genes 40 E. Mapped dominant ataxia genes 47 F. Epidemiology of the autosomal dominant ataxias 49 111. Friedreich ataxia 49 A. Pathology 50 B. Clinical features 51 C. Friedreich ataxia gene 52 D. Frataxin 53 E. Point mutations in the frataxin gene 54 55 F. FRDA-associated GAA triplet repeat expansion G. Molecular mechanisms of disease in Friedreich ataxia 57 H. Phenotype-genotype correlations in Friedreich ataxia 58 60 References One of us (MP) learned about the mapping of Huntington disease gene to chromosome 4 from the late Dr. Anita Harding. She got the news over the phone from her London office during a visit to Italy for a meeting on hereditary ataxias. In Advances In Genetics, Vol. 38 Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved. @@65-266@/98 $25.00

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Britain, they receive Nature at least a week earlier than us. Dr. Harding was very excited, and she immediately said that that was the way to go if we wanted to understand the causes of hereditary ataxias, classify these diseases in a rational way, and eventually find a treatment. At that time, the challenge seemed, and indeed was, formidable. No clue was then available about the genetic basis of what Dr. Harding aptly called “hereditary ataxias of unknown cause,” their classification was confused and controversial, and all attempts to find specific biochemical abnormalities had failed. Fourteen years later, the success of the molecular genetic studies is astounding. The defective genes have been identified for Friedreich ataxia, the major recessive “hereditary ataxia of unknown cause,” and for five dominantly inherited “hereditary ataxias of unknown cause.” Three more dominant ataxia genes have been mapped. The molecular pathogenesis of the dominant ataxias begins to be unraveled and animal models have been and are being developed. Information is also quickly accumulating about the defective protein in Friedreich ataxia. Direct molecular diagnosis is now possible. Classification has been revolutionized. Diagnostic criteria are being redefined in the light of the molecular discoveries. The goal of this review, dedicated to the memory of the late Dr. Harding, is to offer a concise summary of current knowledge about the molecular genetics of some of the hereditary ataxias that used to be classified as of “unknown cause.”

The hereditary ataxias are a heterogeneous group of inherited neurodegenerative diseases whose clinical presentation is characterized by loss of coordination of movements (ataxia) as a main feature. Ataxia may be part of the picture in many neurological disorders, but it may be a late, variable, or minor problem compared to other symptoms. Conversely, in the hereditary ataxias, it is a cardinal feature appearing early in the course of the disease. The common pathological substrate of the inherited ataxias consists in the atrophy of parts of the neural system formed by the cerebellum and its connections. Many clinical and pathological variations occur on these common themes. As a consequence, classification of the hereditary ataxias has long been a problem, raising endless controversies. Many older classifications focused on pathological findings, difficult to univocally relate to clinical manifestations and impossible to substantiate in the living patient, at least before the development of the modern imaging techniques. Harding’s classification ( 1983) represented a major effort to systematize the field (Table 2.1). In her scheme, hereditary ataxias are classified according to the genetic defect when known or according to the salient clinical features. Four groups are defined: congenital ataxias, ataxias due to known metabolic defect; early-onset (<20 in most cases) ataxias of unknown

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Table 2.1. Classification of Hereditary Ataxiasa,b Congenital ataxias Congenital ataxia with mental retardation 2 spasticity (AD, AR, XR) Congenital ataxia with episodic hyperpnea, abnormal eye movements, and mental retardation (Joubert syndrome, AR) Congenital ataxia with mental retardation and partyial aniridia (Gillespie syndrome, AR) Dysequilibrium syndrome (uncertain inheritance) Inherited ataxic syndromes with known metabolic defects Intermittent ataxias with Hyperammoniemia (urea cycle defects) Aminoacidurias Disorders of pyruvate and lactate metabolism Progressive ataxias with Hexosaminidase deficiency Sphingomyelin storage disorders Cholestanolosis Leukodystrophies Mitochondria1encephalomyopathies Abeta- and hypobetaliproteinemia Isolated vitamin E deficiency Wilson disease Cerodi lipofuscinosis Sialidosis Arylsulfatase C deficiency Ataxias with defective DNA repair (ataxia teleangiectasia, xeroderma pigmentosum, Cockayne syndrome) Early onset ataxic disorders of unknown etiology Friedreich ataxia (AR) Early onset AR cerebellar ataxia with Retained reflexes (EOCA) Hypogonadism Myoclonus (Ramsey Hunt syndrome) Childhood deafness Congenital deafness Optic atrophy ? mental retardation (including Behr syndrome) Cataract and mental retardation (Marinesco-Sjogren syndrome) Pigmentary retinopathy Late onset ataxic disorders of unknown etiology A D cerebellar ataxia (ADCA) with Ophtalmoplegiajoptic atrophy/dementia/extrapyramidalfeatures (including Machado-Joseph disease) ( A K A I) Pigmentary retinopathy ? ophtalmoplegia/extrapyramidal features (ADCA 11) ‘‘Pure” ADCA of late onset (after age 50) (ADCA 111) Other progressive dominant ataxic disorders (ADCA IV) Periodic A K A ( A K A V) AR late onset ataxia “From Harding (1983). bAbbredations: AR, autosomal recessive; AD, autosomal dominant; XR, X-linked recessive.

34

Pandolio and Montemini

cause; and late-onset (>20 in most cases) ataxias of unknown cause. The great value of this classification is that it is based on data that can be obtained by clinical examination and laboratory testing, so it can be applied to the living patient. Furthermore, it is a dynamic classification,designed to change as our understanding of these diseases increases as a consequence of the progress of molecular genetic research. In this review, Harding’s classification will be utilized as reference framework, relating to it all the genetic entities that are being defined. The focus will be on some diseases that changed status from “ataxia of unknown cause” to diseases whose genetic defect has been identified. These include several dominantly inherited, progressive, usually late-onset ataxias and Friedreich ataxia, generally an early-onset disorder. Congenital, metabolic and intermittent ataxias, as well as ataxia-teleangiectasia, will not be discussed here.

II. THE AUTOSOMAL DOMINANT PROGRESSIVE ATAXIAS The dominantly inherited degenerative ataxias are probably the group whose classification has been most confused and controversial. Harding (1982, 1983, 1984) used the acronym ADCA (autosomal dominant cerebellar ataxia) to label these disorders and distinguished several types according to clinical features that are consistently observed within families. This choice led to the gathering of several genetically distinct entities under the same label, but generally prevented the opposite from happening; that is, to classify the same disease under different headings. Consequently, as ataxia genes are being mapped and cloned, it becomes possible to single-out specific genetic diseases from the each of the broader clinically defined groups identified in the Harding classification. Most dominant ataxias belong to the ADCA I group. Patients with ADCA I may show, in addition to progressive ataxia usually developing after age 20, ophtalmoplegia, optic atrophy, extrapyramidal signs, and dementia. Machado-Joseph disease was explicitly included in this group. The association of pigmentary retinopathy defines ADCA 11. This feature is either present or absent in all affected members of a family, appearing therefore to be a specific and universal manifestation of the gene defect of ADCA 11. ADCA 111is a “pure” cerebellar ataxia; that is, not associated with other neurological signs or symptoms that may indicate involvement of other structures, usually appearing after age 50. This is again a syndrome showing consistency within affected families. Other ADCAs are rare disorders, among which the intermittent ataxias (ADCA V) are notable for their clinical peculiarity of presenting as recurring acute episodes of imbalance and incoordination. The diseases included in the ADCA V group are due to mutations in specific ion channels and will not be discussed here. For dominant ataxias, it is useful to also briefly summarize other classifications, particularly those based on neuropathological findings, as they are still

2. Hereditary Ataxias

35

commonly referred to in the literature. One problem with these classifications, in addition to the practical inapplicability in the living patient, is the frequent lumping of genetic cases with sporadic, probably nongenetic cases and sometimes the lack of consideration of the type of inheritance of the genetic cases. The main neuropathological distinction is made between degenerative processes involving the cerebellar cortex and the inferior olives (cerebellar cortical or cerebelloolivary atrophy, CCA) and multisystem degenerations involving cerebellum, brain stem, pyramidal tracts, spinocerebellar tracts, and posterior columns of the spinal cord (olivopontocerebellar atrophy, OPCA) (Critchley and Greenfield, 1948). In addition, degenerative processes involving pontine nuclei, pyramidal tracts, spinocerebellar tracts, and posterior columns of the spinal cord but sparing the cerebellar cortex have been recognized (spinopontine atrophy, SPA). Each of these groups has been further subdivided. A much followed classification of OPCAs is that of Koenigsmark and Wiener (1970), who identified five subtypes (OPCA I to OPCA V). OPCA I1 is autosomal recessive, OPCA 111 corresponds to Harding’s ADCA 11, while the other hereditary OPCAs fit in the ADCA I group.

A. Genetic classification of the autosomal dominant ataxias We now know the mutated genes in several ADCAs and the chromosomal localization of a few more. These discoveries have been accompanied by the development of a system of classification in which each entity is defined on the basis of the involved gene, and it is often difficult to relate this genetic nomenclature to the previous clinicopathological classifications. Most ADCA genes are named SCA (for spinocerebellar ataxia), followed by a progressive number indicating the various involved loci, approximately in the order by which they were mapped. So, SCAl is the first ADCA gene that was localized to a specific chromosome, SCA2 the second, and so on, with the exception of SCA6, which was mapped after SCA7. Somewhat confusingly, SCA8 is instead a recessive ataxia gene. Some ADCA genes have also retained the name given to the disease when it was originally described, as is the case for Machado-Joseph disease (MJD), which coincides with SCA3. At the time of writing of this chapter, four ADCA genes have been identified (SCA1, SCA2, SCA3, and SCA6), and three more have been mapped to specific chromosomal regions (SCA4, SCA5, and SCA7).

6. Triplet repeat expansions in the dominant ataxias All so-far identified genes for ADCAs show the same type of mutation (the unstable expansion of a CAG trinucleotide repeat polymorphism localized in the protein-coding region of the respective gene). In this regard, ADCAs are akin to other neurodegenerative disorders such as Huntington disease (HD), dentatorubropallidoluysian atrophy (DRPLA), and spinobulbar muscular atrophy

36

Pandolfo and Montermini (CAG)n

(CAG)n

-rl

-

SCA 1

+

SCA 2

(CAG)n

SCA 3

-1Kb

Figure 2.1. Position of CAG trinucleotide repeats within three cDNAs from genes involved in autosomal dominant degenarative ataxias. The protein coding region in each cDNA is shown as a box, the 5‘ and 3‘ untranslated regions are shown as lines. In all three cases, the CAG repeat is localized within the protein coding region.

(SBMA, Kennedy disease) (Willems, 1994). Figure 2.1 shows the location of the SCA1, SCA2, and SCA3 repeats within the respective genes. The normal and disease-associated size ranges for each of these repeats are shown in Table 2.2. The CAG triplet repeats associated with dominant ataxias share many common features. In normal chromosomes the repeats are polymorphic, although less so in SCA2 (see below). Normal alleles are transmitted from parent to child without changes in size (i.e., they are meiotically stable). Analysis of different tissues from the same individual shows that normal alleles are of the same size in all samples (i.e., they are mitotically stable). Polymorphism in normal alleles likely originated from rare events in which, during replication of the DNA segment corresponding to the repeat, the newly synthesized strand momentarily dissociated from the template strand and reassociated out of register. This “slippage” mechanism has probably generated all simple sequence repeat polymorphisms (Wells, 1996). Disease-associated repeats contain more triplets then normal ones, from a few more to 2-3 times as many, up to about 10 times in some SCA3 cases. They are highly unstable during parent-to-child transmission, so much that size changes are seen in essentially all parent-child pairs. Mitotic instability has also been observed, so far for SCAl and SCA3, resulting in heterogeneity in the size of the expanded repeat in most tissues (Chong e t al., 1995; Lopez-Cendes et al., 1996). Although the size of disease-associated repeats varies among SCAl, SCA2, and SCA3, in these and in the other CAG repeat-containing disease genes (HD, SBMA, and DRPLA), normal alleles always have less than 35-40 triplets, suggesting that a general threshold exists beyond which CAG repeats become unstable and are liable to larger expansions. It has been proposed that such a threshold coincides with the acquisition of a secondary structure, possibly a hairpin, by the DNA strand containing a repeat of that length (Gacy et al., 1995; Wells, 1996). When this occurs in the newly synthesized strand during DNA replication, it causes a large slippage of the polymerase and reiterative DNA synthesis result-

Table 2.2. Autosomal Dominant Ataxias with Identified Triplet Repeat Expansions ~~~~

~~

~~

~

Repeats (CAG) Name

Harding classification

Chromosomal localization

Normal

Expansion

Interruptions in normal alleles

SCAl

AKA1

6p23

Ataxin 1

<38

40-85

(CAT),-,

SCA2

AKAI

12~24.1

Ataxin 2

15-32

36-55

(Cfi),-,

SCA3 & MJD

AKAI

14~32.1

Ataxin 3

<41

60-85

SCA6

AKA1

19~13

mlA

4-16

21-27

Protein

voltage dependent calcium channel

Reference(s) Orr et al. (1993) Chung et d.(1993) Puist et d.(1996) Sanpei et al. (1996) Imbert et d.(1996) Kawaguchi et al. (1994) Schols et d.(1995) Zhuchenko et al. (1997)

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Pandolfo and Montermini

ing in the addition of many repeat units. Conversely, if the template strand folds into a hairpin structure, the resulting slippage will cause the deletion of many repeat units. This hypothesis for repeat instability is supported by studies in a bacterial model (Kang et al., 1995) and by analyses of the DNA structure of triplet repeats of different lengths (Ohshima et d., 1996). Some very interesting recent studies have begun to explore triplet repeat instability in the mouse, an animal in which they were initially thought to show remarkable stability, by transgene technology (Gourdon e t al., 1997; Monckton et al., 1997; Mangiarini et al., 1997). These studies, including one on a CAG repeat from the human HD gene (Mangiarini e t al., 1997), suggest that the sequence context of a triplet repeat and the transcriptional activity of the transgene in which it is contained may play a role in its stability. The CAG repeats in normal SCAl and SCA2 alleles are almost invariably interrupted by 1-3 different triplets, CAT triplets in normal SCAl alleles (Chung et al., 1993), and CAA triplets in SCA2 alleles (Pulst et al., 1996; Sanpei et al., 1996; Imbert et d., 1996). These different triplets are likely to provide anchor points for the correct annealing of the newly synthesized and the template DNA strands during replication, preventing slippage. Instability occurs only when a single, uninterrupted run of CAG triplets exceeds the threshold. Some large normal SCAl alleles containing more than 35 triplets, for example, remain stable because CAT interruptions break their CAG repeat into several smaller repeats, each well below the instability threshold. Meiotic instability of expanded CAG repeats results in different members of the same family harboring repeats of different length. In many instances the average size of the expanded repeat tends to increase from generation to generation. Clinically, this is reflected in the phenomenon of anticipation-in the progressively earlier onset of the disease in younger generations. Indeed, in all triplet repeat-associated diseases, including the ADCAs, an inverse correlation has been demonstrated between the size of the expanded repeat and age of onset (Figure 2.2). Before the discovery that SCA genes contained expanded triplet repeats, the occurrence of anticipation in dominant ataxias was a matter of debate. While some authors believed it could be demonstrated in several families, others warned that ascertainment bias could have played a role in making it appear that the younger generations became affected at an earlier age. While the reality of anticipation in ADCAs is now generally accepted, genotype-phenotype correlation studies have shown that mechanism are not as simple as a general expansion size increase occurring at every generation. A general bias is introduced by the parental origin of the expanded repeat, as all expanded CAG repeats tend to increase in size after paternal transmission more than after maternal transmission. Accordingly, heterogeneity in repeat sizes and tendency to expansion has been confirmed in sperm analysis. Additional factors may play a role in specific diseases and will be discussed in the corresponding sections.

39

2. Hereditary Ataxias 60 -55 -50 --

15 10 5

--

--

--

A A A

+.

SCA2

*

A

8

I C A G REPEATS[

Figure 2.2. Scattergram showing the correlation between age of onset and size of the expanded CAG trinucleotide repeat in patients with SCA1, SCAZ, and SCA3.

Mitotic instability has been demonstrated for the expanded CAG repeats of SCAl and SCA3 (Chong et al., 1995; Lopez-Cendes et al., 1996). As all so-far identified SCA genes are ubiquitously expressed, mitotic instability has been evaluated as a possible cause of the specific neuropathological involvement (Lopez-Cendeset al., 1996). No evidence that affected structures harbor larger repeats was obtained for SCAl and SCA3, in accordance with the observations in similar studies of other expanded CAG repeat diseases. In fact, a slightly smaller and more homogeneous repeat was consistently found in the cerebellar cortex of both SCAl and SCA3 cases. The cerebellar cortex contains a largely predominant neuronal cell type, the cerebellar granule. I t is possible that expanded CAG repeats are more mitotically stable in neurons, while in glial cells, composing the majority of extracerebellar samples, they may be more unstable and subject to slight further expansions. In any case, this does not seem to influence the neuropathology.

C. Polyglutamine proteins in the dominant ataxias The expanded CAG repeats in SCA genes lay within their protein coding regions, in register with the open reading frame (ORF) encoding the amino acid sequence. CAG triplets correspond to the amino acid glutamine, so expansion results in the abnormal elongation of a run of glutamines in the protein product. Such an expanded polyglutamine tract is thought to confer a new activity to the protein, resulting in toxicity for some neuronal cell types (Housman, 1995). Therefore, a common pathogenetic mechanisms appears to underlay the various SCAs, which,

40

Pandolfo and Montermini

being a gain of function, is more related to the nature of the causative mutation than to the normal activity of each involved protein. However, each polyglutamine disorder has distinctive clinicopathological characteristics and a specific relationship between the length of the polyglutamine trait and the age of onset and severity of the disease (see below). Such unique features of each disease very likely depend on the nature of the protein produced by the involved gene. Recent experiments showed how, in transgenic mice, a stretch of 79 glutamines induced neuronal malfunction and death if expressed alone, but had no deleterious effect when included in the SCA3 gene product (Ikeda et al., 1996).Thus, the context in which the polyglutamine trait is located strongly affects its toxic activity. But this may be just one factor determining the specificity of each polyglutamine disease. The developmental pattern and level of expression of the protein are as well likely to affect the timing, pattern, and extent of neuronal degeneration. The mechanisms of toxicity of the polyglutamine proteins have not yet been clarified. Fibrillar precipitates have been shown to be generated in vitro by protein fragments containing an expanded polyglutamine trait (Scherzinger e t al., 1997).These may be the main component of intranuclear inclusion bodies found in transgenic mice expressing expanded polyglutamine proteins (Davies et al. 1997),as well as in Huntington disease (DiFiglia et al., 1997)and SCA3 patients (Paulson et al., 1997).It is possible that protelytic processing produces small fragments containing the expanded polyglutamine trait that are then targeted to the nucleus where they form these fibrillar aggregates together with other proteins, including ubiquitin. The mechanisms and regulation of such protelytic processing and nuclear targeting are unknown, as are the reasons why the nuclear aggregates may then be toxic for the cell. However, intranuclear inclusions seem to affect those neurons which will eventually degenerate, a process better demonstrated in the transgenic models. The role in pathogenesis, if any, of several proteins found to specifically interact with the various polyglutamine proteins (Bao et d. 1996, Burke et al., 1996, Goldberg et al., 1996) is also unknown. In this general picture, SCA6 may represent an exception (Zhuchenko et al. 1997).Despite its association to a CAG expansion, this disease may result from an abnormal function, or a loss of function, of the specific involved protein, a calcium channel subunit, rather than from the general toxic activity of polyglutamine domains, as detailed below.

D. Cloned dominant ataxia genes

1. SCAl This is the first mapped SCA, whose gene is localized on the short arm of chromosome 6. In 1974,Yakura et al. found a form of dominantly inherited ataxia to be linked with HLA. The finding was confirmed in 1977 by Jackson et al., who studied a family with ataxia and extrapyramidal features and obtained a lod score

2. Hereditary Ataxias

41

(z) of 3.15 at a recombination fraction (0) of about 0.12. Subsequent studies confirmed the existence of ADCA families showing linkage to HLA, along with others that appeared unlinked (Koeppen et al., 1980). The term SCAl was introduced after the studies of Morton et al. (1980), who identified 9 HLA-linked families out of 13 included in their analysis. Clinically, the linked families could not be unequivocally distinguished from the unlinked ones. According to Harding’s classification, they belonged to the ADCA I group, with ataxia often associated with hyperreflexia, and sometimes spasticity, scanning and explosive speech, bulbar palsies, and occasionally signs of peripheral neuropathy (Nino et al., 1980; Pedersen, 1980). Neuropathologically, atrophy of the cerebellum, with loss of Purkinje cells, of the pons, and of the inferior olives was consistently observed, suggesting that patients with SCAl have OPCA, one of the pathologically defined types of degenerative ataxia. Both OPCA I and OPCA IV, according to Koenigsmark and Wiener ( 1970), have been pathologically diagnosed in members of SCAl families. Other involved structures included the lower motor cranial nerve nuclei, the posterior columns of the spinal cord, the corticospinal tracts, and in some cases the basal ganglia. Despite their consistency, the neuropathological findings were by no means exclusive for SCA1; however, as similar findings were reported in HLA-unlinked families as well. Positional cloning of the SCAl locus gained momentum after the discovery of anonymous DNA markers, and in particular of microsatellites (Rich and Orr, 1989). Despite their high informativity, the serological polymorphisms at the HLA locus had limited power to detect linkage because of the considerable genetic distance between HLA and SCAl (Zoghbi et al. 1988). The availability of microsatellite markers on the short arm of chromosome 6 rapidly allowed to identify those closely linked to SCA1, considerably increasing the power to detect SCAl families and also allowed to precisely map the locus between flanking markers (Ranum et al., 1991; Keats et al., 1991; Zoghbi et al. 1991; Khati et al., 1993;Jodice et al., 1993). The SCAl mutation was demonstrated to consist in the expansion of a polymorphic CAG repeat by Orr et al. in 1993. Some clinical similarities between SCAl and HD, which was already known to be due to an expanded CAG repeat, prompted these authors to look for such a repeat in the candidate region. These essentially consisted in the fact that HD and SCAl are both dominant neurodegenerative disease with variable age of onset and possible occurrence of anticipation (i.e., of earlier onset of the disease in successive generations). SCAl expanded CAG repeats contain more than 40 triplets, up to about 85 (Orr et al., 1993). Normal alleles have up to 38 triplets; therefore, only a very narrow interval separates normal from pathological alleles. However, almost all normal SCAl normal alleles (98%) differ from pathological ones not only for their overall length, but also because they are interrupted by one or more CAT triplets (Chung et al., 1993). Interruptions in the run of CAGs are thought to exert a stabilizing effect by acting as anchors that prevent strand slippage during DNA replication, a likely mechanism of instability of simple repeat sequences (Wells, 1996).The repeat is meiot-

42

Pandolfo and Montermini

ically unstable; that is, it often changes in size during parent-to-child transmission (Chung e t al., 1993). Larger repeats, with more than 54 triplets, are preferentially transmitted by affected fathers (Jodice e t al., 1994), and paternal transmission more often results in a further increase in size (Chung e t al., 1993; Matilla et al. , 1993). As previously shown for HD and SBMA, in SCAl a strong inverse relationship exists between the size of the CAG repeat and age of onset. This finding, already described by Orr et al. (1993), has been confirmed in subsequent studies in diverse populations (Matillaet al., 1993;Ranum et al. , 1994;Jodice et al., 1994; Giunti et al., 1994, Suzuki et al., 1995; Goldfarb e t al., 1996). However, some residual interfamilial variability persists after correcting for CAG repeat size (Ranum e t al., 1994), suggesting that other factors may play a role in determining the severity of phenotype. The availability of a direct molecular test for SCAl allowed to identify several autopsy specimens with SCAl and to determine the associated neuropathology. SCAl cases appear to be rather homogeneous in their pathological features, all having the typical findings of OPCA (Genis e t al. , 1994; Robitaille et al., 1995). The SCAl gene is transcribed into a 10,660-nt mRNA and encodes an 87-kDa protein, ataxin-1 (Banfi et al., 1994). The gene has 9 exons, and it its genomic organization is very peculiar, as most exons contain untranslated sequences, including a 7,277-bp 3’ untranslatedregion (UTR) in exon 9, one of the longest 3’ UTR described so far. The 5 ’ UTR is spread over the first seven exons, which also undergo alternative splicing in different tissues, suggesting a complex regulation of the expression of the SCAl gene (Banfi e t al., 1994). The CAG repeat is in the first part of the coding region. It encodes a stretch of glutamines in ataxin-1. The corresponding mouse gene (Banfi e t al., 1996) is very similar to its human counterpart, but has virtually no CAG repeat, suggesting that it is not necessary for the evolutionarily conserved function of ataxin-1. The mouse gene is expressed during development, with a peak at embryonic day 14 (E14), its mRNA being most abundant in the cerebellum and in the vertebral column (Banfi et al. , 1996). In adult humans, the ataxin-1 transcript is found ubiquitously (On e t d., 1995). The development of anti-ataxin-1 antibodies has allowed us to determine its intracellular localization. Ataxin-1 is nuclear in all cell types with the exception of Purkinje cells in the cerebellar cortex, where it is cytoplasmatic as well as nuclear (Servadio et al., 1995). Purkinje cells degenerate in SCA1, so this finding may bear some relation with the pathogenesis of the disease, although other degenerating cells apparently express ataxin- 1 in the nucleus only. Western blot studies also revealed that, in patients, abnormal ataxin- 1, containing the expanded polyglutamine trait, is expressed along with the protein encoded by the normal allele, in comparable amounts (Servadio et d., 1995). Transgenic mice expressing either the normal human SCAl gene or a

2. Hereditary Ataxias

43

human gene containing an expanded CAG repeat have been generated (Burright et al., 1995). The CAG repeat appeared to be stable during parent-to-child transmission in the mouse. All mice strains bearing a normal human ataxin-I transgene remained well and had no evidence of ataxia or cerebellar degeneration. Conversely, five out of six strains expressing human ataxin-l with an expanded polyglutamine trait developed ataxia with cerebellar atrophy and Purkinje cell death during adult life.

2. SCA2 The term SCA2 was initially used for families unlinked to chromosome 6p (Lazzarini et al., 1992). However, it designated a precise locus on chromosome 12q23q24.1 after the mapping to this region of a form of dominant ataxia observed in the vicinity of Holguin, Cuba (Gispert et al., 1993). The Cuban families were all related and had a common Spanish ancestry (Orozco-Diazet al., 1989). Clinically, they had a progressive ataxia with peripheral neuropathy, slow ocular movements, and no extrapyramidal signs (Orozco-Diaz et al., 1990; Auburger et al., 1990). Additional families of Italian (Pulst et al., 1993), German, French-Canadian (Lopes Cendesetal., 1994),Tunisian (Belal etal., 1994), and Japanese (Ihara et al., 1994) origin were subsequently linked to the chromosome 12 locus. There phenotype did not appear to have specific features, as some subjects were indistinguishable from individuals with proven SCA 1, others had been diagnosed with Machado-Joseph disease (see below), and a few had a prominent dementia that was absent in most cases. Anticipation could be demonstrated in SCAZ pedigrees (Pulst et al., 1993). Tissue extracts from SCAZ patients were analyzed by Westem blotting using a monoclonal antibody (1C2) directed against proteins with an expanded polyglutamine trait (Trottier et al., 1995). These studies revealed that SCAZ patients produce one such protein, indicating that this disease is caused by the expansion of a CAG (or CAA) trinucleotide repeat in the coding region of the corresponding gene. Recently, three research groups independently identified the SCAZ gene, confirming that in patients it carries an expanded CAG repeat. Pulst etal. (1996) cloned the 1.1-megabase (Mb) SCA2 candidate region into P1 and bacterial artificial chromosome (BAC) clones, and searched for expanded CAG repeats. Sanpei et al. (1996) utilized a novel approach that they called DIRECT (direct identification of repeat expansion and cloning technique) to detect and clone an expanded CAG repeat in SCAZ patients. DIRECT consists of the identification in Southern blots of patients’ genomic DNA of expanded CAG repeats by using a high-specific-activity probe that will not hybridize to normal length repeats, followed by the recovery from gels and cloning of the hybridizing fragment. Finally, Imbert et d. (1996) utilized the above-mentioned 1C2 monoclonal antibody to screen expression cDNA libraries constructed from lymphoblasts of SCAZ patients. Although, unexpectedly given the characteristics of

44

Pandolfo and Montermini

the antibody, from their screenings they only obtained clones with up to 22 CAG repeats; one of these resulted to be expanded in SCA2 patients. The SCAZ CAG repeat shows limited polymorphism in normal chromosomes, with most alleles containing 22 triplets (90%), a few containing 23 repeats (5%), and the remaining ones subdivided into more than 15 alleles, ranging from 15 to 32 repeats (Pulst et al., 1996; Sanpei et al., 1996; Imbert et al., 1996). Normal SCAZ alleles are interrupted by 1-3 CAA triplets, resembling the SCAl normal alleles, except that in that case the interrupting triplet is CAT. Disease-associated alleles have 36 or more triplets, up to about 55, and longer alleles are associated with earlier onset and more severe course. It is interesting that expanded SCA2 alleles appear on average to be shorter than SCAl (and SCA3, see below) alleles. Also the length threshold for SCAZ repeats to exert their pathological effect seems lower than in the case of SCAl and SCA3. Testing autopsy specimens for the SCA2 repeat revealed a great heterogeneity in the neuropathological findings, ranging from severe OPCA with involvement of additional structures, as the substantia nigra, to an almost pure cerebellar atrophy (Lopes Cendes, personal communication). Such heterogeneity of pathological findings may explain why no distinct clinical feature could be found in SCAZ patients.

3. SCA3/Machado-Joseph disease (MJD) The case of SCA31MJDwell illustrates the difficulties and shortcomings that clinicopathological classifications of dominant ataxias necessarily had before gene information became available. SCA3 was introduced by French researchers who mapped a dominant ataxia locus to chromosome 14q24.3-qter by performing linkage analysis in phenotypically ADCA I families that were unlinked to either chromosome 6 or chromosome 12 (Stevanin et al., 1994). Those authors made the explicit remark that no specific clinical feature allowed to differentiate the chromosome 14-linked, SCA3 families from other ADCA I families. At about the same time, a locus for MJD was mapped to the same region of chromosome 14 (Takiyama et al., 1993). MJD is an autosomal dominant ataxia originally described in Portuguese-American families (Woods and Schaunburg, 1972; Rosenberg et al., 1976; Romanul et al., 1977; Rosenberg and Fowler, 1981). The Portuguese families came from the Azorean islands, particularly those of Flores and Sao Miguel, and from Northern mainland Portugal. The presence of eyelid retraction, causing the appearance of “bulging”eyes, and of facial and lingual fasciculations was considered characteristic of MJD (Lima and Coutinho, 1980). Clinically, three forms were distinguished (Coutinho and Andrade, 1978): type 1 with ataxia, ophtalmoplegia, and pyramidal signs; type 2 with ataxia and extrapyramidal signs; and type 3 with ataxia and neuropathy causing distal amyotrophy. The three forms could be observed in the same family, with type 2 characterizing ear-

2. Hereditary Ataxias

45

ly-onset severe cases and type 3 late-onset milder cases. There was evidence for anticipation. Based on the above clinical features, the disease was subsequently diagnosed in families of non-Portuguese descent, including Italian-American (Livingstone and Siqueiros, 1984), Japanese (Sakai et al., 1983), and AfricanAmerican (Healton et al., 1980).It has been speculated that the presence of MJD in Japan is the consequence of the introduction of the defective gene by Portuguese sailors. Pathologically, MJD was considered to be a spinopontine atrophy; that is, an atrophy of nuclei pontis, cranial nerves, spinocerebellar tracts, Clarke’s column, and anterior horns often involving also the substantia nigra and characteristically sparing the inferior olives and the cerebellar cortex (Coutinho et al., 1982). The possibility of an unequivocal clinical diagnosis of MJD was, however, questioned by several authors. In particular, Harding (1983) classified MJD as an ADCA I, feeling that a clinical differentiation from other entities in the group was impossible. The results of linkage analysis immediately suggested that SCA3 and MJD map to the same locus. The discovery that SCA3 and MJD patients carry the same CAG trinucleotide repeat expansion confirmed that the two entities are due to the same mutation in the same gene. The MJD gene was identified by Kawaguchi et al. (1994, who mapped a cDNA containing a CAG repeat to chromosome 14 and found the CAG repeat to be expanded in Japanese patients diagnosed with MJD. The same CAG trinucleotide expansion was then found in patients from families classified as SCA3 (Schols et al., 1995). The SCA3/MJDCAG repeat normal and expanded alleles are characteristically larger than SCAl or SCA2 alleles. Patients have more than 60 triplets, up to about 85. Normal alleles can be as large as 41 repeats (Giunti et al., 1995). A large gap is present between the distributions of normal and expanded alleles (Takiyama et al., 1995; Ranum et al., 1995), contrary to SCA1, where the two distributions touch each other. Expanded alleles are meiotically unstable and may further expand, particularly after paternal transmission (Maruyama et al. , 1995; Igarashi et al., 1996). A correlation exists between age of onset and severity of disease and size of the expanded allele, as earlier onset, type 3 cases have significantly larger expansions (Giunti et al., 1995; Maruyama et d., 1995; Takiyama et al., 1995). However, other factors contribute to clinical variability, including gender, which appears to act independently of expansion size in determining age at onset (Kawakami et al., 1995). In addition, a bias seems to exist that favors the transmission of mutant alleles in male meioses (Ikeuchi et d . , 1996). A few homozygous patients have been observed for the SCA3/MJDexpanded repeat. These patients have much earlier onset and more severe disease then predicted by the size of their expanded alleles, indicating a dosage effect (Takiyama et al., 1995). This characteristic differentiates SCA3/MJDfrom other dominant CAG repeat expansion disease, as HD, where homozygotes appear to be as affected as heterozygotes. Availability of a direct molecular test for SCA3/MJD allowed to establish the diagnosis in large series of patients and also in autopsy cases (Takiyama et

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al., 1994). Some studies of this kind concluded that no specific clinical or pathological feature could be associated to this mutation (Giunti et al. 1995; Cancel e t al., 1995; Diirr e t al., 1996). Some authors found that even signs considered almost pathognomonic, as bulging eyes, were not significantly more frequent than, for example, in SCAl cases (Giunti e t al., 1995; Durr et al., 1996). Neuropathological analysis revealed cases with confirmed molecular diagnosis and unusual features, including atrophy of the inferior olives and of Purkinje cells in the cerebellar cortex. It is still, however, debated if MJDhas specific features allowing differentiation from other dominant ataxias and even from SCA3. Accepting that MJDpatients have a specific phenotype calls for an explanation of why SCA3 patients, despite harboring the same mutation, should be different. The effect of polymorphisms in the same gene has been invoked, but not yet demonstrated, by supporters of this distinction. The SCA3/MJD gene contains the CAG repeat in the coding region, so it also encodes a polyglutamine trait. Differently from SCAl and -2, the repeat is close to the 3’ end of the gene. The encoded protein, ataxin-3, does not bear any resemblance either to ataxin-l or -2 or to a n y protein of known function. Like ataxins-1 and -2, it is ubiquitously expressed. An apoptosis-inducing effect has been demonstrated in cultured cells for a portion of ataxin-3 that includes the expanded repeat. Transgenic mice expressing the gene with an expanded repeat (79 CAGs) did not develop ataxia, but those expressing only a polyglutamine stretch of the same length did (Ikeda et d., 1996). These experiments, while confirming that a toxic gain-of-function conferred by expanded polyglutamine traits is very likely to play a critical role in the pathogenesis of neurodegeneration in this and other CAG repeat-associated diseases, underscore the importance of the context where the polyglutamine trait is located. The presence of larger size expansions in SCA3/MJD can thus find an explanation in the “protective”effect exerted by the ataxin-3 protein, resulting in a shift of the expansion size-age at onset regression curve.

4. SCA6 The SCA6 symbol for some time has not designated any specific ataxia gene, as it was occasionally used for families unlinked to the SCA1, -2, or -3 loci (Lopes Cendes et al. 1994). It now designates a specific ataxia locus on chromosome 1 9 ~ 1 3corresponding , to an a l Avoltage-dependent calcium channel subunit gene (CACNLlA4) (Zhuchenko et al., 1997). The SCA6 gene was identified by screening a human brain cDNA library with a (CGT), oligonucleotide to isolate clones containing transcribed CAG repeats. One of the positive clones was shown to correspond to an isoform of the CACNLlA4 mRNA and had a CAG repeat in its open reading frame. The repeat contained 4 to 16 triplets in control subject, but had 21 to 27 repeats in eight unrelated individuals with late-onset ataxia.

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Linkage analysis of their families confirmed the segregation of ataxia with a larger repeat. Very interestingly, two different human disorders are due to mutations in the same gene: hereditary paroxysmal cerebellar ataxia (HPCA), or episodic ataxia (EA), and familial hemiplegic migraine (FHM). In these disorders, either missense mutations (in FHM) or mutations causing premature termination of the coding sequence (in HPCA/EA) are found (Ophoff et al., 1996). Additionally, the tottering ( t g ) mouse, affected by seizures and ataxia, has mutations in the homologous gene that cause abnormal splicing and protein truncation (Fletcher e t al., 1996). SCA6 patients have a slowly progressive ataxia with mild proprioceptive and vibratory sensory loss. The pathology consists in severe cerebellar atrophy with loss of Purkinje and granule cells and mild atrophy of the brainstem, with limited involvement of the inferior olives (Subramony et al., 1996; Zhuchenko et al., 1997). Therefore, the clinical and pathological features in SCA6 patients are different from those observed in HPCA/EA and in FHM, although some FHM patients may develop a progressive ataxia with cerebellar atrophy (Vahedi et al., 1995). Disease-associated SCA6 repeats are much smaller than those found in other SCAs. No transmission instability is observed, consistent with pathological SCA6 repeats being in the same size range as normal, stable repeats in other loci. However, affected individuals in families segregating larger repeats had onset of symptoms at a significantly younger age than those from families with smaller repeats, confirming also in this case the generally observed inverse correlation between repeat size and age of onset (Zhuchenko et al., 1997).The pathogenesis of SCA6 is still unclear. The question remains open if the expanded polyglutamine trait in the a I Avoltage-dependent calcium channel subunit exerts a toxic effect similar to that of other polyglutamine traits or if it directly interferes with the function of this ion channel. Voltage-dependent calcium channels have an important role in excitability, neurotransmitter release, and control of gene expression in a variety of cells (Catterall, 1995). The involvement of the aIAvoltagedependent calcium channel subunit in HPCA/EA, FHM, and in the tg mouse, as well as a wealth of neurophysiological and neuropharrnacological observations underscore its specific importance in the normal functioning and trofism of the cerebellar system.

E. Mapped dominant ataxia genes

1. SCA4 This form of dominant ataxia was mapped to chromosome 16q in a single, fivegeneration family (Gardner et al., 1994). The clinical phenotype seems characteristic, as all affected individuals have ataxia associated with a peripheral neuropathy of the sensory axonal type (Flanigan et al., 1996). Age of onset is variable. The SCA4 gene is still unidentified.

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2. SCAS Linkage analysis of a very large family, related to President Lincoln’s, led to the localization of an autosomal dominant ataxia locus, called SCA5, to the pericentromeric region of chromosome 11 (1l p l l - q l l ) (Ranum et al., 1994). The clinical picture is that of an almost pure cerebellar ataxia, usually not severely disabilitating. Age of onset was variable and anticipation clearly occurred. Attempts to clone the relative gene are under way.

3. SCA7 Autosomal dominant ataxia with retinal degeneration has long been recognized as a distinct entity because all patients in affected families show both manifestations, while retinal degeneration is seldom, if ever, observed in other ADCA families (Froment et al., 1937; Havener, 1951; Bjork et al., 1956). Harding classified this disease in a specific group, ADCA 11. So far, all studies of ADCA I1 families, regardless of ethnic background, have shown linkage to markers on chromosome 3p21.1-pl2, defining a locus called SCA7 (Benomar et al., 1995; Gouw et al., 1995; Holmberg et al., 1995). Clinically, ADCA I1 patients present with progressive ataxia, dysarthria, and pyramidal signs rarely with extrapyramidal signs, peripheral neuropathy, or dementia (Anttinen et al., 1986; Enevoldson et al., 1994, Gouw et al., 1994). Visual impairment is typically an early manifestation, more so in patients with lower age of onset. Tritan (blue-yellow) color blindness has been reported to be an initial symptom (Gouw et al., 1994). As such impairment is otherwise very rare, it seems to be a rather specific manifestation of this disease. Even in members of the same family, retinopathy may be macular or peripheral (Enevoldson et al., 1994). Pathologically, the picture is that of an OPCA with associated loss of retinal ganglion cells (Koenigsmark and Wiener, 1970). Variability of age of onset and rate of progression may be striking in ADCA I1 families. Some cases, although obligate carriers of the mutation, have no symptoms, others have a mild, late-onset disease, and, at the other extreme, others have an extremely severe, childhood-onset disease leading to early death. Anticipation is obvious in some pedigrees, particularly after paternal transmission (Enevoldson et al., 1994, Benomar et al., 1994)). Western blot analysis of cell extracts from two unrelated SCA7 patients using the 1C2 monoclonal antibody revealed the presence of a 130-kDa protein in both of them (Trottier et al., 1995). This experiment indicated that this disease is highly likely to be caused by the expansion of a glutamine-encoding CAG or CAA repeat. Expansion of a CAG repeat was subsequently found in SCA7 patients using a method that detects such expansions regardless of their genomic location (Lindblad et al., 1996). This method (repeat expansion detection, RED) makes use of a modified ligase chain reaction to selectively amplify large repeats

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of a specific simple sequence from genomic DNA. Recently the gene for SCA7 has been identified (Davis et al., 1997).

F. Epidemiology of the autosomal dominant ataxias The overall frequency of ADCA has been estimated between 0.3 and 1.2 X lop5 in various populations (Filla et al., 1993), with the exception of the already-mentioned Holguin region in Cuba, where a founder effect for SCA2 results in a much higher prevalence (41 X (Orozco-Diazet al., 1990). The availability of direct molecular tests for several ADCAs has allowed us to conduct surveys, both in living patients and in pathology specimens, to establish the proportion of dominant ataxia cases attributable to each identified entity. In general, the cloned ADCA genes now account for the majority of ADCA cases. Results of these studies differed when different populations were examined. Ranum et al. (1995) investigated patients from 31 1 families of varied origin and found 3% of them carrying the SCAl mutation and 21% carrying the SCA3 mutation. Among 92 unrelated ADCA cases, Silveira et al. (1996) found the SCAl mutation in 3% and the SCA3 mutation in 41%. In the latter study, when patients of Portuguese ancestry were excluded, the percentage of SCAl cases increased to 10% and that of SCA3 decreased to 17%. SCAl seems to be particularly common among Italian patients (Jodice et al., 1994; Giunti et al., 1994), accounting for up to 30% of the cases, while no SCA3 cases have so far been identified in Italy. Conversely, up to half of the ADCA cases in patients of German background carry the SCA3 mutation (Schols et al., 1995). Preliminary results with the recently-isolated SCA2 gene suggest that this is the second most common form of dominant ataxia in essentially all populations. Expanded CAG repeats in the SCAl and SCA3 genes have also been searched in sporadic cases of degenerative ataxia, whose clinical and pathological features resemble those of the hereditary cases. These studies excluded that sporadic ataxia cases result from new expansions of these triplet repeats (Ranum et al., 1995). ~~

111. FRlEDRElCH ATAXIA Friedreich ataxia (FRDA) is the most common of the hereditary ataxias, which seems to have a fairly similar prevalence of around 2 X in almost all studied populations, with local clusters due to a founder effect (Skre, 1974; Bouchard et al., 1979; Romeo et al., 1983; Harding, 1983; Leone et al., 1990;Hirayama et al., 1994). The disease was first described in 1863 by Nicholaus Friedreich, Professor of Medicine in Heidelberg. Friedreich’s papers already reported all the essential clinical and pathological features of the disease, a “degenerative atrophy of the

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posterior columns of the spinal c o r d leading to progressive ataxia, sensory loss, and muscle weakness, often associated with scoliosis,foot deformity, and heart disease. For many years, however, a clear definition of FRDA remained a matter of debate. Only in the late 1970s did a renewed interest in the disease lead to the establishment of diagnostic criteria, first by the Quebec Collaborative Group (Geoffroy e t al., 1976), then by Harding (1981). The authors of these rigorous clinical studies, while attempting to define the constant features of FRDA, noticed how the disease may be variable in its manifestations, sometimes even within the same sibship, a rather uncommon finding for recessive disorders. Variability includes age of onset, rate of progression, severity, and extent of disease involvement. Unusual for a recessive disease, variability within the same sibship is not uncommon. Atypical cases, missing one or more of the essential diagnostic features, but with an overall FRDA-like phenotype, can also be identified. If these occur in sibs of typical FRDA patients, they are clearly extreme examples of the clinical spectrum of FRDA, but their classification is uncertain when they occur as isolated cases or cluster in families. Examples include Acadian FRDA, observed in a specific population of French origin living in North America, which has a milder course than classical FRDA and is rarely accompanied by a cardiomyopathy (Barbeau e t al., 1984; Richter et al., 1996); late-onset Friedreich ataxia (LOFA), a disease with all FRDA features but onset after 25 years of age (Klockgether et al., 1993; De Michele et al., 1994); and Friedreich ataxia with retained reflexes (FARR), a variant in which tendon reflexes in the lower limbs are preserved (Palau e t al., 1995). After the identification of the FRDA gene and of its most common mutation, the unstable hyperexpansion of a GAA triplet repeat polymorphism, genotype-phenotype correlations became possible, clarifying these issues and bringing important consequences to the diagnostic criteria for FRDA.

A. Pathology The neuropathological features of FRDA are specific for this disorder and differ from those found in other hereditary ataxias (Lamarche e t al., 1984). Demyelination, loss of fibers, and fibrillary gliosis are observed in the posterior columns of the spinal cord, particularly in the medial tract (originating at the lumbar level), in the spinocerebellar tracts, and in the crossed and uncrossed corticospinal tracts. Clarke’s columns are atrophied, while motor neurons in the ventral horns are not affected. The long tracts of fibers in FRDA, particularly the corticospinal fibers, are more severely affected in their distal portions, suggesting a “dying back” process (Said e t al., 1986). In the brainstem, the gracile and cuneate nuclei, where the dorsal columns of the spinal cord terminate, are severely atrophic. The entering roots of cranial sensory nerves are affected, as well as the auditory and vestibular systems, while cranial motor nuclei are normal. In the cerebellum, the

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cortex is spared until late in the disease course, when Purkinje cell loss can be observed, but the dentate nuclei are severely atrophied. In the peripheral nervous system an axonal sensory neuropathy is the characteristic finding. There is loss of large primary sensory neurons in the dorsal root ganglia, which is considered an early finding (Hughes et al., 1968; Ouvrier et al., 1982). The heart is typically affected by an hypertrophic cardiomyopathy (Harding and Hewer, 1983) that becomes dilatative in some advanced cases (Casazza and Morpurgo, 1996). Microscopically, normal and hypertrophic cardiomyocytes are intermingled with atrophic fibers (Pentland and Fox, 1983).Calcium and iron deposits have been reported (Lamarche et al., 1993).

8. Clinical features The usual age of onset of FRDA is around puberty, but it may vary from 2-3 years to later than 25. Variability is more pronounced among families than within families (Andermann et al., 1976; Winter e t al., 1981), but occasionally large differences among sibs are observed. Progressive, unremitting ataxia is the cardinal feature of the disease. Dysarthria follows within 5 years. Progressive muscular weakness is mostly a consequence of the degeneration of the corticospinal tracts. Loss of position and vibration senses reflect the degeneration of sensory fibers in peripheral nerves and in the spinal cord. Absence of tendon reflexes in the lower limbs has been included in the diagnostic criteria both by the Quebec Collaborative Group (Geoffroy et al., 1976) and by Harding (1981). However, a minority of patients, who recently were shown to carry the molecular abnormality of FRDA, maintain elicitable reflexes for several years after diagnosis (Friedreich ataxia with retained reflexes, FARR) (Diirr et al., 1996; Montermini et al., 1997b). Rare, molecularly proven cases even have exaggerated reflexes and spasticity (Montermini et al., 199713).Extensor plantar responses (Babinski sign), due to corticospinal tract involvement, are essential for the diagnosis. Variable neurological findings include optic atrophy in 30% of patients and sensorineural hearing loss in 20% of patients. They tend to be associated in the same patients, probably reflecting a more severe, widespread disease (Harding, 1981). More than half of the patients have scoliosis, usually slowly progressive, and associated with a kyphotic curve (Allard et al., 1982; Labelle et al., 1986). About half of the cases have pes cavus, pes equinovams, and clawing of the toes (Harding, 1981). Amyotrophy of small hand muscles and of distal leg and foot muscles is common (Harding, 1981). About 10% of FRDA patients are diabetic (Harding, 1981; Finocchiaro et al., 1989), and show a loss if islet cells without the signs of autoimmune aggression found in type I diabetes (Schonle et al., 1989).

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Only some of the patients with cardiomyopathy have clinical manifestations of heart disease. The electrocardiogram (EKG), showing widespread Twave inversions and signs of ventricular hypertrophy, seems to be the most sensitive test for FRDA cardiomyopathy (Harding and Hewer, 1983; Aboliras e t al., 1986). Echocardiography shows concentric hypertrophy of the ventricles (62%) or asymmetric septa1 hypertrophy (29%) (Harding and Hewer, 1983). Magnetic resonance imaging (MRI) or computerized tomography (CT) show atrophy of the cervical spinal cord, with essentially normal brainstem, cerebellum, and cerebrum (Wullner et al., 1993). Interestingly, PET studies have revealed an increase in brain glucose metabolism in patients who can still walk. This yet-unexplained finding appears to be specific for FRDA (Gilman et al., 1990). Nerve conduction studies indicate the presence of an axonal sensory neuropathy by showing very small or absent sensory action potentials (SAPS), with normal or almost normal motor and (when measurable) sensory conduction velocities (Ackroyd et al., 1984).

C. The Friedreieh ataxia gene The FRDA gene was found after a long search that lasted for more than 7 years after linkage to chromosome 9 was established (Chamberlain et al., 1988; Fujita et al., 1989). The now-classic positional cloning approach was followed. The FRDA locus was finely mapped in relation to the closely linked markers (Fujita et al., 1990; Pandolfo e t al., 1990; Chamberlain et al., 1993), a candidate region was defined and cloned (Rodius et al., 1994; Duclos et al., 1994; Montermini et al., 1995), and genes were searched using exon trapping, cDNA direct selection, and computer analysis of genomic sequences (Campuzano et al., 1996). Eventually, only one gene, called X25, was found in the minimum genetically defined candidate interval (Campuzano et al., 1996). X25 is composed of seven exons spread over 95 kb of genomic DNA. The first five exons, 1 to 5a, are within a 40-kb interval. Exon 5b follows after a large intron of 40 kb, then exon 6 is a further 15 kb downstream. The gene is transcribed in the centromere to telomere direction. The first exon has a transcription start site preceded by several in-frame stop codons, and it harbors an unmethylated CpG island, containing several rare restriction sites. Alternative splicing occurs at the 3’ end of the gene. The most abundant transcript goes from exon 1 to 5a. Less-abundant isoforms contain exon 5b instead of 5a, followed or not by exon 6. The use of exon 5a or 5b introduces differences in the amino acid sequence of the encoded protein at its carboxy terminus. Exon 6 is entirely noncoding. The FRDA gene demonstrates tissue-specific expression (Campuzano et al., 1996; Jiralerspong et al., 1997). In human adult tissues, its mRNA is most abundant in heart, followed by liver, skeletal muscle, and pancreas (Campuzano e t d., 1996). Expression is minimal in other tissues, including whole brain. The

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major transcript has a size of 1.3 kb, corresponding to an exon 5a-containing mRNA. Fainter bands of 1.05- and 2.0-kb bands correspond to exon 5b- and exon 5bf6-containing mRNAs, respectively (Campuzano et al., 1996). Two larger bands, of 2.8 and 7.3 kb, appear to derive from a transcribed pseudogene. In the human adult central nervous system (CNS), the 1.3-kb transcript is highly expressed in the spinal cord, at lower levels in the cerebellum, and very little in the cerebral cortex. Overall, expression of X25 appears to be highest in the primary sites of degeneration in FRDA, both within and outside the CNS. The developmental expression of the gene has been investigated in the mouse by RNA in situ hybridization (Jiralerspong et al., 1997). Expression is negligible until embryonic day 10 (ElO), then it progressively increases until E14, remaining stable afterward. The transcript is present in the brain, both in proliferating cells in the subependimal layer, and in more mature cells in the developing forebrain. The spinal cord shows a high expression, particularly in the toracolumbar region, starting around E12.5. Active transcription also occurs in large neuronal cells in the dorsal root ganglia (DRG), starting at E12.5. Among extraneural tissues, the mRNA is found in the developing heart , in groups of pancreatic cells, in the skin, and in the developing teeth.

D. Frataxin Frataxin is the protein encoded by the FRDA gene. The isoform encoded by exon 5a-containing mRNA appears the most abundant and physiologically significant, as it shows a striking degree of evolutionary conservation (Campuzano et al., 1996). Frataxin’s function could not be inferred by its amino acid sequence, as no similarity with protein domains of known function could be detected. The impressive evolutionary conservation of a portion of the frataxin molecule became more and more apparent as its homologs in different species were identified. A stretch of 27 amino acids, encoded by exons 4 and 5a, is entirely identical in the mouse, has 25 identical amino acids in D. mehogaster and in C . elegans, and 22 identical amino acids in S. cerevisiue, with most substitutions being conservative changes. In addition to this highly conserved domain, most of the frataxin sequence is conserved in evolution, with several invariant amino acids throughout its length. Most divergence are observed in the amino-terminal, exon 1-encoded portion. The CyaY proteins from Gram-negative bacteria also show a significant degree of similarity to frataxin’s most conserved region. The degree of similarity of these bacterial proteins is too low to indicate conservation of function, but it has suggested a common origin. Since, according to a prevailing theory, mitochondria derive from an ancestral intracellular symbiont related to purple bacteria, it has been proposed that frataxin might be a mitochondria1 protein whose gene has moved to the nuclear genome during evolution, as happened for most

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originally mitochondrial genes (Gibson et al., 1996). In support of this hypothesis, computer analysis has suggested the presence of a mitochondrial targeting signal in the frataxin homologs from most species, despite the heterogeneity of their amino-terminal sequences. Experimental evidence is now confirming the mitochondrial localization of frataxin (Campuzano et al., 1997). Anti-frataxin antibodies, both polyclonal and monoclonal, have recently been generated and used for immunofluorescenceand immunoelectron microscopy studies, confirming the mitochondrial localization. Experiments not relying on antibodies have also indicated that frataxin is targeted into the mitochondria. They consisted in adding tags as P-galactosidase (p-Gal) or green fluorescent protein (GFP) to the frataxin molecule in order to trace it within cells. Western blot analysis showed in all examined tissues (brain, cerebellum, spinal cord, skeletal muscle, and lymphoblasts) a single band of about 21 kDa. This is slightly less than the predicted sizes of frataxin based on the amino acid sequence (23.5 kDa), in accordance with a posttranslational processing involving the removal of a signal peptide. Western analysis with a monoclonal antibody directed against an exon 1 epitope has confirmed its absence from the mature protein. Accordingly, addition of frataxin exon 1 to the amino terminus of P-Gal or GFP is sufficient to target these proteins to the mitochondria. By disrupting the yeast homologous gene (yeast frataxin homolog, YFHI), which also encodes a mitochondrial protein, it appears that the protein is involved in iron homeostasis (Babcock et al., 1997). The yeast strain with deleted YFHl (AYFHI ) becomes unable to carry out oxidative phosphorylation. Total iron content is souble in AYFHI cells compared to wild-type and mitochondrial iron is 10-fold higher than in wild-type cells. No differences are observed in the mitochondrial content of copper or calcium. Notably, the AFT-1”P strain that has constitutive expression of the high affinity iron uptake system due to a mutation in the transcriptional regulator AFT1 and shows intracellular iron levels comparable to AYFHI, does not show an increase in mitochondrial iron nor a respiratory growth defect. This indicates that the accumulation of mitochondrial iron is specifically associated with the deletion of YFHI, and is not simply a consequence of increased cellular iron uptake. Excess iron in mitochondria, by reacting with oxygen, causes the oxidation of cellular components and ultimately irreversibly damages the cell. Several clues indicate that the yeast model may be relevant for the human disease. Iron deposits are a specific finding in myocardial cells from FRDA patients (Lamarche et al., 1993). Vitamin E deficiency produces a phenotype that resembles FRDA. Vitamin E localizes in mitochondria and is effective in protecting against the free radicals generated by iron. The observed dysfunction of iron-sulfer center containing enzymes in tissues affected by FRDA (Rotig et al., 1997) represent another expected consequence of oxidative stress.

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E. Point mutations in the frataxin gene Point mutations in the frataxin gene are in only about 2% of the FRDA chromosomes (Campuzano et al., 1996). Missense, nonsense, and splice site mutations have been identified (Campuzano et al., 1996; CossCe et al., 199713). Despite their rarity, the identification of these point mutations have been essential in establishing that X25 is the FRDA gene. In all cases characterized so far, affected individuals were heterozygous for their point mutation, with a normal frataxin coding sequence on the other homolog of chromosome 9. None of these patients had distinctive phenotypic features. The lack of homozygotes for point mutations is most likely a consequence of their rarity, but there is the possibility that homozygotes for frataxin null mutations are not observed because they have a very severe or lethal phenotype not directly resembling FRDA.

F. The FRDA-associated GAA triplet repeat expansion The vast majority of FRDA chromosomes (98%) harbor an abnormal GAA repeat expansion within the 12-kb first intron of the frataxin gene (Campuzano et al., 1996; Filla et al., 1996; Durr et al., 1996; Montermini e t al., 1997b). The triplet repeat is localized 1.4 kb after exon 1, in the middle of a an Alu-Sx repetitive sequence (Figure 2.3). It apparently derived from the expansion of the canonical A5TACA6 sequence linking the two halves of Alu sequences. The repeat length in normal chromosomes is less than 42 triplets, while FRDA chro-

Normal

Exon 1

(GAA),,,

.......

Exon 1

.......

Exon 2

.......

(GAA) 100- 1700

Exon 2

.......

Figure 2.3. Localization of the GAA trinucleotide repeat in the first intron of the frataxin gene. Size range is indicated in normal and FRDA chromosomes.

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mosomes have from 120 to > 1700 triplets. The triplet expansion in FRDA has three novel, and so far unique features: it involves GAA trinucleotides, is in an intron, and is associated with an autosonla1 recessive disease. It results in a long DNA segment containing only purines on one strand and pyrimidines on the complementary strand. Repeats in normal chromosomes are stable when transmitted from parent to offspring and of equal size in all tissues. Hyperexpanded, disease-associated repeats show meiotic as well as mitotic instability. Instability during parent-offspring transmission was directly demonstrated by analyzing FRDA families, and additional, indirect evidence came from the detection of two distinct alleles in affected children of consanguineous parents, who are expected to be homozygous by descent at the FRDA locus (Campuzano et al., 1996). Expanded GAA repeats were shown to undergo further expansion or contraction with equal frequency in maternal transmission, while paternal transmission almost always results in contraction (Pianese et al., 1997). In this regard, FRDA-associated GAA repeats behave like the other very large disease-associated expansions in noncoding regions, as fragile X and myotonic dystrophy. Conversely, as discussed in the dominant ataxia section of this review, the smaller expansions of CAG repeats in coding regions tend to undergo size increases during paternal transmission. Mitotic instability in FRDA-associated GAA expansions could be demonstrated by the detection of ample size variations in different cell types or tissues from the same patient (Montermini et al., 1997a,b). Furthermore, heterogeneity in expansion sizes occurs at a variable degree in different tissues. Among those so far examined, cultured fibroblasts and cerebellar cortex show very little heterogeneity in expansion sizes, lymphocytes are more heterogeneous, and most brain regions show a quite complex pattern of allele sizes, indicating extensive heterogeneity. Considering that the estimated carrier frequency for FRDA is about 1:120 in most populations, and that 98% of these individuals carry the GAA expanded repeat, this is the most common triplet repeat expansion so far identified. How did expanded alleles originate? Two hypotheses are possible: 1. from a single, or very few ancestral events or 2. from recurrent events involving at-risk alleles (Imbert et al., 1993). Analysis of the polymorphism of normal alleles of the FRDA-associated GAA repeat and linkage disequilibrium analysis yield support to the second hypothesis (Cossee et al., 1996a; Montermini et al., 1 9 9 7 ~ )Two . classes of normal alleles can be found: “small normal” alleles, averaging 9 repeats, and “large normal” alleles, averaging 18 repeats. Preliminary linkage disequilibrium results revealed the association of different marker haplotypes to each group, and, very interestingly, the FRDA-associated haplotypes are also found in association with some “large normal” alleles. Hyperexpansions may have originated from alleles of the “large normal” group that, after a number of small increases in size because of DNA polymerase slippage events, reached the threshold for instability. We could directly observe the hyperexpansion of an uninterrupted

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(GAA)34 repeat into one containing more than 650 triplets. This is probably the threshold above which this sequence can form secondary structures promoting strand displacement during DNA replication, leading to reiterative synthesis resulting in large size increases (Wells, 1996). Interestingly, stable alleles in the same size range, and up to 42 triplets in length, have also been observed. Sequence analysis revealed that these very large stable alleles carry several (5-10) repetitions of the GAGGAA hexanucleotide interrupting the run of (GAA)n toward its 3’ end. Hexanucleotide repeats are expected to be intrinsically more stable than trinucleotides and exert an “anchor” activity preventing DNA polymerase slippage. Therefore, the FRDA-associated GAA repeat in some large normal alleles is stabilized by interruptions, resembling in this regard other disease-associated trinucleotide repeats.

G. Molecular mechanisms of disease in Friedreich ataxia The expanded GAA repeat has been shown to suppress frataxin gene expression (Campuzano et al., 1996).This type of loss-of-function pathogenetic mechanism is in accordance with the recessive nature of the disease. Evidence of frataxin deficiency was obtained both at the RNA and at the protein level. A severe reduction in the level of mature frataxin mRNA could be demonstrated in cells from FRDA patients by ribonuclease (RNAse) protection (Cosske et al., 1997). Heterozygous carriers have intermediate levels between affected individuals and healthy controls. All portions of frataxin mRNA are reduced in abundance to the same extent, and no evidence of partially processed transcripts is found. These data suggest inhibition of transcription as the most likely mechanism leading to frataxin deficiency, but other possibilities, as abnormal excision of the expansion containing intron, cannot be excluded at this time. Western blot analysis of CNS, lymphoblast, and skeletal muscle samples from FRDA patients confirmed a severe frataxin deficiency (Campuzanoet al., 1997). In patient samples, the 21-kDa band recognized by an anti-frataxin monoclonal antibody is very faint, but still detectable, in agreement with the finding of reduced but not absent frataxin mRNA. The residual amount of frataxin mRNA and protein is inversely proportional to expansion sizes, at least for smaller (<300 triplets) expansions. Such graded effect provides a biological basis for correlations between expansion sizes and phenotypic features. The FRDA-associated hyperexpanded GAA repeat is a polypurinepolypyrimidine (R-Y) sequence; that is, a DNA segment containing only purines (R) in one strand and only pyrimidines (Y) in the other strand. R-Y sequences may adopt a non-B DNA structure in the form of an intramolecular triple helix (Wells et al., 1988; Htun and Dalberg, 1989). Such structure was shown to be highly likely for GAA repeats containing 38 or more triplets in supercoiled plas-

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mids, both at pH 4.5 and a t pH 8.1 (Ohshima et al., 1996). In physiological pH and salt conditions, the most likely conformation is an R-R-Y intramolecular triple helix, where the purine.rich strand of the Watson-Crick R*YDNA duplex dissociates and winds back down the DNA major groove, pairing in an antiparallel orientation with the purine-rich strand, via reverse Hoogsteen hydrogen bonds (-). The formation of an intramolecular triplex by an R-Y sequence requires DNA supercoiling. A wave of local negative supercoiling which could trigger triplex formation is generated behind the polymerase during transcriptional elongation in vivo (Liu and Wang, 1987). In turn, triplex structures have been shown to effectively inhibit transcription (Duval-Valentin et al., 1992). Triplex formation after the passage of RNA polymerase could inhibit further transcription until the structure dissociates, allowing again the passage of the transcription complex. The overall effect of such a mechanism would be a lower frequency of transcription and, consequently, a lower fiataxin mRNA level. Accordingly, the observed proportionality between the length of the expanded GAA tract and the reduction of frataxin mRNA may be explained by the higher stability of triplexes formed by longer R-Y tracts, which would maintain transcription inhibited for a longer time. Although the evidence at present is circumstantial, this is would be the first example of a directional blockade to transcriptional elongation associated with a human genetic disease, defining a novel mutational mechanism.

H. Phenotype-genotype correlations in Friedreich ataxia The dynamic nature of the GAA expansion offers an unexpected way to explain some phenotypic heterogeneity in FRDA that was previously attributed to mutation heterogeneity or, to explain variability within families, to the effect of modifier genes and/or environmental factors. As indicated by three studies (Filla et al., 1996; Diirr et al., 1996; Montermini et al., 1997b), there is a correlation between certain clinical parameters and GAA expansion sizes in FRDA. Earlier age of onset, earlier age when confined in wheelchair, more rapid rate of disease progression, and presence of nonobligatory disease manifestations indicative of more widespread degeneration showed a very good correlation particularly with the size of the smaller repeat (GAA-1) carried by the patient (Figure 2.4). Genotypephenotype studies have also demonstrated a wider clinical variability in homozygous individuals for the FRDA-associatedGAA expansions than expected and allowed by the generally accepted diagnostic criteria. For example, homozygosity for expanded GAA repeats was found in patients with very late onset, up to the fifth decade (LOFA), and in patients with retained reflexes (FARR). The size of the larger allele (GAA-2) also showed some correlation with the clinical parameters. In particular, LOFA patients had both expanded alleles significantly smaller than patients with onset before age 25. The very late onset in LOFA pa-

59

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1200

T 0 0 0.00 0 b b 0 b b b be b 0 0 b bob 0 b 0 0 0 OOb00.0 b 0 b0.0 000 b Ob 0 000 0 0 b b O O O b O b O O b 0 0 b 0 0 0.000 0 0 0 0 b 0 b b 00.0 0 0

i 2 1000

lu

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I 0

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20

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Age of onset Figure 2.4. Scattergram showing the correlation between age of onset and size of the smaller expanded GAA trinucleotide repeat (GAA-1) in 155 FRDA patients.

tients may reflect a milder frataxin deficiency due to a still significant contribution of both alleles to residual frataxin expression. In genotype-phenotype studies, correlation coefficients between disease severity parameters and expansion sizes in peripheral blood lymphocytes were not very high, indicating the existence of additional sources of phenotypic variation. Somatic mosaicism for expansion sizes may be one of these factors (Montermini et al., 1997a,b). As analysis of lymphocytes only provides one sample of the repeat size distribution occurring within a patient, correlations with the phenotype can only be approximate. One study revealed the existence of clinical variability in FRDA which cannot be accounted for by GAA repeat length variations (Montermini et al., 1997a,b). This emerged particularly from the analysis of Acadian FRDA. Despite their phenotypic peculiarities, all Acadian FRDA patients were found to carry two expanded GAA repeats, whose size distribution did not significantly differ from that observed in typical FRDA group. A founder effect in Acadians was demonstrated by linkage disequilibrium analysis, suggesting that the distinctiveness of the Acadian phenotype is linked to the FRDA gene itself rather than to modifying genes which happen to be prevalent in that population. However, no variation in the frataxin coding sequence, as well as no change in the intron sequences directly involved in the splicing process, could be identified in Acadian patients. Differences affecting the level of frataxin expression might be present in the asyet-undefined frataxin promoter/regulatory regions or within the expanded repeat sequence itself.

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Overall, clinical variability in FRDA appears to be largely the consequence of the dynamic nature of the causative mutation, with the possible additional effect of yet-unidentified polymorphisms within or near the FRDA gene. Of course, the concurrence of environmental factors and of modifier genes may still have a role that adds to the variability deriving from the disease gene itself.

Acknowledgments This work was supported by grants from the National Institute of Neurological Diseases and Stroke (NINDS) and by the Muscular Dystrophy Association (MDA) of the USA.

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Liu, L. E,and Wang, J. C. (1987). Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. USA 84, 7024-7027. Livingstone, 1. R., and Sequeiros, J. ( 1984). Machado-Joseph disease in an American-Italian family. 1.Neurogenet. 1, 185-188. Lopes-Cendes, I., Andermann, E., Attig, E., et al. (1994). Confirmation of the SCA-2 locus as an alternative locus for dominantly inherited spinocerebellar ataxias and refinement of the candidate region. Am. J. Hum. Genet. 54, 774-781. Lopes-Cendes, I., Maciel, P., Kish, S. J., et al. (1996). Somatic mosaicism in the central nervous system in spinocerebellar ataxia type 1 and Machado-Joseph disease. Ann. Neurol. 40, 199-206. Mangiarini, L., Sathasivam, K., Mahal, A., et al. (1997). Instability of highly expanded CAG repeats in mice transgenic for the Huntington's disease mutation. Nature Genet. 15, 197-200. Maruyama, H., Nakamura, S., Matsuyama, Z., et al. (1995). Molecular features of the CAG repeats and clinical manifestation of Machado-Joseph disease. Hum. Mol. Genet. 4,807-812. Matilla, T., Volpini, V., Genis, D., etal. (1993). Presymptomatic analysis of spinocerebellar ataxia type 1 (SCAl) via the expansion of the SCAl CAG-repeat in a large pedigree displaying anticipation and parental male bias. Hum. Mol. Genet. 2, 2123-2128. Monckton, D. G., Coolbaugh, M. I., Ashizawa, K. T., Siciliano, M. I., and Caskey, C.T. (1997). Hypermutable myotonic dystrophy CTG repeats in transgenic mice. Nature Genet. 15, 193-196. Montermini, L., Kish, S. J., Jiralerspong, S., et al. (1997a). Somatic mosaicism for the Friedreich ataxia GAA triplet repeat expansions in the central nervous system. Neurology, 49,606-610. Montermini, L., Richter, A., Morgan, K., et al. (1997b). Phenotypic variability in Friedreich ataxia: role of the associated G A A triplet repeat expansion. Ann Neurol., 41,675-682. Montermini, L., Rodius, F., Pianese, L., et al. (1995). The Friedreich ataxia critical region spans a 150kb interval on chromosome 9q13. Am. J. Hum. Genet. 57, 1061-1067. Montermini, L., Andermann, E., Labuda, M., et al. (1997~).The Friedreich ataxia GAA triplet repeat: premutation and normal alleles. Hum. Mol. Genet. 6, 1261-1266. Morton, N. E., Lalouel, 1.-M., Jackson, J. F., Currier, R. D., and Yee, S. (1980). Linkage studies in spinocerebellar ataxia (SCA). Am. ]. Med. Genet. 6 , 251-257. Nino, H. E., Noreen, H. J., Dubey, D. P., et al. (1980). A family with hereditary ataxia: HLA typing. Neurology 30, 12-20. Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D. (1996). Cloning, characterization and properties of seven triplet repeat DNA sequences. ]. Biol. Chem. 271, 16773-16783. Ophoff, R. A., Tenvindt, G. M., Vergouwe, M. N., etd. (1996). Familial hemiplegic migraine and episodic ataxia type 2 are caused by mutations in the Ca2+channel gene CACNLlA4. Cell 87,543-552. Orozco-Diaz, G., Estrada, R., and Perry, T.L. (1989). Dominantly inherited olivopontocerebellar atrophy from eastern Cuba: Clinical, neuropathological, and biochemical findings.]. Neurol. Sci. 93, 37-50. Orozco-Diaz, G., Nodarse Fleites, A., Cordoves Sagaz, R., and Auburger, G. (1990). Autosomal dominant cerebellar ataxia: Clinical analysis of 263 patients from a homogeneous population in Holguin, Cuba. Neurology 40,1369-1375. Orr, H. T., Chung, M., Banfi, S., et al. (1993). Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nature Genet. 4, 221-226. Ouvrier, R. A., McLeod, J. G., and Conchin, T. E. (1982). Friedreich's ataxia-early detection and progression of peripheral nerve abnormalities. ]. Neurol. Sci. 55, 137-145. Palau, F., De Michele, G., Vilchez, J. J., et al. (1995). Early-onset ataxia with cardiomyopathy and retained tendon reflexes maps to the Friedreich's ataxia locus on chromosome 9q. Ann. Neurol. 37, 359-362. Pandolfo, M., Sirugo, G., Antonelli, A., et al. (1990). Friedreich ataxia in Italian families: Genetic homogeneity and linkage disequilibrium with the marker loci D9S5 and D9S15. Am. J. Hum. Genet. 47,228-235.

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Paulson, H. L., Perez, M. K., Trottier, Y., et al. (1997). lntranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. Neuron 19,333-344. Pedersen, L., Platz, P., Ryder, L. P., Lam, L., and Dissing, J. (1980). A linkage study of hereditary ataxias and related disorders: Evidence of heterogeneity of dominant cerebellar ataxia. Hum. Genet. 54, 371-383. Pentland, B., and Fox, K. A. A. (1983). The heart in Friedreich's ataxia. J. Neurol. Neusosurg. Psychian. 46,1138-1 142. Pianese, L., Cavalcanti, F., De Michele, G., et al. (1997). The effect of parental gender on the GAA dynamic mutation in the FRDA gene. Am. J. Hum. Genet. 60,460-463. Pulst, S.-M., Nechiporuk, A., Nechiporuk, T., et al. (1996). Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nature Genet. 14,269-276. Pulst, S.-M., Nechiporuk, A., and Starkman, S. (1993). Anticipation in spinocerebellar ataxia type 2. Nature Genet. 5, &lo. Ranum, L. P. W., Chung, M., Banfi, S., et al. (1994). Molecular and clinical correlations in spinocerebellar ataxia type I: Evidence for familial effects on the age at onset. Am. J. Hum. Genet. 55, 244-2 52. Ranum, L. P. W., Duvick, L. A., Rich, S. S., et al. (1991). Localization of the autosomal dominant HLA-linked spinocerebellar ataxia (SCA1) locus, in two kindreds, within an 8-cM subregion of chromosome 6p. Am. J. Hum. Genet. 49,31-41. Ranum, L. P. W., Lundgren, J. K., Schut, L. J., et al. (1995). Spinocerebellarataxia type 1 and Machado-Josephdisease: incidence of CAG expansions among adult-onset ataxia patients from 31 1 families with dominant, recessive, or sporadic ataxia. Am. J. Hum. Genet. 57,603-608. Ranum, L. P. W., Schut, L. J., Lundgren, I. K., Orr, H. T.,and Livingston, D. M. (1994). Spinocerebellar ataxia type 5 in a family descended from the grandparentsof President Lincoln maps to chromosome 11. Nature Genet. 8,280-284. Rich, S. S., and Orr, H. T. (1989). A linkage map of the short arm of human chromosome 6: Location of the gene for autosomal dominant ataxia (SCA1). Cytogenet. Cell Genet. 51, 1066. [Abstract] Richter, A., Morgan, K., Poirier, J., et al. (1996). Friedreich ataxia in Acadian families from Eastern Canada: Clinical diversity with conserved haplotypes. Am. J. Med. Genet. 64,594-601. Robitaille,Y., Schut, L., and Kish, S. J.( 1995). Structural and immunocytochemical features of olivopontocerebellar atrophy caused by the spinocerebellar ataxia type 1 (SCA-1) mutation define a unique phenotype. Acra Neuropathol. 90,572-581. Rodius, E, Duclos, F., Wrogemann, K., et al. (1994). Recombinations in individuals homozygous by descent localize the Friedreich ataxia locus in a cloned 450-kb interval. Am. J. Hum. Genet. 54, 1050-1 059. Romanul, F. C. A., Fowler, H. L., Radvany,J., Feldman, R. G., and Feingold, M.(1977). Azorean disease of the nervous system. N.End.1. Med. 296, 1505-1508. Romeo, G., Menozzi, P., Ferlini, A,, et al. (1983). Incidence of Friedreich ataxia in Italy estimated from consanguineousmarriages. Am. J. Hum. Genet. 35,523-529. Rosenberg, R. N., and Fowler, H. L. (1981) Autosomal dominant motor system disease of the Portuguese: A review. Neurology 31, 1124-1 126. Rosenberg,R. N., Nyhan, W. L., Bay, C., and Shore, P. (1976). Autosomal dominant striato-nigral degeneration: A clinical, pathologic and biochemical study of a new genetic disorder. Neurology 26, 703-714. Rotig, A., de Lonlay, P., Chretien, D., et al. (1997). Aconitase and mitochondria1 iron-sulpher protein deficiency in Friedreich ataxia. Nat. Genet. 17, 215-217. Said, G., Marion, M. H., Selva, J., and Jamet, C. (1986). Hypotrophic and dyingback nerve fibers in Friedreich's ataxia. Neurology 36, 1292-1298. Sakai, T., Ohta, M., and Ishino, H. (1983).Joseph disease in a non-Portuguese family. Neurology 33, 74-80.

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Sanpei, K., Takano, H., Igarashi, S., et al. (1996). Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT. Nature Genet. 14,277-284. Scherzinger, E., Lurz, R., Turmaine, M., et al. (1997). Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in wino and in wiwo. Cell 90, 549-558. Schols, L., Vieira-Saecker, A. M. M., Schols, S., et al. (1995). Trinucleotide expansion within the MJD1 gene presents clinically as spinocerebellar ataxia and occurs most frequently in German SCA patients. Hum. Mol. Genet. 4, 1001-1005. Schonle, E. J., Boltshauser, E. J., Baekkeskov, S., et al. (1989). Preclinical and manifest diabetes mellitus in young patients with Friedreich's ataxia: No evidence of immune process behind the islet cell destruction. Diabetohgia 32,378-381. Servadio, A., Koshy, B., Armstrong, D., AntalfTy, B., Orr, H. T., and Zoghbi, H. Y. (1995). Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals. Nature Genet. 10, 94-98. Skre, H. (1975). Friedreich's ataxia in Western Norway. Clin. Genet. 7, 287-298. Stevanin, G., Le Guern, E., Ravise, N., et al. (1994). A third locus for autosomal dominant cerebellar ataxia type 1 maps to chromosome 14q24.3-qter: Evidence for the existence of a fourth locus. Am. J. Hum. Genet. 54, 11-20. Subramony, S. H., Fratkin, J. D., Manyam, B. V., and Currier, R. D. (1996). Dominantly inherited cerebello-olivary atrophy is not due to a mutation at the spinocerebellar ataxia- 1, Machado-Joseph disease, or Dentato-Rubro-Pallido-Luysian Atrophy locus. Mow. Disurd. 11, 174-180. Suzuki, Y., Sasaki, H., Wakisaka, A., et al. (1995). Spinocerebellar ataxia 1 (SCAl) in the Japanese: Analysis of CAG trinucleitide (sic) repeat expansion and instability of the repeat for paternal transmission.Jpn. J. Hum. Genet. 40, 131-143. Takiyama, Y., Igarashi, S., Rogaeva, E. A,, et al. (1995). Evidence for intergenerational instability in the CAG repeat in the MJDl gene and for conserved haplotypes at flanking markers amongst Japanese and Caucasian subjects with Machado-Joseph disease. Hum.Mol. Genet. 4, 1137-1 146. Takiyama, Y., Nishizawa, M., Tanaka, H., et al. (1993). The gene for Machado-Joseph disease maps to human chromosome 14q. Nature Genet. 4,300-304. Takiyama, Y., Oyanagi. S., Kawashima, S., et al. (1994). A clinical and pathologic study of a large Japanese family with Machado-Joseph disease tightly linked to the DNA markers on chromosome 14q. Neurology 44, 1302-1308. Trottier, Y., Lutz, Y.,Stevanin, G., et al. (1995). Polyglutamine expansion as a pathological epitope in Huntington's disease and four dominant cerebellar ataxias. Nature 378,403406. Vahedi, K., et al. (1995). A gene for hereditary paroxysmal cerebellar ataxia maps to chromosome 19p. Ann. Neurol. 37,289-293. Wells, R. D., Collier, D. A,, Hanvey, J. C., et al. ( 1988). The chemistry and biology of unusual DNA structures adopted by oligopurine.oligopyrimidinesequences. FASEB J. 2,2939-2949. Wells, R. D. (1996). Molecular basis of genetic instability of triplet repeats. J. Biol. Chem. 271, 2875-2878. Housrnan, D. (1995). Gain of glutamines, gain of function. Nature Genet. 10,3-4. Winter, R. M., Harding, A. E., Baraitser, M., and Bravery, M. B. (1981). Intrafamilial correlation in Friedreich's ataxia. Clin. Genet. 20, 419-427. Woods, B. T., and Schaumburg, H. H. (1972). Nigro-spino-dentatal degeneration with nuclear ophthalmoplegia: A unique and partially treatable clinico-pathological entity. J. Neurol. Sci. 17, 149-166. Wullner, U., Klockgether, T., Petersen, D., et al. (1993). Magnetic resonance imaging in hereditary and idiopathic ataxia. Neurology 43,318-325. Yakura, H., Wakisaka, A., Fujimoto, S., and Itakura, K. (1974). Hereditary ataxia and HL-A genotypes. N. Engl. J. Med. 291, 154-155.

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Zhuchenko, O., Bailey, J., Bonnen, P., et al. (1997). Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the aIA-voltage-dependent calcium channel. Nature Genet. 15,6249. Zoghbi, H. Y., Jodice, C., Sandkuijl, L. A,, et al. (1991). The gene for autosomal dominant spino. cerebellar ataxia (SCA1) maps telomeric to the HLA complex and is closely linked to the D6S89 locus in three large kindreds. Am. 1. Hum. Genet. 49,23-30. Zoghbi, H. Y.,Pollack, M. S., Lyons, L. A., et al. (1988). Spinocerebellarataxia: Variable age of onset and linkage to human leukocyte antigen in a large kindred. Ann. Neurol. 23,580-584.

The Minute Genes in Drosophila and Their Molecular Functions Andrew Lambertsson Department of Biology, Division of General Genetics, University of Oslo, N-0315 Oslo, Norway

I. Introduction 70 11. Historical review 76 A. The pioneering work of Jack Schultz 76 B. The ecdysone hypothesis 78 C. The oxidative phosphorylation hypothesis 79 D. The Minute-tRNA hypothesis 81 E. Mutations in rDNA genes produce Minute-likephenotypes 111. Minutes as genetic tools in studies of growth and development 85 A. Mitotic recombination in Minute mutants B. Minutes as suppressors 86 88 IV. Minutes and the protein synthesis theory A. Molecular biology comes of age in Minute research 89 92 B. Use of transposons to clone Minute genes C. Recessive Minutes 116 118 D. RP mutations without Minute phenotype E. Ribosomal protein genes of special interest 119 121 F. Mutations in nonribosomal protein genes V. Conclusions and prospects 122 References 124

83

84

T h e behavior of genes in combination with each other forms a bridge between the description of development in the different mutant races-what may be called Mendelian embryologyand the study of gene action proper. Jack Schultz (1935)

Advances In Genetics, Vol. 38 Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved. 0065-2660198 $25.00

69

70

Andrew Lambertsson

1. INTRODUCTION Minutes are without doubt one of the most intriguing and evasive group of mutations in Drosophila and maybe in all eukaryotes for that matter. The Minute mutants are of great interest and importance with respect to genetic control of development since, despite the great number of and widely varying chromosomal loci, the phenotypes of different Minute mutants are practically identical. In 1919 Bridges found the first Minute mutant in Drosophila melunogaster, M(3)f or M(3)99B, as it is called today after its location, and within a year he found several more. (Hereafter, all Minutes will be called according to Lindsley and Zimm, 1992.) The Minute mutants were later described by Bridges and Morgan (1923) and characterized in more detail by Schultz (1929). At that time the Minute mutant phenotype was one of the most frequent in Drosophila and today more than 50 Minute loci have been mapped throughout the Drosophila genome (Table 3.1; Lindsley and Zimm, 1992).They are associated with similar dominant visible phenotypes, such as short slender bristles, delayed development, and recessive lethality. Many Minutes are haploinsufficient; that is, they are correlated with a cytologically visible deficiency. In his classic and impressive study Schultz (1929) observed that Minutes are nonadditive in their phenotypic effect; that is, the phenotype of a MI/+;M2/+ fly is not more severe than the phenotype of any of the single mutants. Possible molecular explanation(s) to this and the synergistic effects between Minutes that have been reported will be discussed later. Unfortunately, this nonadditive property of the Minute mutations makes it impossible to determine if a deletion uncovers one or more closely linked Minute loci. He also concluded that while the function of each Minute gene is unique, different Minutes code for products with similar function(s). During the subsequent 40 years, Schultz’s notion produced only three proposals for Minute function: ( 1) Minute genes encode products that are involved in the production or metabolism of or response of target cells to ecdysone, the molting hormone (Brehme, 1939), (2) Minutes code for enzymes involved in the terminal respiratory system (Farnsworth, 1965; Goldin, 1968; Farnsworth and Jozwiak, 1969), and ( 3 ) Minute loci code for the various tRNA species (Ritossa et al., 1966). However, none of these proposals has any experimental support. A much stronger case can be made for the recent proposal that Minutes correspond to mutations in ribosomal protein genes (for review, see Kay and Jacobs-Lorena, 1987), and it is very encouraging to note that today several Minute genes have been shown to code for a ribosomal protein. This idea was first s u g gested by Huang and Baker (1976), who found that independent of the EMS dose there are about 68 Minute mutants induced for every 5000 recessive lethals induced. These data were well in agreement with the number of existing Minutes and did not support the hypothesis that Minutes are the sites of redundant tRNA genes (see below; Ritossa et al., 1966).

3. The Minute Genes in Drosophila

71

Table 3.1. Drosophila Minute Mutations

Locus; synonym

RpL36b; M(1) 1B Sta

M(1)3E M( 1)5A M( 1 )5D6A M( 1 )7BC M(1)7C M(1)8F M(I)IIF M(1)13A M(1)14C M(1)14E RpS5'; M(1)15D M(1) 18C M(2) 21AB M(2)21C; RpAZ? oho23Bb; RpS21 M(2)24D Mf2)24F M(2)25C sop"; M(2)30D/E M(2)31A RpSl3 h; M(2)32A RpL9b; M(2)32D M(2)36F M(2)39F M(2)41A M(2)44C M(2)53 M(2)56CD M(2)56F M(2)58F M(2)60B RpL19"; M(2)60E M(3)62A M(3)62F M(3)63B M(3)65F RpL14b; M(3)66D M(3)67C M(3)69E M(3)76A M(3)80 M(3)82BC

Genetic location

Cytological location

Known gene product

1-0.1 1-0.3 1-15] 1-113) 1-13.7 1-1211 1-1221 1-30.0 14421 1-1491 1454) 11-56.6 1-62.7 2-10] 2-0.0 224121 2-12.9 2-15.0 22-40.5 2-42 22-54 2-54.3 2-55.1 2-57 2-77.5 2-87.5 2-92.3 2-101.2 22-108 3-10) 3-12] 3451 3-1231 33-28.9 3-40.2 3-44.3 3-1471 3-47

1B11-lB12 2A3-2B2 3E3-3E4 5A65A13 5D5-6A 7&7C 7c5-7c9 8F1-8F2 11F1-11F4 12Fb13B6 14B17-14C6 14E 15E1-15E7 18B9-18D 21A-21B 21c1-2 1c 2 23B 24D4-24D8 24F1-25A1 25B9-29C3 30D-30E 3 1A 1-3 1E7 32A1-32A5 32D1-32D5 36F2-36F6 39E-4OA 41A 44c 52D-53E 56C-56D 56F5-56F15 58F 60B 60E343Ell 61F-62A 62E-63A 63B6d3C1 65F10-65Fll 66D1-66D11 67C1-67ClO 69E2-70A8 76A3-76B2 79E5-80F 82A-82C

RPW6 D-p40; RPSa?

RP7/8! RPS6 or RPS14?

RPS5

RPAZ? RPS2l

RPS2 RPS13 RPL9

RPL19

RPL14

Reference"

1 1, 18 1 1 1,2 1 1,3,4,5,6 1 1 1 1, 7 7 1,7 1 1 1,8 9 1 1 1 10 1 11 12 1 1 1 1 1 1 1 1 13 14 1 1 1 1 15 1 1 1 1 1 (continues)

72

Andrew Lambertsson

Table 3.1. (Continues)

Locus; synonym

Genetic location

Cytological location

M(3)85E M(3)86D RpS3 b; M(3)95A M(3)96A M(3)96C M(3)96CF M(3)99B RpL32b;M(3)99D M(3)99E M(3) JOOCF RpS3A”; M(4) 101

3-{50) 3-50.0 3-79.7 3484) 3-84.5 3-90.2 3-101.2 3-(101) 3-106.2 3-105 4-0

85E2-85Fl 86D8-86D10 95A1-95Al 95E 6 9 6 A 5 96C1-96C5 96C-97A 99B5-99B9 99D1-99D9 99E4-99Fl 100C-tip 101F2-102A5

Known gene product RPW? RPS3

RPL32

RPS3A

Referencea

1 1, 19 1, 16 1 1 1 1 1, 17 1 1 1, 20,21

“1, Lindsley and Zimm (1992); 2, Bums et al. (1984); 3, Watson et al. (1992); 4, Stewart and Denell (1993); 5, Andersson and Lambertsson (1990); 6, Dorer et al. (1991); 7, McKim et al. (1996); 8, Wigboldus (1987); 9, B. Mechler, personal communication; 10, Crarnton and Laksi (1994); 11, SrebseLarssenandLambertsson (1996); 12,Schmidtetal. (1996); 13, B.Reed,FlyBase; 14, Hartetal. (1993); 15, Sreboe-Larssen et al. (1997); 16, Andersson et al. (1994); 17, Kongsuwan et al. (1985); 18, Melnicketal. (1993); 19, C. J. O’Kane, FlyBaseUID: FBgn0020910; 20,Reynaudetal. (1997); 21,J. Kronharnrn and A. Rasmuson-Lestander, personal communication. bNew gene names; see text for explanation.

Due to the long history and the evasive nature of the Minutes this chapter will include a review of Minute research up to the 1980s. The most important theories will be highlighted and the latter part of the chapter will be devoted to the past 15 years of research in this field. This period is dominated by the entree of molecular biology and state-of-the-art molecular genetic techniques in Drosophila research. To date more than 40 Drosophila ribosomal protein (rp)-genes have been cloned and cytologically localized (Table 3.2) but not more than 13 have been unambiguously correlated to Minute loci. The majority of the latter have been cloned by P-element mutagenesis, and the rest have been fortuitously stumbled over. Many of the other cloned rp-genes map cytologically at or near the sites of earlier localized Minutes. This lends indirect support to the notion that Minute loci encode ribosomal proteins. I have aimed at making this review on Minutes and their functions as comprehensive as possible. However, even today, almost 80 years after Bridges discovered the first Minute, only 13 of the -55 established Minute genes have been unambiguously correlated to ribosomal proteins; this makes it somewhat difficult to discuss the Minute genes and their functions at full length. This review will therefore contain data on cloned ribosomal protein genes that have not been correlated to Minutes. Furthermore, the use of Minutes in mitotic recombination analysis and in developmental genetic studies and Minutes as suppressors will be

Table 3.2. Cloned ribosomal protein genes in Drosophila melanogaster

Gene; synonym

Cytological position

Protein

Mutant phenotype

GenBank accession number

sopa;M(2)30D/E

RPS2

30&30E

Minute

RpS3"; M(3)95A

RPS3

95A1-95A1

Minute

RpS3A; M(4)101

RPS3A

101F1-101Fl

Minute

U01334; U01335; X69120; P31009 L13690; X72921; Qo6559 Y10115; P55830

RpS4

RPS4

?

D16257; P41042

RpS5; M( 1) 15D RpS6"; air8; hen; M(1)7C? RpS9 RpSl2

RPS5 RPS6

1BI-lB2 3A3-3A6 15E1-15E7 7c5-7c9

Minute Minute; tumors in hematopoietic system

RPS9 RPS 12

69Al49F7

?

?

?

U48394; Q24186 L02075;L02074; L07881; LO1658 X69677;A56687 X89659; P80455

RpS13"; M(2)32A

RPS13

32A1-32A5

Minute

RpS14a, M(1)7C?

RPS14

7c5-7c9

Minute?

RpS14b; M(1)7C? RpS 15A RpS17; M(2)67C? RpS18; M(2)56F? RpS J 9 RpS20 oho23B"; RpS21

RPS14 RPS15A RPSl7 RPS18 RPS19 RPSZO RPS2l

7c5-7c9

Minute?

?

?

67Cl47C10 56F1-56F17 15A1-15A11 92F-93A 23B1-23B8

Minute? Minute?

RpS25

RPS25

?

?

+

? ?

X91854; X91853; 219052; Q03334 M21045; P14130 M21045; P14130 221673; P48149 M22142; P17704 L22959; P41094 X73153; P39018 Y11119

Minute; tumors in hematopoietic system; U03289; P48588

Reference Barrio et nl. (1993) Cramton and Laski (1994) Wilson er al. (1993) Andersson et al. (1994) E. Reynaud et al. (1997), J. Kronhamm and A. Rasmuson-Lestander, personal communication Yokokura et al. (1993) McKim et al. (1996) Watson et al. (1992) Stewart and Denell (1993) Zhang and Bownes (1993) S. R. Greig (1995). FlyBase UID: FBgn0014027 Saeb~e-Larssenand Lambertsson, (1996) Brown et al. (1988); Andersson and Lambertsson (1990) Lavoie et al. (1994) Maki et al. (1989) Garwood and Lepesant (1994a) Baumgarmer et nl. (1993) Chan et al. (1997) Hemnann et al. (1995); B. Mechler, personal communication B. Edgar (1994), FlyBase UID: 0010413 (continues)

Table 3.2. (Continues) Cytological position

Gene; synonym

Protein

RpS26

RPS26

?

?

RpS27A; Ubi-fS0 RpPO; Ape RpP1

RPS27A

31E1-31E7

Overexpressed in

PO RPAl

79GD 53C1-53C6

RpP2; RpA2; M(2)21C?

RPA2

2 1C1-2 1C3

Antisense: female sterility Minute?

~ t a ;D

SA

2A3-B2

stubarista; Minute

RpL 1

RPLl

98A1-98B8

?

RpL3

RPL3

86D

Minute?

RpL7

RPL7

?

?

X82782; P32100

RpL7A; Surf-3 RpL9; M(2)32D

RPL7A RPL9

6AdB 32D1-32D5

?

Minute

X82782; P46223 X94613; 283384;

RpLl 1 RpLl2; M(3)62F? RpLl3; Bbcl RpL14"; M(3)66D

RPLll RPLl2 RPL13 RPL14

56D1-56D15 62Eld2E9 29E1-29F8 66D146D15

?

U15643; P46222

-N

Mutant phenotype

mouse tumors ?

GenBank accession number X13625; X14247; P13008 M22536; X69119; 271103; P15357 M25772; P19889 X05466; X05016; PO5389 Y00504; S62170; PO8570 M77133; M90422; P38979 X13382; e1906; X13382; PO9180 AF016835; ~2384754

Minute? ?

Minute

X77926; P41126 Y10017; Y10015;

Reference Itoh et al. (1989a,b) Lee et al. (1988); Barrio et al. (1994) Kelley et al. (1989) Qian et al. (1987, 1988) Wigbolduset al. (1987); Olson et al. (1993) Melnick et al. (1993) Rafti et al. (1988,1989) H. Y. E. Chan, Y. Zhang, J. D. Hoheisel, and C. J. O'Kane (1997), FlyBase UID: FBgn0020910 F. Quan and M. A. Forte (1992), FlyBase UID: FBgn0005593 Armes and Fried (1995) Schmidt et al. (1996) P50882 hochelle and Suter (1995) Bums et al. (1984) Helps et al. (1995) Saebgx!-Larssen et al. (1997) Y09766; P55841

zjn"; RpL15; fs(3)02729 RpL 1 7A; M(2)58F? RpL I 8An RpL19"; M(2)60E

RPL15

80AX

Female sterile

RPL17A

58F659A3

Minute?

M85295; P48159

RPL18A RPL19

54B1-54B18 60E6-60E11

?

Minute

RpL22

RPL22

?

?

X75339; P41093 X74776; L32181; P36241 U42587; P50887

RpLZS

RPL25

62C

?

RpL27Aa RpL27Ab RpL29 RpL30 RpL32; M(3)99D; RP49 RpL36; M(I)IB

RPL27A RPL27A RPL29 RPL3O RPL32

8 7 F 1 4 8 A 12 24F-25A

?

?

56FC56F15 99D4-99D8

? ?

Minute

RPL36

1B13-lB13

Minute

RpL40; Ubi-f52

RPL40

97A1-97A10

?

Rp7/8; M(1)5D6A Rp2 1 Rp34

?

5D5-5D8

Minute?

? ?

"Isolated by P element mutagenesis.

X74484; P41092 U66357; Q94530 U40226; Q24154 X00848; X00848; U92431; PO4359 U20543; P49630

X53059; X59943; P18101; PO2248

K. Dej and A. Spradling (personal communication) Noselli and Vincent (1992) Ntwasa et al. (1994) Hart et al. (1993) C. V. C. Glover (1996), FlyBase UID: 0015288 D. Hush (1995), FlyBase UID: FBgn0014878 Ganvood and Lepesant (1994b) Fox and Gaynor (1996) Kania et al. (1995) Kongsuwan et al. (1985) Voelker et al. (1989); Duffy et al. (1996); W. R. Steinhauer (1999, FlyBase UID: FBgn0002579 Cabrera et al. (1992) Bums et al. (1984) FlyBase UID: FBgn0003273 FlybBase UID: FBgnOO17546

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discussed. I have deliberately chosen to classify some mutants as Minutes where the authors’ classification is recessive Minutes or non-Minute. In doing so I may inadvertently have tread on someone’s toes, and I apologize for that.

II. HISTORICAL REVIEW A. The pioneering work of Jack Schultz In the 1920s geneticists and biologists began to turn their attention from the empirical study of phenotypes to the experimental study of gene action and interaction using genetic and environmental factors to influence gene expression and development. Early work in this field on lower eukaryotes implied that the expression of an adult trait is dependent upon a balanced interaction between different growth processes and that these and the trait could be affected by genetic and/or environmental factors (Goldschmidt, 1927; Ford and Huxley, 1927; Plunkett, 1926). Similar results were obtained by Wright (1925, 1927) on the color factors in guinea pig and the notion was that if one mutant gene modifies the effect of another, they are likely to be involved in some way in the same process in development. By the mid-1920s Morgan and others realized that the future of genetics lay in its connections with development and evolution. At that time the Minutes were one of the most common mutations in D. melanogaster and they all modified the rate of development; they were thus well suited for experiments in developmental genetics. In 1929 Schultz published his work on 16 Minutes and their interactions with each other and other dominant genes. By then about 20 different Minute loci had been mapped (Figure 3.11, and today more than 50 Minutes have been found (Lindsley and Zimm, 1992).

1. The Minute reaction a. The Minute phenotype As pointed out by Schultz (1929), there are three main characteristics of the Minute phenotype, namely delayed larval development, short thin bristles, and recessive lethality. Minutes may also display several other dominant traits, including larger and rough eyes, abnormal wings, defective abdominal segmentation, reduced fertility and viability, small body size, and the elimination of chromosomes in somatic cells. It was striking that so many different genes should produce such similar phenotypic effects. The Minute loci are scattered throughout the genome and they are haploinsufficient; that is, flies having only one dose of a chromosome segment containing a wild-type Minute gene show the same phenotype as Minute point mutations (Lindsley et al., 1972). The Minutes qualified as material for studies of gene interaction and development. The central question asked by Schultz

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0 10 20 30 40 50 60 70 80 90 100 Figure 3.1. Chromosome map, showing the Minute loci and a few of the mutants of Dosophila in 1929. Reprinted with permission from Schultz (1929).

was whether the similar phenotypes of Minutes were caused (1) by the same mechanism in all cases implying that the Minute loci encoded different products with related functions, (2) by the absence of unrelated functions, and ( 3 )by the lack of the same genetic material at different loci.

b. The primary and secondary reactions of the Minutes To test whether Minutes at different loci represented the same kind of primary effect, Schultz (1929) used 16 different Minutes in 55 combinations. O n the basis of these tests he found that at least two reactions were involved in the production of the Minute phenotype. First, a primary reaction, different in different Minutes, involved in effecting the Minute reaction and second, a reaction he called the Minute reaction proper. This observation was based on the fact that no combination with two or three Minutes produced a cumulative effect regarding the appearance of the Minute phenotype. Examination of the interaction of Minutes with Delta andJummed showed that all the Minutes studied produced a similar effect, but with differences in intensity approximately proportional to the differences in viability, bristle reduction, and duration of development. These observations further supported Schultz’s notion about the Minute reaction proper and that this was concerned with one or more of the numerous growth processes of the fly. Thus, he proposed the hypothesis that the function of each normal Minute gene is unique, but different Minutes encode products with similar functions. A Minute mutation will lead to a decrease in the rate in one of the growth processes, which then becomes a limiting reaction. At the time, this idea explained why

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Minutes are nonadditive in their phenotypic effect (see above), and also the lethal effects of Minutes when homozygous; in the latter case a necessary process does not take place. Today, accumulating data strongly indicate that Minute loci encode a ribosomal protein; so far, 13 Minutes have been shown to do this (see below and Table 3.2). This offers a molecular explanation(s) to the Minute phenotype: reduced amount of an rp results in an insufficient number of functioning ribosomes, which in turn leads to deficient protein synthesis. Although rp-genes are constitutively expressed, we may assume that various rp-genes are differentially expressed and/or that the threshold requirement is different for each component needed for ribosome assembly and function. Therefore, the loss of an allele of one ribosomal protein gene will (in most cases) result in a Minute phenotype different from that produced by a mutation in an other rp-gene. This may thus explain, at the molecular level, the nonadditive property of the Minute mutations. O n the other hand, should two rps have essential interaction in the ribosome, or otherwise in the cell, it may also explain why two Minutes have synergistic lethal effects (see below). Schultz (1929) also showed that two doses of a single dominant Minute are lethal in triploids. In contrast, Schellenbarger and Duttagupta (1978) did not find a similar effect with M(2)56F when a single dose of M+ was added as a small duplication to the Y chromosome. Schultz also concluded that most, if not all, of the Minutes are deficiences. Unfortunately, the nonadditive effect of the Minute mutations makes it impossible to determine if a deletion uncovers one or more closely linked Minute loci.

B. The ecdysone hypothesis Focusing on the effect of Minutes on development, studies were made by Dunn and Coyne (1935) and Dunn and Mossige (1937), who found that the delay in development of Minute flies is chiefly due to a retardation in the egg-larval period. Dunn and Coyne (1935) suggested that Minutes do not act by simply slowing the whole developmental process, but through a change in rate of development at some stage in the egglarval period. Accordingly, the effect of Minutes on viability and other characters sensitive to growth rate would be smaller when the development was slow, greater when the development was rapid. This agrees well with the observations that Minutes have a higher survival rate at low temperatures and that the male, with a lower growth rate, is less adversely affected. In an attempt to elucidate how Minutes prolong development and the production of smaller flies and how and in which processes the gene products function, Brehme (1939) established that (1) the time of puparium formation is delayed, (2) there is a delay in the occurrence of the first and second larval molts, (3) at the time puparium formation, the Minute larva is markedly narrower and

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slightly shorter than the wild-type, and (4) certain of the Minute imaginal disks are retarded in growth and differentiation. Brehme suggested that the delay in time of pupation of heterozygous Minutes is due to an insufficient or delayed secretion of the pupation hormone ecdysone or to the inability of the tissues to react to the hormone. As to the delay in time of the larval molts Brehme (1939) suggested that in Drosophila the stimuli to molting and pupation are one and the same. This had in fact already been put forward by Buddenbrock (1930), who believed that in the Lepidoptera the molting and pupation hormones are qualitatively the same. Today we know this to be the case. As to larval growth, imaginal disk development, and differentiation, very few studies had been made at that time. Brehme (1939) found that growth rate is retarded in both larval and imaginal tissues in Minutes. She also realized that although there might be a close relationship between the four characteristics of the development of Minute heterozygotes, no known mechanism could account for their occurrence as pleiotropic effects of the same genetic factors. It was suggested that there exists some material in the larva which is used by growing tissues and in manufacturing ecdysone and of the stimulus to molting. The normal Minute factor may control the production of this material and in heterozygous Minutes the amount of it is reduced, with the result that the rate of tissue growth is altered and molting and pupation are delayed. As mentioned earlier, all Minutes are characterized by delayed larval development, short and thin bristles, dominance over wild-type, and homozygous lethality. Despite the prolonged larval period heterozygous Minute larvae are smaller than their wild-type siblings and this size difference also extends into the adult stage. In her continuous work on Minutes Brehme (1941a,b) was able to show that this size reduction is due to a decrease in cell size and not in cell number and that homozygous Minute larvae grow only very slightly and do not molt. Furthermore, transplantation of eye disks of M(3)95A' heterozygotes to wild-type larvae showed that the more rapid growth rate of the host has no effect on the growth rate of the transplant, thus showing that the Minute mutation is autonomous. In these experiments Brehme also showed that the facet number of adult Minute flies is not significantly lower than that of controls, but the size of individual facets is smaller. These observations clearly supported Schultz's notion that the Minute factors are concerned primarily with growth and that the Minute reaction reflects an aspect of growth common to all flies (cells). If this is interfered with in any one of a variety of ways it will produce the same result, the Minute phenotype.

C. The oxidative phosphorylation hypothesis In the middle of the 1950s Farnsworth (1957a,b) began a series of experiments in an attempt to explain the Minute syndrome. In these early papers Farnsworth

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(1957a,b) was interested in determining whether different Minutes, so similar in heterozygous phenotype, also were alike with respect to homozygous phenotype. In the first two papers she analyzed the factors responsible for the death of individuals homozygous for seven different Minutes, located on the X, second, and third chromosomes, namely M( 1)15D, M(2)58F, M(2)58F1, M(3)95A', M(3)95A2, M(3)95A3, and M(3)95A4. Famsworth (1957a) was able to show that the time of onset of death as well as the specific organs affected was the same in eight different homozygous Minutes. Thus, all homozygotes except M(2)58F', which dies in the egg stage, die in the larval stage. At 12 hr of embryonic development the first morphological anomaly is observed and involves the midgut which is retarded in formation and differentiates more slowly than is the case in controls. In later stages, the only abnormality usually observed involves the midgut. Although cardia and caecae develop normally, the anterior and often the middle regions of the midgut remain large, rounded, and filled with yolk. The elongation of this entire structure is retarded and even at hatching, yolk is found in the gut. It is also interesting to note that hatched homozygous Minute larvae may live for several days but that neither growth nor molt was observed. However, there were always a few homozygotes that failed to hatch and these individuals frequently had abnormalities of the forgut, midgut, and nervous system. All these observations supported the earlier work of Schultz (1929), Brehme (1939, 1941a,b), Dunn and Coyne (1935), and Dunn and Mossige (1937) that the Minute factors are concerned essentially with growth. Further work by Farnsworth in the 1960s focused on physiological grounds to explain the Minute syndrome. In the first of a series of papers the amounts of total soluble protein and cytochrome c oxidase activity in different larval stages were determined for controls and for nine heterozygous Minutes (Famsworth, 1964,1965a). Specific delays in growth rate were detected based on total soluble protein content but these restrictions were not indispensably correlated with a reduction of the concentration. Instead, enzyme activity equal to or higher than that of the controls was observed in periods of retarded growth. M(2)24F' was the only Minute with a distinctly low cytochrome c oxidase activity in the early stages of development. Later in development, however, this activity was not significantly different from the controls, since cytochrome c oxidase activity in the normal organism declined with increasing larval age. The observation by Brehme (1941a,b) that cell size in Minutes is smaller than in normal animals shows that the Minute aberration is common to all cells and organs. Famsworth (1965a) postulated that the biochemical basis for the Minute phenotype was to be found in some process that is (1) characteristic to all cells, (2) involved in protein synthesis, (3) involved in cellular respiration, and (4) sufficientlycomplex to offer enough sites for control so that many qualitatively distinct lesions could each cause a similar effect. The one process that met all four criteria was that of oxidative phos-

3. The Minute Genes in Drosophila

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phorylation in which ATP is generated. A deficiency of any one or several of the enzymes involved or at any other step where the high energy phosphatase bonds are generated would produce lower levels of ATP in the cells. This would, in tum, affect any cellular processes requiring energy in the form of ATP, among them protein synthesis. In the following study Farnsworth ( 1965b) analyzed oxidative phosphorylation in mitochondria isolated from larvae heterozygous for nine different Minutes on the second and third chromosome. She observed that uncoupling of phosphorylation from oxidation took place in each of these Minutes, since less ATP was formed from the same amount of substrate than in the control. The result was taken to support Schultz’s (1929) postulated Minute reaction: a single response to variety of fundamental stimuli. In this case the single response is a decrease in ATP production suggested to be caused by different basic biochemical lesions. According to Famsworth (1965b) a defective ATP production could partially explain the slow growth and development of the Minutes, since protein synthesis is a dominant physiological activity in larvae and is particularly dependent on a flawless ATP supply. In support of her hypothesis Famsworth referred to experiments made by Wolsky (1937), who used carbon monoxide to block the activity of cytochrome c oxidase in Drosophila pupae. The most conspicuous result of this treatment was short bristles in hatching flies, a typical Minute trait. Farnsworth found additional support of her hypothesis in the work of Wilson and King (1955) and Fleischer et al. (1957). These authors demonstrated that feeding adult females 2,4-dinitrophenol, a known uncoupling agent, slows down maturation of the ovaries and eventually stalls ovarian development. Furthermore, feeding larvae with 2,4-dinitrophenol was earlier shown to slow down growth and delay molting and pupation, thus producing a phenocopy of the Minute condition (Thomton, 1947; Wilson, 1947). Intermediary metabolism and terminal respiration are extremely complex processes and are subject to control at many sites. A very attractive hypothesis was therefore that all Minute genes were involved at some point or other in energy metabolism (Farnsworth, 1965b), but later studies were unable to corroborate the data (Goldin, 1968; Farnsworth and Jozwiak, 1969; Famsworth, 1970). It is important to keep in mind that most, if not all, of the Minutes used in these and later studies were deficiencies. Thus, it was impossible to make any genetic fine analysis and the results were uncertain as to whether the deleted region contained genes other than the Minute gene, something which researchers were aware of.

D. The MinutetRNA hypothesis At about the same time as Famsworth published her papers on the oxidative phosphorylation, Spiegelman and his coworkers published a series of papers on Drosophila DNA-RNA hybridization experiments (Ritossa and Spiegelman, 1965; Ritossa et al., 1966a,b). Hybridization with labeled ribosomal RNA (rRNA) with

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Drosophila DNA revealed that approximately 0.27% of the DNA is complementary to rRNA (Hall and Spiegelman, 1961;Vermeulen and Atwood, 1965; Ritossa and Spiegelman, 1965). This DNA constitutes the transcription units for rRNA and is referred to as rDNA, and the amount per haploid genome specifies 130 molecules each of 18s and 28s rRNA. Ritossa et al. (1965a) showed that, as opposed to 5.8S, 18S, and 28s rDNA, the tRNA genes are not localized in the nucleolar organizer regions and not restricted to the X and Y chromosomes but spread all over the genome. They found that approximately 0.015% of the Drosophila genome is complementary to tRNA, which leads to an average copy number of 13-15 for each of the -60 different tRNA species. Based upon these results and the attractive fact that the number of Minutes and tRNA genes is about the same, Ritossa et al. (1966a) proposed the hypothesis that the Minute loci harbor the redundant tRNA genes. This redundancy implies that one or two point mutations in a tRNA gene cluster will have no effect. What made the tRNA-Minute hypothesis so attractive was the fact that most, but not all, Minutes are associated with a deficiency. This is the only type of mutation that would inactivate tandemly repeated transcription units and thus produce a detectable phenotype. Furthermore, deletions for different tRNA loci would lead to similar resultsthe Minute reaction proper (Schultz, 1929). One way to test this hypothesis was to hybridize 3H-labeled tRNA to salivary gland chromosomes and compare labeled regions with the genetically mapped Minute loci. This approach was used by Steffensen and Wimber (1971), but a convincing correlation could not be made, mostly because several Minute loci were not accurately mapped, and some of the cytological mapping of the radioactivity appeared to be in error. It was later shown by in situ hybridization that tRNA coding sites do not map near known Minute genes (Grigliatti et al., 1977; Elder et al., 1980a,b; Hayashi et al., 1980). Thus it appeared that the Minute-tRNA connection could not be corroborated and additional evidence strongly suggested that Minutes do not correspond to tRNA loci. First, the amounts of 20 aa-tRNAs were assayed in four Minute mutants, and the only difference in comparison with wild-type was found in M(3)69E, where the tRNAThr was reduced to about 60% (White, 1975). Second, by using ethyl methanesulfonate (EMS), which primarily induces point mutations (and sometimes small deletions), it was shown that EMS readily induces Minute mutations (Stone, 1974; Huang and Baker, 1976; Schellenbarger and Duttagupta, 1978). This would not be possible if Minutes are the sites of redundant tRNA genes. Huang and Baker (1976) also found that independent of EMS dose there are about 68 Minute mutants induced for every 5000 recessive lethals induced. These numbers fit well with the relative numbers of existing Minutes and lethal loci in the genome. Since the average size of the Minute locus is the same as the average locus capable of mutating to a lethal, this essentially eliminated the hypothesis that Minute loci are the sites of tRNA.

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E. Mutations in rDNA genes produce Minufe-like phenotypes A number of genetic loci that modify Drosophila wild-type to a Minute-like phenotype by affecting the protein synthesis have been described. Thus, the suppressor offorked [sulf)] was suggested as a putative ribosomal protein mutant (Dudick et al., 1974; Lambertsson, 1975) but this gene was later shown to encode at least three transcripts, one 2.6-kb RNA and two minor RNAs of 1.3 and 2.9 kb (Mitchelson et al., 1993). The major deduced su(f)gene product is an 84-kDa protein that is homologous to RNA14 of yeast, a vital gene involved in RNA stability (Mitchelson et al., 1993). Falke and Wright (1975) described several separate X-linked cold-sensitive female sterile mutants that were proposed to have defective ribosome assembly. However, no further molecular characterization of these mutants has been reported.

1. Mini (min) The eukaryotic ribosome consists of about 80 proteins and 4 types of rRNA: 5S, 5.8S, 18S, and 28s RNA. Ordinarily, the haploid complement of D. mehogaster has about 165 copies of 5s rRNA genes, all clustered on the second chromosome at map position 2-90; cytological position 56El-E6 (Kania et al., 1995; Lindsley and Zimm, 1992). mini (min) is a mutation in Drosophila that deletes almost 50% of the 5s genes; according to Procunier and Tartof (1975) this number is increased by compensation to 265 in hemizygotes for the normal allele. Not surprisingly, the phenotype of min mutants is very similar to that of mutants affecting genes for other rRNAs (bobbed, bb); see below) and the Minute phenotype. Thus, eclosion of min hemizygotes is delayed by about 2 days and viability is reduced, more so at 29°C than at 25°C. Since min deletions include not only 5s rRNA genes but also adjacent tRNA genes, and deletions internal to the 5s rRNA array are not min, the relationship between min and the 5s rRNA genes is unclear (Tschudi et al., 1982).

2. Bobbed (bb) The bobbed locus harbors the structural genes for 5.8S,18S, and 28s rRNA. bobbed mutants are characterized by small bristles, delayed development, and etched abdominal tergites. The mutant alleles of bb can described as hypomorphs (Muller, 1932): they do the same thing as the wild-type allele, but not so effectively. The rRNA genes are clustered, and the X and Y chromosomes of Drosophila each have a cluster of approximately 225 rRNA genes organized as a large tandem array of transcription units separated by nontranscribed spacers. bobbed mutations are partial deletions of the rRNA genes (Ritossa et al., 1966b), and the severity of the phenotype is dependent on the number of rRNA genes present. A 50% reduction

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in the number of rRNA genes results in a bb phenotype whereas a 90% reduction is lethal. In the early studies rRNA gene number was measured by saturation hybridization, a method that was not always reliable (Shermoen and Kiefer, 1975). It was later shown that many of the rRNA genes contain two types of insertions and that these transcription units are not transcribed and consequently not functional (Long and Dawid, 1979; Long etal., 1980;Jamrich and Miller, 1984). Thus, it is the number of functional rRNA genes that is important rather than the absolute number. The rate of oogenesis is slowed down in bb flies, i.e., the time of each stage of oogenesis is increased. However, despite the deletion of rRNA genes in bb flies it has been shown that the total RNA content of developing and mature oocytes is the same in bb as in wild-type flies. It is very likely that in bobbed flies accumulation of rRNA becomes a rate-limiting factor in development. Another intriguing observation is that although the rate of protein synthesis must be lowered in bb oocytes, because of the above-mentioned factors, the proportion of polysomeassociated ribosomes is the same in ovaries of bb and wild-type flies. In addition, there was no difference in average size of polysomal and postpolysomal fractions. These observations led to the proposal that the reduction in the rate of protein synthesis takes place by a concurrent decrease in the rates of initiation, elongation, and termination of translation (Kay and Jacobs-Lorena, 1985).A reduction in the rate of protein synthesis is likely to lengthen the cell cycle and to impair bristle formation and gametogenesis, which are postulated to require maximum rates of synthesis. Apart from thinning and shortening of the bristles and etching of the abdomen the body of bb mutants is relatively normal. This suggests that most developmental programs are safely guided through development despite the reduced capacity in protein synthesis (Kay and Jacobs-Lorena, 1987); it is only those body parts/organs which require maximum rates of protein synthesis that will be affected. Therefore, lowering the temperature minimizes the effects of bb mutants.

111. MINUTESAS GENETIC TOOLS IN STUDIES OF GROWTH AND DEVELOPMENT Apart from the typical Minute characters mentioned above Minutes also present exceptional genetic properties. Thus Stern (1936) and Kaplan (1953) showed that Minutes had an increased rate of spontaneous mitotic recombination in their own chromosome arm. Miklos (1972) showed another peculiar genetic property of Minutes, that of inducing nondisjunction of the X chromosome. An important finding was made by Stern and Tokunaga (1971), who showed in genetic mosaics that bristle differentiation and cell lethality of some Minutes are cell autonomous in their expression (also see above).

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A. Mitotic recombination In Minufe mutants

1. Parameters in development Ferrus (1975) analyzed and correlated mitotic recombination parameters with other developmental parameters in 16 Minutes in an attempt to understand the basis of the Minute phenotype and to explore the usefulness of Minutes in clonal analysis. The Minutes represented 12 loci distributed over all chromosome arms and included three pairs of alleles and four deficiences. In each Minute both spontaneous and X-ray-induced mitotic recombination frequencies as well as clone sizes of the different cell marker clones were analyzed. These parameters were studied both in wing disks and in abdominal histoblasts. The frequencies of spontaneous mitotic recombination were not affected by the presence of the Minute mutations studied. O n the other hand, most of the different Minutes significantly increased the frequency of induced mitotic recombination relative to wild-type. The analysis of different parameters revealed that developmental delay, increase in M clone size, bristle length, and mitotic recombination frequencies are all correlated. These results have been useful in developmental studies of imaginal disks. +

2. Cell competition It was shown by Stern and Tokunaga (1971) in genetic mosaics that bristle differentiation and lethality of some Minutes are cell autonomous in their expression. The retarded development in Minutes was studied by Morata and Ripoll (1975), who showed that their extended development was the result of a cellautonomous slower rate of cell division. They found that when clones of Minute cells are produced by inducing mitotic recombination in non-Minute animals they do not survive. This elimination of slowly dividing but viable cells was termed cell competition. It must be pointed out, however, that the Minute cells are inviable only when growing next to non-Minute cells. When Minute+ clones are produced in a Minute background they grow to be very large as a consequence of their advantageous growth rate. However, these clones always respect compartment boundaries (Garcia-Bellido et al., 1973,1976). Two models were proposed for the effect of Minute mutations on larval development. 1. Minutes do not directly affect larval cells, but larval delay is a consequence of delayed imaginal development. However, as Sheam et al. (1971) had shown that the presence of imaginal discs is not necessary for larval development, this model would require additional both complicating and confusing assumptions. 2. Minute mutations affect both larval and imaginal cells at the same metabolic level. Minutes are dosage sensitive, suggesting that the presence in both larval and imaginal cells of normal levels of Mf products is a limiting factor required for development to proceed. This product might be required for cell growth and the rate of cell growth could regulate both the rates of larval development and imaginal disk cell division.

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The autonomous nature of both normal and mutant Minutes on the division rate of proliferating cells has been taken advantage of in developmental studies. This makes it possible to study both normal and mutant development in the same individual and has been successfully used to study clonal restrictions and growth in compartments in the wing imaginal disks (Garcia-Bellido et al., 1973). Using two different Minutes Simpson and Morata (1981) showed that cell competition results from differences in growth rate between cells and that the intensity is greater in the more extreme Minute with the slowest rate of cell division. These authors also showed that cell competition mainly occurs in the center of compartments and not across compartment boundaries.

3. Maternal effects of minutes on early development Females heterozygous for Minute M(3)67C were used to study the effect of maternal haploinsufficiency on protein synthesis and segmentation in embryos. Embryos from M(3)67C/+ females were shown to have lowered rates of protein synthesis by =30% during the syncytial nuclear cycles of early embryogenesis (Boring et al., 1989). The maternal effect of the Minute mutation also produces segmentation defects in the posterior abdomen of developing larvae. In addition, embryos from Minute females revealed abnormal expression patterns of the segmentation gene fwhi turazu (ftz) at the cellular blastoderm stage of embryogenesis (Boring et al., 1989). The effects of the Minute mutation could be phenocopied by injecting wild-type embryos with cycloheximide at concentrations which lowered protein synthesis rates by -30% (see above). The results were taken to show that M(3)67C is involved in protein synthesis and that a general decrease in protein synthesis rates during early embryogenesis results in abnormal determination of the posterior abdominal segment primordia (Boring et al., 1989). While this may be true it would be interesting to study other Minutes for these effects.

B. Minufes as suppressors Drosophikamekanogaster females homozygous for the X-linked mutant gene zeste (z; Gans, 1948,1953) have lemon yellow eye color, whereas hemizygous males have the normal red eye color. The yellow zeste phenotype can be evoked in males by duplicating the rightmost part of the white locus (the we and wsP sites). In contrast, the zeste phenotype is suppressed in homozygous z females that are heterozygous for a deficiency or a mutant allele of the same part of the white locus. Mutagenesis experiments, using EMS, ICR- 170, and X-rays, were performed to induce modifiers of the zeste expression (Green, 1967; Kalisch and Rasmuson, 1974). Several dominant autosomal modifiers, suppressors as well as enhancers, of the zeste eye color phenotype were isolated, and notably some of these had the Minute phenotype. In an extensive study of modifiers of zeste Persson (1976a,b)

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showed that one, namely Su(d5, had typical Minute characteristics; for example, homozygous lethal, shorter and thinner bristles, and about 3 days delayed larval development at 25°C. The females were also practically sterile at temperatures above 18°C. Most interestingly, this stock reverted frequently to wild-type, suggesting that Su(z)5 was induced by a mobile element; today, several Minutes have been isolated by using P-element mutagenesis (see below). Persson (197615) also tested the ability of several Minutes and Su(z) genes to suppress the zeste eye color in three different types of zeste males: wis, z Dp(J ;I)wG, and z Tr(w+R). Surprisingly, all Minutes, except M(3)40/30 (cytological position unknown), were clearly able to suppress zeste only in the 2: Dp( J ;J) wG stock whereas only a slight suppressor effect was observed in the Tr(w+R) . Both M(3)40/30 and the Su(7) genes, including Su(z)5 which has Minute phenotype, suppress zeste in all three stocks. It is important and intriguing to note that no modifier effects were observed in females. Thus, the ability of Minutes to suppress the zeste eye color is exclusively revealed in males with a duplication (or triplication) of the chromosomal segment reaching from the zeste locus up to and including the white locus, as in Dp(J; J)wG and Tr(l; J)wTG.However, no correlation between severity of the Minute and degree of suppression of zeste was observed. In contrast, for the Su(z) genes to suppress zeste only an intralocus duplication for a right nondose compensated sublocus of the white locus is required. The zeste gene interacts not only with the white locus but also with the bithorax and decapentaplegic complexes, changing the phenotypic expression of these loci (for references, see Lindsley and Zimm, 1992). The zeste transcript is most abundant in maternal FWA, declines during larval growth and increases again in third instar larvae and pupae (Pirrotta et al., 1988). The gene product is found in the nucleus and functions as a transcription factor. The protein binds directly to specific DNA sequences of white, Ultrabithorax, decupentapkgic, Antennapedia, and engraikd regulatory regions (Benson and Pirrotta, 1987,1988; Biggin et al., 1988; Mansukhani et al., 1988). Females deficient for z survive and are fertile, indicating that the zeste gene product is not required for viability or female fertility (Goldberg et al., 1989). So, how can we explain the suppressor effect of Minutes on zeste? No doubt, the regulatory gene zeste has a complex network of genetic interactions as indicated by the fact that anti-zeste antibodies interact with approximately 60 specific bands in polytene chromosomes; this number is dramatically increased by heat-shock (Pirrotta et al., 1988). The expression of the mutant lemon yellow eye color requires two proximate or paired copies of w+, whereas in hemizygous males or in females with unpaired copies of w+ the eye color is wild-type red. Thus, duplicating (or triplicating) the nondose compensated right part of the white locus changes the eye color to lemon yellow in males and in heterozygous &+females. Persson ( 1976b) suggested that the nondose compensated right part produces some factor that regulates the zeste locus or interacts with its product. The white

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product must also be present at a critical level to evoke the zeste eye color. A duplication of the right part of the white locus will lead to an overproduction of the white product resulting in the zeste eye color in z males and in dz+ females. Interestingly, the Su(z) genes are able to suppress the zeste eye color to wild type, and the smaller the duplication in the right part of the white locus the better the suppression. In contrast, the suppressor effect of the Minutes requires longer aberrations, like ZIP( 1;1)zffi and Tr(1; 1)zG where the whole white locus is duplicated. The reason for this is not understood at present. However, assuming that the white locus produces a zeste-interacting factor when the cells synthesize the eye pigments, the amount of this factor may be reduced in Minutes due to the lowered rate of protein synthesis. When the amount of the white product falls below the minimum level required for evoking the ZeSte eye color the wild-type eye color is restored. In his study Persson (1976b) also found that different Minutes effected the zeste phenotype differently. This observation is very likely explained by different Minute genes being transcribed in different amounts above the minimum levels required for normal function. This in tum may explain why different Minutes have such varied phenotypic expression (see under “Antisense mRNA Approach”). Another explanation could be that Minutes delay the pupal period and thus allowing more pigment to be accumulated. zeste flies reared at lower temperatures develop darker eyes than at 25°C and lighter eyes when reared at higher (than 25°C) temperatures (Jan Larsson, personal communication). This explanation may seem less likely mainly because Minutes are generally known not to affect the length of the pupal development. However, Brehme (1939) showed that M(3)95A1/+ heterozygotes in addition to forming puparia 41-43 hr later than wild type also have the pupal period prolonged by 12 to 21 hr. Whether all Minutes have a prolonged pupal period is probably not tested.

IV. MlNUTES AND THE PROTEIN SYNTHESIS THEORY The general nature of the Minute mutations was noted already by Schultz (1929), who postulated a primary and a secondary effect (action) to explain this. Later studies by others gradually focused on lesions in the protein synthesis machinery as the primary effect of Minute mutations. Earlier work by Famsworth and Goldin (see above) suggested that protein synthesis was impaired in Minutes, but the primary action was believed to affect the terminal respiratory system. In fact, Goldin (1968) suggested and also rejected the possibility that their experiments were testing the result of some more general and primary metabolic disturbance such as a general block of protein synthesis. In a study of the relationship between Minutes and suboptimal translation Walker ( 1985) showed that newly emerged Minute females bearing the M(3)95A, M(3)69E, and M(1)18C contained a lower level of

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yolk proteins than their non-Minute sisters. This low amount of yolk polypeptides correlated with a lower abundance of cytoplasmic RNA in Minutes compared to non-Minutes. However, by one week of age both Minutes and non-Minutes contained the same amount of yolk proteins and the corresponding RNA. These observations lead to the conclusion that adult Minutes have a slower maturation (just as Minute larvae); and there was no evidence to suggest that the primary lesion in Minutes affects the translational machinery (Walker, 1985). As mentioned above, the first really interesting hypothesis was the one suggested by Ritossa et al. (1966) that Minutes are the genes for tRNAs and that mutations in these genes would affect protein synthesis. This hypothesis was mainly based on the similarity in number of Minute loci and tRNAs and the phenotypic abnormalities associated with the Minutes. As mentioned already, this theory was proven wrong but the notion that Minutes were involved in some way or other in protein synthesis was not abandoned. The rRNA genes have already been discussed and we know that deficiencies for these genes produce a Minute or Minute-like phenotype. So, if Minute mutations affect protein synthesis the ribosomal proteins are the only likely candidates remaining as products encoded by the Minute genes. The eukaryotic ribosome contains about 80 different proteins, a number that could match the number of Minutes, although the existing numbers of Minutes were only 50. This number is probably too small since most Minutes supposedly are deficiencies, some of which could cover more than one Minute gene.

A. Molecular biology comes of age in Minute research The introduction of hybrid DNA techniques in the 1970s revolutionized molecular biology research. This technique allows the researcher to clone genes and transform them back into the organism to study the effect of changes (small or large) made in vitro on gene expression.

1. Rp49-the

first rp-gene to be cloned

To isolate segments of D. melanoguster DNA containing ribosomal protein (rp) genes, Vaslet et al. (1980) purified 6s-12s poly(A)+ mRNA, the likely size for rp mRNA. This RNA was then labeled to a high specific activity with reverse transcriptase and used to screen a genomic D. melanogaster lambda library. Potential clones were then analyzed for the presence of rp genes. One clone, C25, was shown to contain the structural gene for D. melanoguster rp49. This clone was then mapped to its genomic position 99D on the third chromosome by in situ hybridization. As pointed out by the authors this map position is close to the genetic locus of the first Minute locus discovered, namely M(3)99B (Lindsley and Zimm, 1992). This Minute maps 0.3 map units to the right of the claret eye color gene,

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which was cytologically mapped to region 99C-E (Bridges and Morgan, 1923). The cloning of a Drosophila rp gene that mapped very close to a Minute locus led Vaslet et al. (1980) to suggest that Minute genes may encode ribosomal proteins. Later, several rp genes were cloned and characterized by Chooi and coworkers (Bums et al., 1984, and references therein). Many of the clones containing rp genes were cytologically mapped within the genetic loci of Minute mutations (Table 3.2); for example, Rp7/8 hybridized to 5D on the X chromosome, RpSl8 to 15B on the same chromosome, and RpLl2 hybridized to 62E on the second chromosome. This further strengthened the notion postulated by Vaslet et al. ( 1980) that Minute genes encode ribosomal proteins. However, these correlations between location of rp genes and the mapped Minute loci were circumstantial in that they could have been coincidental.

2. The breakthrough a. M(3)99D encodes RPL32 [RP49] To prove that a Minute gene encodes a ribosomal protein required more advanced genetic and molecular biological methods. The methods for DNA-mediated germline transformation in Drosophila, based on the behavior of cloned and manipulated P-elements, were introduced in 1982 (Spradling and Rubin, 1982; Rubin and Spradling, 1982) and are now standard procedure for analyzing many problems in Drosophila molecular genetics. The technique was used by Kongsuwan et al. (1985), who used the already cloned gene for RP49 (Vaslet et al., 1980) to transform M(3)99D/+ heterozygotes. (The mammalian homolog to RP49 is L32 and RP49 should therefore be called RPL32). Transformed flies were “rescued” in the sense that they no longer had the Minute phenotype, unambiguously demonstrating that the M(3)99D gene encodes RP49. So, it appeared that the best criterion to establish the Minute-to-ribosomal-protein correspondence would be the rescue of a Minute by a cloned rp gene, cytologically mapping in the region, by P-element-mediated transformation.

b. The RpL32 [Rp49] gene The RpL32 gene, cytologically mapped to 99D2-9 (Kongsuwan et al., 1985), shares most of its characteristics with other rp genes found in eukaryotes (O’Connell and Rosbash, 1984). The 5’ end of Rp49 has possible CAT and TATA box-like sequences, ATGCATTAG at position -90 and ATATTT at -50, respectively, and a very short (9-bp) 5’ UTR. The putative transcription start site is not in a pyrimidine rich tract, however, which is usually the case for eukaryotic rp genes (see below). The first exon is 102 bp long, followed by a short intron of 59 bp, and a 420 bp 3’ exon, encoding a 132-amino-acid-long protein. It is interesting to note that RpL32 has an alternative uninterrupted open reading frame in ad-

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dition to the putative RPL32 coding sequence (OConnell and Rosbash, 1984). This extra reading frame has not been observed to produce any polypeptide.

c.

M(1 ) 1B encodes RPL36

This mutation was discovered by Patterson (1932) as a typical Minute with the characteristic short slender bristles, delayed development, and homozygous lethality. Heterozygotes deficient for the locus are extreme Minute of low viability and 75 hr delayed eclosion (Ferrus, 1975). M( I ) I B has been reported to affect posterior pattern formation and to act, probably indirectly, as a dosage-sensitive maternal modifier of runt (run) (Duffyet al., 1996). It also interacts genetically with eve, Kr, and kni (Duffyet al., 1996). The M( I ) IBB46allele also interacts with runt and causes tergite defects in greater than 65% of run3 heterozygotes. The penetrance and severity of the segmental defects seen in larvae heterozygous for the Kr or kni mutations increase with reduced maternal M(f)IB activity (Duffy et al., 1996). Reduced maternal M(f)IB activity has no effect on the larval or adult phenotypes of twi or Dl heterozygotes or on the viability of Kr heterozygotes. Both the recessive lethality and the Minute phenotype of M(1) I B were rescued by a 10-kb genomic DNA fragment that was reintroduced by P-element transformation (Voelker et al., 1989). The M( I ) I B locus was suggested to encode a 3.5-kb message that would produce an =110,000-Da protein (Voelker et al., 1996). This is far larger than any known ribosomal proteins, which range from 3,454 Da (rat L41) to 47,280 Da (rat L4) (Wool et al., 1995), and it seems unlikely, therefore, that the 3.5-kb transcript encodes a ribosomal protein. A more likely candidate is a 1898-bp genomic DNA sequence from the same region that contains a potential reading frame for a 115-amino-acids-long protein that has homology to rat L36 (W. R. Steinhauer, FlyBase; accession numbers: U20543; P49630). The correct name for M(1) I B now is RpL36. Since RpS4 also has been mapped to 1B (and to 3A; see below; Yokokura et al., 1993), it is likely that the 1B region contains at least two rp genes.

d. Antisense mRNA approach Several attempts were made to rescue other Minutes with the transformation technique but these experiments failed (Kay and Jacobs-Lorena, 1987; Qian et al., 1988; Kay et al., 1988; Dorer et al., 1991). The main reason for this failure is probably that most Minutes represent chromosomal deletions and that these may encompass more than one rp gene. Another reason is that genes may integrate in or close to heterochromatic regions resulting in no or insufficient product being made to rescue the mutation. Yet another explanation may be that the various r p s are produced in amounts with different “safety margins” with respect to the minimum level at which they are required. According to this model, rps which are

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normally produced close to their threshold requirement will be present in insufficient amounts after a reduction by =50% (haploinsufficiency), with a concomitant reduction in ribosome assembly and protein synthesis, whereas a similar reduction of r p s that normally are overexpressed has less effect. It is important to understand that the threshold level for sufficient production of a ribosomal protein must involve the combined efficiences of mRNA accumulation, translation, transport from the nucleus to the cytoplasm and back to the nucleolus, and assembly into ribosomes. Thus, high abundance of a specific rp mRNA does not necessarily mean that it is overproduced, but can be compensatory for a low efficiency in some other process. Recent results in our laboratory show that the severity of the Minute phenotypes in three mutants is inversely correlated with the relative abundance of their respective mRNAs in wild-type flies (Saeboe-Larssen, 1997; S. Saeboe-Larssen, E-E. Aas, and A. Lambertsson, manuscript in preparation). These results are supported by the observations that heteroalleles in a single rp locus can mimic the phenotypic differences that exist between different Minutelrp gene mutations (Szboe-Larssen et al., 1998). To overcome this flaw attempts were made to inactivate an rp gene by constructing the corresponding antisense gene (Qian et al., 1988; Patel and Jacobs-Lorena, 1992). Cloned RpAI DNA was placed in sense and antisense orientation under the control of the strong heat-shock promoter hsp70 and Pelement transformed into flies. Flies exposed to 37°C for I hr accumulated threeto fourfold of sense or antisense RNA levels as compared to the endogenous rp49 mRNA. Frequent induction of the antisense gene during development caused a slight developmental delay, and some of the hatching “antisense” flies also had thin and short bristles, typical traits of the Minute phenotype. In addition, “antisense” females subjected to heat pulse laid fewer eggs, indicating an affected oogenesis. These females recovered their fertility after two days at normal temperature. Despite the promising results this experimental approach to correlate Minutes and rp genes has not been pursued. One reason for this is that antisense constructs are unstable in flies and that the stability changes with age of the stock (Patel and Jacobs-Lorena, 1992). Another explanation is that not all of the rp genes are equally dosage sensitive, which appears to be a prerequisite for experiments of antisense interference (Patel and Jacobs-Lorena, 1992).

9. The use of transposons to clone Minute genes One of the most important technical innovations in Drosophila genetics in the past decade has been the development of inducing mutations with single modified and marked P-elements (Cooley et al., 1988; for references, see Wolfner and Goldberg, 1994). These P-elements are stable once integrated because they lack transposase-encoding sequences that would cause them to move on their own. A modified P-element contains sequences encoding a dominant visible marker (for

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example, a white+ or rosy+ eye color gene), which allows the detection of flies containing the P-element and screening of transformants. They also have the bacterial plasmid sequences for ampicillin resistance and replication origo. This facilitates the cloning of Drosophila sequences adjacent to the insertion site of the P-element. Another useful sequence often included in the P-element constructs is the lac2 sequence. Fused to a weak but general promoter the lac2 allows the detection of enhancers with a developmental expression pattern. The P-element is mobilized by crossing flies carrying the marked Pelement with others having a transposase gene called 42-3 (Robertson et al., 1988). Among the progeny are flies carrying both the marked P-element and the transposase gene, the “jumpstarter” flies. In the next generation jumps of the marked P-element are identified in flies in which the marker no longer segregates with the chromosome on which the P-element was originally located. These flies are then analyzed for the desired (usually dominant) phenotype or used individually to establish stocks carrying the chromosome with the inserted P-element. Today, 7 Minute genes (all encoding ribosomal proteins; Table 3.2) have been cloned and characterized by this technique. Below is a presentation of these genes, and along with those fortuitously stumbled upon in the search for other genes. (Tables 3.1 and 3.2). It should be noted that genes whose name in FlyBase now is rp or M(*) will become Rp***, where It***” is the S (small subunit) or L (large subunit) designation and the number (as in, for example, RpS3 and RpL14) (M. Ashburner and A. de Grey, personal communication; FlyBase and A. Lambertsson, FlyBase Nomenclature, May 1997). Genes currently named for homology or function will also be renamed according to this scheme. However, genes now named for mutant phenotypes will stay as they are, according to the Drosophila convention. Whereas I have adopted these changes in headings and tables, new and old gene names intermingle in the text.

1. sop (string of pearls) [RpS2; M( 2)3OD/E] a. Gene and phenotype The first allele of string of pearls (sop), sopp, was isolated as a recessive autosomal female sterile mutant in a P-element mutagenesis screen (Cramton and Laski, 1994). The P-element insertion site was mapped by in situ hybridization to the 30D/E border on chromosome 2L. Egg chambers of homozygous sopp ovaries appear to develop normally up to stage 5 (King, 1970), but development is stalled at this point. Nonetheless, each cyst in sopp ovaries appears to properly differentiate 15 polyploid nurse cells and one oocyte, which is positioned correctly at the posterior of each developing egg chamber (Cramton and Laski, 1994). sop‘ males are fertile. Interestingly, homozygous sopp flies (but not heterozygotes), both male and female, have short and thin bristles, delayed larval development, and larval

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lethality. sopp homozygotes have a survival rate of 10-15% of the number expected if the flies were fully viable (Cramton and Laski, 1994). The authors are cautious and suggest that the Minute traits of “homozygous animals qualify sop to begin a subclass of ribosomal protein genes with Minute-like characteristics” (Cramton and Laski, 1994). However, based on the available data, especially Figure 2A, I suggest sopp be classified as a regular dominant Minute allele, M(2)30D/E. sop is a single copy gene and consists of two exons interrupted by a 340nt long intron in the 5’ UTR. The fact that the only intron is in the 5’ UTR is an unusual condition, as most rp genes have one intron just downstream of the translation start site. As mentioned before, the first intron in rp genes may contain sequences important for gene regulation. The transcript is 905 nt long producing a predicted 267-amino-acid protein. The transcription start site is in a polypyrimidine tract, but the CTTTT’sequence is not obvious. The P-element insertion in sopp is located 28 nucleotides upstream of the predicted transcription start site; that is, in the promoter region. The cloning and analysis of the Drosophila S2 cDNA was first reported by Barrio et al. (1993). The sop gene produces a single 0.9-kb transcript that is abundantly expressed at all developmental stages (Cramton and Laski, 1994). It is noteworthy that heterozygous and homozygous flies show a reduction and not a knock-out of sop mRNA, a situation similar to RpS6 and RpL14 (Watson et al., 1992; Stewart and Denell, 1993; Szeboe-Larssenet al., 1997). Quantitation of transcript abundance in sop homozygotes (relative to RP49) demonstrated a 60-70% reduction of sop mRNA relative to wild-type flies. We have observed the same amount of reduction (-70%) in homozygous RpS3W flies, which show a severe Minute phenotype, have very low viability, and both sexes are sterile (SEbae-Larssen et al., 1998;see below). These results eloquently establish a correlation between mRNA abundance and strength of Minute phenotype.

b. The RPS2 protein As mentioned above, the predicted Drosophila DS2 protein is 267 amino acids long and shows 82 and 60% amino acid identity with the rat and yeast homologs, respectively (Cramton and Laski, 1994). Eukaryotic S2 is the homolog of prokaryotic ribosomal protein S5, and there is about 30% identity between Drosophila S2 and the bacterial equivalent (Ramakrishnan and White, 1992; Suzuki e t al., 1991; All-Robyn et al., 1990). The eukaryotic S2 protein belongs to the Group I ribosomal proteins, which have homologs in the eubacterial and archaebacterial kingdoms (Wool et al., 1995; see below). The prokaryotic homolog S2 and the yeast homolog YS2 were first isolated as universal suppressors or ribosomal ambiguity (ram) mutants (Piepersberg et al., 1975; All-Robyn et al., 1990), and it is possible that ribosomal protein S2 has a similar proofreading function in ribosomes of higher eukaryotes. Oogenesis in sopp mutant ovaries stops at stage 5. This phe-

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notype is reminiscent of recessive mutants like Bicaudal-D and egalitarian (Mohler and Wieschaus, 1986; Schupbach and Wieschaus, 1991; Theurkauf et al., 1993). Mutations in these genes arrest oogenesis at stage 5 and either fail to produce differentiating cysts or maintain the identity of the oocyte. In contrast, sopp mutant ovaries develop an oocyte that is correctly positioned and is discernible by the smaller nonpolyploid nucleus (Cramton and Laski, 1994). The low fertility of heterozygous females is a well-known Minute characteristic. The reduction in fertility shows the same quantitative variation as the other Minute somatic characters. Jacobs-Lorena and coworkers used antisense constructs for ribosomal proteins A1 and 49 and were able to produce a “small egg” female sterile phenotype and a transient stall of oogenesis, respectively (Qian et al., 1988; Pate1 and Jacobs-Lorena, 1992). These results suggest that oogenesis is sensitive to a reduction in the amount of a ribosomal protein. Considering the extreme requirement for efficient protein synthesis during oogenesis these results are not surprising. Even a modest decrease in the level of a ribosomal component that will lead to a reduction of complete ribosomes needed for efficient protein synthesis is likely to slow down or arrest oogenesis. If each ribosomal component has a threshold requirement and a level of expression that may vary in different tissues, then the amount of any one component that is close to the essential threshold in a particular tissue may make that tissue more impressionable to a reduction in expression and produce a phenotype that appears to be tissue specific. This may explain the great variation of the somatic characters observed among the Minute mutations. Alternatively, Drosophila RPS2 may have a nonribosomal role (cf., RPS3 and RPS6) during oogenesis.

2. RpS3 [M(3)95A] a. The RpS3 gene and mutant phenotype M(3)95A has a severe Minute phenotype and was one of the first Minutes to be isolated and characterized (Bridges and Morgan, 1923; Schultz, 1929).Several alleles have been described, but most of them appear to have the same severe Minute phenotype (see Lindsley and Zimm, 1992); they are probably deficient for M(3)95A and the surrounding DNA regions. M(3)95A heterozygotes have very short and thin bristles, and eclosion is delayed 40-42 hr at 25°C;homozygotes die in first instar. M(3)95A also enhances L and B (Dunn and Coyne, 1935), Bx3, Nco, Nfa-1, ap4, Jag, Ser, Ly, and apXa (Bryson, 1939). In a P-element mutagenesis experiment using the Plhc921 construct described by O’Kane and Gehring (1987), one Minute male was obtained from approximately 10,000 progeny (Anderson et al., 1994). Recombination analysis mapped the insertion and the mutant phenotype to map position 111-73,two map units to the right of ebony. Genomic DNA 5’ to the insertion site was obtained by inverse PCR, and this DNA was then used to screen a Drosophila genomic A li-

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brary. DNA from this region was also used as probe for in situ hybridization to salivary gland polytene chromosomes, and the hybridization was localized to region 94F/95A on the third chromosome. Minute M(3)95A was earlier mapped to this region, and in a complementation analysis the P-element-induced Minute, now called P{Iac921M(3)95A, was shown to be allelic to M(3)95AJ and M(3)95A2. The gene was cloned, and sequencing genomic and cDNA material revealed that Minute M(3)95A encodes the Drosophila homolog to mammalian ribosomal protein RPS3 (Anderson et al., 1994). Sequence analysis also showed that the M(3)95A gene is a typical ribosomal protein gene as it lacks TATA or CAAT motifs frequently found in promoter regions of genes transcribed by RNA polymerase 11. Furthermore, the transcription start site is in a polypyrimidine rich tract, which is typical for mammalian and many Drosophila rp gene cap sites (Wool et al., 1995; Stewart and Denell, 1993; Mager, 1988). There are two open reading frames, 36 and 702 nucleotides long, respectively, separated by a single 256nucleotide intron. The deduced protein is 246 amino acids long and has a predicted molecular mass of 27.5 kDa. In comparison, the calculated molecular mass of human and rat RPS3 is 26.7 kDa, and that of Xenopus is 27.0 kDa. The amino acid sequence of the Drosophila RPS3 protein is strikingly conserved when compared to these species; the overall identity is ~ 7 8 % for all three. As expected for a ribosomal protein gene, the RpS3 gene is abundantly expressed in all developmental stages. The level of transcript is reduced by about 50% in P{hc921M(3)95A heterozygotes, showing that the P-element insertion disrupts the locus encoding RPS3. The amount of transcript is restored to wildtype levels in homozygous revertants. Interestingly, the high-resolution Northern gels used by Anderson et al. (1994) revealed the presence of two RpS3 transcripts, differing in size by approximately 40 nucleotides. The proportion of the transcripts does not change during development. Whether the two transcripts are generated by alternative splicing or by other means is not known.

b. The RPS3 protein The RpS3 gene was first cloned from a Drosophila cDNA library using the rat S3 homolog (Wilson et al., 1993). These authors were interested in ribosomal protein genes that were overexpressed in colorectal cancers and that might have alternative roles for their encoded proteins. RPS3 had been shown to cross-link to eukaryotic initiation factors eIF-2 (Westermann et al., 1979) and eIF-3 (Tolan et al., 1983). Wilson et al. (1994) showed that RPS3 is also strongly associated with the nuclear matrix, a result which is consistent with the presence of a nuclear localization signal for S3 (Chelsky et al., 1989).The Drosophila S3 protein was postulated to have a role in DNA metabolism, and it was subsequently shown to cleave DNA containing an apurinic/apyrimidinic site via a p-elimination reaction (Wilson et al., 1994); that is, it behaves as an AP lyase. It was recently shown that Drosophila RPS3 efficiently cleaves DNA con-

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taining 8-oxoguanine residues (Yacoub et al., 1996a). The importance of this DNA repair activity acting on 8-oxoguanine is emphasized by the ability of S3 to rescue the H,O, sensitivity of an E. coli mutM strain, which is defective for the repair of 8-oxoguanine. RPS3 is capable of fully abolishing the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-to-T transversions. RPS3 is also capable of rescuing the alkylation sensitivity of an E. coli mutant deficient for the AP endonuclease activities associated with exonuclease I11 (xth) and endonuclease IV (nfo). These results are very interesting and suggest that an AP lyase may represent a significant source of DNA repair activity for the repair of AP sites. The results also raise the possibility that DNA repair and protein translation are coupled. Thus, RPS3 appears to belong to the intriguing class of ribosomal proteins that may have other roles in the cell (see below). Another Drosophila ribosomal protein, PO, has recently been found to have AP endonuclease activity, suggesting that this protein represents a multifunctional protein with a role in DNA repair (Yacoubet al., 199613).The Drosophila PO acts on abasic DNA, a lesion known to block DNA replication and cause mutations in E. coli. Notably, PO also contains vigorous DNase activity for both single- and double-stranded DNA. PO can be found in the nucleus tightly associated with the nuclear matrix.

3. Homozygous viable RpS3 alleles To revert the severe Minute phenotype of P{Iac92m/1(3)95A to wild type, the Pelement was remobilized and apart from wild-type revertants several partial revertants were also found (Saebfle-Larssenet al., 1998). Two of these, M(3)95Aprv9 and M(3)95Apv", were found to survive in homozygous condition and were further characterized genetically and molecularly.

'

a. RpS3p"l [M(3 )95APrv1'1 In the heterozygous condition this allele is a weak Minute with respect to the bristle and the delayed development phenotype. Somewhat surprisingly we found homozygous RpS3p"" flies that are viable and fertile. The Minute phenotype of these flies is more severe than that of heterozygous RpS3P"" flies; we classify the phenotype as medium Minute. RpS3p"" is semilethal in combination with the P{Iac92N(3)95A allele; the survival rate of these heterozygotes is only 3-5% of the number expected if the flies were fully viable. Most animals die as pupae or are too weak to break out of the pupal case. The heterozygous flies that hatch show a drastically increased Minute phenotype and also have severe morphologic lesions such as missing antennas and legs, very small and rough eyes, and they usually live for only 2 or 3 days. R P S ~ ~ " ~ / R P S heterozygotes ~~~"'' have a severe Minute phenotype. Again, there is a significant correlation between phenotypes and abundance of RpS3 mRNA in these lines.

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When analyzed at the nucleotide level we found that the P-element had left a 32-nt long insertion in the 5’ UTR upon excision (SEbeie-Larssen et al., 1998). This insertion consists of the P-element’s 5’ (17 nt) and 3’ (15 nt) ends, including the inverted repeats that may form a hairpin structure at the DNA and/or RNA level. In addition, this sequence also contains four uATGs (upstream ATGs), two of which are at the 5’ most end of the UTR. These two are followed almost immediately by a stop codon. The 3’ penultimate uATG is out of frame and has a stop codon at +863 when the original splice sites are used; if these are not used there is a stop signal at position +653. The 3’most uATG is in frame with the downstream ORE It had been shown earlier that the P-element insertion in the 5’ leader sequence caused an -50% reduction of RpS3 transcript in P{Iac92M(3)95A heterozygous flies (Andersson et al., 1994). Quantitative Northern analysis of homozygous and heterozygous RpS3pw” flies revealed that the amount of RpS3 mRNA is reduced to =80 and 90%, respectively (Saebae-Larssen et al., 1998). Thus, a reduction by as little as =10% of RpS3 transcript is enough to produce a weak but clear Minute phenotype in heterozygous flies. This may appear as a marginal reduction that should not have any effect on the organism, but we have to keep in mind that P{hc92)M(3)95A (with a 4 0 % reduction in mRNA) as well as the M(3)95A1 and M(3)95A2 (deficiencies?)alleles are severe Minutes. This may indicate that the normal expression level of RpS3 is close to the relevant threshold and, thus, that the slightest reduction will produce the Minute phenotype.

b. RpS3Pw9 [M(3)95Aprvl Heterozygous RpS3pV9/+flies have a medium Minute phenotype and homozygous RpS3pw9/RpS3pv9animals show an extreme Minute phenotype with both sexes completely sterile (Saebae-Larssen et al., 1998). The sterility is caused by a very early disruption of gametogenesis, resulting in no or very small gonads with no functional cells (Figure 3.2). Homozygous RpS3pw9flies also have morphological lesions indicating defective imaginal disk development. Another conspicuous effect observed is the uncompleted rotation of segment A9 in males, which carries the external genitalia. During normal male development the genitalia (A9) rotate 360” in the pupal stage so that the vas deferens loops once about the intestine (Gleichauf, 1936). In homozygous RpS3pv9 males this rotation is uncompleted in about 65% of the eclosed males, and several different degrees of uncompleted rotation are observed. Little is known about the mechanisms behind this process. RpS3Prv9 is lethal in combination with P{hc92M(3)95A and RpS3pw9/RpS3w1flies are severely defective but fertile Minutes. Only 50% of the expected number of homozygous RpS3pV9flies hatch (with twice as many males) and these animals are generally very weak. Under crowded conditions only 10-15% RpS3pwgflies hatch.

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Figure 3.2. Scanning electron microscopy of half of a wild-type ovary (A) and a whole ovary from a homozygousM(3)95Apv9 female (B). There are no ovarioles in the mutant ovary (B) and the network of connective tissue, the peritoneal sheath, that holds the ovarioles together is not recognizable. ( 0 ) Ovary; (01) ovariole; (ps) peritoneal sheath; (t) trachea; (uo)undeveloped ovary.

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Southern analysis had shown that the P-element excised imprecisely, and subsequent sequence analysis revealed that 102 nt of the P-element were left behind: 18 bp from the 5' inverted repeat, the intact 3 1-bp 3' inverted repeat and 53 bp from the internal P-element. The 102-bp insert may form a base-paired stem with a single loop and in addition to the four extra ATGs present in the 32-bp insert the 102-bp sequence contains a fifth ATG in-frame with the downstream ORE Quantitative Northern analysis of homozygous RpS3pv9 females revealed that the amount of RpS3 mRNA is only 30% of that of wild-type females. In comparison, the amount of RpS3 transcript is -55% in homozygous RpS3pv9 males. this may explain why approximately 50% more homozygous males hatch. These results reveal the straightforward correlation between amount of rp transcript and the strength of the M(3)95A phenotype. It is most likely, therefore, that the reduction of mRNA abundance needed to produce a detectable Minute phenotype is different for different genes, because each rp gene may be transcribed in different amounts above the minimum levels required and because each ribosomal protein has a different function in the ribosome and maybe elsewhere, too. The abundance of cytoplasmic mRNA in a cell is determined by both the rate of transcription and the rate at which the mRNA is degraded; both processes contribute equally to gene expression. The most likely effect of the small insertions is on transcription, given the good correlation between decrease of mRNA and phenotype. However, it is becoming clear that protein synthesis may play a role in mRNA degradation (Theodorakis and Cleveland, 1996,and references therein). This may be accomplished by message-instabilityelements carried by specific mRNAs or by a global mechanism for degradation of mRNA. The presence of upstream AUGs (uAUGs) in general in rp mRNAs will very likely upset translation by decreasing the frequency of initiation at the downstream AUG, and out-of-frame uAUGs in particular may also initiate nonsensemediated mRNA degradation. mRNAs might have in their coding regions sequences that mark the transcript for degradation unless those sequences are translated (Zhang et al., 1995). Premature termination would then expose those sequences to a machinery that would degrade the mRNA. Another possibility is that the process of nuclear transport and cytoplasmic translation is coupled and that mRNAs would exit the nucleus 5' end first. The ribosomes would then associate with the mRNA before transport is completed. Encountering a premature terminator the ribosomes will terminate translation before transport is complete, and the mRNA could be degraded in the nucleus or at the nuclear pore. Since both RpS3pW9and RpS3~""have two consecutive uAUGs close to the 5' terminus followed by a stop codon after one triplet, this may constitute a possibility for nonsense-mediated degradation. However, since there is a >50% difference in reduction of RpS3 transcript between the two alleles this cannot be a likely explanation. Nor would it seem possible that the 3' penultimate uAUG with its premature terminator codon is responsible for the reduced RpS3 mRNA in RpS3pv9.

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Instead, it is more likely that it is the inserts per se that cause the reduction in transcript amount; the longer the insert the stronger the phenotype/suppression. We are presently analyzing nuclear and cytoplasmic RNA to try to pinpoint at what level these inserts affect gene expression.

4. RpS5 [M(1)15D] a. Gene and mutant phenotype In a genetic and molecular analysis of region 15E on the X chromosome, the gene encoding ribosomal protein RPS5 was identified as M(1)15D (McKim e t al., 1996). The two alleles investigated, M(f)f5D’ and M(1)f5D2, are associated with a 6-kb insertion of unknown DNA and a breakpoint rearrangement, respectively, that affect the genomic region that produces the 0.8 kb RpS5 transcript. A third allele, M(f ) 15DR28t”, was EMS-induced. Since RpS5 was mapped to 15E, perhaps the correct name of M( I ) 15D should be M( I ) 15E. The Drosophila S5 protein shows a very high conservation, with 86% sequence identity to the rat protein (McKim et d., 1996). Ribosomal protein S5 has homologs both in the eubacterial and archaebacterial kingdom and thus belongs to the Group I ribosomal proteins (Wool et al., 1995). McKim et al. (1996) also studied X-ray-induced lethal mutations in region 14 and two of the lethal complementation groups defined genetically separable Minute loci, M(1)14C andM(1)fqE; the M(f)14C locus most likelycorresponds to a Minute locus mapped to this region by Schalet (1986) and Stanewsky et al. (1993). Some interesting observations were made regarding combinations with these three Minutes. Whereas double-mutant heterozygous females with the genotype M(1)14E +/+ M(1)15D (both point mutations according to McKim et al., 1996) were fully viable, the M(1) f4C +/+ M(1) f4E heterozygotes were lethal. However, M(1) 14Cas well asM(1)14E were X-ray induced (McKim etal., 1996) and may therefore have overlapping deletions which could explain the lethality ofM(f)14C M(1)14E. With respect to M(1) 14E and M(f ) 15D, heterozygosity for either a single deficiency uncovering both Minutes [Df(l )BK14/+; no breakpoint was given for this deficiency] or two deficiencies affecting them separately [Df(1) I9 +/+ Df(1 )BK28] were lethal. Since Df( f ) f9/M(f) f4E female heterozygotes are viable and Minute (the deficiency uncovers M(f ) 14C, at least, but should also uncover M(f)14E according to Kim e t al., 1996; no data are presented on the status of Df(l)BK28/+ heterozygotes), Df(l)BKI4 and Df(I)BK28 may both uncover an essential gene. It is therefore noteworthy that yet another rp gene has been mapped to this region, namely RpS19 in 15A (Baumgartner et al., 1993).No Minute has been mapped to 15A,and this may be due to the deficiency uncovering 14E and/or 15D, which at the same time uncovers a Minute locus at 15A. Alternatively, it is pos-

+/+

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sible (but not likely) that a mutation in RpS19 may not be hemizygous lethal or even show a Minute phenotype. This is a reminder of the danger of using deficiencies when mapping Minutes. It should also be mentioned that an RPS18 cDNA clone was previously mapped by in situ hybridization to 15B (Bums et al., 1984). This was later contradicted by Ganvood and Lepesant (1994), who mapped RpS18 to region 56F on the second chromosome. These contradictory results do not exclude the possibility that 15B harbors an rp gene. To explain the survival of M(1) 14E +/+ M(1) 15D heterozygotes, as opposed to the lethality of the deficiency heterozygotes, the authors suggested that this might be due to one of the Minute alleles being a hypomorph (McKim et d., 1996). While hypomorphy has been shown to be true in some developmental stages for P-element-induced Minutes encoding ribosomal proteins (transcript variation between 30 and 80% of wild type; Szbpre-Larssenet al., 1997), we may assume that a 50% reduction (null allele) in expression of an rp gene is sufficient to produce a Minute phenotype (weak, medium, or strong). We may also assume that the sensitivity to transcript reduction varies among rp genes and we cannot rule out the possibility that two Minute mutations have a synergistic effect, as suggested by the lethality ofM(1)14C +/+ M(1)14E heterozygotes (McKim et al., 1996). However, since deficiencies were involved in this analysis some caution should be observed when interpreting these results. Interaction between two Minutes was first reported by Morgan (1929, who showed that M(2)d/+ ;M(3)d/+ heterozygotes were Minute, whereas M(2)d/+ and M(3)d/+ were wild type. Much later, Minute interaction had been reported to occur between M(3)65F and M(3)69E (Del Prado and Ripoll, 1983). These Minutes were first believed to be alleles, because they did not complement for lethality. By using a series of duplications in the region they showed that these Minutes were separated by approximately four subdivisions (65F-69E). These cases of Minute interaction should be investigated further.

5. RpS6 [M( 1)7C?] a. Gene and mutant phenotype The Drosophila RpS6 gene had previously been sequenced and shown to have the potential to encode two isoforms of the RPS6 protein via differential splicing (Stewart and Denell, 1993a). In P-element mutagenesis experiments, four lethal mutations (hen', hen2, WG1288, and aid) were isolated that affect the larval hemocytes, mediators of the insect immune response (Watson et al., 1992; Stewart and Denell, 1993b). Mutant larvae show melanotic tumors, and dying animals develop grossly hypertrophied hematopoietic organs because of overproliferation and aberrant development of hemocytes. Surprisingly, it was found that the Pelement had inserted in the 5' regulatory region of the RpS6 gene, which maps to position 20.45 on the standard genetic map and to chromosome position 7C5-Dl.

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This region harbors another ribosomal protein gene, namely RpS14 (Brown et al., 1988). This gene is, as far as we know, the only duplicated ribosomal protein gene in Drosophila. Haploinsufficiencyfor RpS14 was suggested to cause the Minute phenotype of M( J)7C (Anderson and Lambertsson, 1990), but this was later shown to be wrong (Doreretal., 1991). It wassuggested that M(1)7C is adigenic Minute, which can only be produced by the interaction between two Minutes or one Minute and another gene (Dorer et al., 1991). Such an interaction was reported by Morgan et al. (1925) for M(2)d and M(3)d (both lost), which produced a Minute phenotype only when the flies were heterozygous for both mutations. Heterozygotesfor either one alone were wild type, whereas homozygotes for either one were lethal. Interactions between Minutes have been suggested to take place also between M(1)14C and M(1)J4D (McKim et al., 1996) and M(3)65F and M(3)69E (Shellenbarger and Duttagupta, 1978).However, in the former case the Minutes were deficiencies, and therefore we cannot rule out the possibility that they uncovered more than one Minute or some other essential gene. Therefore, these results should be interpreted with caution. All hemizygous RpS6 mutant males are developmentally delayed at all stages and can remain living as third instar larvae for up to 3 weeks (Stewart and Denell, 199313).Lethality has been observed at all developmental stages but generally occurs during the extended third instar period. In air8 hemizygotes the ring gland, salivary glands, and most imaginal disks fail to reach normal size, and in the late-larval lethal phase large melanotic tumors can be seen in the body cavity (Watson et al., 1992). The fact that RpS6 mutants fail to pupate but can live as larvae for up to 3 weeks may suggest that the ring gland fails to produce enough ecdysone or that the tissues are unable to react to the hormone (cf., Brehme, 1939).It was recently shown by Herrmann et al. (1995) that mutation of the gene encoding RPS2l in Drosophila also causes tissue overgrowth of the hematopoietic organs and considerably delayed larval development. Therefore, RpS2 I may also be classified as a tumor-suppressor gene. Northern analysis of hemizygous hen’ and hen2 third instar larvae revealed that the RpS6 transcript is quantitatively reduced (apparently 0 > mRNA < 50%, but no quantitative analysis was made) in these animals (Stewart and Denell, 1993b). The level of RpS6 transcript is restored to wild-type levels in hen’ and hen2 revertant male larvae, indicating that the P-element insertions disrupt the gene. Northern analysis of mRNA from air8 hemizygotes revealed an almostcomplete absence of RpS6 transcript (Watson et al., 1992; no quantitative analysis was made). As for the hen revertants the level of RpS6 mRNA was restored to a level indistinguishable from wild-type in air8 revertants. The difference between the alleles in the expression of RpS6 may be due to the fact that the P-element in air8 inserted 10 bp from the potential transcription start site, whereas the Pelement inserted 10-20 bp further downstream in the hen alleles (Watson et al., 1992; Stewart and Denell, 1993b). The difference is quite small but emphasizes

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the importance of analyzing more than one mutant allele in studies of gene regulation and effect on phenotype. What is notable about the RpS6 insertion alleles is that the P-element insertions do not completely knock out RpS6 gene activity. The explanation for this is very likely that the P-element inserts 5’ to the transcription start site, which may allow a reduced rate of transcription. Similar results were observed in M(3)66D (Szebae-Larssen et al., 1997) and M(2)30D/E (sop; Cramton and Laski, 1994) where the P-element inserted in the same region. In contrast, insertion of the P-element in the 5’ UTR resulted in complete disruption of gene expression; that is, M(2)32A and M(3)95A (Szebae-Larssen and Lambertsson, 1996; Anderson et al., 1994). Therefore, the low rate of transcription of RpS6 in hemizygous mutant animals makes them survive up to and including the late third larval instar. Surprisingly, though, no mutant male develops to the adult stage (cf., RpS3 revertants below). It is also noteworthy that none of the mutant heterozygous females were reported to have a Minute phenotype (Watson et al., 1992, Stewart and Denell, 199313). Admittedly, weak Minutes may be difficult to classif y (and the classification is more often than not biased), but after having examined the alleles I have come to the conclusion that all heterozygous mutant females are weak Minutes (A. Lambertsson, unpublished results). Considering that the P-element mutations are not null alleles, the amount of RpS6 mRNA in heterozygous females could be around 75% of wild type, a level that may result in a weak Minute phenotype, if not wild type (cf., M(3)95A revertants below). Transcriptional control of rp genes in higher organisms is generally executed by sequences in the first intron but also by sequences both upstream and downstream of the mRNA start site (Atchison et al., 1989; Hariharan and Perry, 1990). RpS6 has a CAGCCAAC sequence at position +21 to 28 which is identical at 6 of 8 bases to the internal promoter sequence of the mouse RpL32 gene (Atchison et al., 1989). In addition, RpS6 also has a number of motifs in the 5’ region that are known parts of internal promoters of developmentally regulated Drosophila genes lacking functional TATA boxes; for example, Ultrabithorax, Deformed, Antennapedia, engaikd, white, and zeste (Arkhipova and Ilyin, 1991). The cDNA sequence for Drosophila RpS6 was also reported by Spencer and Mackie (1993). Transcription initiation of mammalian and several Drosophila ribosomal protein genes typically starts in a pyrimidine-rich tract (Mager, 1988; Stewart and Denell, 199313; Anderson et al., 1994). As already mentioned, the presence of a polypyrimidine tract has been found to be important for both promoter function and translational regulation (Chuang and Perry, 1989; Moura-Net0 et al., 1989; Hariharan and Perry, 1990; Levy e t al., 1991). It should be noted, though, that not all Drosophila ribosomal protein genes have this motif (Mager, 1988). RpS6 has three transcription start sites, two of which have the pyrimidine rich tract, and there are no TATA or CAAT motifs present in the promoter region. These

+

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transcription start sites contain the typical CTTTT sequence where transcription usually starts at the C (Andersson et al., 1994; Saebgie-Larssen and Lambertsson, 1996; Schmidt et al., 1996). Of 75 rat rp cDNAs analyzed, 40 have the pyrimidine-rich tract, which range from 4 to 20 nucleotides (Wool et al., 1995). Ten of the 40 have the sequence CTTTCC and a variant (CT, or C , or 2 ) in the others. This motif may function as a promoter that binds a warn-acting factor that regulates and coordinates translation of rp mRNAs (Wool et al., 1990; Perry and Meyuhas, 1990; Levy et al., 1991). It is interesting to note that the Drosophila RpS6 gene has the capacity to encode two isoforms of the RPS6 protein via alternative splicing (Stewart and Denell, 1993a). The predicted -0.75-kb alternate transcript was never detected on Northern blots (Stewart and Denell, 1993b), suggesting that its abundance is low or its expression is temporally and/or spatially restricted or that differential splicing does not take place. It should be recalled that RpS3 produces two equally abundant transcripts differing by about 50 nt (Andersson et al., 1994). In this case the origin of the second transcript is still not known and nothing is known about whether two proteins are produced (M. Lyamouri and A. Lambertsson, work in progress).

b. The RPS6 protein S6 is the major, but not the only, phosphorylated eukaryotic ribosomal protein (Wool, 1979; Leader, 1980); the others are PO, P1, and P2 (Tsurugi et al., 1978; Towbin et al., 1982). In the rat S6 protein there are five serines at the carboxyl end that are known to be phosphorylated (Gressner and Wool, 1974a; Chan and Wool, 1988). There is an extraordinary number and variety of agents and of alterations in the cellular environment that affect the phosphorylation of S6 (Chan and Wool, 1988). These include hormones, viral infection, serum treatment, fertilization, heat-shock, and more. This response to mitogenic stimulation is highly conserved and occurs in all systems examined so far (Sturgill and Wu, 1991). The phosphorylation of S6 is supposed to trigger or facilitate the high rates of protein synthesis required for cell division (Pardee, 1989) and to alter the patterns of translation (Palen and Traugh, 1987; Thomas et al., 1981). Unfortunately, the function(s) of S6 is not known, and it is therefore not possible to foresee how its function(s) may be altered by phosphorylation. Even though there is a general correlation between an increase in the phosphorylation of S6 and an increase in protein synthesis, known inhibitors of protein synthesis such as cycloheximide and puromycin are very effective stimuli for the phosphorylation of S6 (Gressner and Wool, 1974b). Two suggestions for the consequences of the phosphorylation of S6 have been put forward. The first one proposes that S6 forms part of the mRNAbinding domain in the 40s subunit and that the attachment of certain mRNAs is favored by phosphorylation (Wool, 1979; Terao and Ogata, 1979; Duncan and

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McConkey, 1982; Takahashi and Ogata, 1981; Thomas et al., 1982). However, there is no substantial evidence that phosphorylation of S6 in any way affects the activity of the ribosome (Leader et al., 1981). The second proposal is that the phosphorylation of S6 has no purpose at all and that S6 is just a fortuitous substrate for protein kinases that have other and more specific substrates (Chan and Wool, 1988). Indirect evidence supports this negative hypothesis. The yeast homolog YS6 has only two phosphorylation sites instead of the five present in the mammalian S6, but the phosphorylation of YS6 is affected by the same stimuli; for example, growth, sporulation, heat-shock, and other conditions (Johnson and Warner, 1987).After inactivating the two genes encoding YS6 cells were transfected with a plasmid containing a mutant YS6 cDNA in which the two serines that are phosphorylated were replaced with alanines (Johnson and Warner, 1987). The mutant YS6 was not phosphorylated but was nonetheless incorporated into ribosomes. The mutant strain grew and responded normally to the induction of sporulation and heat-shock. In a competitive growth experiment equal numbers of mutant and wild-type cells were mixed together and diluted into fresh medium, which is a strong stimulus for growth and phosphorylation of YS6, when growth became stationary (Johnson and Warner, 1987). Wild-type YS6 is phosphorylated and dephosphorylated during each cell cycle; after 70 generations the fraction of mutant and wild-type cells was unchanged. This experiment demonstrated that there was no selection against cells with the mutated YS6 that could not be phosphorylated, and that the phosphorylation of the two serines in YS6 serves no observable purpose. Thus, our knowledge of the functions of S6 is meager, and the somewhat obscure role of S6 phosphorylation in physiological processes is puzzling. The observation that mutations in the Dosophila S6 gene cause hypertrophy of the larval hematopoietic organs may therefore be important in our efforts to gain more information about this protein.

6. RpS13 [M(2)32A] a. Gene and mutant phenotype Using the P{aCWI mutagenesis procedure described by Bier et al. (1989),we have induced and cloned two previously undescribed Minutes, one on the second and one on the third chromosome. The second-chromosome Minute, PflacWM(2)32A1, is characterized as an intermediate-level defective phenotype, with respect to abnormal bristle formation and retardation of larval development (Szb@e-Larssen and Lambertsson, 1996).The one on the third chromosome, P{IacWM(3)66D1, is a severe Minute (Saebae-Larssen et al., 1997;see below). P{aCW)M(2)32A1 was mapped to region 32A on the second chromo-

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some by in situ hybridization to salivary gland chromosomes and by recombination and deficiency mapping. Remobilization of the P-element restored the phenotype to wild type, showing that the Minute phenotype was caused by the P-element insertion. After cloning the Minute gene via plasmid rescue, M(2)32A was then characterized and sequenced. Comparisons of M(2)32A genomic and cDNA clones with the sequences in the EMBL and GeneBank databases revealed a very high level of identity (98.9% in a 564-bp overlap) to the D. rnelanoguster RPZ7 cDNA (McNabb and Ashbumer, 1993) and to RPSZ3 cDNA sequences from Musca domestzca (76%; 2. H. Zhou and M. Syvanen, unpublished results), Rattus rattus (73%; Suzukietal., 1990), andHomosapiens (71%;Chadeneauetal., 1993). The very high level of identity between the RPZ 7 cDNA and the one reported by our laboratory and the fact that both cDNAs derived from single-copy genes suggested that both originated from the same gene. However, the Rp17 gene was cytologically mapped to 29A (McNabb and Ashbumer, 1993), a region not containing a Minute locus (Lindsley and Zimm, 1992). Based on our genetic data and the high level of identity to RPSZ 3 cDNA sequences from various species, we concluded that our cDNA corresponds to the eukaryotic RpSl3. Given the high identity between RPSI3 and RPI7, we also believe that RP17 corresponds to this locus. Comparing the RPS 13 cDNA sequence with the corresponding genomic sequence M(2)32A revealed two introns of 226 and 62 bp, respectively. There is one major transcription start site at position -30 relative to the open reading frame, with additional minor transcription start sites at positions -31, -33, and -34. Transcription is initiated in a polypyrimidine tract, typical for mammalian and several Drosophila ribosomal protein gene cap sites (Mager, 1988; Stewart and Denell, 1993; Andersson et al., 1994).The presence of a polypyrimidine tract has been found to be important for both promoter function and translational regulation (Chung and Perry, 1990;Moura-Net0 et al., 1989; Hariharan and Perry, 1990; Levy et al., 1991). Also, the promoter of M(2)32A has no TATA or CAAT motifs typically found in promoters of genes transcribed by RNA polymerase 11. The exact location of the inserted P-element was found to be in the middle of the 5’ untranslated leader between nucleotides - 15 and - 16 relative to the translation initiation codon. Northem blot analysis, using a single stranded cDNA probe (antisense), revealed an abundantly and in all developmental stages expressed transcript of 0.6 kb. This transcript is -3.5 times more abundant in females (ovaries) than in males when compared with total poly(A)+ RNA. The abundance of this transcript is reduced by -50% in P[IacWm(2)32A1 heterozygotes, implying that the Pelement insertion completely disrupts the M(2)32A locus. The amount of transcript is restored to the wild-type level in homozygous male and female revertants (Szbfle-Larssen and Lambertsson, 1996).

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b. The RPS13 protein The Drosophila RPSl3 cDNA is 629 bp long, including the poly(A) tail, and contains 17 nucleotides of untranslated leader, a 453-bp open reading frame encoding a protein of 151 amino acids and a 123-bp untranslated trailer (poly(A) tail not included) containing two polyadenylation signals ( AATAAA). Conceptual translation of the open reading frame predicts a protein with 151 amino acids and a MW of =17 kDa. As a comparison, rat S13 has 150 amino acids and a MW of 17,080 Da (Wool et al., 1995). Drosophila S13 is well conserved and shows 87% identity to both human and rat S13 (Suzuki et al., 1990; Chadeneau 1993), 78% to the parasitic nematode B. pahngi S13 (Ellenberger et al., 1989), 69% to the fission yeast Schizosacchromyces pombe S13 (Marks and Simanis, 1992), 71% to the monocot plant 2. mays S13 (Joanin et al., 1993), and 32% identity to the archaebacterium Haloarcula marismortui S15 (Scholzen and Amdt, 1992). Consistent with the need for transportation to the nucleolus, Drosophila S13 has the bipartite nuclear targeting signal IKK{ll}RKDKD in position 92-1 10 (Saebae-Larssen and Lambertsson, 1996; Dingwall and Laskey, 1991).

7. oho23B [RpS21; M(2)23B] a. Gene and mutant phenotype The RpS2I gene was isolated in a large-scale P-element mutagenesis screen of the second chromosome as a lethal with overgrown hematopoietic organs; the mutant is called oho23B or l(2)kl68-14 (Herrmann et al., 1995; I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). The P-element had inserted in the promoter region, causing a 30- to 40-fold reduction of the RpS2l transcript in homozygous mutant larvae. Heterozygous flies show a weak Minute bristle phenotype. This phenotype is more severe (severe Minute) in an allele where the P-element deleted the RpS21 promoter region upon excision. Pelement-mediated transformation of a 2.5-kb genomic DNA fragment containg the RpS21 gene rescues both mutant phenotypes (I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). The development of mutant animals is arrested at the larval to pupal transition and the larvae may survive for 2 weeks after the pupation of the heterozygous siblings. The most striking phenotype is the massive overgrowth of the hematopoietic organs and particularly of the most anterior lobes, which become as big as the brain. It is interesting to note there is no release of hemocytes from the hematopoietic organs into the hemolymph; all cells remain in the glands. At the time of normal pupation the imaginal disks of the mutant larvae are smaller than normal, but in older mutant larvae the disks become much larger with duplicated structures and the leg and wing disks tend to fuse together, although they keep a more or less apparent folding pattern.

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The RpS2I gene consists of four exons of 41,60,192, and 75 bp in length separated by three relatively small introns of 71, 71, and 63 bp. The major form of the RPSZI transcript has a size of about 0.4 bp. There is also a minor 0.7-kb transcript representing about 5% of the total level. Investigation of a cDNA corresponding to this transcript revealed that it contains an extension of the 3' UTR without any additional intron (Herrmann et al., 1995; I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). Using the yeast two-hybrid system it was shown that the S21 protein strongly interacts with the ribosome-associated protein p40 encoded by the stubarista ( s t a ) gene (I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). This result is certainly encouraging in that it very likely will allow future detection of interactions between other ribosomal proteins or between ribosomal and nonribosomal proteins.

8. RpL9 [M(2)32D] a. Gene and mutant phenotype In experiments analyzing male-sterile mutations caused by the integration of the PfhcWl enhancer trap P-element, Schmidt et al. (1996) stumbled on a new Minute locus at 32D on the second chromosome. Analyzing the cloned region they found, among others, an abundantly expressed 0.8-kb transcript 5' to the Pelement insertion site. Searching the data bases the authors found significant matches to eukaryotic ribosomal protein L9 and the corresponding L6 in bacteria and organelles. No Minute had been mapped to 32D before (Lindsley and Zimm, 1992; the M(2)32A paper was published at about the same time); so to find out whether this region harbored a Minute locus the P-element was remobilized to generate deletions in that area. Among the white-eyed male offspring one was isolated that had the typical Minute phenotype: short and thin bristles and slightly rough eyes. The mutant line, M(2)32D, also showed delayed development, and the females, but not the males, had severely impaired fertility (Schmidt et al., 1996).The mutation also enhanced the wing vein defects of Delta mutants, as was also shown for M(2)60E (Hart et al., 1993). Southern analysis indicated that an imprecise excision of the P-element had truncated the 3' end of the RpL9 gene. Since the remobilization of the P-element caused the Minute phenotype it was, of course, impossible to use this approach to restore the normal one. Therefore, the authors had to perform transformation rescue experiments. By introducing a genomic DNA fragment (3.9 kb) containing the RpL9 transcription unit the wild-type phenotype was restored, showing that the M(2)32D gene encodes RPL9. A more detailed genetic and molecular analysis of the 32D region has been made by Schmidt (1996). Interestingly, there is a lethal PZ element insertion, l(2)01501 (Karpen

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and Spradling, 1992), about 900 bp upstream of M(2)32D (Schmidt et al., 1996). Since M(2)32D/l(2)01501 flies are fully viable these mutations are not alleles, and the lethality associated with l(2)01501 must be caused by a mutation in a gene 5’ to the M(2)32D gene. Remobilizing the PZ element generated yet another M(2)32D allele, M(2)32D2, which was lethal in trans with the original M(2)32D mutation (Schmidt et al., 1992). The authors also used the Df(2L)/39 deficiency with breakpoints in 3 1AB and 32D-E and showed that it uncovers the male-sterile mutation associated with the P{lacW} insertion at 32D, the M(2)32D gene as well as the l(2)01501 mutation. The Minute phenotype of Df(2L)]39 has been ascribed to another Minute, namely M(2)30A, and introducing the 3.9-kb genomic fragment into Df(2L)J39 flies did not restore the wild-type phenotype. We now know that there is at least one other Minute in this region, namely M(2)32A (Szbae-Larssen and Lambertsson, 1996). This emphatically shows why it has been impossible to use Minutes caused by deficiencies in fine genetic and molecular genetic/biology studies. Whether there is a Minute at 30A remains to be shown. The RpL9 gene is relatively complex consisting of two short exons (18 and 50 nucleotides) and two long ones (25 1 and 398 nucleotides) separated by three introns of 73, 243, and 275 nucleotides (Schmidt et al., 1996). Drosophila ribosomal protein genes usually have one or two introns. The estimated length of the RpL9 mRNA, including the poly(A) tail, is 800 nucleotides.

b. The RPL9 protein Conceptual translation of the RpL9 cDNA generates a polypeptide of 190 amino acids and a molecular weight of 21406 Da. The RPLB is homologous to rat L9, and this protein has been conserved during evolution, having homologs both in archaebacteria and eubacteria (Wool et al., 1995). Together with more than 30 other eukaryotic ribosomal proteins L9 phylogenetically belongs to Group I (Wool et al., 1995) that have homologs in the eubacterial and archaebacterial kingdom. It should be noted that in cultured human cells RPLB has been implicated in the delivery and binding of the ricin A chain to the ribosome where the toxin attacks the large rRNA (Vater et al., 1995).

9. RpL14 [M(3)66D] a. Gene and mutant phenotype In the same P-element mutagenesis experiment described above we isolated P{IaCWM(3)66DJ,a severe Minute with respect to defective bristle formation and retardation of larval development; fertility and viability of heterozygotes are good (Szbae-Larssen et al., 1997). The bristles of P(lacW)(M3)66DJ heterozygous flies are extremely thin and short, and in the two to three generations after the mutant was isolated the Minute flies also very often had notched wings, missing or

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Figure 3.3. Scanning electron micrograph of Drosophila head (frontal view) from (A) wild-type and (B)P/hcWIM(3)66D'/+ flies. T h e malformed and undeveloped antennae (arrowheads), the missing maxillary palp (open short arrow) and the malformed right side of the head (open short arrow) with the much smaller right eye. (a2-a3) Second and third antenna1 segment; (ar) arista; (ce) compound eye; (mpl) maxillary palp.

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malformed antennae, maxillary palps, ocelli, eyes, and legs (Figures 3.3 and 3.4). However, after some more generations these extreme characters disappeared in the balanced stock and only reappear when P{kxWm/i(3)66D1flies are mated with unrelated flies. The M(3)66D gene was cloned, sequenced, and characterized in a fashion analogous to that described above for M(2)32A. Sequence comparisons performed by Dr. A. Gluck (personal communication) revealed that the Drosophila sequence was homologous to the rat L14 sequence (Chan et al., 1996). The exact location of the inserted P-element was found to be in the promoter region 52 nucleotides upstream of the M(3)66D transcription start site (cf., RpS6 and sopp). The M(3)66D gene contains 5 nucleotides of untranslated leader, a 498bp long open reading frame, and a 92-bp untranslated trailer with a polyadenylation signal (AATAAA) located 27 bp upstream of the polyadenylation site (Szbpe-Larssen et al., 1997). There are three introns of 276,232, and 260 bp (5‘ to 3’).The most upstream intron intersects the open reading frame immediately after the ATG start codon, making the first intron only 19 bp long. There is one major transcription start site within a pyrimidine-rich tract at position - 16 relative to the open reading frame and the transcript is -650 nucleotides. The M(3)66D promoter region contains putative upstream elements in the form of two consensus Drosophila CAAT motifs, AGCAACA and TGCAACT [consensus: (A/T)GCA(A/T)N(A/T); O’Connell and Rosbash, 19841. Similarly positioned CAAT boxes are also present in the promoters of the rp genes encoding RPS17, RPL7, and RPL32 (Szbpe-Larssen et al., 1997). There is, however, no apparent TATA box present in the promoter region. The latter is usually not present in rp genes. The insertion of the P-element in P{kxWIM(3)66DJ 52 bp upstream of the transcription start site causes an overall reduction RpLI 4 mRNA abundance. There are, however, significant differences in percentage reduction between the various developmental stages. The decrease is most manifest in third-instar larvae, 54% of wild type, and substantial reductions are also noticed in 1 to 3-dayold pupae and adult females with 60-70% and 63% of wild-type, respectively. The reduction is less apparent in second-instar larvae and adult males, each with -80% of wild-type RpS6 mRNA. These results may indicate that the P-element is interfering with a promoter function that is employed with different efficiency at various developmental stages.

b. The RPL14 protein The M(3)66D cDNA encodes a 19-kDa protein with a large excess of positively charged amino acids. The protein has a calculated net charge of +30 at physiological pH and is increasingly polar toward the C-terminal end (Szbpe-Larssen et al., 1997). The Drosophila RPL14 shares 42% identity with rat and human ribosomal protein L14 (Chan et al., 1996; Li et al., 1993; Aoki et al., 1996), al-

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Figure 3.4. Scanning electron microscopy of DTosophila head (viewed from above) from (A) wildtype and (B) PlhcWlM(3)66D’/+ flies. Notice the small and thin bristles, the deformed and misplaced ocellus (arrowhead), and the undeveloped antenna (open short arrow) lacking segments 3-5 and the arista in the mutant (B). (a2) Second antenna1 segment; (oc) ocelli; (ocb) ocellar bristles; (orb) orbital bristles; (pvb) postvertical bristles; (vb) vertical bristles.

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though the fly protein has 165 residues compared to 214 in rat L14 (Chan et al., 1996); most of the missing amino acids are at the carboxyl terminus. The yeast L14 hasonly138residuescompared to the214ofratL14 (Chanetal., 1996);yeast L14 lacks the carboxylterminal84 amino acids. The large variation in the number of residues in homologous eukaryotic L14 is unusual among ribosomal proteins. It appears that amino acids have been added to the C-terminal end during evolution (Chan et al., 1996). The eukaryote RPL14 has no counterpart in prokaryotes and thus belongs to the Group I11 ribosomal proteins (Wool et al. , 1995).

10. A homozygous viable RpL14 allele As mentioned above, M(3)66D is the middle gene in a tight cluster of three genes at 66D, very closely linked to hairy ( h , 111-26.5; Szbae-Larssen et al., 1997). In a remobilizationexperiment several revertants were isolated (A. Lambertsson,unpublished results). Among them was one white-eyed female that had therefore lost the P-element marker and showed a weak Minute phenotype, P{IacW&l(3)66DJPU3. The next generation revealed homozygous white-eyed flies with a more severe Minute phenotype. These flies were also fertile and can be maintained as a homozygous stock. Sequencing the region of the insertion revealed that the P-element had excised imperfectly and left 30 nucleotides. This small insertion in the promoter is thus capable of upsetting the expression of the RpL14 gene, suggesting that this region is sensitive to small sequence changes.

11. RpL19 [M(2)60E] a. Gene and mutant phenotype M(2)60E was first isolated as a recessive lethal P-element insertion line called E(DlJKP135 (Klein and Campos-Ortega, 1992; Hart et al., 1993) with a dominant enhancing effect on the phenotype of Delta, a gene encoding a surface transmembrane protein with EGF repeats in its extracellular domain (e.g., Vassin et al., 1987; Kopczynski et al. , 1988). Being a P-element induced allele the correct name should be PlhcWhl(2) 60E (Flybase “Genetic Nomenclature for Drosophila melunogaster,” March 23, 1995). The gene appears to have the typical polypyrimidine tract, where transcription usually starts in rp genes, and the Pelement inserted between this stretch of pyrimidines and the tentative translation start codon (Hart et al., 1993). Having three exons and two introns the RpL19 gene should produce a transcript of about 700 nt, including the 3’ trailer (Hart et al., 1993). The L19 ribosomal protein has a homolog in archaebacteria and thus belongs to Group I1 ribosomal protein (Wool et al., 1995). E(Dl)KP135 [M(3)60E] also interacts with the if mutation of the PS2a (position-specific) integrin gene. The PS integrin family contains a-and P-genes, and heterodimers are formed between the common PSP subunit and either PSla

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or PS2ol. The integrins function as cell-adhesion molecules throughout development and are probably involved in the adhesion of the two epithelial surfaces of the developing wing blade (Wilcox e t al., 1989). Most likely, the if mutation causes wing blistering by impairing the mutual adhesion of the cell layers that form the wing (Brower and Jaffe, 1989; Wilcox et al., 1989; Zusman e t al., 1990; Brabant and Brower, 1993). P{hcWb4(2)60E is characterized as a severe Minute. The P-element insertion in the 5' UTR of the RpL19 gene probably reduces gene expression by about 50% (see SaebQe-Larssenand Lambertsson, 1996). It is therefore interesting to note that the Minute phenotype of the two deficiences, Df(2R)ESI and M(2)60E, which are haploinsufficient for RpLl9 and therefore should have no more than 50% of wild-type RpLI9 expression, is slightly less severe than that of PIlacWN(2)60E (Hart et al., 1993). This is also reflected in the interaction with nd' and if, where P{lacWb4(2)60E strongly enhances the notched and blistered phenotypes, respectively; M(2)60E less so and Df(2R)ESI only marginally. Thus, the severity of the Minute alleles appears to correlate with the effects on wing development. The authors offer several explanations for this, and although it seems implausible that any of them can apply, alone or in combination, the differences between the alleles may be ascribed to different genetic backgrounds. Many, but not all, Minutes are known to affect wing morphology (Lindsley and Zimm, 1992). Several Minutes enhance dominance of such venation characters as px and net as well as the notched phenotypes (see above) and bristle characters as sc. In addition, dominant lethal effects are frequent in combination with Delta, Jammed, and in some cases withDichaete.One explanation for this would be that the reduced rates of protein synthesis in Minutes result in reduced levels of these gene products. Normal development is dependent on a critical threshold level of many proteins, and in Minutes the levels of these proteins may fall below this absolute minimum resulting in the described effects, and the more severe the Minute, the stronger the effect.

12. Stuburista ( s t a ) The D. mehmgmter gene stubarista ( s t a ) , which maps to cytological position 2A3B2 on the X chromosome, was shown to encode the highly conserved ribosome associated protein p40 [D-p40; Melnick et al., 1993). Using immunochemistry (McCafferey et al., 1990) as well as sucrose-gradient analysis (Auth and Brawerman, 1992) it was revealed that this protein is associated with polyribosomes in the cytoplasm. After ribosome dissociation p40 remains bound to the 40s ribosomal small subunit. A n association between p40 and the cytoskeleton has also been revealed (Keppel and Schaller, 1991), and this is consistent with studies showing an association between polyribosomes and the cytoskeleton (Cervera et al., 1981; Howe and Hershey, 1984).

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The viable stubarista allele staJ is associated with a number of phenotypes that qualifies stu as a Minute; these include malformed antennae, all bristles short and thin, and female sterility (Melnick et al., 1993). This mutant phenotype and the fact that D-p40 is required during oogenesis and imaginal development (Melnick et al., 1993) suggests that this protein has basic cellular function reminiscent of a true ribosomal protein. The s t a gene produces a single l-kb transcript expressed maternally and throughout development (Melnick et al., 1993). There is a single intron of 192 bp and an open reading frame of 270 amino acids. The Drosophila p40 shows a high degree of identity with p40 proteins from the two evolutionary distant species human (63%) and hydra (58%) and a 27% homology to E. coli S2 (Melnick et d.,1993). E. coli S2 corresponds to mammalian ribosomal protein Sa (Wool et al., 1995), is found on the surface of the small subunit where it helps stabilize conformation (Marion and Marion, 1988), and is also involved in tRNA binding (Shimizu and Craven, 1976). It is possible that D-p40 corresponds to mammalian Sa.

C. Recessive Minutes The dominant visible phenotypes and the recessive lethality have been associated with the Minute mutations since their discovery. In addition, several Minutes have low fertility, especially the females. The dominant Minute phenotype has made it relatively easy to isolate these mutations. In theory, the reduction in mRNA abundance needed to produce a detectable a Minute phenotype will be different for different rp genes, and we would therefore expect to find recessive Minutes. In addition, mutations in some rp genes may not result in a Minute phenotype. In describing the first Minutes, Bridges and Morgan (1923) mentioned that “also a few recessive small-bristle mutants were found and recovered in F,, notably tiny bristles.” And later Mohr (1925) described what he called a “Minutelike recessive.” Unfortunately, it is not clear to what extent these recessive mutants exhibited the other effects typical of the dominant Minute phenotype. These older observations are important, because we now know that recessive or homozygous viable Minutes do exist (see below). These Minutes are interesting, because they lack the characteristic haploinsufficiency but show the dominant Minute traits (small and thin bristles and delayed development) in both heterozygotes and homozygotes, with a clear synergistic effect in the latter; both fertile and sterile recessive Minutes have been isolated. The molecular explanation is that they are not null alleles and that the M/+ heterozygotes thus produce >50 but
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Below follows a description of the phenotypes and genes of the present recessive Minutes. It should be noted that while the published mutants were classified as “Minute-like”their classification as recessive Minutes in this chapter is the author’s decision; based on the information given in the papers and the fact that recessive alleles of both P{lac92/M(3)95A and P{lacW)h4(3)66D have been isolated (see below; Szbae-Larssen et al., 1997; A. Lambertsson, unpublished results).

1. Short-bristle, a Minute-like 111-chromosome recessive The short-bristk (sb) character was first observed in a culture in which fat females were tested by dumpy males (Mohr, 1924). This cross gave mostly wild-type flies; but a small number of the flies, both males and females, had thinner and shorter bristles (Mohr, 1925). The same type of flies were later found in a dumpy culture, which led Mohr to conclude that this mutation must have occurred in this stock. The sb locus was mapped to the third chromosome at about 46 or about midway between scarkt (3-44.0; 73A3-73A4) and pink (3-48.0; 85A6-85A6). It is noteworthy that Minute M(3)80 (3-47; 79E5-80F; see below) maps to the same region, and we may speculate that they represent two different alleles of the same gene. Homozygous sb mutants also had about 2 days delayed development, another typical Minute trait. Most interestingly, while short-bristle females showed normal fertility when mated to unrelated males they were practically sterile when mated inter se. A similar phenotype had been observed for the X-linked gene fused and the second chromosome recessive morula (Lynch, 1919). The short-bristle mutation of Mohr (1925) was later lost, and the short bristk (stb) mutation listed in “The Red Book” (Lindsley and Zimm, 1992) is not the same, even though it appears to have the same phenotype. stb was discovered by Fahmy and Fahmy (1958) in a mutagenesis experiment and maps to the X chromosome.

2. M(3)80 [M(3)Q-III] M(3)80 is a temperature-sensitive third-chromosome Minute that was isolated and characterized by Sinclair et al. (1981). Its cytological map position is at 79E5-80F near the centromere in 3L (left arm of this chromosome). At 29” heterozygous M(3)80 flies exhibit all the dominant traits of Minutes while homozygous animals are lethal. Raised at 22” heterozygotes are phenotypically normal and homozygotes show moderate Minute traits (Sinclair et al., 1981). M(3)80 also displays temperature-sensitive sterility and maternal-effect lethality. Temperatureshift experiments revealed that the temperature-sensitive period of M(3)80 lethality is polyphasic, starting in the first instar and ending in the latter half of pupation. Heat pulses produced a large number of remarkable phenotypes in

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M(3)80 flies, including morphological defects in structures produced by the imaginal disks (Sinclair et al., 1981). Behaving as recessive Minute at the permissive temperature, the characteristics of M(3)80 suggest that this gene has an essential function in the cell. As suggested by the authors (Sinclair et al., 1981) the M(3)80 may specify a component required for protein synthesis. This is likely but can only be demonstrated by hard-core molecular and genetic techniques. Nevertheless, M(3)80 qualifies as a recessive Minute and should be useful in elucidating the molecular basis for the Minute reaction and the regulation of Minute genes.

D. rp mutations without Minute phenotype Mutations in the RpS6 gene (see above) were initially reported to have a nonMinute phenotype (Watson et al., 1992; Stewart and Denell, 1993). As already mentioned, this classification may not be altogether correct, and the RpS6 mutant alleles have been classified as Minutes in this chapter. Another example of a mutation in an rp gene reported to result in a non-Minute phenotype is fs(3)02729, encoding RPL15 (K. Dej and A. Spradling, personal communication).

1. ziti (fs(3)02729; RpL15) A P-element-induced insertional mutation, fs(3102729, has a single insert at 80A-C and renders homozygous females nearly sterile. This mutation indentifies a new gene, ziti (Dej and Spradling, 1997), which encodes ribosomal protein L15 (see below). An examination of the nurse cell chromosome morphology of this line indicates that polytene chromosomes persist longer than in wild-type egg chambers such that some cells with a ploidy greater than 32C are polytene or condensed. Late-stage egg chambers tend to degenerate (after vitellogenesis). Sequencing of the genomic regions flanking the P-element insert revealed the presence of two ORFs: the first containing a translation initiation site at 52 bp away from the 3’ end of the P-insert. The peptide predicted by the two ORFs shows strong homology to ribosomal protein L15 of many species, including Rattus norvegicus , Chironumus tentans, Saccharomyces cerevisiae , and Saccharomyces pombe.The genomic sequence in Drosophila has consensus splice sites that indicate the presence of an intron of 173 bp between two exons of 309 bp each. The deduced polypeptide, to be referred to as RPL15, is thus 206 amino acids in length, which is consistent with previously described L15 proteins. Northern analysis of total RNA revealed a single transcript of -900 bp that is more abundant in females than in males, with the highest levels seen in ovaries. Neither fs(3)02729 nor the derived excision alleles show the somatic Minute phenotypes characteristic of mutations in previously described ribosomal protein genes of Drosophila. It should be noted that fs(3)02729 maps within

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the 79E5-80F region defined by M(3)80 (Sinclair et al., 1981), a temperaturesensitive Minute and the short-bristle (sb) mutation described by Mohr (1925). Furthermore, cloned Rp21 DNA was reported to hybridize to this region (Kay et al., 1988) but the transformed Rp21 gene did not complement M(3)80 (Kay et al., 1988).

E. Ribosomal protein genes of special interest It is obvious from the above that the majority of the Minute loci have not been correlated to ribosomal proteins or to any other gene product. However, several Drosophila rp genes have been cloned and shown by in situ hybridization to map in a region where a Minute has been located. This is circumstantial evidence, and to corroborate this connection it would be necessary to use P-element mutagenesis and plasmid rescue and/or transformation rescue experiments with the cloned rp genes. While these time-consuming tasks have not been carried out, it is nevertheless pertinent to describe two of these rp genes.

1. RpS4 The cDNA-encoding ribosomal protein S4 in D. melanogaster was isolated, sequenced, and its expression analyzed by Yokokura et al. (1993). The =l-kb mRNA is expressed throughout development and encodes a 260-amino-acid-long polypeptide, which shows 75% sequence identity to human ribosomal protein S4. Interestingly, the cDNA hybridized to two sites on the X chromosome, 1B1-2 and 3A3-6. As described above, region 1B has been reported to contain M(1) 1B (Patterson, 1932; Voelker et al., 1989), whereas a Minute locus has not been found in 3A (Lindsley and Zimm, 1992). It is not known if both or only one of the two RpS4 loci are active. While most rp genes in higher eukaryotes also exist as pseudogenes this is not the case in Drosophila (Ashbumer, 1993; Wool et al., 1995). O n the other hand, Drosophila ribosomal protein gene RpS14 exists as a functional tandem duplication (Brown et al., 1988), and RpS6 has the potential to encode two isoforms of the RPS6 protein via differential splicing (Stewart and Denell, 1993a). In humans the sex-linked genes HS4X and HS4Y encode distinct isoforms of ribosomal protein S4 (Fisher et al., 1990; Watanabe et al., 1993; Zinn et al., 1994). Haploinsufficiency of HS4X and HS4Y has been suggested to play a role in the development of the Ullrich-Tumer syndrome (UTS; Ullrich, 1930; Turner, 1938; Fisher et al., 1990; Watanabe e t al., 1993; Zinn et al., 1994), the complex human phenotype associated with monosomy X. Both HS4X and HS4Y are widely transcribed in human tissues, suggesting that the ribosomes of human males and females are structurally distinct. One of the arguments for genes being involved in the Turner syndrome is that they should escape X inactivation; oth-

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envise differences in the phenotypes of 4 5 3 and 46,XX females would not be expected. The observation that HS4X escapes X inactivation bears on the hypothesized role of HS4 deficiency in Turner syndrome (Fisher et al., 1990). By using isoform-specific antisera it was possible to show that human S4X and S4Y are both present in translationally active ribosomes (Zinn et al., 1994). In normal 46,XY cells S4Y is about 10-15% as abundant as S4X in ribosomes; in 49,XYYYY cells S4Y is about half as abundant as S4X; and in 49,XXXXY cells S4Y is barely detectable (Zinn et al., 1994). These results further strengthened the notion that haploinsufficiency of S4 plays a role in Turner syndrome. O n the other hand, patients with a 46,X,i(Xq) (X chromosome with two long arms, iso-X) karyotype carry three gene copies of HS4X and cannot be distinguished phenotypically from 45,X UTS patients (Geerkens etal., 1996). These authors investigated the HS4X expression in four 46,X,i(Xq) patients and found significantly increased levels of S4X transcript in all patients. Based on these results the authors concluded that haploinsufficiency of HS4X is not the cause of UTS (Geerkens et al., 1996). Very recently Rao et al. (1997) presented data that implicated a homeobox-containing gene, called SHOX (short stature homeoboxcontaining gene), to be involved in idiopathic growth retardation and the short stature phenotype of Turner syndrome patients. (Short stature and gonadal dysgenesis are consistent findings that are considered to be the leading symptoms of the Turner syndrome; Turner, 1938; Rao et al., 1997). The gene is located within the pseudoautosomal region (PAR1) of the human sex chromosomes and has at least two alternatively spliced forms, encoding proteins with different patterns of expression. It is very likely that the Turner syndrome is due to monosomy of genes common to the X and Y chromosomes.

2. RpS14A and B [M( 1)7C?] Drosophila ribosomal proteins S14A and S14B are encoded by two nearly identical adjacent genes (RpS14A and RpS14B) on the X chromosome (Brown et al., 1988); both genes are active throughout development (Brown et al., 1988; Andersson and Lambertsson, 1990). The genes were mapped to the 7C5-9 region, which contains a previously described Minute, M(1)7C (Lefevre and Watkins, 1986). This locus was reported earlier as a haploinviable region by Lefevre and Johnson (1973), and when RpS14A and -B were cloned it was proposed that they were identical to M(1)7C (Brown et al., 1988). This proposal was supported by Northern analysis of deficiency-heterozygotesfor RpS14A and -B showing a severe Minute phenotype that revealed greatly reduced amount of RpS14 mRNA (Anderson and Lambertsson, 1990). Admittedly, these deficiencies are large and based on non-Minute progeny obtained from crosses with the small deficiency D f ( l ) ~ n that + ~ ~deletes ~ RpS14A and -B and at least part of the sn locus, this proposal was rejected (Dorer et d., 1991). As already discussed above it was suggest-

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ed that M(1)7C is a single gene completely distinct from RpS14 and that it may be a digenic Minute (Dorer et al., 1991). These results may be easier to understand today when we know that the ribosomal protein gene RpS6 is located =40 kb distal to RpS14. Females of the Pelement-induced RpS6 mutant alleles are weak Minutes (see above), and we may expect that a null allele would produce a more severe phenotype. Remobilization of the P-element has so far been unsuccessful in producing null alleles of RpS6 (Kellie Watson, personal communication). As far as we know the only isolated ~ ~deficien~ . mutation for the RpS 14 genes is the small-deficiency Df( 1 ) ~ n +The cy heterozygotes were reported to be fully viable and fertile and without any detectable Minute trait (Dorer et al., 1991). This may be explained by the presence of the two active RpS14 alleles on the same chromosome and calls attention to the necessity of obtaining several mutant alleles. The possibility that RpS6 and RpS14 may interact to produce the severe Minute in region 7C [cf., M(3)65F and M(3)69E; Del Prado and Ripoll, 19831 is intriguing and should be analyzed further.

3. Other cloned Rp-genes In addition, several other rp genes have been cloned and characterized and their cytological map position identified by in situ hybridization. In many cases the map position was in a region where a Minute had been genetically mapped. In other cases rp genes were cytologically mapped to chromosomal regions not harboring any Minute locus. The cloned Drosophila rp genes known today are listed in Table 3.2.

F. Mutations in nonribosomal protein genes We cannot exclude the possibility that mutations in genes other than ribosomal protein genes may lead to the Minute phenotype. However, a nonribosomal mutation has yet to be reported to cause the Minute phenotype. Complete or partial inactivation of genes involved in protein synthesis such as aminoacyl-tRNA synthetases or protein synthesis factors and mutations that affect ribosome synthesis and transport may lead to a Minute phenotype or to a similar one. We have already discussed the bobbed and mini mutations in this respect. Another possible candidate should be mentioned here. The enzyme Sadenosylmethionine decarboxylase (SAMDC) is together with ornithine decarboxylase involved in the synthesis of polyamines (spermidine and spermine), compounds that have been implicated in protein synthesis. Two recessive mutations in the Drosophila SAMDC gene (in 3 1D-E) result in homozygous flies that have Minute-like bristles and larval development that is delayed -2 days (Jan Larsson, personal communication). The SAMDC transcript is constitutively pro-

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duced throughout development, and Northern analysis revealed that mutant males and females have reduced levels of the mRNA. The expression of SAMDC is not significantly higher in ovaries than in other tissues. Homozygous mutants of both sexes are weak but fertile, and no heterozygous phenotype has been observed. It is interesting to note that inhibition of polyamine synthesis markedly altered the length, diameter, and composition of the hair fiber of sheep (Hynd and Nancarrow, 1996). Further studies of the SAMDC mutants and of the role of polyamines in protein synthesis will be necessary before any conclusions can be drawn about the Minute-like phenotype produced by these mutants in Drosophila.

V. Conclusions and prospects About 80 years after the detection of the first Minute locus and almost as many years of research into their function(s), it seems, at last, that the Minute riddle will be solved. The evasive nature of the Minutes is emphasized by the fact that during the first 40 years after Schultz’s paper (1929) only three theories about the functions of the Minute genes were launched, none of which could be experimentally supported. Almost 50 years later Huang and Baker (1976) were the first to suggest that Minutes are the structural loci for ribosomal proteins, and the first Minute-to-rp correlation [M(3)99D and RPL321 was made in 1985 (Kongsuwan et al., 1985). It then took 8 years before the next correlation was achieved, M(2)60E and RPL19 (Hart et al., 1993). Today 13 Minute loci have been shown to encode a ribosomal protein, and most of these correlations have been made by direct or indirect use of Pelement mutagenesis. It may thus be argued that the Minute-to-rp correlation may be of general validity. Apart from the rp genes now correlated to Minutes, at least 30 other rp genes in D. melanogaster have been cloned and many of these map cytologically at or close to known Minute loci, thus indirectly supporting the Minuteto-rp correspondence. However, much work remains before we may know the function of all Minutes. One of the most recent and important findings described in this chapter is the isolation of viable homozygous Minutes, both fertile and sterile ones. These alleles show a synergistic effect as homozygotes; that is, their phenotype is more severe than in heterozygotes. Northern analysis of all possible combinations of alleles of the same Minute locus is consistent with this observation that less mRNA correlates with a more severe mutant phenotype and vice versa. Recessive alleles will provide us with a means not only to understand the Minute syndrome but also to study how the Minute genes are regulated. Another important finding is that several ribosomal proteins appear to have other functions apart from being part of the translational machinery; for example, apurinic/apyrimidinicendonuclease activities and tumor-suppressor functions. These results suggest that ribosomal proteins

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are involved in regulatory processes that may be important in normal development. Future research on the multifunctional character of ribosomal proteins will indeed be interesting to follow. Although deficiencies are very useful in gene mapping, the fact that most Minutes are associated with deficiencies has hampered genetic fine analysis and molecular approaches. It is also impossible to know whether a deficiency uncovers more than one Minute or some other essential gene. This renders interpretation of genetic analysis of interactions between Minutes difficult, and this calls for caution when working with Minute-related deletions. Ideally, these kinds of studies should be performed with indisputable nondeficiency Minutes. Therefore, Pelement mutagenesis seems to be the method of choice in the effort to clone and establish an unambiguous correspondence between a Minute gene and its product and in studying interactions between Minutes. Theoretically the Minute phenotype should be found in organisms other than Drosophila. The Turner syndrome (45,X) in humans has been compared to the Minute phenotype and suggested to be due to haploinsufficiencyfor HS4 (Fisher et al., 1990; Watanabe et al., 1993; Zinn et al., 1994). This proposal has later been disputed (Geerkens et al., 1996), and several genes are likely to be involved in the Turner syndrome (see above; Rao et al., 1997). It is also likely that the symptoms of the Minute/Turner syndrome will show differently in different species, and this reinforces the necessity of having more than one model organism. Minutes or Minute-like phenotypes are likely to exist in higher organisms, and efforts should be made to design screening techniques for their detection. In doing so we must be aware of that mutations in ribosomal protein genes may not always result only in dominant Minute phenotypes but also in recessive factors or no Minute phenotype at all. In addition, mutations in genes other than rp genes may result in the Minute phenotype. The Drosophila SAMDC mutant, which has a Minute-like phenotype, is a potential candidate in this respect. To date, all of the rat ribosomal proteins and their genes have been characterized (I. Wool, personal communication), and 73 of the human rp genes have been mapped more precisely on the physical map (Kenmochi et al., 1997). This map of human rp genes may serve as a tool for finding human diseases which might be caused by rp gene mutations or chromosome aberrations. Despite all the work going on in different laboratories very little is known about the regulation of eukaryotic rp genes and about the functions and interactions of ribosomal proteins; extraribosomal functions have been shown for several ribosomal proteins (see above; Wool, 1996), and models for the regulation of some r p s have been suggested (Vilardell and Warner, 1994, 1997; Tasheva and Roufa, 1995). Over the next decade we will certainly learn more about these and other processes, probably with major contributions coming from model organisms. Interestingly, the ongoing genome projects in each of the model organisms have shown that the gene systems in Drosophila show the highest degree of structural conservation to those

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of humans (Miklos and Rubin, 1996).We may thus expect that many components of biological processes, and their interactions, will be conserved between flies and humans. Therefore, the information obtained from experimental work on ribosomes and their components in Drosophila may be transferred to human biology.

Acknowledgments I thank Kimberley Dej, Allan Spradling,JanLarsson, Bernard Mechler, and Istvan Toeroek for allowing me to quote their unpublished findings. I also thank Jan Larsson and Marcel0 Jacobs-Lorena for advice and support. Special thanks to Torill Rolfsen for the scanning electron micrographs,to Edward B. Lewis and Linda Treeger for trying to track down information about M(3)40UO, to Stein Saeboe-Larssen for compiling most of the data in Table 3.2, and to Anton Cluck for identifying the Drosophila RpLJ4 gene sequence. I am also grateful to Kellie Watson for valuable discussions regarding RpS6. Michael Ashburner and Aubrey de Grey are acknowledged for their help with the new names of Minutes and rp genes. 1 thank Eva Lambertsson Bjork for linguistic improvements. Genetics is acknowledged for allowing me to reprint Figure 1 from the paper by Jack Schultz (1929). Finally, special thanks to Jeffrey Hall for his patience and support during this period. I have endeavored to make this review as comprehensive as possible and I therefore apologize to colleagues whose work has not been cited. Work in the author’s laboratory is supported by the Norwegian Research Council.

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Genetics of Biological Rhythms in Dousuphila Jeffrey C. Hall Department of Biology and NSF Center for Biological Timing, Brandeis University, Waltham Massachusetts 02254

I. Introduction 135 11. Chronogenetics 136 136 A. Elementary genetics of Drosophih rhythm mutants B. Entrainment and phase-resettings 142 C. Implications of period-altered rhythms or arrhythmicity 146 1157 D. Other phenotypes affected by rhythm mutations 111. Chronogenetic biology 163 163 A. Brain-behavioral studies of genes influencing fly rhythms B. per-Expression rhythmicities studied in situ 167 C. Extraocular photoreception of clock-resetting signals 171 D. Constitutive expression of the period gene? 172 IV. A final chronogenetic thought 173 References 173

1. INTRODUCTION Molecular chronobiology started with rhythm mutations insofar as studies of gene actions that seem at the heart of clock function are concerned. This chapter will therefore discuss rhythm mutants and to a more limited extent clock molecules; including how several of the former led to examples of the latter, and how certain aspects of mutationally altered rhythms connect with studies revolving around the molecularly based control of the fly’s biological oscillations. However, most of this Advances in Genetics, Vol. 38 Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved. 0065-2660198$25.00

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discussion will involve genetics only, in that the molecular biology of circadian rhythms has been reviewed to death: Dunlap (1996) provides the most recent comprehensive summary of this subject along with specifying and poking some fun at of all the other such reviews. For many years, the most salient rhythm variants in Drosophila were the period mutants. Instead of starting again from the beginning of this genetic story (“there are per mutants with fast clocks, slow clocks, and no clocks,” etc.), some rhythm-mutant phenotypes that have been examined or reevaluated recently will be discussed. The genetic subtopics to be presented include several puzzlesinteresting or anomalous rhythm phenotypes that are not yet explainable in “any” terms, let alone the molecular ones, reviewed as indicated above.

II. CHRONOGENETICS A. Elementary genetics of Drosophih rhythm mutants The first phenotypic subject to be considered may, nevertheless, have a nice molecular correlate. This refers to per mutations that cause alterations of the circadian clock‘s pace and that they seem to interfere with the normal gene’s expression (or its ultimate effects on the rhythm). For example, when a long-period per‘- mutation is present in a female-heterozygous for this X-chromosomal mutation and per+, the flies’ periods are longer than in females whose other period gene is nonfunctional. This result was obtained at the dawn of chronogenetics (Konopka and Benzer, 1971) and was confirmed 20 years later (Gailey et al., 1991). The latter study also showed that a male, carrying p e L on his X chromosome and the normal allele of this gene as a duplication, also exhibits a longer rhythm period than a companion fly whose X carries a per-null mutation as its sole genetic variant. Thus, in both kinds of heterozygous situations (in males and females), the effects of the per‘- mutation are “worse than nothing.” The most recent long-period per mutant, peTSLrH,is also semidominant and causes a slower clock pace when heterozygous with per+ than perSLrH/per- flies (Hamblen et al., 1998). It would be valuable in this regard to test the second p d - mutant-called per-lag-variegated (Konopka, 1988)-in flies that also carry per+. Note for the record that the so-called “per‘-2’’ mutant (e.g., Jackson et al., 1983; Saunders et al., 1994) turned out to be molecularly identical to (and possibly a mere re isolation of) the original p e L mutation (Gailey et al., 1991). Further arguments involving heterozygous combinations suggest that two short-period mutations are doing something other than causing the clock to “run too fast,” owing to some sort of hyperactivity associated with this allele (cf., Cot6 and Brody, 1986). That pe? (19-hr cycle durations in constant darkness) and perT (16-hr in that condition) could be regarded as hypermorphic mutations

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is based on the fact that extra copies of the normal allele of this gene lead to shorter-than-normal rhythm periods (Smith and Konopka, 1982; Konopka et al., 1994). This refers to both circadian 7 values and a periodically fluctuating parameter associated with the male’s courtship song (Hamblen et al., 1986; also see below). But if pe@ and perT were merely “hyper,” then one imagines that a female heterozygous for that mutation and the normal allele would have a faster rhythm than in the case of pe?s/per- or perT/per- females (“-” in these cases referring to a deletion of the gene). However, the periods of these hemizygous mutant females are 20 and 17-18 hr, respectively; yet, pe@/+ and perT/+ females exhibit appreciably longer periods: 21.5 and 20 hr, respectively (Konopka and Benzer, 1971; Dushay et al., 1990; Konopka et al., 1994). These values are squarely between those for wild-type and the mutant homozygotes. Hence, the normal per allele can be thought of as interfering with the action of these short-period mutations or vice versa (see Cot6 and Brody, 1986, for further discussion). Incidentally, two copies of a fast-clock per mutation routinely yield shorter periodicities than are measured for perF/per- (Smith and Konopka, 1982; Dushay et al., 1990; Konopka et al., 1994). (“F” generically designates any kind of short-period per mutation.) This phenotypic effect could, and did (see below), make a case of putative noncomplementation between a somewhat fast mutant and pero1 impossible to interpret. perclk/per- came out the same as perclk/+ (Dushay et al., 1990; also see below). In this context, keep in mind that this “zero” allele (01)-the first and, as it turned out, the sole truly per-null point mutant isolated-is almost identical in its effects to homozygosity for a deletion of the locus (a fully per- genotype). Note that this was one of the first instances of investigators realizing the power of overlapping deletions: either of such deficiencies (Df’s) by themselves is a recessive lethal; but when crossed to each other, the result in the DflDfanimal can be removal of a very small chromosomal segment that creates an utterly null genotype for the locus of interest (for per: Smith and Konopka, 1981; see Kingsley et al., 1990, for a mammalian example). It is also notable that homozygosity for per- causes hyperactivity at the behavioral level (number of locomotor activity events per unit of time, exemplified in this chapter in Figure 4.2, below), compared to the effects of per‘“ (Hamblen et al., 1986; Hamblen-Coyle et al., 1989). An almost-arrhythmic mutant known as p e P 4 leads to a similar kind of excess locomotion (Hamblen-Coyle et al., 1989) as does a new rhythm mutant called Toki (Matsumoto et al., 1994), which is located on a separate chromosome (see below). For perF/peP”s part, the phenotypes were essentially identical to perF/+ in a case where “F” (by itself, in a perF/Y male) caused a relatively slight shortening of the period. This caused the so-called “Clock” mutation to be ambiguous as to whether it is a per allele (Dushay etal., 1990). Just to make sure that that’s clear: Clk by itself: mild shortening; Clk/+: even milder, but enough to infer semidomi-

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nance; hence, the uppercase “C.” [The relentless semidominance of period-altering rhythm mutations has also been reviewed to death (e.g., Dunlap, 1990,19931.1 It seemed as if Clk could be a per mutation by virtue of genetic map position, but not by noncomplementation (see above). In particular, peFIk was mapped by meiotic recombination very close and to the right of pepJ on the standard X-chromosomal map (Dushay et al., 1990). To determine whether it was indeed a per allele or is mutated at a nearby locus required the cloning of the per DNA Clk mutant and transforming it into a per(“ genetic background; this led to an -1.5-hr period of shortening like that exhibited by the straight p e p k mutant (Dushay et al., 1992). Subsequently, the sequencing of (Hamblen et al., 1998) confirmed that it is mutated within the per gene at a site downstream of pep’; this is also to the right of the null mutation on the aforementioned standard map of this chromosome; given the direction of per transcription, the 5’ end of the gene is leftward on that map (Figure 4.1; see color plate). The amelioration of perF effects on rhythms by dropping the mutation’s dose from 2 to 1 (in females) suggests further that the mutant proteins are interfering with other processes. First, pe6,perT, and perck are all accounted for by amino acid substitutions in the PER protein (Baylies et al., 1987; Yu et al., 198713; Hamblen et al., 1998;Figure 4.1). Second, recall that any of these mutations when heterozygous with the normal allele causes distinctly different period changes than does per-/+, in a manner that suggests these p e p s are “antimorphic” mutations. Antimorphs, classically, are cases of interference with the normal gene’s action in heterozygotes; “morphies” of Drosophila mutations in general are most recently reviewed by Greenspan (1996). The definition of antimorph includes the titrating out of the mutation’s effect by adding extra copies of the allele (compared to genotypes carrying in the cases at hand only one dose of per+). That kind of test has not been performed for these per mutants, however. Nevertheless, the interference hypothesis can be extended in terms of peF/per-F being more mutant phenotypically than are the corresponding peF/perheterozygotes (see above). Here, one imagines that twice as much of the bad protein effects a more severe interference with other clock-related gene products than would occur when only one mutant allele is pumping out less of the amino-acidsubstituted PER polypeptide. Bear in mind that pitting the effects of the two genotypes under consideration (homozygous vs hemizygous) cannot involve any interference with the per gene’s normal product. Also, consider an analogy to a (actually the) clock mutation in mouse and the longer-than-normal periods caused by it. This includes Clock’s semidominance to the allele, naturally (Vitatema et d., 1994; and see below). Clock has been shown to be a classic antimorph (Antoch et d., 1997; King et d., 1997a); and the idea that the mutant CLOCK protein would also interfere with heterologous factors is supported by Clk/CUc being more abnormal (longerperiod) thanClk/Clk- (Kingetd., 1997a).Theclockmutant indeed turned out to produce a qualitatively altered CLOCK polypeptide (King et d., 1997b).

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Two relatively new autosomal rhythm mutants in Drosophila, each of which exhibit longer-than-normal circadian (behavioral) periods, seem semidominant, although this is difficult to discern. One such case is the chemically induced, 2nd-chromosomal Toki mutant: 7 for the mutant homozygote, -25.5 hr; and for the Toki/+ heterozygote, 24.5 hr (Matsumoto et d., 1994). The other mutant, Ritsu, was isolated from the wild (Murata et al., 1994): 7 for the mutant homozygote, -27.5 hr; and for the Ritsu/+ heterozygote, 25 hr; there are factors on both the large autosomes underlying the 7-lengthening effects of Ritsu; it was reported that more than half the effect is due to a mappable factor on chromosome 2 (Murata et al., 1994). Deletions corresponding to these Toki and Ritsu factors are not yet available, and the mutants have so far been studied only in terms of their behavior-rhythm abnormalities. per mutations affect eclosion as well as the locomotor rhythms of adults (Konopka and Benzer, 1971, though Hamblen et al., 1998). A third autosomal mutant has been delved into molecularly more than genetically. This is timeless, which is a recessive arrhythmic, both in terms of eclosion (the isolation phenotype) and locomotor activity (Sehgal et al., 1994). tim was mapped to a 2nd-chromosomal site that is separate from that of Toki, although it could be near or at the altered factor on this same chromosome carried in the Ritsu strain. It was not reported whether tim/+ flies exhibit any noticeable abnormalities in their rhythms. pep1 or per-/+ females do produce locomotor periods that are slightly but consistently 0.5 to l hr longer than normal (Konopka and Benzer, 1971; Smith and Konopka, 1982; Citri et al., 1987; Hamblen-Coyle etal., 1989). The arrhythmic disconnected mutant, in contrast, is thoroughly recessive (Dushay et al., 1989); this is the exception that proves the rule, as disco is not a “clock-operation” mutant but a brain-damaged one (see below). The tim mutant’s arrhythmicity was quickly tied to clock operation as opposed to having an anatomical etiology. This is because tirn interacts with per: The initial tim-arrhythmic mutation (now tim“’ ) caused per+-mediated mRNA cycling (reviewed in general by Hall, 1995; Dunlap, 1996) to be essentially aperiodic (Sehgal et al., 1994). One set of studies resulting from cloning tirn (reviewed by Sehgal et al., 1996; Young et al., 1996) was to show that TIM and PER physically interact. W h y ? See the two reviews just cited, along with Rosbash et al. (1996) for more on the relevant cellular and molecular biology. Given these two levels of molecular interactions, per and tirn mutations could interact genetically. This was shown to be so when per-L was mutated, leading to recovery of an autosomal-dominant suppressor of per% period-lengthening effects (Rutila et al., 1996). The suppressor proved to be the first period-altered tim mutant recovered: timSL, the superscript standing for “suppressor-of-long.” Interactions between timSLand per alleles are specific, in that the new mutation does not by itself (in a per+ genetic background) lead to appreciable shortening; and in combination with pe$, timsLmakes the period slightly longer than pe$ alone (Rutila et al.,

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1996). The putatively anomalous interactions between the TIMSL and PERL forms of these proteins are appreciable only in a formalistic sense: TIMSLcarries an amino acid substitution that is not located in the regions of this protein that were shown biochemically (in extracts from transfected tissue-cultured cells) to interact with PER (Saez and Young, 1996; Figure 4.1). In terms of finding flies with rhythm defects, the X chromosome has been battered with chemical mutagens for almost 30 years (Konopka, 1987, 1988; Hamblen-Coyle et al., 1989; Dushay et al., 1990; Konopka et al., 1991, 1994). This has led mostly to per mutants (n = 8 independently isolated alleles, one of which appeared to occur spontaneously: Hamblen et al., 1998); also one Andante mutant, with its moderately slow clock, by definition (see below); and a newly isolated quasiarrhythmic. The latter was mapped (roughly) to the middle of the X chromosome, but is not allelic to Andante or disco mutations (Konopka et d.,1994). disco mutations lead to arrhythmicity as well as a disconnection between the visual system and the brain; it was on that anatomical criterion that the discos were isolated (Steller et al., 1987) and then subsequently tested for eclosion and locomotor rhythms (Dushay et al., 1989; Hardin et d., 1992a). Notwithstanding all this time during which searches for such X-linked rhythm variants have been carried out, it is not possible to say whether that chromosome is “saturated” for the identification of genes acting in this area of the fly’s biology. The genetic picture is far dimmer for the autosomes: one mutant allele has been found at two of the three loci mentioned above, with two tim mutations reported so far (see above). Three older rhythm variants-one on chromosome 2, the other on 3-were reported many years ago (Jackson, 1983); but they disappeared from the literature from that point onward. The two 2nd-chromosomal cases could have been allelic to one another based on rough map positions (Jackson, 1983), but probably not to either Toki, Ritsu, or tim. A screen whose genetic strategy differed from all the others because it involved transposon mobilization resulted in an early-phase mutant (Newby and Jackson, 1993). Its eclosion phenotype in this regard was similar to two of the seemingly lost cases just mentioned. The new mutant lark is interesting for (at least) two reasons: (1) It is a dominant phase-altered variant and was, in fact, recovered in tests of strains carrying newly mobilized transposons heterozygous with normal autosomes; had the mutant not been looked for in this manner it would not have been isolated, because lark is a recessive lethal (Newby and Jackson, 1993); all other rhythm-mutant screens in Drosophila (and Neurospora for that matter) demanded that any new variant strain be viable (e.g., Konopka, 1987; Dunlap, 1993). (2) lark affects only the phase of eclosion rhythmicity, not that associated with adult locomotion (Newby and Jackson, 1993). In contrast to how most rhythm mutants in Drosophila were identified, the two extant single-gene rhythm mutants in mammals were found as heterozygotes-one serendipitously (Ralph and Menaker, 1988)-and the other resulting

Figure 4.1. The period gene transcript and the protein it encodes. D. mekmogaster’s per mRNA is diagrammed at the top; the exons are in pink (2 through 8 contain the protein-coding open reading frame); introns are designated by upward-pointing partial triangles. Intragenic sites of the three classic per mutations (Konopka and Benzer, 1971) are shown below the top diagram (Yu et al., 1987b; Baylies et al., 1987). per-mutated sites (Hamblen et al., 1998) from sequence analysis of newer mutants (Hamblen-Coyle er al., 1989; Dushay et al., 1990; Konopka et al., 1994; Hamblen et al., 1998) are shown above the diagram; the numbers in parentheses refer to the amino acids substituted in three of these mutants (the N-terminal residue being 1); the lowercase letters associated with per04 refer to the nucleotide change in this mutant (adenine+thymine) at a splice-junction site (Hamblen et al., 1998). At the bottom are depicted the “domains” of the approximately 1200amino-acid PER protein. These include a nuclear localization signal (black vertical stripe), cytoplasmic localization domain (shaded red), and two (red) regions of PER-with-TIM protein interactions (Vosshall et al., 1994; Saez and Young, 1996). The meaning of the TG repeat domain (of -20 alternating threonine-glycine pairs; dark purple) is reviewed by Kyriacou et al. (1996). The PAS region (green), now known in many kinds of proteins, is involved in dimerization interactions with other polypeptides (reviewed by Schmidt and Bradfield, 1996, albeit not specifically in the context of clock genes). The PER gene from a silkmoth species (Reppert et al., 1994) is only -800 amino acids long and is in the main strongly homologous to the N-terminal two-thirds of PER from D. mehnogasrer (as indicated in yellow).

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from induction by ethyl-nitroso-urea and behavioral screening of the immediate progeny of mutagenized parents (Vitaterna et al., 1994). Thus, the short-period tau mutant of hamster and the long-period Clock mutant of mouse (see above) could have been recessive lethals but turned out not to be: instead, the homozygotes are viable and both exhibit more substantial alterations of circadian periodicity than are observed when tau or Clock are heterozygous (Ralph and Menaker, 1988; Vitaterna et al., 1994). Thus, lark is unique, including that it is the only vital locus known, so far, to be a rhythm-related gene as well-although null mutations at most of the loci mentioned in this chapter are unknown and could if they are isolable prove to be lethal. An additional genetic property of the gene is that extra copies of lark+ (in a normal genetic background) lead to later and later eclosion-peak phases (Newby and Jackson, 1996). [Thus: increasing per+ dosage pushes the flies in a mutant direction (like perCk or peg); whereas the same genetic test of lark+ function acts in opposite direction of the mutation.] Since lark affects only eclosion rhythms, the mutation could define a rhythm factor solely concerned with output from the pacemaker. Another recently isolated eclosion mutant seems as if it could be defective mainly (or exclusively) with light input (Lankinen, 1993a). This is light-independent-neat-eclosion(lime) a single-gene, autosomal mutation in D. subobscura, which leads to nearly arrhythmic eclosion in LD. But exposing linne to temperature cycks readily entrained the mutant cultures, and periodic eclosion then continued in constant temperature; the emergence rhythm even persisted (after high-low entrainment) in constant light, which is a periodic phenotype that seems unique to this strain (Lankinen, 1993a). It was not reported whether lime affects adult activity rhythms. An additional autosomal locus in D. melanoguster has some chronobiological significance for the locomotor behavior of adults. This was uncovered in activity monitoring of ebony body color mutants. In general, e mutants are pleiotropic (exhibiting a variety of visible, biochemical, and behavioral defects: reviewed by Black, 1988).This now includes the fact that some alleles at the locus lead to circadian behavioral arrhyhmicity while leaving periodic eclosion untouched (Newby and Jackson, 1990).This is roughly the opposite of the eclosionaltered but behaviorally normal lark mutant, although both genes may control output-only functions. There is, however, another element of rhythmicity that is disrupted by (at least some of the) e mutations: cyclical variations of tyrosine aminotransferase activity (Tauber and Hardeland, 1977). The oscillating level of this enzyme happens to be one the few catalytic-factor rhythms (perhaps the only one) known in this organism. e became in part a rhythm-related gene in the same spirit as did disco; that is, by testing a visibly abnormal mutant for its rhythmicity or lack thereof. Similarly, certain new Andante mutations were found by merely looking at the descendants of mutagenized flies (Newby et al., 1991). This was based on the fact that the -25- to 26-hr And mutant is also a dusky wing variant, and And’s rhythm

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defect indeed maps to the classical dy locus (Konopka et d., 1991). Thus, several new dy mutants were isolated by eye (the observers’), and some but not all of these newly induced mutants were also And-like in their behavioral rhythms (Newby et al., 1991). This is uninterpretable in terms of how wing development and rhythms may be in part controlled by the same factors. It was hypothesized that small wing size, as caused by dy mutants, might have a concomitant in the brain, which would somehow slow down the clock‘s pace (Newby et d., 1991); but no evidence for this could be obtained in histological analyses of adult CNS sections of dyAd and other mutants involving this gene (van Swinderen and Hall, 1995). dy, by the way, seems on phenogenetic grounds as if it may be a complex locus; but possible molecular corollaries have been presented only in a sketchy manner (review: Jackson and Newby, 1993). Screens for mutants that would, in a manner analogous to dy, e , and the like, be considered “partly rhythm variants” have been performed systematically in Drosophila: nearly all such candidate mutants were normal for their circadian behavior rhythms (Vosshall and Young, 1995). This survey was carried out in the spirit of those previously performed in Neurospora. Thus, a fairly wide array of phenotypically aberrant strains-again, originally isolated on rhythm-unrelated criteria-were examined for their patterns of (daily) conidial banding; only a few of them turned out to exhibit circadian rhythm defects, in some cases dramatic ones (Feldman and Dunlap, 1983; Lakin-Thomas and Brody, 1985; Lakin-Thomas et al., 1990).

B. Synchronization to environmental cycles and phase resettings In addition to isolating some of the Drosophila mutants as phase-angle variants (see above), those found by virtue of period alterations were subsequently shown also to exhibit anomalous, albeit expected, phases associated with their entrained rhythms. Thus, in 1ight:dark cycles, the behavioral rhythms of long-period per variants (a mutant and certain transgenic strains carrying truncated per-locus DNA fragments or amino acid substitutions effected by in vim0 mutagenesis) have delayed evening peaks of locomotor activity (Hamblen-Coyle et al., 1992; Saunders et al., 1994). The usual such peak is at about lights-off in tests of wild-type, which flies nevertheless exhibit a considerable anticipation of that L-to-D transition (Saunders, 1982; Hamblen-Coyle et d., 1992). The opposite phase alteration occurred in tests of short-period mutants and per transgenics (HamblenCoyle et al., 1992; Saunders et al., 1994). These effects could readily be rationalized in formal terms (given Drosophila‘s phase-response curve: Dushay et al. , 1990; Saunders et d., 1994). However, it was perhaps odd that the morning locomotor peak was moved only slightly earlier or later than usual (this being about the time of lights-on for wild-type) in LD tests of, say, pe? or per-L adults (Hamblen-Coyle et al., 1992).

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It was notable that all individuals expressing period-altering mutations were able readily to entrain to cycles of 12:12 hr LD (Hamblen-Coyle et al., 1992; Konopka et al., 1994), even though their free-running periods are 19,29, and 16 hr (Hamblen-Coyle et al., 1992; Konopka et al., 1994). Thus, these mutants can reset their clocks by 5 to 8 hr per day. This is not necessarily because of hyperphase shiftability (see below), because wild-type adults can entrain to 8:8 LD cycles, although their evening peaks were substantially later than the time of L-toD (Wheeler et al., 1993). This is consistent with the fact that the super-short perT mutant behaves in synchrony with 12:12 LD cycles with a much earlier-than-norma1 evening peak (Konopka et al., 1994). So, the limits of entrainment for this organism seem very wide (in these experiments, the light intensities employed were not especially intense, implying that unnatural hyper-shiftings were not being effected). Finally, when the Zeitgeber’s cycle durations were squeezed down to 5 1 2 hr (e.g., LD 6:6) genetically normal individuals were found to free-run (Wheeler et al., 1993). Note that mammals in general have narrower entrainment limits (e.g., Aschoff and Pohl, 1978), as exemplified by the behavior of one of the aforementioned hamster mutants (reviewed by Menaker and Refinetti, 1993; Menaker and Takahashi, 1995): the homozygous “20-hour” tau mutant free-runs in conditions of 12:12 LD, although tau/+ individuals (7= 22 hr) do entrain with an earlier-than-normal onset of nocturnal wheel-running; this being the phenotype with respect to which the mutant was noticed in the first place (Ralph and Menaker, 1988). Clock does not fit with these mammalian dicta: mutant homozygotes appear to entrain to 24-hr LD cycles (Menaker and Takahashi, 1995). Arrhythmic per mutants are clearly cyclical in their LD behavior. But these pep’ or per- adults do not entrain, in the sense that they are likely to be merely forced into action by the D-to-L and L-to-D transitions (Wheeler et al., 1993). In addition, these mutants’ behaviors often include quasiplateaus of locomotor activity throughout most of the light and dark periods at high and low levels of locomotion, respectively. Behavior in these conditions may be a sensitive indicator of a very weak circadian pacemaker, because two variants that are essentially DD-arrhythmia (peP4 and one of the per-truncated transgenic types mentioned above) are not that much different from wild-type in LD in that they anticipate the environmental transitions (Hamblen-Coyle et al. , 1989; Frisch et al., 1994). Their activity also subsides to a deep locomotor trough near the middle of the light period, which is distinctly un-peP-like (see above). Another DD arrhythmic type, disco, seemed superficially to be entraining; for example, its cyclical behavior in LD did not include abrupt behavioral transitions occurring near the times of the environmental changes (Dushay et al., 1989). That this nominally arrhythmic mutant can indeed entrain was proved in a double-mutant: perS was combined with a mutant disco allele, and the former mutation moved the evening peak of activity to a distinctly earlier time than was observed in singlemutant disco flies (Hardin et al., 1992a). It could have been predicted that disco

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would be epistatic to p e 6 , but the opposite result was obtained. These results also imply that the disco mutant is not devoid of pacemaker function (discussed further in a neurobiological context, below). Mirroring old results from eclosion experiments (e.g., Zimmerman et al., 1968), wild-type Drosophila have been shown to entrain to temperature cycles (Wheeler et al., 1993) and be phase-shiftable by temperature pulses (Edery et al., 1994). In the former of these tests, adult behavior was observed to peak approximately 3 hr in advance of warmer-to-cooler transitions; in the latter, the PRC resulting from heat-pulses was found to be very similar to the so-called classical light-pulse PRC. “So-called” is the phrase invoked, because PRCs for wild-type Drosophila behavior did not appear in the literature until the 1990s (against a background of the many eclosion PRC experiments performed in the old days on Drosophila pseudoobscura). The shapes-including durations and amplitudes-of the behavioral PRCs are not necessarily of great intrinsic interest in that these data, from tests of genetically normal D. mlanoguster, are conventional ( h s h a y et al., 1990; Edery et al., 1994; Saunders et al., 1994). Some implications and intrigues come from comparing effects on wild-type results of the temperature pulses to parallel experiments involving a “per-inducible” transgenic type (Edery et al., 1994) and from comparing light-pulse experiments performed on genetically normal adults, to those involving period mutants. Such mutant-vs-normal comparisons occurred, first of all, in eclosion tests: the resetting amplitude for pe? was found to be considerably greater than in the case of developing wild-type cultures (Konopka, 1979). Thus, the mutant exhibited “type 0” resetting, compared to a “type 1”curve (with its relatively smaller degrees of phase advances and delays) that was determined for genetically normal Drosophila (see Winfree and Gordon, 1977). A further component of this eclosion-PRC analysis revealed that the “subjective day” was the only portion of the curve affected by this mutation in the sense that the segment of the plot corresponding to light insensitivity was diminished by the same 5-hr value as for the effects of pe? on free-running periods (Konopka, 1979; Konopka and Orr, 1980). It was puzzling that a comparison of behviural PRCs, involving wild-type adults and others expressing this same period mutation, indicated type- 1 resetting for both genotypes, though pe? adults did (as in the eclosion experiment) have all their cycle-shortening limited to the subjective day (results of R.J. Konopka and D. Orr, presented in Hall and Rosbash, 1987). Moreover, estimations of a vs pthe amounts of activity vs rest exhibited by adult flies in conjunction with their free-running behavioral cycles-suggested that only the active (a)part of the cycle is affected by pe? (Konopka and Orr, 1980). One problem with these older data is the apple-and-oranges matter of different light-pulse qualities and durations, as applied in the eclosion-vs-behave

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ioral experiments. These difficulties have been partially rectified by the most recent foray into fly PRCs (Saunders et al., 1994). Here, the selective pe? effects on 01 were confirmed in phase-shifting experiments involving adult behavior. This mutant was also found, after all, to have heightened light responsiveness: a direct comparison of short- vs longer-duration light pulses showed the increases in PRC amplitude for this mutant to be relatively greater than in the case of wild-type (Saunders et al., 1994). The same hypersensitivity and type-0 PRC is observed in light-pulse experiments on the tau mutant, with the proviso that this fast-clock mutant hamster has first been placed for many days in constant darkness (Menaker and Takahashi, 1995). The accentuation of peg’s light sensitivity (albeit not to intensity variations: cf., Winfree and Gordon, 1977) may be connected with the fact that this Drosophila mutant is hypersensitive in comparison to wild-type to the arrhythmiainducing effects of constant dim light (Konopka et al., 1989). In the relevant PRC experiments, all genotypes tested ( n = 3 ) changed from type 1 to type 0 with increased duration of the pulses (Saunders et al., 1994). The mutant newly analyzed in this manner was perL, whose PRCs were found to exhibit a lengthening of the late subjective day and early subjective night-roughly the same region of the relevant behavioral PRCs that was affected by pe? (Saunders et al., 1994; contra Konopka’s eclosion PRC). With respect to 01 in these recent experiments onpe&, that segment of the behavioral cycles was not stretched out (or otherwise altered) by this mutation (Saunders et al., 1994). In contrast, the slightly long-period Toki mutant has a larger-than-normal a / p ratio (Matsumoto et al., 1994). Yet, the analogous data for p e 4 indicate that a portion of the formally determined subjective day and the active subset of its actual behavioral cycles can be independently affected. It also should be mentioned that, as noted for pe?, both per‘L and Toki are somewhat hypersensitive to LL exposure, insofar as “going arrhythmic” at relatively low light intensities is concerned (Konopka et al., 1989; Matsumoto et al., 1994). It is not self-evident how these detailed features of Drosophila‘s restactivity cycles, and the resettings thereof, are going to promote understanding of pacemaker function at the level of cellular biochemistry. Nevertheless, there are some apparent connections of these PRC concerns with elements of the period gene’s expression, as it is mentioned molecularly (see below). And those molecular analyses may also wish, one day, to cope with such basic matters as how do perS and the like shorten circadianperiodicity ?--even before one grapples with things like this mutant’s higher-amplitude PRC. The same question is necessarily applicable to perL and peTSLIH in the temporally opposite direction. Yet, before considerations of per’s molecular expression explain all of this, some further aspects of mutationally induced changes of 7 and rhythm defects caused by other kinds of mutations will be discussed from a more biological and formalistic perspective.

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C. Implications of period-altered rhythms or arrhythmicity 1. Long-period phenotypes The per-L mutants have, as it were, long been thought to be severe hypomorphs. For instance, recall the result mentioned above, in which a slight drop in dosage of the normal per+ allele leads to -24.5- to 25-hr periodicities. per% (n = 3, then 2: see above) are much longer than that, so they would suffer a >> 2X decrement. However, these mutants would be imagined to produce the period gene product(s) at levels appreciably above nothing (cf., Cot6 and Brody, 1986), at least in part because of p e e ’ s behavior. Some individuals expressing this nominally zero allele are arrhythmic, but a few exhibit extremely long free-running periodicities (Hamblen-Coyle et al., 1989); so per04 would be hypothesized to express near a threshold value, below which is equivalent to genetic amorphy for certain of these mutant adults. That p e L seems hypomorphic on formal grounds jibes with certain geneexpression results: an antibody against the protein product of this gene (PER) led to much weaker-than-normal staining of the cells and tissues that express PER in wild-type (Zerr et al., 1990; also see below). However, Western blot experiments suggested that PERL levels may not be subnormal (Rutila et al., 1996). At the RNA level, a series of per transgenics-each carrying an incomplete form of the gene in a different genomic location-was found to exhibit wide variations in grossly determined levels of the per transcript (Baylies et al., 1987); the lower was this value, from Northern blot experiments, the longer was the freerunning behavioral period; the extreme lines were >40 hr. How would a pe4 fly generate, it seems, such a low level of its protein (if that is the correct biochemical phenotype)? It does not merely dribble out a tiny bit of the mRNA; per% peak levels are about the same as in wild-type (Hardin et al., 1990). Instead, it would appear that the amino acid substitution responsible for p e p s nominally 29-hr rhythm (see below) leads to a polypeptide that acts at an effectively low level or perhaps is at such a level. Therefore, the “stability”of PERL would be low; the quoted noun implies anomalously easy degradability. Alternatively, PERLwould be present at quasinormal levels (cf., Rutila et al., 1996) with this mutated polypeptide presenting poor antigenicity to the particular antibody applied (Zen et al., 1990). Perhaps the former of these hypotheses has greater force, owing to the facts that PERL interacts weakly with TIM (Gekakis et al., 1995), and light-induced depletion of TIM (e.g., Young et al., 1996) or elimination of that protein in the timol mutant (Price et al., 1995) cause normal PER to go to, or constitutively stay at, very low levels. One more feature of the circadian behavior of per-L flies is registered: as was alluded to above, this mutant is not (so to speak) chronically a “29 hour” fly, because its free-running periods are quite temperature sensitive; for example, at 30”, its rest-activity cycles stretch out to almost 35 hr (Konopka et al., 1989; Ewer

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et al., 1990). In the strictest sense, this would be called “temperature overcompensation,” as opposed to a loss of temperature compensation. The latter would be inferred if a mutated clock ran faster and faster as the temperature was raised. This does occur in nontemperature-compensated frequency mutants of Neurospora (reviewed by Hall, 1997). Also, peG, perT, and peGLrH exhibit similar temperature dependences (Konopka et al., 1989,1994; Ewer etal., 1990; Hamblen et al., 1998); that is, opposite to that of pe+ but not as dramatic heat-induced increases in clock pace as in some of the fiq mutants. A further feature of PERL’s instability involves the fact that the PERLwith-TIM interactions are weakened further as the temperature is raised (Gekakis et al., 1995). This would make the former (mutated) polypeptide even more subject to degradation than it is at milder temperatures. A curiosity in this respect is that timsL suppresses per% heat sensitivity in terms of slowing down the behavioral rhythm; but does not alter the degree to which TIM and PERL can interact at elevated temperatures (Rutila et al., 1996). Perhaps PERL, once it is released from its TIM interaction as the temperature goes up, also exhibits an intrinsic abnormality of the PER polypeptide’s conformation. This could render it (even) more susceptible to degradation. An analogous heat lability could obtain in another kind of temperature-sensitive, long-period per mutant. This is an in uiao-mutated transgenic, in which per’s theonine-glycine (TG) amino acid repeat was deliberately removed (Yu et al., 1987a); this repeat, involving some 20 TG dimeric pairs (Figure 4.1), has a variety of implications (see below). In the transgenic at hand, this partly PER-deleted product functioned quite well as a circadian clock component at medium temperatures (Yu et al., 1987a); but further experiments showed that the period lengthened significantly in tests of heated, repeat-deleted individuals (Ewer et al., 1990). “Some 20 TG pairs” means that the length of this intra-PER repeat varies among strains of D. mehnoguster (initially reported by Yu et al., 1987a). These natural variations in the length of D. mehnogmter’s TG repeat, and additional ones generated by in vitro mutagenesis, were correlated with varying degrees of temperature sensitivity (Ewer et al., 1990; Sawyer et al., 1997): the longer the repeat length, the less of heat-induced increase in behavioral cycle durations. In this context, the intraspecific variations in question form a geographic cline in the northern hemisphere (Kyriacou et al., 1996; cf., Allemand and David, 1976; Lankinen, 1993b; Kliman and Hay; 1993; also see Dover, 1989,1993; and Coyne, 1992, for perspectives). This permitted the authors of the most recent investigation of this matter (Sawyer et al., 1997) to suggest a possible selection-based explanation for the distribution of this per polymorphism. Another aspect of the per gene’s evolution is that the length of the TG repeat region coevolves with a stretch of amino acids immediately flanking the repeat (review: Kyriacou et al., 1996). This has intriguing functional consequences for clock function in a situation where the flanking region from one species is jux-

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taposed with the repeat region of another in chimeric per gene constructs transformed into flies (Peixoto et al., 1998). This digression is not straying too far from the subject at hand, because one anomalous feature of the interspecific chimeric per gene function is pelJ--like (or TG deletion-like) temperature sensitivity of behavioral periodicity, in a situation where the repeat-flanking region from D. mehogaster is juxtaposed with D. pseudoobscura’s repeat (Peixoto et al., 1998). A region within the per gene separate from the T G repeat has been shown to exhibit length polymorphisms (Ford et d., 1994). The relatively Nterminal region (of the protein)--called the PAS domain-is homologous to a portion of the product encoded by Drosophila’s single-minded gene; and just as provocatively to certain vertebrate genes (see below). This -260-amino-acid part of PER is believed more and more to function as a polypeptide dimerization domain (first such experiment: Huang e t al., 1993; also see Saez and Young, 1996; Rosbash et al., 1996). Few biochemical insights are at hand for the domain that includes the enigmatic TG repeat (although see Castiglione-Morelli et al., 1995).

2. Short-period phenotypes As was discussed above, the hypermorphic hypothesis for pe? and other fast(F)clock mutants became oversimplified as a result of testing various peF heterozygous types. Short-period phenotypes involving the per gene have also been reinterpreted more radically. They are now imagined to be partial loss-of-function mutants, whereby the region of the gene at and around the site of the amino acid substitution in pe? loses some or all of its function, as a result of that specific mutation and a fairly large collection of others (Rutila et d., 1992; Baylies et d., 1992). First, note that the intragenic region including the pe?-defined serine residue (Yu et al., 1987b; Baylies et al., 1987-mutated at the site shown in Figure 4.1) and the perT-defined Gly one (Hamblen et al., 1998)-is a highly conserved one evolutionarily (Colot et al., 1988; Figure 4.1). This region, which is between PAS and the TG repeat (Figure 4. l),could be important for the polypeptide’s structure or function. Changing the pep-defined serine to “anything” (almost) always led to shorter-than-normal circadian behavioral rhythms (Rutila et al., 1992). Moreover, other in uitro-effected amino acid substitutions scattered arbitrarily around and fairly near this site also led in the main to shorter-than-norma1 rhythms (Baylies et al., 1992). Overall, it did not seem as if there was much of a pattern to these relationships between genotype and phenotype in that no necessity for a particular quality or fine-level location of a given substitution-or, in a few instances, multiple such changes-was discernible. Again: damage this region of PER, speed up the clock. A vague parallel from mammals might be mentioned: if the clock in those kinds of organisms is anatomically damaged, then a similarly short-period circadian rhythm is the result. Here, however, damage refers to the anatomy of the

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circadian pacemaker structure: when that hypothalamic SCN is partially lesioned (e.g., Pickard and Turek, 1983; 1985; Davis and Gorski, 1984; Ruis et al., 1987) or deteriorates because of age (e.g., Pittendrigh and Daan, 1974; Davis and Menaker, 1980; Brock, 1991), the usual result is a shorter-than-normal freerunning period; although this is not necessarily the case for all types of vertebrates (Pohl, 1993). At all events, a pacemaker that runs relatively fast should not be viewed as being under the control of an overly robust clock. Moreover, a fast-running clock does not always do that: a small percentage of pe? flies, reared then maintained and locomotor-tested in DD (Power et al., 1995b), exhibited very long periods (>> 24 hr, let alone longer than the canonical value of 19 hr for this mutant). This is not dissimilar to the ability of the homozygous tau mutant to manifest 7’s much greater than the “20-hour mutation” would have it do (Menaker et al., 1994)-after, in fact, the hamsters were maintained in DD for several cycles; or in other experiments, after they were pretreated by LL conditions for many days. The anomalously long periods (up to 28 hr) often reverted to conventional values (for this genotype) but then might lengthen again for many days (Menaker et al., 1994; Menaker and Takahashi, 1995). These results from the mammalian mutant were taken to imply destabilizing effects of mu on the “circadian system,” as opposed to this gene being solely concerned with pacemaker components. Yet, the destabilization that also can be exhibited by peg (Power et al., 1995b)-which is mutated at or near the pacemaker itself-suggests that tau at least in part acts at that central focus.

3. Arrhythmic phenotypes The absence of eclosion or behavioral rhythmicity is not a completely straightforward matter, whereby one can state simply that “no clocks are present or functioning.” The latter could come from a thoroughgoing lesion of cellular pacemaker structures (see above), possibly one that is effected genetically (see below). In Drosophila, the supposedly arrhythmic mutants were reinterpreted when it was found that relatively high-frequency cycles could be teased out of the behavioral records. These ulhzldian rhythms, which only sometimes are overt in such records (Hamblen-Coyle et al., 1989), had periods ranging from 5 to 15 hr (Dowse et al., 1987). Such periodicities have been found in the analysis of both peF point mutants and the per- type (Dowse et al., 1987); moreover, the rather sloppily rhythmic perL mutant exhibits ultradian components (Dowse and Ringo, 1987) in conjunction with its most well known 29-hr period (at 25”). One cannot resist another mammalian parallel: the first deliberately induced and isolated rhythm mutant in mouse exhibits an -27- to 28-hr period (when the autosomal mutation is homozygous); these rhythms are not only somewhat weak (as they appear in the behavioral plots), but they also can break up into “ultradians” after the mutant mice have been in DD for several days (Vitaterna et al., 1994).

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Two ways to interpret the ultradian periodic components that are uncovered by the effects of these rhythm mutations are as follows: (1) By knocking out function of the circadian clock, or nearly so doing, they reveal the capacity that lurks in these organisms to behave in a periodic manner; whereby such behavioral cycles are neither especially impressive (are sloppily rhythmic) nor tightly controlled (have widely varying periodicities); yet, when the circadian clock is robustly functioning (in wild-type), then the output from this pacemaker into behavior overrides the possibility of manifesting the weak ultradian cycles. (2) By eliminating a fundamental element of the circadian clock‘s mechanism, an arrhythmic mutation causes the high-frequency pacemakers of which that clock is composed to control directly the organism’s periodic behavior; this hypothesis views the overall nature of the circadian pacemaker to be a population of coupled, high-frequency oscillators (Dowse and Ringo, 1992, 1993; Helfrich-Farster and Diez-Noguera, 1994). The latter of these two notions has support in some cherished oscillator formalisms (e.g., Klevecz, 1992; Klevecz and Bolen, 1993). Thus, these examples of clock theory have long held that the fundamental unit of not only circadian but also other biological cycles are composed of oscillations that are only a few hours in their cycle durations. Such high-frequency oscillators would be coopted for the organism’s daily clock function and, more specifically, be coupled in a particular manner, such that an (overall) low-frequency oscillation results; this could readily be in the circadian realm. A model of this kind goes on to predict that certain rhythm mutations would befoul or even eliminate the coupling components of the circadian clock. This, then, could be the nature of the defects in mutants such as pe& and per0, which in turn suggests that the n o m l function of this gene is to encode a coupling factor. Let us hold onto this thought-and recall it when certain physiological studies of per mutants are discussed below-but also keep in mind that the coupled-oscillator hypothesis is so formalistic that it could easily permit the components in need of being coupled for 24-hr pacemaking to be a collection of separate cells , on the one hand, or inmacellular entities, on the other. Recent studies of mutationally induced arrhythmicity in Drosophiila lead to the suggestion that the ultradian rhythms being discussed are an epiphenomenon. Thus, I favor the first of the two interpretations of these behavioral anomalies (hypothesis 1, above). The results that are now emerging involve the fact that all known genetic etiologies of circadian arrhythmic behavior bring out ultradian components as well (Table 4.1). Take the disco mutant; owing to the fact that its nonrhythmic behavior has an anatomical etiology, the cells of the fly’s clock structure could be thoroughly eradicated in most disco individuals. Whether the relevant oscillator components (under the second of the two interpretations) are these various cells, or are contained within each such cell, disco flies lacking these cells should be arrhythmic in a profound manner: no ultradian components

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Table 4.1. Ultradian Behavioral Rhythms in Drosophila “Arrhythmic” Mutantsa

Genotype

Source of mutant

Number tested

%with ultrasians

peP’ perper04 disco restless

R. J. Konopka Genetic cross R. J. Konopka K.-E Fischbach R. Mestel

34 16 41 49 48

59 44 46 29 31

AC72

R. Mestel

24

54

EJl2

M. S. Dushay

14

79

E64 time' cl-7 (D.P.) cI-8 (D.P.)

R. J. Konopka

33 436 33 46

33 2-10 45 33

See the citation C. S. Pittendrigh C. S. Pittendrigh

Source of data Dowse et al. (1987) Dowse et al. (1987) Hamblen-Coyle et al. (1989) Dowse et al. (1989) P. E. Hardin, M. Kaneko, and M. J. Hamblen-Coyle (unpub.) M. Kaneko, J. E. Rutila, and M. J. Hamblen-Coyle (unpub.) M. S. Dushay and M. J. Hamblen-Coyle (unpub.) Konopka er al. (1994) Sehgal et al. (1994) see Table 4.2 see Table 4.2

T h e s e mutants, as adult flies, yield locomotor activity records, the great majority of which are arrhythmic by inspection and elementary periodogram analysis. Yet, an appreciable subset of the data records contain high-frequency (ultradian) components. Some of these are self-evident, but they usually need to be revealed by “higher resolution” analyses (MESA and autocorrelation: see Dowse and Ringo, 1992,1993 for reviews). A majority of the mutants were induced by chemical mutagenesis, the exceptions being per- (as indicated), three transposon-mobilization-induced mutants (restless, AC72, EJlZ), and one that came from transposon-mobilized crosses but seemed to have occurred spontaneously (time'). Flies expressing the timeless mutation (time') were implied by Sehgal et al. (1994) to exhibit “ultradians” in the approximate percentage indicated; although this is likely to be an underestimate, owing to the absence (so far) of high-resolution analyses of the behavioral records.

would appear in the behavior records. Yet, disco has the same fairly high proportion of ultradian-rhythmic individuals as does peP and the like (Dowse et al., 1989; Table 4.1). From the standpoint of hypothesis 2, the situation worsened when it was found that all the arrhythmic mutants known exhibit these ultradian periodicities in a significant proportion of the individual-fly tests (Table 4.1). This includes the aforementioned mutant (Konopka et al., 1994), recently isolated on the X chromosome of D. mehogaster (which is neither per nor disco nor And);the timekss mutant (Sehgal et d., 1994); and also all four of the extant arrhythmic mutants in D. psewloobscura (Table 4.1). The latter, incidentally, were chemically induced many years ago (there were five of them then), isolated on the basis of eclosion arrhythmicity (Pittendrigh, 1974), and are now shown to be behaviorally arrhythmic (in the circadian domain) as well (Figure 4.2). Complementation tests of these mutants (Table 4.2) indicate two mutations at each of two loci, con-

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Table 4.2. Arrhythmicity and Elementary Genetics of Putative Clock Mutants in D. pseudoobscura" Complementation tests

Cross

Arrhythmic [n]

cI-7 X cl- 10 cI-7 X cI-8 cl-7 x cl-9

cI-8 X cI-9 cI-8 X cl-10 cI-9 (=7) X &I0 (=8)

19 4 0 14 3 3

Rhythmic [n (avg. T

* SEM)]

0 9 (24.7 ? 0.2) 9 (24.8 ? 0.1) 0

10 (25.6 2 0.4) 13 (25.0 ? 0.2)

Male segregants (from mutation/+ heterozygous females) ~~

Mutant cl-7

cLJO ( = 7 ) ~1.8 cI-9 (=8)

Arrhythmic [n]

Rhythmic [n (avg. T ? SEM)]

14 14 20 13

12 (24.7 t 0.2) 15 (24.2 ? 0.2) 16 (24.6 ? 0.2) 19 (24.8 2 0.2)

OFour of the five eclosion-arrhythmic mutants of Pittendrigh (1974) were tested and genetically analyzed with regard to locomotor behavior. The T values were determined by periodogram analyses. Konopka (1979) briefly noted that individual adults from these strains tested as behaviorally arrhythmic. The data tabulated here confirm (and document) this statement and extend it in two ways: By revealing that the four (extant) mutations define two complementation groups and by demonstrating that the generic variant present in each strain is a single-locus, X-chromosomal mutation. T h e complementation tests imply that cl-10 should be renamed as an allele of cl-7, hence cl-7'O; similarly, cl-9 now = ~ 1 - (also 8 ~ see Figure 4.1).

Figure 4.2. Locomotor activity records of the recently analyzed D. pseudoobscura arrhythmic mutants. These data were collected, double-plotted, and analyzed by periodogram as described in Hamblen et al. (1986) and Hamblen-Coyle et al. (1989). ( A ) Arrhythmic records of females heterozygous for a given two noncomplementing mutations, whose genotypes are indicated; based on the original designations of Pittendrigh (1974),as modified by the results of these complementation tests (see Table 4.2 for numerical details). (B) Examples of rhythmic records, produced by females heterozygous for a given two complementing mutations; the period estimates came from periodogram analyses. (C) Examples of rhythmic (plus periodogram-derived T , mean circadian period, values); and of arrhythmic segregants from among the offspring of females heterozygous for a given X-chromosomal mutation (see Table 4.2 for the overall numbers of males exhibiting the two kinds of phenotype, exemplified here with respect to progeny from each of the four relevant crosses).

A C/-7/C/-710 FEMALES

B

cI-71~1-8FEMALE

Figure 4.2-Continued

154

T = 25.5 h

C RHYTHMIC MALE SEGREGANTS FROM:

C/-7/+ FEMALE

T = 24.0 h

ARRHYTHMIC MALE SEGREGANTS FROM: C / - 7 / + FEMALE

155

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Jeffrey C. Hall

firming the initial but purely verbal description of these mutants’ genetics; and, indeed, all of these rhythm variants segregates as a single-gene, X-chromosomal mutation (Table 4.2). One feature of these experiments is that normal activity-rhythm periods in this species are in our hands (M.J. Hamblen and J.C. Hall, unpublished) routinely not less than 24 hr (as exemplified in Fig. 4.2 and summarized in Table 4.2). This is in contrast to what was noted in a study that compared eclosion and adult locomotor periodicities of D. pseudoobscura (Engelmann and Mack, 1978). Stepping back from the details of these rhythm mutants, the actions of the genes defined by such mutations are connected with oscillator coupling loses most of its force, given the genetic nonspecificity of ultradian rhythms that can be discerned at a glance (Table 4.1). That is, if one is to cling to hypothesis 2in general terms and with respect to the effects of mutations-then one has to imagine that all of the genetic loci implied by these tabulated mutants have something to do with oscillator coupling. It now seems more compelling to embrace hypothesis 1: scratch a circadian rhythm, uncover ultradians. One further way in which these high-frequency cycles have been permitted to well up from the fly’s behavioral capacities involves environmental treatments: Rearing Drosophila, and always keeping the adult animals, in constant darkness leads to phenocopies of mutants such as per0 and disco; the parallels to these mutants’ circadian arrhyhmicities included ultradian periodicities (Dowse and Ringo, 1989). These results were tacitly challenged by a subsequent study, which reported that dark-reared and behaviorally tested flies were nearly always rhythmic, meaning the manifestation of clear circadian cycles (Sehgal et al., 1992). A problem is that one set of these experiments involved chronic DD for some generations (Dowse and Ringo, 1989) with the other imposing that condition only at the (embryonic) beginning of a given life cycle (Sehgal et al., 1992). In addition, it was suggested-in conjunction with replications of the original results (Power et al., 1995a,b)-that the “one-generation DD” experiments allowed for unwanted exposure of the animals to pacemaker-influencing light: nonsafe safelights. Whether this disquieting possibility was an actual problem, the experiments in which embryos emanating from LD-reared parents were put into DD included the following augmentations: light pulses were delivered to the cultures at various stages, and it was found that early ones would cause the eventual adults to be not only rhythmic but relatively in phase with one another; whereas the allDD flies were unphased (Sehgal et al., 1992). It was further suggested that the embryonic neural expression of per (cf. James et al., 1987; Liu et al., 1988; Siwicki et al., 1988) may have something to do with this “time memory”: storage of the lateembryonic light-pulse information for behavioral usage If weeks later (also see discussion in Kaneko et al., 1997). This is a reasonable supposition; but it should be mentioned that the studies in which per gene products were found within (and

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in fact throughout) the embryonic nervous system were never followed up in terms of cellular details or definitive assessments of this early expression’s functional significance (if any). From the older investigations of per expression, the gene’s products were said to disappear from most, possibly all, larval tissues, as of the early stages of the first instar period (James et al., 1986; Bargiello et al., 1987; Siwicki et d., 1988). As a potential biological corollary, no circadian rhythms of larval locomotion were detectable in one concerted effort to find them (Sawin et al., 1994). However, it has turned out that Drosophila clock genes products are detectable within the larval nervous system (Kaneko et al., 1997). In certain of DD experiments introduced above-those which involved generation’s worth of that condition (Power et al., 1995a,b)-light pulses given to larvae yielded no behaviorally rhythmic adults (i.e., the same result, in these investigators’ hands, as found when no light pulses were delivered). In contrast, several old experiments involving D. pseudoobscura, and one in which light manipulations were performed on developing D. melanogaster (Brett, 1955), showed that pulses given to larvae are readily able to start and synchronize the circadian clock underlying eclosion (review: Saunders, 1982). The time-memory for that phenotype was thought to have nothing to do with the clock controlled by period gene expression-assuming the negative results from attempts to find larval per expression (in any tissues except the salivary glands: see below) were definitive; they were not (Kaneko et al., 1997).

D. Other phenotypes affected by rhythm mutations

1. Various temporal features of Drosophila biology Most of the fly’s rhythm mutants exhibit defects in eclosion rhythms. In this respect, reappearance of per mRNA and protein during metamorphosis is readily detectable (James et al., 1986; Bargiello et al., 1987; Liu et al., 1988; Siwicki et al., 1988). These mutants also are usually defective in adult locomotion. Now, mutations such as per, And, and tim had to affect eclosion, adult behavior, or both, because it was on those criteria that the genetic variants were isolated. Subsequent tests of these mutants-involving temporal variations other than circadian rhythms of eclosion and locomotion-have been performed based on the idea that those phenotypes could also be within the domains of these genes’ act ions. Given the prominent expression of per in the adult visual system, including and especially the photoreceptors, it seemed as if the following should have been controlled by this gene: a free-running circadian rhythm of rhabdomere turnover (Stark et al., 1988), which is analogous to rhythms within photorecep-

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Jeffrey C. Hall

tor subcellular compartments in other invertebrates and in vertebrates as well (e.g., Arikawa etal., 1988; LaVail, 1976,1980; Korenbrot and Fernald, 1989). Yet, application of all three per mutant types failed to reveal any systematic effects of the mutations on the presence of, or periodicity associated with, this visual phenotype (Chen et al., 1992). The biological significance of per products in the eye thus remains completely unknown; though the presence of the gene products there has been useful in molecular descriptions and manipulations of per expression and function (see later section). Another candidate for a visual-system rhythm in Drosophila is suggested by fluctuating frequencies of synapses and of axonal diameters in the optic ganglia of houseflies (Pyza and Meinertzhagen, 1993, 1995; Meinertzhagen and Pyza, 1996).per in Drosophila is expressed in those ganglia, with the protein detectable only in glia (Siwicki et al., 1988; Ewer et al., 1992; Frisch et al., 1994). So it seems possible that one or more of the Musca rhythms in question would be controlled by this gene’s action in the optic lobe glia (see below for further discussion). In females, the per mRNA and protein are prominent in the ovary (Liu et al., 1988; Saez and Young, 1988).That there is a free-running ovarian rhythm (Allemand, 1976a,b) made it seem as if this, too, would connect with expression of the clock factor in that organ. Yet, biological rhythmicity in question (periodically occurring vitellogenesis) has never been tested in per-mutant females. In the meantime, the distinctly cytoplasmic expression of PER in the relevant ovarian follicle cells (Liu et al., 1988, 1992; Saez and Young, 1988) has made it seem less likely that the gene influences an endogenously controlled rhythm in this tissue. The reason for such an inference, dovetailing with experiments in which the time course of ovarian per expression has been monitored, will become clear below. Another time-associated phenotype related to the female’s reproductive system is the ovarian diapause that occurs when the amount of daytime per 24-hr cycle shortens below a “critical” period (called critical day length, or CDL). This kind if diapause was unknown in D. melanogaster until shutdown of ovarian function was discovered to occur at quite low temperatures (Saunders and Gilbert, 1990; Williams and Sokolowski, 1993). Once again, per could have influenced this biological phenotype, especially inasmuch as the determination of critical daylength is controlled by a circadian pacemaker (Saunders, 1990; Gillanders and Saunders, 1992). Yet, CDLs were found to be normal inpe? orperLfemales (Saunders et al., 1989; Saunders, 1990). The curious effect of genetic variation at this locus involved a shortening of CDL in per-null females, which was especially pronounced in the per- type (Saunders, 1990). This is not interpretable in any particular manner, except to say that ovarian expression of the gene could somehow play a role in the control of diapause (reviewed by Hall, 1996). Also, the geographical cline associated with per’s TG repeat variation (Costa et al., 1992) may relate in some way to detection of the differing amounts of daylight that are ex-

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tant (during a given time of year) at different latitudes (cf. Lankinen, 1993b); for now, this is just as good a hypothesis for the biological significance of the cline as is the one involving temperature compensation (see above). The eye and ovarian rhythms under discussion may be underpinned by pacemakers that act autonomously. This would in principle fit with expression of a clock gene in peripheral effector organs as well as in a central location, such as the brain (see below). These Drosophila possibilities (though the ones just described did not pan out) bring to mind a peripheral insect organ-the moth testis-which operates a thoroughly autonomous circadian pacemaker (Giebultowicz et al., 1989, 1994; Giebultowicz and Joy, 1992). One of the few per-expressing tissues in which the matter of gene expression autonomy has been delved into involves a noncircudian behavioral rhythm. These are the courtship song cycles, whose periodicities are in the range of 30-70 sec, depending on the Drosophila species. This superultradian rhythmicity has been reviewed several times, from the standpoint of its (very) existence and also from a variety of analytical angles (Hall and Kyriacou, 1990; Kyriacou, 1990, 1993; Kyriacou et al., 1992, 1993). The current discussion will concern itself only with certain recent facts, and a new issue or two, related to the male’s rhythmic singing behavior. A genetic mosaic analysis of song rhythmicity as affected by a per mutation led to the tentative conclusion (as alluded to above) that the gene controls the “song circuitry” via action only in the fly’s thorax, probably the thoracic ganglia located there (Konopka et al., 1996). It should therefore be noted that the only cell types in these ganglia to express the gene are glia; no per-expressing neurons were detectable in the four fused ganglia of the ventral thorax (Ewer et al., 1992). This leads to the notion that thoracic expression of per indirectly modulates function of the aforementioned neuronal circuitry in some manner (cf., Barres, 1991; Laming, 1989), as opposed to controlling its operation directly. Relatively new genetic findings on the courtship song rhythms are as follows. (1) A controversy over the temporal fluctations in the relevant song parameter (reviewed by Hall and Kyriacou, 1990) seems finally to have been resolved in favor of the reality of such ultradian behavioral rhythmicity (Ah et al., 1998). (2) One gene beyond per has been looked into with regard to an influence on song, as well as circadian, rhythms: the original And mutation, when hemizygous in males, was found to cause a mild lengthening of the usual 55-60 sec song-rhythm period (Kyriacou et al., 1993). (3) Period differences in this oscillating character have been known for some time to have functional significance in matings between males that sing to females (Kyriacou and Hall, 1982, 1984), with the latter presumably interpreting the former’s song to determine if it is “good for her;” these earlier results (from “playback” experiments) proved reproducible (Griffith et al., 1993); the singing periodicity needs to be like that of her species for optimal mating receptivity (Kyriacou and Hall, 1982, 1984), suggesting that the evo-

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Jeffrey C. Hall

lution of differences in this element of courtship behavior bears a relationship to species-isolatingmechanisms. (4) It could have followed that intraspecific period differences would lead to the same kinds of variations in mating-initiation latencies; for this, females expressing pe4 or peg were tested to see if they are receptive to mating attempts of the corresponding male most readily; this would be analogous to D. melanoguster females responding better to (electronically mimicked) songs with 55-sec periods, compared to the effects of stimulating such females with songs varying according to the 35-sec rhythm that is characteristic of D. sirnulam (Kyriacou and Hall, 1982, 1984); yet there was no intraspecific parallel to this behavior: the D. melanoguster per mutants just noted were most receptive when stimulated by songs containing the normal 55-sec rhythm (Greenacre et d., 1993); stated another way: it seems as if the per gene does not act in the female to influence her interpretation of the acoustical stimuli that are important for courtship. A molecularly based experiment involving the two (closely related) species being discussed effected a transfer of the entire per gene from D. sirnulam to a D. melanoguster per‘“ “host” (Wheeler et al., 1991). The transfer brought along with it the shorter period of the “donor” species. This is consistent with experiments on hybrid males, which showed that the entirety of the genetic etiology for this interspecific behavioral difference maps to the X chromosome (Kyriacou and Hall, 1986). The intragenic etiology of this species difference was mapped (by analysis of further transgenic types) to an -700 basepair subset of per (Wheeler et al., 1991). That region is, in general, an evolutionarily diverged portion of the gene, which includes coding sequences for the TG repeat (e.g., Costa et al., 1991, 1992; review: Kyriacou et al., 1996). Previously, a deletion of that repeatencoding region was found (Yu et al., 1987a) to cause a shortening of the D. melanoguster male’s song period, while leaving circadian periods either normal in duration or slightly longer than normal at elevated temperatures (see above). There is no explicit connection of such simulans-like song behavior to the bona fide D. sirnuhm rhythmicity that is controlled by an intact version of the TG repeat-encoding region (and the -300 bp flanking that region on either side). For example, this repeat within D. sirnulam’s PER is not shorter (and in facts tends to be longer) in comparison to D. melanoguster (Peixoto et al., 1992; Rosata et al., 1994). More generally, no mechanistic insights have been forthcoming as to the manner in which the period gene can affect biological cycles whose time constants are 103 shorter than circadian periodicities. The molecular underpinnings of such daily rhythms are beginning to be understood with regard to the expression of per and the function of the products it encodes. Yet, the circadian molecular cyclings (Hall, 1995; Rosbash, 1995) and the models that emerged in this regard (e.g., Dunlap, 1993) would not appear to have anything to do with 1-minute periods. As these models necessarily include the role played by the timeless gene (e.g., Sehgal et a!., 1996), it will be interesting to determine whether tirn mutants affect song rhythmicity.

-

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One final phenotype that is both related to time and (insofar as the current status of the literature goes) affected by per is the duration of egg-to-adult development (Kyriacou et al., 1990).The most convincing component of these results was the longer-than-normal time required for pe& animals to develop. Once again, per’s molecularly monitored expression (within a given day: see below) bears no noticeable relationship to the control of this “infradian” (>>24-hr) component of the fly’s biological timing (cf. Poodry and Woods, 1990). Some additional temporally based phenotypes have been claimed to be aberrant in one per mutant type or the other; but these did not stand up to reexamination (review: Hall, 1990). The most recent experiment in this regard looked again at the larval heartbeat, which had been noted to be erratic in animals expressing pep1 (review: Hall and Kyriacou, 1990). Not only was this irreproducible in a quantitatively detailed analysis, but the other per mutants were also normal for this -3-Hz rhythm (Dowse et al., 1995).

2. Non-temporal phenotypes Learning in Drosophila involves phenomena that are on the cusp of being temporally related, though the relevant behavioral testing regimes do not always (or quintessentially) involve a sustained time base. In any event, it was thought that timing defects harbored by rhythm mutants could cause learning abnormalities as a reasonable extension of their main impairment (Jackson et al., 1983). The evidence reported to buttress this notion involved abnormalities of conditioned courtship behavior, as exhibited specifically by Iong-period mutants. However, a replication and extension of these experiments failed to show any consistent or meaningful connection of the per-L mutation, or of per transgenes associated with slow-running clocks, with either courtship-related learning or classical conditioning (Gailey et al., 1991). The most severely leaming-impaired rhythm mutant in the earlier study was Andante (Jackson et d., 1983), notwithstanding its rather mild period-lengthening (Konopka et al., 1991). When this particular matter was reassessed, by testing the original dyAd mutant and the new slow-clock mutants at this locus, no learning deficits were found (van Swinderen and Hall, 1995). Consider this for an irony, however: certain of the centripetal axon pathways, projecting from what appear to be per-expressing neurons (see below), end near the dendritic regions of the mushroom bodies (demonstrated by HelfrichFoster and Homberg, 1993, and implied by Nassel et al., 1993). These paired structures in the dorsal brain are involved in learning (review: Davis, 1993; also see de Belle and Heisenberg, 1994; Connolly et al., 1996). One reason to believe that this is so concerns spatial expression of the dunce gene: the CAMP phosphodiesterase encoded within this locus (review: Nighorn et al., 1994) seems especially prominent in the mushroom bodies (Davis, 1993), although it is present in other regions of the CNS in head and thorax (Nighorn et d . , 1991). Lest one wonder

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why this little discourse on a “learning gene,” it has turned out that dunce is a clock-related one as well (Levine et al., 1994): The adult fly’s free-running 7 is mildly shortened by dnc mutations; the PRC is altered, such that the “delay region” (early in the subjective night) is accentuated compared to wild-type. This study went on to show CAMP levels rhythmically fluctuate in constant darkness (this was most convincing in a dnc mutant); and CAMP-dependent kinase mutants (cf. Davis, 1996) were (as individual flies) often arrhythmic when behaviorally tested in DD. In summary, learning mutants can be rhythm ones, acting on the input side of the circadian system and in terms of ongoing pacemaker function; but rhythm mutants do not seem to be learning impaired. Recall that certain per mutants exhibit altered light sensitivity with regard to their circadian system itself. It thus might follow that, in general, these mutants would exhibit altered visual responses. This was reported to be the case, based on phototaxis tests of pe? and per‘“ (Palmer et al., 1985). But subsequent experiments of this kind showed these (and other) per mutants to be normal in such visual responses; their optomotor behavior was normal as well (Dushay et al., 1989). The most provocative of the nontemporal per mutant phenotypes concerned larval salivary gland cells. They were reported to exhibit very weak intercellular communication in peP’ and stronger-than-normal physiological coupling in glands dissected from pe? larvae (Bargielloet al., 1987). These findings seemed for all the world to be congruent with the notions, discussed above, that pep’ is an oscillator-decoupling mutation; and the grander possibility that the circadian clock is constructed by certain degrees of coupling strengths among component, ultradian oscillators. Thus, the physiological data from the salivary gland dye-fills and electrical recordings would extend this pacemaker hypothesis, encouraging it to specify that the components in question are separate cells (Bargiello et al., 1987). Implicit in this extension is an additional one: per mutations would affect communication amongst brain pacemaker cells of the adult fly in a manner that parallels their effects on the (nonneural) organ in larvae. The problem was that the seminal observations, in which extents and rapidities of dye-transfer were monitored in the various larval genotypes, proved to be irreproducible in the hands of two sets of investigators (Siwicki et al., 1992a); the irreproducibility was subsequently documented in a comprehensive manner (Flint et al., 1993). The original authors of the study reporting the differences among the three per genotypes in the meantime withdrew the claim (Saez et al., 1992). The original report had also depicted expression of both per mRNA and protein in the developing salivary glands; PER, moreover, was shown at the boundaries of this organ’s cells (Bargiello et al., 1987). This, too, has been a controversial chapter in the per story, in the sense that another set of observations on the developing animal failed to reveal per products-of any kind and in any subcellular location-within the salivary gland (Liu et al., 1988; Siwicki et al., 1988; as reviewed in tabular form by Hall and Kyriacou, 1990). As this disagreement

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still lurks in the literature (see Baylies et al., 1993), it may serve to introduce a more general discussion of the period gene’s spatial expression.

II. CHRONOGENETIC BIOLOGY A. Brain-behavioral studies of genes influencing Drosophila rhythms 1. Spatial expression of the period gene Most assessments and manipulations of the spatial expression of per, as augmented by neurological experiments using other rhythm mutants, have concentrated on the adult. In that stage, per mRNA and PER are found in a host of tissue types (tabulated extensively in Hall and Kyriacou, 1990; also shown diagramatically in Hall, 1995).This includes the CNS and PNS; for the latter, eye expression is conspicuous (see above), but other sensory organs (or at least the appendages carrying them) possess per products as well (most recently: Plautz et al., 199715). The significance of this gene’s expression in all tissues except the CNS is unknown (as implied by the foregoing discussion of per products in the visual system). Yet, brain expression has been found to be necessary for locomotor circadian rhythms. This was shown by behavioral analysis of genetic mosaics in which each animal was part pero* and per+ (Ewer et al., 1992). These results are generally congruent with a previous study of pe?//per+ mosaics (Konopka et al., 1983),although the earlier experiments did not include a cellular marker for the genotype of different brain regions in a given genetically mixed fly. Ewer et al. (1992) introduced such a marker, a per-lacZ fusion transgene, linked on the X chromosome to per’s normal allele (cf. Liu et al., 1988). Perhaps the most salient conclusion from this brain-behavioral study was that certain lateral neurons ( L N s ) - o f which there are -12-15 pairs of per expressors in wild-type-must be genetically normal for a mosaic to exhibit a clear circadian rhythm of locomotion; per+ LNs in but one side of the head were sufficient for that behavioral phenotype (Ewer et al., 1992). There were, in addition, a handful of curious mosaics in which per+ was present in only a subset of the 1800 brain glia; which make up the majority cell type in which the gene is normally expressed; the curiosity is that these particular individuals exhibited weak, long-period rhythms, even though all per neurons carried only the null allele (Ewer et al., 1992). Thus, it can be suggested that glial cells contribute to pacemaker function. A further intrigue is that certain of the “per glia” in the head are found in close association with axons that project from per neurons (HelfrichForster, 1995: see below). As for the thorax, recall that many additional glia (and that cell type only) express per in the thoracic CNS and that this may be part of the system of song-rhythm control (see above). Also, consider the current perspective on glial cells, which are now generally viewed as functional entities as opposed to merely

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structural ones in the nervous system in general (Laming, 1989; Barres, 1991), including the mammalian SCN in particular (e.g., van den Pol et d., 1992; Prosser et al., 1994). Glial expression of per is not necessary for circadian behavioral rhythms: A certain transgenic type (missing the “S’-flanking”region of the gene) exhibits robust rhythmicity of this kind (Frisch et al., 1994); CNS expression of the transgene was detectable only in a subset of per neurons (cf., Liu et al., 1991). In one of the rhythmic strains of this type (n = 2 overall), PER was also in the photoreceptors (Frisch et al., 1994). The rhythms exhibited by these per-promoterless transgenics were not, however, fully wild-type in character (Liu et d., 1991; Frisch et al., 1994; cf., Vosshall and Young, 1995); this leaves the door open for a glial contribution to normal rhythmicity. per-expressing neurons include a set of dorsal cells (DNs) in the adult brain, which are variable in number: 3-10 pairs per hemibrain (Ewer et al., 1992; Frisch e t al., 1994). One of the rhythmic, but per-promoterless transgenic strains had no DN expression (Frisch et al., 1994); the other did (the latter also being the eye-expressor: see above). Thus, per products in these dorsal cells would seem to be unnecessary for fairly strong behavioral rhythms (which were much more evident in the transgenics than in the case of the “glial-only” mosaics described above). Nor are the DNs sufficient: this conclusion stems from brain-behavioral studies of the disco mutant, which was introduced above in terms of its basic genetic, neuroanatomical, and arrhythmic phenotype. It is also known that few or no LNs are detectable in (anti-PER-mediated) immunohistochemical examinations of disco adults (Zerr et al., 1990). Yet, in some recent observations, nearnormal numbers of DNs in the brains of this mutant were counted (M. Kaneko and J.C. Hall, unpublished). Thus, if these cells are involved in the cellular control of circadian behaviorial rhythms, the notion would be that they play only a contributory role-as may be so for per glia. Additional per-related results from disco histology showed that the former gene’s expression is readily detectable in both photoreceptors and glia (Zerr et al., 1990). Perhaps the latter cells can provide a fairly robust (behavioral) pacemaker in the brain when recurring boosts are delivered to clock‘s functioning; that is, in conditions of light-dark cycling. Recall that in LD disco adults entrain and also exhibit a clear influence of the period gene’s action on the clock that runs that rhythm (not a mere response to the Zeitgeber). As will be discussed in more detail below, the perS mutation that affected disco’s behavioral rhythmicity (Hardin et al., 1992a) had also been found to affect its own expression; and this included an influence on PER production in glial cells of the adult CNS (Zerr et al., 1990).

2. Humoral relevance of per effects on rhythms? The per-DNs in the fly’s brain could correspond to certain brain neurosecretory cells that were once reported to sometimes be in ectopic locations in per(“

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adults-and, for that matter, in D. pseudoobscura flies expressing arrhythmic mutations at either of the aforementioned X-chromosomal loci (Konopka and Wells, 1980; cf. Table 2). The functional meaning of those cells is in general unknown (cf. Rensing, 1964, 1971). Yet, it is notable that other per-expressing cells are being suggested to have some sort of neurohumoral significance. Several cells right in the vicinity of the LNs stained with a fucsin reagent (Helfrich-Forster, 1991), which is taken to mark neurosecretory cells (this reagent was also used to show that the dorsomedial cells mentioned above can be in the wrong locations in arrhythmic mutants). It was not proved that these laterally located, fuscin-stained cells are per LNs, but it was interesting that disco caused a large decrease in their numbers (Helfrich-Forster, 1991). A further study in this area applied antibodies against a substance known (albeit in a misstated manner for flies) as “pigment-dispersinghormone” (see Rao and Riehm, 1989, 1993, for reviews). Once, again cells suspiciously near the LNs were PDH positive, and the same kind of disco-induced depletion of them was determined (Helfrich-Forster and Homberg, 1993). Detailed scrutiny of the PDHpositive cells indicated that only one of the two clusters ofper neurons in this lateral brain region (cf., Ewer et al., 1992; Frisch et al., 1994) contain this hormone (Helfrich-Forster and Homberg, 1993). The colocalization implied by that statement has now been backed up by showing that anti-PDH and PGAL histochemistry-in flies carrying the per-lacZ fusion construct mentioned above-label the same cells (Helfrich-Forster, 1995). Unlike what is the case for the fusion protein produced by the transgene just noted, or for native PER itself, the PDH polypeptide more or less fills the neuronal processes projecting from per LNs (Helfrich-Forster and Homberg, 1993). Thus, one can now observe that the axons projecting from some of these cells go out into the optic lobes and that others send processes into the brain itself. These data from staining for PDH suggest that per LNs fall in the category of “medulatangential neurons” (MTNs), a supposition that is consistent with observations made of signals mediated by the (multipurpose) PER-P-GAL fusion protein; in the relevant transgenic adults, the a-P-GAL immunoreactivity extended some distance away from per-lateral neuronal cell bodies in both directions (B. Frisch and J.C. Hall, unpublished). The MTN cells have been described on basic anatomical criteria in various dipteran species, including Drosophila (Fischbach and Dittrich, 1989). One wonders what the centrifugal subset of these projections are for, especially because there are no known visual system (biological) rhythms for that system in Drosophila (see above). However, perhaps the Pyza-Meinertzhagen rhythms in the optic lobes of houseflies are controlled not by endogenous oscillations in the relevant peripheral ganglion but are instead under efferent control, whose specific anatomical substrate would be the outwardly projecting axons from these per-LN cells. Central control of this sort, subserving peripherally occurring rhythms in arthropod visual systems, is well known (e.g., Fleissner et al., 1993, and references

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therein). The housefly-fruitfly analogy cannot be pushed very far, however, because the rhythms in Musca occur in the most distal optic ganglion (Pyza and Meinertzhagen, 1993,1995), to where the centripetal per neuronal projections (as marked by PDH) do not reach (Helfrich-Forster and Homberg, 1993;Nassel et al., 1993). Yet, in another fly, Phormia, some of the PDH-immunoreactive neurons do project all the way out to the distally located lamina in the optic lobe (Nassel et al., 1993); although a relationship between such cells and their processes to rhythms in this large fly-and Musca for that matter-is speculative. That M u ca per has been cloned (Nielsen et al., 1994) will permit the spatial expression of this gene eventually to be assessed; this should include asking whether that largefly PER protein and PDH are colocalized (cf., Helfrich-Forster, 1995). The MTNs in general project not only centrifugally (as just discussed) but also into the CNS proper (Fischbach and Dittrich, 1989). If one could determine the ultimate (if not immediate) cellular targets of centripetally projecting per LNs in pupue, then these should in principle include eclosion hormone-containing cells (Horodyski et al., 1993). Such central-brain neurons would presumably be among those influenced by per’s action, since mutations in that gene affect the pupal-to-adult emergence rhythm (Konopka and Benzer, 1971; Dushay et al., 1990; Konopka et al., 1994). The subset of MTNs that contains the putative hormone under discussion could also release it-and do so in a circadianly variable fashion (cf., Rensing, 1971). This would be analogous to rhythmic release into the cerebrospinal fluid of a mammalian peptide hormone known as arginine-vasopressin or AVP (reviews: Reppert et al., 1987; Inouye, 1996); the source if this substance rhythm is almost certainly the cells of the SCN, in which expression of the AVP-encoding gene happens to be cyclical (Uhl and Reppert, 1986; Robinson et al., 1988; Burback et al., 1988; Carter and Murphy, 1991; Majzoub et al., 1991; Inouye, 1996). However, the biological significance of the clock cell-produced oscillation of AVP levels is unknown. Yet, closing the connections between a hypothetical PDH rhythm and Drosophila‘s rhythmic behavior is within the realm of possibility. One reason to state this hope is that an apparently humoral influence on the fly’s rest-activity cycles has long been known: implanting a pe?-expressing brain into the abdomen of an arrhythmic pep1 adult caused the host to become rhythmic in a few instances-with appropriately short periods (Handler and Konopka, 1979). This result has never been confirmed or denied; so it is not known whether the material that one imagines to be dribbling out of the implanted brain reflects the bona fide presence of the same substance(s) in the hemolymph (blood) of an intact animal; nor can one deduce whether this humoral signal exerted a direct influence on the host’s thorax (which mediates the fly’s “walkingrhythm” in a proximate manner), as opposed to traveling to per LNs in the brain; those neurons might then send some other kind of signal to more posterior regions of the CNS.

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3. Miscellaneous results related to per neurons 1. One more small fact that tends to connect per expression in these cells to behavioral rhythmicity is that an increase in the dosage of per+ was found both to shorten this rhythm’s period (as found in the early genetic experiments on this gene: see above) and also to effect stronger-than normal staining in the LNs (Zerr et al., 1990). 2. The lark mutant, isolated as an eclosion-phase variant, was induced by transposon mobilization. The mobile piece of DNA carried a reporter factor (hZ,as usual), and the expression of lark that could be inferred from PGAL histochemistry performed on pharate adults included (though was not limited to) the brain region which includes the LNs (Newby and Jackson, 1993). For per’s part, it is expressed in these late-pupal cells-and in they alone, insofar as the head tissues of pharate adults are concerned (Siwicki et al., 1988; Konopka et al., 1994). These findings do not necessarily mean that lark influences per itself (or vice versa: Newby and Jackson, 1996). But at least the former gene’s product may be present in the right cells, if indeed it is proper to infer that the LNs contribute to periodic eclosion as well as to the rhythmic behavior of adults. There is at least something periodic about per expressing in the late-pupal LNs: it has recently been shown that the levels of staining, observed after application of anti-PER serum, are cyclical (Konopka et al., 1994). This introduces the broader aspects of time-dependent variations in per expression.

B. per-expression rhythmicities studied in sifu

1. Basics of PER’Scircadian cycling of abundance Rhythmic fluctuations in the levels of this clock gene’s products were discovered immunohistochemically (Siwicki et al., 1988). This story has matured dramatically, as will be discussed briefly below. Here, per cycling is considered from the standpoint of cells, tissues, and certain manipulations of the period gene’s structure. Cyclical variations in the amounts of per products extends to the mRNA level (initially: Hardin et al., 1990; reviews: Hall, 1995; Rosbash, 1995). The abundance oscillations of this transcript occur in all body regions of the adult, with the notable exception of the female abdomen (Hardin, 1994). The temporally constant levels of per mRNA there-when combined with the transcript taken from other body regions and from males-was enough to obscure the cyclical variations everywhere else in experiments that simply extracted RNA from sexually mixed populations of whole flies at different times of day and night (Ready et al., 1984; Young et al., 1985). Incidentally, both the transcript and PER protein cyclings persist in constant darkness, although the robustness of these variations

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(notably, the amplitude) is diminished in DD (Hardin, 1994). In LL, PER cycling was looked for (Zerr et d . , 1990); there was not only none of it, but also almost no immunohistochemically detectable signal (also see Price et d., 1995). The temporally constant level of female abdominal per mRNA had its source narrowed down to the ovaries (Hardin, 1994). PER staining has not been monitored in that tissue at different time points. But immunohistochemical studies have shown that PER cycles nicely in all the neuronal and glial cells in which the gene is expressed (see above), in all photoreceptors (Siwicki et al., 1988, Zerr et al., 1990); and, moving posteriorly, in the gut and the corpora cardiaca (Frisch et al., 1994). These two thoracic tissues bear comment: the latter constitutes one of the few places in which per expression is detectable in pupae (Liu et d . , 1988), and PER seems to cycle there (Konopka et al., 1994). As for rhythmically fluctuating immunoreactivity in the alimentary system, its status is somewhat shaky, because it was reported not to occur in one study (Zerr et al., 1990), but to exhibit an apparent peak and trough in another (Frisch et d., 1994). Those times, for any tissue or cell type in which PER cycling has been observed, are toward the end of the night and day, respectively, in LD experiments (Zerr et al., 1990; Frisch et al., 199b). More recent experiments have detected PER cycling just about any place (or life-cycle stage) where one looks for it (Emery et al., 1997; Kaneko et al., 1997; Giebultowicz and Hege, 1997; Hege et al., 1997; Stanewsky et al., 1997a; Plautz et al., 199713). The techniques for this have involved quantitative immunohstochemistry (cf. Zerr et al., 1990) as well as a more convenient and powerful way to monitor cyclical gene expression over long time courses: fusion of parts of the per gene to sequences encoding firefly luciferase (Brandes et al., 1996). This allows for oscillation clock gene expression to be nicely monitored in real time in studies of live flies (Plautz et al., 1997a, Stanewsky et al., 199713) and parts of them, including internal organs (Emery et al., 1997) and appendages (Plautz et al., 199b). Certain such luciferase-reported rhythms are sustained in DD (Emery et al., 1997); others are not (Plautz et al., 1997b).

2. Subcellular locations of PER protein Another aspect of anti-PER-mediated staining, and (in effect) of its rhythmic variations, is that the strong signals are largely nuclear in the cells of a given cycling tissue (Siwicki et al., 1988; Saez and Young, 1988; Zerr et al., 1990; Liu et al., 1988,1992; Stanewsky et al., 1997b). The exception that proves the rule involves follicle cells of the ovary (the one per-expressing cell type in that organ); here, PER (or the aforementioned PER-PGAL fusion protein) appears to be an exclusively cytoplasmic signal (Liu et al., 1988,1992;Saez and Young, 1988). What rule? Here, it is mentioned again (also see below) that PER levels not only define a rhythm, but also that that protein influences its o m rhythmicity (Zerr et al., 1990;

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Hardin et al., 1990). This includes feedback effects of PER on per transcription (e.g., Hardin et al., 1992b). This would seem to demand PERs presence in (or at least an influence of the protein on) the nucleus for at least part of the cycle (see Curtin et al., 1995, for details). For PER or any translated polypeptide to find its way to the nucleus is not necessarily an automatic event. In this regard, the possibility that timeless gene action contributed to nuclear localization of PER was put forth (Vosshall et al., 1994). This study did not nail the point, however: No PER signals at all were detectable immunohistochemically in timOl mutant adults. This was taken to mean that the PER protein was too spread out in the mutant cells observed (photoreceptors) for staining to be elicited. But that particular feature of the hypothesis was wrong (Price et al., 1995). Effects of tim on subcellular localization of a per product were demonstrated via a PER-PGALfusion protein (produced by the selfsame transgene used in other contexts mentioned above): the PGAL reporter signal was present at a chronically robust level with a cellularly diffuse appearance (Vosshall et al., 1994). Subsequently, native PER and TIM proteins were analyzed for their physical interactions (reviews: Young et al., 1996; Rosbash et al., 1996), including those involved in moving heterodimers into the nucleus. The latter experiments (with PER, TIM, and fragments thereof) have been performed only in nonneurally derived tissue-culture cells (Saez and Young, 1996). PER-like immunoreactivity has been found in organisms outside of Drosophila by application of the original anti-PER antibody known as “ a n t i 3 (e.g., Siwicki et al., 1989,1992b; Frisch et al., 1996). That reagent was given this name because it was generated by first producing a synthetic peptide, deliberately chosen from the vicinity ofper% amino acid substitution (Siwicki et al., 1988). This region, in turn, is an evolutionarily conserved one at least among and beyond Drosophih species (Colot et al., 1988; Reppert et d., 1994; Kyriacou et d., 1996). Thus, perhaps the anti-S-mediated staining of pacemaker structures in molluscs and a mammal should not be considered a coincidence or a miracle. There are, however, at least two problems with these potentially provocative results: (i) In the gastropods Aplysiu and Bulla (two species of marine mollusc), the staining elicited by anti-PER seemed exclusively cytoplasmic (Siwicki et al., 1989); hence, not analogous to the intracellular location of this clock protein in what are now presumed to be the fly’s circadian pacemakers (although there may be anti-S-elicited nuclear signals in some of the cells within a beetle’s nervous system, which are stained via application of this antibody: Frisch et al., 1996). (ii) The cyclical variation in levels of the PER-like protein, shown by Western blottings in accompaniment to the initial report of the pacemaker cell stainings in Aplysia (Siwicki et al., 1989) proved to be irreproducible (Strack and Jacklet, 1993). For the mammal’s part, staining of cells within the SCN observed by ap-

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plication of anti-S was not, in one study, correlated with any detectable variations in levels of a PER-like biochemical signal; also, there was insufficient resolution of the staining within the rat SCN to tell whether the signal might include the nucleus (Siwicki et al., 1992b). However, another investigation of this kind reported cycling of PER-like immunoreactivity in rat SCN by Western blotting (Rosewell et al., 1994). A notion that emerges from these various studies of PER expression is that the protein’s presence exclusively in a nonnuclear compartment might mean that per will not cycle in such a tissue type (note that the staining signal once reported to be at cell boundaries within the larval salivary gland was not monitored temporally). This is not universally the case: cloning ofger from a silkmoth (Reppert et al., 1994) followed by assessing the lepidopteran’sPER expression both spatially and temporally revealed thoroughgoing cytoplasmic signals in adult brain neurons; this immunoreactivity did exhibit daily fluctuations in apparent abundance (Sauman and Reppert, 1996). Such results are not yet interpretable (reviewed by Hall, 1996). Also, they involve different patterns of expression of the same gene products in the silkmoth eye (Reppert et al., 1994; Sauman and Reppert, 1996). Those visual system patterns are more conventional; that is, similar to what one finds in both brain and photoreceptor cells of adult Drosophih (Siwicki et al., 1988; Zerr et al., 1990; Frisch et al., 1994; Curtin et al., 1995). What could “cytoplasmic-only”expression of PER mean (Sauman and Reppert, 1996; Hall, 1996)? Such a subcellular location might suggest that the protein is involved only in carrying environmental signals from the outer regions of the cell into the nucleus. This is what silkmoth PER may be doing, such that it would not be an actual clock protein in brain cells (as opposed to eyes ones) of this species. A n analogy could be provided by PER as it is expressed in the cytoplasm of certain ovarian cells in Drosophih females (see above). Consider that periodic egg-hying in this species is not controlled by a clock but must be driven by light4ark cycles (e.g., Allemand and David, 1976; Fleugel, 1981). This makes one wonder if PER’S subcellular location in the ovary is poised to participate in the intracellular trafficking of externally originating signals. Constant light is known to exert a substantial (negative) effect on the steady-state level of immunohistochemically detected PER (Zerr et al., 1990). This almost certainly occurs by light-induced degradation of TIM protein-PER’S partner-leading to the latter being unprotected from protease activity (e.g., Price et al., 1995; Rosbash et al., 1996). Yet, if PER-TIM dimers are able to form and hold themselves together, the complex moves into the nucleus (see above). Connecting this phenomenon with the (indirect) light effects on PER levels strengthens one’s feelingaerived from the early studies of PERs subcellular localization (Hall, 1995)-that this protein is a part of the machinery involved in mediating environmental inputs into the clock, as well as functioning the operation of the pacemaker itself.

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C. Extraocular photoreception of clock-resetting signals The way that light inputs itself to the circadian system of Drosophila (as in many organisms) involves photoreception that can be mediated by something other than the standard external eyes (see Fleissner et al., 1993, for an example). Most of the evidence for this in Drosophila is genetic and negative (reviewed by Hall and Kyriacou, 1990): The ability of flies to entrain and have the phase of their behavioral rhythm is largely unaffected when they are rendered anatomically or physiologically blind by a given visual system mutation that affects the development or function of all external photoreceptors (yes, including the ocelli). The anatomical mutant used in several of these experiments is sine oculis, which also leads to anomalous free-running rhythms (e.g., Helfrich, 1986). It has been argued that this could be because of more central defects in so, caused either by that mutation directly or by a “cascade” effect into the brain, resulting from the eye’s absence. It has turned out that, in fact, so is a somewhat broadly expressed gene whose pleiotropy includes that it can mutate to developmental lethality (Cheyette et al., 1994). However, the sine oculis allele used in all the rhythmrelated experiments-so’-turns out to be somewhat limited in its effects, in the sense that only the gene’s eye-imaginal disk expression is knocked out. Thus, C N S - o r at least optic lobe-abnormalities in so* can be interpreted as being caused solely by an indirect effect of the absent communication from peripheral afferents (also see Fischbach and Technau, 1984). The absence of effects on circadian light-responsiveness of the physiological eye mutations implies that these specific pieces of the “phototransduction machinery” (e.g., a phospholipase-C encoded by the no-receptm-potentl-A gene) are not used by the clock-system photoreceptors; indeed nmpA-null mutants are in no way “circadian blind” (Dushay et al., 1989; Hamblen-Coyle et al., 1992; Wheeler et al., 1993). It also remains a mystery as to what photoreceptive molecule subserves circadian light reception in Drosophila; it is known only that the substance seems not to be rhodopsin (reviewed by Zimmerman and Ives, 1971). These authors, and others (Klemm and Ninnemann, 1976), tentatively suggest that the relevant substance may be a flavinlik molecule, but this is not a strong inference, based as it is solely on action-spectral data. Moreover, all of the relevant experiments have been performed only in D. pseudoobscura and only with regard to phase shifts of that species’s eclosion rhythms. There are also some provocative, albeit once again nondefinitive, findings about what and where the circadian photoreceptor cells may be: the so-called 6th and 7th eyes of Drosophila; these paired structures are located between the retinal layer and the first optic ganglion, are not eliminated by anatomical eyeremoving mutations, and seem to send processes to the vicinity of per lateral neurons (Hofbauer and Buchner, 1989; van Swinderen and Hall, 1995). It is intriguing, furthermore, that PER is expressed in eyes 6 and 7 (B. Frisch and J.C.

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Hall, unpublished), as in all other known photoreceptors (see above). These descriptive studies dovetail with genetic experimental ones, in which an (externally) blind mutant called glass was applied; gl mutations happen to knock out the internal eye structures as well as ruining all the standard retinal cells. But such mutant adults were still entrainable and phase shiftable (Helfrich-Forster, 1997; B. Frisch, M.S. Dushay, and J.C. Hall, unpublished). This leads to the hypothesis that the 6th and 7th eyes have circadian-photoreceptive function, and they do send such inputs to the LNs (van Swinderen and Hall, 1995; B. Frisch and J.C. Hall, unpublished). But the latter (CNS neuronal) cells also possess the capacity to receive and process light insofar as circadian clock resetting is concerned. Thus, the pacemakers would themselves be the ultimate back-up receptor cells in terms of the circadian system’s light responsiveness. Yet, photoreceptors in the compound eye are now known to be (it seems) the principal workhorse light-receptor cells subserving circadian function (even though they are not required): Recent application of visual mutants to eliminate or inactivate external photoreceptors dropped the sensitivity of the fly’s circadian system dramatically (Helfrich-Forster, 1997).

D. Constitutive expression of the periodgene? Recall the per transgene that is devoid of the gene’s 5’-flanking region and that its expression can mediate by gene-product and behavior rhythms (Frisch et al., 1994). The current genetic understanding of these phenomena is as follows: the promoterless per’s products cycle in LD conditions (Frisch et al., 1994), but the mRNA may not do so in DD. mRNA cycling that would only be light driven (hence not autonomous) has been observed for mammalian genes expressed in the hypothalamic clock structure (e.g., Inouye, 1996), and this is also seen for an opsin-encoding gene in Drosophila (Cheng and Hardin, 1998). If per mRNA (again, as transcribed from the transgene lacking a 5’4anking region) does not cycle in constant conditions, how are these flies rhythmic behaviorally? It appears as if PER cycling can occur all by itself by temporally varying degradation of the protein, which is systematic enough to support a relatively weak circadian molecular cycle (Dembinska et al., 1997; Cheng and Hardin, 1998). These results and the associated hypothesis are not quite compelling: temporal assessments of per mRNA levels in flies carrying the promoterless form of the gene have not been carried out in DD; noncycling of per-coding (mRNA) sequences, under the control of an opsin gene promoter that acts constitutively in DD, nevertheless allows for PER to cycle (Cheng and Hardin, 1998); but this is not connected to any known biological rhythm in the eye (see above), whereas (presumed) cycling of PER in brain neurons in this transgenic type does underlie and known biological (behavioral) rhythm in constant conditions. These questions and empirical inadequacies notwithstanding, the sce-

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nario outlined above (and by Dembinska et al., 1997; Cheng and Hardin, 1998) speaks to the fact that the molecular clockworks in this organism-in terms of identifying all of the factors involved in circadian cycling of the relevant gene products and how the results of such oscillations find their way out into control of the organism’s biological rhythms-are far from being comprehensively defined. Thus, as merely one inadequacy: How would the “PER protease” be identified-starting with genetics or instead by biochemistry and cloning? And how would the putative cycle of this enzyme level or activity be controlled?

One way to extend our knowledge of and insights about these circadian processes will be to continue efforts in the area of pure chronogenetics: to isolate novel rhythm mutants and determine whether the genes specify suspected parts of the pacemaker machinery or whether some of the newly identified loci encode heretofore unanticipated molecular contributors. This kind of clock gene hunting, by mutations and position cloning, is how molecular chronobiology started and is the main theme of this review.

Acknowledgments Some of the findings reviewed here have stemmed from experiments of the author and his co-workers; these were supported by grants from the US.Public Health Service: NIH GM-33205 and NIMH MH-51573.1 thank Melanie Hamblen for generating the data in Table 4.2 and Figure 4.2.1 appreciate comments on the manuscript from Ralf Stanewsky.

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P. A. Jeggo MRC Cell Mutation Unit, University of Sussex, Brighton BN19RR, United Kingdom

I. Introduction 186 11. Formation of DNA strand breaks 186 111. Mechanisms for the repair of DNA DSBs 188 A. Homologous recombination 188 B. Single-strand annealing (SSA) pathway 190 C. Nonhomologous end-joining (NHEJ) 190 191 IV. Identification of genes required for NHEJ A. Studies with mammalian cells 191 B. Studies with yeast 193 V. Characterization of proteins involved in NHEJ by biochemical, molecular, and genetic analysis 196 A. Components of DNA-PK 196 B. XRCC4 and DNA ligase IV 200 VI. DNA-PK as a protein kinase 200 VII. The mechanism of NHEJ 201 201 A. Studies with yeast B. Studies with mammalian cells 203 C. Studies examining V( D)J recombination 204 VIII. The contribution of NHEJ and HR to DSB rejoining in yeast versus mammalian cells 205 IX. DNA-PK-defective mice 206 X. Radiosensitivity and immunedeficiency 207 XI. A Model for NHEJ 208 XII. Summary 210 References 2 11 Advances in Genelics, Vol. 38 Copyright 0 1998 by Academic Press All rights of reproduction in a n y form reserved. 0065-2660/98 $25.00

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1. INTRODUCTION Double-strand breaks (DSBs) arise in DNA both on exposure to exogenous DNA damaging agents and endogenously during certain cellular processes. The repair of such DSBs is imperative to maintain the integrity of the DNA molecule and to limit the mutational and oncogenic potential of such lesions. From recent studies it has become evident that all cells have multiple mechanisms for the repair of DSBs and that the absence of these repair mechanisms results in enhanced sensitivity to DNA-damaging agents. In addition, several molecular processes, such as mating type switching in yeast and V(D)J recombination in mammals, introduce DSBs into DNA during processes involving specific and controlled genetic rearrangements that serve to enhance the genetic diversity of the organism. Defects affecting specific DSB repair mechanisms can result in loss of function of these processes. Identification of the first mechanism involved in the repair of DSBs was, like many repair studies, initiated by an examination of lower organisms, from which defective mutants were isolated with relative ease. More recently, an important DSB repair mechanism was identified through studies with mammalian cell lines and has subsequently been identified and characterized in yeast. These studies have exposed surprising differences in the way DSBs are repaired in higher and lower eukaryotes. Here I will review the DSB repair mechanisms which have been identified, with major emphasis on the more recently identified mechanism of nonhomologous DNA end-joining (NHEJ).I will consider the different utilization of the repair mechanisms between species and discuss the potential consequences of lack of repair for human disorders.

II. FORMATION OF DNA STRAND BREAKS Strand breaks may involve only a single strand of the DNA duplex (single-strand break (ssb)) or both strands may be broken at identical or closely opposed sites (DSB). Ssbs are frequently encountered by cells since they can arise by endogenous DNA damage, a wide variety of exogenous DNA damaging agents, and as an intermediate in many repair processes. They are efficiently and rapidly repaired and do not represent a serious lethal lesion to a cell. In contrast, most cells probably encounter DSBs infrequently but the potential lethal consequences of unrepaired DSBs are highly significant. DNA DSBs are very rarely, if at all, generated by endogenous oxidative damage and therefore are unlikely to represent a spontaneous lesion of a n y significance. DSBs are, however, introduced by endonucleases during several metabolic processes. These breaks are usually site specific and occur in defined cell types in higher organisms and are repaired by specific DSB repair mechanisms. Mutants defective in defined DSB repair mechanisms may therefore be defective in these metabolic processes. One such process is mating

5. DNA Breakage and Repair

Cleavage reaction during V(D)J recombination

187 Cleavage reaction during retroviral integration

Figure 5.1. Formation of DNA DSBs during V(D)J recombination and retoriviral integration. See text for a description of the process. The large triangle depicts the RGAI/2 complex; the large circle depicts the integrase complex.

type switching in yeast, in which a site-specificDSB is introduced by a specific endonuclease (for example the HO endonuclease in Saccharomyes cereuisiae). Such a DSB is lethal to a yeast cell in a repair-deficient background. In higher organisms, V( D)J recombination also involves the introduction of a site-specific DSB. This process requires the products of two genes, termed recombination activating genes (RAG1 and RAGZ), and the initial step is a single-strand nicking reaction yielding a 3’-OH terminus (van Gent et al., 1995) (Figure 5.1) followed by a direct nucleophilic attack on the phosphodiester bond of the opposite strand by the 3’-OH group formed. The result of this transesterification reaction is a hairpin structure at the nonprotein-bound end and a blunt 5’ phosphorylated end bearing the recombinase protein (van Gent et al., 1996). Transposition by bacteriophage Mu and retroviral integration are also initiated by a single-strand break and followed by a transesterification reaction resulting in the formation of similar but not identical end structures. In these cases however, the 3’-hydroxyl is used as a

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nucleophile to attack a new DNA molecule resulting in transposition rather than hairpin formation (van Gent et al., 1996) (Figure 5.1). Recombination during meiosis is a further metabolic process involving the introduction of DSBs. In yeast at least nine genes have been identified as playing a role in DSB formation and repair and some of these function additionally in the repair of DSBs in nonmeiotic cells (Keeney and Giroux, 1997). The mechanism of break formation occurs somewhat differently from that arising during V(D)J recombination and transposition (Keeney and Giroux, 1997). Primary cleavage occurs by two transesterification reactions involving nucleophilic attack from a side chain on the recombinase onto the DNA phosphodiester backbone. This creates a pair of breaks closely spaced on opposite strands with a free 3’-OH terminus and a protein-bound 5’ terminus. This reaction is analogous to those catalyzed by DNA topoisomerases. In all these cases it is evident that the breaks are, at least in their initial stages, protein bound, which may serve to some extent to limit nucleolytic degradation and facilitate the rejoining process. Ionizing radiation (IR) is probably the most significant exogenous agent inducing DSBs. Although one DSB is introduced per 20 SSBs, a DSB probably represents the most significant lethal lesion induced by IR (Frankenberg et al., 1981). The type of DSB introduced by IR will depend on the nature of the radiation (for example, whether it involves high or low energy transfer). The breaks formed are generally complex and may involve damaged bases at their termini. Thus, defined mechanisms may be needed for their repair rather than simple end ligation. Notwithstanding the complexity of radiation-induced breaks, restriction enzyme (RE)-induced breaks are frequently used to study DSB repair and appear to represent a good model system since mutants which are unable to repair radiation-induced breaks are also unable to repair RE-induced breaks (see for example Bryant et al., 1987).

A. Homologous recombination In lower organisms homologous recombination (HR) represents the major mechanism for the repair of DSBs. HR requires an extensive region of homology between the strand under repair and an undamaged homologous strand, and since any sequence information lost at the site of the break is repaired using information from the undamaged homolog, it represents a very efficient, high-fidelity repair mechanism. HR has been amply described previously (Friedberg et al., 1995) and only a brief overview will be given here. Elegant work by Szostak et al. (1983) and Resnick and Martin (1976) led to the proposal of a model for HR in which a DSB was an initiating event (Resnick

189

5. DNA Breakage and Repair

Yeast RADIATION

SWITCHING

DSBs --YKu70. YKu80, DNA ligase

NH\s,

R A D 5 0 - 5 ~ S S i Radl/RadlO

:and 4 end-end joining

ACCURATE

DELETION FORMATION

ACCURATE

ACCURATE

DELETION FORMATION

ACCURATE??

\

ERCCl/XPF

SSA!

.HE/

XRCC4, K ~ 7 080, , DNA DNA-PKCS ligase 4

RAD51.52and 54

DSBs --

IONlSlNG RADIATION

77

V(D)J RECOMBINATION

MAMMALIAN CELLS Figure 5.2. Comparison of DSB rejoining in yeast and mammalian cells. Heavy arrows depicts major usage of the rejoining pathway; lighter arrows depict less significant usage.

and Martin, 1976; Szostak et al., 1983). Single-strand regions derived from the initial break by exonucleolytic digestion of one strand were proposed to invade the homologous intact strand creating two regions of heteroduplex DNA. The gaps are then filled in by DNA synthesis using the intact strand as a template (see Figure 5.2). A Holliday junction, the point at which the strands cross over, is resolved by exonucleolytic cleavage and ligation to the original strand. Variations to the process occur depending on whether the Holliday junctions are resolved with or without crossovers. The biochemistry of this process has been examined in detail and some steps have been reconstituted in vitro (see, for example, Eggleston et al., 1997)). In S . cerevisia, HR requires the products of the RAD52 epistasis group, represented by RAD50-58. The phenotype of the rad50-58 mutants differ considerably with some showing sensitivity to IR, defects in DSB rejoining,

190

P. A. Jeggo

decreased spontaneous and radiation-induced mitotic recombination, and decreased meiosis, while others show only some of these defects (see Friedberg e t al., 1995 for further discussion). Rud.51, -52, and -54 are exquisitely y-ray-sensitive mutants and clearly play a critical role in DSB repair in S. cereoisiae. The ability of mammalian cells to carry out HR and the significance of this pathway in higher organisms will be discussed in Section VIII.

B. Single-strand annealing (SSA) pathway Considerable evidence has now accumulated for an alternative pathway of DSB repair which utilizes short direct repeat sequences (Fishman-Lobell et al., 1992; Lin et al., 1990a). Fraying of the DSB ends results in exposed regions of ss DNA and the annealing of short regions of homology promotes the rejoining step. In yeast the products of RADJ and R A D J O act to digest the single-strand tail regions generated (Fishman-Lobe11and Haber, 1992). Mammalian homologs of radl and radl0, XPF and ERCCl , have been identified and likely operate in a similar way (Brookman et al., 1996; Sijbers et al., 1996; van Duin et al., 1986). Extensive 5'to-3' degradation of DNA can occur prior to annealing, occasionally generating large deletions. Thus, in contrast to HR, which is potentially a mechanism ofhigh fidelity, the SSA mechanism results in the deletion of sequences between the direct repeats (see Figure 5.2) and is therefore inherently a process of low fidelity. This process was originally proposed on the basis of transformation experiments in mammalian cells (Lin et al., 1984, 1990b) and subsequently direct repeat sequences have been found at the junctions of radiation-induced deletions in human cells (Morris and Thacker, 1993). The process has also been characterized in yeast, particularly in tandemly repeated genes such as the ribosomal DNA array, and occurs independent of RAD5.2 (Fishman-Lobe11et al., 1992; Ozenberger and Roeder, 1991)

C. Nonhomologous end-joining (NHEJ) It has long been evident that, in comparison with yeast, mammalian cells have a greater ability to rejoin DNA ends in the absence of any homology (Roth et al., 1985). The majority of integration events of exogenous DNA into chromosomal DNA occur in the absence of any apparent homology (Roth and Wilson, 1985). Additionally, the analysis of plasmid rejoining using extracts of Xenopus laevis has identified a mechanism of NHEJ, as well as the existence of an alignment protein which promotes the rejoining of nonhomologous ends (Pfeiffer et al., 1994; Thode et al., 1990). More recently genes involved in the NHEJ process have been identified (see Section IV). The significant advances which have taken place in the characterization of NHEJ in recent years will be the focus of the ensuing sections.

5.

DNA Breakage and Repair

191

IV. IDENTIFICATION OF GENES REQUIRED FOR NHEJ A. Studies with mammalian cells One route to identify repair genes has been the isolation and characterization of repair-defective mutants. Cultured rodent cell lines have been particularly valuable for this approach partly because they have extensive hemizygosity, making it easier to isolate defective mutants, and partly due to their ease of handling. At least nine complementation groups of rodent mutants sensitive to ionizing radiation have been described (Jeggo et al., 1991; Thacker and Wilkinson, 1991; Thompson and Jeggo, 1995). Members of three of these groups (IR groups 4, 5 , and 7), which include the most markedly y-ray-sensitive rodent lines, have defects in their ability to rejoin DNA DSBs (Jeggo, 1997). The members of these complementation groups are listed in Table 5.1 and share strikingly similar phee notypes including an inability to carry out V( D)J recombination. This surprising phenotype, which will be discussed in more detail in Section VIIC, is not a property found in radiosensitive mutants belonging to the other complementation groups, suggesting that the proteins defined by groups 4,5, and 7 might operate in a single pathway (Pergola et al., 1993; Taccioli et al., 1993; Thacker et al., 1994). Two different approaches led to the identification of the first of the genes defined by these complementation groups, namely XRCCS, the gene defective in IR group 5 mutants. Attempts to clone XRCCS by complementation of a defective rodent mutant with human DNA localized a complementing gene to the region 2q33-34 (Hafezparast et al., 1993; Jeggo et al., 1992). Ku is a highly abundant heterodimeric nuclear protein, which was originally identified as an antigen present in the sera of certain autoimmune patients (Mimori et al., 1981). Ku is composed of 70- and 80-kDa subunits and, significantly, has a characteristic ability to bind to double-stranded (ds) DNA ends without sequence specificity, rendering it a candidate complementing gene. Colocalization of the gene encoding the Ku80 protein to the same chromosomal segment as XRCCS was one route leading to its identification as the complementing gene (Cai et al., 1994; Taccioli et al., 1994b). Parallel studies on the IR group 5 mutants showed that they lack DNA end-binding activity with properties identical to those of the Ku protein (Getts and Stamato, 1994; Rathmell and Chu, 1994). The ability of Ku80 cDNA but not Ku70 cDNA to complement the group 5 mutants and subsequent sequence analysis of some of the group 5 mutants (see below) verified Ku80 as the product of XRCCS (Boubnov et al., 1995; Errami et d., 1996; Singleton et al., 1997; Smider et al., 1994; Taccioli et al., 1994b). In 1991, Ku was recognized as a component of DNA-dependent protein kinase (DNA-PK), a kinase activity that is specifically activated by ds DNA ends due to their similar DNA binding properties (Dvir et al., 1992; Gottlieb and Jackson, 1993).Thus, Ku is the DNA targeting component of DNA-PK, and its cat-

Table 5.1. Rodent Cell Lines with Defects in DNA Double-Strand Break Repair Phenotype

IR group

Gene defective

4 5

xRcc4 Ku80IXRCCS

6

Ku70IXRCC6 DNA-PKCS xRcc7

7

Cell lines defective

XR-1 xrsl-7 XRV-15B; XRV-9B sxi-1 and 3 Ku80 KO ES cells Ku70 KO ES cells Mouse scid cell line

V(D)J recombination

DNA end-bindmg

DNA-PK activity

Deffective signal and coding joints Defective signal and coding joints

Normal Defective

Normal Defective

Defective signals and coding joints Defective coding; normal signal

Defective Normal

Defective Defective

Defective signal and coding

Normal

Defective

v-3 in-20 sx9, SXlO Equine scid cell line

5. DNA Breakage and Repair

193

alytic subunit is a 450-kDa protein termed DNA-PK catalytic subunit (DNAPKcs). Binding of Ku to ds DNA ends recruits DNA-PKcs and serves to activate the kinase activity. All the group 5 mutants also lack DNA-PK activity consistent with the predictions of these in v i m biochemical observations (Finnie et al., 1995). The identification of XRCC.5 as encoding one component of the DNAPK complex, therefore raised the possibility that IR complementation group 4 or 7, members of which display similar properties to the group 5 mutants, might define one of the other components of this complex, namely Ku70 or DNA-PKcs. In contrast to the group 5 mutants, electrophoretic mobility shift analysis (EMSA) of groups 4 and 7 mutants revealed parental levels of ds DNA end-binding activity. Significantly, however, group 7 mutants also lack DNA-PK activity and have undetectable or severely reduced levels of DNA-PKcs protein by Westem blot analysis (Blunt et al., 1995; Kirchgessner et al., 1995; Peterson et al., 1995). Verification that XRCC7 encodes DNA-PKcs was demonstrated by the ability of YACs encoding DNA-PKcs to complement the group 7 mutant V-3 and by the identification of a mutation in DNA-PKcs in the mouse scid cell line, another group 7 mutant (Blunt et al., 1995, 1996). These significant results therefore provided a function for DNA-PK as well as identifying three new genes involved in the cellular response to radiation and V(D)J recombination. Rodent mutants defective in Ku70 have not been isolated as radiosensitive mutants but Ku70 knock-out cell lines have now been constructed and display the anticipated radiosensitivity and V(D)J recombination defects (Gu et al., 1997). Shortly after the identification of XRCC.5 and -7, XRCC4, the gene defective in IR group 4, was cloned but, in this case, the DNA sequence provided little clue to the likely function of the encoded protein (Li et al., 1995). Recently, however, XRCC4 has been shown to be extremely tightly associated with the recently discovered DNA ligase IV. The two proteins copurify following a number of chromatographic steps, coimmunoprecipitate and interact in the yeast twohybrid system (Critchlow et al., 1997). Additionally, XRCC4 stimulates the ligation of DNA DSBs but not single-strand breaks by DNA ligase IV in a cell-free system (Grawunder e t al., 1997). These results, together with studies with defective yeast mutants (to be discussed in Section IV,B), suggest that XRCC4 functions as a cofactor for ligase IV and provide evidence for a role of DNA ligase IV in NHEJ.

B. Studies with yeast A second successful route for the identification of genes involved in DNA repair processes has been the isolation of mammalian homologs of yeast genes known to be involved in repair processes. The further characterization of genes and proteins involved in NHEJ demonstrates the benefits of characterizing both lower and higher eukaryotic systems. Until the identification of the role of DNA-PK in

194

P. A. Jeggo

mammalian cells, the role of NHEJ as a DSB repair mechanism in yeast was barely recognized. A S. cerevisiae Ku70 null mutant (Hdfl ) had been characterized and had a surprising temperature-sensitive phenotype for growth, but a defect in DSB repair had not been recognized (Feldmann and Winnacker, 1993). This was not surprising, since an hdfl mutant fails to display any y-ray sensitivity, but a subsequently constructed hdflrd.52 double-mutant showed greater y-ray sensitivity than a rad52 single-mutant, consistent with the notion that Ku operates in a DSB repair mechanism distinct from homologous recombination (Boulton and Jackson, 199613; Milne et al., 1996; Siede et al., 199613). More recently an assay was developed for NHEJ in yeast and Ku70 proved to be defective in this process (see Section VI1,A). A yeast Ku80 homolog has now also been identified based on its homology with the Caenorhabditis elegans and mammalian Ku80 proteins and null mutants proved to have a similar phenotype to hdfl (Boulton and Jackson, 1996a; Milne et al., 1996). Yeast homologs of DNA-PKcs and XRCC4 have not yet been reported. Tellp and meclp represent proteins closely related to DNA-PKcs and may fulfil an overlapping function. The inability to detect an XRCC4 homolog may be the result of poor sequence conservation between species and the fact that the important functional domains in XRCC4 have not yet been identified. A DNA ligase IV yeast homolog has recently been identified and null mutants display the anticipated y-ray sensitivity in a rad52 background and defective NHEJ (see Section VII,A for further details), consolidating the evidence described above that DNA ligase IV is involved in the NHEJ process (Schar et al., 1997; Teo and Jackson, 1997; Wilson et al., 1997). More recently, using the yeast two-hybrid system, Sir4 was identified as a protein interacting with Hdfl (Tsukamoto et al., 1997). Sir4 (silencing information regulator protein) is a regulatory factor in transcriptional silencing at telomeres and HM mating-type switching loci in yeast. Additional Sir proteins (Sirl,-2, and -3) have been identified. Sir2 and Sir3 are also necessary for silencing at telomeres but Sirl makes only a small contribution to silencing at HM loci and has no effect at telomeres. Significantly, sir2, -3, and -4, but not sirl , are yray sensitive in a rud52 background and have defects in an assay for NHEJ identical to the phenotype of the yeast hdfl mutant (see Section VI1,A). hdfl sir double mutants have the same level of defect as either isogenic single mutant. Thus Sir2, -3, and -4 but not Sirl are concluded to play a role in NHEJ. These proteins do not operate in HR. Other studies with yeast have shown that RadSO, Mrell, and Xrs2 are also involved in a process of homology-independent end-joining (Milne et al., 1996; Moore and Haber, 1996; Schiestl et al., 1994; Tsukamoto et al., 1996). rad50, mrel 1 , and ms2 mutants are defective in meiotic recombination but are proficient in mitotic recombination (Malone et al., 1990). They also have defects in their ability to repair DNA dsbs. They are able to carry out mating type switch-

Table 5.2. Genes Involved in NHEJ Gene

Mathod of identification

Reference

Mammalian cells Ku80 (XRCCS) Ku70 (XRCC6) DNA-PKCS(XRCC7) XRCC4 DNA figase IV

Defective rodent cell lines Analysis of KO cell lines and KO mice Analysis of KO cell lines and KO mice Defective rodent cell lines Defective rodent cell lines protein interacting with XRCC4 supportive evidence in yeast

Jeggo (1990) Nussenzweig et al. (1996); Zhu et al. (1996) Jeggo (1990) Li et al. (1995); Stamato et al. (1983) Critchlow et al. (1997); Grawunder (1997) Schar et al. (1997); Wilson et al. (1997); Teo (1997) Yeast

Ku70; Ku80 DNA ligase N sh2, -3, and -4

mad50 xrs-2

mrel I

null mutants y-ray sensitive in rad52 background and defective in NHEJ assay null mutants y-ray sensitive in rad52 background and defective in NHEJ assay sir 4 interacts with YKu70 null mutants y-ray sensitive in rad52 background and defective in NHEJ assay mutants defective in illegitimate recombination and/or NHEJ epistatic with KU70 for MMS sensitivity and NHEJ mutants defective in illegitimate recombination and/or NHEJ interacts with rad 50 mutants defective in illegitimate recombination and/or NHEJ interacts with rad 50

Boulton and Jackson (1996b) Boulton and Jackson (1996a); Milne et al. (1996) Teo and Jackson (1997); Wilson et al. (1997) Tsukamoto et al. (1997)

Milne et al. (1996); Moore and Haber (1996); Shiest1 et al. (1994); Tsukamoto et al. (1996a) Moore and Haber (1996); Tsukamoto et d. (1996a)

Moore and Haber (1996); Tsukamoto et al. (1996a)

196

P. A. Jeggo

ing but with delayed kinetics. In addition to these similar phenotypes Rad5O interacts with Mrel 1 and Xrs2, suggesting that they operate in the same pathway and possibly as a protein complex (Johzuka and Ogawa, 1995). These mutants therefore differ from but overlap with the RAD52 epistasis group of mutants in their phenotype and differ from the mutants defective in the two Ku subunits and sir2, -3 and -4 mutants. Nevertheless a r&OA mutant was shown to be epistatic with the Ku mutants for methyl methane sulphonate (MMS) sensitivity and for NHEJ, suggesting that they are operating in a Ku-dependent pathway (Milne et al., 1996). Additionally, rad50, mrell, and xrs2 have all been shown to be defective in illegitimate recombination or NHEJ using the same assays that exposed defects in hdfl and the sir mutants (Moore and Haber, 1996; Tsukamoto et al., 1996). Taken together, there is strong evidence showing that in yeast Ku70, Ku80, DNA ligase IV, and Sir2, -3, and -4 function in NHEJ and more indirect evidence implicating the involvement of Mrell, Rad50, and Xrs2 (Table 5.2). In mammalian cells there is strong evidence for the involvement of Ku70, Ku80, DNA-PKcs, XRCC4, and DNA ligase IV (Table 5.2).

V. CHARACTERIZATION OF PROTEINS INVOLVED IN NHEJ BY BIOCHEMICAL, MOLECULAR, AND GENETIC ANALYSIS A. Components of DNA-PK

1. Kuprotein As mentioned above, Ku comprises a 70- and 80-kDa subunit and acts as the DNA binding component of DNA-PK. Ku binds strongly to ds DNA ends, but in vitro studies have shown that it can also bind to gapped and nicked molecules and to closed DNA hairpins (Blier et al., 1993; Falzon et al., 1993). Consistent with these studies on DNA binding, the kinase activity of DNA-PK is also activated by similar structures (Morozov et al., 1994). These findings have led to the suggestion that Ku recognizes, and hence DNA-PK is activated by, ss-to-ds transitions in DNA. The DNA end-binding activity requires the presence and interaction of both Ku subunits (Griffith et al., 1992). Intriguingly, Ku has the ability to translocate along the DNA molecule to internal positions, forming a multimeric proteinDNA complex (Zhang and Yaneva, 1992). The ability of multiple Ku molecules to bind to a single ds DNA end may be important in the recruitment of additional proteins such as the Sir4 protein, which has been identified as playing a role in the NHEJ process on the basis of its ability to bind to Ku70 (Tsukamoto et al., 1997). Studies with the Ku80-defective cell lines support these biochemical findings and provide further information about the functioning of the DNA-PK complex. The defective cell lines display their major sensitivity to agents which

5. DNA Breakage and Repair

197

induce DNA DSBs, suggesting that the most significant interaction of Ku is with ds DNA ends (Jeggo, 1990). All the Ku80-defective mutants also lack Ku70 protein and Ku70 ES cell lines lack Ku80 protein, suggesting that in vivo the two subunits of Ku are only stable when coexpressed (Chen et al., 1996; Gu et al., 1997; Singleton et al., 1997). However the two subunits have been expressed separately in a Baculovirus expression system (On0 et d., 1994) indicating that the lack of stability in mammalian cells may reflect the proteolytic degradation of either subunit when not associated with the other. In contrast, Western blot analysis of Ku and DNA-PKcs in the defective cell lines shows that Ku is stable in the absence of DNA-PKcs and vice versa. Ku70 and -80 are encoded by cDNAs of 1.8 and 2.2 kb respectively (Reeves and Sthoeger, 1989; Yaneva et al., 1989). Neither DNA sequence contains any significant homology to other proteins or domains and the homology between species occurs throughout the length of the proteins without any obviously highly conserved regions. A leucine zipper motif has been reported but the sequence suggests that it would be unable to form an amphipathic-coiled coil structure, which is essential for the protein interaction function of a leucine zipper motif. Ku80 has been reported to have ATPase- and ATP-dependent helicase activity although neither a convincing ATP binding domain nor a helicase motif are evident (Cao et al., 1994; Tuteja et al., 1994). Site-directed mutations in putative ATP binding domains in Ku80 do not impair protein function in NHEJ (Singleton et al., 1997). Mutations in Ku80 have been identified in a number of group 5 mutants. The cDNAs of XR-V15B and XR-V9B have in-frame deletions of 46 and 84 amino acids respectively, and ms-4 has an out-of-frame deletion resulting in the loss of two exons (Errami et al., 1996; Singleton et al., 1997). These deletions probably arise from mutations in splice site sequences, which has been confirmed for xrs-4. xrs-6 has a 13-bp insertion near its 5’ terminus, again as a result of a mutational change in a splice site sequence located within an intron (Mizuta et d., 1996; Singleton et al., 1997). Two additional x r s mutants have very low transcript levels but the sequence appears to be wild type, suggesting that the mutational change may lie within regulatory sequences (Singleton et al., 1997). These results are important in confirming that Ku80 is the product of XRCC.5 but provide few clues to important functional sites within the protein. In v i m studies on sites required for Ku70/80 interaction using the yeast two hybrid system have suggested that a single domain is required for their interaction, but, in contrast, site directed mutational studies on Ku80 have shown that mutational changes over a large portion of the protein result in loss of a stable Ku70/80 interaction, suggesting that the two subunits require multiple sites for effective interaction (Wu and Lieber, 1996; unpublished observations from the laboratories of Jeggo and Jackson). Surprisingly a second transcript, designated KARP- 1, which overlaps with Ku80, has recently been described (Myung et al., 1997). The initiation

198

P. A. JegfiO

codon and two additional exons for KARP-1 lie upstream of the Ku80 initiation codon, resulting in a protein 9 kDa larger than Ku80. The KARP-1-specific sequences encode a leucine zipper motif, which does conform to the requirements to produce a likely functional coiled coil structure. Antisense constructs confer radiosensitivity to cells, and antibodies raised to the Karp- 1-specificregion, which do not cross-react with Ku80, neutralize DNA-PK activity in uitro. These results implicate the involvement of Karp-1 in a response to ionizing radiation. O n the other hand, Karp-1 appears to be primate specific even though NHEJ clearly operates in nonprimates.

2. DNA-PKCS DNA-PKcs has one of the largest cDNAs known and is a member of the phosphatidylinositol (PI) 3-kinase superfamily (Figure 5.3) (Hartley et d., 1995).This family of large proteins is characterized by the presence of a kinase domain at their 3’ terminus but can be divided into two subfamilies. One group, which include the PI 3- and PI 4-kinases, have lipid kinase activity, and a second group, termed PIK-related kinases, have protein rather than lipid kinase activity, although this has not been confirmed for all the proteins in this subgroup (Hunter, 1995; Keith and Schreiber, 1995). The lipid kinases tend to be smaller proteins, normally around 100 kDa, whereas the PIK-related kinases are generally greater than 200 kDa. In addition to the conserved kinase domain, this subgroup has a highly conserved region at their extreme 3’ terminus. A further subset of PIK-related kinases has another conserved domain, termed the RAD3 domain, just downstream of the kinase domain. Members of these families are shown in Figure 5.3 and include ATM, the gene defective in ataxia telangiectasia (A-T)cell lines, and other genes which function in cell cycle checkpoint control or other DNA damage response pathways. DNA-PK is a serine-threonine kinase and lacks detectable lipid phosphorylation activity. The large size of the DNA-PKcs cDNA has limited a structure/function mutational analysis but examination of defective rodent cell lines suggests that the highly conserved 3’ terminus is essential for kinase activity and is required for NHEJ. The DNA-PKcs cDNA has been sequenced from the mouse scid cell line and the only mutation present is a single base change creating an ochre stop codon at a site downstream of the PI 3-kinase domain, but upstream of the highly conserved 3’ extreme terminal region (the PIK-related region in Figure 5.3) (Araki e t al., 1997; Blunt et al., 1996; Danska et al., 1996). The resulting protein is truncated by 83 amino acids. Mouse scid fibroblast cells have very low levels of DNA-PKcs protein, indicating that this mutation decreases the protein’s stability. Analysis of different scid cell lines, some of which have higher residual DNA-PKcs levels, has shown that the mutant protein can bind to DNA but cannot function as a protein kinase (Danska e t al., 1996; our unpublished ob-

Sp Rad3p Dm Mei4l p Sc Mecl D PIK related H~ ATM kinases H~ ATR 200kDa Sc Tor1p Sc ToRp Hs FRAP HS DNA-PK

-

Hs P14Ka PI4 kinases Sc S7T4p Sc PIK1D

- lOOkDa

Hs ~ 1O1a HS p i 1op PI3 kinases H~ 106 sc vps34p

1 RAD3

f

kinase

PIK-related

protein kinase

I Lipid kinase activity

Lipid kinase activity (8-position on inositol rung)

Figure 5.3. Members of the PI 3-kinase superfamily and their structural relationship. Sp, Sacchurmyces pumbe; Em, DTosophih mehnogaster; Sc. Sacchuromyces cereuisiae; Hs, human.

200

P. A. Jeggo

servations). Irs-20 is another group 7 mutant which also has decreased protein levels compared with parental cells. Again, the residual protein can bind to DNA but is unable to function as a protein kinase. A mutation creating a single-residue change in the 3’ terminus has been identified, which is a likely candidate mutation, but unfortunately, only the terminal 1.3 kb of the cDNA has been sequenced and firm conclusions will have to await further sequencing (Priestley et al., in press). A SCID disorder has also been identified in Arabian foals and shown to result from a mutation in DNA-PKcs, yielding a protein truncated at residue 3155 upstream of the PI 3-kinase domain (Shin et al., 1997; Wiler et al., 1995). Thus the C-terminal region is inactivated in both alleles. It may be significant that equine scid cells have a more severe V(D)J recombination phenotype (resulting in loss of signal and coding joint formation (see Section VII,C)), compared with a “leaky”phenotype for mouse scid and irs-20 cells, both of which retain the PI 3kinase domain. Analysis of DNA-PKcs knock out mice and cell lines will be required to assess the scope of DNA-PKcs function. DNA-PKcs also possesses a leucine zipper motif, which may be important for interaction with Ku or additional proteins, and a n autophosphorylation site (Hartley et al., 1995). Autophosphorylation of DNA-PKcs, in contrast to Ku phosphorylation, has been suggested to control DNA-PK activity by affecting dissociation of DNA-PKcs from the Ku-DNA complex (Chan and Lees-Miller, 1996). Further discussion of DNA-PK activity is presented in Section VI.

B. XRCC4 and DNA ligase IV DNA ligase IV has a core ligase catalytic domain at its N-terminus, which displays a high level of sequence similarity with other DNA ligases (Wei et al., 1995). Additionally, there is a carboxy-terminus extension which possesses two tandem copies of a BRCT domain that was identified in the BRCAl breast cancer susceptibility protein and is present in a series of DNA repair and cell cycle checkpoint proteins (Callebaut and Momon, 1997). This extension is not found in other ligases with the exception of DNA ligase 111which has a single copy of a BRCT domain. Interaction studies of the different domains of DNA ligase IV have shown that it is the carboxy-terminal part of DNA ligase IV comprising the BRCTl domains that interacts with XRCC4 (Critchlow et al., 1997).

VI. DNA-PK AS A PROTEIN KINASE DNA-PK is unique as a protein kinase in being activated by the presence of ds DNA ends, thus potentially providing the cell with a signaling mechanism to re-

5. DNA Breakage and Repair

20 1

spond to DSBs in its DNA. In vitro DNA-PK can phosphorylate many DNA binding proteins including transcription factors such as Spl, c-Jun, and p53 (for additional proteins phosphorylated by DNA-PK, see Anderson, 1993). However most of these candidate proteins are phosphorylated in DNA-PK-defective mutants. Therefore although they might be phosphorylated by DNA-PK in vivo, it is unlikely that DNA-PK is the unique kinase executing their phosphorylation and that this represents the unique function of DNA-PK. Phosphorylation by DNAPK occurs most efficiently when the substrate is bound to the same DNA molecule as DNA-PK, suggesting that the relevant in vivo substrate might be a protein bound close to the DSB. Significantly, several components of the NHEJ process can be phosphorylated by DNA-PK in vitro, including the components of DNAPK itself and XRCC4. Moreover, autophosphorylation of DNA-PKcs results in loss of DNA-PK activity due to dissociation of the complex from DNA (Chan and Lees-Miller, 1996). Thus one function of the kinase activity may be regulation of the NHEJ process. DNA-PKcs has also been shown to interact with c-ah1 constitutively, and following irradiation this interaction is stimulated and an association of c-abl with Ku can also be seen (Kharbanda et al., 1997). C-ah1 can also phosphorylate DNA-PK and thereby inhibit its ability to associate with DNA. Conversely, DNA-PK can phosphorylate c-abl and thereby activate its kinase activity. However, c-abl also interacts with ATM, and A-T-defective cell lines also show decreased activation of c-abl following irradiation. The functional significance of these phosphorylation events is unclear but they raise the possibility that DNA-PK and ATM may converge at least for some aspects of their response. The groups 4,5, and 7 mutants are all able to arrest at cell cycle checkpoints showing that the unique function of DNA-PK is not to effect cell cycle arrest (Jeggo, 1985; Nussenzweig et al., 1996; Rathmell et d., 1997; Weibezahn et d., 1985). It cannot be ruled out, however, that DNA-PK may participate in a signal transduction pathway involved in cell cycle control but that this aspect of its function overlaps with other kinases such as the related ATM protein. Although the analysis of DNA-PKcs-defective mutants suggests that specific loss of the kinase activity results in loss of function in NHEJ (our unpublished observations), it cannot currently be determined whether the kinase activity of DNAPKcs represents the unique function or whether this large protein has some other role.

VII. THE MECHANISM OF NHEJ A. Studies with yeast Although the existence of NHEJ was identified in mammalian cells, yeast genetic systems have been exploited to provide further information and several plas-

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mid based assays have been established to examine the process. One of these is a recircularization assay, which involves the rejoining of restriction-cut plasmids. Rejoining by HR is precluded, as the plasmid does not bear any homology with yeast chromosomal sequences. Since the plasmid can replicate in yeast, the rejoining process can be monitored in the yeast cells by selection for a marker present on the plasmid, or alternatively, the rejoined plasmid can be rescued in Escherichia coli and the junctions sequenced. Using this assay, NHEJ is decreased around 10- to 20-fold in Ku70, Ku80, DNA ligase lV, and sir2, -3, and -4 mutant backgrounds (Boulton and Jackson, 1996a,b; Teo and Jackson, 1997; Tsukamoto et al., 1997; Wilson et al., 1997). The assay therefore monitors aprocess overlapping genetically with NHEJ in higher organisms. Significantly, however, some rejoining remains, suggesting the presence of a third Ku-independent rejoining process separate from SSA and HR. While wild-type yeast rejoin their breaks, accurately enabling the plasmid to be redigested with the restriction enzyme originally utilized to induce the break, the breaks rejoined in Ku mutants suffer deletions of varying sizes. The junctions in Ku mutants are frequently at the sites of short direct repeat sequences. These results suggest that, at least for restriction enzyme induced breaks, the Ku-dependent process repairs breaks accurately and that the Ku-independent mechanism is error prone. It could be argued, however, that in Ku-defective mutants the same rejoining mechanism operates albeit at reduced frequency and that the function of Ku is merely to protect the ds DNA ends. Interestingly, in the DNA ligase IV-defective mutants, in contrast to the Ku-defective mutants, some repair products are rejoined accurately, although many do suffer deletions. The accurate junctions could reflect the ability of DNA ligase I to substitute for DNA ligase IV in NHEJ or could indicate a distinct rejoining mechanism which requires Ku but not DNA ligase IV. The presence of deletions, however, substantiates the evidence for a Ku- and DNA ligase IV-independent errorprone rejoining mechanism. Similar studies using a vector containing the ADE2 gene on a URA3 selectable plasmid have given slightly different results and suggest that Ku and DNA ligase IV function in a microdeletion/insertion end-processing pathway as well as the accurate rejoining pathway identified above (Wilson et al., 1997). A further but slightly different assay monitoring “illegitimate” recombination in yeast has also shown a Ku dependence. This assay utilizes a yeast plasmid bearing both negatively and positively selectable markers. Selection for the presence of the plasmid, but loss of two of the markers, results in the selection of plasmids which harbor deletions. Analysis of the junctions has shown that rejoining occurs at short regions of homology, suggesting that the process monitors an “illegitimate” recombination process. The frequency of these deletion events is decreased in Ku70, Ku80, and the sir2, -3, and -4 mutants. It is not clear how this relates to the studies described above and why here Ku appears to be required for an errorprone (deletion-generating) repair mechanism (Tsukamoto et al., 1996, 1997).

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In addition to these studies with plasmid DNA, the rejoining of breaks induced in chromosomal DNA by the HO endonuclease at the MAT locus has shown that Ku can also function in the rejoining of chromosomal breaks. In this case, it is necessary to utilize a rad 524 background since the dominant mechanism for rejoining these breaks is Rad52-dependent HR (Milne e t al., 1996).

B. Studies with mammalian cells The integration of exogenous DNA during the process of DNA transfection in mammalian cells has been shown to occur by a mechanism that requires little or no homology (Roth e t al., 1985; Roth and Wilson, 1985, 1986). Significantly, therefore the rodent x r s mutants, which are defective in Ku80, have a 4-10-fold decreased transfection frequency consistent with a role of DNA-PK in NHEJ (Hamilton and Thacker, 1987;Jeggo and Smith-Ravin, 1989;Moore et al., 1986). However, this is a relatively modest decrease, especially considering the 1000 fold decrease in ability of these mutants to carry out V(D)J recombination (see Section VI1,C) and may indicate the existence of other DNA-PK-independent illegitimate recombination mechanisms. Several studies have also examined HR in the ms mutants and show that the absence of Ku80 does not impair the efficiency of this process. Two early studies examined extrachromosomal recombination between plasmids bearing nonoverlapping deletions in selectable markers (the guanine phosphoribosyl transferase gene (gpt) and the neomycin resistance gene). Recombination was scored by selection for stable transfectants that had integrated an intact gene. Thus, these studies require both an HR event as well as an integration event involving NH recombination. One study reported a 4-fold decrease in HR in xrs cells, while the other obtained parental HR frequencies (Hamilton and Thacker, 1987; Moore e t al., 1986). A more recent and elegant study has examined HR in a construct integrated into chromosomal sequences (Liang et al., 1996). This construct contained the 18-bp recognition site for the rare-cutting I-Scel endonuclease flanked by two incomplete fragments of the neoR gene. DSBs were introduced specifically at this site by cotransfection with plasmids encoding the I-Sce I endonuclease. Intrachromosomal HR between the fragments was monitored by selection for neoR clones. The xrs mutants yielded wildtype frequencies of HR in this assay. A novel system has also been designed to examine rejoining using short direct repeat sequences. A construct containing an I-Scel site flanked by short direct repeat sequences was integrated into chromosomal DNA such that rejoining utilizing the direct repeat sequences following the introduction of breaks at the IScel site reconstituted an intact neoR gene (Liang et d., 1996). The ms cells showed a dramatic decrease in the frequency of neoR colonies compared with parental cells. One explanation is that this reflects a role of Ku in this rejoining process, which resembles SSA, but an alternative explanation is the Ku helps to

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protect the ends and, in its absence, excessive degradation removes the repeat sequences thus preventing the rejoining process. Further studies are in progress to determine the precise role of Ku and whether the process is also DNA-PKcs dependent. Plasmid based assays for NHEJ, similar to those utilized in yeast, have so far failed to show a DNA-PK dependence. For example, the rejoining of restriction-cut plasmids monitored following their integration into chromosomal DNA or by rescue of the plasmids into E. coli occurs at parental frequencies in xrs and scid cell lines (Chang et al., 1993; Smith-Ravin and Jeggo, 1989). One study suggested that Ku plays a role in protecting the plasmid ends, but the rejoining process per se was unimpaired (Liang and Jasin, 1996). These results therefore contrast with those obtained with the yeast Ku mutants. One difference is that the plasmids utilized in mammalian cells are nonreplicating and therefore may not have the same higher-order structure of chromosomal sequences, which will be discussed in Section XI. Taken together these studies show that DNA-PK does not function in HR but does play a role in the nonhomologous integration of exogenous DNA sequences in mammalian cells. At present the studies on mammalian cells do not allow an assessment of whether the rejoining process occurs accurately or fiequently results in deletion formation.

C. NHEJ in V(D)J recombination V( D)J recombination is a site-specificrejoining process that occurs during development of the immune response and serves to enhance the diversity of the immunoglobulin and T cell receptor genes. For excellent reviews giving further details of this process see Alt et al. (1992), Gellert (1997), and Lewis (1994). Here I will focus only on the involvement of DNA-PK and the information it can provide concerning NHEJ. In germ line cells the variable (V), diversity (D), and joining (J) segments occur in tandem arrays and during T- and B-cell development are rearranged into an assortment of contiguous units. Each V, D, or J segment, termed the coding sequence, is associated with a partially conserved signal sequence (RSS) and the recombination process is initiated by a site-specific DSB at the junction of the RSS and coding sequence. Rejoining takes place between two such breaks to produce a coding junction at which two coding sequences have rejoined and a signal junction involving the two RSSs. The signal junctions are normally precise, whereas the coding junctions have specific modifications, involving the addition and deletion of nucleotides. To examine V(D)J recombination in cultured cells, plasmids encoding RAG1 and -2, two gene products essential for the process that are not expressed in somatic cells, are introduced into the cells by DNA transfection together with constructs bearing signal or coding sequences. Effective rejoining results in the activation of a reporter gene (the CAT gene) and

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the rearranged products are rescued and analyzed in E. coli. For further details of this assay see Hesse et al. (1987) and Taccioli et al. (1993). The groups 4,5, and 7 mutants are defective in their ability to carry out V(D)J recombination but there are important differences between them. Groups 4 and 5 mutants are 100- to 1000-fold defective in both signal and coding join formation and the junctions that form have large deletions at their termini (Pergola et al., 1993;Taccioli et al., 1993). Bona fide coding junctions have, in fact, not been recovered from group 5 mutants. In contrast, group 7 mutants show a major defect in coding join formation but only a minor (two- to fivefold) defect in signal join formation (Pergola et al., 1993; Taccioli et al., 1994a). (There are also additional differences in the modifications found at the coding junctions which will not be discussed here). Although it cannot be excluded that the ability of DNA-PKcs mutants to rejoin signal ends arises due to the leakiness of the mutants, the fact that it is shown by all three of the group 7 mutants so far examined suggests that it represents a difference in the requirement for Ku and DNA-PKcs in signal-versus-codingjoin formation. Recent in vitro studies on the early stages of the recombination process have confirmed that the initial cleavage reaction catalyzed by RAG1/2 produces blunt-ended signal termini, while the coding ends are formed as hairpin structures. It has therefore been suggested that a possible role of DNA-PK is to phosphorylate and activate a protein capable of resolving the hairpin structures (Roth et al., 1995). While this suggestion is plausible and interesting, it does not provide an explanation for the requirement of DNA-PK in the rejoining of radiation-induced breaks, which do not have hairpin structures at their termini.

VIII. THE CONTRIBUTION OF HR AND NHEJ TO DSB REJOINING IN YEAST VERSUS MAMMALIAN CELLS The exquisite sensitivity of yeast mutants defective in HR and the enhanced sensitivity to IR of haploid compared to diploid yeast attest to the major role of HR in the repair of DSBs in lower organisms (Saeki et al., 1980). Indeed, the contribution of NHEJ is only evident in a rad52 background or in situations where an undamaged homolog is not present. The majority of integration events in S. cerevisiae occur at sites of homology demonstrating the involvement of HR in this process (Orr-Weaver et al., 1981) and mating-type switching, which represents a developmentally site-specific recombination mechanism, also occurs by a HR mechanism. In contrast, NHEJ plays the dominant role in rejoining DBSs in mammalian cells, demonstrated in part by the marked radiation sensitivity of DNA-PK-defective cell lines. However, although earlier studies suggested that HR occurred infrequently, if at all, in mammalian cells (Rosenstauss and Chasin, 1978; Tarrant and Holliday, 1977),more recent studies have presented a different view. Homologs of the yeast RAD genes have been identified in higher organisms

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and are ubiquitously expressed (Kanaar et al., 1996; Muris et al., 1993; Shinohara et al., 1992). Assays for HR show that the process takes place at measurable frequencies in somatic cells, and RAD.54 knock-out cell lines display moderate radiosensitivity (Essers et al., 1997). Finally, XRCCZ and XRCC3, the genes defective in the ionizing radiation-sensitive mutants from groups 2 and 3 (irs-l and irslSF, respectively), both belong to the recA protein family and there is circumstantial and preliminary evidence to suggest that they may function in a recombinational repair mechanism (see, for example, Thompson, 1996). Both mutants show moderate y-ray sensitivity though less dramatic than that seen with the Ku defective mutants. The groups 4,5, and 7 mutants all display elevated y-ray sensitivity and ability to repair DNA DSBs in late S/G2 phase of the cell cycle (Jeggo, 1990; Whitmore et al., 1989). In this context it is maybe significant that irs-l cells do not show any change in resistance through the cell cycle and thus appear to lack the late S/G2-specific repair pathway (Cheong et al., 1994). One possibility,, therefore, is that the S/G2 response represents HR and that in mammalian cells the process is restricted to operate at a unique stage (S/G2) of the cell cycle. In conclusion, therefore, the mechanisms of NHEJ and HR appear to be conserved between species but the contribution of the two processes to overall DSB rejoining differs dramatically between yeast and mammalian cells (depicted in Figure 5.2). The selective pressures driving this change are unclear but it is noteworthy that yeast cells harboring yeast artificial chromosomes frequently undergo rearrangements at alu repeat sequences (Bennett et al., 1996), raising the possibility that HR may enhance genetic instability in an organism with extensive repetitive sequences.

IX. DNA-PK-DEFECTIVE MICE Mice homozygous for the scid (severe combined immunodeficiency) mutation were originally identified by their inability to rearrange correctly their immunoglobulin and T-cell receptor genes, resulting in severely reduced levels of mature T and B lymphocytes (Bosma et al., 1983).The defect is due to an inability to carry out V(D)J recombination, the process responsible for the assembly of the immunoglobulin and T-cell receptor genes (Bosma and Carroll, 1991). Subsequently, scid mice were found to have a defect in DSB rejoining and to be defective in DNA-PKcs (Biedermann et al., 1991; Blunt et al., 1995; Hendrickson et al., 1991; Kirchgessneretal., 1995; Petersonetal., 1995). The analysis ofscidmice has revealed considerable information about the consequences of defects in NHEJ (Bosma and Carroll, 1991). More recently, knock out Ku70 and Ku80 mice have provided further insight (Nussenzweig et al., 1996; Zhu et al., 1996). The Ku80, Ku70, and scid mice all display a profound severe combined immunodeficiency

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(SCID) phenotype which represents the most significant consequence of a lack of NHEJ. Some mature T and B cells develop in all these mice, indicating that an alternative rejoining process can operate during V( D)J recombination. Here, however, I will not focus on the immunological details of these animals. Scid mice are fertile, develop normally, and have only a very low elevated frequency of spontaneous tumors (Custer et al., 1985). In contrast, Ku80 mice are severely growth retarded and show premature ageing (Nussenzweig et al., 1996; Zhu et al., 1996). Scid mice have a small elevated frequency of thymic tumors following radiation exposure (Bosma and Carroll, 1991), but the response of Ku mice to such treatment has not yet been reported. The difference between the scid and Ku mice most probably reflects function(s) of Ku that is independent of its role as a component of DNA-PK but could also be attributed to some remaining function carried out by the residual mutant DNA-PKcs present in the scid cells. The analysis of DNAPKcs knock-out mice, which are currently being constructed, will therefore be of interest. Yeast disrupted for Ku80 and Ku70 contain shortened telomeres (Boulton and Jackson, 1996a; Porter et al., 1996) and it has therefore been proposed that Ku functions in the maintenance of telomere length. While this phenotype is observed for both Ku70 and Ku80 yeast mutants, it is not a property displayed by the yeast DNA ligase IV mutants, suggesting that it is a consequence of a loss of Ku function rather than loss of NHEJ and that Ku has a function beyond its role in NHEJ. The retarded growth and premature ageing features which are unique to the Ku knock out mice may also reflect a role for Ku separate from its function in NHEJ and the potential role in telomere maintenance is an obvious and interesting possibility (Nussenzweig et al., 1996).

X. RADIOSENSITIVITY AND IMMUNE DEFICIENCY A-T is a human immunodeficiency disorder with a variety of associated features including clinical radiosensitivity and cell lines derived from these patients display pronounced radiosensitivity (Taylor et al., 1996). The ATM gene is a member of the PI 3-kinase superfamily and thus a close relative of DNA-PKcs (Savitsky et al., 1995). In yeast, tellp is the likely structural homolog of ATM, but, based on sequence comparison, it is also one of the closest homologs identified for DNAPKcs (Greenwell et al., 1995; Keith and Schreiber, 1995). Tellp plays a role in telomere maintenance, a function that is also ascribed to the Ku protein in yeast (Boulton and Jackson, 1996a; Greenwell et al., 1995). Another set of related proteins are the homologs Rad3p, Mei4lp, and Meclp that operate in checkpoint control but might also function in a DNA repair pathway (Bentley et al., 1996; Hari et al., 1995; Siede et al., 1996a). It is possible, therefore, that these proteins have shared or overlapping functions, which may differ between yeast and mam-

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malian cells. There are some additional intriguing overlaps between A-T- and DNA-PK-defective cell lines. Ten percent of T lymphocytes from A-T patients show translocations and inversions which frequently involve TCR genes (Taylor e t al., 1996). Although A-T cells do not have the same magnitude of defect in DSB rejoining as the DNA-PK defective cell lines, a small fraction of breaks remain unrejoined in all A-T cell lines analyzed, and the fraction of unrejoined breaks correlates with the level of radiosensitivity (Foray et d., 1995; Foray et al., 1997). Additionally, neither A-T nor the NHEJ defective cell lines show any repair of potentially lethal damage (RPLD), a recovery process that is observed when cells are irradiated and held for a period under nongrowing conditions prior to plating in growth medium (Arlett and Priestley, 1984; Iliakis and Okayasu, 1990; Thacker and Stretch, 1985). A-T cells differ significantly from the groups 4, 5, and 7 mutants in displaying pronounced defects in their ability to arrest at cell cycle checkpoints (Jeggo, 1990; Kastan et al. 1992; Nussenzweig et d. 1996; Rathmell et al., 1997; Weibezahn e t d., 1985). However, there is evidence to s u g gest that A-T cells have both a DNA repair and checkpoint defect (Thacker, 1994;Jeggo et al., submitted). The marked SCID phenotype of DNA-PK defective mice raised the possibility that such defects may contribute to human SCID disorders. Although the majority of scid patients have defects in adenosine deaminase (ADA), a small percentage are ADA proficient. Cell lines derived from a significant percentage of these patients are radiosensitive but surprisingly those examined to date are proficient in their ability to rejoin DNA DSBs (CavazzanaCalvo et d., 1993; Nicolas et al. 1996; Sproston et al., 1997). These patients therefore represent a new radiosensitive disorder overlapping with immunodeficiency. In addition to these cell lines, we have characterized two further radiosensitive lines from patients with forms of immunodeficiency, though not T-B- scid. Nijmegen Breakage Syndrome (NBS) represents a further disorder in which these two phenotypes overlap (van der Burgt et al., 1996). These results therefore expose a surprisingly complex overlap between DNA repair and the development of the immune response likely representing the involvement of multiple gene products in both pathways. One possibility is that these cell lines are defective in additional gene products operating in NHEJ which do not impinge upon the efficiency of the process but, for example, its fidelity. Intriguingly, studies have suggested that A-T cells may be defective in the fidelity of DNA rejoining (Cox et d., 1986; Debenham et al., 1988; Ganesh et al. 1993). Alternatively, there may be other repair processes which are utilized during immune development.

XI. A MODEL FOR NHW Two intriguing features of NHEJ need to be considered when evaluating the mechanism. First, in mammalian cells restriction-cut plasmid DNAs can be re-

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Figure 5.4. Model of NHEJ. (Step 1 ) Ku binds to the DNA ends and recruits DNA-PKcs thus activating DNA-PK activity. (Step 2) Ku translocates along the DNA molecule and recruits Sir2,-3 and -4. This causes chromatin condensation and decreases accessibility of the DNA ends. (Step 3) Additional factors bind to Sir2,-3, and -4 resulting in chromatin decondensation and facilitating the rejoining by XRCC4/DNA ligase IV. The role of DNAPK is unclear but may regulate the binding/release of Ku to DNA ends and other proteins during the process.

joined both in v i m and in vivo by a DNA-PK independent process (Chang et al., 1993).One possibility is that the role of DNA-PK is therefore connected with the higher-order structure of chromosomal DNA, and the involvement of the Sir proteins adds fuel to this speculation. Second, DNA-PKcs is required for the rejoining of radiation-induced breaks but is only required for coding join formation and not for signal join formation in V(D)J recombination. Thus, DNA-PKcs appears to play a somewhat different role to Ku in the process. A model for NHEJ which attempts to incorporate these features is outlined in Figure 5.4. The ability of Ku to bind to ds DNA ends suggests that it is a likely first step in the DSB repair process. Ionizing radiation-induced breaks are likely to have damaged bases at their termini and V(D)J coding ends arise as hairpin structures, but in vim0 studies suggest that Ku is able to bind to these type of ends. The second step may then involve Ku’s translocation along the DNA and association with Sir2, -3, and -4 via Sir45 interaction with Ku. This complex formed along the DNA alters the higher-order chromatin structure enhancing chromatin con-

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densation. This may serve several functions, including the prevention of nucleolytic degradation of the ends, the inhibition of transcription in the vicinity of the break, holding the ends in a tight complex preventing nonspecific rejoining to other broken ends, and enhancing the rejoining of the correct DNA ends. However, by making the DNA less accessible it will also hinder the repair of the DNA DSB. Recently, another protein termed DISl has been reported to interact with Sir4 and to modify the silenced chromatin structure (Zhang and Buchman, 1997). It is therefore possible that DISl or a similar protein may interact with the Sir/Ku complex to enhance the accessibility of the condensed chromatin to repair enzymes and specifically to XRCC4/DNA ligase IV. The ability to condense and decondense chromatin during the process may be important to achieve initially the arrest of ongoing metabolic processes and to prevent aberrant recombination events and, subsequently, to allow the repair process to proceed. The binding of DNA-PKcs to the Ku/DNA complex and activation of DNA-PK can occur independently of the interaction of Ku with the Sir proteins and may or may not precede it. The function of the kinase activity is another uncertain feature of the process. One interesting possibility is that it phosphorylates components of the NHEJ machinery and helps to drive the change from a condensed to a noncondensed state. Autophosphorylation may additionally causes release of the complex from DNA when the ligation step is complete. It is also possible that DNA-PKcs acts as a framework for recruiting proteins involved in the modificationlrepair of the ends or impinges upon these processes by preventing the switch from a condensed to an DNA open structure. The signal ends formed during V(D)J recombination do not require any form of processing and may therefore be rejoined in the absence of DNA-PKcs.

For many years it has been evident that mammalian cells differ dramatically from yeast and rejoin the majority of their DNA DSBs by a nonhomologous mechanism, recently termed NHEJ. In the last few years a number of genes and proteins have been identified that operate in the pathway providing insights into the mechanism. These proteins include the three components of DNA-PK, DNA ligase IV, and XRCC4. In yeast Sir2, -3, and -4 proteins are also involved in the process and therefore are likely to play a role in higher organisms. Studies with yeast suggest that NHEJ is an error-free mechanism. Although the process is far from understood, it is likely that the DNA-PK complex or Ku alone acts in a complex with the Sir proteins possibly protecting the ends and preventing random rejoining. Further work is required to establish the details of this mechanism and to determine whether this represents an accurate rejoining process for a complex break induced by ionizing radiation. It will be intriguing to discover how the cell

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achieves efficient and accurate rejoining without the use of homology. Interactions between the components of DNA-PK and other proteins playing a central role in damage response mechanisms are beginning to emerge. Interestingly, there is evidence that DNA repair and damage response mechanisms overlap in lower organisms. The overlapping defects of the yeast Ku mutants, tell mutants, and AT cell lines in telomere maintenance further suggest overlapping functions or interacting mechanisms. A challenge for the future will be to establish how these different damage response mechanisms overlap and interact.

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Antisense mRNA, protein synthesis theory, 91 Ataxias, see Hereditary ataxias ATM gene, DNA repair mechanisms, 207-208 Autosomal dominant progressive ataxias, 3 4 4 9 classification, 35 clones, 40-47 SCAJ gene, 4 0 4 3 SCA2 gene, 4 3 4 4 SCA3 gene, 4 4 4 6 SCA6 gene, 4 6 4 7 epidemiology, 49 Machado-Joseph disease, 44-46 mapped genes, 4 7 4 9 SCA4 gene, 47-48 SCAS gene, 48 SCA7 gene, 48-49 polyglutamine proteins, 3 9 4 0 triplet repeat expansions, 35-39

Biological rhythms, see Chronogenetics Bobbed gene, rDNA gene mutations, 83-84 Body plan genes, human malformation body axes establishment, 3-16 domains, 3 dorsoventral patterning, 14-16 floor plate role, 14-15 forebrain region, 3-7 Hox gene cluster, 8-14 isthmic region, 7-8 midbrain region, 3-7 notochord role, 14-15 rhombospinal region, 8-14 rostrocaudal patterning, 3-14 sonic hedgehog gene, 15-16 limb patterning, 16-21 homeobox genes, 18-21 limb development, 16 secreted molecules, 16-18 ocular anlage patterning eye formation, 21 homeobox genes, 21-22 overview, 1-3

tooth anlage patterning homeobox genes, 23 tooth development, 22-23 Brain behavior, biological rhythm studies, 163-1 67 Brain patterning, homeobox genes, rostrocaudal axis, 3-7

CAG triplet repeat expansions, autosomal dominant progressive ataxias, 35-39 Chronogenetics arrhythmic phenotypes, 149-157 brain-behavioral studies, 163-167 clock-resetting signals, 171-172 courtship song cycle, 159-160 egg-to-adult development, 161 environmental synchronization, 142-145 fundamentals, 135-142 long-period phenotypes, 146-148 non-temporal phenotypes, 161-163 ovarian diapause, 158-159 overview, 135-136, 173 period gene expression, 163-168, 172-173 short-period phenotypes, 148-149 visual rhythms, 157-158 Circadian rhythms, see Chronogenetics Cloning, see Autosomal dominant progressive ataxias, clones; Transposon cloning method Courtship song cycle, Drosophih rhythm genetics, 159-160

Deformation genetics, see Human malformation genes Development, see specific types DNA break formation, 186-188 [DNA gene mutations bobbed gene, 83-84 mini gene, 83

219

220

Index

DNA (cont.) repair mechanisms, 185-21 1 DNA-protein kinase characteristics, 200-201 components, 196-200 defective mouse models, 206-207 DNA ligase IV, 200 Ku protein, 191, 196-198 repair model, 208-210 mcc4,200 double-stranded break repair, 188-190, 205-206 homologous recombination, 188-190, 205-206 immune deficiency, 207-208 nonhomologous end-joining DNA ligase IV, 200 DNA-protein kinase components, 196-200 Ku protein, 191, 196-198 mammalian cell studies, 191-193, 203-206 mechanisms, 190 model, 208-2 10 variablediversity-joining recombination, 204-205 XRCC4,194,200 yeast studies, 193-196,201-203,205 overview, 186,208-211 radiosensitivity,207-208 single-strandedbreak annealing pathway, 190 DNA ligase IV, DNA break repair mechanisms, nonhomologous end-joining, 200 DNA-protein kinase, DNA break repair mechanisms characteristics, 200-201 components, 196-200 defective mouse models, 206207 DNA ligase IV, 200 Ku protein, 196-198 repair model, 208-210 xRcc4,200 Dorsoventral patterning, human malformation genes floor plate role, 14-15 notochord role, 14-15 sonic hedgehog gene, 15-16 Drosophila biological rhythm genetics arrhythmic phenotypes, 149-157

brain-behavioralstudies, 163-167 clock-resettingsignals, 171-1 72 courtship song cycle, 159-160 egg-to-adult development, 161 environmental synchronization, 142-145 fundamentals, 135-142 long-period phenotypes, 146-148 non-temporal phenotypes, 161-163 ovarian diapause, 158-159 overview, 135-136, 173 period gene expression, 163-168,172-173 short-periodphenotypes, 148-149 visual rhythms, 157-158 gene models, see specifi genes

Ecdysone hypothesis, minute genes, 78-79 Emu genes, brain patterning role, rostrocaudal axis, 3-7 End-joining technique, see Nonhomologous end-joining Eye development, human malformation, ocular anlage patterning eye formation, 21 homeobox genes, 21-22

Floor plate, dorsoventral patterning, human malformation genes, 14-15 Frataxin gene, see Friedreich ataxia Friedreich ataxia, 49-60 clinical features, 5 1-52 disease mechanisms, 57-58 frataxin gene GGA triplet repeat expansion, 55-57 point mutations, 54-55 frataxin protein, 53-54 gene characteristics, 52-53 pathology,50-51 phenotype-genotype correlations, 58-60 fs(3JO2729 gene, protein synthesis theory, 118-119

GGA triplet repeat expansion, Friedreich ataxia association, 55-57

Hereditary ataxias autosomal dominant progressive ataxias, 34-49

22 1

Index classification, 35 clones, 40-47 SCAJ gene, 40-43 SCA2 gene, 43-44 SCA3 gene, 44-46 SCA6 gene, 46-47 epidemiology, 49 Machado-Joseph disease, 4 4 4 6 mapped genes, 47-49 SCA4 gene, 47-48 SCA.5 gene, 48 SCA7 gene, 48-49 polyglutamine proteins, 39-40 triplet repeat expansions, 35-39 Friedreich ataxia, 49-60 clinical features, 51-52 disease mechanisms, 57-58 fraraxin gene GGA triplet repeat expansion, 55-57 point mutations, 54-55 frataxin protein, 53-54 gene characteristics, 52-53 pathology, 50-51 phenotype-genotype correlations, 58-60 overview, 3 1-34 Homeobox genes, see also specific types human malformation brain patterning, rostrocaudal axis, 3-7 limb patterning, 18-21 ocular anlage patterning, 21-22 tooth anlage patterning, 23 Homologous recombination, DNA break repair mechanisms, 188-190,203-206 Hox genes gene expression, 8-10 gene structure, 8-10 limb development, 18-21 mutations, 10-13 regulation, 13-14 rhombospinal region patterning, rostrocaudal axis, 8-14 Human malformation genes body axes establishment, 3-16 dorsoventral patterning floor plate role, 14-15 notochord role, 14-15 sonic hedgehog gene, 15-16 rostrocaudal patterning, 3-14 domains, 3 forebrain region, 3-7 Hox gene cluster, 8-14

isthmic region, 7-8 midbrain region, 3-7 rhombospinal region, 8-14 limb patterning, 16-21 homeobox genes, 18-21 limb development, 16 secreted molecules, 1 6 1 8 ocular anlage patterning eye formation, 21 homeobox genes, 21-22 overview, 1-3 tooth anlage patterning homeobox genes, 23 tooth development, 22-23

Immune deficiency, DNA repair mechanisms, radiosensitivity, 207-208 Isthmic region patterning, rostrocaudal axis,

7-8

Joseph-Machado disease, autosomal dominant progressive ataxia role, 44-46

Ku protein, DNA break repair mechanisms, nonhomologous end-joining, 191,

196-198

Limb patterning, human malformation genes,

16-21 homeobox genes, 18-21 limb development, 16 secreted molecules. 16-18

MachadeJoseph disease, autosomal dominant progressive ataxia role, 44-46 Malformation genes, see Human malformation genes; specific types M(I) genes, protein synthesis theory M(I)JB gene, 91 M(J)7C gene, 102-106,120-121 M(J)15Dgene, 101-102

222 M(2) genes, protein synthesis theory M(2)23B gene, 108-109 M(2)30D/E gene, 93-95 M(2)32A gene, 106-108 M(2)32D gene, 109-1 10 M(2)60E gene, 114-1 15 M(3) genes, protein synthesis theory M(3)95A gene, 95-97 M(3)95A@"" gene, 97-101 M(3)66D gene, 110-1 14 M(3)99D gene, 90 M(3)80 gene, 117-1 18 M(3)Q-III gene, 117-118 Mini gene, rDNA gene mutations, 83 Minute genes development studies, 84-88 mitotic recombination cell competition, 85-86 maternal effects, 86 parameters, 85 suppressor function, 86-88 future research directions, 122-124 historical perspectives, 76-84 ecdysone hypothesis, 78-79 minute phenotype, 76-77 minute reaction, 76-78 minute-tRNA hypothesis, 81-82 oxidative phosphorylation hypothesis, 79-81 rDNA gene mutations bobbed gene, 83-84 mini gene, 83 overview, 70-76, 122-124 protein synthesis theory, 88-122 molecular research, 89-92 antisense mRNA, 91 M(1)JB gene, 91 M(3)99D gene, 90 Rp49 gene, 89-91 RpL32 gene, 90-91 RpL36 gene, 91 non-minute phenotype ribosomal protein mutations, 118-1 19 nonribosomal protein genes, 121-122 recessive minutes, 116-118 M(3)80 gene, 117-118 M(3)Q-III gene, 117-1 18 short-bristle gene, 117 ribosomal protein genes, 119-121 transposon clone method, 92-1 16

Index M(1)7C gene, 102-106 M(1)ZSD gene, 101-102 M(2)23B gene, 108-109 M(2)30D/E gene, 93-95 M(2)32A gene, 106-108 M(2)32D gene, 109-1 10 M(2)60E gene, 114-115 M(3)66D gene, 110-114 M(3)95A gene, 95-97 M(3)95Am" gene, 97-101 oho23B gene, 108-109 RpL9 gene, 109-110 RpL14gene, 110-114 RpL19gene, 114-115 RpS2 gene, 93-95 RpS3 gene, 95-97 RpS3m" gene, 97-98 R P S ~ @ *gene, ~ 98-101 RpSSgene, 101-102 RpS6 gene, 102-106 RpS13 gene, 106-108 string of pearls gene, 93-95 stubarista gene, 115-1 16 Mitotic recombination, minute genes cell competition, 85-86 maternal effects, 86 parameters, 85

Nonhomologous end-joining, DNA repair mechanisms DNA ligase IV, 200 DNA-protein kinase components, 196-200 Ku protein, 191, 196-198 mammalian cell studies, 191-193, 203-206 mechanisms, 190 repair model, 208-210 variable-diversity-joining recombination, 204-205 XRCC4, 194,200 yeast studies, 193-196, 201-203, 205 Notochord, dorsoventral patterning, human malformation genes, 14-15

Ocular anlage patterning, human malformation genes eye formation, 21 homeobox genes, 21-22

223

Index Otx genes, brain patterning role, rostrocaudal axis, 3-7 Ovarian diapause, Drosophila biological rhythm genetics, 158-159 Oxidative phosphorylation hypothesis, minute genes, 79-81

Pax genes eye adage patterning, 21-22 isthmic region development, 7-8 Period gene, chronogenetics regulation arrhythmic phenotypes, 149-157 brain-behavioral studies, 163-167 clock-resetting signals, 17 1-1 72 courtship song cycle, 159-160 egg-to-adult development, 161 environmental synchronization, 142-145 expression, 163-168, 172-173 fundamentals, 135-142 long-period phenotypes, 146-148 non-temporal phenotypes, 161-163 ovarian diapause, 158-159 overview, 135-136, 173 short-period phenotypes, 148-149 visual rhythms, 157-158 Phosphorylation hypothesis, minute genes,

79-81 Polyglutamine proteins, autosomal dominant progressive ataxias association, 3 9 4 0 Progressive ataxias, see Autosomal dominant progressive ataxias Protein, see specific types Protein kinase, see DNA-protein kinase Protein synthesis theory, minute genes, 88-

122 molecular research, 89-92 antisense mRNA, 91 Rp49 gene, 89-91 RpL36 gene, 91 non-minute phenotype ribosomal protein mutations, 118-1 19 nonribosomal protein genes, 121-122 recessive minutes, 116-1 18 M(3)80 gene, 117-1 18 short-bristle gene, 117 ribosomal protein genes, 119-121 RpS4 gene, 119-120 RpS14 gene, 120-121

transposon clone method, 92-1 16 M(1)7C gene, 102-106

M(1)15Dgene, 101-102 M(2)23B gene, 108-109 M(2)30D/E gene, 93-95 M(2)32A gene, 106-108 M(2)32D gene, 109-1 10 M(2)60E gene, 114-115 M(3)66Dgene, 110-114 M(3)95A gene, 95-97 M(3)95Apv I gene, 97- 101 oho23B gene, 108-109 RpL9 gene, 109-1 10 RpL14gene, 110-114 KpL19 gene, 114-115 RpS2 gene, 93-95 KpS3 gene, 95-97 RpS3w" gene, 97-98 RpS3pu99gene, 98-101 KpS.5 gene, 101-102 RpS6 gene, 102-106 RpS13 gene, 106-108 string ofpearls gene, 93-95 stubarista gene, 115-1 16

Radiosensitivity, immune deficiency role, DNA repair mechanisms, 207-208 Recombination homologous recombination, DNA break repair mechanisms, 188-190,203-

206 mitotic recombination, minute genes cell competition, 85-86 maternal effects, 86 parameters, 85 variable-diversity-joining recombination, DNA break repair mechanisms break formation, 186-188 nonhomologous end-joining, 204-205 Repeat expansions, see Triplet repeat expansions Retinoic acid, Hox gene regulation, 14 Rhombospinal region patterning, rostrocaudal axis, Hox genes role, 8-14 gene expression, 8-10 gene structure, 8-10 mutations, 10-13 regulation, 13-14

224

Index

Ribosomal protein genes, protein synthesis the. ory

Rp49 gene, 89-91 RpL genes RpL9 gene, 109-110 RpLJ4 gene, 110-114 RpLJ5 gene, 118-1 19 RpLJ 9 gene, 114-1 15 RpL32 gene, 90-91 RpL36 gene, 91 RpS genes RpS2 gene, 93-95 RpS3 gene, 95-97 RpS3m" gene, 97-98 RpS3mq9gene, 98-101 RpS4 gene, 119-120 RpS5 gene, 101-102 RpS6 gene, 102-106 RpSJ3 gene, 106-108 RpSJ4 gene, 120-121 RNA antisense mRNA, protein synthesis theory,

91 tRNA, minute gene hypothesis, 81-82 Rostrocaudalpatterning, human malformation genes, 3-14 domains, 3 forebrain region, 3-7 Hox gene cluster, 8-14 isthmic region, 7-8 midbrain region, 3-7 rhombospinal region, 8-14

SCA genes, autosomal dominant progressive ataxias clones SCAJ gene, 40-43 SCA2 gene, 43-44 SCA3 gene, 44-1.6 SCA6 gene, 4647 mapped genes SCA4 gene, 47-48 SCA5 gene, 48 SCA7 gene, 48-49 triplet repeat expansions, 35-39 Short-bnstfe gene, protein synthesis theory, 117 Song cycles, Drosophila rhythm genetics,

159-160 Sonic hedgehog gene, human malformation role

dorsoventralpatterning, 15-16 limb patterning, 17-18 Spinal development, see Body plan genes String of pearls gene, protein synthesis theory,

93-95 S t h r i s t a gene, protein synthesis theory,

115-116

Tooth anlage patterning, human malformation genes homeobox genes, 23 tooth development, 22-23 Transposon cloning method, minute genes, protein synthesis theory, 92-1 16 M(J)7C gene, 102-106

M(J)J5Dgene, 101-102 M(2)23B gene, 108-109 M(2)30DlE gene, 93-95 M(2)32A gene, 106-108 M(2)32D gene, 109-1 10 M(2)60E gene, 114-115 M(3)66D gene, 110-1 14 M(3)95A gene, 95-97 M(3)95Am" gene, 97-101 oho23B gene, 108-109 RpL9 gene, 109-110 RpLJ4 gene, 110-1 14 RpL19 gene, 114-115 RpS2 gene, 93-95 RpS3 gene, 95-97 RpS3m" gene, 97-98 RpS3M9 gene, 98-101 RpSSgene, 101-102 RpS6 gene, 102-106 RpSJ3 gene, 106-108 smng of pearls gene, 93-95 stubarista gene, 115-1 16 Triplet repeat expansions autosomal dominant progressive ataxias, CAG repeat, 35-39 Friedreich ataxia, GGA repeat, 55-57 tRNA, minute gene hypothesis, 81-82

Variable-diversity-joining recombination, DNA break repair mechanisms break formation, 186-188 nonhomologous end-joining, 204-205

Index

225

Visual rhythms, Drosophih chronogenetics, 157-158

Yeast, DNA break repair studies, nonhomologous end-joining, 193-196,201-203,205

XRCC4, DNA break repair mechanisms, nonhomologous end-joining, 194, 200

Ziti gene, protein synthesis theory, 118-1 19 Zone of polarizing activity, limb development role, 16-18

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