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Suppressors of position-effect variegation and the cdc2Dm gene in Drosophila Melanogaster Clegg, Nigel 1992

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SUPPRESSORS OF POSITION-EFFECT VARIEGATION AND THE cdc2Dm GENE IN DROSOPHILA MELANOGASTER By NIGEL JOHN MIATT CLEGG B.Sc.(Hon.)/ Queen's University, 1983 M.Sc, Queen's University, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1992 © Nigel John Miatt Clegg, 1992 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of t&3^<3Çl \ The University of British Columbia Vancouver, Canada Date 2 3 / 4 / 9 ^ DE-6 (2/88) ABSTRACT Suppressor of Position-Effect-Variegation (Su(var)) mutations i n Drosophila melanogaster are believed to i d e n t i f y genes that p a r t i c i p a t e i n the establishment or maintenance of heterochromatic domains. A cytogenetic analysis of region 31 of the Drosophila melanogaster polytene chromosome map was undertaken to c l a r i f y the number and p o s i t i o n of several previously i d e n t i f i e d Su(var) mutations. Ten d e f i c i e n c i e s were used to divide the 31 region i n t o 15 separate subintervals. Results of t h i s analysis suggest that there i s a s i n g l e Su(var) locus (Suvar(2)1) i n the 31A-B region. Two recessive suppressors of p o s i t i o n - e f f e c t variegation reside i n the 31E-32A region. A fourth locus, Su(var)216, was positioned i n region 3IE. Additional mutations were sought throughout the 31 region. In t o t a l , one hundred and twenty-one new EMS, gamma-i r r a d i a t i o n , and P element induced mutations were tested f o r complementation and mapped using d e f i c i e n c i e s . None of the mutations had a Su(var) phenotype, but 8 a l l e l e s f a i l e d to complement the l e t h a l phenotype associated with the Su(var)216 chromosome. A P element induced a l l e l e of Su(var)216 was cloned and sequenced. The P i s adjacent to cdc2Dm, the Drosophila homologue of the f i s s i o n yeast cdc2 gene. The kinase encoded by cdc2 i s required f o r proper progression through the c e l l c y c l e . The l e t h a l phenotype of Su(var)216 can be rescued by an i i e c t o p i c a l l y placed cdc2Dm gene construct; however, the Su(var) phenotypes are not rescued. Deficiency mapping of Su(var)216 with a cdc2Dm gene construct i n the genetic background suggests that the Su(var)216 and cdc2Dm mutations may be t i g h t l y linked (<0.5 cM) but separable. Six EMS induced missense mutations of cdc2Dm were sequenced. One mutation, cdc2E1~*, i s located within the PSTAIR sequence of cdc2Dm, a region believed to i n t e r a c t with c y c l i n s . A second mutation, cdc2D5? i s within a region highly conserved amongst kinases. Hemizygous cdc2D5? mutants die as embryos i f they i n h e r i t the mutation from t h e i r mothers, but die as larvae i f they i n h e r i t the mutation from t h e i r fathers. Most of the other mutant a l l e l e s of cdc2Dm die predominantly during the pupal stage. i i i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES i x LIST OF BIOCHEMICAL ABBREVIATIONS x i LIST OF GENETIC ABBREVIATIONS x i i ACKNOWLEDGEMENTS x i v GENERAL INTRODUCTION 1 CHAPTER 1 - LITERATURE REVIEW A. Euchromatin and Heterochromatin 5 B. P o s i t i o n - e f f e c t Variegation 7 General Genetics and Cytology 7 The Timing of Po s i t i o n - e f f e c t Variegation 11 Molecular Aspects of P o s i t i o n - e f f e c t Variegation 13 Modifiers of Po s i t i o n - e f f e c t Variegation 15 C. Suppressors and Enhancers of P o s i t i o n - e f f e c t Variegation 17 Cloned Genes 21 CHAPTER 2 INTRODUCTION 25 MATERIALS AND METHODS 31 Mutant s t r a i n s 31 Culture conditions 32 Eye pigment assessment 32 Cytology 34 Ethyl methanesulphonate (EMS) mutagenesis 34 i v Hybrid dysgenesis screen 34 Deficiency mapping and complementation te s t s 36 Lethal phase analysis 38 RESULTS Cytogenetics 39 Genetic Screens 47 General Complementation 48 Suppressor Loci D i s t a l to Df(2L)J27 52 Non-suppressor L o c i D i s t a l to Df(2L)J27 53 Lo c i Uncovered by Df(2L)J27 54 Sub-interval 5 54 Sub-interval 6 55 Sub-interval 7 55 Sub-interval 8 55 Sub-interval 9 61 Sub-interval 10 63 Sub-interval 11 66 Lo c i Proximal to Df(2L)J27 67 DISCUSSION 70 CHAPTER 3 INTRODUCTION 75 MATERIALS AND METHODS 77 Genetics 77 Molecular Biology 80 In situ hybridization to polytene c hromosomes 80 I s o l a t i o n of genomic DNA 82 Iso l a t i o n of bacteriophage lambda DNA 83 Is o l a t i o n of plasmid DNA 84 DNA r e s t r i c t i o n digests and gel electrophoresis 86 Transfer of DNA to hy b r i d i z a t i o n membrane 87 Hybridization of r a d i o l a b e l l e d probe to transferred nucleic acids 87 Lab e l l i n g of DNA 88 Library construction 88 Screening l i b r a r i e s of recombinant bacteriophage lambda 90 Subcloning bacteriophage and cosmid lambda 91 Bl o t t i n g DNA from b a c t e r i a l colonies 92 RNA extraction 93 RNA gel electrophoresis and Northern b l o t t i n g 94 DNA Sequence Analysis 95 Directed deletions 97 RESULTS 99 Genetics 99 Cloning a P element i n Region 3IE 106 Transcripts Originating Near the Cloned P Element 117 Alignment of Genomic, cDNA, and P Element Sequences 127 The Cloned P element i s Adjacent to cdc2Dm 128 cdc2Dm Transcription 134 v i cdc2Dm Gene Structure 134 Sequencing the 5' End of a cda2-like Gene from D. v i r i l i s 138 DISCUSSION 145 CHAPTER 4 INTRODUCTION 151 MATERIALS AND METHODS 154 C u t i c l e Preparations 155 Primers 155 Cloning Using the Polymerase Chain Reaction 156 DNA Sequence Analysis 159 RESULTS 160 A l l e l e s of cdc2 160 Adult Phenotypes 165 Lethal Phases 166 Embryonic and Larval Phenotypes 170 The Relationship between Su(var)216 and cdc2Dm 173 The Sequences of cdc2Dm Mutant A l l e l e s 178 DISCUSSION 183 APPENDIX 1 192 APPENDIX 2 198 APPENDIX 3 204 APPENDIX 4 207 REFERENCES 214 v i i LIST OF TABLES TABLE PAGE 1 Summary of mapping r e s u l t s involving region 31 deletions and Su(var) mutations 28 2 Cyt o l o g i c a l l i m i t s and origi n s of chromosomal rearrangements 42 3 Previously described mutations i n region 31 49 4 Complementation data f o r mutations i n the same deficiency i n t e r v a l as the mutation 24-127 56 5 Complementation matrix f o r the El-13 locus 57 6 Complementation pattern amongst a l l e l e s of the DG25 locus 59 7 Lethal phases f o r mutations which f a i l to complement Df(2L)JRl6 62 8 Complementation matrix f o r a l l e l e s of the B35 locus 64 9 Complementation matrix for da a l l e l e s 65 10 Complementation pattern of a l l e l e s of 1(2)54 68 11 Phenotypic rescue of three mutant a l l e l e s of cdc2Dm 161 12 Phenotypic rescue of h e t e r o a l l e l i c combinations of cdc2Dm mutations 163 13 Complementation crosses for cdc2E20Dm 164 14 Complementation matrix for a l l e l e s of cdc2Dm 167 15 Lethal phases of cdc2Dm mutants raised at 22 169 16 Mapping a second-site mutation on the cdc2Su(var>216 chromosome 176 v i i i LIST OF FIGURES FIGURE PAGE 1 Su(var) l o c i and the Sandler c l u s t e r 27 2 Summary of hemizygous l e t h a l mutations i s o l a t e d i n region 31 33 3 EMS mutagenesis screen 35 4 P element mutagenesis scheme (Df(2L)J77) 37 5 Cyt o l o g i c a l extent of d e f i c i e n c i e s i n region 31 40 6 Lo c i d i s t a l to Df(2L)J27 44 7 Lo c i uncovered by Df(2L)J27 45 8 L o c i centromere proximal to Df(2L)J27 46 9 P element screen (Df(2L)J2) 78 10 Genetic crosses to i s o l a t e the OK15A second chromosome 79 11 Phenotypic reversion of the homozygous l e t h a l i t y of Su(var)216p mutant. 100 12 P elements i n OK15A derived s t r a i n s 103 13 L o c a l i z a t i o n of a P element i n the 3IE region by in situ hybridization 105 14 A r e s t r i c t i o n map of DNA flanking the P element i n the 3IE region 108 15 Subclones from the 31 region 109 16 pR3.8, pRP4.2, and a wild-type P element 111 17 pR3.8 and pRP4.2 probed with a 1.5 kb EcoRI/Hindlll fragment from pR3.8 112 18 pR3.8 and pRP4.2 probed with a 500 bp HindIII/EcoRI P element fragment from the construct HBA89 114 19 A Southern b l o t of genomic DNA from the mutants Su(var)216p and R28 digested with EcoRI 118 i x FIGURE PAGE 20 Embryonic t r a n s c r i p t s that hybridize to pR3.8 120 21 cDNA clones that hybridize to pR3.8 121 22 pc800 hybridized to subcloned DNA from the 3IE region 123 23 pcllOO hybridized to subcloned DNA from the 31E region 125 24 The nucleotide and deduced protein sequence of the product of the cdc2Dm locus 129 25 Comparison of cdc2Dm with other members of the CDC2 protein family 132 26 The sequencing strategy f o r cdc2Dm 135 27 P o t e n t i a l upstream regulatory sequences of cdc2Dm 137 28 A subclone of D. v i r i l i s DNA with homology to the f i r s t and second exons of cdc2Dm 140 29 Sequence comparison of cdc2Dm and a s i m i l a r gene i n D. v i r i l i s 141 30 DNA sequence of the 5' flanking region of a cdc2-like gene from Drosophila v i r i l i s 143 31 Mutations i n cdc2Dm 180 x LIST OF BIOCHEMICAL ABBREVIATIONS APS ammonium persulphate bp base pairs BCIP 5-bromo-4-chloro-3-indolyl phosphate DTT d i t h i o t h r e i t o l EDTA ethylene-diaminetetra-acetic a c i d EMS ethylmethane sulfonate kb kilobase pairs lambda-dil 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10 mM MgCl 2 LM 10 g/1 tryptone, 5 g/1 yeast extract, 2 g/1 MgCl 2, 5 g/1 NaCl MOPS 3-[N-morpholino]propanesulfonic a c i d NBT nitro-blue tetrazolium nt nucleotides PEG polyethylene g l y c o l 8000 pfu plague forming units PVP p o l y v i n y l p y r r o l i d i n e SSC standard s a l i n e c i t r a t e (0.15 M sodium chloride, 0.015 M sodium c i t r a t e ) TE 10 mM Tris-HCl (pH 8.0), ImM EDTA TEMED N,N,N',N'-tetramethylethylenediamine X-gal 5-bromo-4-chloro-3-indolyl-beta-D-galactoside x i LIST OF GENETIC ABBREVIATIONS Balancers CyO SMI TM3 TM6 Mutants b cn Cy G la w*4 Ly nw° P[ry+ 2-3](99B) pr PinYt ryS06 S Sp Curly derivative of Oster In(2LR)0, dplvI Cy pr cn2 Second Multiple In(2LR)SMl, al2 Cy cn2 sp2 In(3LR)TM3, ry** Sb In(3LR)TM6, ss- Ubx"b black (2-48.5); body colour cinnabar (2-57.5); eye colour Curly (2-6.1); wings curled upward Glazed; ommatidia a smooth sheet; p e r i c e n t r i c inversion on Chromosome 2 Infl)*/*4; variegated f o r white Lyra (3-40.5); l a t e r a l margins of wing excised narrow-Dominant (2-83); long, t h i n wings abbreviated A2-3(99B) ; P element i n s e r t i o n that activates other elements i n the soma and germline (see Robertson et a l . , 1988) purple (2-54.5); eye colour Pin-Yellow tip (2-107.3); d i s t a l t h i r d of thoracic b r i s t l e s t h i n and yellow rosy (3-52); eye colour Star (2-13); i r r e g u l a r eye facets, eye reduced Sternopleural (2-22); sternopleural b r i s t l e s increased i n number x i i Sb Stubble (3-58.2); short thick b r i s t l e s Sbv Stubble-Variegated; associated with T(2;3)Sbv =T(2;3)41A-C;88;89B: displays p o s i t i o n e f f e c t variegation Tft Tuft (2-53.2); extra b r i s t l e s on mesothorax x i i i Acknowledgements I wish to thank my supervisor, Dr. T. G r i g l i a t t i , and Dr. G. Spiegelman fo r advice and f i n a n c i a l support. I also thank members of my committee, Drs. H. Brock, R. MacGillivray, and D. Moerman for h e l p f u l discussions. I would l i k e to thank M. 0'Grady f o r a s s i s t i n g with molecular aspects of the project, and R. Mottus f o r a s s i s t i n g with the genetics. At some time or another, v i r t u a l l y every member of the F l y Group p a r t i c i p a t e d i n the genetic analysis of the 31 region and I am g r a t e f u l for t h e i r e f f o r t s . I am p a r t i c u l a r l y indebted to Hugh Brock, Ian Whitehead, and Don S i n c l a i r . In many discussions and a b r i e f c o l l a b o r a t i v e e f f o r t with Hugh, I've learned how research can and should work. Ian and I collaborated on numerous aspects of t h i s project including genetic screens, a c y t o l o g i c a l analysis of the 31 region, and the analysis of t r a n s c r i p t i o n units i n 3IE. Many tedious evenings of v i r g i n c o l l e c t i n g were r e l i e v e d by our discussions. Don S i n c l a i r ' s enthusiasm, knowledge, and willingness to discuss and t e s t ideas were i n s p i r a t i o n a l . Much of the work described here was b u i l t on his e f f o r t s . F i n a l l y , none of the work would have been completed without the understanding and support of my wife Kathy. x i v GENERAL INTRODUCTION In the interphase nucleus of a t y p i c a l eukaryotic c e l l such as that of Drosophila melanogaster, approximately 1.4 X 10s base pairs of DNA measuring over 30 centimetres i n length must be folded to occupy a spherical nucleus only 10 um i n diameter. This remarkable compaction i s thought to involve several h i e r a r c h i c a l l e v e l s of chromatin organization. The lowest l e v e l of organization consists of approximately 145 bp of DNA wrapped around a histone octamer (consisting of one H3-H4 tetramer and two histone H2A-H2B dimers ) to form a nucleosome. Adjacent nucleosomes are separated from each other by roughly 30-50 bp of l i n k e r DNA (McGhee and Felsenfeld, 1980). A second, less well defined l e v e l of organization i s the subsequent fol d i n g of the DNA/histone complex into a h e l i c a l or solenoid-like 30 nm f i b e r (Felsenfeld and McGhee, 1986). These f i r s t two l e v e l s of f o l d i n g r e s u l t i n roughly a 40-fold compaction of the DNA. To a t t a i n the 100- to 200-fold compaction required to package the DNA complement in t o the nucleus, a d d i t i o n a l l e v e l s of organization are required. These add i t i o n a l l e v e l s of organization are poorly understood, but i n most current models the chromatin f i b e r i s sequestered into a series of discrete and t o p o l o g i c a l l y independent domains (Jackson, 1991). The establishment and maintenance of these higher l e v e l s of organization requires a d d i t i o n a l non-histone chromosomal proteins (NHPs). The organization of chromosomes i n t o highly compact 1 structures influences the u t i l i z a t i o n of the genetic material. This i s best exemplified by experiments involving histone gene stoichiometry. In in vitro assays, i f nucleosomes are formed on genes p r i o r to the assembly of a t r a n s c r i p t i o n complex that includes e i t h e r RNA polymerase I ( S c h l i s s e l and Brown, 1984) or RNA polymerase II (Workman and Roeder, 1987), then the genes become r e s i s t a n t to t r a n s c r i p t i o n . In S. cerevisiae, changes i n histone stoichiometry a l t e r t r a n s c r i p t i o n patterns (Clark-Adams et al., 1988), and nucleosome depletion increases t r a n s c r i p t i o n i n i t i a t i o n in vivo (Han and Grunstein, 1988). Nucleosomes may also repress t r a n s c r i p t i o n i f they are positioned at s p e c i f i c s i t e s on the chromosome (Roth et a l . , 1990). In addition to simply blocking the access of t r a n s c r i p t i o n factors to DNA, protein complexes that condense DNA may also have more subtle and s p e c i f i c e f f e c t s on gene regulation. For example, sequences i n the amino terminal domain of the histone H4 gene of Sacharomyces cerevisiae are necessary to repress (Kayne et a l . , 1988) or to activate (Durrin et a l . , 1991) the t r a n s c r i p t i o n of s p e c i f i c genes in vivo. Hence, histones are mu l t i f u n c t i o n a l . NHPs involved i n e s t a b l i s h i n g and maintaining chromosomal architecture are also l i k e l y to have both global and gene-s p e c i f i c e f f e c t s on t r a n s c r i p t i o n a l regulation. NHPs constitute approximately 1/3 of the mass of chromatin, yet how they i n t e r a c t to form higher orders of structure i s l a r g e l y 2 unknown. Although NHPs are now being i d e n t i f i e d at a rapid rate, the sheer numbers and po t e n t i a l interactions between proteins as they assemble i n t o functional chromatin complexes pose a problem f o r the biochemical study of chromatin structure and function. In m u l t i c e l l u l a r organisms, most studies of NHPs have focused on the i s o l a t i o n of proteins which remain associated with DNA following various types of biochemical extraction (e.g. Alfageme et al., 1980). This biochemical approach has been very successful i n i d e n t i f y i n g a subset of proteins that associate with DNA, but i n many instances the b i o l o g i c a l function of the proteins i s unclear. A complementary approach to the in vitro biochemical analysis of chromatin structure i n m u l t i c e l l u l a r organisms i s to i d e n t i f y g e n e t i c a l l y genes that a f f e c t chromatin condensation. With the exception of dominant mutations i n the tandemly r e i t e r a t e d histone genes, mutations that a f f e c t chromatin condensation are l i k e l y to a r i s e i n genes that encode NHPs or proteins that modify e i t h e r NHPs or histones. A genetic analysis of such mutations may reveal the developmental consequences of impaired or alt e r e d chromatin structure. Also, interactions between mutations at d i f f e r e n t l o c i may reveal previously unsuspected r e l a t i o n s h i p s between components of chromatin. A further advantage to the genetic approach i s that some mutations, such as those induced by transposable elements, can f a c i l i t a t e gene cloning. Thus, genetical and biochemical studies can proceed i n concert. 3 This thesis i s concerned with genes that a f f e c t two c y t o l o g i c a l l y d i f f e r e n t i a b l e states of chromatin compaction: euchromatin versus heterochromatin. Euchromatin décondenses during interphase and contains the majority of t r a n s c r i p t i o n a l l y active gene species while heterochromatin f a i l s to décondense during interphase and i s considered, for the most part, to be t r a n s c r i p t i o n a l l y quiescent. Herein I describe studies to clone one of several Suppressor-of-position-effect-variegation (Su(var)) genes located i n region 31 on the Drosophila melanogaster polytene chromosome map. These genes are believed to help to maintain or e s t a b l i s h heterochromatic and/or euchromatic domains. The hypothesis that Su(var) genes encode NHPs or modifiers of NHPs that a f f e c t chromatin condensation i s discussed i n Chapter 1, a survey of the current l i t e r a t u r e on p o s i t i o n - e f f e c t variegation and i t s modifiers. A cytogenetic analysis of a region that contains several genes that suppress p o s i t i o n -e f f e c t variegation i s described i n Chapter 2. In Chapter 3, the transposon tagging and cloning of the putative suppressor cdc2DnfSu<y,kr)216F i s presented. F i n a l l y , Chapter 4 describes the phenotype of mutants at the cdc2Dm locus and the r e l a t i o n s h i p of cdc2Dm to suppressors of p o s i t i o n - e f f e c t variegation. 4 CHAPTER 1 LITERATURE REVIEW A. EUCHROMATIN AND HETEROCHROMATIN Eukaryotic chromatin assumes i t s most condensed state i n metaphase chromosomes; however, t h i s state i s quite transient i n most t i s s u e s . During telophase, some chromosomal regions unravel to form euchromatin, while others r e t a i n a dense stai n i n g , compact morphology i n the interphase nucleus. The l a t t e r regions constitute heterochromatin. In Drosophila, m i t o t i c chromosomes possess conspicuous heterochromatic regions adjacent to t h e i r centromeres. These regions are best v i s u a l i z e d during prophase before euchromatic chromatin becomes condensed. In Drosophila melanogaster, heterochromatin comprises the proximal half of the acrocentric X chromosome, the proximal quarters of each arm of the metacentric second and t h i r d chromosomes, and the e n t i r e Y chromosome. Most of the fourth chromosome i s also considered to be heterochromatic. These heterochromatic regions r e p l i c a t e l a t e r i n S phase than euchromatin (Barigozzi et al., 1966). In interphase n u c l e i i n the somatic t i s s u e s of most organisms, euchromatin i s generally a very l o o s e l y packaged e n t i t y which i s morphologically d i f f u s e and hence i s e a s i l y defined or delineated. An exception to t h i s general rule i s the polytene chromosomes from the s a l i v a r y glands of mature larvae of dipteran insects such as Drosophila. During polytenization i n Drosophila euchromatin undergoes up to ten 5 rounds of DNA r e p l i c a t i o n without chromatid asynapsis or cytokinesis. The r e s u l t i s a set of euchromatic chromosome arms with a c h a r a c t e r i s t i c banded morphology. Extensive biochemical and genetic data accummulated over the l a s t h a l f -century indicate that the majority of genes are located i n t h i s polytenized material. The heterochromatin of polytene chromosomes can be subdivided i n t o two c y t o l o g i c a l l y d i f f e r e n t categories: a- and (3-heterochromatin (Heitz, 1934). Alpha-heterochromatin surrounds the centromere of each chromosome and mingles with s i m i l a r regions of other chromosomes to form the chromocenter. Beta-heterochromatin i s the poorly banded material which connects the chromocenter to the polytene chromosome arms (Heitz, 1934). The two types of heterochromatin also have d i s t i n c t biochemical and b i o l o g i c a l properties. Alpha-heterochromatin i s not polytenized (Rudkin, 1969; G a l l et al., 1971). I t consists predominantly of s a t e l l i t e DNA sequences and contains few, i f any, transcribed genes. In contrast, p"-heterochromatin — l i k e euchromatin — i s endoreduplicated (Gall et al., 1971; Lakhotia, 1974). I t i s t r a n s c r i p t i o n a l l y active (Biessman et al., 1981; Miklos et al., 1984; Devlin et al., 1990), although i t contains fewer unique genes than euchromatin (see H i l l i k e r et al., 1980). Beta-heterochromatin contains a higher concentration of middle r e p e t i t i v e elements than e i t h e r euchromatin or a-heterochromatin (Spradling and Rubin, 1981; Healy et al., 1988; Devlin et al., 1990). 6 The formation of heterochromatin takes place during early embryogenesis. During the i n i t i a l , very rapid nuclear d i v i s i o n s i n the s y n c i t i a l blastoderm, the n u c l e i appear homogeneous (Mahowald, 1963); however, by cycle 10-13 heterochromatin i s v i s i b l e using Hoechst 33258 s t a i n i n g (Foe and Alberts, 1985). The appearance of heterochromatin i n d i v i s i o n cycle 10 of Drosophila melanogaster i s correlated with the recruitment of heterochromatic proteins encoded by maternal mRNAs (James et al., 1989). The cytogenetic d i s t i n c t i o n between heterochromatin and euchromatin i s also correlated with the a c q u i s i t i o n of t r a n s c r i p t i o n a l competence i n d i v i s i o n cycle 10 (Edgar and Schubiger, 1986). B. POSITION-EFFECT VARIEGATION General Genetics and Cytology P o s i t i o n - e f f e c t variegation (PEV) i s the i n a c t i v a t i o n of a gene by an adjacent chromosomal rearrangement. In i t s most common form, PEV involves euchromatic genes which are inact i v a t e d by a genetic rearrangement with a breakpoint i n a heterochromatic region. I f one chromosome bears a mutant a l l e l e (g) and the other the relocated (J?) wild-type a l l e l e (g +), then the hétérozygote J?(g +;/gwill show a mosaic of wild-type and mutant gene expression within the t i s s u e affected by t h i s gene. In contrast, an R(g)/g+ w i l l be wild-type. This type of variegated p o s i t i o n e f f e c t has been observed i n a wide v a r i e t y of organisms, including mice (Cattanach, 7 1974) and plants (Catcheside, 1947). Hence, PEV seems to be a phenomenon that i s widespread among eukaryotes. Nonetheless, i t has been given the most attention by f r u i t - f l y g e n e t i c i s t s , who have been studying i t f o r nearly a century (see Karpen and Spradling, 1990). Therefore, with rare exceptions, t h i s survey w i l l confine i t s e l f to drosophilids and predominantly Drosophila melanogaster. That an observed variegation i s caused by a p o s i t i o n e f f e c t can be demonstrated i n two ways. The f i r s t i s by reversion of the variegated gene i n a c t i v a t i o n phenotype with reversion of the rearrangement (e.g. Hinton and Goldsmith, 1950). The second i s by recovering a wild-type phenotype when a mutant a l l e l e i s exchanged for the ina c t i v a t e d a l l e l e by crossing over between the locus and the breakpoint (e.g. Judd, 1955). The l a t t e r r e s u l t demonstrates that the gene i n a c t i v a t i o n r e s u l t i n g from PEV does not r e s u l t from gene loss or mutation, at l e a s t i n the meiotic stem c e l l s . In Drosophila, most euchromatic and heterochromatic sequences appear susceptible to, or capable of causing, PEV. Only ebony (Brosseau, 1970) and the Bithorax Complex (E.B. Lewis, c i t e d i n Henikoff, 1990) have not yielded variegating a l l e l e s upon a search. When euchromatic genes variegate, the rearrangement breakpoint associated with, and presumably causing, PEV i s t y p i c a l l y i n a-heterochromatin (Tartof, et al. 1989). Unique genes that normally reside i n p-heterochromatin 8 are also able to variegate i n response to a change i n chromatin environment (Hess1er, 1958; Wakimoto and Hearn, 1990). Heterochromatic l o c i d isplay mosaicism when moved adjacent to d i s t a l euchromatin and thus t h i s form of variegation i s d i f f e r e n t from the more common form of PEV i n that i t does not involve a-heterochromatin. Two examples of genes which variegate under these circumstances are light (It) which i s normally located i n 2L heterochromatin (Hessler, 1958; Wakimoto and Hearn, 1990) and cubitis interruptus (ci) on chromosome 4 (Stern and Kodani, 1955). In these instances, f o r reasons unknown (but see Wakimoto and Hearn, 1990), the variegating rearrangements are r e s t r i c t e d to those with breakpoints i n the very c e n t r i c or d i s t a l portions of the euchromatic region of the chromosomes. Only r a r e l y has PEV been associated with euchromatin/euchromatin rearrangement breakpoints. One such case i s i n Drosophila hydei (van Breugel, 1988). There i s usually a p o l a r i t y of gene i n a c t i v a t i o n associated with PEV. Whenever an affected locus i s several bands from the rearrangement breakpoint, intervening l o c i are also affected. An example i s the t r a n s l o c a t i o n Tfl^Jw*258'18 (Demerec and Slizynska, 1937: c i t e d i n Cohen, 1962). In t h i s case, the roughest eye-morphology locus i s c l o s e r to the variegation-inducing rearrangement breakpoint than the white eye-colour locus. In eyes displaying mosaicism, the rough patches of the eye are larger than, and completely include, 9 a l l areas of the eye that are white. In t h i s variegating system, whenever the white gene i s repressed, roughest i s always inactivated. The order of gene repression r e f l e c t s the gene order on the chromosome suggesting a polarized spreading e f f e c t of an i n a c t i v a t i n g substance along the chromosome. The polarized spreading e f f e c t associated with PEV i s correlated with a c y t o l o g i c a l disruption of the banding pattern i n polytene chromosomes. This "heterochromatization" i s suggestive of a spread of heterochromatin along the chromosome. Using tr a n s l o c a t i o n T( 1; 4 Jw*258-21, which breaks c l o s e r to the white gene (i n 3C2) than to the Notch gene (in 3C7), Hartmann-Goldstein (1967) showed that band 3C2 was c l e a r l y v i s i b l e i n more s a l i v a r y gland c e l l s than was 3C7, and was always v i s i b l e when 3C7 was v i s i b l e . The extent of c y t o l o g i c a l variegation correlated well with the extent of white and Notch variegation i n adults. In Drosophila hydei, t h i s spreading e f f e c t has also been observed c y t o l o g i c a l l y for heterochromatic genes which variegate near euchromatic breakpoints (Hess, 1970). Exceptions to the r u l e that i n a c t i v a t i o n of more proximal l o c i accompanies the i n a c t i v a t i o n of l o c i further from the breakpoint are rare. Clark and Chovnick (1986) reported a gene that f a i l s to variegate although i t l i e s c l o s e r to a rearrangement breakpoint than a variegating rosy a l l e l e . However, subsequent studies have refuted these claims (A. Chovnick, pers. comm.). 10 Variegating l o c i have been observed up to 80 polytene chromosome bands distant from the associated rearrangement breakpoint (Schultz, 1950). Since each c y t o l o g i c a l l y v i s i b l e band on a polytene chromosome contains, on average, 20-30 kilobases of DNA (Spierer et a l . , 1983), over 1500 kilobases of DNA separate the affected locus from the breakpoint. In most cases, however, the observed distances are not so great. The distance over which the i n a c t i v a t i o n extends seems to depend on the p a r t i c u l a r variegation inducing region and the p a r t i c u l a r euchromatic region involved (Spofford, 1976). The Timing of P o s i t i o n - e f f e c t Variegation For some variegating rearrangements, such as T(l; 4)w*258~18, the mosaicism i s so f i n e grained that the f i n a l decision whether or not the relevant locus i s to be a c t i v e must occur l a t e i n development. Nonetheless, i n several instances investigators have argued f o r an e a r l y i n a c t i v a t i o n event. For example, embryos hemizygous fo r variegating rDNA cis t r o n s (In( 1 fs^/O embryos) synthesize s i g n i f i c a n t l y l e s s rRNA during the f i r s t f i v e hours of embryogenesis, r e s u l t i n g i n 14% less rRNA i n newly hatched l a r v a than i n controls (Puckett and Snyder, c i t e d i n Spofford, 1976). Based on the data of Noujdin (1936) and several developmental studies (Bryant, 1970; Garcia-Bellido and Merriam, 1969 and 1971), Spofford (1976) hypothesized that the time of decision of y ac variegation i n In(l)sc8 was e a r l y i n development: perhaps as e a r l y as the 11 c e l l u l a r blastoderm stage. The roughest locus, which variegates when adjacent to In(l)rst3, may also be inact i v a t e d as e a r l y as the time of blastoderm formation (Spofford, 1969; Spofford, 1976). Later estimates of the timing of the i n a c t i v a t i o n event are derived from c e l l lineage analyses i n the eye. For the variegating rearrangement T(Y;SJpe"1 i n Drosophila v i r i l i s , the boundaries between mosaic patches of peach (pe) and pe+ eye ti s s u e (Baker, 1967) are very s i m i l a r to the outlines of cl o n a l twin spots generated by somatic crossing over i n f i r s t -i n s t a r larvae (Baker, 1967). This suggests that the time of i n a c t i v a t i o n i s no l a t e r than the f i r s t l a r v a l i n s t a r . Similar r e s u l t s have been obtained f o r variegating eye-colour phenotypes i n Drosophila melanogaster (Baker, 1967; Becker, 1960 c i t e d i n Baker, 1967). Studies of twin spots i n a variegating background have, i n general, confirmed that i n a c t i v a t i o n of the w locus i n Drosophila melanogaster occurs i n the f i r s t l a r v a l i n s t a r (Baker, 1967; Janning, 1970). In addition to de l i m i t i n g the developmental i n t e r v a l during which the t r a n s c r i p t i o n a l fate of a variegating a l l e l e i s determined, these c e l l lineage studies demonstrate that the i n a c t i v a t i o n decision must be c l o n a l l y i n h e r i t e d because progenitor c e l l s undergo many more rounds of d i v i s i o n before an eye i s formed. 12 Molecular Aspects of P o s i t i o n - e f f e c t Variegation For rosy (Rushlow et al., 1984), Hsp70 (Henikoff, 1981), Sgs4 (Kornher and Kauffman, 1986), and brown (Henikoff and Dreesen, 1989), the gene i n a c t i v a t i o n associated with PEV has been shown to r e s u l t i n a reduction i n nascent t r a n s c r i p t accumulation. This apparent reduction could be a consequence of reduced a c c e s s i b i l i t y of the template DNA to t r a n s c r i p t i o n f a c t o r s . A l t e r n a t i v e l y , i n polytene t i s s u e s , i t might r e f l e c t a reduced number of gene copies. Position-effect variegation associated with euchromatic l o c i requires that the affected locus be adjacent to a rearrangement breakpoint i n heterochromatin. Since a-heterochromatin i s under-replicated i n polytene t i s s u e s , the variegating phenotype and i n d i s t i n c t cytogenetic morphology associated with variegating l o c i might be a consequence of underreplication. Using cloned DNA sequences, the degree of r e p l i c a t i o n at variegating l o c i has been compared to that at non-variegating euchromatic l o c i . No s i g n i f i c a n t underreplication was detected f o r the white gene (Hayashi et al., 1990), the heatshock locus (Henikoff, 1981), or the rosy locus (Rushlow et a l . , 1984). However, the Sgs4 gene i s underreplicated i n T(l;4)w^58'21 (Kornher and Kauffman, 1986), as i s the yellow gene i n Dp(l;f)1187 (Karpen and Spradling, 1990). Thus, i n some instances, PEV i s correlated with underreplication i n polytene c e l l s . Although underreplication might explain variegation of some l o c i i n polytene t i s s u e s , i t cannot explain variegation i n d i p l o i d 13 t i s s u e s . The pigment c e l l s of the eye are not thought to be polytene or po l y p l o i d (Karpen and Spradling, 1990), yet eye-colour genes variegate. S i m i l a r l y , germline t i s s u e i s not pol y p l o i d , yet the nod locus i s subject to PEV (Zhang and Hawley, 1990). Gene t r a n s c r i p t i o n near euchromatin/heterochromatin rearrangement breakpoints may be repressed because of altered a c c e s s i b i l t y of the chromatin to t r a n s c r i p t i o n f a c t o r s . In situ h y b r i d i z a t i o n with cloned probes suggests that i n polytene chromosomes the DNA of a variegating white a l l e l e i s less accessible to a molecular probe than i s a white a l l e l e d i stant from the rearrangement breakpoint (Hayashi et al., 1990). Thus, PEV may be a consequence of an a l t e r e d chromatin conformation. Biochemical studies have also focused on the structure of rearrangement breakpoints associated with PEV. Tartof et al. (1984) cloned three rearrangement breakpoints associated with variegation at the white locus, w"51" and w™"0 d i s p l a y f i n e -grained mosaicism suggestive of a l a t e determinitive event, while w*4 i s coarsely mottled with large c l o n a l patches of w t i s s u e suggesting an e a r l y decision. In a l l three cases the rearrangement i s very close to the white gene, w™511' and w""0 share nearly i d e n t i c a l euchromatic breakpoints, which may be of importance i n determining t h e i r fine-grained pattern of variegation. When w*4 was reinverted to re-activate the w gene, adjacent r e p e t i t i v e sequences were s t i l l associated with the 14 white gene. This r e s u l t suggested that the phenomenon which causes PEV originates not at the breakpoint i t s e l f , but deep i n heterochromatin from whence i t i s propagated. However, Reuter et al. (1985) have re-activated the white gene i n a s i m i l a r fashion and have shown that such "wild-type" revertants often do s t i l l variegate i f a strong modifier of PEV (see "Modifiers of PEV") i s present i n the genetic background. Tartof has claimed that the revertants induced i n his laboratory do not respond to modifiers of PEV (T. G r i g l i a t t i , pers. comm.). Thus, i f s p e c i f i c DNA segments are required f o r PEV, they are unknown. Modifiers of P o s i t i o n - e f f e c t Variegation Higher temperatures usually suppress PEV, while lower temperatures enhance the variegating phenotype (Gowen and Gay, 1934). Crowding of larvae during e a r l y development also enhances variegation (Hinton, 1949). F l i e s reared at pH 2.6 develop more slowly than f l i e s reared at higher pH. Since t h i s treatment also enhances white eye-colour variegation i n In(l)w*4 , M i c h a i l i d i s et al. (1988) have suggested that many environmental e f f e c t s are a consequence of prolonged development. They further speculate that prolonged development i s responsible f o r the enhancement of PEV by some agents which i n t e r f e r e with DNA synthesis (Schultz, 1956). Some chemical agents, however, have e f f e c t s which are separable from t h e i r e f f e c t s on development time. For example, 15 both butyrate and proprionate suppress white i n a c t i v a t i o n associated with In(l)w*4, although they s i g n i f i c a n t l y prolong development (Mottus et al., 1980; Rushlow et al., 1984). The mechanisms whereby exogenous chemicals influence PEV are i l l - d e f i n e d . Sodium butyrate i s hypothesized to induce suppression by i n h i b i t i n g histone deacetylation, thereby ameliorating a l t e r a t i o n s i n chromatin structure at or near variegating l o c i (Mottus et al., 1980). However, while sodium butyrate does a f f e c t chromatin compaction (Annunziato et al., 1988), i t also a f f e c t s several other c e l l u l a r processes (see Boffa et al., 1981; Christman et al., 1980). Another chemical agent, DMSO, which has no known e f f e c t on histone modification, also suppresses PEV (cited i n M i c h a i l i d i s et al., 1988). Several genetic factors have been shown to modify PEV. Extra heterochromatin, located elsewhere i n the genome, af f e c t s the expression of variegating genes. The presence of a Y chromosome suppresses variegation; i t s absence enhances i t (Gowen and Gay, 1934). Thus, XXY females and XYY males are almost wild-type f o r l o c i near a variegating rearrangement, while XO males show more extreme variegation than XY males (Spofford, 1976). Deletions and duplications f o r autosomal heterochromatin have s i m i l a r e f f e c t s on PEV (Spofford, 1976). In the case of the Y chromosome, an analysis of numerous large d e f i c i e n c i e s suggest that suppression i s a function of the amount of Y heterochromatin i n the genome and that i t i s not 16 a t t r i b u t a b l e to any dis c r e t e region of the Y chromosome (Dimi t r i and Pisano, 1989). Deletion of histone genes suppresses variegation (Moore et a l . , 1979; Moore et al., 1983; Khesin and Bashkirov, 1979), as do deletions f o r numerous other genetic l o c i (e.g. Wustmann et al., 1989). These modifiers are discussed i n d e t a i l i n the section e n t i t l e d "Su(var)s and E(var)s". C. SUPPRESSORS AND ENHANCERS OF POSITION-EFFECT VARIEGATION There are many euchromatic l o c i which can be mutated to generate suppressors (Su(var)s) (Spofford, 1967; Reuter and Wolff, 1981; S i n c l a i r et al., 1983; Reuter et al., 1986) or enhancers (E(var)s) of PEV ( S i n c l a i r et al., 1989; Reuter and Wolff, 1981; Locke et al., 1988). The greatest number of modifiers has been i n f e r r e d from the deficiency/ duplication mapping of haplo- and triplo-abnormal l o c i . From a study of 12 mutations which i d e n t i f i e d 4 independent l o c i , Locke et al. (1988) proposed that 20-30 dosage-sensitive l o c i existed. However, Wustmann et al. (1989) have suggested that there may be as many as 120-150 l o c i , since 38 haplo-dependent modifiers of PEV were i d e n t i f i e d i n approximately 30% of the autosomal complement. This number i s i n good agreement with estimates based on cytogenetic analyses of modifiers of PEV i n regions 87C (Henikoff, 1979), 86-88 (Reuter et al., 1987), and 24D4-25F2 (Szidonya and Reuter, 1988). The 42 known dosage-dependent l o c i can be divided into 17 four classes: (1) haplo-abnormal suppressor l o c i with a triplo-abnormal enhancer function ( 2) haplo-abnormal enhancer l o c i with a triplo-abnormal suppressor function (3) haplo-abnormal enhancer l o c i , and (4) haplo-abnormal suppressor l o c i (Locke et al., 1988; Wustmann et al., 1989). To explain the dosage-dependence of so many l o c i a f f e c t i n g the same phenomenon, Locke et al. (1988) elaborated upon e a r l i e r hypotheses which suggested that modifiers of PEV encode chromatin proteins or modifiers of chromatin proteins (Zuckerkandl, 1974; Spofford, 1976; S i n c l a i r et al., 1983). They suggested that these l o c i encode proteins which p a r t i c i p a t e i n a large macromolecular complex, chromatin, and that the observed dosage e f f e c t s were a r e s u l t of the law of mass action. S p e c i f i c a l l y , a large assemblage such as chromatin would be ex q u i s i t e l y s e n s i t i v e to small changes i n the concentration of any one constituent, since any such a l t e r a t i o n would drive the assembly/disassembly of chromatin away from i t s normal equilibrium. Since only eight l o c i are both haplo- and triplo-abnormal, these l o c i are l i k e l y to play p i v o t a l roles i n the PEV phenomenon. In addition to duplication/deficiency analyses, a large number of Su(var) and E(var) mutations have been i s o l a t e d which a f f e c t single l o c i (Reuter and Wolff, 1981; Reuter et al., 1986; S i n c l a i r et al., 1983). Reuter and his colleagues have i d e n t i f i e d 12 dominant Su(var) mutations on the second chromosome and 11 on the t h i r d , as well as two E(var) 18 mutations (see Wustmann et al., 1989). Many of the mutations described by S i n c l a i r et al. (1983) are l i k e l y to represent add i t i o n a l a l l e l e s of these l o c i . A comprehensive study of a l l e l i s m between these two sets of mutants i s i n progress (T. G r i g l i a t t i , pers. comm.). In general, there i s a good c o r r e l a t i o n between Su(var) and E(var) mutants, and dosage-s e n s i t i v e genes which modify PEV (summarized i n Wustmann et al., 1989), suggesting that most Su(var) and E(var) mutations are amorphs or hypermorphs. Su(var) and E(var) mutations a f f e c t PEV generally since they suppress or enhance the i n a c t i v a t i o n of l o c i associated with d i f f e r e n t rearrangements (Hayashi et al., 1990; Reuter et al., 1982; S i n c l a i r et al., 1989, 1991). The extent to which they modify PEV i s a l l e l e s p e c i f i c . One mutation might suppress i n a c t i v a t i o n of the white gene i n In(l)w™4 by 90%, while another might suppress the i n a c t i v a t i o n by only 50%. The effectiveness of a p a r t i c u l a r Su(var) or E(var) mutation can vary depending on the rearrangement involved. Thus, a strong suppressor of white gene i n a c t i v a t i o n i n In(l)w*4, could be a weak suppressor of Stubble gene i n a c t i v a t i o n i n T(2;3)Sbv. F i n a l l y , genetic modifiers of PEV do not always d i s p l a y the same trends i n t h e i r a b i l i t y to a f f e c t variegating l o c i . One Su(var) mutation might suppress i n a c t i v a t i o n of white better than i n a c t i v a t i o n of brown, while another might have more profound e f f e c t s on brown variegation than on white (e.g. Hayashi et al., 1990). 19 Mutations at some, but not a l l , Su(var) l o c i are sexually dimorphic, each sex suppressing the same variegating locus to a d i f f e r e n t extent (Hayashi et al., 1990; Reuter et al., 1986; S i n c l a i r et al., 1991). It remains uncertain whether or not the majority of genetic modifiers of PEV are e s s e n t i a l f o r normal development. In one study, most suppressor mutants i s o l a t e d on the t h i r d chromosome were homozygous l e t h a l or s t e r i l e (Reuter et al., 1986), while i n another, a l l were homozygous v i a b l e ( S i n c l a i r et al., 1983). The homozygous via b l e mutants may simply be hypomorphic a l l e l e s of e s s e n t i a l l o c i , or they may represent d i f f e r e n t , non-essential l o c i . Only mutations i n Su-var(2)l and Su-var(3) have been tested f o r t h e i r e f f e c t s i n the germ-line (Szabad et al., 1988). Germ-line recombination studies with mutant a l l e l e s Su-var(2)l01 and Su-var(3)03 indicate that wild-type functioning of both Su-var(2)1 and Su-var(3) i s required f o r normal development of the germ-line as well as f o r the soma. The most extensively studied Su(var) locus i s Su-var(2)l, located i n region 31 on the polytene chromosome map. A l l e l e s of Su-var(2)l display a general e f f e c t on PEV (Reuter et a l . , 1982; Hayashi et al., 1990; S i n c l a i r et al., 1991), and reduce the c y t o l o g i c a l heterochromatization associated with variegating rearrangements (Reuter et al., 1982; Hayashi et al., 1990). The Su-var(2)l01 a l l e l e increases the in vitro t r a n s c r i p t i o n a l capacity of polytene chromosomes i n intersexes 20 (Khesin and Bashkirov, 1979), suggesting that i t also a f f e c t s euchromatic regions. Larvae heterozygous or homozygous fo r the Su-var(2)l01 a l l e l e also e x h i b i t s i g n i f i c a n t hyperacetylation of histone H4 and an increased a c c e s s i b i l i t y of DNA to endogenous endonucleases (Dorn et al., 1986). These findings suggest that Su-var(2)1 encodes a modifier of an NHP such as a histone de-acetylase (Dorn et al., 1986), or an NHP which a l t e r s the a c c e s s i b i l i t y of heterochromatin to modifiers such as acetylases. Su-var(2)l i s also s e n s i t i v e to known modifiers of PEV. Mutant a l l e l e s e x h i b i t strongly reduced v i a b i l i t y as homozygotes or transheterozygotes when grown on medium containing sodium butyrate (Reuter et al., 1986; S i n c l a i r et al., 1991). They also i n t e r a c t with the Y chromosome, such that XXY; Su-var/Su-var progeny die but X/0; Su-var/Su-var progeny survive. In contrast, a l l e l e s of Su-var(3)l, Su-var(3)2 and Su-var(3)9 are not s e n s i t i v e to sodium butyrate or Y heterochromatin (Reuter et al., 1986). Thus, modifiers of PEV only modify the phenotypes of mutations at some Su(var) l o c i . Presumably these al t e r e d phenotypes are the r e s u l t of additive e f f e c t s on chromatin assembly or compaction. Cloned Genes Two unique genes that a f f e c t PEV have been cloned. HPl was o r i g i n a l l y i d e n t i f i e d as a gene encoding a heterochromatin binding protein (James et al., 1986). Subsequently, two 21 a l l e l i c Su(var) mutations were found to have lesions i n the gene (Eissenberg et al., 1990). Su(var)205 ( S i n c l a i r et al., 1983) contains a G to A t r a n s i t i o n at the f i r s t nucleotide of the l a s t i n t r o n of HP-1, causing m i s s p l i c i n g of the mRNA. The HP1 sequence of the Su(var)2S mutant contains a nonsense mutation. The phenotypic e f f e c t s of a s p l i c i n g defect are hard to predict, but nonsense mutations usually r e s u l t i n loss of protein a c t i v i t y . Since a los s - o f - f u n c t i o n mutation i n HPl suppresses PEV, while a du p l i c a t i o n for the gene enhances variegation (Wustmann et a l . , 1989), HPl i s a haplo- t r i p l o -abnormal locus with respect to PEV. The HPl protein has sequence s i m i l a r i t y with the protein encoded by the Polycomb {Pc) gene (Paro and Hogness, 1991). Like HPl, Polycomb protein i s also a component of chromatin (Zink and Paro, 1989), and possibly part of a heterochromatin-l i k e complex (see Gaunt and Singh, 1990; see Paro, 1990). The region of s i m i l a r i t y between the two proteins i s 37 amino acids long; and within t h i s chromo domain (chromatin organization modifier) 24 amino acids are p r e c i s e l y conserved. Since both HPl (T.C. James, c i t e d i n Singh et a l . , 1991) and Pc (Paro, 1990) proteins f a i l to bind DNA, the chromo-domain could be involved i n protein-protein interactions which permit packaging i n heterochromatin or heterochromatin-like complexes (Singh et al., 1991). The Polycomb gene i s a member of the Polycomb-Group, a set of genes which act as dosage-sensitive, negative regulators of homeotic genes (Jurgens, 1985). Thus, PEV and the negative regulation of homeotic gene expression might represent analagous mechanisms of gene repression. In fact, some members of the Polycomb-Group are modifiers of PEV (D. S i n c l a i r , N. Clegg, T. G r i g l i a t t i , and H. Brock, submitted). Hence, some modifiers of PEV may act not only as general regulators ( i . e . influencing the euchromatin/heterochromatin dec i s i o n ) , but also as regulators of s p e c i f i c , developmentally important genes. Using a DNA probe from the region of HP-1 that encodes the chromo domain (the chromo box), Singh et a l . (1991) i s o l a t e d murine and human cDNAs with sequence s i m i l a r i t y to the Drosophila gene. These clones may represent t r a n s c r i p t s from genes with analagous modes of action to the HPl gene i n f r u i t f l i e s . Cross-hybridization was also detected to the DNA from other plant and animal species. Since one of these species, Caenorhabditis elegans, has no c y t o l o g i c a l l y v i s i b l e heterochromatin (Wood, 1988), chromo domains might be involved i n other instances of heritable gene repression (Singh et al., 1991). The other cloned modifier of PEV i s Suvar(3)7. Deletion of one copy of Suvar(3)7 suppresses variegation of the white gene i n In(l)w*4, but duplications of the locus enhance variegation of white (Reuter et al., 1987). Reuter et al. (1990) deficiency mapped the suppressor locus, then transformed wild-type f r u i t f l i e s with pieces of DNA within the 23 appropriate de f i c i e n c y i n t e r v a l . A DNA fragment containing the Suvar(3)7 locus was i d e n t i f i e d by i t s a b i l i t y to enhance white variegation i n In(l)w*4. Suvar(3)7 encodes a 932 amino-acid deduced protein sequence with 5 p o t e n t i a l DNA-binding z i n c -fingers of the Cys 2-His 2 type. Unlike the zinc-fingers found i n t r a n s c r i p t i o n f a c t o r s , the Suvar(3)7 motifs are separated from each other by 40-107 amino-acids. The zinc-fingers of Suvar(3)7 may serve to bind and draw together r e l a t i v e l y d istant DNA sequences for packaging, perhaps at s c a f f o l d attachment s i t e s (Reuter et al., 1990). The cloning of Su(var) genes extends the scope of genetic studies on PEV and i n the future w i l l be of cardinal importance i n a genetic/biochemical d i s s e c t i o n of chromatin structure. 24 CHAPTER 2 INTRODUCTION The genetics and cytology of region 31 on the l e f t arm of chromosome 2 has not been i n t e n s i v e l y studied; however, several suppressor of p o s i t i o n - e f f e c t variegation (Su(var)) mutations have been l o c a l i z e d to t h i s region ( S i n c l a i r et al. 1983, 1991; Wustmann et al. 1989; Reuter et al., 1982). The most extensively researched Su(var) locus (see "Literature Review"), Suvar(2)l, has been de f i c i e n c y mapped to 31A-D (Reuter et al., 1982; Wustmann et a l . , 1989; S i n c l a i r et al., 1991). At l e a s t 13 a l l e l e s of t h i s locus have been reported. Suvar(2)l a l l e l e s display a range of phenotypes. Some mutants are homozygous l e t h a l , while others are only semi-l e t h a l . H e t e r o a l l e l i c combinations of Suvar(2)l mutations are also semi-lethal. This r e s u l t s from a marked reduction i n the numbers of males. The su r v i v a l of h e t e r o a l l e l i c females i s only s l i g h t l y affected, but such females lay no eggs. The same sexual dimorphism i s observed amongst mutation-bearing hemizygotes: males die and females are infecund. However, unlike mutant hemizygotes, f l i e s heterozygous f o r any two Suvar(2)l mutations also display a red-brown eye phenotype and held-out wings ( S i n c l a i r et al., 1991; Brock, 1989). The difference i n phenotype between mutant hemizygotes and f l i e s bearing h e t e r o a l l e l i c combinations of Suvar(2)l a l l e l e s suggests that mutations at the Suvar(2)l locus may a l t e r the function of the wild-type gene product ( S i n c l a i r et al., 25 1991). Three other dominant Su(var) mutations have also been mapped within region 31 using t h e i r suppressor phenotypes and secondary s t e r i l e or l e t h a l phenotypes (summarised i n Figure 1 and Table 1; S i n c l a i r et al., 1991). Recombination studies, based on suppressor phenotypes, place Su(var)204 and Su(var)207 0.5-1.0 cM to the l e f t (centromere-distal) of Jammed, while the l e t h a l phenotype of Su(var)216 maps to the r i g h t . A l l three mutations are uncovered by Df(2L)J2, which i s deleted f o r region 31. Each mutation can be further positioned by d e f i c i e n c i e s which p a r t i t i o n region 31 in t o several genetic subregions. Both Su(var)204 and Su(var)207 map to the same i n t e r v a l delimited by the d i s t a l breakpoints of Df(2L)J39 and Df(2L)J77 (Figure 1). However, Su(var)204 and Su(var)207 f u l l y complement each other. The mutation Su(var)216 f a i l s to complement Df(2L)J27, placing i t i n an e n t i r e l y d i f f e r e n t d e f i c i e n c y i n t e r v a l (Figure 1). Remarkably, Su(var)216, Su(var)207, and Suvar(2)l, d i s p l a y intergenic e f f e c t s when combined i n trans. These include male sem i - l e t h a l i t y , female infecundity, and the red-brown eye-colour phenotype seen i n some homozygotes (Brock, 1989; S i n c l a i r et al., 1991). The close proximity of these mutations on the chromosome and t h e i r phenotypic interactions might indicate some common o r i g i n or shared function ( S i n c l a i r et al., 1983). Although most f u n c t i o n a l l y r e l a t e d genes are not clustered, several exceptions have been reported (Karch et 26 Table 1: Summary of mapping r e s u l t s involving region 31 deletions and Su(var) mutations. Data summary = male v i a b i l i t y / f e m a l e f e r t i l i t y ; v= v i a b l e , 1= l e t h a l , sl= semi-l e t h a l (<15% of expected progeny); f= female f e r t i l e , s= female s t e r i l e (no eggs produced). Deletion Su(var) mutation Su(var)204 Su(var)207 Su(var)216 Suvar(2)l Df(2L)J2 v/s v/s 1/1 1/s Df(2L)J39 v/s 1/1 1/1 v/f Df(2L)J77 v/f v/f 1/1 v/f Df(2L)J27 v/f v/f 1/1 v/f Df(2L)Jl06 v/f v/f 1/1 v/f 28 al. 1985; Kaufman et al., 1980; Spradling et al., 1980; K a r l i k et al., 1984). Equally p l a u s i b l e , however, i s that the dominant mutations represent a random sampling of the estimated 120-150 Su(var) l o c i (Wustmann et al., 1989) i n the Drosophila genome. Two recessive suppressors of p o s i t i o n - e f f e c t variegation (PEV), each represented by a sing l e a l l e l e , have also been i d e n t i f i e d i n region 31 ( S i n c l a i r et al., 1991). One, mfs48 (Sandler, 1977), i s located i n 31E while the other, wavoid-like (wdl; Sandler, 1977), i s i n the i n t e r v a l 31F-32A. The number and chromosomal d i s t r i b u t i o n of recessive suppressors of PEV i s not known; therefore, l i k e the dominant suppressor mutations, the presence of two recessive su(var) l o c i i n region 31 might be eith e r coincidental or of functional s i g n i f i c a n c e . The two recessive suppressors of PEV are part of another group of genes. Sandler and his colleagues (Sandler, 1977; Lindsey et al., 1980) proposed that daughterless (da), hold-up (hup), wavoid-like (wdl), mfs48, daughterless-abo-like (dal) and abnormal oocyte (abo) constitute a c l u s t e r of f u n c t i o n a l l y r e l a t e d genes i n 31E-32B. Excluding mfs48, which cannot be tested, mutant a l l e l e s of these l o c i are a l l s e n s i t i v e to the i n t r a c e l l u l a r l e v e l s of heterochromatin (Sandler, 1977), a property shared by dominant modifiers of PEV (Reuter et al., 1982; S i n c l a i r et al., 1991). Thus, some members of the Sandler c l u s t e r might p a r t i c i p a t e i n the establishment or 29 maintenance of the gross structure of chromatin. A l l together, the a v a i l a b l e dominant and recesssive mutations suggest that as many as s i x l o c i a f f e c t i n g PEV might reside i n region 31. A comprehensive genetic analysis of region 31 was undertaken with the following objectives: (1) to determine the number of Su(var) l o c i that e x i s t i n t h i s region, (2) to i s o l a t e a d d i t i o n a l a l l e l e s of Su(var)/su(var) l o c i represented by single mutations, (3) to obtain n u l l mutations at Su(var) l o c i f o r which there are putative gain-of-function mutations, and (4) to examine the nature of the genes flanking the Su(var)/su(var) l o c i . This chapter extends the deficiency mapping of 64 mutations previously i s o l a t e d i n the G r i g l i a t t i laboratory (Figure 2; see Brock, 1989). In addition, i t describes the i s o l a t i o n of 57 new mutations within a sub-interval of the cytogenetic region 31 delimited by the centromere-distal breakpoint of Df(2L)J77 and the centromere proximal breakpoint of Df(2L)Jl06. The Df(2L)J77-Df(2L)Jl06 sub-i n t e r v a l was i n t e n s i v e l y analysed because the suppressors Su(var)216 and mfs48 reside i n that region (Brock, 1989; S i n c l a i r et al., 1991), and the region was s u f f i c i e n t l y small to permit a thorough search f o r ad d i t i o n a l a l l e l e s at these l o c i . 30 MATERIALS AND METHODS Mutant strainst The chromosomal d e f i c i e n c i e s used i n t h i s study are l i s t e d i n Table 2. Df(2L)J2, Df(2L)J27, and Df(2L)J39 are described i n Mange and Sandler (1973) and Sandler (1977). F l i e s bearing Df(2L)J39 are female s t e r i l e , but not infecund. Both sexes display a Minute phenotype when heterozygous f o r Df(2L)J39. A stock was maintained by outcrossing Df(2L)J39/SM5 or Df(2L)J39/bwVDe2 males to bwVDe2/SM5 females each generation. Df(2L)J77 and Df(2L)J106 were provided by J. Lengyel (see Salas and Lengyel, 1984). The origins of mutants previously reported to be i n region 31 are summarized i n a composite table i n the Results section (Table 3). Several recessive l e t h a l and/or female s t e r i l e mutants were provided by L. Sandler: da, dal, wdl, hup, mfs48 and 1(2)54 (see Mange and Sandler, 1973; Sandler, 1977). The mutant da2 was obtained from C. Cronmiller. The following female s t e r i l e mutants were provided by T. Schupbach: erratic (err) mat(2)earlyQM47, mat(2)synPJ50, trk, PI23, RU26, and DG25 (Table 3; see Schupbach and Wieschaus, 1989). Female-sterile-2-rosy-4, (fs(2)ry4), which i s female infecund, was provided by A. Spradling. Su(var)204 and Su(var)207 were induced i n the G r i g l i a t t i laboratory ( S i n c l a i r et al., 1983) and are described i n d e t a i l i n S i n c l a i r et al. (1991). Suvar(2)l01 (Reuter et al., 1982) was provided by G. Reuter. A summary of mutations i s o l a t e d i n the G r i g l i a t t i lab between 1983 and 1988 appears i n Figure 2. From 5000 second chromosomes tested, S i n c l a i r , Kafer, Camfield, and G r i g l i a t t i i s o l a t e d 16 gamma-radiation induced mutations which were l e t h a l i n trans with Df(2L)J2 ( c i t e d i n Brock, 1989). Brock (1989) i s o l a t e d 40 ethyl methanesulphonate (EMS) induced mutations that were l e t h a l i n trans with Df(2L)J2 from amongst 1484 second chromosomes. Harrington (1990) i s o l a t e d 8 gamma-rad i a t i o n induced mutations that were l e t h a l i n trans with Df(2L)J27 from amongst 5000 chromosomes. S i n c l a i r (unpublished) reverted the crumpled wing phenotype of the neomorphic mutation Jammed-34e (J34a) according to the method of Salas and Lengyel (1984) and obtained 18 J* s t r a i n s . Culture conditionsi F l y cultures were maintained at 22° on corn-meal sucrose Drosophila medium supplemented with 0.04% Tegosept as a mould i n h i b i t o r . Except where indicated, experiments were performed at 25°. Eye pigment assessment: The a b i l i t y of a mutation to suppress the i n a c t i v a t i o n of the white gene i n the variegating rearrangement In(l)w™4 was assessed v i s u a l l y . In(l)w*4/Y; mutant/CyO males were mated to In(l)w*4/In(l)yf4; +/+ females. Mutation bearing progeny with pigment l e v e l s less than approximately 70% of wild-type l e v e l s were scored as non-suppressors. Progeny were observed i n p a r a l l e l with In(l)wm4/In(l)w*4 f l i e s which had 10-30% of wild-type pigment l e v e l s , and with suppressed f l i e s previously shown (Brock, 1989) to have approximately 70% pigment. 32 33 Cytology: Males bearing balanced chromosome rearrangements were crossed to wild-type Oregon-R females and the of f s p r i n g raised at 18°. Sa l i v a r y glands from t h i r d i n s t a r larvae were dissected i n Drosophila s a l i n e and f i x e d i n 45% a c e t i c acid. The chromosomes were then squashed i n acetic a c i d , water and l a c t i c acid (3:2:1), or stained i n lacto-aceto-orcein (Yoon et al., 1973) and then squashed. Chromosomes were examined under phase contrast optics and interpreted according to the revised map of Bridges (Lefevre, 1976). Ethyl methanesulphonate (EMS) mutagenesis: Two independent sets of experiments were conducted to i s o l a t e EMS induced mutations (Fig. 3). In both sets of experiments, homozygous b pr cn males were mutagenized with ethyl methanesulfonate (0.025 M) by the method of Lewis and Bacher (1968). These males were mated en masse to Tft/In(2LR)CyO, dplvI Cy pr cn2 v i r g i n females at 22° (Figure 3). Male progeny with a b pr cn chromosome balanced over CyO were i d e n t i f i e d by a curly-wing phenotype and pr cn eyes. Each male was i n d i v i d u a l l y mated at 29° to 3-5 v i r g i n s from stocks of e i t h e r Df(2L)J27/CyO or Df(2L)J106/CyO (two separate experiments). The presence of a recessive l e t h a l mutation on the marked chromosome was indicated by the absence of the Jb pr cn/deficiency ( s t r a i g h t -winged) class of f l i e s amongst the progeny. To determine whether any of the mutants were temperature s e n s i t i v e , the tes t s were repeated at 22°. Hybrid dysgenesis screen: The i s o l a t i o n of P transposable 34 Figure 3. E M S mutagenesis sc reen . Males Females E M S b pr cn Df(2L)J C y O b pr cn C y O Tft b m a s s m a t i n g s p a i r - w i s e m a t i n g s b pr cn Df(2L)J S c o r e a b s e n c e of t h i s c l a s s 35 element induced mutations i n region 31 was accomplished as follows (Figure 4). Homozygous b pr cn males were mated en masse to Sp/CyO; Sb A2-3/TM6, Ubx females. Males of the genotype b pr cn/CyO; Sb A2-3/+ were mated at 18° to females homozygous for the Birmingham second chromosome and ry506. The A2-3 locus provides an active source of P transposase, while the Birmingham chromosome c a r r i e s 13 p a r t i a l l y deleted P elements. Males of genotype b pr cn/Birm2; Sb A2-3/ry506 were mass mated at 18° to Tft/CyO females, b pr cn/CyO male progeny were then i n d i v i d u a l l y mated to 3-5 Df(2L)J77/CyO females. A l e t h a l mutation was indicated by the absence of b pr cn/Df(2L)J77 f l i e s i n the f i n a l cross. Deficiency mapping and complementation tests : F i r s t , each EMS, gamma-ray, and P element l e t h a l or s t e r i l e mutation was l o c a l i z e d to a sub-interval of cytogenetic region 31 by i t s f a i l u r e to complement a battery of d e f i c i e n c i e s f o r v i a b i l i t y or f e r t i l i t y . Once t h i s was accomplished, mutations within each sub-i n t e r v a l were tested f o r a l l e l i s m v i a inter se complementation anal y s i s . Pairs of mutants, maintained as hétérozygotes with CyO, were mated. F a i l u r e to complement was based on the absence of Cy* f l i e s amongst at l e a s t 50 Fj progeny, or the detection of a s t e r i l e or v i s i b l e phenotype amongst Cy* f l i e s . For l e t h a l mutations which complement each other, 33% of the t o t a l progeny were expected to be Cy*. At l e a s t 50 f l i e s were scored to ascertain that two mutations complemented each 36 Figure 4. P element mutagenesis screen . Males Females b pr cn . b pr cn ' + X Sp ; 2~3Sb m a s s CyO TM6 UbX m a t i n g s b pr cn CyO b pr cn birm b pr cn CyO 2-3Sb 2-3Sb birm ; ry birm ' ry Tft CyO + + Df(2)J77 CyO b pr cn CyO Df(2)J77 . _*_ CyO ' + Df(2)J77 S c o r e a b s e n c e of t h i s c l a s s m a s s mat i n g s m a s s mat i n g s p a i r - w i s e m a t i n g s 37 other. When a deficiency sub-interval contained a large number of mutants, a single representative a l l e l e from each complementation group i d e n t i f i e d i n e a r l y rounds of crosses was used as a t e s t e r s t r a i n i n subsequent crosses. For some s t e r i l e mutations, f a i l u r e to complement a s i n g l e a l l e l e of a series was used as the c r i t e r i o n f o r i n c l u s i o n i n the complementation group. Lethal phase analysis: The p r i n c i p a l time at which hemizygous mutants die was determined by mating mutant/+ males to Df(2L)JR16/CyO females. A f t e r 72 hours, the parents were transferred to bottles with p l a i n agar medium covered with a smear of yeast paste. A f t e r several hours of egg deposition, the eggs were transferred to t h i n s t r i p s of construction paper and counted. The paper s t r i p s were placed on the surface of regular cornmeal/agar medium. Two to three days l a t e r , the construction paper was removed from the v i a l and the unhatched eggs were counted. Light eggs displayed no discernable development and were assumed to be u n f e r t i l i z e d . Dark eggs represented dead embryos. The difference between the number of hatched eggs and the number of pupae indicated the extent of l a r v a l death. Pupal death was determined by counting the un-eclosed f l i e s . 38 RESULTS Cytogenetics The region deleted by Df(2L)J2 defines the physical bounds of t h i s a nalysis. Df(2L)J2 extends from 31A to 32A, a region that encompasses approximately 43 bands. Several smaller d e f i c i e n c i e s further subdivide t h i s segment of the chromosome int o a number of sub-regions (Figure 1 and Figure 5 ). In polytene chromosomes the banding pattern within regions 31A and 3IF i s d i s t i n c t i v e , and rearrangement breakpoints can be determined with reasonable accuracy. In contrast, the banding pattern i n the 31B-E i n t e r v a l i s p a r t i c u l a r l y unclear (see Lefevre, 1976). In view of t h i s d i f f i c u l t y , there i s some uncertainty associated with the assignment of d e f i c i e n c y breakpoints. The approximate locations of the d e f i c i e n c y breakpoints are shown i n Figure 5 and Table 2. In addition to the previously i d e n t i f i e d d e f i c i e n c i e s Df(2L)J2, Df(2L)J39, Df(2L)J27, Df(2L)J77, and Df(2L)Jl06), s i x a d d i t i o n a l d e f i c i e n c i e s were i d e n t i f i e d amongst the gamma-ra d i a t i o n induced J revertants i s o l a t e d by S i n c l a i r (see Materials and Methods). Df(2L)JRl, Df(2L)JR3, Df(2L)JR4, Df(2L)JRll Df(2L)JR16, and Df(2L)JRl7 were a l l l e t h a l with numerous complementation groups (see below). Df (2L)JR11 i t s e l f was female s t e r i l e and was not investigated i n d e t a i l . The approximate c y t o l o g i c a l breakpoints of these d e f i c i e n c i e s are shown i n Figure 5. To order the r e l a t i v e breakpoints of the 10 d e f i c i e n c i e s 39 Figure 5. The c y t o l o g i c a l extent of d e f i c i e n c i e s i n region 31. Dashed l i n e s indicate the uncertainty associated with each breakpoint. Deficiency names have been shortened f o r c l a r i t y ; f u l l names appear i n the text. 40 41 TABLE 2. Cyt o l o g i c a l l i m i t s and o r i g i n s of chromosomal rearrangements. Rearrangement Reference Comments Df(2L)J2 Mange and Sandler (1973) Df(2L)31A3;32A Df(2L)J39 Mange and Sandler (1973) Df(2L)31D;32B female s t e r i l e Df(2L)J27 Mange and Sandler (1973) Df(2LJ31D-31E Df(2L)J77 Salas and Lengyel (1984) Df(2L)3ID;3IE Df(2L)J106 Salas and Lengyel (1984) Df(2L)31D;31E Df(2L)JRl S i n c l a i r ; This study Df(2L)31B;31D Df(2L)JR3 S i n c l a i r ; This study Df(2L)31D;31F Df(2L)JRl6 S i n c l a i r ; This study In(2L)30C-D;3lE associated with a defic i e n c y i n 3 IE Df(2L)JRl7 S i n c l a i r ; This study not v i s i b l e Df(2L)G2 S i n c l a i r ; This study Df(2L)31D;31F Df(2L)JRll S i n c l a i r ; This study female s t e r i l e 42 more accurately, genetic and c y t o l o g i c a l data were compared. A l l e l e s from each complementation group within region 31 were defi c i e n c y mapped (Appendix 1), r e s u l t i n g i n the i d e n t i f i c a t i o n of 15 small deficiency sub-intervals (Figure 6, Figure 7, Figure 8). The Df(2L)JRl chromosome complements da, but f a i l s to complement fs(2)ry4 (Figure 6; Appendix 1). These l o c i have been mapped by in situ hybridization to 3 IE (Cronmiller et al., 1988) and 31B (Spradling, pers. comm.; N. Clegg and I.P. Whitehead, unpublished), respectively. Thus, Df(2L)JRl must extend as f a r as 3IB, but does not delete sequences i n 3IE. Contrary to the c y t o l o g i c a l analysis of Sandler (1977), Df(2L)J39 cannot extend past 31B, since Df(2L)J39/fs(2)ry4 females lay eggs, but hemizygous Df(2L)JRl/fs(2)ry4 f l i e s do not. Df(2L)JR3 and Df(2L)JR4 delete the same complementation groups. C y t o l o g i c a l l y , Df(2L)JR3 appears to be s i m i l a r to Df(2L)Jl06, but complementation data (Figures 6, 7, and 8) indicate s l i g h t differences. The Df(2L)JR3 chromosome i s deleted f o r 3IE, but normal p a i r i n g of homologues i s cons i s t e n t l y disrupted throughout the region 30D-31E. Thus, the d e f i c i e n c y may be associated with a small, paracentric inversion i n t h i s region. Df(2L)JR17 i s not c y t o l o g i c a l l y v i s i b l e . A s i n g l e deficiency, Df(2L)G2, was i s o l a t e d i n a gamma-i r r a d i a t i o n screen f o r a d d i t i o n a l a l l e l e s of Su(var)216 (data not shown). I t deletes a large portion of the 31 region. 43 4 Hi b Genetically, i t uncovers the same mutations as the centromere d i s t a l end of Df(2L)J39. Genetic screens This genetic characterization of region 31 incorporates data from previous screens (see Materials and Methods) i n addition to the r e s u l t s from three new screens. The r e s u l t s of the l a t t e r are described below. Five thousand EMS-mutagenized second chromosomes were examined f o r new hemizygous l e t h a l mutations within the bounds of Df(2L)Jl06 (Screen 1). Twenty-seven mutations were recovered. From 10000 EMS mutagenized chromosomes an add i t i o n a l 26 mutations were recovered that were l e t h a l i n trans with Df(2L)J27 (Screen 2). F i n a l l y , 4 hemizygous l e t h a l P element induced mutations which f a i l to complement Df(2L)J77 were i s o l a t e d from amongst 12500 mutagenized chromosomes (Screen 3). The frequencies of i s o l a t i o n of mutations were 0.0054, 0.0026, and 0.0003 f o r Screens 1, 2, and 3, respectively. The new mutants, which were o r i g i n a l l y i d e n t i f i e d as hemizygous l e t h a l s at 29°, were retested and found to be l e t h a l at 25°. Mutants i s o l a t e d i n Screen 2 were also tested at 22°, but no temperature-sensitive mutations were detected. No se x - s p e c i f i c mutants were recovered, nor were any dominant suppressors of PEV. Brock (1989) described the i s o l a t i o n of 16 gamma-ray induced mutations and 39 EMS induced mutations which f a i l e d to complement Df(2L)J2 at 29°. Amongst the EMS induced mutants, two mutations (E20 and A65) were temperature s e n s i t i v e : hemizygotes raised at 25° survived more frequently than f l i e s r a i s e d at 29°. The mutations i s o l a t e d i n each of the above screens were placed i n t o complementation groups along with a l l other mutations previously shown to map i n region 31. General complementation In t o t a l , one hundred and twenty-one hemizygous l e t h a l mutants were assigned to 43 d i f f e r e n t complementation groups (Figures 6, 7, and 8). Thirty-seven were new a l l e l e s of 7 d i f f e r e n t mutations i d e n t i f i e d i n other studies (Table 3); the remainder are d i s t r i b u t e d amongst 36 new complementation groups (Figures 6, 7, and 8). In addition, 10 hemizygous v i a b l e revertants of the J34e mutant a l l e l e were recovered amongst the 18 J revertants recovered by S i n c l a i r (see Materials and Methods). Since these revertants are hemizygous v i a b l e , and they do not a f f e c t any known complementation groups other than J, they are l i k e l y to be "point mutation" revertants. There are at l e a s t 12 l o c i from the region f o r which no new mutant a l l e l e s were recovered (Table 3). Several of these l o c i were o r i g i n a l l y i d e n t i f i e d by c r i t e r i a other than hemizygous l e t h a l i t y . These include a l l e l e s of the maternal 48 Table 3. Previously described mutations i n region 31. Mutation Comments Reference No. New A l l e l e s Suvar(2)l hemizygous male l e t h a l , female s t e r i l e Su(var)204 hemizygous female s t e r i l e Su(var)207 hemizygous l e t h a l Su(var)216 hemizygous l e t h a l bsk da dal DG25 err fs(2)ry4 hup J 1(2)54 mat(2) synPJSO mat(2) earlyQM4 7 zygotic l e t h a l ; c u t i c l e defects p l e i o t r o p i c zygotic l e t h a l homozygous mothers lay small eggs hemizygous l e t h a l ; maternal e f f e c t s t e r i l e female s t e r i l e ; P element i n s e r t wings held up neomorphic mutation; wings crumpled hemizygous l e t h a l ; see mat(2)earlyQM4 7 homozygotes arrest i n s y n c i t i a l blastoderm maternal e f f e c t s t e r i l e ; p r e s y n c i t i a l arrest i n homozygotes Reuter et al., 1982 S i n c l a i r et al., 1983 S i n c l a i r et al., 1983 S i n c l a i r et al., 1983 Nusslein-Volhard et al., 1984 see Cline, 1989 Sandler 1977 T. Schupbach, pers. comm. Schupbach and Wieschaus, 1989 A. Spradling, pers. comm. Sandler, 1977 see Lindsley and G r e l l , 1968 Sandler, 1977 Schupbach and Wieschaus, 1989 Schupbach and Wieschaus, 1989 0 0 8 0 9 0 7 0 10 ( see 1(2)54 49 Mutation Comments Reference No. New A l l e l e s mfs48 hemizygous i n v i a b l e ; c e n t r i o l e segregation defect i n spermatids Lindsley et al., 1980 3 PI23 hemizygous s t e r i l e ; T. Schiipbach, pers. comm. 0 pirn Nusslein-Volhard et al., 1984 1 RU26 homozygous females lay collapsed eggs T. Schiipbach/ pers. comm. 0 trk hemizygous female s t e r i l e ; terminal group mutant Schiipbach and Wieschaus, 1986 0 wdl wavy wings Sandler, 1977 0 50 e f f e c t female s t e r i l e mutants PI23, RU26, trk, and mat(2)synPJ50, a l l of which are located i n the in t e n s i v e l y mutagenized region between the d i s t a l breakpoint of Df(2L)J77 and the centromere proximal breakpoint of Df(2L)J27 (Figure 7). However, addit i o n a l a l l e l e s of the homozygous s t e r i l e mutants mat ( 2 ) earlyQM4 7, err, and DG25 were i d e n t i f i e d (Figure 7), i n d i c a t i n g that these l o c i have e s s e n t i a l functions not r e s t r i c t e d to the female germline. In accord with d e t a i l e d studies of other regions (see Lefevre and Watkins, 1986), the t o t a l number of complementation groups i d e n t i f i e d i n the region 31A-32A (53) roughly corresponds to the t o t a l number of bands deleted by Df(2L)J2 (43). As i n other studies (e.g. Lasko and Pardue, 1988), the pattern of complementation associated with l o c i i n each of the def i c i e n c y i n t e r v a l s was generally simple. For non-complementing mutations, e i t h e r there were no surviving transheterozygotes, or the f l i e s displayed a v i s i b l e or s t e r i l e phenotype. In a few cases, there were deviations from t h i s simple complementation pattern, with one or more mutations displaying apparent intragenic complementation. These cases probably involve haplo-specific l e t h a l mutations with s u f f i c i e n t r e s i d u a l a c t i v i t y to permit i n t e r a l l e l i c complementation (Nash and Janca, 1983). They are noted below, as s p e c i f i c deficiency i n t e r v a l s are discussed. 51 Suppressor l o c i d i s t a l to Df(2L)J27 Three dominant Su(var) l o c i (Suvar(2)l, Su(var)204, and Su(var)207) have been mapped by recombination and deletion analyses centromere d i s t a l to Df(2L)J27 (see Figure 1). Complementation analysis with the EMS and gamma-radiation induced a l l e l e s c i t e d i n Brock (1989) d i d not reveal any new l e t h a l a l l e l e s of these l o c i . An a d d i t i o n a l screen of 20000 chromosomes also f a i l e d to detect any P element induced recessive l e t h a l a l l e l e s of Su(var)214, although several trans-acting second s i t e enhancers of p o s i t i o n - e f f e c t variegation were recovered (Whitehead and Clegg, data not shown). Mapping with new d e f i c i e n c i e s indicates that Su(var)207 must be assigned to a new lo c a t i o n . F l i e s of genotype Df(2L)JRl/Su(var)207 are v i a b l e and f e r t i l e (Appendix 1), yet Df(2L)J39/Su(var)207 f l i e s die and Df(2L)J2/Su(var)207 f l i e s are female s t e r i l e and male semi-lethal (Table 1). Since Df(2L)JR1 deletes J and extends further towards the telomere than Df(2L)J39 (Figure 6), the l e t h a l phenotype of Df(2L)J39/Su(var)207 must map centromere proximal to J. This i s not consistent with recombination mapping which positions Su(var)207 to the l e f t of J ( S i n c l a i r , unpublished). The ri g h t breakpoint of Df(2L)J39 extends further toward the centromere than that of Df(2L)J2. Therefore, a l i k e l y explanation of the genetic and recombination data i s that the l e t h a l mutation i s a second s i t e mutation on the Su(var)207 chromosome which maps 52 outside the confines of Df(2L)J2, but which i s within the region deleted by Df(2L)J39. Since Df(2L)J2/Su(var)207 females are s t e r i l e , the Su(var)207 mutation has been re-assigned to the d e f i c i e n c y i n t e r v a l centromere d i s t a l to Df(2L)JRl. Deficiency mapping r e s u l t s suggest Su(var)204 i s not i n i t s previously reported p o s i t i o n (Brock, 1989). Female f l i e s of genotype Df(2)J2/Su(var)204, Df(2L)J39/Su(var)204, or Df(2L)G2/Su(var)204, are a l l s t e r i l e . In contrast, females of genotype Df(2L)JRl/Su(var)204 are f e r t i l e . Df(2L)JRl deletes J, and extends beyond the centromere d i s t a l breakpoint of Df(2L)J39; therefore, the female s t e r i l e mutation on the the Su(var)204 chromosome cannot be located to the l e f t of J . Recombination mapping, however, positions the Su(var) suppressor phenotype to the l e f t of J. The female s t e r i l e mutation resides within region 31, centromere proximal to Df(2L)J106, but i t s l o c a t i o n has not been investigated further. Hence no phenotype other than suppression i s d i r e c t l y a t t r i b u t a b l e to Su(var)204, and the mutation cannot be positioned by deficiency mapping. Non-suppressor l o c i d i s t a l to Df(2L)J27 Six new recessive l e t h a l complementation groups were i d e n t i f i e d between the centromere d i s t a l breakpoint of Df(2L)J2 and Df(2L)J27 (Figure 6). The d i s t r i b u t i o n of a l l e l e s amongst the various screens performed i n the region i s l i s t e d i n Appendix 2. With the exception of C98 and B149 each 53 complementation group contains a s i n g l e a l l e l e . The complementation pattern amongst B149 a l l e l e s was previously determined by Brock (1989). A l l three C98 a l l e l e s were l e t h a l i n pair-wise combinations (Appendix 3). No recessive l e t h a l a l l e l e s of trk or fs(2)ry4 were recovered. A trk mutation provided by T. Schupbach was mapped to deficiency sub-interval 4 (Figure 6) based on hemizygous female s t e r i l i t y . The fs(2)ry4 locus was also d e f i c i e n c y mapped based on hemizygous female infecundity rather than recessive l e t h a l i t y . fs(2)ry4 complements Df(2L)G2 f o r f e r t i l i t y . I t also complements Df(2L)J39, based on the observation that Df(2L)J39/fs(2)ry4 females lay eggs. Df(2L)J39/fs(2)ry4 females lay eggs that f a i l to hatch, whereas fs(2)ry4/Df(2L)J2 and fs(2)ry4/Df(2L)JRl females are infecund. L o c i uncovered bv Df(2L)J27 The majority of mutants described i n t h i s study f a i l to complement Df(2L)J27. The region spanned by Df(2L)J27 has been divided i n t o 8 sub-intervals by a l l the a v a i l a b l e d e f i c i e n c i e s (Figure 7). The o r i g i n and d i s t r i b u t i o n of mutations amongst the region 31 screens i s given i n Appendix 2 and s a l i e n t aspects are described below. Sub-interval 5 : One large complementation group, 24-127, deficiency maps to t h i s i n t e r v a l . The recessive l e t h a l mutation 24-127 was induced by gamma-irradiation ( c i t e d i n 54 Brock, 1989) and i s associated with a p e r i c e n t r i c inversion with breakpoints i n 3ID and region 51. Eleven mutations f a i l to complement the 24-127 mutation. Juter se crosses have been performed between two subsets of mutants, and mutant A141 was tested with both subsets (Table 4). Amongst the f i r s t set of crosses, only one combination of a l l e l e s was v i a b l e and f e r t i l e . In crosses between mutations E56 and A63 approximately 25% of the expected number of h e t e r o a l l e l i c progeny survive to adulthood. In the second set of crosses A141/E2-13 progeny also survive. These complementation patterns are probably a r e s u l t of a l l e l e s encoding p a r t i a l l y functional products which, although mutant, can rescue the l e t h a l phenotype. Thus, our analysis i d e n t i f i e s only a single locus associated with the l e f t breakpoint of the inversion associated with 24-127. Sub-interval 6: Four l e t h a l mutations mapped within t h i s subinterval and a l l four f a i l to complement each other (Table 5), thereby i d e n t i f y i n g a s i n g l e complementation group designated the El-13 gene. Sub-interval 7: Three newly described complementation groups, plus a fourth defined by the single RU26 mutation (mapped on the basis of hemizygous female s t e r i l i t y ) i s o l a t e d by T. Schiipbach, have been i d e n t i f i e d i n t h i s region. Sub-interval 8i Two independent complementation groups, bsk and DG25, have been positioned i n t h i s segment. This region may also include a t h i r d locus, namely, that defined by J 55 Table 4. Complementation data for mutations i n the same deficiency i n t e r v a l as the mutation 24-127. Ratios represent the number of Cy to straight-winged progeny recovered from the cross mutantl/CyO X mutant2/CyO 24-127 A63 A141 C35 E56 H30 El 5 A63 94:0 A141 176:0 154:0 C35 118:0 156:0 294:0 E56 H30 El 5 104:0 228:0 460:0 437:51 148:0 86:0 102:0 158:0 n.d. 165:0 180:0 n.d. - 218:0 n.d. mm n.d. A141 E2-1 E2-12 24-127 A141 E2-1 E2-12 E2-13 E2-32 E2-42 176:0 242:0 226:0 399:0 138:0 262:0 E2-13 E2-32 E2-42 339:0 580:0 211:0 210:77 199:0 249:0 328:1 225:0 n.d. 294:0 n.d. 343:0 - 245:0 164:0 n.d. 56 matrix mutations. No new a l l e l e s of bsk were recovered. The o r i g i n a l bsk mutation (Niisslein-Volhard et al., 1984) was mapped on the basis of hemizygous l e t h a l i t y . The o r i g i n a l DG25 mutant i s homozygous s t e r i l e , laying t i n y eggs (T. Schiipbach, pers. comm.). When t h i s mutant i s crossed to Df(2L)J27, less than 40% of the expected number of hemizygous f l i e s survive and they are female s t e r i l e . Seven putative a l l e l e s of t h i s locus were i s o l a t e d : 4 were EMS induced and three were gamma-ray induced (Appendix 2). Only f i v e of the new mutations have been studied i n d e t a i l . Their pattern of complementation i s presented i n Table 6. In inter se crosses, these f i v e a l l e l e s produce a spectrum of abnormalities. H e t e r o a l l e l i c mutants have wavy-wings with a wax-like appearance and are female s t e r i l e and semi-lethal (Table 6). Some a l l e l i c combinations have rough eyes and/or missing or damaged macrochaetes. Mutant hemizygotes that eclose have the same phenotypes as h e t e r o a l l e l i c mutants. Two less well studied mutations, E2-5 and E2-21, complement the other DG25 a l l e l e s f o r v i a b i l i t y and f e r t i l i t y , but not fo r other v i s i b l e phenotypes. Both mutations cause a wavy, waxy wing phenotype when heterozygous f o r any of the other DG25 a l l e l e s . Also, when crossed to Df(2L)JRl6, a few hemizygous mutants eclose. These hemizygotes have rough eyes and waxy wings s i m i l a r to those observed amongst DG25 a l l e l e s . These phenotypes suggest that E2-5 and E2-21 are weak a l l e l e s of DG25, although t h e i r complete l e t h a l i t y i n trans with each 58 Table 6. Complementation pattern amongst a l l e l e s of the DG25 locus. Ratios above the matrix diagonal represent the proportion of Cy to straight-winged progeny i n crosses between mutants heterozygous f o r CyO. The s t e r i l i t y (S) or f e r t i l i t y (F) of h e t e r o a l l e l i c mutant females i s noted beneath the matrix diagonal. 23-127 29-142 25-159 D22 DG25 C93 13-117 23-127 - 60:10 597:22 98:3 503:97 226:12 92:0 29-142 S - 375:12 157:38 n.d. 98:18 125:84 25-159 S S - 62:19 n.d. 273:19 46:22 D22 S S S - n.d. 160:33 331:148 DG25 s n.d n.d. n.d. - n.d. 423:184 C93 s S S S n.d. - 234:101 13-117 s F F F F F — Cross Progeny 23-127 X Df(2L)J27 424:24 29-142 X Df(2L)J27 464:57 25-159 X Df(2L)J27 1134:26 D22 X Df(2L)J106 145:0 DG25 X Df(2L)JRl 448:0 C93 X Df(2L)Jl06 232:11 13-117 X Df(2L)J27 779:0 59 other i s hard to reconcile with t h i s view. One a l l e l e of DG25, 23-1273, i s l e t h a l i n trans with 13-117. The mutation 13-117 i s an a l l e l e of the El-13 complementation group, which i s located i n deficiency sub-i n t e r v a l 6 (Figure 7). While 13-117 i s l e t h a l i n trans with 23-127, f l i e s of genotype Df(2L)JR17/13-117 and Df(2L)JR3/13-117 are v i a b l e and f e r t i l e . F l i e s heterozygous f o r 23-127 and other a l l e l e s of El-13 are vi a b l e , suggesting that 23-127 and 13-117 may share a common second s i t e mutation outside region 31. A l t e r n a t i v e l y , the two mutations may i n t e r a c t i n some unknown fashion to cause a l e t h a l phenotype. The A8 mutation, which i s not included i n the a l l e l e s shown i n Figure 7, may also be an a l l e l e of the DG25 locus. Expression of t h i s mutation i s temperature dependent; at 25° t h i s mutation complements the 23-127 a l l e l e , but at 29° the hétérozygote has a wavy-winged phenotype. The precise l o c a t i o n of the J locus remains unknown. I t should l i e within the region of overlap shared by a l l of the J-derived d e f i c i e n c i e s ; however, Df(2L)JRl6 s t i l l expresses J i n some genetic backgrounds. This suggests that the d i s t a l breakpoint of t h i s deficiency may be adjacent to J rather than simply deleting i t . New sequences juxtaposed to the locus might p e r i o d i c a l l y i n a c t i v a t e J creating a euchromatic p o s i t i o n e f f e c t , although t h i s does not explain why a Jammed phenotype i s r a r e l y observed i n some backgrounds. Conservatively, J must be located i n the i n t e r v a l between the 60 d i s t a l breakpoint of Df(2L)JR3 and the proximal breakpoint of Df(2L)JRl (sub-intervals 7+8). Its close proximity to DG25, which also imparts a recessive wing phenotype, may indicate some connection between the l o c i . Inter se crosses between these two sets of mutants did not produce a l e t h a l or any strong v i s i b l e phenotype. However, J i t s e l f i s hemizygous v i a b l e . Thus, the absence of any phenotype i n DG25/J i n d i v i d u a l s i s not conclusive. No hemizygous l e t h a l mutations of J were recovered i n any of the region 31 EMS-mutagenesis screens. Amongst the 18 gamma-induced mutations that revert the J" phenotype, s i x are associated with d e f i c i e n c i e s i n region 31; and two others segregate only i n males, suggesting that they are T(Y;2) translocations. The remaining revertants are hemizygous vi a b l e and f e r t i l e . Thus, the J product may not be e s s e n t i a l for v i a b i l i t y . The two p o t e n t i a l Y;2 translocations may be useful i n p o s i t i o n i n g J, but were not examined cyt o g e n e t i c a l l y . Sub-interval 9: There are two l o c i i n t h i s segment. One i s the zygotic l e t h a l mutation pirn (Figure 7). A s i n g l e a l l e l e of pirn was recovered. Unlike the o r i g i n a l a l l e l e , which causes embryonic death (Nusslein-Volhard et al., 1984), piwF1'15 hemizygotes die predominantly during the l a r v a l stages (Table 7). A few hemizygous progeny even survive to adulthood at 22°. The E2-15 mutation, which defines the second locus within t h i s sub-interval, causes predominantly embryonic l e t h a l i t y i n hemizygotes (Table 7). 61 Table 7. Lethal phases f o r mutations which f a i l t o complement Df(2L)JR16. Mutant hemizygotes should represent 25% of the progeny from the cross mutation/* X Df(2L)JR16/Cy0. Mutant X JR16/CyO % Embryonic L e t h a l i t y % Larval L e t h a l i t y % Pupal L e t h a l i t y % Mutant Adults E1-1E2-16/+ 3 (5/247) 28 (68/247) 5 (10/247) 0 E1-3/+ 23 (106/459) 6 (26/459) 0 0 E1-15/+ 5 (18/439) 18 (79/439) 0 5 (17/342) E2-17/+ 1 (4/426) 28 (119/426) 3 (9/426) 0 E2-15/+ 28 (107/388) 5 (19/388) 1 (3/388) 0 E2-43/+ 2 (4/353) 26 (91/353) 3 (3/353) 0 62 Sub-interval 10: The i n t e r v a l defined by the proximal breakpoints of Df(2L)JRl7 and Df(2L)J16 contains 9 i d e n t i f i e d l o c i . Lethal phases have been determined for several mutations within t h i s i n t e r v a l (Table 7). Amongst these mutations, only El-3 i s l e t h a l i n embryos as a hemizygote. Five of the complementation groups i n t h i s region, El-3, El-12, El-19, El-28, and E2-17 a l l have simple complementation patterns (Appendix 2). The largest complementation group i n the region i s represented predominantly by a l l e l e s which f a i l to complement (Table 8); however, a few f l i e s do survive i n some h e t e r o a l l e l i c combinations (Appendix 3). A l l h e t e r o a l l e l i c survivers are v i a b l e and f e r t i l e without any obvious mutant phenotypes. Three of the complementation groups within t h i s region, da, Su(var)216, and mfs48, were of s p e c i a l i n t e r e s t as known or p o t e n t i a l suppressors of PEV: da: Nine new da a l l e l e s were i s o l a t e d . Two are hemizygous l e t h a l but homozygous v i a b l e . A t h i r d , daE1~21, i s e s s e n t i a l l y hemizygous l e t h a l , but produces a few v i a b l e and f e r t i l e progeny (Table 9). None of the a l l e l e s i s a suppressor of PEV. One second chromosome bearing an a l l e l e of da i s l e t h a l i n combination with a chromosome bearing an a l l e l e of 1(2)54. The two l o c i are separable by deficiency mapping and the e f f e c t i s a l l e l e s p e c i f i c . This suggests that the two mutants may share a common l e t h a l mutation outside region 31. Table 8. Complementation matrix f o r a l l e l e s of the B35 locus. The r a t i o indicates the number of Cy to s t r a i g h t winged progeny from the cross mutantl/CyO X mutant2/CyO. B35 E2-16 13-83 B26 465:0 n.d. 284:0 El-1 240:0 240:27 n.d. E2-16 95:0 - 617:0 E2-31 138:0 290:0 268:2 E2-38 104:0 244:0 n.d. G2-3 118:0 G2-7 156:0 13-47 631:0 n.d. 13-83 193:0 617:0 -64 Table 9. Complementation matrix for da a l l e l e s . Ratios represent the proportion of Cy to straight-winged progeny recovered from the cross mutantl/CyO X mutant2/CyO. da2 F75 310:0 G2-10 66:0 77-11 201:0 El-21 53:10 El-2 6 152:0 E2-20 536:0 E2-24 119:0 E2-30 64:0 E2-35 137:0 Cross El-21 X Df(2L)J27 El-21 X Df(2L)Jl06 El-21 X Df(2L)J77 El-21 X Df(2L)JRl6 El-21 X El-26 E2-35 E2-24 61:0 129:0 n.d. 148:0 59:0 146:0 126:4 n.d. 69:0 n.d. 65:0 103:0 57:0 -206:0 122:0 n.d. Progeny 166:5 107:0 176:15 447:25 385:11 65 Su(var)216i Eight new mutations were recovered that are l e t h a l with the o r i g i n a l Su(var)216 chromosome. A l l 8 were EMS induced. None i s a dominant suppressor of PEV. The genetics of these mutations are discussed i n Chapter 4. mfs48: Three new a l l e l e s of t h i s recessive suppressor of PEV were recovered. Each a l l e l e i s hemizygous l e t h a l , but h e t e r o a l l e l i c survivors from inter se crosses a l l display the short b r i s t l e phenotype observed by Lindsley et al. (1980). F l i e s bearing a l l h e t e r o a l l e l i c combinations suppress PEV (I.P. Whitehead, personal communication). One of the a l l e l e s was induced i n a P element mutagenesis screen (Screen 3), while the other two a l l e l e s were EMS induced. Sub-interval 11: Ten genes have been positioned to t h i s i n t e r v a l . Four of the complementation groups are represented by sing l e mutations {E2-22, El-8, 77-12, 77-14). Three of these mutants (El-8, 77-12, and 77-14) display reduced v i a b i l i t y when crossed pairwise. Hence, they may a c t u a l l y be weak a l l e l e s of the same locus. Two of the mutations, 77-12 and 77-14, were induced i n a P element mutagenesis scheme. Since these genes may contain P elements, they may be useful f o r further cytogenetic and molecular studies of the region. No new a l l e l e s of PI23 or PJ50 were i s o l a t e d . Both mutations were mapped to deficiency sub-interval 11 based on hemizygous female s t e r i l i t y . Three new hemizygous l e t h a l a l l e l e s of err were recovered. One of these a l l e l e s , A76, i s weak. I t i s not l e t h a l with the o r i g i n a l err mutation, but t h i s combination i s female s t e r i l e at 29° (Appendix 3). No v i s i b l e phenotypes were observed amongst these mutant progeny. One gene i n t h i s region, 1(2)54, was of s p e c i a l i n t e r e s t because i t , along with da and mfs48, was proposed to be part of a gene c l u s t e r (Sandler, 1977). The homozygous female s t e r i l e mutation mat(2)earlyQM4 7 (Schupbach and Wieschaus, 1989) f a i l s to complement 1(2)54 (Sandler, 1977). Several a l l e l e s of 1(2)54 are vi a b l e i n h e t e r o a l l e l i c combinations with mat(2)earlyQM4 7 (Table 10). In each instance, mutant adults have (with variable expressivity) disorganized eye facets and wings held up over the body i n a very sharp "V" formation. Approximately 50% of mutants with rough eyes also have scalloped wings. A l l surviving h e t e r o a l l e l i c combinations of mutations are female s t e r i l e . Thus, the 1(2)54 product i s required for both v i a b i l i t y and female f e r t i l i t y . L o c i proximal to Df(2L)J27 Thirteen l o c i map between the centromere proximal breakpoints of Df(2L)J27 and Df(2L)J2. Their cytogenetic d i s t r i b u t i o n i s shown i n Figure 8. Four complementation groups, El-5, El-18, El-25, and E3, map to an i n t e r v a l between the centromere proximal breakpoints of Df(2L)J27 and Df(2L)J106 (sub-interval 13). Three of the l o c i are represented by a single mutation. The fourth i s represented by four a l l e l e s which are l e t h a l i n h e t e r o a l l e i c combinations (Appendix 3). 67 Table 10: Complementation pattern of a l l e l e s of 1(2)54. The a l l e l e mat(2)earlyQM4 7 i s abbreviated to QM47. Ratios above the matrix diagonal are the proportion of Cy progeny r e l a t i v e to straight-winged progeny i n a cross between mutants heterozygous f o r CyO. Female s t e r i l i t y (S), where applicable, i s noted below the matrix diagonal. QM4 7 HI 13 C70 G2-4 El-2 El-16 1(2)54 317:0 438:0 345:0 126:0 313:0 209:0 QM4 7 - 770:52 n.d. 279:12 274:143 165:33 H113 S - 520:0 68:0 203:0 118:0 C70 n.d. - 180:0 164:0 354:0 G2-4 S - 54:0 433:0 El-2 S - 309:0 El-16 S -68 In sub-interval 14, there i s a sing l e locus represented by three a l l e l e s . A l l three a l l e l e s are hemizygous l e t h a l (Brock, 1989), but are semi-lethal i n h e t e r o a l l e l i c combinations (Appendix 3). Survivors have etched t e r g i t e s and are male and female s t e r i l e . Eight complementation groups map to de f i c i e n c y sub-i n t e r v a l 15. Five are represented by sing l e a l l e l e s that have no phenotypes other than hemizygous l e t h a l i t y (Brock, 1989). The hup, wdl and dal mutations have previously been reported to map i n the region deleted by Df (2L)J2, but centromere proximal to Df(2L)Jl06 ( S i n c l a i r et. al., 1991). Neither hup nor wdl display t h e i r respective "wings held up" or "wavy wing" phenotypes with Df (2L)JR3 or Df(2L)G2. hup and dal were not included i n the complementation analysis because they are only weakly i n v i a b l e , but wdl was included because i t i s also hemizygous semi-lethal. No new a l l e l e s of wdl were recovered. 69 D I S C U S S I O N This study adds region 31-32A to a growing number of regions which have been i n t e n s i v e l y characterized at the genetic l e v e l . Throughout the region deleted by Df(2L)J2, 136 mutations were deficiency mapped and tested for complementation. Included i n t h i s number were 57 new hemizygous l e t h a l mutations which map between the d i s t a l breakpoint of Df(2L)J77 and the proximal breakpoint of Df(2L)J106. T h i r t y - f i v e new complementation groups have been described, and 15 previously described l o c i have been more p r e c i s e l y mapped within region 31. Three new a l l e l e s of the recessive suppressor mfs48 were recovered. S i m i l a r l y , eight new a l l e l e s of Su(var)216 were i s o l a t e d . The new a l l e l e s of Su(var)216 a l l f a i l to suppress, a point which w i l l be elaborated on i n Chapter 4 when the genetics of the putative Su(var)216 a l l e l e s are discussed. The genetics of mfs48 i s being investigated i n d e t a i l by I.P. Whitehead. Genetic recombination experiments p o s i t i o n the Suvar(2)l, Su(var)204, and Su(var)207 mutations very close to each other (32-35 cM; S i n c l a i r et al., 1983). I n i t i a l d e l e t i o n analyses suggested that they were separate l o c i . However, dominant mutations are d i f f i c u l t to map by d e f i c i e n c i e s because secondary, recessive phenotypes must be ascribed to the same locus. The preliminary conclusion that Su(var)207 and Suvar(2)l are separate l o c i i s now i n doubt. Complementation 70 analysis with a new deficiency, i n combination with previous recombination data, suggests that Su(var)207 and Suvar(2)l map to the same small deficiency i n t e r v a l . Su(var)207 i s v i a b l e i n trans with Df(2L)JRl, but maps to the r i g h t of J by recombination. Assuming that the hemizygous s t e r i l i t y of the Su(var)207 chromosome i s a true secondary phenotype of the suppressor mutation, these data can only be reconciled i f Su(var)207 i s located d i s t a l to Df(2L)JRl. Two observations suggest that Su(var)207 i s an a l l e l e of Suvar(2)l. F i r s t , Su(var)207 and a l l e l e s of Suvar(2)l i n t e r a c t . Transheterozygotes display a red-brown eye phenotype, reduced male v i a b i l i t y , and female infecundity c h a r a c t e r i s t i c of h e t e r o a l l e l i c combinations of Suvar(2)l a l l e l e s . Second, l i k e Su(var)207, a l l e l e s of Suvar(2)l are infecund i n trans with Df(2L)J2. Previously, the l e t h a l i t y of Su(var)207/Df (2L)J39 was attributed to an exacerbation of the s t e r i l i t y phenotype caused by the large semi-lethal deficiency. However, t h i s i n t e r p r e t a t i o n , i n conjunction with recombination data, would position Su(var)207 i n the region deleted by Df(2L)JRl. Since the d e f i c i e n c y and Su(var)207 complement, the Df(2L)J39 results are probably caused by a second s i t e recessive l e t h a l mutation on the Su(var)207 chromosome. Deficiency mapping i n combination with previous recombination data also suggests that the i n i t i a l l o c a l i z a t i o n of Su(var)204 may have been incorrect. A second s i t e l e s i o n , 71 which on the basis of deficiency mapping appears to be located to the r i g h t of J, can account f o r the s t e r i l i t y of Su(var)204 females over Df(2L)G2 and Df(2L)JR3. Su(var)204 has been recombination mapped to the l e f t of J" ( S i n c l a i r , unpublished). Hence the suppression of p o s i t i o n - e f f e c t variegation and the s t e r i l i t y phenotypes are separable. The Su(var)204 mutation has no other secondary phenotypes which can be used to ascertain i t s true l o c a t i o n . A du p l i c a t i o n f o r region 31 cannot be used to map the Su(var) because the only extant dup l i c a t i o n acts as an enhancer ( S i n c l a i r et al., 1991). This enhancer, l i k e most others, ameliorates the suppression phenotype of numerous d i s t a n t l y located Su(var) mutations. At present the only way to locate Su(var)204 appears to be by recombination mapping with very t i g h t l y linked markers. The suggestion that there are t i g h t l y linked second s i t e mutations on both the Su(var)204 and Su(var)207 chromosomes underscores the need for extreme care i n i n t e r p r e t i n g Su(var) phenotypes or recessive phenotypes associated with any dominant mutant a l l e l e . U n t i l further d e t a i l e d mapping experiments are performed, a conservative i n t e r p r e t a t i o n of the available deficiency mapping data i s that there i s a single dominant suppressor locus i n 31A-B. By t h i s i n t e r p r e t a t i o n , region 31 may contain four genes that a f f e c t PEV: Suvar(2)l, Su(var)216, mfs48, and wdl. However, data presented i n Chapter 4 suggest that Su(var)216 i s also an a l l e l e of Suvar(2)l and that the l e t h a l phenotype of the 72 suppressor-bearing chromosome i s caused by a t i g h t l y linked second-site l e t h a l mutation i n the 3IE region. Suvar(2)l i s separated from the recessive suppressor l o c i mfs48 and wdl by numerous complementation groups; and mutations i n the intervening complementation groups do not cause dominant suppression of PEV. Furthermore, wdl and mfs48 map to opposite sides of da (D. S i n c l a i r , unpublished). Mutations i n the da gene do not suppress PEV. Thus, t h i s study suggests that suppressors of PEV i n region 31 are dispersed among genes with other functions. Sandler and his colleagues (Sandler, 1977; Lindsley et a l . , 1980) proposed, by analogy with the Bithorax Complex (Lewis, 1979), that regions 31 and 32 contain a c l u s t e r of f u n c t i o n a l l y r e l a t e d genes that interact with heterochromatin and that act early i n development. Two members of the proposed c l u s t e r (mfs48 and wdl) are recessive suppressors of PEV. Because we have not i d e n t i f i e d any new homozygous via b l e mutants, we do not know i f additional recessive su(var) l o c i reside i n the 31E-32A region. More important, however, i s the question of whether there exists a functional c l u s t e r of genes. I t i s i n t e r e s t i n g i n t h i s regard that f l i e s bearing h e t e r o a l l e l i c combinations of 1(2)54 d i s p l a y a held-up wing phenotype s i m i l a r to that of hup. They might, therefore, have s i m i l a r functions. Nonetheless, deficiency and complementation mapping indicate that the proposed c l u s t e r of r e l a t e d genes would have to be very large: wdl i s separated from da and 73 mfs48 by at l e a s t 15 complementation groups. Additional l o c i probably map between wdl, i n region 31, and abo, a dis t a n t member of the c l u s t e r i n region 32. Further analyses w i l l be necessary to t e s t the c l u s t e r i n g hypothesis, but such a large number of f u n c t i o n a l l y r e l a t e d e s s e n t i a l l o c i would be extraordinary. 74 CHAPTER 3 INTRODUCTION Throughout the past decade, genetic observations have suggested that many l o c i associated with supression of PEV encode non-histone chromosomal proteins or proteins associated with chromatin assembly or modification. A d i r e c t t e s t of t h i s hypothesis i s to clone Su(var) l o c i and use biochemical assays to determine the function of the gene product. During the time period i n which t h i s thesis was progressing, two Su(var) genes were cloned. DNA sequence analysis of both Su(var) genes suggests that they encode NHPs, thus substantiating one portion of the o r i g i n a l hypothesis. A d d i t i o n a l genes need to be characterized to begin a comprehensive biochemical study of chromâtin-associated proteins and t h e i r e f f e c t s on gene t r a n s c r i p t i o n and chromosome architecture. This chapter describes a P element mutagenesis screen for hemizygous l e t h a l mutations i n region 31. One mutation recovered from t h i s screen was l e t h a l with the Su(var)216 chromosome. The P element transposon associated with t h i s l e t h a l i t y was cloned and the adjacent DNA was sequenced to determine the loc a t i o n of flanking t r a n s c r i p t i o n u n i t s . The P element was located i n the untranslated 5' sequence of the cdc2Dm gene, a gene which encodes a serine/threonine kinase required f o r progression through the c e l l c ycle (Lehner and O' F a r r e l l , 1990b), and which has histone Hi as one of i t s targets f o r phosphorylation. Although suppressors of PEV are frequently thought of as NHPs, some Su(var) genes might encode products which modify NHPs or are involved i n the biochemical pathways that lead to chromatin assembly. In f a c t , a phosphatase 1 gene of Drosophila may be a suppressor of PEV (Axton et a l . , 1990). This gene i s also implicated i n the control of mitotic progression, suggesting a l i n k between PEV and the c e l l c ycle. The properties of cdc2Dm are discussed i n t h i s context. 76 M A T E R I A L S AND METHODS Genetics A genetic screen was performed to i d e n t i f y Su(var) genes i n region 31 (Figure 9). Males from three s t r a i n s with P elements (OK, Harwich, and n2) were mated, i n separate sets of experiments, to homozygous b pr cn females without P elements (M s t r a i n s ) . In such crosses, there i s a marked increase i n the frequency of P element transposition within the germline of the F x males and females. Thus, the mobile elements serve as a mutagen i n the F x hybrids. Mutagenized b pr cn second chromosomes were captured over a balancer chromosome i n a P element-containing s t r a i n . P element s t r a i n s contain a repressor that reduce the frequency of tra n s p o s i t i o n events, thereby s t a b i l i z i n g the new s i t e s of P element i n s e r t i o n . Male progeny from such crosses were i n d i v i d u a l l y mated to 3-5 Df(2L)J2/CyO females. The appropriate chromosomes, from l i n e s that were hemizygous l e t h a l i n trans with Df(2L)J2, were re-balanced i n a P element containing background to prevent further P element excision events. Subsequently, the mutations were tested for f a i l u r e to complement i n d i v i d u a l Su(var) genes that map under Df(2L)J2. Putative Su(var) mutants were out-crossed f o r several generations to progressively replace Chromosomes 1, 2, and 3 with homologues that did not contain P elements. The protocol i s i l l u s t r a t e d i n Figure 10. Lyra (Ly) i s a dominant t h i r d 77 Figure 9. P element screen Females Males b pr cn b pr cn X (P s t r a i n ) (P s t r a i n ) X b pr cn SM1 Df(2L)J2 SM1 X b pr cn SM1 mated singly SM1 SM1 d i e s b pr cn * SMI Df(2L)J2 SM1 C u r l y w i n g e d C u r l y w i n g e d c n e y e d b pr cn * Df(2L)J2 S t r a i g h t w i n g e d red e y e d 78 Genetic crosses to i s o l a t e the chromosome mutation that causes a wing-shape change. TM3 and CyO are multiply inverted t h i r d and second chromosomes marked with the dominant mutation Stubble and Curly, r e s p e c t i v e l y . Sb causes a short, t h i c k b r i s t l e phenotype; Cy causes a curly wing phenotype. In addition to providing phenotypic markers to score chromosome segregation, balancer chromosomes (TM3 and CyO) were necessary to prevent meiotic recombination i n females. Male Drosophila melanogaster do not undergo meiotic recombination. Molecular Biology In situ Hybridization to Polytene Chromosomes In situ hybridization to Drosophila s a l i v a r y chromosomes with b i o t i n y l a t e d DNA probes was performed e s s e n t i a l l y according to Whiting et al. (1987). S a l i v a r y glands from mature t h i r d i n s t a r larvae were dissected i n 0.8% s a l i n e then moved to a droplet of 45% acetic acid f o r approximately 20 s. The glands were then incubated i n f i x a t i v e ( 1 part l a c t i c a c i d : 2 parts d i s t i l l e d water: 3 parts g l a c i a l a c e t i c acid) for 2-3 min. The glands were placed on a clean microscope s l i d e , covered with an ethanol-washed ( a i r dried) c o v e r s l i p , and squashed. The s l i d e s were frozen i n l i q u i d nitrogen, then the coverslips were removed using a sharp razor blade. The s l i d e s were immediately placed i n col d (-50°) ethanol which was l e f t to gradually warm to room temperature. The s l i d e s were heated for 30 min at 70° i n 2X SSC, then dehydrated i n 80 pre-warmed 70% and 95% ethanol f o r 20 and 10 min, respec t i v e l y . The ethanol solutions were pre-heated to 65° so that the s l i d e s slowly cooled to room temperature. The s l i d e s were stored at 4°. A nick-translated probe (2 u.g) was prepared using b i o t i n -11-dUTP (Bethesda Research Lab.) and the BRL nick t r a n s l a t i o n k i t , according to the manufacturers i n s t r u c t i o n s . Two-hundred micrograms of sonicated herring sperm DNA were added to the ni c k - t r a n s l a t i o n mixture. The DNA was ethanol p r e c i p i t a t e d and resuspended i n 117 u l of water. Immediately p r i o r to use, the probe was heat denatured i n b o i l i n g water f o r 5 min and plunged i n t o an i c e bath. The following was added to the denatured probe solu t i o n : 40 u l 10% dextran sulphate, 40 u l 2OX SSC, and 4 u l 5OX Denhardt's solution. Immediately p r i o r to hybridization with the DNA probe, the chromosomes on stored s l i d e s were denatured i n 70 mM NaOH and washed i n 70% ethanol f o r 4 min and 95% ethanol f o r 4 min. Twelve m i c r o l i t r e s of probe solution was placed on each s l i d e , and covered with an acid-washed c o v e r s l i p . The s l i d e s were placed i n a sealed box with f i l t e r paper saturated i n 2X SSC and the box was incubated at 58.5° f o r 6-12 hours. Once the hybridization was complete, the s l i d e s were washed twice i n 2X SSC at 54° for 20 min and once i n SSC at room temperature f o r 20 min. The BRL BluGene k i t was used f o r detecting the b i o t i n y l a t e d probe. Slides were washed i n Buffer 1 (0.1 M 81 Tris-HCl, pH 7.5, 0.15 M NaCl) supplemented with 1% BSA for one hour. Then they were drained and 200 \il of d i l u t e d streptavidin-poly(AP) conjugate (2 | i l of BRL stock solution i n 1.5 ml Buffer 1) was placed over the chromosomes. A co v e r s l i p was placed on each s l i d e and the s l i d e s were placed i n a sealed box with a buffer saturated towel. The box was l e f t at room temperature f o r 2 hours. The coverslips were removed by dipping the s l i d e s i n Buffer 1. The s l i d e s were washed twice i n 25 ml of Buffer 1 at room temperature f o r 10 min each, then once i n Buffer 3 (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 0.05 M MgCl2) f o r 10 min. The s l i d e s were drained. About 100-200 \il of dye s o l u t i o n (3.3 u l NBT i n 0.75 ml Buffer 3 and 2.5 u l BCIP solution) were pipetted over each s l i d e . A c o v e r s l i p was placed over the dye solution and the s l i d e s were placed i n a sealed box and incubated at room temperature overnight. The coverslips were removed by dipping the s l i d e s i n d i s t i l l e d water. The s l i d e s were washed i n water f o r 3 hours. The chromosomes were photographed with water under the co v e r s l i p . I s o l a t i o n of Genomic DNA High molecular weight DNA was p u r i f i e d by a modification of the procedure of Jowett (1986). Several hundred adult f l i e s were flash-frozen i n l i q u i d nitrogen, then ground to a powder i n a cooled mortar and pestle. The powder was transferred to 82 10 ml of l y s i s buffer (100 mM Tris-HCl (pH 8.0), 50 mM EDTA, 1% SDS, 0.15 mM spermine, 0.5 mM spermidine) containing 100 Hg/ml proteinase K. Afte r incubation at 65° f o r 2 hours, the mixture was extracted once with an equal volume of TE e q u i l i b r a t e d phenol (pH 8.0), twice with phenol/chloroform (1:1), and once with chloroform. The aqueous phase was transferred to a fresh tube, and overlaid with two volumes of i c e - c o l d 95% ethanol. DNA was spooled around a heat-sealed Pasteur pipette and re-dissolved i n TE. I s o l a t i o n of Bacteriophage Lambda DNA Bacteriophage lambda were i s o l a t e d according to the plate lysate method of F r i t s c h (Maniatis et a l . , 1982). Approximately 3 X 106 bacteriophages were mixed with 1.6 X 108 b a c t e r i a l c e l l s . A f t e r incubating the mixture f o r 15 min at 37°, the c e l l suspension was d i l u t e d to 5 ml with LM. The l i q u i d culture was incubated at 37° for 6-9 hours u n t i l the culture cleared. B a c t e r i a l debris was removed from the lysate by centrifugation. RNase A and DNase I were added to the supernatant, each to a f i n a l concentration of 50 jig/ml, and the mixture was incubated f o r 30 min at 37°. An equal volume PEG solu t i o n (2.5 M NaCl and 20% (w/v) PEG i n lambda-dil) was added, and the mixture was incubated at 4° f o r one hour. P r e c i p i t a t e d bacteriophage p a r t i c l e s were recovered by c e n t r i f ugation at 10,000 rpm i n an SS34 rotor f o r 30 min. The supernatant was poured o f f , and the tubes were drained i n an 83 inverted p o s i t i o n overnight. Any f l u i d remaining i n the tube was absorbed with a paper towel. The bacteriophage p e l l e t was resuspended i n 0.4 ml of TE and transferred to a microfuge tube. Residual debris was removed by cen t r i f u g a t i o n at 8000g. Twenty m i c r o l i t r e s of 0.5 M EDTA (pH 8.0) was added, and the s o l u t i o n was extracted twice with phenol/chloroform (1:1), and once with chloroform. Bacteriophage DNA was p r e c i p i t a t e d with 2.5 volumes of 95% ethanol. The DNA was p e l l e t e d by centrifugation, vacuum dried, and resuspended i n TE. I s o l a t i o n of Plasmid DNA Plasmid DNA was i s o l a t e d by the a l k a l i n e l y s i s method. Small, medium and large scale i s o l a t i o n s were performed. The bacteria p e l l e t e d from 2, 50, or 500 ml of LM (containing 0.005% amp i c i l l i n ) were resuspended i n 0.2, 2.5, or 10 ml of Solution 1 (50 mM glucose, 20 mM Tris-HCl (pH 8.0), 10 mM EDTA), respectively. One volume of Solution 2 (0.4 N NaOH, 2% SDS) was added, and the s o l u t i o n l e f t at 4° f o r 10 min to lyse the bacteria. One-half volume of 3 M potassium acetate solu t i o n was added to the lysed b a c t e r i a l suspension and the mixture was incubated f o r an a d d i t i o n a l 10 min at 4°. To remove b a c t e r i a l debris, small scale preparations were microfuged at 12000 xg f o r 5 min; medium and large scale preparations were centrifuged f o r 20 min i n a S o r v a l l ss34 rotor at 8000 rpm and 15000 rpm, r e s p e c t i v e l y . The supernatant was transferred to a new tube. DNA was p r e c i p i t a t e d d i r e c t l y 84 from the supernatant of large scale preparations by the addition of isopropanol (60% v/v). The supernatant from small and medium scale preparations was extracted once with phenol/chloroform (1:1); and then plasmid DNA was pr e c i p i t a t e d with 2 volumes of i c e - c o l d 95% ethanol. The DNA was pel l e t e d as previously described, then vacuum dried. For small scale plasmid preparations the DNA p e l l e t was resuspended i n 50 u l TE, ready for use. For medium scale preparations, the DNA was resuspended i n 300 ul of TE. One m i c r o l i t r e of RNAse A (10 mg/ml) was added and the soluti o n was incubated at 22° for 30 min. One half volume of 7.5 M ammonium acetate was added and RNAse was pre c i p i t a t e d f o r 10 min at -80°. A f t e r c e n t r i f u g a t i o n (12000 xg f o r 5 min), the supernatant was transferred to a new microfuge tube. Plasmid DNA was pr e c i p i t a t e d with 2 volumes of ic e - c o l d 95% ethanol. The DNA was p e l l e t e d (12000 xg f o r 10 min), dried under vacuum, then resuspended i n 900 u l of TE. The DNA was again p r e c i p i t a t e d by the addition of 600 u l of PEG solution (20% PEG 6000, 2.5 M NaCl) and incubation at 4° for 1 hour. The plasmid DNA was p e l l e t e d (12000 xg f o r 10 min), dried, and resuspended i n 100 u l of TE. For large scale preparations, the DNA was resuspended i n 8 ml TE buffer. Eight grams of CsCl and 0.8 ml of ethidium bromide (10 mg/ml i n water) were added. The sol u t i o n was poured in t o a Beckman polyallomer 16 X 76 mm Quick-Seal centrifuge tube and the tube was heat-sealed. Equilibrium 85 c e n t r i f ugation was at 45,000 rpm f o r 36 h (at 22°) with a S o r v a l l Tft65.13 rotor i n a Beckman L8-80 u l t r a c e n t r i f u g e . Supercoiled plasmid DNA was removed from the gradient using an 18 gauge needle attached to a 3 ml syringe. Ethidium bromide was extracted with sec-butanol, then the solu t i o n was d i l u t e d 3 times with water. Plasmid DNA was p r e c i p i t a t e d by the addition of 2 volumes of cold 95% ethanol. The DNA was p e l l e t e d by centrifugation, vacuum dried, and resuspended i n TE. DNA R e s t r i c t i o n Digests and Gel Electrophoresis For most plasmid and lambda phage digestions, 1-2 \ig of DNA i n 17 u l of water were mixed with 2 u l of the appropriate BRL core buffer and 1-2 u l of r e s t r i c t i o n endonuclease (3-10 u n i t s ) , then incubated at 37° for 3 hours. Some samples were incubated with heat-treated RNAse A (100 fig/ml, 15 min, room temperature) to hydrolyze contaminating RNA. Digestions were stopped by adding 0.2 volumes of loading buffer (6 M urea, 25% sucrose, 50 mM EDTA, and orange G dye). Genomic DNA (2-5 \ig) was made more d i l u t e (<100 u-g/ml) and digested overnight, then ethanol p r e c i p i t a t e d and redissolved i n 25 u l of TE. Digested DNA was fractionated on 0.8-1.2% agarose gels containing Tris-acetate buffer (40 mM Tris-HCl, 5 mM sodium acetate, 1 mM EDTA, pH adjusted to 7.8 with a c e t i c a c i d ) , and 0.1 u,g/ml ethidium bromide. Gels were electrophoresed at 1.8-3.7 V/cm f o r 2-12 hours, then photographed by transmitted 86 u l t r a v i o l e t l i g h t . Transfer of DNA to Hybridization Membrane DNA was transferred to Hybond-N nylon membrane according to a procedure adapted from Southern (1975). The gel was incubated i n 0.4 N NaOH, 0.6 M NaCl f o r 30 minutes at room temperature with gentle a g i t a t i o n . Then i t was neutralized i n 1.5 M NaCl, 0.5 M Tris-HCl (pH 5.5) f o r 30 minutes with shaking. For b l o t t i n g , the gel was placed on a pre-soaked Whatman 3MM paper wick suspended above a r e s e r v o i r of 5X SSC tra n s f e r s o l u t i o n . A pre-wetted membrane was placed on the uppermost gel surface and o v e r l a i d f i r s t with dry Whatman 3MM paper and then with a th i c k stack of paper towels. A glass plate with a small weight on i t was placed over the top to ensure even t r a n s f e r . Transfer was complete within 6-12 hours, a f t e r which time the bl o t was washed i n 2X SSC and dried at room temperature. DNA was permanently a f f i x e d to the membrane by exposure to a 254 nm u l t r a - v i o l e t l i g h t f o r 5 minutes. Hybridization of Radiolabelled Probe to Transferred Nucleic Acids Southern blots (DNA) were incubated i n a sealed p l a s t i c bag with 10 ml of Hybridization Solution (6X SSC, 5X Denhardt's and 0.1% SDS: 100X Denhardt's i s 2% BSA, 2% F i c o l l , 2% PVP) f o r 2 hours at 65° with a g i t a t i o n . Radiolabelled probe was added and allowed to hybridize to the f i l t e r f or 12-36 87 hours at 65° with shaking. High stringency washes were: three times 30 min i n 0.1X SSC, 0.1% SDS at 65° with shaking. To detect sequences with a low degree of homology to the probe, low stringency washes were performed i n Hybridization Solution f o r 2 h at 65°. Lab e l l i n g of DNA DNA (50-200 ng) was r a d i o l a b e l l e d to a s p e c i f i c a c t i v i t y of approximately 1.8 X 109 dpm/ug by random hexamer-primed DNA l a b e l l i n g (Feinberg and Vogelstein, 1983, 1984) using a Boehringer Mannheim Random Primed DNA L a b e l l i n g k i t . Library Construction Two micrograms of lambda EMBL4 DNA were digested i n a 40 Hi volume with an excess of EcoRI and BamHI (20 u n i t s ) . A f t e r 1 hour, the enzymes were inactivated by heating at 72° f o r 15 min. Sodium acetate was added to a f i n a l concentration of 300 mM, and the DNA was pr e c i p i t a t e d with 0.6 volumes of isopropanol for 15 min at room temperature. The p r e c i p i t a t e was washed with 70% ethanol and vacuum dried. These conditions excised the c e n t r a l s t u f f e r region of the vector and exposed the EcoRI cloning s i t e . Digestion with EcoRI and BamHI prevents the s t u f f e r fragment from r e - l i g a t i n g i n t o the vector i n subsequent reactions. Since the very short EcoRI-BamHI l i n k e r fragments do not p r e c i p i t a t e i n the isopropanol step, the p r o b a b i l i t y of a r e - l i g a t e d s t u f f e r 88 fragment i s further reduced. R e s t r i c t i o n endonuclease digested Drosophila genomic DNA was prepared as follows. Twenty micrograms of uncut genomic DNA was resuspended i n 200 u l digestion buffer (100 mM NaCl, 10 mM MgCl 2, 1 mM DTT, Tris-HCl (pH 7.5)) heated to 37°. Ten units of EcoRI were added and the time-course of digestion was monitored by removing 40 (il (2 ng) samples. Each sample was made 10 mM with respect to EDTA, and stored on i c e u n t i l a l l samples were c o l l e c t e d . The enzyme was then heat i n a c t i v a t e d at 72° for 15 min. One half microgram of DNA from each sample was electrophoresed i n a 0.3% agarose g e l . The lane with the maximum i n t e n s i t y of fluorescence i n the 15-20 kb range was determined, and the sample with the maximum number of molecules i n that s i z e range was prepared f o r l i g a t i o n . The p a r t i a l l y digested genomic DNA was p r e c i p i t a t e d with ethanol, vacuum dried, and resuspended i n phosphatase buffer (50 mM Tris-HCl (pH 9.0), 1 mM MgCl 2, 0.1 mM ZnCl 2, and 1 mM spermidine). One half unit of c a l f a l k a l i n e phosphatase was added and the reaction was incubated at 37° f o r 1 h. The mixture was extracted sequentially with phenol, phenol/chloroform (1:1), and chloroform, then made 100 mM with respect to sodium acetate. The DNA was p r e c i p i t a t e d with 2 volumes of col d 95% ethanol. Approximately 0.5 jig of genomic DNA p a r t i a l l y digested with EcoRI and 2 u,g of EcoRI digested vector were resuspended i n 10 [il l i g a s e buffer (10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 89 5 mM (3-mercaptoethanol and 1 mM ATP ). One unit of T4 DNA li g a s e (BRL) was added and the l i g a t i o n proceeded overnight at 15°. Ligated DNA was packaged using a Gigapack Plus (Stratagene) packaging k i t according to the manufacturer's i n s t r u c t i o n s . Screening of L i b r a r i e s of Recombinant Bacteriophage Lambda Four lambda-libraries were screened. An unamplified p a r t i a l EcoRI l i b r a r y from Drosophila was constructed i n EMBL4 (Frischauf et al., 1983), as described above. The host bacterium was NM539. The same b a c t e r i a l s t r a i n was used to amplify a Drosophila v i r i l i s p a r t i a l Sau3A l i b r a r y (Dr. J . Tamkun) cloned i n EMBL3. A Xgtll 0-12 hour embryonic cDNA l i b r a r y (Dr. J . Tamkun) was propagated on Y1090hsdR. A t h i r d i n s t a r imaginai disc cDNA l i b r a r y (Dr. G. Rubin) constructed i n kgtlO was amplified on C600hflA. Recombinant X. bacteriophage were propagated on E. coli s t r a i n s prepared as follows. A single h o s t - c e l l colony was grown overnight i n LM, and then 5 ml were used to inoculate a fresh 50 ml culture. A f t e r 3 hours at 37°, the c e l l s were pe l l e t e d i n a bench-top centrifuge (10 min at 3000 xg) and resuspended i n 25 ml of s t e r i l e 10 mM MgS02. These c e l l s were viab l e f o r several weeks. Approximately 3-5 x 104 pfu, suspended i n 2-50 u l of lambda-dil (100 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10 mM MgCl 2), were added to 100 u l of p l a t i n g c e l l s and incubated at 90 37° f o r 15 min. The bacteria were then mixed with 8 ml of molten 50° top agar (LM + 7 g/1 Bacto-agar). The agar was poured onto the surface of a 150 X 15 mm LM agar plate (LM + 14 g/1 Bacto-agar) and allowed to co o l . Plates were incubated at 37° f o r 6-12 hours to allow the b a c t e r i a l lawn to grow. Lambda phage DNA was transferred from plagues on LM-plates to pre-cut n i t r o c e l l u l o s e f i l t e r s by a modification of the procedure of Benton and Davis (1977). Each plate with bacteriophage plaques was blotted with a dry n i t r o c e l l u l o s e f i l t e r . The f i l t e r was peeled o f f the b a c t e r i a l lawn, and placed i n 0.4 N NaOH f o r 30 seconds. The f i l t e r was then washed f o r 60 seconds i n 1.5 M NaCl, 0.5 M Tris-HCl (pH 5.5), and f i n a l l y for 30 seconds i n 2X SSC. F i l t e r s were a i r dried on Whatman 3MM paper and baked f o r 2 hours at 80°. The f i l t e r s were hybridized with a r a d i o l a b e l l e d probe to i d e n t i f y DNA with sequence s i m i l a r i t y . Several rounds of rep l a t i n g and re-probing at successively higher phage d i l u t i o n s were necessary to p u r i f y each lambda clone. Subcloning Bacteriophage and Cosmid DNA Bacteriophage or cosmid DNA was digested with an appropriate r e s t r i c t i o n endonuclease and the DNA was size fractionated on an agarose g e l . A gel s l i c e containing the fragment to be subcloned was transferred to a microfuge tube, and the DNA fragment was p u r i f i e d using glass beads (Gene Clean; Bio 101) according to the manufacturer's i n s t r u c t i o n s . 91 F i f t y to f i v e hundred nanograms of r e s t r i c t i o n endonuclease digested DNA was co-precipitated with 50 ng of l i n e a r i z e d pUCl9 with compatible ends. The DNA was resuspended i n a 20 (il reaction volume containing 10 mM Tris-HCl (pH 8), 10 mM MgCl 2 / 1 mM DTT and 1 mM ATP. One unit of T4 DNA lig a s e was added and the sample was incubated f o r 8-12 h at 16°. pUC plasmids were trans fected by adding 3 u l of the l i g a t i o n reaction mixture to 50-200 u l of thawed competent DH5-a (subcloning e f f i c i e n c y ; Bethesda Research Laboratories). A f t e r 30 min on i c e the c e l l s were heat shocked at 37° f o r 20 s. The c e l l s were placed on i c e for 2 min, then d i l u t e d with 500 (il of LM and incubated at 37° f o r 1 h. C e l l s (50-100 (il) were spread over a 10 cm 0.005% a m p i c i l l i n (w/v) LM-plate, whose surface had been coated with 50 (il of 2% X-gal. White colonies were selected as recombinants, and t h e i r i n s e r t s were screened by either mini-plasmid preparation or colony hyb r i d i z a t i o n . B l o t t i n g DNA from B a c t e r i a l Colonies Plasmid or cosmid DNA from lysed b a c t e r i a l colonies was blotted d i r e c t l y onto the surface of n i t r o c e l l u l o s e f i l t e r s by a modification of the procedure of Grunstein and Hogness (1975). A pre-cut n i t r o c e l l u l o s e f i l t e r was placed over colonies grown on an a m p i c i l l i n LM plate. The f i l t e r was peeled o f f and placed bacteria-side up on Whatman 3MM paper soaked with denaturing solution (0.5 M NaOH, 1.5 M NaCl). 92 A f t e r 5 min i t was then transferred to n e u t r a l i z i n g s o l u t i o n (1.5 M NaCl, 0.5 M Tris-HCl pH 8.0) f o r 5 min. F i l t e r s were dried and baked at 80° f o r 2 hours. RNA extraction RNA was extracted e s s e n t i a l l y according to Jowett (1986). Approximately 200 u l of 0-6 hour o l d embryos were flash-frozen i n l i q u i d nitrogen and ground to powder with a mortar and pes t l e . The powder was brushed in t o 4 ml of phenol saturated with 200 mM sodium acetate (pH 5.0) at 65°. A f t e r vortexing, an equal volume of Extraction Buffer (150 mM sodium acetate (pH 5.0), 2% SDS) was added. The sol u t i o n was incubated at 65° for 5 minutes, then allowed to cool to 22°. Subsequently, 4 ml of chloroform was added, vortexed, and centrifuged to separate the aqueous and organic phases. The organic phase was removed, and the aqueous phase was re-extracted: f i r s t with an equal volume of phenol/chloroform (1:1), then with chloroform. RNA was p r e c i p i t a t e d overnight at -20° by the addition of 2.5 volumes of 95% ethanol. The RNA was p e l l e t e d , washed with 70% ethanol, then re-dissolved i n diethylpyrocarbonate-treated water. Poly (A) + RNA was p u r i f i e d from t o t a l RNA by chromatography on oli g o ( d T ) - c e l l u l o s e . Oligo(dT)-cellulose was e q u i l i b r a t e d i n s t e r i l e loading buffer (20 mM T r i s (pH7.6), 0.5 M NaCl, 1 mM EDTA, 0.1% SDS) and poured i n t o a disposable, s t e r i l e column. The column was washed sequentially with the following: (1) 3 volumes of s t e r i l e water, (2) 3 volumes of 0.1 M NaOH and 5 mM EDTA, (3) 3 volumes of s t e r i l e water, and (4) 5 volumes of loading buffer. Total RNA dissolved i n water was heated to 65° for 5 min, then an equal volume of 2X loading buffer was added. The RNA sol u t i o n was loaded onto the column followed by 10 volumes of loading buffer. Next 4 volumes of washing buffer (20 mM T r i s (pH 7.6), 0.1 M NaCl, 1 mM EDTA, 0.1% SDS) were passed through the column. Poly (A) + was eluted from the column with 2-3 column volumes of s t e r i l e e l u t i o n buffer (10 mM T r i s (pH 7.5), 1 mM EDTA, 0.05% SDS). Sodium acetate (3 M, pH 5.8) was added to a f i n a l concentration of 300 mM and the RNA was pr e c i p i t a t e d with 2.5 volumes of ethanol at -70°. RNA Gel Electrophoresis and Northern B l o t t i n g RNA was fractionated by s i z e i n a formaldehyde, denaturing g e l . Two grammes of agarose, 20 ml 10X MOPS (0.2 M 3-(N-morpholino) propanesulfonic acid, 50 mM sodium acetate, 10 mM EDTA, adjusted to pH 7.0), and 174 ml d i e t h y l pyrocarbonate (DEP) treated autoclaved water was added to a 500 ml f l a s k . The agarose was melted by b o i l i n g , then the sol u t i o n was cooled to 50°. In a fumehood, 10.2 ml 37% formaldehyde was added to the agarose s o l u t i o n . The gel mixture was poured i n t o an 20 X 15 cm gel t r a y and allowed to set f o r one hour. Approximately 5 |xg of poly(A) + RNA, resuspended i n 5 u l 94 of water was mixed with 25 u l electrophoresis sample buffer (0.75 ml deionized formamide, 0.15 ml 10X MOPS, 0.24 ml formaldehyde, 0.1 ml water, 0.1 ml g l y c e r o l , 0.08 ml 10% (w/v) bromophenol blue. The sample was heated at 65° f o r 15 min. Ethidium bromide (1 u l of a 1.0 mg/ml solution) was added, and the denaturing gel was loaded. The formaldehyde gel was immersed i n IX MOPS/EDTA, the sample wells were flushed with electrophoresis buffer, and the prepared samples were loaded. Electrophoresis was at 30 V at room temperature. Following electrophoresis, the gel was soaked fo r two 20 min periods i n 10X SSC at room temperature with shaking. The gel was blotted as previously described f o r DNA, except that the t r a n s f e r solution was 10X SSC. RNA was fi x e d to the Hybond-N membrane (Amersham) by exposure to short wave rad i a t i o n (254 nm) for 5 min. Radiolabelled DNA probes were hybridized to Northern blots exactly as described f o r Southern b l o t s . DNA Sequence Analysis Fragments of DNA were subcloned i n t o pUC19, then sequenced using a modification of the procedure of Tabor and Richardson (1987). Four micrograms of double-stranded plasmid DNA template was denatured for 2 minutes at 65° i n 400 mM NaOH, then made 300 mM with respect to sodium acetate. The DNA was p r e c i p i t a t e d with 2 volumes of i c e - c o l d 95% ethanol and 95 p e l l e t e d by cen t r i f u g a t i o n . DNA primer (3 pmol) was annealed to the template f o r 20 minutes at 37°, i n 10 u l of Annealing Buffer (50 mM NaCl, 10 mM MgCl 2 / 40 mM Tris-HCl (pH7.5)). The annealing reaction was d i l u t e d to a f i n a l volume of 13.5 u l by the addition of 1 [il of 0.1 M DTT, 0.5 u l of (a- 3 5S)dATP, and 2 u l of nucleotide mix (1.5 uM each of dGTP, dCTP, and dTTP). Following the addition of 2 u l (1.8 Units) of T7 DNA polymerase, the l a b e l i n g mix was incubated at 22°. After 8 min, 3.5 u l of the reaction mix was added to each of four microfuge tubes. Each microfuge tube contained 2.5 ul of a d i f f e r e n t termination mix: one f o r each of the four deoxyribonucleotides found i n DNA. The termination mixes contained 80 uM each of three dNTPs (dATP, dCTP, dGTP, or dTTP) and 8 uM of the fourth dNTP. Termination reactions were performed at 37° f o r 10 minutes. Reactions were stopped by the addition of 4 u l of Stop Solution (95% formamide, 20 mM EDTA, 0.5% bromophenol blue, 0.5% xylene cyanol). Sequencing reactions were loaded i n t o sharktooth sample wells i n a 33 X 40 X 0.04 cm 6% polyacrylamide/urea sequencing ge l . Electrophoresis was at approximately 45 Watts f o r 1-6 hours. Ninety-nine m i l l i l i t r e s of gel so l u t i o n was prepared by mixing 15 ml of 40% (w/v) acrylamide stock (acrylamide:bisacrylamide; 19:1), 50 g of urea, 10 ml of 10X TBE (121.1 g/1 T r i s base, 55 g/1 boric acid, 7.4 g/1 Na2EDTA, pH 8.3) and 35 ml of water. Polymerization was induced by the addition of 1 ml 10% (w/v) ammonium sulphate and 20 u l TEMED. 96 Directed Deletions Exonuclease III directed deletions were performed using a protocol based on the procedure developed by Henikoff (1984). Five micrograms of plasmid DNA was double-digested with two r e s t r i c t i o n endonucleases to produce double-stranded DNA with one 5' protruding end and one 4-base 3'protruding end. The DNA was p r e c i p i t a t e d and re-dissolved i n 57 u l of 66 mM Tris-HCl (pH 8.0), 6.6 mM MgCl 2, at 35°. Exonuclease III (325 Units) was added to increase the reaction volume to 60 u l . A f t e r a delay of 20 seconds, and every 30 seconds thereafter, 2.5 u l samples of t h i s reaction were mixed with 7.5 u l of i c e - c o l d S i mix (40.5 mM potassium acetate (pH 4.6), 338 mM NaCl, 1.4 mM ZnS04, 6.6% g l y c e r o l , 3.5 units SI nuclease) i n separate microfuge tubes. When a l l of the desired samples had been c o l l e c t e d , the reactions were transferred to 22° f o r 30 minutes. The SI enzyme was i n a c t i v a t e d by heat-denaturation at 70° f o r 10 minutes following the addition of 1 ul of stop mix (300 mM Tris-HCl (pH 8.0), 50 mM EDTA). To assess the extent of exonuclease III digestion, 2 u l (approximately 40 ng DNA) of each sample was run on a 0.8% agarose g e l . A f t e r the addition of 0.2 units of Klenow DNA polymerase, the remainder of each reaction (9 ul) was incubated at 37° f o r 3 minutes. Next, 1 u l of dNTP mix (0.125 mM each of dATP, dCTP, dGTP, dTTP) was added to each tube. The tubes were incubated at 37° for an a d d i t i o n a l 10 minutes. Subsequent DNA l i g a t i o n s to vector and transformation were performed as previously described. 98 R E S U L T S Genetics Fourteen thousand second chromosomes were screened f o r P element induced hemizygous l e t h a l mutations i n region 31 using the protocol i n Figure 9. Four P element induced mutations were i s o l a t e d . Three f u l l y complemented the suppressors Su(var)207, Su(var)204, Su(var)216, and Suvar(2)l. (These stocks subsequently died of a mould i n f e c t i o n ) . The fourth, 0K15A, f a i l e d to complement Df(2L)J27, Df(2L)Jl06, and Su(var)216. Hybridization of a cloned P element probe to squashes of 0K15A s a l i v a r y gland polytene chromosomes established the presence of a P element i n region 3IE (see Figure 13). However, P elements frequently transpose in t o a gene and then excise again, deleting adjacent sequences. Such mutations are not r e a d i l y clonable, since no P element resides i n the gene. To t e s t f o r the presence of a functional P element at the Su(var)216 locus, the l e t h a l phenotype of 0K15A was reverted according to the protocol i n Figure 11. The t h i r d chromosome ca r r i e s A2-3(99B), a g e n e t i c a l l y engineered P element which does not i t s e l f transpose but which provides a very active source of transposase (Robertson et al., 1988). Amongst 5000 progeny from the dysgenic cross, 38 survived i n trans with Su(var)216. T h i r t y - s i x of these were s t e r i l e , but two, R34 and R28, were f e r t i l e . Both of these putative revertants were also v i a b l e when hemizygous for region 31. Thus, a mobile P element Figure 11. Phenotypic reversion of the homozygous l e t h a l i t y of i s associated with the l e t h a l phenotype of Su(var)216. The o r i g i n a l OK s t r a i n contains approximately 30 i n t a c t or p a r t i a l l y deleted P elements d i s t r i b u t e d over the f u l l chromosome complement. The abundance of elements i n OK15A made i t d i f f i c u l t to i d e n t i f y the Su(var)216p transposon; therefore, the X and t h i r d chromosomes were replaced with homologues without P elements. The genetic crosses are i l l u s t r a t e d i n Figure 10. Each cross was designed to introduce non-P element bearing chromosomes from males, since t h i s causes a lower incidence of transposition than the r e c i p r o c a l cross (see Kidwell, 1986). In addition to reducing the t o t a l number of P elements i n the stock, the crosses removed the second chromosome from a dysgenic background. Many P elements have a defective transposase gene and are only able to transpose i f an a l t e r n a t i v e source of transposase i s present. Thus, i f f u l l y functional P elements are removed from the genetic background, the remaining elements are not able to transpose. The loss of transposon mobility can be tested by crossing P element bearing males to non-P element bearing females at 29°. Under these extreme conditions, dysgenic female progeny are s t e r i l e (see Kidwell, 1986). OK15A females were s t i l l f e r t i l e at 29° a f t e r the replacement of both sets of X and t h i r d chromosomes, suggesting that none of the remaining P elements was capable of transposition. Once the mutagenized t h i r d and X chromosomes had been 101 replaced, a Southern blot of digested 0K15A genomic DNA was hybridized with a cloned P element. The pattern of bands obtained suggested that numerous P elements s t i l l remained i n the stock (Figure 12). To reduce the number of elements further, the r i g h t arm of Chromosome 2 was replaced by genetic recombination. Su(var)216F b pr cn/S Sp Tft nw° PinYt females were crossed to Gla/CyO males, and recombinant Tft nw° Pinrt/CyO male progeny were c o l l e c t e d and tested f o r the Su(var)216p l e s i o n . Each male was crossed to 4-5 females of genotype Df(2L)J27/CyO. In 150 such p a i r matings, two recombinants, OK#l and OK#2, were obtained that were l e t h a l i n trans with Df(2L)J27. Assuming that only a single cross-over event occurred, the entir e r i g h t arm of the chromosome should have been replaced. Approximately 5 DNA fragments homologous to a 900 bp P element probe are detectable on Southern blots of EcoRI-cut OK#l genomic DNA (Figure 12). A s i m i l a r number of bands hybridize to a 500 bp P element probe when OK#l DNA i s digested with BamHI and Xbal. BamHI and Xbal do not cut within the DNA of f u l l length P elements (O'Hare and Rubin, 1983); t h i s suggests that approximately 5 d i s t i n c t P elements are present i n OK#l. The OK#l s t r a i n was re-tested f o r the presence of a P element i n region 31 by in situ h y b r i d i z a t i o n (Figure 13), and then an OK#l genomic DNA l i b r a r y was constructed. 102 Figure 12. P elements i n 0K15A derived s t r a i n s . A. P elements on the second chromosome detected with a 950 bp HindiII fragment from pn 25.1 (O'Hare and Rubin, 1983). B. P elements on the second chromosome a f t e r genetic recombination. The probe was a 500 bp EcoRI/HindIII P element fragment from HBA-89 (Simon et al., 1985). Panel A. Lane: 1. Sail 2. BamHI 3. EcoRI 4. Xhol 5. Xbal 6. SstI Panel B. Lane: 1. BamHI 2. Xbal 3. EcoRI 103 104 Figure 13. L o c a l i z a t i o n of a P element i n the 3 IE region by in situ h y b r i d i z a t i o n . An arrow indicates the s i t e of P element hy b r i d i z a t i o n . 105 Cloning a P element i n Region 3IE A p a r t i a l EcoRI l i b r a r y was constructed i n EMBL4 as described i n Materials and Methods. The unamplified l i b r a r y contained approximately 7 x 105 recombinant phage, or 70 genomic equivalents of Drosophila melanogaster DNA, assuming an average i n s e r t s i z e of 17 kb and equal representation of a l l sequences. A portion of the unamplified l i b r a r y (3 x 105) was screened with a r a d i o l a b e l l e d P element probe. Twenty recombinant phage were plaque p u r i f i e d and t h e i r DNA i s o l a t e d . Since the chromosome bearing Su(var)216p contained approximately 5 P elements, several of the recombinant bacteriophage were expected to have genomic DNA ins e r t s from locations other than region 3IE. To i d e n t i f y genomic DNA inse r t s with sequence s i m i l a r i t y to region 31, the clones could have been tested by in situ h y b r i d i z a t i o n . Instead, P containing clones were i d e n t i f i e d by cross-hybridization with an overlapping series of cosmids containing sequences from region 31. Su(var)216 maps very close to da i n genetic recombination experiments (0.003 cM ; S i n c l a i r , unpublished); therefore, cosmid clones were obtained for the da region. One set of cosmid clones, the JT s e r i e s , was provided by C. Cronmiller. The second set, cl to clO, was i s o l a t e d using a cDNA (adm 134G6; Carlson, 1982) which maps by in situ h y b r i d i z a t i o n to region 3 IE. The two sets of cosmids overlap and span approximately 60 kb of DNA around da (only a small portion of 106 which has been mapped). Two overlapping cosmids that include da and the regions immediately proximal and d i s t a l to the da locus are shown i n Figure 14. Recombinant clones from the Su(var)216p l i b r a r y were divided i n t o groups based on cross-hybridization of the genomic DNA i n s e r t s , then a representative clone from each group was r a d i o l a b e l l e d and hybridized to Southern blots of JT35 and c7. Four independently i s o l a t e d , 17 kb recombinant phage cross-hybridized to the cosmid clones. A 4.2 kb EcoRI fragment containing the P element was i s o l a t e d from one of the recombinant phage clones (X#6.1) and inserted i n t o pUC19 to create pRP4.2. Hybridization of the 4.2 kb fragment to Southern blots of JT35 and c7 cosmid DNA indicated that the P element i s inserted i n a unique 3.8 kb "wild-type" EcoRI fragment (Figure 15). In Figure 15 Panel B, a P element probe hybridizes to pRP4.2 but not to the 3.8 kb EcoRI fragment i n JT35 or c7 fragment. Hybridization of the P element probe to a large fragment i n each of the cosmid lanes i s caused by P element sequences i n the vectors CosPer (JT35) and CosPneo (c7). Figure 15 Panel C demonstrates that the 3.8 EcoRI fragment hybridizes to pRP4.2. The 3.8 kb EcoRI was subcloned from JT35 to create pR3.8. Additional hybridization experiments and comparison of the r e s t r i c t i o n endonuclease maps of the two subclones positioned the P element within 3 kb of the da gene. However, genetic complementation data (Chapter 1) c l e a r l y demonstrate 107 X that the P element does not disrupt the da gene (data not shown). Disregarding the presence of a P element, the genomic i n s e r t i n pRP4.2 i s s l i g h t l y d i f f e r e n t from that i n pR3.8 (Figure 16). Digestion of the two plasmids with EcoRI and Sstll revealed that pRP4.2 lacks approximately 180 bp of DNA that i s present i n pR3.8. Sequence analysis of pRP4.2, pR3.8, and overlapping DNA fragments from the P element-mutated chromosome ( see Chapter 4 ) indicates that the b pr cn chromosome has an extra EcoRI s i t e , caused by a single basepair t r a n s i t i o n , located between the two s i t e s i n pR3.8. This extra s i t e served as one of the cloning s i t e s for pRP4.2. R e s t r i c t i o n endonuclease mapping and cross-hybridization experiments positioned the P element i n s e r t i o n of pRP4.2 i n a fragment with homology to a 1.5 kb Hindlll/EcoRI fragment from pR3.8 (Figures 16-18). Comparison of the s i z e of the wild-type fragment with the transposon-containing fragment suggests the element i s approximately 600 bp long. This i n t e r p r e t a t i o n i s consistent with the observation that the P element i s missing several i n t e r n a l r e s t r i c t i o n endonuclease s i t e s that are normally present i n f u l l length (2.9 kb) P elements (Figure 16). A s i n g l e Hindlll s i t e not present i n pR3.8 was present i n pRP4.2. When pRP4.1 was digested with Hindlll and probed with r a d i o l a b e l l e d P element DNA, only DNA fragments from one side of the enzyme recognition s i t e hybridized (Figure 18). There 110 1 1 112 \ 114 s are two Hindlll s i t e s i n f u l l length P elements (Figure 16): one i s located 38 bp from one end of the element, and the other i s approximately 840 bp di s t a n t . Therefore, the hybridization data suggest that the conserved Hindlll s i t e i n pRP4.2 i s the one cl o s e s t to the end of the P element. The precise l o c a t i o n and siz e of the P element i n s e r t was determined by sequencing. A 1.4 kb Hindlll fragment that cross-hybridized to the P element was subcloned and then sequenced on a single DNA strand along with 300 bp of DNA from the adjacent 850 bp Hindlll/EcoRI fragment (see Figure 16). The sequence of the P element, i n f e r r e d from a comparison of the genomic DNA with a cloned P element (O'Hare and Rubin, 1983), appears i n Appendix 4. The P element i n pRP4.2 i s 609 bp long and i s located approximately 950 bp from the nearest wild-type Hindlll s i t e and 200 bp from the adjacent Sstll s i t e . The centre of the element i s deleted with respect to a f u l l length P element, but i t retains the 31 bp perfect inverse terminal repeats required f o r t r a n s p o s i t i o n . The same 8 bp of genomic DNA are r e i t e r a t e d on both sides of the element: a c h a r a c t e r i s t i c sequence d u p l i c a t i o n caused by P element i n s e r t i o n (O'Hare and Rubin, 1983). The open reading frames i n i n t a c t P elements a l l extend 5 ' to 3 ' away from the Hindlll s i t e that i s retained i n pRP4.2. In vivo t r a n s c r i p t i o n from the P element i n 31E i s therefore predicted to be oriented towards da. Since the tr a n s c r i p t i o n i n i t i a t i o n and termination s i t e s are s t i l l 116 present i n the truncated transposon, a mRNA of less than 600 bp i s predicted. Because of the presence of additional elements of unknown siz e i n the OK#l s t r a i n , I have not searched for t h i s t r a n s c r i p t . If the l e t h a l phenotype of Su(var)216p i s associated with the P element i n region 3IE, then revertants of Su(var)216* should be a l t e r e d at the s i t e of transposon i n s e r t i o n . Su(var)216p revertants R28 and R34 were tested for the loss of the P element Hindlll endonuclease recognition s i t e . Figure 19 demonstrates the absence of the Hindlll s i t e when R28 genomic DNA was digested with EcoRI/HindIII and probed with a flanking 350 bp Sstll/EcoRI fragment from pRP4.2. M o b i l i t y differences between the balancer and the b pr cn chromosome fragments are caused by the previously described EcoRI polymorphism. I d e n t i c a l r e s u l t s were obtained f o r R34. Subsequent cloning and sequencing experiments (data not shown) have confirmed that the P element i s e n t i r e l y absent from both revertants. Furthermore, no flanking wild-type sequences were deleted when the element excised. Taken together, the cloning and reversion studies support the hypothesis that the P element i n pRP4.2 causes the Su(var)216P phenotype in vivo. Transcripts Originating Near the Cloned P element Cronmiller et al. (1988) observed three t r a n s c r i p t s that hybridized to a 4.7 kb Sail fragment immediately adjacent to da. The P element resides within t h i s fragment, so smaller 117 118 i n t e r n a l fragments were used to map the t r a n s c r i p t i o n units further and to obtain cDNA clones f o r each t r a n s c r i p t . To search f o r putative t r a n s c r i p t i o n units that might be disrupted by the Su(var)216p P element i n s e r t , Northern blots of e a r l y embryo mRNA (0-6 h) were probed with DNA fragments adjacent to the transposon. In t h i s study only two tr a n s c r i p t s were detected when Northern blots were probed with fragments from t h i s region (Figure 20). R e s t r i c t i o n endonuclease fragments to the l e f t of the s i t e of P element i n s e r t i o n hybridized to an 800 nt t r a n s c r i p t , while sequences to the ri g h t hybridized to an 1100 nt t r a n s c r i p t . A wild-type 1.0 kb Sstll/Hindlll fragment which encompasses the s i t e of P element i n s e r t i o n hybridized to both mRNAs. Neither t r a n s c r i p t i s altered i n siz e i n the mutant stra i n s Su(var)216 and Su(var)216* (I.P. Whitehead, personal communication). To locate the t r a n s c r i p t i o n units adjacent to the P element more pr e c i s e l y , cDNA clones were i s o l a t e d from a t h i r d l a r v a l i n s t a r imaginai disc cDNA l i b r a r y constructed i n XgtlO. This method detected clones representing three t r a n s c r i p t i o n units within the 4.7 kb Sail fragment. I n i t i a l l y , the l i b r a r y was probed with two fragments adjacent to the P element. One was a 1.5 kb EcoRI/HindIII fragment, which hybridized to both the 800 nt and 1100 nt t r a n s c r i p t s . The second was a 1.0 kb Hindlll fragment that hybridized only to the 800 nt message. Twelve 1200 bp and eight 800 nt cDNAs were i s o l a t e d . R e s t r i c t i o n maps of the two types of cDNA appear i n Figure 21. 119 Figure 20. Embryonic t r a n s c r i p t s that hybridize to pR3.8. Northern blots of poly(A +) RNA (5 ^g/lane) were probed with (A) a 1.4 kb Bglll/Sstll fragment, (B) a 450 bp Sstll/Hindlll fragment, and (C) a 900 bp Hindlll fragment (see Figure 16). Transcript sizes were i n f e r r e d using high and low molecular weight RNA ladders (BRL) as siz e standards. A. B. C. 1100 nt-800 nt-120 Figure 21. Restr ict ion maps of the three cDNA c lones that hybridize to pR3.8. (Symbols: R, EcoRI; H, Hindl l l ; K, Kpnl ; K, Kpnl; P, Pst l ; S, Sail) p c 8 0 0 R H R L J I PC1100 R R R R I I L J cBgl4 R P K S R I I I I I 600 bp 121 Copy DNAs of s i m i l a r s i z e hybridized to each other, but the two classes of cDNA d i d not cross-hybridize. The same two classes of cDNA were also present i n a l i b r a r y made from embryonic RNA. One representative clone from each s i z e class was hybridized to a Northern b l o t of early embryo mRNA. Each clone hybridized to a t r a n s c r i p t i o n unit s i m i l a r i n s i z e to the cDNA probe, suggesting that the 800 and 1200 bp cDNAs are nearly f u l l length. A representative 800 nt cDNA , pc800, hybridized only to sequences on the side of the P element nearest to da (Figure 22). The 1.1 kb species, represented by pel 100, hybridized to sequences on the opposite side of the P element (Figure 23). In addition to hybridizing to the 3.8 kb EcoRI fragment, the larger cDNA also hybridized to the adjacent 0.5 kb and 5.8 kb EcoRI fragments (Figure 23). To search f o r the t h i r d t r a n s c r i p t near da (Cronmiller et a l . , 1988) that was not detected on Northern b l o t s , the imaginai d i s c l i b r a r y was screened with a subclone from the 4.7 kb Sail fragment that f a i l e d to hybridize to e i t h e r pcllOO or pc800. Using a 600 bp Sall/Bglll fragment centromere d i s t a l to the s i t e s of hybridization f o r pcllOO and pc800, a set of three homologous clones was i d e n t i f i e d . These clones hybridize to several r e s t r i c t i o n endonuclease fragments within the larger 5.8 kb EcoRI fragment, but they do not hybridize to pcllOO. The longest cDNA recovered (cBgl4) i s 3.2 kb (Figure 21). This i s s i g n i f i c a n t l y larger than the 1900, 1100, and 800 122 123 Figure 23. pcllOO hybridized to subcloned DNA from the 31E region. pHc2.7 i s a 2.7 kb Hindi subclone that spans the s i t e of P element i n s e r t i o n (see Figure 14). pR5.8 i s an EcoRI subclone that overlaps pHc2.7 (see Figure 14). Lanes : 1. pR3.8 HindIII/SstlI 2. pRP4.2 HindIII/SstlI 3. pHc2.7 EcoRI/Bglll 4. pR5.8 EcoRI/Bglll 5. X Hindlll 125 nt t r a n s c r i p t s observed by Cronmiller et al. (1988). Perhaps our cDNA represents an unprocessed message or an imaginai d i s c - s p e c i f i c t r a n s c r i p t . In any event, t h i s 3.2 kb cDNA helps to define the probable extent of the regulatory sequences associated with the 1100 nucleotide t r a n s c r i p t i o n u n i t . Thus, both candidates for the Su(var)216 gene (represented by pcllOO and pc800) have been positioned between flanking t r a n s c r i p t i o n u n i t s . Alignment of Genomic, cDNA, and P element Sequences Since both the pcllOO and pc800 cDNA clones hybridized to sequences adjacent to the s i t e of P element i n s e r t i o n , the pattern of hybridization alone did not resolve which t r a n s c r i p t i o n unit encoded Su(var)216p. To address t h i s question further, the DNA sequences of the pc800 and pcllOO were determined. These sequences were then compared with the DNA adjacent to the s i t e of P element i n s e r t i o n . The cDNA pc800 was sequenced on a single strand. Subsequent analyses demonstrated that t h i s t r a n s c r i p t i s not encoded by Su(var)216p. The sequence of the 800 bp cDNA i s presented i n Appendix 4, along with the genomic sequence of the gene i t represents. The cDNA pcllOO was digested with EcoRI, and three fragments, 200, 450, and 450 bp long, were subcloned i n t o pUC19 and then sequenced inward from the pol y l i n k e r on both strands. The ori e n t a t i o n and order of the EcoRI fragments was determined by comparing the cDNA sequence 127 with the sequence of cloned genomic DNA (see below) and by sequencing across the EcoRI recognition s i t e s i n the parent phage clone using synthetic primers (see Chapter 4). Comparison of the cDNA sequences with the genomic DNA flanking the P element revealed that the genes represented by pc800 and pcllOO are divergently transcribed. The or i e n t a t i o n of the genes was i n f e r r e d from the following properties of the cDNAs r e l a t i v e to the genomic sequence : (1) the l o c a t i o n of the poly (A) + t a i l , (2) a long open reading frame, and (3) the ori e n t a t i o n of the s p l i c e s i t e s , and (4) the p o s i t i o n of the polyadenylation s i g n a l . These features are discussed i n d e t a i l i n a subsequent section. The s i t e of P element i n s e r t i o n i s 2 bp upstream of the 5' end of the 1100 bp cDNA and approximately 560 bp from the 5' end of the 800 bp cDNA (see Figure 26, Appendix 4). These data suggest that the P element most l i k e l y a f f e c t s the expression of the larger t r a n s c r i p t i o n u n i t . The proof of t h i s assertion i s presented i n Chapter 4. The Cloned P element i s Adjacent to cdc2Dm The sequence and conceptual t r a n s l a t i o n of pcllOO i s presented i n Figure 24. Excluding the poly(A) t a i l , the cDNA i s 1035 nucleotides long and contains 32 nucleotides of 5' flanking sequence, an open reading frame of 891 nucleotides, and a 3' flanking region of 106 nucleotides. A p o t e n t i a l polyadenylation signal (AATAAA; Proudfoot and Brownlee, 1976) i s located 88 nucleotides beyond the ochre stop codon of the 128 Figure 24 . The nucleotide and deduced protein sequence of the product of the cdc2Dm locus. Amino acids that are invariant among kinases are indicated i n bold-face type; highly conserved residues are i n i t a l i c s . Thin l i n e s highlight conserved kinase sequences; a th i c k l i n e shows the location of the PSTAIR sequence. GGTGGCTTGCAAAGAAATAGCTTAATAAATT 15 Met Glu Asp Phe Glu Lys I l e Glu Lys I l e Gly Glu Gly Thr Tyr ATG GAG GAT TTT GAG AAA ATT GAG AAG ATT GGC GAG GGC ACA TAT 30 Gly Val Val Tyr Lys Gly Arg Asn Arg Leu Thr Gly Gin I l e Val GGC GTG GTG TAT AAA GGT CGC AAT CGC CTG ACG GGC CAA ATT GTG 45 Ala Met Lys Lys I l e Arg Leu Glu Ser Asp Asp Glu Gly Val Pro GCA ATG AAG AAA ATC CGC TTG GAG TCC GAC GAC GAA GGC GTT CCA 60 Ser Thr Ala I l e Arg Glu I l e Ser Leu Leu Lys Glu Leu Lys His TCA ACC GCG ATC AGA GAA ATT TCG TTG CTT AAG GAG TTG AAA CAT 75 Glu Asn I l e Val Cys Leu Glu Asp Val Leu Met Glu Glu Asn Arg GAA AAC ATT GTC TGT TTG GAG GAT GTT TTG ATG GAG GAG AAC CGC 90 I l e Tyr Leu I l e Phe Glu Phe Leu Ser Met Asp Leu Lys Lys Tyr ATA TAC TTC ATC TTT GAA TTC CTA TCG ATG GAC CTC AAG AAA TAC 105 Met Asp Ser Leu Pro Val Asp Lys His Met Glu Ser Glu Ser Val ATG GAT TCG CTG CCA GTT GAT AAG CAC ATG GAG AGT GAA TTG GTC 120 Arg Ser Tyr Leu Tyr Gin I l e Thr Ser Ala I l e Leu Phe Cys His CGT AGC TAT TTG TAC CAA ATA ACT AGC GCC ATT CTT TTC TGC CAT 135 Arg Arg Arg Val Leu His Arg Asp Leu Lys Pro Gin Asn Leu Leu CGT CGG CGA GTA CTT CAC CGT GAT CTT AAG CCG CAG AAC TTA CTA 129 150 I l e Asp Lys Ser Gly Leu I l e Lys Val Ala Asp Phe Gly Leu Gly ATC GAC AAG AGT GGC CTC ATA AAA GTC GCC GAC TTT GGA CTT GGC 165 Arg Ser Phe Gly I l e Pro Val Arg I l e Tyr Thr His Glu I l e Val CGA TCC TTT GGC ATT CCG GTG CGC ATT TAT ACG CAC GAG ATT GTT 180 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gly Ser Pro Arg ACC TTG TGG TAC AGA GCG CCG GAG GTG CTA CTG GGT TCA CCC CGG 195 Tyr Ser Cys Pro Val Asp I l e Trp Ser I l e Gly Cys I l e Phe Ala TAT TCC TGT CCC GTC GAT ATC TGG TCC ATT GGA TGC ATA TTC GCG 210 Glu Met Ala Thr Arg Lys Pro Leu Phe Gin Gly Asp Ser Glu I l e GAG ATG GCA ACG AGA AAG CCG CTA TTC CAG GGT GAC TCG GAA ATT 225 Asp Gin Leu Phe Arg Met Phe Arg I l e Leu Lys Thr Pro Thr Glu GAC CAG TTG TTT AGA ATG TTT AGA ATT CTG AAA ACA CCT ACC GAA 240 Asp I l e Trp Pro Gly Val Thr Ser Leu Pro Asp Tyr Lys Asn Thr GAC ATT TGG CCG GGC GTT ACT TCG CTA CCC GAC TAT AAG AAC ACG 255 Phe Pro Cys Trp Ser Thr Asn Gin Leu Thr Asn Gin Leu Lys Asn TTC CCC TGC TGG TCC ACG AAC CAA TTG ACC AAT CAG TTA AAG AAT 270 Leu Asp Ala Asn Gly I l e Asp Leu I l e Gin Lys Met Leu I l e Tyr CTC GAT GCG AAT GGT ATT GAT CTC ATA CAA AAG ATG TTA ATC TAC 285 Asp Pro Val His Arg I l e Ser Ala Lys Asp I l e Leu Glu His Pro GAT CCA GTT CAT CGC ATT TCC GCC AAG GAC ATT TTG GAG CAT CCC Tyr Phe Asn Gly Phe Gin Ser Gly Leu Val Arg Asn Oc TAT TTC AAT GGT TTT CAA TCG GGC TTA GTT CGA AAT TAACGTTCGGT ATTCTCGTTTGACTTTAACTAAGAATTTTAAAACAAGAGATCTTGGTATCTAA TCTAAAGCAAAATAGCCGTAAATAAAACTAAGGGTGTAAAAC[poly(A)] 130 open reading frame. The open reading frame i n pel100 codes f o r a 297 amino aci d polypeptide (Mr 34,442). Comparison of the predicted protein sequence with sequences i n the EMBL/Genbank data base suggests that pel100 encodes a kinase. The conceptually translated protein contains the sequences Arg 1 2 7-Asp-Leu 1 2 9, Asp 1 4 6-Phe-Gly 1 4 8, Ala 1 7 1-Pro-Glu 1 7 3, and Gly n-Glu-Gly-Thr-Tyr-Gly 1 6. The f i r s t three sequences are t y p i c a l of protein kinase c a t a l y t i c domains (Hanks et al., 1988; Hunter, 1987); the l a s t sequence matches a consensus motif (Gly-X-Gly-X-X-Gly) found i n protein kinases and other nucleotide binding proteins (Hanks et al., 1988). Furthermore, when the predicted protein i s aligned with known kinase sequences, i t contains 9 amino acid residues which are invariant amongst kinases (Gly 1 6, Lys 3 3, Glu 5 1, Asp 1 2 8,Asn 1 3 3, Asp 1 4 6, Gly 1 4 8, Glu 1 7 3, and Arg 2 7 5 ) plus 5 other residues which are highly conserved (Hanks et al., 1988). A variant of the consensus Asp-Leu-Lys-Pro-Glu-Asn, Asp 1 2 8-Leu-Lys-Pro-Gln-Asn 1 3 3, suggests that the putative kinase has serine/threonine s p e c i f i c i t y (Hanks et al., 1988). Sequence comparisons indicate that the deduced protein i s a member of the CDC28-cdc2+ kinase subfamily, and i s most s i m i l a r to the highly conserved CDC2 proteins. I t contains a 16 residue PSTAIR sequence (EGVPSTAIREISLLKE; Figure 24, Figure 25) i d e n t i c a l to those found i n a l l homologues of the S. pombe cdc2+ gene product (Norbury and Nurse, 1989). There i s 72% sequence s i m i l a r i t y between the Drosophila protein and 131 132 sequence i d e n t i t y ; 133 the human CDC2 gene product, approximately 70% s i m i l a r i t y with the mouse and chicken CDC2 proteins, and 68% s i m i l a r i t y with the Schizosaccharomyces pombe cdc2 gene product (see Figure 25). The most c l o s e l y related protein i n Drosophila i s the cdc2c gene product (Lehner and O ' F a r r e l l , 1990) which shares 58% sequence i d e n t i t y with the deduced protein product. These s i m i l a r i t i e s suggest that the putative kinase gene i s the Drosophila homologue of the f i s s i o n yeast cdc2 gene. Since t h i s work was completed, Lehner and O' F a r r e l l (1990b) and Jimenez et al. (1990) have independently confirmed t h i s speculation by rescuing the phenotype of a yeast cdc2 mutant with a cloned cdc2Dm gene. cdc2Dm Transcription Northern blots of mRNA from d i f f e r e n t developmental stages were probed with the cdc2Dm cDNA (data not shown). An 1100 nt t r a n s c r i p t i s present e a r l y i n development (0-9 hours), but i s rare i n larvae, pupae and adult males. Some tr a n s c r i p t i o n i s evident i n adult females, presumably because of the maternal contribution of cdc2Dm t r a n s c r i p t i n developing eggs. cdc2Dm Gene Structure To deduce the structure of the cdc2Dm t r a n s c r i p t i o n unit, a 2.9 kb HincII/Sall fragment of genomic DNA was sequenced (Figure 27). The fragment contains sequences homologous to 134 Figure 26. The sequencing strategy for cdc2Dm. A. Extent and d i rect ion of sequencing the directed delet ions is shown by arrows. B. The locat ions of cdc2Dm (pc1100) and f lanking t ranscr ip t ion units. Th ick lines below the restr ict ion map represent exons. Thinner lines represent introns. (Symbols : B, Bgl l l ; R, EcoRI; He, H i n d i ; St, Sstl l ) A. He St R R B S B. P element He St R R B S p c 8 0 0 cdc2Dm cBgl4 t o o bp 135 cDNAs that hybridize to opposite sides of the cdc2Dm t r a n s c r i p t i o n unit; therefore, the e n t i r e regulatory region of cdc2Dm has probably been sequenced. The complete sequence appears i n Appendix 4. A comparison of the cDNA and genomic sequences indicates that the cdc2Dm gene has two introns (see Figure 26 and Appendix 4). Intron 1 i s 385 bp long, and i n t r o n 2 i s 57 bp long. Both introns have t y p i c a l GT/AG s p l i c e junctions. Intron 1 interrupts the codon that s p e c i f i e s Arg 5 0 within the PSTAIR sequence of the cdc2Dm protein. Intron 2 interrupts the codon that s p e c i f i e s Arg 2 1 8. Many regulatory elements that influence t r a n s c r i p t i o n by polymerase II are located just 5' to the coding region. If cBgl4, the clone that hybridizes immediately 3 ' to cdc2Dm, represents a bona fide t r a n s c r i p t i o n unit, i t seems u n l i k e l y that cdc2Dm possesses 3' regulatory sequences because the polyadenylation s i t e of cBgl4 i s only 14 bp from the 3' end of cdc2Dm (Figure 26 and Appendix 4). Since the t r a n s l a t i o n i n i t i a t i o n s i t e s of cdc2Dm and the gene represented by pc800 are separated by less than 550 bp (Figure 26 and Appendix 4), t h i s region was searched f o r regulatory motifs. A TATA box-l i k e sequence (AATAAA) i s located 115 bp 5' to the t r a n s l a t i o n a l s t a r t s i t e of the cdc2Dm gene (Figure 27). Twenty-two basepairs downstream from t h i s TATA-like sequence i s a sequence (ATCGTTT) s i m i l a r to the consensus insect cap s i t e ATCAG/TTC/T (Hultmark et al., 1986). However, at p o s i t i o n 136 Figure 27. P o t e n t i a l upstream regulatory sequences of cdc2Dm. Symbols: * TATA-like sequences; ++ a cap s i t e sequence; =, -r e i t e r a t e d motifs. GAAACAACAA AAATGAAGTA AAACAGTTGC GGTATTCCAA TTACATTTTT TAAATTAATT TCTTTAGTAC CGTACTACTG GTACTCACCT TCAAAAGATA TAAAATAGAA ATTAATTGCA CCAAAAACTC ATAAGTTAAT TAATAGTATA TTAGCAGAAA CGTTTGTCTC CGAACTCAAA CAAAGTGATG TCTTAATTAA TTGAAATCAC TATAAAAAAA AGCGTGGAAT CAATAAGTTG CCTGAATATT * * * * * * * * * * * * i i m n GAGTTTCATT CCCACATTCC AAATGAATAA ATGTAGCTAG CTTAGCATCG TTTAAACTGT CTGGTAATAC TAGAGCATAT ACGTCAAAAA CGCGCTAATT TAAAAGTCGG TGGCTTGCAA AGAAATAGCT TAATAAATT ATG GAG GAT Met Glu Asp TTT GAG AAA ATT GAG AAG ATT GGC GAG GGC ACA TAT GGC Phe Glu Lys I l e Glu Lys I l e Gly Glu Gly Thr Tyr Gly 137 175 upstream of the t r a n s l a t i o n s t a r t s i t e there e x i s t s a sequence (TATAAA) which i s a better match with the canonical TATA-box. Therefore, i t w i l l be necessary to determine the i n i t i a t i o n s i t e f o r t r a n s c r i p t i o n i n order to i n f e r the l o c a t i o n of the true TATA sequence. Three r e - i t e r a t e d sequence motifs occur i n the region immediately 5' to cdc2Dm. The sequence TGAATA i s repeated twice (at positions -117 and -148; Figure 27), and so i s the sequence CATTCC (positions -126 and -134). The sequence AATTAATT/A occurs four times i n the region between the t r a n s l a t i o n i n i t i a t i o n s i t e s of the two genes. The s i g n i f i c a n c e of these short motifs, i f any, i s not currently known. Sequencing the 5' End of a cdc2-like Gene from D. v i r i l i s A d e t a i l e d mapping of upstream t r a n s c r i p t i o n a l control elements associated with cdc2Dm was beyond the scope of t h i s work. Nonetheless, the central r o l e of t h i s gene i n c o n t r o l l i n g c e l l cycle and i n i t i a t i o n of chromosome condensation, as well as the r e l a t i v e l y small region between cdc2Dm and i t s 5' neighbour (less than 600 bp), makes such studies i n t r i g u i n g . To obtain preliminary data f o r future studies on the control of cdc2Dm expression, a gene with sequence s i m i l a r i t y to cdc2Dm was cloned from Drosophila v i r i l i s . The sequence 5' to t h i s gene was then compared to that of D. melanogaster to i d e n t i f y conserved regulatory 138 motifs. A D. v i r i l i s genomic DNA l i b r a r y constructed i n EMBL3 was screened with a cdc2Dm cDNA using conditions that permit complementary DNA strands to anneal even i f t h e i r nucleotide sequences d i f f e r by as much as 28%. Ten recombinant bacteriophage were recovered, a l l of which share e s s e n t i a l l y i d e n t i c a l r e s t r i c t i o n endonuclease digestion fragments. A single fragment which cross-hybridized to the f i r s t 180 bp of pcllOO was subcloned. The r e s t r i c t i o n map of the cloned fragment i s completely d i f f e r e n t from that of the region surrounding cdc2Dm (Figure 28). The clone was sequenced i n one d i r e c t i o n only to i d e n t i f y the 5' end of the D. v i r i l i s gene (see Figure 27). The sequence encoding the f i r s t 217 amino acids of the cdc2-like gene was determined along with 300 bp 5' to the i n i t i a t i o n codon (Figure 29, Figure 30). To ensure the accuracy of the 5' sequence, t h i s region was also sequenced on the opposite DNA strand using synthesized DNA primers. Several l i n e s of evidence suggest that the cloned and p a r t i a l l y sequenced gene i s the Drosophila v i r i l i s cdc2 gene. F i r s t , the D. v i r i l i s nucleotide sequence i s 75.5% i d e n t i c a l to that of the cdc2Dm coding region (Figure 29). Second, the inf e r r e d s p l i c e s i t e s f or the f i r s t and second introns are located at the same points i n the DNA sequence. Third, the predicted amino acid sequence of the D. v i r i l i s protein d i f f e r s from the cdc2Dm sequence by only 9 residues (Figure 139 Figure 28. Restr ic t ion map of a subclone of D. v i r i l is DNA with homology to the first and second exons of cdc2Dm. (Symbols : B, Bgl l l ; R, EcoRI; K, Kpnl; S, Sa i l ; St, Sst l l ) R L B H e B I » St _ L c d c 2 D m 5 ' h o m o l o g y 5 0 0 bp 140 Figure 29. Sequence comparison of 142 Figure 30. DNA sequence of the 5' flanking region of a cdc2-l i k e gene from Drosophila v i r i l i s . Underlined sequences ind i c a t e the lo c a t i o n of DNA primers. GATTTAAAAA GTTGTGGCCT GCCGGCGTTC AAAATGGTCG TGCATTAAAA ATAGTGTATG CGATTGAAAT AACTGTGGCA TGCATACATA TAGCGAGATG GGCTGCCACC CAGGTACCAT TTCCCATTTG TTTATTTTCA TTTCATAAAT ACCTTAAAAA CATATATTAA CGGCTTCATA CTCAGCAGTC TGGGGTCGCC GTATGTCTAT GTGTTTCAAT ATATTTAATA AAAAACCTGC GAAAACGCAT CTGAACGTTC TTACTCTGTC TCGTTTCGAT ACATTGTATG TATCGGTGCT AGCTACCGTG ATTTTTTCGG 50 CAGCCGTGAC 100 GGCTTTTGAG 150 TGGGCACAGT 200 TTTTCTTGTT 250 GTTAGTGGTA 300 TATAAATATA 350 GTATTCTAAA 400 AAACTACCCG 4 8GGAGTAAAATGGAAGACTTCGAAAAAATTGAAAAGATTGGAGAAGGTACTTATGGCGT MetGluAspPheGluLysIleGluLysIleGlyGluGlyThrTyrGly 143 29). Each s u b s t i t u t i o n , with the exception of Glu 9 7, has an i d e n t i c a l amino ac i d at the same p o s i t i o n i n at l e a s t one other member of the CDC2 protein family. The cdc2Dm Asp 9 7 and D. v i r i l i s Glu 9 7 residues are s i m i l a r i n charge. F i n a l l y , cBgl4, the cDNA that hybridizes to DNA adjacent to cdc2Dm also hybridizes to a DNA fragment adjacent to the D. v i r i l i s gene (data not shown). Unfortunately, a comparison of the 5' region of the D. v i r i l i s and D. melanogaster clones reveals no strong sequence s i m i l a r i t i e s . In p a r t i c u l a r the r e i t e r a t e d sequence motifs adjacent to the cdc2Dm gene are not found near the D. v i r i l i s sequence (Figure 30). 144 DISCUSSION This chapter described the cloning and sequencing of a gene with strong sequence s i m i l a r i t y to the cell-division-cycle-2 {cdc2) gene of humans and S. pombe. Recently, two research groups have reported the i s o l a t i o n of cDNA clones from Drosophila melanogaster which, when placed i n an expression vector, are able to rescue c e l l c ycle progression i n yeast defective f o r the cdc2 product (Jimenez et a l . , 1990; Lehner and O' F a r r e l l , 1990). These cDNA clones hybridize to 3IE on Drosophila polytene chromosomes and are i d e n t i c a l i n sequence to the gene adjacent to the Sufvar;.2I6-associated P element. The cDNA sequence reported by Jimenez et al. (1990) extends 23 bp further 5' than our cDNA. This suggests that the P element i n Su(var)216p i s within the transcribed portion of cdc2Dm, although t h i s was not apparent on Northern blots of adult mRNA (I.P. Whitehead, personal communication). We are currently r e - i n v e s t i g a t i n g the expression of Su(var)216p because the mutant i s not completely l e t h a l as a homozygote ( see Chapter 3 ). This suggests that some Su(var)216p mRNA may be transcribed and t h i s mRNA could be used as a molecular marker to te s t f o r the perdurance of the cdc2Dm mRNA. In Drosophila, some maternally i n h e r i t e d messenger RNAs are degraded at the mid-blastula t r a n s i t i o n when t r a n s c r i p t i o n i s i n i t i a t e d from the zygotic genome. Jimenez et al. (1990) have suggested that cdc2Dm mRNA may be 145 excluded from the developing zygote a f t e r the mid-blastula t r a n s i t i o n . If a P element can be detected i n a mutant cdc2Dm t r a n s c r i p t , then i t may be possible to ask whether maternally derived cdc2Dm mRNA l o c a l i z e s to post-mid-blastula t r a n s i t i o n embryos that do not i n h e r i t the Su(var)216p a l l e l e from eith e r parent. The cdc2Dm gene i s simple i n i t s organization. I t has two introns, unlike the S. pombe gene which has four introns (Hindley and Phear, 1984), and the S. cerevisiae gene which has none (Lorincz and Reed, 1986). The s i t e s of the introns within the coding sequence of cdc2Dm, are e n t i r e l y d i f f e r e n t from those i n S. pombe. Homologues of cdc2 have been found i n a v a r i e t y of organisms, and i t seems l i k e l y that the product of the cdc2 gene i s required f o r normal mitotic progression i n a l l eukaryotes (see Nurse, 1990). The highest l e v e l of cdc2Dm mRNA i s detected i n the ear l y embryo when c e l l d i v i s i o n i s most frequent. During l a t e r stages f a r less mRNA i s detectable. Fine-grained analyses indicate that cdc2Dm mRNA i s expressed i n the d i v i d i n g nervous system of larvae, but not i n other m i t o t i c a l l y i n a c t i v e tissues (Jimenez et al., 1990). In adults, cdc2Dm mRNA i s rare i n males but somewhat more abundant i n females, probably because they sequester cdc2Dm mRNA i n t h e i r eggs (Jimenez et al., 1990). The presence of cdc2Dm cDNA clones i n an imaginai d i s c cDNA l i b r a r y suggests, not s u r p r i s i n g l y , that cdc2Dm i s also expressed i n t h i s 146 p r o l i f e r a t i n g t i s s u e . Observation of cdc2Dm mRNA i n whole-mount embryos suggests that cdc2Dm may be t r a n s c r i p t i o n a l l y regulated at d i v i s i o n 14 (Jimenez et al., 1990). At t h i s stage, the ear l y synchronous c e l l d i v i s i o n s of the embryo give way to a s p a t i a l l y and temporally r e s t r i c t e d sequence of c e l l d i v i s i o n s (Foe, 1989). At a gross l e v e l , cdc2Dm t r a n s c r i p t i o n increases i n the regions that are about to di v i d e . Several genes that display s p a t i a l l y r e s t r i c t e d patterns of t r a n s c r i p t i o n have complex upstream regulatory sequences that permit the binding of diverse t r a n s c r i p t i o n f a c t o r s . In at le a s t one case, engrailed, these sequence elements have been conserved through evolution (Kassis et al., 1989). As a prelude to promoter deletion studies we looked f o r 5' sequence conservation i n a Drosophila v i r i l i s homologue of cdc2Dm. When cdc2Dm and a s i m i l a r sequence from Drosophila v i r i l i s were aligned to t e s t f o r sequence s i m i l a r i t y 5' to the t r a n s l a t i o n a l s t a r t s i t e of the genes, no strong s i m i l a r i t y was found. Drosophila melanogaster, and Drosophila v i r i l i s diverged approximately 60 m i l l i o n years ago (Beverley and Wilson, 1984), and t h i s evolutionary distance should be s u f f i c i e n t to di s t i n g u i s h between functional and nonfunctional sequences (estimated unconstrained divergence time, 1% per m i l l i o n years: Hayashida and Miyata, 1983; Pe r l e r et al., 1980). This suggests that the three r e - i t e r a t e d sequences 5' to the t r a n s l a t i o n a l s t a r t s i t e of cdc2Dm might not be 147 important f o r t r a n s c r i p t i o n a l regulation. However, t h i s i n t e r p r e t a t i o n i s subject to q u a l i f i c a t i o n . I t i s possible that the cloned gene from D. v i r i l i s i s i n a c t i v e , or i s a gene with s l i g h t l y d i f f e r e n t functions from cdc2Dm. I f t h i s were the case, then the regulatory regions associated with the D. v i r i l i s and D. melanogaster genes might be quite d i f f e r e n t . Further experiments are necessary to determine the si g n i f i c a n c e of the r e - i t e r a t e d sequences adjacent to cdc2Dm. In yeast, the cdc2 gene product functions i n the Gx to S and G2 to M phase t r a n s i t i o n s of the c e l l c y c l e . Thus, i t i s perhaps s u r p r i s i n g that a P element i n cdc2Dm i s associated with suppression of PEV. However, p34 c d c 2 kinase a c t i v i t y has been implicated i n the control of chromatin condensation during mitosis. In the macroplasmodium of Physarum polycephalum, histone HI phosphorylation i s strongly correlated with chromatin condensation (Bradbury et al., 1973, 1974). Phosphorylation of histones i s probably brought about by p34 c d c 2, since p34 c d o 2 phosphorylates the same s i t e s on histone Hi in vitro as are phosphorylated in vivo (Langan et al., 1989). Furthermore, i n Drosophila melanogaster, a l t e r a t i o n s i n the a v a i l a b i l i t y of histones f o r DNA binding, e i t h e r by a l t e r i n g the amount of template or a l t e r i n g histone modifications, appears to suppress PEV (Moore et al., 1979; Mottus et al., 1980). The cdc2 gene product may also act as a regulator of gene expression v i a i t s e f f e c t s on chromatin. In the macronucleus 148 of Tetrahymena, a growth-associated histone Hi kinase a c t i v i t y has been observed with strong enzymatic and physical properties to cdc2 Hi kinase i n other eukaryotes. The macronucleus undergoes d i v i s i o n without mitosis; therefore, t h i s a c t i v i t y must serve some function other than simply being part of a mitosis-associated chromatin condensation pathway. Since phosphorylation of histones i n Tetrahymena can be induced by phys i o l o g i c a l stresses, p34 may play a r o l e . Consistent with e i t h e r a gross or more subtle r o l e for p34 kinase i n chromatin condensation i s the observation that the recognition s i t e f o r phosphorylation by cdc2 kinase i s found most frequently i n DNA binding proteins (Suzuki, 1989). The structure of phosphorylation s i t e s on known p34 c d c 2 substrates i s predicted to form a beta-turn that could bind DNA i n the minor groove. Phosphorylation of these s i t e s would disrupt the structure, thereby preventing DNA binding. Such a mechanism could r e l i e v e constraints on chromatin at the time of mitosis, thereby permitting some other mechanism to condense the chromatin (Moreno and Nurse, 1990), perhaps at the l e v e l of the nuclear s c a f f o l d . The same loosening of protein-DNA interactions might f a c i l i t a t e the passage of polymerase during S phase, or i t could be exploited by the c e l l to regulate gene expression t r a n s i e n t l y i n r e s t r i c t e d chromatin domains (Roth et al., 1991; Roth et al., 1988). Thus, a Su(var)-like phenotype might r e s u l t i f the s p e c i f i c i t y or regulation of the cdc2Dm encoded kinase were subtly 149 a l t e r e d . These p o s s i b i l i t i e s made the d e t a i l e d i n v e s t i g a t i o n of cdc2Dm p a r t i c u l a r l y desirable. The analysis of cdc2 mutants both with respect to the c e l l cycle and the suppression of p o s i t i o n - e f f e c t variegation are reported i n the following chapter. 150 CHAPTER 4 INTRODUCTION Drosophila melanogaster i s one of many eukaryotes i n which a homologue of the f i s s i o n yeast cdc2+ gene has been i d e n t i f i e d . In yeast p34 c d c 2 kinase i s required f o r the i n i t i a t i o n of DNA synthesis (Gi-S) and f o r the proper execution of mitosis (G2-M). The two temporal requirements are g e n e t i c a l l y separable (Booher and Beach, 1987), and at each point p34 d i f f e r s i n terms of i t s a c t i v i t y (Moreno et al., 1989), i t s phosphorylation state (Gould and Nurse, 1989), and i t s association with other proteins (Booher et al., 1989). Two l i n e s of evidence suggest that homologues of cdc2* have s i m i l a r functions i n c e l l cycle regulation throughout the animal kingdom. F i r s t , the human cdc2+ gene can substitute for the f i s s i o n yeast gene i n both i t s G^S and G2-M roles (Lee and Nurse, 1987). Although the converse experiment has not been performed, p34 stimulates DNA r e p l i c a t i o n i n extracts from mammalian G1 c e l l s (D'Urso et al., 1990) and Xenopus eggs (Blow and Nurse, 1990). Second, the kinase encoded by the cdc2 homolog of Xenopus laevis i s a component of M-phase promoting fa c t o r (MPF) (Gautier et al., 1988; Dunphy et al., 1988) and a component of a s t a r f i s h M-phase-specific histone HI kinase (Arion et al., 1988; Labbé et al., 1988). Thus cdc2 gene a c t i v i t y i s associated with the co n t r o l of c e l l cycle events i n a wide v a r i e t y of taxa. Entry i n t o mitosis i s believed to be brought about by the 151 stimulation of p34 o d c 2 kinase a c t i v i t y , which r i s e s to a peak at M-phase (reviewed i n Nurse, 1990). p34 c d c 2 phosphorylates other proteins to i n i t i a t e such major M-phase events as chromosome condensation, c y t o s k e l e t a l reorganization, nuclear envelope breakdown, and changes i n c e l l shape. In S. pombe, the a c t i v a t i o n of p34 c d c 2 requires a c y c l i n - l i k e protein, p 5 g c d c i 3 , a n ( j t j j e cdc25* gene product. This a c t i v a t i o n i s i n h i b i t e d by the weel+ gene product, which i s a putative serine/threonine kinase. The balance of these independent a c t i v a t i o n / i n h i b i t i o n pathways modulates p34 function, r e s u l t i n g i n the advancement or delay of mitosis. Genes with sequence s i m i l a r i t y to cdcl3, cdc25, and weel have been found i n several eukaryotes (Hadwiger et al., 1989; Surana et al., 1991; Igarashi et al., 1991; Sadhu et al., 1990; Lehner and O ' F a r r e l l , 1989; Edgar and O ' F a r r e l l , 1989) suggesting that they too are part of a universal regulatory mechanism. In f i s s i o n yeast, the onset of mitosis can also be al t e r e d by d i r e c t mutation of cdc2. Loss of function mutations delay or prevent mitosis, while dominant altered-function mutations advance the onset of mitosis. In Drosophila melanogaster, genetic studies on the regulation of entry into S phase and mitosis are only beginning (see Glover, 1991), but several regulatory genes have been i s o l a t e d . String, the homolog of the yeast cdc25 gene, has been cloned. During e a r l y embryogenesis, 152 overexpression of string* advances the onset of mitosis, suggesting that the string product i s a mi t o t i c a c t i v a t o r (Edgar and O ' F a r r e l l , 1989). Two genes with sequence s i m i l a r i t y to the cdcl3 gene product have also been cloned (Lehner and O ' F a r r e l l , 1989). The product of one such gene ( c y c l i n A) i s e s s e n t i a l f o r c e l l cycle progression i n the developing nervous system (Lehner and O ' F a r r e l l , 1990a). In addition to the work described i n the previous chapter, two other groups have cloned cdc2Dm. The sequence of a cDNA and the wild-type d i s t r i b u t i o n of cdc2Dm mRNA has been determined (Jimenez et a l . , 1990; Lehner and O ' F a r r e l l , 1990), but no mutants have yet been reported. This chapter describes the preliminary genetic and molecular analysis of ten cdc2Dm mutations, with s p e c i a l reference to Su(var)216. 153 MATERIALS AND METHODS C u t i c l e Preparations Embryos were c o l l e c t e d overnight on a p e t r i dish spread with yeast paste (yeast mixed with 5% (w/v) a c e t i c a c i d and 5% ethanol). The dish was maintained at 25° f o r 24-48 hours to allow wild-type embryos to hatch. Mutant embryos that f a i l e d to hatch were c o l l e c t e d f o r examination. The surface of the p e t r i plate was washed with water, and the embryos were c o l l e c t e d with a sieve made of nitex netting. The embryos were thoroughly washed; then they were placed i n 50% bleach for 3 min. The bleach was removed by washing several times with a soluti o n of 0.8% NaCl, 0.1% T r i t o n X-100. The embryos were drained by placing absorbent paper under the sieve; then they were transferred to a tube containing 700 u l of PEMFA (lOOmM Pipes (pH 6.5), 2 mM EGTA, 5 mM MgS04, 1% formaldehyde) and 700 fil of heptane. The tube was rotated f o r 30 min at room temperature. The lower phase was removed, 700 u l of methanol was added, and the mixture was vortexed f o r 15 s. The embryos were allowed to sink. The overlying l i q u i d was removed, and the embryos were washed 3 times with 700 u l of methanol. The methanol was replaced with acetic a c i d : g l y c e r o l (4:1), and the tube was incubated at 60° for at l e a s t 1 hour. The soluti o n was d i l u t e d 1:1 with Hoyer's mountant, trans f e r r e d to a microscope s l i d e , o v e r l a i d with a c o v e r s l i p , and l e f t on a warming tray f o r 24-48 hours before viewing under a microscope. 154 Primers For sequencing, eight primers were designed that spanned the cdc2 gene. The primer sequences were: BB1 ACGTTTGTCCTCCGAACTC; BB2, GTCTGGTAATACTAGAGC; BB3, TTCCATCAACCGCGATCA; BB4 , ACAATCCTATATGCGTAC; BB5 , ATGGAGGAGAACCGCATA; BB 6, GTCGCCGACTTTGGACT; BB7,TGTTTAGGTAACCACAGG; BB8, CGATCCAGTTCATCGCAT. The f i r s t primer was i d e n t i c a l to a genomic DNA sequence located approximately 250 bp upstream from the t r a n s l a t i o n s t a r t s i t e of the gene. The remainder were i d e n t i c a l to sequences spaced at approximately 180 bp i n t e r v a l s along the genomic DNA. The eighth primer hybridized to a sequence i n the t h i r d exon of cdc2, approximately 180 bp from the 3' end of the gene. Thus, the e n t i r e coding region of cdc2Dm could be sequenced with the primers. The primers were synthesized i n the U.B.C. Oligonucleotide Synthesis Laboratory. Primer purification: A Sep-pak C 1 8 column ( M i l l i p o r e , #51910) was prepared by washing f i r s t with 10 ml of 100% a c e t o n i t r i l e (HPLC grade), then with 10 ml of water. The DNA primer, resuspended i n 1.5 ml of 0.5 M ammonium acetate, was passed over the column. The column, with DNA bound, was washed with water; then the oligonucleotide was eluted from the column i n 3 ml of 40% a c e t o n i t r i l e . The DNA was p e l l e t e d by l y o p h i l i z i n g the a c e t o n i t r i l e s o l u t i o n f o r approximately 4 hours. The p e l l e t was resuspended i n 300 mM sodium acetate and 155 p r e c i p i t a t e d with two volumes of 95% ethanol. Afte r centrifugation, the p e l l e t was vacuum dried, and resuspended ready f o r use. Cloning using the polymerase chain reaction Mutant a l l e l e s of cdc2 were cloned by amplifying the gene-encoding sequences from t o t a l genomic DNA using the polymerase chain reaction. The amplified DNA was subcloned and i n d i v i d u a l clones were sequenced to determine the s i t e of the mutation. Five f r u i t f l i e s , a l l heterozygous f o r a mutant a l l e l e , were homogenized i n 200 u l of buffer (100 mM Tris-HCl pH 8.0, 50 mM NaCl, 50 mM EDTA, 1% SDS, 0.15 mM spermine, 0.5 mM spermidine) with 100 fig/ml proteinase K. The mixture was incubated at 65° for 1 hour. T h i r t y f i l of 8 M potassium acetate were added and the mixture was l e f t on i c e f o r 1 hour. The sample was centrifuged (10,000 rpm for 10 min) and the p e l l e t was discarded. Genomic DNA was p r e c i p i t a t e d from the supernatant by the addition of 2 volumes of c o l d 95% ethanol. Following centrifugation, the p e l l e t was resuspended i n 300 mM sodium acetate. RNA was hydrolyzed with RNAse at 100 fig/ml. The sample was mixed with an equal volume of phenol/chloroform (1:1) and centrifuged. DNA was r e -p r e c i p i t a t e d from the aqueous phase by the addition of 2 volumes of cold 95% ethanol. The DNA was p e l l e t e d by cent r i f u g a t i o n , vacuum dried, and re-suspended i n water. 156 To amplify cdc2Dm sequences from genomic DNA, two.primers were chosen from opposite sides of the transcribed region. Primer 1 (GGCTGCAGGTAGCTAGCTTAGCATCG) had a GG clamp, a PstI r e s t r i c t i o n endonuclease recognition s i t e , and 18 bp of sequence i d e n t i c a l to that found 107 bp upstream of the cdc2 t r a n s l a t i o n s t a r t s i t e . Primer 2 (GGGGATCCGCTAGCAGTGCTCTCTAT) had a GG clamp, a BamHI r e s t r i c t i o n endonuclease s i t e , and 18 bp of sequence i d e n t i c a l to that found approximately 150 bp beyond the 3 ' terminus of the cdc2 gene. The primers were synthesized i n the U.B.C. Oligonucleotide Synthesis Laboratory. Polymerase chain reactions were performed using the GeneAmp PCR reagent k i t (Cetus Corp.). Three independent reactions were performed f o r each mutant. Each 99.5 u l PCR reaction mix contained 10 u l of 10X reaction buffer (100 mM Tris-HCl (pH 8.3, at 25°), 500 mM KC1, 15 mM MgCl 2, 0.1% (w/v) g e l a t i n ) ; 2 (xl each of 10 mM dATP, dGTP, dCTP, and dTTP; 30 pmol each of primers #1 and #2; and 100 ng of Drosophila genomic DNA. The reaction mix was o v e r l a i d with p a r a f f i n o i l (Mallinckrodt, #6357), then placed at 100° f o r three minutes to denature any contaminating proteases. AmpliTaq DNA polymerase (0.5 u l ; 2.5 Units) was added to the heated so l u t i o n . DNA amp l i f i c a t i o n reactions were performed immediately i n a SingleBlock System thermal c y c l e r (Ericomp, Inc.). Twenty-six cycles of amp l i f i c a t i o n were performed. The 157 f i r s t 25 were as follows: double stranded DNA was melted at 94°; the primers were annealed to the template at 56°; and the polymerase extended the primers at 72°. Each step i n the cycle proceeded f o r 30 s. The twenty-sixth c y c l e consisted of an i d e n t i c a l melting step, followed by a 2 min annealing reaction and a 5 min primer extension reaction. The annealing temperature was determined empirically, by increasing the annealing temperature u n t i l only a sing l e band of DNA a m p l i f i c a t i o n products was v i s i b l e on agarose gels. The sol u t i o n containing the amplified gene product was extracted once each with equal volumes of phenol, phenol/chloroform, and chloroform. The DNA was p r e c i p i t a t e d with ethanol and resuspended i n 100 n i TE. Yields were 70 to 100 jig of amplified product. The amplified DNA was cloned i n t o a plasmid vector (pUC19) using methods that are described i n d e t a i l i n Chapter 3. One uq of amplified DNA was digested with the r e s t r i c t i o n endonucleases P s t J and BamHI. The DNA was electrophoresed for several hours on a 1% agarose g e l , then a portion of the gel containing the DNA band was excised with a razor-blade. The DNA was p u r i f i e d using glass beads and resuspended i n water. Approximately 500 ng of amplified DNA was mixed with 50 ng of Pstl/BamffJ-digested pUC19. The DNA l i g a t i o n procedure and subsequent transformation of a b a c t e r i a l host were done as described i n Chapter 3. 158 DNA Sequence Analysis Sequencing reactions were performed as described i n Chapter 2, except that i n t e r n a l cdc2Dm primers were used. Each gel-cloned mutant was sequenced only once. For PCR amplified products, f i v e d i f f e r e n t clones were sequenced to ensure that the mutation present in vivo could be distinguished from random mutations that arose during the am p l i f i c a t i o n reaction. The f i v e clones were chosen from amongst the products of three independent PCR reactions. 159 RESULTS A l l e l e s of cdc2 Eight hemizygous l e t h a l mutations which map to region 31 f a i l t o complement the l e t h a l i t y associated with the Su(var)216 chromosome (Chapter 1, see Table 14). These are: B47, D57, E10, El-4, El-9, El-23, El-24, and 216P. To t e s t for a l l e l i s m with cdc2Dm, mutant stra i n s were mated with f l i e s bearing cdc2.12, an X-linked construct containing the cdc2Dm* gene, which was generated by transformation (C. Lehner, personal communication). cdc2.12/cdc2.12 ; Df(2L)J27/CyO females were crossed to Su(var)216/CyO, 216P/CyO and D57/CyO males (Table 11). In each case, the e c t o p i c a l l y placed w i l d -type function of cdc2.12 rescued the l e t h a l i t y of mutant/Df(2L)J27 progeny of both sexes. Similar r e s u l t s were obtained i n an analogous t e s t using the 4.1 s t r a i n (Table 11), which contains a cdc2Dm* gene construct inserted on the t h i r d chromosome (C. Lehner, personal communication). Thus, Su(var)216, 216P and D57 can be rescued by a single wildtype a l l e l e of cdc2Dm. The other f i v e putative cdc2Dm mutants were crossed to cdc2.12/cdc2.12; cdc2Su<var>2ie/CyO f l i e s and the progeny were examined f o r the presence or absence of cdc2Su(yaI)216/mutant survivors. (This t e s t f o r a l l e l i s m was used instead of the previous one because the Df(2L)J27 stock was nearly i n f e r t i l e i n the presence of ei t h e r cdc2.12 or 4.1.) When the construct-bearing suppressor stock was crossed to each of the remaining 160 Table 11. Phenotypic rescue of three mutant a l l e l e s of mutants, the v i a b i l i t y of the mutant trans-heterozygotes was restored (Table 12). Although the l e t h a l phenotype was a l l e v i a t e d , the majority of crosses showed a marked reduction i n the t o t a l number of male progeny. The reason f o r t h i s reduction i s unclear, since crosses in v o l v i n g a deficiency do not have a s i m i l a r e f f e c t (Table 11). One addi t i o n a l putative a l l e l e of cdc2Dm, E20, i s vi a b l e and f e r t i l e i n combination with cdc2Su(var)216 but f a i l s to complement cdc2D5V and cdc2E1° (Table 13). Trans-heterozygous cdc2D57/cdc2E20 females are infecund at 25° and both males and females are i n v i a b l e at 29°. Each of these phenotypes i s rescued by a s i n g l e copy of cdc2.12. Heterozygous cdc2E10/cdc2E2° mutants are e s s e n t i a l l y l e t h a l at 25° unless they also carry the cdc2.12+ construct. S u r p r i s i n g l y , the cdc2.12+ construct has l i t t l e e f f e c t on the v i a b i l i t y of eith e r cdc2E10/cdc2E20 or cdc2E2°/Df(2L)JRl6 progeny at 29°. These e f f e c t s might be caused by second s i t e mutations i n the stocks or they may r e f l e c t unusual properties of cdc2E20. Some cdc2E20/CyO hétérozygotes have mildly etched t e r g i t e s . Although t h i s may be a consequence of a second s i t e mutation, other cdc2Dm mutants have a s i m i l a r but more severe recessive phenotype (see below). Taken together these observations suggest that cdc2E2° may be semi-dominant. 162 Table 12. Phenotypic rescue of h e t e r o a l l e l i c combinations cdc2Dm mutations. Table 13. Complementation crosses with cdc2l Adult Phenotypes The cdc21sp and cdc2E2° mutations are both weak a l l e l e s of cdc2Dm with respect to v i a b i l i t y . At 22°, rare cdc2216P/cdc2216P homozygotes eclose (less than 1 homozygote per 300 hétérozygotes). These f l i e s have damaged, duplicated or missing machrochaetes, gnarled l e g j o i n t s , and l i t t l e or no abdominal c u t i c l e . They do not survive f o r more than a few days, hence they have not been tested f o r f e r t i l i t y . Hemizygous cdc2216P progeny do not survive to adulthood. At 22°, homozygous cdc2E2° mutants comprise approximately 8% of the adult f l i e s i n our cdc2E20/CyO stock. These f l i e s have mil d l y etched ste r n i t e s and t e r g i t e s but do not display any of the other cdc2216p/cdc2216p phenotypes. Male homozygotes are s t e r i l e and female homozygotes are infecund. A s i m i l a r phenotype i s observed for hemizygous cdc2E20 males, but hemizygous females are infrequently f e r t i l e (1 i n 10). I t i s possible, though u n l i k e l y , that these females may a c t u a l l y be cdc2E20/CyO hétérozygotes with poor Cy expression. A cdc2.12/cdc2.12; cdc2E20/CyO stock has been established, but the frequency of homozygous cdc2E20/cdc2E2° progeny remains low, suggesting that the mutant chromosome c a r r i e s at l e a s t one addit i o n a l l e s i o n that a f f e c t s homozygous v i a b i l i t y . Occasional cdc2E1~* and cdc2E1~24 hemizygotes survive to adulthood when reared at 22° (see Table 15). These have severely etched s t e r n i t e s and t e r g i t e s but are f e r t i l e . The same phenotype i s observed amongst almost a l l progeny-bearing 165 h e t e r o a l l e l i c combinations of cdc2Dm mutations (see Table 14), e s p e c i a l l y those involving cdc2E1~*. Thus, a recessive abdominal phenotype i s associated with most mutant a l l e l e s of cdc2Dm. One h e t e r o a l l e l i c combination that expresses only very weak abdominal etching i s cdc2D57/cdc2E20. At moderate temperatures (18°, 22° and 25°), cdc2D57/cdc2E2° females are infecund and males are f e r t i l e . The same phenotype i s observed regardless of the parental source of each mutant a l l e l e . The ovaries of 10 infecund cdc2D57/cdc2E20 females raised at 25° were dissected and examined. In each case no ovarioles were v i s i b l e within the ovary (data not shown); however, the p o s s i b i l i t y that some germline p r o l i f e r a t i o n has occurred cannot be excluded. Lethal Phases Excluding cdc2E2°, a l l of the cdc2Dm mutants are e s s e n t i a l l y l e t h a l as hemizygotes; however, the pattern of complementation between d i f f e r e n t mutant a l l e l e s (Table 14) suggests that several of the mutant gene products s t i l l r e t a i n some a c t i v i t y . These p a r t i a l l y functional products may provide clues as to the function of the wild-type gene product, i f the f l i e s that carry them die at recognizable stages of development (e.g. gastrulation) or when s p e c i f i c t issues are being elaborated (e.g. imaginai disc growth). 166 Table 14. Complementation matrix f o r a l l e l e s of cdc2Dm. Ratios indicate the number of c u r l y to straight-winged f l i e s amongst progeny from the cross cdc2*/CyO X cdc2r/CyO Su(var) 216 B4 7 D57 E10 El-4 Su(var) 216 1716:0 177:0 1220:0 136:50 B4 7 460:0 - 283:0 516:0 114:29 D57 258:0 308:0 - 631:0 102:0 E10 248:0 775:0 523:0 - 562:44 El-4 439:115 156:10 359:0 186:0 -El-9 131:0 342:0 161:0 150:0 375:77 El-23 730:0 1021:0 857:0 831:0 636:14 El-2 4 509:23 1496:0 1368:0 1405:0 173:24 216V 551:1 1508:0 407:0 892:0 607:267 El-9 El-23 El-2 4 216P Su(var) 216 217:0 215:0 1577:0 215:10 B4 7 211:0 279:0 290:0 125:0 D57 140:0 107:0 842:0 253:0 E10 226:0 194:0 416:0 389:0 El-4 319:0 207:4 451:21 432:102 El-9 - 346:0 357:1 132:0 El-23 933:0 - 641:0 199:0 El-2 4 1514:7 492:0 - 1366:344 216P 799:1 387:20 721:179 -167 To begin a more d e t a i l e d analysis of the roles of cdc2Dm i n a g e n e t i c a l l y t r a c t a b l e metazoan, the l e t h a l phase f o r each cdc2Dm mutant was determined (Table 15). Each cross was designed so that a l l progeny classes but one, the mutant/deficiency c l a s s , p o t e n t i a l l y survived and were phenotypically d i s t i n c t . In p i l o t studies, f i v e of the mutants, cdc2Su<var>216, cdc2E1° , cdc2E1~4, cdc2E1~9, and cdc2E1-24 died most frequently as pupae, regardless of the parental source of the mutation-bearing chromosome (Table 15). In two cases, cdc2E1~23 and cdc2B4?, progeny that i n h e r i t e d the mutation from t h e i r fathers died most frequently as larvae, while i n the r e c i p r o c a l cross they died most frequently as pupae. However, the most dramatic r e s u l t s were obtained f o r cdc2D5?. In three independent experiments, hemizygous cdc2D57/Df(2L)JRl6 progeny of cdc2D57/CyO mothers died as embryos, whereas hemizygous progeny that i n h e r i t e d cdc2DS? from t h e i r fathers died as larvae. Thus, the cdc2D5? a l l e l e displays a maternal e f f e c t . To confirm the embryonic l e t h a l i t y of cdc2D5?, the cdc2D5? and Df(2L)JRl6 second chromosomes were re-balanced over a multiply inverted chromosome marked with Black-cells (Be). In f l i e s bearing the Bc-balancer, dark pigment grains are v i s i b l e underneath the c u t i c l e . When cdc2D57/Bc mothers were crossed to Df(2L)JR16/Bc males, no non-Bc larvae were observed amongst 484 progeny. A s i m i l a r r e s u l t was obtained i n the cross cdc2D57/Bc X cdc2D57/Bc. 168 Table 15. Lethal phases of cdc2Dm mutants ra i s e d at 22 0 c. Male X Df(2L)JRl6 /CyO % Embryonic l e t h a l i t y % Larval l e t h a l i t y % Pupal l e t h a l i t y % Adult v i a b i l i t y Su(var)216 /+ 3 (10/408) 9 (35/408) 25 (105/408) 0 B47/+ 3 (10/433) 19 (81/433) 13 (53/433) 0 D57/+ 2 (6/383) 28 (107/383) 5 (19/383) 0 E10/+ 2 (6/502) 5 (24/502) 24 (117/502) 0 El-4/-h 5 (17/394) 8 (31/394) 25 (97/394) 2 (4/249) El-9/-h 4 (13/424) 12 (49/424) 25 (105/424) 0 El-23/+ 2 (7/340) 25 (83/340) 9 (29/340) 0 El-24/+ 3 (13/446) 13 (54/446) 21 (91/446) 6 (16/288) Female X % % Larval % Pupal % Adult Df(2L)JRl6 Embryonic L e t h a l i t y L e t h a l i t y V i a b i l i t y L e t h a l i t y Su(var)216 /CyO 5 (21/461) 14 (61/461) 19 (85/461) 0 B47/CyO 1 (4/399) 11 (42/399) 22 (87/399) 0 D57/CyO 31 (127/414) 6 (25/414) 12 (47/414) 0 El0/CyO 5 (17/406) 10 (37/406) 23 (91/406) 0 El-4/CyO 10 (44/453) 17 (75/453) 30 (135/453) 1 (1/199) El-9/CyO 5 (21/479) 10 (45/479) 26 (121/479) 0 El-23/CyO 2 (5/440) 12 (49/440) 22 (93/440) 0 El-24/CyO 4 (16/449) 2 (9/449) 31 (139/449) 4 (10/285) 169 While cdc2D5? hemizygotes die most frequently as embryos or larvae, the majority of cdc2E10 hemizygotes die as pupae (Table 15). This i s s u r p r i s i n g since cdc2D57/cdc2B20 hétérozygotes are i n v i a b l e only at 29°, while cdc2E10/cdc2E2° mutants are strongly i n v i a b l e at lower temperatures. Furthermore, cdc2D57/cdc2E20 females are always infecund, whereas cdc2E10/cdc2E2° females are fecund. The d i f f e r e n t phenotypes of cdc2D5? and cdc2E1° hemizygotes suggest that the two mutants are q u a l i t a t i v e l y d i f f e r e n t . I t i s curious i n t h i s regard that progeny from the cross cdc2D57/CyO X cdc2E10/CyO die most frequently as embryos, even i f cdc2E1° i s maternally i n h e r i t e d (data not shown). In contrast, progeny from the cross cdc2D57/CyO X Df(2L)JRl6 die as larvae. These observations suggest that cdc2E1° may have stronger e f f e c t s on v i a b i l i t y than a n u l l mutation. I t i s possible that both cdc2D5? and cdc2E1° have additional recessive l e t h a l mutations on the second chromosome. However, i t seems u n l i k e l y that both would share the same second s i t e l e t h a l mutation. Hence the odd pattern of l e t h a l i t y remains i n t r i g u i n g . Embryonic and Larval Phenotypes If cdc2Dm exerts i t s influence predominantly through i t s e f f e c t s on c e l l cycle progression, then the terminal phenotypes of cdc2Dm mutants might be expected to exhibit gross defects i n c e l l p r o l i f e r a t i o n . In t h i s respect, two periods of development are of p a r t i c u l a r i n t e r e s t : the 170 embryonic and pupal stages. During embryogenesis the tissues of the l a r v a p r o l i f e r a t e . Most of these tissues (with the exception of a few tissues such as the nervous system) are histolysed at the pupal stage. The majority of adult tissues originate from s p e c i f i c f o c i of p r o l i f e r a t i n g c e l l s within the larvae which only d i f f e r e n t i a t e at the pupal stage during metamorphosis. To t e s t f o r p o t e n t i a l d i s r u p t i o n of c e l l p r o l i f e r a t i o n i n l a r v a l and adult precursor c e l l s , the embryonic phenotype of cdc2D57 hemizygotes was investigated along with the pupal l e t h a l i t y of cdc2E10/Df(2L)JRl6 hemizygotes and cdc2E10/cdc2B47 hétérozygotes. Since Df(2L)J27 deletes cdc2Dm and Df(2L)J27/Df(2L)J27 homozygotes survive u n t i l a f t e r the embryonic epidermis secretes a c u t i c l e (at approximately 15-18 hours i n the 22 hour embryonic development at 25°), the c u t i c l e s of hemizygous cdc2D5? females were examined for pattern defects. Sixty-eight c u t i c l e s were observed from embryos which f a i l e d to hatch when cdc2D51/+ females were mated to Df(2L)J27/CyO males. Only nine embryos had mild head or c u t i c l e defects. In every other case the c u t i c l e appeared normal with no obvious holes or missing s t r u c t u r a l landmarks. Thus, the strongest of our a l l e l e s does not grossly disrupt t h i s aspect of development. This i s i n contrast to homozygous Df(2L)J27/Df(2L)J27 embryos which have misshapen heads and holes i n t h e i r c u t i c l e s (Niisslein-Volhard et al., 1984). A high proportion of mutants bearing h e t e r o a l l e l i c 171 combinations of cdc2 mutations arrest at the pupal stage. Many of these appear to cease development predominantly as pharate adults. Dissection of the pupal cases of these mutants again reveals severe s t e r n i t e and t e r g i t e etching. The precursors of these tissues divide very r a p i d l y during the e a r l y pupal stage and therefore t h i s phenotype i s not s u r p r i s i n g f o r a mutant with a p o t e n t i a l defect i n c e l l cycle regulation. In addition to developmental arrest l a t e i n the pupal period, some combinations of mutant cdc2Dm a l l e l e s die somewhat e a r l i e r without any overt d i f f e r e n t i a t i o n . One such combination, cdc2E10/cdc2B47, arrests at Stage P3 (bubble prepupa) to P4 (Bainbridge and Bownes, 1981). At the l a t t e r stage, the imaginai discs of the larvae usually evert to elaborate the structures of the adult f l y ; however, no discs are v i s i b l e i n the cdc2E10/cdc2B47 larvae. A s i m i l a r phenotype i s observed f o r cdc2E10/Df(2L)JR16 larvae. These observations suggest that cdc2Dm mutations prevent the p r o l i f e r a t i o n of at lea s t some tissues that give r i s e to adult structures. These tissues normally undergo rapid p r o l i f e r a t i o n during the l a r v a l period. In addition to mitosis, cdc2 has been implicated i n progression i n t o S phase. In Drosophila, S phase i s uncoupled from mitosis during the polytenization of many l a r v a l tissues (including the s a l i v a r y glands, which reach t h e i r maximum s i z e i n t h i r d i n s t a r larvae). I f the cdc2Dm mutants a f f e c t S phase, then the extent of polytenization might be reduced. Therefore, 172 I examined l a r v a l s a l i v a r y glands i n cdc2E10/Df(2L)JR16 hemizygous progeny from the cross cdc2B10/Bc cf X Df(2L)JR16/Bc Î . The s a l i v a r y glands dissected from wandering t h i r d i n s t a r cdc2E10/Df(2L)JR16 larvae were normal i n s i z e , and polytene chromosomes were v i s i b l e i n squash preparations. Similar r e s u l t s were obtained with cdc2E1~". Thus, cdc2E10 and cdc2E1~" do not appear to a f f e c t polytenization, although I cannot exclude the p o s s i b i l i t y that maternally i n h e r i t e d cdc2 mRNA rescues progression i n t o S phase. The Relationship Between Sufvar)216 and cdc2Dm Several observations suggest that cdc2 homologues may play a r o l e i n the compaction and modification of chromatin, properties also hypothesized f o r suppressors of PEV. In numerous systems, the a c t i v i t y of p34 c d c 2 kinase i s strongly correlated with chromosome condensation during mitosis (see Nurse, 1990; Moreno and Nurse, 1990). p34 c d c 2 phosphorylates histones, which are the p r i n c i p a l proteins of chromatin. Also, chromatin modification by p34 c d c 2-like a c t i v i t y has been observed i n the absence of mitosis (Roth et a l . , 1991). Furthermore, suppressors of PEV a f f e c t chromatin condensation (e.g. Dorn et a l . 1986), and t h e i r e f f e c t s may be brought about by histone modification (Dorn et a l . , 1986; Mottus et a l . , 1980). Taken together these observations r a i s e the p o s s i b i l i t y that cdc2Dm mutants might suppress PEV by a l t e r i n g the normal pattern of chromatin modification. 173 While the cdc2Dm locus i s a good candidate f o r a Su(var) gene, i t was e s s e n t i a l to demonstrate that the Su(var) mutation i s within the cdc2Dm locus. Sequence analysis of Su(var)216 (see below) demonstrates that a si n g l e point mutation causes an a l t e r a t i o n i n the cdc2Dm protein. So the Su(var)216 chromosome c l e a r l y has a mutation i n cdc2Dm. I t remained to be determined, however, whether cdc2 mutations suppress PEV. Amongst 10 mutants, only the Su(var)216 a l l e l e strongly suppresses PEV. When v/°4/w"4; Su(var)216/CyO males are outcrossed to \^4/\t4 females, both male and female progeny have approximately 80% red pigment compared with 10% i n w*4/w™4 controls ( S i n c l a i r et a l . , 1991). Scored v i s u a l l y , no other cdc2Dm mutant suppresses PEV when compared to contro l s . Furthermore, a single copy of cdc2Dm* inserted on the X chromosome rescues the l e t h a l phenotype of Su(var)216/Su(var)216, but has no e f f e c t on the Su(var) phenotype (data not shown). This r e s u l t may not be too su r p r i s i n g since the amount of functional p34 expressed i n the construct-bearing s t r a i n i s not known (euchromatic p o s i t i o n e f f e c t s are well documented i n Drosophila). These observations can be explained i f cdc2Su<vaT)216 i s hypothesized to be a rare gain-of-function mutation, and the wild-type gene, unlike many other Su(var) l o c i , i s not dose s e n s i t i v e . However, other circumstantial evidence i s less easy to explain. Hemizygous Su(var)216/Df(2L)J2 males do not survive to 174 adulthood, while females display reduced v i a b i l i t y and are infecund ( S i n c l a i r et al., 1991). The same phenotypes p r e v a i l i n the presence of e i t h e r one or two copies of the X linked cdc2Dm* gene construct, cdc2.12 (Table 16). However, when Su(var)216 was made heterozygous with Df(2L)J77, Df(2L)J27, Df(2L)G2, or Df(2L)J106 i n the presence of cdc2.12 (cdc2.12/+; Su(var)216/deletion), mutant/deficiency progeny were male v i a b l e and female f e r t i l e (Table 16). The mutant phenotype of cdc2.12/+; Su(var)216/Df(2L)J2 i s u n l i k e l y to be caused by a combination of poor cdc2.12 expression exacerbated by deletion fo r many l o c i , since cdc2E10/Df(2L)J2 progeny survive and are f e r t i l e i n the presence of an ectopic copy of cdc2Dm+. An alternate explanation i s that the Su(var)216 s t r a i n c a r r i e s a cdc2Dm mutation and a second s i t e male-lethal, female-sterile mutation which maps very near to cdc2Dm and was not removed by the recombination replacement protocols. The f i n d i n g that cdc2.12/+; Su(var)216/Su(var)216 females are v i a b l e (Table 13) but s t i l l infecund with a wings-held-out phenotype also supports the second-site hypothesis. Since some Su(var)216/Su(var)216 males are via b l e i n the presence of cdc2.12, the second s i t e mutation appears to be quite weak with respect to i t s e f f e c t s on v i a b i l i t y . The p o s s i b i l i t y of a c l o s e l y linked second s i t e mutation led us to examine the interactions between Su(var)216 and the other Su(var) l o c i hypothesized to map i n region 31. Even i n the presence of cdc2.12, recessive i n t e r a c t i o n s are observed 175 Table 16. Mapping a second-site mutation on the chromosome. between Su(var)216 and members of the Suvar(2)l complementation group (Table 16). Transheterozygotes of genotype cdc2.12/+; Su(var)216/Su(var)214 are female infecund and e s s e n t i a l l y male l e t h a l , just as they are i n the absence of cdc2.12. S i m i l a r l y , transheterozygous Su(var)216/Su(var)207 and Su(var)216/Su(var)210 females are infecund, with or without cdc2.12. In each case, the trans-heterozygotes have a wings-held-out phenotype and a red/brown eye colour s i m i l a r to that of h e t e r o a l l e l i c combinations of Suvar(2)l. Thus, cdc2.12 f a i l s to a l l e v i a t e any of the hypothesized interactions between Su(var)216 and Suvar(2)l. I n i t i a l l y , the phenotype of Su(var)216/Suvar(2)l hétérozygotes was interpreted as an i n t e r a c t i o n between the mutant products of two separate l o c i (Brock, 1989); however, the second mutation on the Su(var)216 chromosome might simply be an a l l e l e of Suvar(2)l. To t e s t t h i s hypothesis, a colleague (I.P. Whitehead) attempted to separate the suppression phenotype from the l e t h a l phenotype of cdc2Su(var>216. In a large scale recombination experiment (>100,000 f l i e s ) , f i v e recombinants were i s o l a t e d which mapped to the r i g h t of J and which were v i a b l e with Df(2L)G2 but which s t i l l suppressed PEV. One recombinant, Rl, behaved i n a manner s i m i l a r to the putative second s i t e mutation on the Su(var)216 chromosome. Homozygotes were via b l e but female s t e r i l e , and Rl/Df(2L)J2 progeny were male l e t h a l and female infecund (Table 16). The remaining recombinant s t r a i n s have s i m i l a r 177 phenotypes (I.P. Whitehead and G. Stromatich, personnal communications). Taken together, these findings suggest that the Su(var)216 chromosome suppresses PEV because of a mutation that i s t i g h t l y linked to cdc2Dm. They do not, however, formally exclude the p o s s i b i l i t y that cdc2Su(vaT)216 also acts as a suppressor of PEV. The Sequences of cdc2Dm Mutant A l l e l e s Regardless of whether or not al t e r e d expression of cdc2Dm protein acts as a suppressor of PEV or not, CDC2 proteins play a c e n t r a l r o l e i n the control of c e l l d i v i s i o n . Since we have a large c o l l e c t i o n of cdc2Dm mutants, I conducted studies to co r r e l a t e s p e c i f i c mutations with mutant phenotypes. To further characterize cdc2Dm, 6 mutant a l l e l e s were cloned. Two of the mutations, cdc2E1~9 and cdc2E1~23 were subcloned into pUC19 a f t e r a Hindlll/Sail fragment containing the e n t i r e gene was excised from s i z e fractionated genomic DNA. The remainder of the mutations were cloned by am p l i f i c a t i o n of the cdc2Dm gene using the polymerase chain reaction, followed by subcloning of the product DNA. In both cases, the DNA i s o l a t e d contained two d i f f e r e n t a l l e l e s of cdc2Dm. Wildtype and mutant chromosomes were d i f f e r e n t i a t e d based on the presence or absence of an EcoRI s i t e i n Intron 1 of cdc2Dm. Mutant a l l e l e s induced on e i t h e r the multiply marked b pr cn or b It rl chromosomes have an extra EcoRI r e s t r i c t i o n endonuclease s i t e , 178 while mutants i s o l a t e d on the cn bw chromosome do not. By mating mutant bearing f l i e s to the appropriately marked second chromosome i t was possible to generate heterozygous progeny f o r both the Intron 1 polymorphism and a recessive l e t h a l cdc2Dm mutation. Thus, i t was possible to i d e n t i f y cloned mutant a l l e l e s from wildtype a l l e l e s . Each of the cloned cdc2Dm a l l e l e s has a singl e basepair missense mutation caused by a nucleotide t r a n s i t i o n within the coding region of the gene. Conceptual t r a n s l a t i o n reveals that the resultant amino acid substitutions are not clustered, but are d i s t r i b u t e d throughout the predicted protein (Figure 32). The amino acid s u b s t i t u t i o n Gly to Asp i n cdc2DmEl-4 i s within the PSTAIR sequence, a motif that i s absolutely conserved amongst functional homologues of cdc2. In both cdc2DmSu(var)216 and cdc2DmD57, the unique amino acid s u b s t i t u t i o n i s within the c e n t r a l c a t a l y t i c core of the enzyme (see Hanks et a l . , 1988). The two respective a l t e r a t i o n s are only three residues apart. However, the amino aci d s u b s t i t u t i o n i n cdc2DmD57 (Arg) replaces Gly 1 4 8, a residue which i s invariant not only within the CDC2 family of proteins, but amongst a l l kinases (Hanks et a l . , 1988). In contrast, A l a 1 4 5 , the amino acid that i s replaced by Val i n cdc2DmSu(var)216, i s not invariant amongst a l l kinases. In f a c t , an alt e r n a t i v e residue i s found at t h i s s i t e i n the homologous protein of S. cerevisiae, although i n t h i s case, the s u b s t i t u t i o n i s conservative. 179 Figure 31. Mutations i n cdc2Dm. Underlining i n the protein sequence indicates amino acids conserved amongst a l l CDC2 proteins. Mutant Mutation Amino Acid Substitution # A l t e r a t i o n Domaina El-4 GGC to GAC 43 G to D 2/3 cdc2Sa<vaT)216 GCC to GTC 145 A to V 8 D57 GGA to AGA 148 G to R 7 El-24 GAG to AAG 196 E to K 9 El-23 GGT to GAT 206 G to D 9 El-9 CCC to TCC 242 P to S 11 D 55 MEDFEKIEKIGEGTYGWYKGRNRLTGOIVAMKKIRLESDDEGVPSTAIREISLL 110 KELKHENIVCLEDVLMEENRIYLIFEFLSMDLKKYMDSLPVDKHMESELVRSYLY V R 165 QITSAILFCHRRRVLHRDLKPONLLIDKSGLIKVADFGLGRSFGIPVRIYTHEIV K D 220 TLWYRAPEVLLGSPRYSCPVDIWSIGCIFAEMATRKPLFQGDSEIDQLFRMFRIL S 275 KTPTE DIWPGVTSLPDYKNTFPCWSTNQLTNOLKNLDANGIPLIOKMLIYDPVHR 297 ISAKDILEHPYFNGFOSGLVRN a The domain number refers to dis c r e t e regions of sequence homology amongst kinases (Hanks et al., 1988). 180 The three remaining mutations, cdc2E1'24, cdc2B1~23, and cdc2B1'9, a l l cause amino acid substitutions at residues that are highly conserved within the CDC2 protein family. Of t h i s group, cdc2E1~9 i s most i n t e r e s t i n g , because i t causes an amino aci d s u b s t i t u t i o n i n a region that i s e i t h e r absent or poorly conserved i n other kinases (Hanks et a l . , 1988). Hence i t may help to determine the s p e c i f i c properties of cdc2-like kinases. Recently, the three dimensional structure of the c a t a l y t i c subunit of c y c l i c AMP-dependent protein kinase (cAPK) has been determined by X-ray crystallography (Knighton et a l . , 1991). Since CDC2 proteins are s i m i l a r i n sequence to cAPK and several biochemical properties and features of kinases are conserved (see Hanks et al., 1988), the structure of cdc2Dm may be s i m i l a r to that of cAPK. Alignment of the two sequences suggests that amino acid substitutions i n cdc2DmEl-4, cdc2DmD57, cdc2El-23, and cdc2DmEl-9 a l l are l i k e l y to be within randomly c o i l e d regions. Similar secondary structures f o r cdc2Dm are predicted using the algorithm of Chou and Fassman (1974). This algorithm also predicts that the a l t e r a t i o n i n cdc2DmEl-9 i s i n a region of random c o i l . Several amino acid residues adjacent to and i n c l u d i n g Pro 2 4 2 have no counterparts i n cAPK. Other members of the kinase family have sequence insertions at the same p o s i t i o n i n the cAPK sequence (e.g. CDC7 260(93)261; see Knighton et al., 1991). Based on t h e i r c r y s t a l l o g r a p h i c data, Knighton et al. 181 (1991) speculated that a d d i t i o n a l amino acids i n t h i s portion of the sequence are located on the outside of the protein. 182 DISCUSSION Ten recessive l e t h a l cdc2Dm a l l e l e s have been described i n t h i s analysis. Lehner and O'Farrel (1990b) i s o l a t e d a second cdc2-like gene, cdc2c, from Drosophila using the polymerase chain reaction. However, t h i s gene shares only 58% amino acid sequence i d e n t i t y with the cdc2Dm gene product and f a i l s to complement cdc2 defects i n f i s s i o n yeast. Our data demonstrate that cdc2c cannot rescue the l e t h a l phenotype of cdc2Dm in vivo. Nine of the ten cdc2Dm mutations do not suppress PEV, and the tenth i s on a chromosome which has a t i g h t l y linked Su(var) mutation. Although the cdc2Su(var>216 a l l e l e has not been recovered without the adjacent Su(var) mutation, there i s no evidence that the cdc2Dm a l l e l e i s a suppressor of PEV. Amongst the ten mutations of cdc2Dm which have been i s o l a t e d , cdc2D57 i s the strongest a l l e l e with respect to l e t h a l i t y . Hemizygous mutant progeny which i n h e r i t cdc2D57 from t h e i r mothers die as embryos, while those that i n h e r i t i t from t h e i r fathers die as larvae. This difference i n l e t h a l phase suggests that despite being a strong a l l e l e , the gene product s t i l l retains some a c t i v i t y and that i t antagonizes the function of the wild-type gene product. In Drosophila, the concentration of gene product i s normally proportional to the number of copies of the gene. Since cdc2 mRNA and p34 protein are sequestered i n the egg (Lehner and O ' F a r r e l l , 1990b; 183 Jimenez et al., 1990), cdc2DS7/CyO mothers should sequester 50% each of mutant and wildtype product. In contrast Df(2L)JRl6/CyO mothers sequester only wild-type product, but half as much as d i p l o i d f l i e s . Given that cdc2DS7/Df(2L)JR16 progeny of cdc2n5? mothers die e a r l i e r than progeny i n the re c i p r o c a l cross, the cdc2D5? product may t i t r a t e some factor required by wild-type cdc2Dm for embryonic development. The nucleotide a l t e r a t i o n i n cdc2D5? i s consistent with t h i s i n t e r p r e t a t i o n . The mutation causes an amino acid s u b s t i t u t i o n i n a s i t e conserved throughout a l l kinases (Hanks et al., 1988). Biochemical analyses suggest that t h i s amino ac i d s i t e i s involved i n ATP binding which i s required f o r proper kinase function (see Hanks et al., 1988). Thus, i t i s possible that the kinase a c t i v i t y of p34 i s i n a c t i v e , yet i t s a b i l i t y to in t e r a c t with other regulatory elements i s s t i l l i n t a c t . Although the morphology of hemizygous cdc2D5V embryos has yet to be investigated, the normal appearance of embryonic c u t i c l e s suggests that hemizygotes are not grossly defective i n c e l l p r o l i f e r a t i o n during e a r l y embryogenesis. In Drosophila, the f i r s t t h i r t e e n rounds of c e l l d i v i s i o n are driven by maternally inherited product (Edgar et al., 1986); however, from d i v i s i o n 14 onward, the zygotic genome i s activ e . In the case of string, the Drosophila homologue of cdc25, maternally endowed message i s degraded at the t r a n s i t i o n to zygotic t r a n s c r i p t i o n a l c o n t r o l , and f a i l u r e to produce a wild-type product i n the zygote r e s u l t s i n mi t o t i c 184 a r r e s t i n G2 of interphase 14, despite normal morphogenetic movements i n the e a r l y embryo (Edgar and O ' F a r r e l l , 1989). The r e s u l t i s a severely deformed embryo that secretes an abnormal c u t i c l e . Pimples homozygotes, which arrest at d i v i s i o n 15 (C. Lehner, personal communication), also have an abnormal c u t i c l e (Niisslein-Volhard et a l . , 1984). Since hemizygous cdc2D57 f l i e s do not have defective c u t i c l e s , the mutant does not appear to cause c e l l cycle arrest at the mid-blastula t r a n s i t i o n . An a l t e r n a t i v e p o s s i b i l i t y to widespread c e l l cycle a r r e s t i s that c e l l d i v i s i o n i s affected i n a c r i t i c a l part of the nervous system. Unlike the c e l l s of other t i s s u e types that only undergo 3-4 d i v i s i o n s following the mid-blastula t r a n s i t i o n , the nervous system of wild-type f l i e s continues to divide throughout embryogenesis and the l a r v a l stages. Such speculation awaits a more de t a i l e d analysis of the mutants (which i s underway i n col l a b o r a t i o n with the laboratories of C. Lehner (Max Planck Institute) and P. O ' F a r r e l l (UCSF)). If cdc2D5? i s not completely i n a c t i v e , then the cdc2Dm n u l l phenotype remains unknown. The maternal dowry of cdc2 mRNA and protein might be s u f f i c i e n t to permit the completion of embryogenesis, or cdc2~ mutants might ar r e s t at the mid-b l a s t u l a t r a n s i t i o n , l i k e string mutants. Two observations suggest that zygotic cdc2Dm a c t i v i t y might be e s s e n t i a l f o r embryogenesis. F i r s t , observations of wild-type embryos suggests that cdc2 t r a n s c r i p t i o n i s activated i n each region of the embryo as that region begins to undergo mitosis 185 (Jimenez et al., 1990). Second, maternal cdc2 product i s l a r g e l y excluded from c e l l s of the developing embryo at the mid-blastula t r a n s i t i o n . This pattern of a c t i v i t y could represent a redundant control mechanism; however, only a few s p e c i f i c maternal t r a n s c r i p t s , string f o r example (Edgar and O ' F a r r e l l , 1989), have been shown to be degraded at the time zygotic t r a n s c r i p t i o n i s activated. Thus, i t i s also possible that zygotic regulation of cdc2Dm t r a n s c r i p t i o n i s a requirement for normal development. A genetic argument fo r or against the need for z y g o t i c a l l y expressed cdc2Dm during embryogenesis w i l l require a true n u l l a l l e l e f o r the locus. The P element induced mutation cdc2216P should be p a r t i c u l a r l y useful f o r i s o l a t i n g an amorphic a l l e l e of cdc2Dm, since the element can be mobilized i n dysgenic crosses. Small intragenic deletions that i n a c t i v a t e the gene could be selected by t e s t i n g f o r l e t h a l i t y i n trans with cdc2E2° or cdc2E1~4. The phenotypes of several less severe mutant a l l e l e s of cdc2Dm are consistent with defects i n c e l l p r o l i f e r a t i o n , although other explanations are not excluded. The absence of obvious imaginai discs and polytene chromosomes i n cdc2B47/cdc2E10 hétérozygotes suggests that both c e l l d i v i s i o n and DNA r e p l i c a t i o n are affected. These observations w i l l , of course, require more de t a i l e d documentation. The absence of imaginai disc t i s s u e might be investigated using a d i s c -s p e c i f i c antibody, and the extent of DNA endoreduplication 186 could be assayed using quantitative dot-blots. The severe s t e r n i t e and t e r g i t e etching associated with hemizygous cdc2Dm a l l e l e s may also r e f l e c t a f a i l u r e of c e l l s to p r o l i f e r a t e . Tergites and s t e r n i t e s are c u t i c u l a r structures secreted by the underling epidermis which covers the abdomen of the adult f l y . Most adult tissues are derived from imaginai discs that divide throughout the l a r v a l stages; however, the abdominal epidermis i s derived from h i s t o b l a s t s which are quiescent during l a r v a l development. At pupariation, h i s t o b l a s t c e l l s p r o l i f e r a t e r a p i d l y f o r several d i v i s i o n s ( c e l l cycle times are 2-3 hours compared with 10-12 hours for imaginai d i s c c e l l s ; Madhavan and Madhavan, 1980) before t h e i r rate of d i v i s i o n slows. Since there are only approximately 6-20 h i s t o b l a s t c e l l s i n mature t h i r d - i n s t a r larvae (Roseland and Schneiderman, 1979) which must divide to cover each abdominal segment, rapid d i v i s i o n i s necessary to generate enough c e l l s . C u t i c l e etching might, therefore, be a consequence of c e l l death, or i t might r e f l e c t a reduction i n the t o t a l number of epidermal c e l l s at the time of e c l o s i o n . Fewer epidermal c e l l s might be present i f the rate of c e l l d i v i s i o n were slowed because of the increased time required to accumulate s u f f i c i e n t functional cdc2 product to i n i t i a t e m itosis. Since the imaginai discs divide at a slower rate over a longer period of time, aspects of adult morphogenesis dependent on these c e l l s might be less susceptible to reduced cdc2 l e v e l s and t h i s would explain the lack of pattern defects 187 i n other c u t i c u l a r structures i n mutant f l i e s . Two of the mutants reported here have amino acid substitutions at the same s i t e as previously reported a l t e r a t i o n s i n the homologous residue from other species. The amino ac i d s u b s t i t u t i o n i n cdc2DmEl-4 (G 4 3 to D43) a l t e r s the 16 amino acid EGVPSTAIREISLLKE sequence that i s absolutely conserved across the CDC2 family of proteins. The S. pombe mutant cdc2-M35 has a glutamate residue at the same p o s i t i o n . In f i s s i o n yeast, the cdc2-M35 protein product i s temperature s e n s i t i v e , but even at the permissive temperature, mutant haploid c e l l s are elongated and undergo d i v i s i o n at a c e l l s i z e approximately 70% larger than wild-type (Nurse and Thuriaux, 1980). The e s s e n t i a l l y l e t h a l phenotype of the Drosophila mutant confirms the importance of t h i s residue to the cdc2 product of m u l t i c e l l u l a r organisms. Since some hemizygous f l i e s do survive to adulthood, t h i s suggests that the gene product i s at l e a s t p a r t i a l l y f u n c t i o n a l , as i n the yeast cdc2-M35 mutant. Several l i n e s of evidence suggest that the PSTAIR region may be the s i t e of i n t e r a c t i o n with some regulator of cdc2 kinase function. F i r s t , microinjection of the PSTAIR peptide induces meiotic maturation i n s t a r f i s h oocytes (Labbé et a l . , 1989), but t h i s phenomenon i s prevented i f the sequence i s truncated or i f amino acid substitutions are introduced (Picard et a l . , 1990). Second, microinjected PSTAIR peptide accelerates nuclear envelope breakdown and chromosome 188 condensation i n Xenopus egg extracts compared to MPF alone (Gautier et al., 1988). F i n a l l y , microinjection of t h i s peptide i n t o both s t a r f i s h and Xenopus oocytes tr i g g e r s a s p e c i f i c increase i n the concentration of i n t r a c e l l u l a r free Ca 2 + (Picard et al., 1990) . Only the i n t a c t , unmutated form of the PSTAIR peptide has t h i s e f f e c t . I t might, therefore, be possible to g e n e t i c a l l y s e l e c t f o r mutations i n Drosophila which suppress the l e t h a l i t y of mutations i n cdc2E1~*. In a s i m i l a r vein, i t would be i n t r i g u i n g to t e s t the ef f e c t s of overexpression of the Drosophila c y c l i n genes on cdc2E1~4. Cyclins form part of the functional cdc2 holoenzyme and some researchers have suggested that the PSTAIR sequence may be important f o r t h i s i n t e r a c t i o n (see Draetta, 1990). I f so, excess c y l i n protein may be able to rescue the cdc2E1~* l e t h a l phenotype. Using antibodies to the c y c l i n proteins i t might also be possible to detect differences i n the concentration of the cdc2Dm/'eyelin complex i n the mutant r e l a t i v e to wildtype f l i e s . The cdc2E1~9 replaces p r o l i n e with serine, while a mutation i n the homologous codon of S. cerevisiae replaces p r o l i n e with leucine (Lôrincz and Reed, 1986). The yeast mutant i s temperature s e n s i t i v e and has an aberrant morphology consistent with c o n s t i t u t i v e l y low cdc2 a c t i v i t y . No unusual phenotypes other than l e t h a l i t y were evident i n a l l e l i c crosses involving the cdc2Dm mutant. In addition to the c y c l i n s , another S. pombe protein, 189 p l 3 s u c l , associates with cdc2. A s i m i l a r protein has been i d e n t i f i e d i n humans (Richardson et al., 1990) and p l 3 s u c l i t s e l f can be used to p u r i f y p34 c d c 2 homologues from other species. These findings suggest that the pl3" u c l protein may also be part of a universal c e l l cycle mechanism. In yeast cdc2, the region between amino acids 177-208 may be the s i t e of i n t e r a c t i o n with p l 3 , u c l because three mutations within t h i s region, cdc2.33, cdc2.56 and cdc2.L7, are reduced i n t h e i r a b i l i t y t o bind pl3" u c l in vitro. The cdc2E1~24 mutation l i e s within the homologous region i n cdc2Dm (amino acids 171-202), and the mutation i n cdc2E1"23 i s only 4 amino acids d i s t a n t . Thus, these mutations may also disrupt the binding of cdc2Dm to p l 3 s u c l . Potential a l t e r a t i o n s i n the binding of p l 3 8 u c l could be tested e i t h e r in vivo or in vitro. An additional cdc2Dm mutation, cdc2E1°, also maps within the p o t e n t i a l p l 3 8 U C l binding region i n f e r r e d from yeast. Only one PCR-cloned product of t h i s mutant has been sequenced i n the current study; however, only a singl e basepair a l t e r a t i o n i s present i n the en t i r e coding region. This mutation causes a s u b s t i t u t i o n for Leu 1 7 6. The e f f e c t s of t h i s s u b s t i t u t i o n could also be tested f o r interactions with pl3" u c l. I f the mutation proved to be a cloning a r t i f a c t , i t should s t i l l be possible to t e s t the e f f e c t of the mutation in vivo or in vitro using the conventional methodologies of molecular biology. My i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of several 190 cdc2Dm mutations has l a i d the foundations f o r future, more det a i l e d , studies of p34 kinase i n the growth and development of Drosophila. 191 APPENDIX 1. Deficiency mapping recessive l e t h a l and s t e r i l e mutations i n region 31. 192 Table 1A. Deficiency mapping of complementation between the centromere d i s t a l breakpoints of Table IB: Deficiency mapping of a representative a l l e l e from each complementation group that i s l e t h a l i n trans with Df(2L)J27. Each cross was mutant/CyO X Df/CyO. The r a t i o presented i s the number of Cy to straight-winged f l i e s ( + , f u l l complementation; S, female s t e r i l e ; n.d., not done). Mutation Df(2L)J27 Df(2L)Jl06 Df(2L)JR3 Df(2L)JRl 24-127 148:0 + + 101:1 E 1 _ 1 3 E 1 - 1 7 91:0 206:0 + 148:0 RU26 n.d. n.d. 144:69 (S) 159:4 (S) El-6 n.d. 164:0 179:0 91:0 El-7 68:0 163:0 157:0 43:0 El-22 169:0 148:1 189:0 103:0 bsk - n.d. 268:0 231:0 D G 2 523-127 424:24 (S) 145:0 225:1 (S) 152:0 pirn 203:0 166:0 205:0 + E2-15 103:0 n.d. 135:0 + da 7 7" 1 1 193:0 150:0 325:0 + mfs48 E 1- 1 0 140:0 212:0 81:0 + Su(var) 216 164:0 268:0 138:0 + El-1 123:0 277:0 170:0 + El-3 168:0 155:0 n.d. + El-12 233:0 170:0 n.d. + El-19 275:1 123:0 53:0 + El-28 211:0 60:0 71:0 + E2-17 70:0 155:0 160:0 + 1(2) 5 4 E 1 - 1 6 180:0 154:0 185:0 + e r r " 6 420:68 (S) 160:0 n.d. n.d. PI23 (S) n.d. (S) + PJ50 (S) n.d. n.d. + El-8 270:0 238:0 91:0 + El-14 229:0 325:0 180:0 + 194 Mutation Table 1C. Deficiency mapping of representative mutations that Table ID. Deficiency mapping of a representative a l l e l e from APPENDIX 2. A summary of hemizygous l e t h a l mutations i n region 31, and the screens i n which they were induced. 198 Table 1. Hemizygous l e t h a l mutants i n region 31. Screens were performed over the following d e f i c i e n c i e s : Screen 1, Df(2L)Jl06j Screen 2, Df(2L)J27; Screen 3, Df(2L)J27; Screen 4, Df(2L)J2 (see Brock, 1989); Screen 5, Df(2L)J2 (Brock, 1989); and Screen 6, Df(2L)J27 (Harrington, 1990). Complementation Deficiency Mutant Mutagen Screen Group Interval A102 1 A102 EMS 5 16-165 1 16-165 GAMMA 4 1-44 1 1-44 GAMMA 4 C98 2 C98 EMS 5 27-168 GAMMA 4 CI 04 EMS 5 F133 3 F133 EMS 5 B149 4 B149 EMS 5 2-119 GAMMA 4 19-153 GAMMA 4 14-140 4 14-140 GAMMA 4 24-127 5 24-127 GAMMA 4 El 5 EMS 5 A63 EMS 5 A141 EMS 5 C35 EMS 5 E56 EMS 5 H30 EMS 5 E2-1 EMS 2 E2-12 EMS 2 E2-13 EMS 2 E2-32 EMS 2 E2-42 EMS 2 El-13 6 El-13 EMS 1 El-17 EMS 1 G2-5 EMS 6 El-6 7 El-6 EMS 1 199 Complementation Deficiency Mutant Mutagen Screen Group Interval El-7 7 El-7 EMS 1 El-22 7 El-22 EMS 1 DG25 8 23-127 GAMMA 4 29-142 GAMMA 4 25-159 GAMMA 4 D22 EMS 5 C93 EMS 5 E2-5 EMS 2 E2-21 EMS 2 pirn 9 El-15 EMS 1 E2-15 9 E2-15 EMS 2 da 10 F75 EMS 5 E2-20 EMS 2 E2-35 EMS 2 E2-30 EMS 2 E2-24 EMS 2 El-21 EMS 2 El-2 6 EMS 1 G2-10 GAMMA 6 77-11 P ELEMENT 3 mfs48 10 E2-43 EMS 2 77-13 P ELEMENT 3 El-12 EMS 1 Su(var)216/cdc2 10 D57 EMS 5 E20 EMS 5 B47 EMS 5 E10 EMS 5 El-4 EMS 1 El-9 EMS 1 El-23 EMS 1 200 Complementation Deficiency Mutant Mutagen Screen Group Interval El-24 EMS 1 El-1 10 El-1 EMS 1 B35 EMS 5 B26 EMS 5 13-47 GAMMA 4 13-83 GAMMA 4 E2-16 EMS 2 E2-31 EMS 2 E2-38 EMS 2 G2-3 GAMMA 6 G2-7 GAMMA 6 El-3 10 El-3 EMS 1 E113 EMS 5 G2- GAMMA 6 El-12 10 El-12 EMS 1 E2-29 EMS 2 E2-33 EMS 2 G2-9 GAMMA 6 El-19 10 El-19 EMS 1 E34 EMS 5 G78 EMS 5 El-2 8 10 El-28 EMS 1 E2-17 10 E2-17 EMS 2 B106 EMS 5 E2-44 EMS 2 G2-6 GAMMA 6 1(2)54 11 H113 EMS 5 E2-24 EMS 2 C70 EMS 5 G2-4 EMS 6 201 Complementation Deficiency Mutant Mutagen Screen Group Interval El-2 EMS 1 El-16 EMS 1 e r r 11 A76 EMS 5 C36 EMS 5 El-2 7 EMS 1 El-8 11 El-8 EMS 1 El-14 11 El-14 EMS 1 E2-9 11 E2-9 EMS 2 E2-23 EMS 2 77-12 11 77-12 P ELEMENT 3 77-14 11 77-14 P ELEMENT 3 E2-22 11 E2-22 EMS 2 29-85 12 29-85 EMS 5 E2-34 EMS 2 E2-2 EMS 2 E2-7 EMS 2 El-5 13 El-5 EMS 1 El-11 EMS 1 A61 EMS 5 14-195 GAMMA 4 El-18 13 El-18 EMS 1 El-2 5 13 El-25 EMS 1 E3 13 E3 EMS 5 H95 14 H95 EMS 5 B100 EMS 5 E73 EMS 5 A65 15 A65 EMS 5 D121 15 D121 EMS 5 E24 15 E24 EMS 5 F15 15 F15 EMS 5 202 Complementation Deficiency Mutant Mutagen Screen Group Interval Gl 15 Gl EMS 5 203 APPENDIX 3. Miscellaneous complementation crosses f o r mutants i n region 31. 204 Table 1. Complementation crosses f o r mutants i n region 31. Ratios represent the proportion of Cy to straight-winged progeny recovered from the cross mutantl/CyO X mutant2/CyO. Deficiency Locus Cross Progeny Interval 2 C98 C98 X 27-168 276:0 C98 X C104 264:0 27-168 X CI04 65:0 10 El-1 13-83 X B26 227:0 B26 X 13-47 107:0 E2-31 X G2-3 163:0 E2-31 X G2-7 143:0 E2-31 X E2-16 446:0 E2-31 X E2-38 79:0 E2-16 X El-1 403:46 E2-16 X E2-38 410:0 G2-3 X E2-38 142:0 El-1 X G2-7 160:11 10 El-3 El-3 X E113 205:0 G2-4 X El 13 _ i 10 El-12 El-12 X E2-29 191:0 El-12 X E2-33 207:0 El-12 X G2-9 _ i 10 El-19 El-19 X G78 84:0 G78 X £34 346:0 10 E2-17 E2-17 X B106" 43:0 £2-44 X B106 190:14 G2-6 X B106" _ i 10 G7S X E34 120:0 El-19 x G75 84:0 10 mfs48 E2-43 x mfs4i? 194:0 £2-43 x 77-13 490:0 E2-43 x £1-10 89:0 205 Deficiency Locus Interval Cross Progeny El-10 X mfs48 199:0 77-13 X mfs48 1270:3982 11 err RE54 X A76 (29°) 244:213 RE54 X C36 208:0 A76 X C36 (29°) 483:0 C36 X El-27 166:0 11 El-14 El-14 x 33-161 209:0 11 E2-9 E2-9 x E2-23 163:0 11 77-14 77-14 x El-8 70:20 11 77-12 77-12 X El-8 67:15 12 29-85 29-85 x E2-34 531:17 29-85 x £2-2 405:2 2°-c?5 x E2-7 400:0 13 El-5 14-195 x A6"5 57:0 El-5 x 14-195 130:0 El-11 x 14-195 229:0 14 H95 H95 x BI00 226:183 J?°5 x £73 387:943 2?73 X B100 57:103 Harrington (1990) 2data of I.P. Whitehead 3female s t e r i l e straight-winged progeny 206 APPENDIX 4. Sequencing data for the region surrounding cdc2Dm. 207 Figure 1. The sequencing strategy for p c 8 0 0 and the gene it represents. Arrows indicate the direct ion of sequencing and the extent of the directed delet ions, (see Chapter 3 for symbols ) . A . Genomic DNA H H Sp He B. cDNA H H Sp He <4 500 bp 2 0 8 Figure 2. The complete genomic sequence of a region encompassing the genes represented by pc800 (underlined; nt 270-1150), pcllOO (cdc2Dmj double underlined; nt 1650-3100),and cBgl4 (underlined; 3100-3250). AAGCTTAAAA CACGACGAAC CAGGTAAATG GGTTGAAGAA ATTTATATAA AAACTTAGTA TAAGTGTAAT AAGAAAAACT TTGGGTTTCA ATAGCTATAA CACCATTTGG AAAATCCAAT TTTCAAATCT ACTATAAACA ACGTATAAGG AAAAATAAAG AAAAAATATT AGAAATGTGT TTTTATAATG GTATCATAGC 200 TTCATACAAT TTTACTAAGG TCCTCGTTTT TAGTCCCCAT AAATCCAGGA ATTATTTCGA TATTCCGCTC TCTTAAGACA TTCCCCTGAC ACAAATCCTA ATCATAGATA TTAACAGGTA TATGAAAAGT TTATTGCATT GGAAAAACAG TTTGTTGTGC ATTTAGCTCC ATCGGTAATG TGCGTAGGCG CGGTTGCTTT 400 CGCACAGCCT GTGCAGATCG TCCTTTCGCT TGATGACCCT TCCCTGGCCA TGGGCTGCGT CGAGAATCTC CCAGGCCAAC TTTTCCGGCA GGGACACCTT ACGCTCCTTC TCGCGGGCAG CTTCCAAGAG CCACTTCATG GCCAGAAAAT ATGACCGCTT CGTGGTGATG GGAACAGGGA CTTGATAGGT GACACCACCA 600 CGTTTTATGG CGGTCACTTG AAGGAGCGGT CGGCAGTTTT CAACCGCTTG TTTCAGAAGC GTTTCGGGAT TGGTGTTTAT GGTTGTCTTC TCCCCTTTGG CCAGATTCAT GTGCTCCGTC TGGGTTCGTT TTATTAGCTC CAGCGTCTTG GACAAAAGCG TTCTGGCCAA GGCACTGTTC CCCTTTTTCG TTATATAATT 800 GATCATTTTA TGTTTTGTAT CGTCGTGAAA GATGGTATCA GACGATTTGT TGACCGCCGC CTTGATGGGC ACATGGTAAA GCTTGGACAG ATCATTCTTT TGCTCTAGTT GTTTATATTT TTGTACAATT GGTTCGACAT AGTGTGTGGG ATACACCGAT ATCAATCGAA GGCAGCTACA ATAAACAAAT GGGTGATCAG 1000 TCATTTGTGT TTTTAGAGAT AAGTAACACT TTAGAGAAGT TTAACTTACÇ TGAGCCTTGA AGTTTTCTCT GCAATCCTAC CTAAAAGCGA CATGTTTACA TCGTCTGCTG TGTCGGCAAA AAATAAAAAA ACTATGTTAT ATATATGTTA 209 TGCACATTTG CGGCATGCAT ATAGGTGTGT CAAGATATCG ACAAAGAGCT TAGTAATTTT GAAACAACAA AAATGAAGTA AAACAGTTGC GGTATTCCAA TTACATTTTT TAAATTAATT TCTTTAGTAC CGTACTACTG GTACTCACCT TCAAAAGATA TAAAATAGAA ATTAATTGCA CCAAAAACTC ATAAGTTAAT TAATAGGATA TTAGCAGAAA CGTTTGTCTC CGAACTCAAA CAAAGTGATG TCTTAATTAA TTGAAATCAC TATAAAAAAA AGCGTGGAAT TTTACAGTAC ACTAAAATTA ACTTTAAAAA AATTAACAAC ATTTTTAAGA TACAGCAATT CAATAAGTTG CCTGAATATT GAGTTTCATT CCCACATTCC AAATGAATAA ATGTAGCTAG CTTAGCATCG TTTAAACTGT CTGGTAATAC TAGAGCATAT ACGTCAAAAA CGCGCTAATT TAAAAGTCGG TGGCTTGCAA AGAAATAGCT TAATAAATTA TGGAGGATTT TGAGAAAAGT GAGAAGATTG GCGAGGGCAC ATATGGCGTG GTGGATAAGG GTCGCAACCG CCTGACGGGC CAAATTGTGG CAATGAAGAA AATCCGCTTG GAGTCCGACG ACGAAGGCGT TCCATCAACC GCTATCAGGT AAAATGCCGC GGCTTGGACG CCCAAGCCTC AAAATGTGTC ATGTTCTTTC GCGACTTTTT TCCATTCAAA TGGCGGATAT GCTTAATTGA AAGTATGTTC CTTGACATCT TATGGGTTGG TTTGAAGATT TTGCAATATT GTTTTTAATT TATACTGTGG AAGTCTAGCA TAATTATGTT ACGGCTTATG TTCATCATAC ATATGTGTGT GTATGTACAA TCCTATATGC GTACTTATGC TCATGTGTAC ATCATCATAC TTTCTATTTA TGTTTATTAA TTGGAAGCCT GCGAATACTT TCTGGTTCAA CTGATAGACC AATAACGAAA ATACTTTAAT CACATGTTTT TTCTATAATG TAGTAATATT TAATATCCAT TACAGAGAAA TTTCGTTGCT TAAGGAGTTG AAACATGAAA ACATTGTCTG GTTGGAATGG AGGAGAACCG CATATACTTG ATCTTTGAAT TCCTATCGAT GGACCTCAAG AAATACATGG ATTCGCTGCC AGTTGATAAG CACATGGAGA GTGAATTGGT CCGTAGCTAT TTGTACCAAA TAACTAGCGC CATTCTTTTC TGCCATCGTC GGCGAGTACT TCACCGTGAT CTTAAGCCGC AGAACTTACT AATCGACAAG 210 AGTGGCCTCA TAAAAGTCGC CGACTTTGGA CTTGGCCGAT CCTTTGGCAT TCCGGTCTCC ATTTATACGC ACGAGATTGT TACCTTGTGG TACAGAGCGC CGGAGGTGCT ACTGGGTTCA CCCCGGTATT CCTGTCCCGT CGATATCTGG TCCATTGGAT GCATATTCGC GGAGATGGCA ACGAGAAAGC CGCTATTCCA GGGTGACTCG GAAATTGACC AGTTGTTTAG AATGTTTAGG TAACCACAGG TAATTTACTT TCTATTCCCT GTGATACTCA CACTCATTGA TTGCAGAATT CTGAAAACAC CTACCGAAGA CATTTGGCCG GGCGTTACTT CGCTACCCGA CTATAAGAAC ACGTTCCCCT GCTGGTCCAC GAACCAATTG ACCAATCAGT TAAAGAATCT CGATGCGAAT GGTATTGATC TCATACAAAA GATGTTAATC TACGATCCAG TTCATCGCAT TTCCGCCAAG GACATTTTGG AGCATCCCTA TTTCAATGGT TTTCAATCGG GCTTAGTTCG AAATTAACGT TCGGTATTCT CGTTTGACTT TAACTAAGAA TTTTAAAACA AGAGATCTTG GTATCTAATC TAAAGCAAAA TAGCCGTAAA TAAAACTAAG GGTGTAAAAC AATCAACGTT TACTTAATTT ATGTAAGTGT ATTTACAGAT TTACGCCAAA TTACCAGCGC TCTAACAGAA TAATAAGGCT TCAAGGCTTT AATTATTATA CAAAAAGAAA GTTAAATATG GAAATCCTCG TTGAAACTAG TCCTATTATA GAGAGCACTG CTAGCACCGC TGTCATGACG TCTAAAGACG CCAAAGCAGC CGCGCCTCTT CATCACGTAA GCACCAATGA TGGTCGGCAC CGCGATGAGC AGGACAACCA AGAGTCCAAC CCATATAAGA CTGCCCGTCG AACTTTCTAT CATATGTTCA TTCGCCACTC TGAGCAGCGT AACATCGTTT AACTTGTTCG GACCGCCACA TTTGACATCT TTGGCCAGAA CGGGCGTGGT CTTGTTGATT CGATCTATCA AGACGTTGAT CAAGAAGTCA TTGGATTCGT CGCAATTCCA CGGGTTGAAT CTAAGATCCA GAGCCTTAAG TTTATCCCAG CGCACTAGTA GTTCTTTCGG CAAAGTCGAC 211 Figure 3. The DNA sequence and conceptual t r a n s l a t i o n pc800. TAACATAGTTTTTTTATTTTTTGCCGACACAGCAGACGATGTAAAC ATGTCGCTTTTAGGTAGGATTGCAGAGAAAACTTCAAGGCTCAGCTGCCTTCGA MetSerLeuLeuGlyArglleAlaGluLysThrSerArgLeuSerCysLeuArg TTGATGTCGGTGTATCCCACACACTATGTCGAACCAATTGTACAAAAATATAAA LeuMetSerValTyrProThrHisTyrValGluProIleValGlnLysTyrLys CAACTATAGCAAAAGAATGATCTGTCCAAGCTTTACCATGTGCCCATCAAGGCG GlnLeuGluGlnLysAsnAspLeuSerLysLeuTyrHisValProIleLysAla GCGGTCAACAAATCGTCTTATACCATCTTTCACGACGATACAAAACATAAAATG AlaValAsnLysSerSerAspThrllePheHisAspAspThrLysHisLysMet ATCAATTATATAACGAAAAAGGGGAACAGTGCCTTGGCCAGAACGCTTTTGTCC IleAsnTyrlleThrLysLysLysAsnSerAlaLeuAlaArgThrLeuLeuSer AAGACGCTGGAGCTAATAAAACGAACCCAGACGGAGCACATGAATCTGGCCAAA LysThrLeuGluLeuIleLysArgThrGlnThrGluHisMetAsnLeuAlaLys GGGGAGAAGACAACCATAAACACCAATCCCGAAACGCTTCTGAAACAAGCGGTT GlyGluLysThrThrlleAsnThrAsnProGluThrLeuLeuLysGlnAlaVal GAAAACTGCCGACCGCTCCTTCAAGTGACCGCCATAAAACGTGGTGGTGTCACC GluAsnCysArgProLeuLeuGlnValThrAlalleLysArgGlyGlyValThr TATCAAGTCCCTGTTCCCATCACCACGAAGCGGTCATATTTTCTGGCCATGAAG TyrGlnValProValProIleThrThrLysAsgSerTyrPheLeuAlaMetLys TGGCTCTTGGAAGCTGCCCGCGAGAAGGAGCGTAAGGTGTCCCTGCCGGAAAAG TrpLeuLeuGluAlaAlaArgGluLysGluArgLysValSerLeuProGluLys TTGGCCTGGGAGATTCTCGACGCAGCCCATGGCCAGGGAAGGGTCATCAAGCGA LeuAlaTrpGluIleLeuAspAlaAlaHisGlyGlnLysArgVallleLysArg AAGGACGATCTGCACAGGCTGTGCGAAAGCAACCGCGCCTACGCACATTACCGA LysAspAspLeuHisArgLeuCysGluSerAsnArgAlaTyrAlaHisTyrArg TGGAGCTAAATGCACAACAAACTGTTTTTCCAATGCAATAAACTTTTCATATAC TrpSer*** CTGTTAA 212 Figure 4. The sequence of the P element associated with cdc2Dm i n region 31E. The 31 bp inverted repeats are underlined. GCTAATTTAA AAGTCGGTGG CATGATGAAA TAACATAAGG TGGTCCCGTC GAAAGCCGAA GCTTACCGAA GTATACACTT AAATTCAGTG CACGTTTGCT TGTTGAGAGG AAAGGTTGTG TGCGGACGAA AAAACATTAA CCCTTACGTG GAATAAAAAA AAATGAAATA TTGCAAATTT TGCTGCAAAG CTGTGACTGG AGTAAAATTA ATTC deletion AATAATAA TAATTTTGAA ATTACAAATA ATGTAAAGGA AAAATTAATA TTAGCAGCGC GAAACGTCGA TGTTGATAAA CAAGTAAAAT CTTTTTATTT TAAAATTAGA ATATATTTTA GAATTAAGTA CTTCAACAAA AAAATTGAAA TTAAAAANCA AAAACAAAAG TTAATTGGAA ACTCCAAATT ATTAAAAATA AAACTTTAAA AATAATTTCG TCTAATTAAT AATTCAAACC CCACGGACAT GCTAAGGGTT AATCAACAAT CATATCGCTG TCTCACTCAG ACTCAATACG ACACTCAGAA TACTATTCCT TTCACTCGCA CTTATTGCAA GCATACGTTA AGTGGATGTC TCTTGCCGAC GGGACCACCT TATGTTATTT CATCATGGTC GGTGGCTTGC 213 REFERENCES Alfageme, C.R., G.T. Rudkin, and L.H. Cohen (1980). I s o l a t i o n , properties and c e l l u l a r d i s t r i b u t i o n of Dl, a chromosomal protein of Drosophila. Chromosoma 78, 1-31. Annunziato, A.T., L.-L.Y. Frado, R.L. Seale, and C L . F . Woodcock (1988). Treatment with sodium butyrate i n h i b i t s the complete condensation of interphase chromatin. Chromosoma 96, 132-138. Arion, D., L. Meijer, L. Brizuela, and D. Beach (1988). cdc2 i s a component of the M phase-specific histone HI kinase: evidence for i d e n t i t y with MPF. C e l l 55, 371-378. Axton, M.J., V. Dombradi, P.T.W. Cohen, and D.M. Glover (1990). One of the protein phosphatase 1 isoenzymes i n Drosophila i s e s s e n t i a l f o r mitosis. C e l l 63, 33-46. Bainbridge, S.P., and M. Bownes (1981). Staging the metamorphosis of Drosophila melanogaster. J . Embryol. Exp. Morph. 66, 57-80. Baker, W.K. (1963). Genetic control of pigment d i f f e r e n t i a t i o n i n somatic c e l l s . Am. Zool. 3, 57-69. Baker, W. K. (1967). A c l o n a l system of d i f f e r e n t i a l gene a c t i v i t y i n Drosophila. Dev. B i o l . 16, 1-17. Baker, W.K. (1968). P o s i t i o n - e f f e c t variegation. Adv. Genet. 14, 133-169. Barigozzi, C , S. D o l f i n i , M. Fraccaro, C.R. Raimondi, and L. Tiepolo (1966). In vitro study of the DNA r e p l i c a t i o n patterns of somatic chromosomes of Drosophila melanogaster. Exp. C e l l Research 43, 231-234. Benton, W.D., and R.W. Davis (1977). Screening Xgt recombinant clones by hybridization to single plagues in situ. Science 196, 180-182. Beverly, S.M., and A.C. Wilson (1984). Molecular evolution i n Drosophila and higher Diptera. I I . A time scale f o r f l y evolution. J . Mol. Evol. 21, 1-13. Biessmann, H., P. Kruger, C. Schroper, and E. Spindler (1981). Molecular cloning and preliminary c h a r a c t e r i z a t i o n of a Drosophila melanogaster gene from a region adjacent to the centromeric beta-heterochromatin. Chromosoma 82, 493-503. 214 Blow, J . J . , and P. Nurse (1990). A cdc2-like protein i s involved i n the i n i t i a t i o n of DNA r e p l i c a t i o n i n Xenopus egg extracts. C e l l 62, 855-862. Boffa, L.C., R.J. Gruss, and V.A. A l l f r e y (1981). Manifold e f f e c t s of sodium butyrate on nuclear functions. J . B i o l . Chem. 256, 9612-9621. Booher, R., and D. Beach (1987). Interaction between cdcl3+ and cdc2* i n the co n t r o l of mitosis i n f i s s i o n yeast; d i s s o c i a t i o n of the Gx and G2 r o l e s of the cdc2* protein kinase. EMBO J. 6, 3441-3447. Booher, R.N., C E . A l f a , J.S. Hyams, and D.H. Beach (1989). The f i s s i o n yeast cdc2/cdcl3/sucl protein kinase: regulation of c a t a l y t i c a c t i v i t y and nuclear l o c a l i z a t i o n . C e l l 58, 485-497. Bradbury, E.M., R.J. I n g l i s , and H.R. Matthews (1974). Control of c e l l d i v i s i o n by very l y s i n e r i c h histone ( f l ) phosphorylation. Nature 247, 257-261. Bradbury, E.M., R.J. I n g l i s , H.R. Matthews, and N. Sarner (1973). Phosphorylation of very l y s i n e r i c h histone i n Physarum polycepharum: c o r r e l a t i o n with chromosome condensation. Eur. J . Biochem. 33, 131-139. Brock, J.-A. K. (1989). A genetic analysis of region 31 on chromosome 2 of Drosophila melanogaster. M.Sc. Thesis. University of B r i t i s h Columbia. Brosseau, G.E. J r . (1970). V-type p o s i t i o n e f f e c t s f o r e* and ro* i n Drosophila. Dros. Inf. Serv. 45, 100. Bryant, P.J. (1970). C e l l lineage r e l a t i o n s h i p s i n the imaginai wing disc of Drosophila melanogaster. Dev. Biol. 22, 389-411. Carlson, J . (1982). Jonah genes. PhD. Thesis, Stanford. Catcheside, D.G. (1947). The P-locus p o s i t i o n e f f e c t of Oenothera. J . Genet. 48, 31-42. Cattanach, B.M. (1974). Posi t i o n e f f e c t variegation i n the mouse. Genet. Res. Camb. 23, 291-306. Caudy, M., E.H. G r e l l , C. Dambly-Chaudiere, A. Ghysen, L.Y. Jan, and Y.N. Jan (1988). The maternal sex determination gene daughterless has zygotic a c t i v i t y necessary f o r the formation of peripheral neurons i n Drosophila. Genes Dev. 2, 843-852. 215 Caudy, M., H. Vassin, M. Brand, R. Tuma, L.Y. Jan, and Y.N. Jan (1988). daughterless, a Drosophila gene e s s e n t i a l for both neurogenesis and sex determination, has sequence s i m i l a r i t i e s to myc and the achaete-scute complex. C e l l 55, 1061-1067. Chou, P.Y., and S.D. Fassman (1974). P r e d i c t i o n of protein conformation. Biochem. 13, 222. Christman, J.K., N. Weich, B. Schoenbrun, N.K. Schneiderman, and A. Acs (1980). Hypomethylation of DNA during d i f f e r e n t i a t i o n of Friend erythroleukemia c e l l s . J . C e l l B i o l . 86, 366-370. Clark, S.H., and A. Chovnick (1986). Studies of normal and po s i t i o n - a f f e c t e d expression of rosy region genes i n Drosophila melanogaster. Genetics 114, 819-840. Clark-Adams, CD., D. Norris, M.A. Osley, J.S. Fassler, and F. Winston (1988). Changes i n histone gene dosage a l t e r t r a n s c r i p t i o n i n yeast. Genes Dev. 2, 150-159. Cl i n e , T.W. (1989). The a f f a i r s of daughterless and the promiscuity of developmental regulators. C e l l 59, 231-234. Cohen, J . (1962). P o s i t i o n e f f e c t variegation at several c l o s e l y - l i n k e d l o c i i n Drosophila melanogaster, Genetics 47, 647-659. Cronmiller, C , P. Schedl, and T.W. Cline (1988). Molecular char a c t e r i z a t i o n of daughterless, a Drosophila sex determination gene with multiple roles i n development. Genes and Development 2, 1666-1676. Demerec, M., and H. Slizynska (1937). Mottled white 258-18 of Drosophila melanogaster. Chromosoma 33, 319-344. Devlin, R.H., B. Bingham, and B.T. Wakimoto (1990). The organization and expression of the light gene, a heterochromatic gene of Drosophila melanogaster. Genetics 125, 129-140. D i m i t r i , P., and C Pisano (1989). P o s i t i o n e f f e c t variegation i n Drosophila melanogaster. Relationship between suppression e f f e c t and the amount of Y chromosome. Genetics 122, 793-800. Doree, M. (1990). Control of M-phase by maturation-promoting f a c t o r . Current Opinion C e l l B i o l . 2, 269-273. Dorn, R., S. Heymann, R. L i n d i g k e i t , and G. Reuter (1986). Suppressor mutation of p o s i t i o n e f f e c t variegation i n Drosophila melanogaster a f f e c t i n g chromatin properties. Chromosoma 93, 398-403. 216 Draetta, G. (1990). C e l l cycle c o n t r o l i n eukaryotes: molecular mechanisms of cdc2 a c t i v a t i o n . TIBS 15, 378-383. Dunphy, W.G., L. Br i z u e l a , D. Beach, and J . Newport (1988). The Xenopus cdc2 protein i s a component of MPF, a cytoplasmic regulator of mitosis. C e l l 54, 423-431. Durrin, L.K., R.K. Mann, P.S. Kayne, and M. Grunstein (1991). Yeast histone H4 N-terminal sequence i s required f o r promoter a c t i v a t i o n in vivo. C e l l 65, 1023-1031. D'Urso, F., R.L. Marraccino, Marshak, D.R., and J.M. Roberts (1990). C e l l cycle control of DNA r e p l i c a t i o n by a homolog from human c e l l s of the p34 cdc2 protein kinase. Science 250, 786-791. Edgar, B.A., and P.H. O'Far r e l l (1989). Genetic control of c e l l d i v i s i o n patterns i n the Drosophila embryo. C e l l 57, 177-187. Edgar, B.A., and P.H. O'Far r e l l (1990). The three postblastoderm c e l l cycles of Drosophila embryogenesis are regulated i n G2 by string. C e l l 62, 469-480. Edgar, B.A., C P . Kiehle, and G. Schubiger (1986). C e l l cycle control by the nucleo-cytoplasmic r a t i o i n e a r l y Drosophila development. C e l l 44, 365-372. Edgar, B.A., and G. Schubiger (1986). Parameters c o n t r o l l i n g t r a n s c r i p t i o n a l a c t i v a t i o n during e a r l y Drosophila development. C e l l 44, 871-877. Eissenberg, J.C. (1989). Position e f f e c t variegation i n Drosophila, towards a genetics of chromatin assembly. Bioessays 11, 14-17. Eissenberg, J . C , T.C James, D.M. Foster-Hartnett, T. Hartnett, V. Ngan, and S. E l g i n (1990). Mutation i n a heterochromatin-specific chromosomal protein i s associated with suppression of p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster. Proc. Nat l . Acad. S c i . USA 87, 9923-9927. Engels, W.R., W.K. Benz, CR. Preston, P.L. Graham, R.W. P h i l l i s , and H.M. Robertson (1987). Somatic e f f e c t s of P element a c t i v i t y i n Drosophila melanogaster. Genetics 117, 745-757. F e i l e r , H.S., and T.W. Jacobs (1990). C e l l d i v i s i o n i n higher plants, a cdc2 gene, i t s 34-kDa product, and histone Hi kinase a c t i v i t y i n pea. Proc. Natl. Acad. S c i . USA 87, 5397-5401. 217 Felsenfeld, G., and J.D. McGhee (1986). Structure of the 30 nm chromatin f i b e r . C e l l 44, 375-377. Feinberg, A.P., and B. Vogelstein (1983). A technique for r a d i o l a b e l l i n g DNA r e s t r i c t i o n endonuclease fragments to high s p e c i f i c a c t i v i t y . Anal. Biochem. 132, 6-13. Feinberg, A.P., and B. Vogelstein (1984). A technique for radiolabbeling DNA r e s t r i c t i o n endonuclease fragments to high s p e c i f i c a c t i v i t y : addendum. Anal Biochem. 137, 266-267. Foe, V.E. (1989). M i t o t i c domains reveal e a r l y commitment of c e l l s i n Drosophila embryos. Development 107, 1-22. Foe, V.E., and B.M. Alberts (1985). Reversible chromosome condensation induced i n Drosophila embryos by anoxia: v i s u a l i z a t i o n of the interphase nuclear organization. J . C e l l B i o l . 100, 1623-1636. Frischauf, A.M., H. Leharch, A. Poustka, and N. Murray (1983). Lambda replacement vectors carrying p o l y l i n k e r sequences. J . Mol. B i o l . 170, 827-842. G a l l , J.G., E.H. Cohen, and M.L. Polan (1971). Repetitive DNA sequences i n Drosophila. Chromosoma 33, 319-344. Garcia-Bellido, A., and J.R. Merriam (1969). C e l l l i n e l a g e of the imaginai disks i n Drosophila gynandromorphs. J . Exp. Zool. 170, 61-76. Garcia-Bellido, A., and J.R. Merriam (1971). Parameters of the wing imaginai d i s c development of Drosophila melanogaster. Dev. B i o l . 24, 61-87. Gaunt, S.J., and P.M. Singh (1990). Homeogene expression patterns and chromosomal imprinting. Trends Genet. 6, 208-212. Gautier, J . , C. Norbury, M. Lokha, P. Nurse, and J . Mailer (1988). P u r i f i e d maturation-promoting fa c t o r contains the product of a Xenopus homolog of the f i s s i o n yeast c e l l cycle control gene cdc2. C e l l 54, 433-439. Glover, D.M. (1991). Mitosis i n the Drosophila embryo - i n and out of c o n t r o l . Trends Genet. 7, 125-131. Grunstein, M., and D. Hogness (1975). Colony h y b r i d i z a t i o n : A method fo r the i s o l a t i o n of cloned DNAs that contain a s p e c i f i c gene. Proc. Natl . Acad. S c i . USA. 72, 3961-3965. Gowen, J.W., and E.H. Gay (1934). Chromosome c o n s t i t u t i o n and behavior i n ever-sporting and mottling i n Drosophila melanogaster. Genetics 19, 189-208. 218 Gowen, J.W., and E.H. Gay (1935). E f f e c t of temperature on sporting eye color i n Drosophila melanogaster. Science 77, 312. Gould, K.L., and P. Nurse (1989). Tyrosine phosphorylation of the f i s s i o n yeast cdc2+ protein kinase regulates entry into mitosis. Nature 342, 39-45. G s e l l , R. (1971). Untersuchungen zur s t a b i l i t a t einer yellow p o s i t i o n - e f f e k t - v a r i e g a t i o n i n imaginalscheiben-kulturen von Drosophila melanogaster. Molec. Gen. Genet. 110, 218-237. Hadwiger, J.A., C. Wittenberg, M.A. de Barros Lopez, H.E. Richardson, and S.I. Reed (1989). A family of c y c l i n homologs that control Gx phase i n yeast. Proc. Natl . Acad. S c i . USA 86, 6255-6259. Han, M., and M. Grunstein (1988). Nucleosome loss activates yeast downstream promoters in vivo. C e l l 55, 1137-1145. Hanks, S.K., A.M. Quinn, and T. Hunter (1988). The protein kinase family, conserved features and deduced phylogeny of the c a t a l y t i c domains. Science 241, 42-52. Harrington, M. (1990). Suppression of p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster by antimorphic mutations of heterochromatin protein components. B.Sc. Honours Thesis. University of B r i t i s h Columbia. Hartmann-Goldstein, I.J. (1967). On the r e l a t i o n s h i p between heterochromatinization and variegation i n Drosophila, with s p e c i a l reference to temperature-sensitive periods. Genet. Res. 10, 143-159. Hayashi, S., A. Ruddell, D. S i n c l a i r , and T. G r i g l i a t t i (1990). Chromosomal structure i s altered by mutations that suppress or enhance p o s i t i o n e f f e c t variegation. Chromosoma 99, 391-400. Hayashida, H., and T. Miyata (1983). Unusual evolutionary conservation and frequent DNA segment exchange i n class 1 genes of the major histo c o m p a t i b i l i t y complex. Proc. Natl . Acad. S c i . USA 80, 2671-2675. Healy, M.J., R.J. Russell, and G.L.G. Miklos (1988). Molecular studies on interspersed r e p e t i t i v e and unique sequences i n the region of the complementation group uncoordinated on the X chromosome of Drosophila melanogaster. Mol. Gen. Genet. 213, 63-71. 219 Heitz, E. (1934). Uber alpha und beta-heterochromatin sowei Konstanz und Bau der Chromeren bei Drosophila. B i o l . Z e n t r a l b l . 54, 588-609. Henikoff, S. (1979). P o s i t i o n - e f f e c t s and variegation enhancers i n an autosomal region of Drosophila melanogaster. Genetics 93, 106-115. Henikoff, S. (1981). P o s i t i o n - e f f e c t variegation and chromosome structure of a heat shock puff i n Drosophila. Chromosoma 83, 381-393. Henikoff, S. (1984). U n i d i r e c t i o n a l digestion with exonuclease III creates targeted breakpoints f o r DNA sequencing. Gene 28, 351-359. Henikoff, S. (1990). P o s i t i o n - e f f e c t variegation a f t e r 60 years. Trends Genet. 6, 422-426. Henikoff, S., and T.D. Dreesen (1989). Trans-inactivation of the Drosophila brown gene: evidence f o r t r a n s c r i p t i o n a l repression and somatic p a i r i n g dependence. Proc. N a t l . Acad. S c i . USA. 86, 6704-6708. Hess, 0. (1970). Genetic function correlated with unfolding of lampbrush loops by the Y chromosome spermatocytes of Drosophila hydei. Molec. Gen. Genet. 106, 328-346. Hessler, A.Y. (1958). V-type p o s i t i o n e f f e c t s at the l i g h t locus i n Drosophila melanogaster. Genetics 43, 395-403. H i l l i k e r , A.J., R. Appels, and A. Schalet (1980). The genetic analysis of D. melanogaster heterochromatin. C e l l 21, 607-619. Hindley, J . , and G.A. Phear (1984). Sequence of the c e l l d i v i s i o n gene CDC2 from Schizosaccharomyces pombe; patterns of s p l i c i n g and homology to protein kinases. Gene 31, 129-134. Hinton, T. (1949). The modification of the expression of a p o s i t i o n e f f e c t . Am. Nat. 83, 69-94. Hinton, T., and W. Goldsmith (1950) An analysis of phenotypic reversions at the brown locus i n Drosophila. J . Exp. Zool. 114, 103-114. Hultmark, D., R. Klemenz, and W.J. Gehring (1986). Tr a n s l a t i o n a l and t r a n s c r i p t i o n a l control elements i n the untranslated leader of the heat-shock gene hsp22. C e l l 44, 429-438. Hunter, T. (1987). A thousand and one protein kinases. C e l l 50, 823-829. 220 Igarashi, M., A. Nagata, S. Jinno, K. Suto, and H. Okayama (1991). Ffeel +-like gene i n human c e l l s . Nature 353, 80-83. Jackson, D.A. (1991). Structure-function re l a t i o n s h i p s i n eukaryotic n u c l e i . Bioessays 13, 1-10. James, T.C., and S.C.R. E l g i n (1986). I d e n t i f i c a t i o n of a nonhistone chromosomal protein associated with heterochromatin i n Drosophila melanogaster and i t s gene. Mol. C e l l . B i o l . 6, 3862-3872. James, T.C., J.C. Eissenberg, C. Craig, V. D i e t r i c h , A. Hobson, and S.C.R. E l g i n (1989). D i s t r i b u t i o n patterns of HPl, a hetrochromatin-associated nonhistone chromosomal protein of Drosophila. Eur. J . C e l l B i o l . 50, 170-180. J a n n i n g , W. ( 1 9 7 0 ) . B e s t i m m u n g d e s heterochromatisierungsstadiums beim white-positionseffekt m i t t e l s rontgeninduzierter mitotischer rekombination i n der augenanlage von Drosophila melanogaster. Molec. Gen. Genet. 107, 128-149. Jimenez, J . , L. Alphey, P. Nurse, and D.M. Glover (1990). Complementation of f i s s i o n yeast cdc2tB and cdc25tB mutants i d e n t i f i e s two c e l l cycle genes from Drosophila, a cdc2 homologue and string. EMBO 9, 3565-3571. Jowett, T. (1986). Preparation of nucleic acids. In, D.B. Roberts (ed.) Drosophila, a p r a c t i c a l approach. IRL Press, Washington D.C. pp 275-286. Judd, B.H. (1955). Direct proof of a variegated-type p o s i t i o n e f f e c t at the white locus i n Drosophila melanogaster. Genetics 196, 739-744. Jurgens, G. (1985). A group of genes c o n t r o l l i n g the s p a t i a l expression of the bithorax complex i n Drosophila. Nature 316, 153-155. K a r l i k , C.C., J.W. Mahaffey, M.D. Coutu, and E.A. Fyrberg (1984). Organization of c o n t r a c t i l e protein genes within the 88F subdivision of the D. melanogaster t h i r d chromosome. Karpen, G.H., and A.C. Spradling (1990). Reduced DNA polytenization of a minichromosome region undergoing p o s i t i o n -e f f e c t variegation i n Drosophila. C e l l 63, 97-107. Kassis, J.A., C. Desplan, D.K. Wright, and P.H. O'F a r r e l l (1989). Evolutionary conservation of homeodomain-binding s i t e s and other sequences upstream and within the major t r a n s c r i p t i o n unit of the Drosophila segmentation gene engrailed. Mol. C e l l . B i o l . 9, 4304-4311. 221 Kassis, J.A., M.L. Wong, and P.H. O ' F a r r e l l (1985). Electron microscope heteroduplex mapping i d e n t i f i e s regions of the engrailed locus that are conserved between Drosophila melanogaster and Drosophila v i r i l i s . Mol. C e l l . B i o l . 5, 3600-3609. Kayne, P.S., U.-J. Kim, M. Han, J.R. Mullen, F. Yoshizaki, and M. Grunstein (1988). Extremely conserved histone H4 N terminus i s dispensible f o r growth but e s s e n t i a l f o r repressing the s i l e n t mating l o c i i n yeast. C e l l 55, 27-39. Khesin, R.B., and B.A. Bashkirov (1979). Influence of deficiency of the histone gene-containing 38B-40 region on X-chromosome template a c t i v i t y and the white gene p o s i t i o n e f f e c t variegation i n Drosophila melanogaster. Mol. Gen. Genet. 162, 323-328. Kidwell, M.G. (1986). P-M mutagenesis. In, D.B. Roberts (ed.) Drosophila, a p r a c t i c a l approach. IRL Press, Washington D.C. pp 59-81. Knighton, D.R., J . Zheng, L.F.T. Eyck, V.A. Ashford, N-H. Xuong, S.S. Taylor, and J.M. Sowadski (1991). Crys t a l structure of the c a t a l y t i c subunit of c y c l i c adenosine monophosphate-dependent protein kinase. Science 253, 407-414. Kornher, J.S., and S.A. Kauffman (1986). Variegated expression of the Sgs-4 locus i n Drosophila melanogaster. Chromosoma 94, 205-216. Krek, W., and E.A. Nigg (1989). Structure and developmental expression of the chicken CDC2 kinase. EMBO 8, 3071-3078. Labbe, J.C., A. Picard, G. Peaucellier, J.C. Cavadore, P. Nurse, and M. Doree (1989). P u r i f i c a t i o n of MPF from s t a r f i s h , i d e n t i f i c a t i o n as the HI histone kinase p34 c d c 2 and a possible mechanism fo r i t s periodic a c t i v a t i o n . C e l l 57, 253-263. Lakhotia, S.C. (1974). EM autoradiographic studies on polytene n u c l e i of Drosophila melanogaster. I I I . L o c a l i s a t i o n of non-r e p l i c a t i n g chromatin to the chromocentre heterochromatin. Chromosoma 46, 145-160. Langan, T.A., J . Gautier, M. Lokha, R. Hollingsworth, S. Moreno, P. Nurse, J . Mailer, and R.A. S c a l f a n i (1989). Mammalian growth associated histone Hi kinase, a homolog of cdc2+/CDC28 protein kinases c o n t r o l l i n g m i t o t i c entry i n yeast and frog c e l l s . Mol. C e l l . B i o l . 9, 3860-3868. Lasko, P.F., and M.L. Pardue (1988). Studies of the genetic organization of the vestigial microregion of Drosophila melanogaster. Genetics 120, 495-502. 222 Lawrence, P.A., S.M. Green, and P. Johnston (1978). Compartmentalization and growth of the Drosophila abdomen. J . Embryol. Exp. Morph. 43, 233-245. Lee, M.G., and P. Nurse (1987). Complementation used to clone a human homolog of the f i s s i o n yeast c e l l cycle c o n t r o l gene cdc2. Nature 327,680-685. Lefevre, G. J r . (1976). A photographic representation and i n t e r p r e t a t i o n of the polytene chromosome of Drosophila melanogaster s a l i v a r y glands, pp. 36-61. In, The Genetics and Biology of Drosophila, V o l . l a , Edited by M. Ashburner and E. No v i t s k i . Academic Press, New York. Lefevre, G., and W. Watkins (1986). The question of the t o t a l gene number i n Drosophila melanogaster. Genetics 113, 869-895. Lehner, C F . , and P. O ' F a r r e l l (1989). Expression and function of Drosophila c y c l i n A during embryonic c e l l cycle progression. C e l l 56, 957-968. Lehner, C F . , and P. O ' F a r r e l l (1990a). The roles of Drosophila c y c l i n s A and B i n mitoti c c o n t r o l . C e l l 61, 535-547. Lehner, C F . , and P. O'Far r e l l (1990b). Drosophila cdc2 homologs, a functional homolog i s coexpressed with a cognate variant. EMBO 9, 3573-3581. Lewis, E.B. (1978). A gene complex c o n t r o l l i n g segmentation i n Drosophila. Nature 276, 565-570. Lewis, E.B., and F. Bacher (1968). Method of feeding ethyl methanesulfonate (EMS) to Drosophila males. Drosophila Inform. Serv. 43, 193. Lindsley, D.E., L.S.B. Goldstein, and L. Sandler (1980). Male s t e r i l i t y i n maternal-effect mutants. Drosophila Inform. Ser. 55, 84-85. Lindsley, D.L., and E.H. G r e l l (1968). Genetic v a r i a t i o n s of Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627. Lindsley, D.L., and G. Zimm (1986). The genome of Drosophila melanogaster.Part 1, Genes A-K. Drosophila Inform. Ser. 64, 1-227. Lindsley, D.L., and G. Zimm (1986). The genome of Drosophila melanogaster. Part 2, l e t h a l s ; maps. Drosophila Inform. Ser. 65, 1-158. 223 Locke, J . , M.A. Kotarski, and K. D. T a r t o f f (1988). Dosage-dependent modifiers of p o s i t i o n e f f e c t variegation i n Drosophila and a mass action model that explains t h e i r e f f e c t . Genetics 1320, 181-198. Lorincz, A.T., and S.I. Reed (1986). Sequence analysis of temperature-sensitive mutations i n the Saccharomyces cerevisiae gene CDC28. Mol. C e l l B i o l . 6, 4099-4103. Madhavan, M.M., and K. Madhavan (1980). Morphogenesis of the epidermis of adult abdomen of Drosophila. J . Embryol. exp. Morph. 60, 1-31. Mahowald, A.P. (1963). Electron microscopy of the formation of the c e l l u l a r blastoderm i n Drosophila melanogaster. Exp. C e l l Res. 32, 457-468. Maniatis, T., E. R. F r i t s c h , and J . Sambrook (1982). Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Mange, A.P., and L. Sandler (1973). A note on the maternal e f f e c t mutants daughterless, and abnormal oocyte i n Drosophila melanogaster. Genetics 73, 73-86. McGhee, J.D., and G. Felsenfeld (1980). Nucleosome structure. Annu. Rev. Biochem. 49, 1115-1156. Meijer, L., D. Arion, R. Golsteyn, J. Pines, L. Brizuela, T. Hunt, and D. Beach (1989). C y c l i n i s a component of the sea urchin egg M-phase s p e c i f i c histone Hi kinase. EMBO J. 8, 2275-2282. M i c h a i l i d i s , J . , N.D. Murray, and J.A. Marshall Graves (1988). A c o r r e l a t i o n between development time and variegated p o s i t i o n - e f f e c t i n Drosophila melanogaster. Genet. Res. 52, 119-123. Miklos, G.L.G., M.J. Healy, P. Pain, A.J. Howells, and R.J. Russell (1984). Molecular and genetic stuies on the euchromatin-heterochromatin t r a n s i t i o n region of the X chromosome of Drosophila melanogaster. I. A cloned entry point near the uncoordinated (une) locus. Chromosoma 89, 218-227. Moore, G.D., J.D. Procunier, D.P. Cross, and T.A. G r i g l i a t t i (1979). Histone gene d e f i c i e n c i e s and p o s i t i o n e f f e c t variegation i n Drosophila. Nature 282, 312-314. Moore, G.D., D.A. S i n c l a i r , and T. G r i g l i a t t i (1983). Histone gene m u l t i p l i c i t y and p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster. Genetics 105, 327-344. 224 Moreno, S., J . Hayles, and P. Nurse (1989). Regulation of p34cdc2 protein kinase during mitosis. C e l l 58, 361-372. Moreno, S., and P. Nurse (1990). Substrates f o r p34 c d c 2: In vivo Veritas? C e l l 61, 549-551. Morla, A., G. Draetta, D. Beach, and J . Wang (1989). Reversible tyrosine phosphorylation of cdc2, dephosphorylation accompanies a c t i v a t i o n during entry i n t o m i t o s i s . C e l l 58, 193-203. Mottus, R., R. Reeves, and T.A. G r i g l i a t t i (1980). Butyrate suppression of p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster. Mol. Gen. Genet. 178, 465-469. Nash, D., and F.C. Janca (1983). Hypomorphic l e t h a l mutations and t h e i r implications f o r the i n t e r p r e t a t i o n of l e t h a l complementation studies i n Drosophila. Genetics 105, 957-968. Norbury, C.J., and P. Nurse (1989). Control of the higher eukaryotic c e l l cycle by p34cdc2 homologues. Biochimica et Biophysica Acta 989, 85-95. Noujdin, N.I. (1936). Genetic analysis of c e r t a i n problems of the physiology of development of Drosophila melanogaster. B i o l . Zh. (Mosk.) 4, 571-624. Nurse, P. (1990). Universal control mechanism regulating the onset of M-phase. Nature 344, 503-508. Nurse, P., and Y. B i s s e t t (1981). Gene required i n Gl for commitment to c e l l cycle and i n G2 for c o n t r o l of mitosis i n f i s s i o n yeast. Nature 292, 558-560. Nurse, P., and P. Thuriaux (1980). Regulatory genes c o n t r o l l i n g mitosis i n the f i s s i o n yeast Schizosaccharomyces pombe. Genetics 96, 627-637. Nusslein-Volhard, C , E. Wieschaus, and H. Kluding (1984). Mutation a f f e c t i n g the pattern of the l a r v a l c u t i c l e i n Drosophila melanogaster, 1. Zygotic l o c i on the second chromosome. Wilhelms Roux's Arch. Dev. B i o l . 193, 267-282. O ' F a r r e l l , P.H., B.A. Edgar, D. Lakich, and C.F. Lehner (1989). Directing c e l l d i v i s i o n during development. Science 246, 635-640. O'Hare, K., and G.M. Rubin (1983). Structures of P transposable elements and t h e i r s i t e s of i n s e r t i o n and excision i n the Drosophila melanogaster genome. C e l l 34, 25-35. 225 Paro, R., and D.S. Hogness (1991). The polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. Proc. N a t l . Acad. S c i . USA 88, 263-267. Paro, R. (1990). Imprinting a determined state i n t o the chromatin of Drosophila. Trends Genet. 6, 416-421. Perler, F., A. E f s t r a t i a d i s , P. Lomedico, W. G i l b e r t , R. Kolodner, and J . Dodgson (1980). The evolution of genes, the chicken preproinsulin gene. C e l l 20, 555-566. Picard, A., J . Cavadore, P. Lory, J . Berenengo, C. Ojeda, and M. Doree (1990). Microinjection of a conserved peptide sequence of p34 c d c 2 induces a Ca 2 + transient i n oocytes. Science 247, 327-329. Pines, J . , and T. Hunter (1989). I s o l a t i o n of a human c y c l i n cDNA, evidence f o r c y c l i n mRNA and protein regulation i n the c e l l cycle and f o r i n t e r a c t i o n with p34 c d c 2. C e l l 58, 833-846. Proudfoot, N.J., and G.G. Brownlee (1976). 3' non-coding region sequences i n eukaryotic messenger RNA. Nature 263, 211-214. Reuter, G., R. Dorn, and H.J. Hoffmann (1982). Butyrate s e n s i t i v e suppressor of p o s i t i o n - e f f e c t variegation mutations i n Drosophila melanogaster. Mol. Gen. Genet. 188, 480-485. Reuter, G., R. Dorn, G. Wustmann, B. Friede, and G. Rauh (1986). Third chromosome suppressor of p o s i t i o n - e f f e c t variegation l o c i i n Drosophila melanogaster. Mol. Gen. Genet. 202, 481-487. Reuter, G., J . Gausz, H. Gyurkovics, B. Friede, R. Bang, A. Spierer, L.M.C. H a l l , and P. Spierer (1987). Modifiers of p o s i t i o n - e f f e c t variegation i n the region from 86-88B of the Drosophila melanogaster t h i r d chromosome. Mol. Gen. Genet. 210, 429-436. Reuter, G., M. Giarre, J . Farah, J . Gausz, A. Spierer, and P. Spierer (1990). Dependence of p o s i t i o n - e f f e c t variegation i n Drosophila on dose of a gene encoding an unusual z i n c - f i n g e r protein. Nature 344, 243-244. Reuter, G., and J . Szidonya (1983). Cytogenetic analysis of variegation suppressors and a dominant temperature-sensitive l e t h a l i n region 23-26 of chromosome 2L i n Drosophila melanogaster. Chromosoma 88, 277-285. Reuter, G., W. Werner, and H.J. Hoffmann (1982). Mutants a f f e c t i n g p o s i t i o n - e f f e c t heterochromatization i n Drosophila melanogaster. Chromosoma 85, 539-551. 226 Reuter, G., and I. Wolff (1981). I s o l a t i o n of dominant suppressor mutations f o r p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster. Mol. Gen. Genet. 182, 516-519. Reuter, G., I. Wolff, and B. Friede (1985). Functional properties of the heterochromatic sequences inducing w"4 p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster. Chromosoma 93, 132-139. Richardson, H.E., C.S. Stueland, J . Thomas, P. Russell, and S.I. Reed (1990). Human cDNAs encoding homologs of the small p 3 4 c d c 2 8 - c d c 2 _ a s s o c £ a t e £ j protein of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Genes Dev. 4, 1332-1344. Robertson, H.M., C.R. Preston, R.W. P h i l l i s , D.M. Johnson-S c h l i t z , W.K. Benz, and W.R. Engels (1988). A stable source of P element transposase i n Drosophila melanogaster. Genetics 118, 461-470. Roseland, C.R., and H.A. Schneiderman (1979). Regulation and metamorphosis of the abdominal h i s t o b l a s t s of Drosophila melanogaster. Wilhelm Roux's Archives 186, 235-265. Roth, S.Y., M.P. C o l l i n i , G. Draetta, D. Beach, and C.D. A l l i s (1991). A cdc2-like kinase phosphorylates histone HI kinase i n the amitotic macronucleus of Tetrahymena. EMBO 10, 2069-2075. Roth, S.Y., A. Dean, and R.T. Simpson (1990). Yeast alpha-2 repressor positions nucleosomes i n TRP1/ARS1 chromatin. Mol. C e l l . B i o l . 10, 2247-2260. Roth, S.Y., I.G. Schulman, R. Richman, R.G. Cook, and C.D. A l l i s (1988). Characterization of phosphorylation s i t e s i n histone Hi i n the amitotic macronucleus of Tetrahymena during d i f f e r e n t p h y s i o l o g i c a l states. J . C e l l B i o l . 107, 2473-2482. Rudkin, G.T. (1969) Non-replicationg DNA i n Drosophila. Genetics (Suppl.) 61, 227-238. Rushlow, C.A., W. Bender, and A. Chovnick (1984). Studies on the mechanism of heterochromatic p o s i t i o n e f f e c t at the rosy locus of Drosophila melanogaster. Genetics 108, 603-615. Russell, P., and P. Nurse (1986). cdc25+ functions as an inducer i n the mitoti c control of f i s s i o n yeast. C e l l 45, 145-153. Russell, P., and P. Nurse (1987). Negative regulation of mitosis by weel*, a gene encoding a protein kinase homolog. C e l l 49, 559-567. 227 Russell, P., S. Moreno, and S.I. Reed (1989). Conservation of mitotic controls i n f i s s i o n and budding yeasts. C e l l 57, 295-303. Sadhu, K., S.I. Reed, H. Richardson, and P. Russell (1990). Human homolog of f i s s i o n yeast cdc25 m i t o t i c inducer i s predominantly expressed i n G2. Proc. Natl . Acad. S c i . USA 87, 5139-5143. Salas, F., and J.A. Lengyel (1984). New Mutants. Drosophila Inform. Ser. 60, 243-244. Sandler, L. (1977). Evidence f o r a set of c l o s e l y - l i n k e d autosomal genes that i n t e r a c t with sex chromosome heterochromatin i n Drosophila melanogaster. Genetics 86, 567-582. S c h l i s s e l , M.S., and D.D. Brown (1984). The t r a n s c r i p t i o n a l regulation of Xenopus 5S RNA genes i n chromatin, the roles of active stable t r a n s c r i p t i o n complexes and histone Hi. C e l l 37, 903-913. Schultz, J . (1950). Interrelations of factors a f f e c t i n g hetrochromatin-induced variegation i n Drosophila. Genetics 35, 134. Schultz, J . (1956). The r e l a t i o n of heterochromatic chromosome regions to the nucleic acid content of the c e l l . Cold Spring Harbor Symp. Quant. B i o l . 21, 307-327. Shupbach, T., and E. Wieschaus (1986). Maternal-effect mutations a l t e r i n g the anteri o r - p o s t e r i o r pattern of the Drosophila embryo. Wilhelms Roux's Arch. Dev. B i o l . 195, 302-317. Schupbach, T., and E. Wieschaus (1989). Female s t e r i l e mutations on the second chromosome of Drosophila melanogaster. I. Maternal e f f e c t mutations. Genetics 121, 101-117. Shuttleworth, J . , R. Godfrey, and A. Colman (1990). p40 M O 1 5, a cdc2-related protein kinase involved i n negative regulation of meiotic maturation i n Xenopus oocytes. EMBO 9, 3233-3240. Simon, J.A., C.A. Sutton, R.B. 1obeli, R.L. Glaser, and J.T. L i s (1985). Determinants of heat shock-induced chromosome puffing. C e l l 40, 805-817. S i n c l a i r , D.A.R., R.C. Mottus, and T.A. G r i g l i a t t i (1983). Genes which suppress p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster are clustered. Mol. Gen. Genet. 191, 326-333. 228 S i n c l a i r , D.A.R., Y.K. Lloyd, and T.A. G r i g l i a t t i (1989). Characterization of mutations that enhance p o s i t i o n e f f e c t variegation i n Drosophila melanogaster. Mol. Gen. Genet. 216, 328-333. S i n c l a i r , D.A.R., A.A. Ruddell, J.K. Brock, N.J. Clegg, V.K. Lloyd, and T.A. G r i g l i a t t i (1991). A cytogenetic and genetic characterization of a group of c l o s e l y - l i n k e d second chromosome mutations that suppress p o s i t i o n - e f f e c t variegation i n Drosophila melanogaster. Genetics, i n press. Singh, P., J.R. M i l l e r , J . Pearce, R. Kothary, R.D. Burton, R. Paro, T.C. James, and S.J. Gaunt (1991). A sequence motif found i n a Drosophila heterochromatin protein i s conserved i n animals and plants. Nucl. Acids Res. 19, 789-793. S l a t i s , H.M. (1955). P o s i t i o n e f f e c t s at the brown locus om Drosophila melanogaster. Genetics 40, 5-23. Southern, E.M. (1975). Detection of s p e c i f i c sequences among DNA fragments separated by gel electrophoresis. J . Mol. B i o l . 98, 503-517. Spierer, P., A. Spierer, W. Bender, and D.S. Hogness (1983). Molecular mapping of genetic and chromomeric units i n Drosophila melanogaster. J . Mol. B i o l . 168, 35-50. Spofford, J.B. (1976). P o s i t i o n - e f f e c t variegation i n Drosophila. In, Ashburner, M., and E. Novit s k i (eds) The genetics and biology of Drosophila, Vol. l c . Academic Press, New York, pp 955-1018. Spofford, J . (1967). Single-locus modification of p o s i t i o n -e f f e c t variegation i n Drosophila melanogaster. I. White variegation. Genetics 57, 751-766. Spofford, J . (1969). Single-locus modification of p o s i t i o n -e f f e c t variegation i n Drosophila melanogaster. I I . Region 3C l o c i . Genetics 62, 555-571. Spradling, A.C., and G.H. Karpen (1990) S i x t y years of mystery. Genetics 126, 779-784. Spradling, A.C., and G.M. Rubin (1981). Drosophila genome organization: conserved and dynamic aspects. Annu. Rev. Genet. 15, 219-264. Stern, C , and M. Kodani (1955). Studies on the p o s i t i o n e f f e c t at the cubitus interruptus locus of Drosophila melanogaster. Genetics 40, 343-373. 229 Strausfeld, U., J.C. Labbe, D. Fesguet, J.C. Cavadore, A. Picard, K. Sadhu, P. Russell, and M. Doree (1991). Dephosphorylation and a c t i v a t i o n of a p3 4 c d c 2 / c y c l i n B complex in vitro by human CDC25 protein. Nature 351, 242-245. S u l l i v a n , W., J.S. Minden, and B.M. Alberts (1990). daughterless-abo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the l a t e blastoderm d i v i s i o n s . Development 110, 311-323. Surana, U., H. Robitsch, C. Price, T. Schuster, I. F i t c h , A.B. Futcher, and K. Nasmyth (1991). The r o l e of CDC28 and c y c l i n s during mitosis i n the budding yeast S. c e r e v i s i a e . C e l l 65, 145-161. Suzuki, M. (1989). SPXX, a frequent sequence motif i n gene regulatory proteins. J . Mol. B i o l . 207, 61-84. Szabad, J . , G. Reuter, and M-B. Schroeder (1988). The e f f e c t s of two mutations connected with chromatin function on female germ-line c e l l s of Drosophila melanogaster. Mol. Gen. Genet. 211, 56-62. Szidonya, J . , and G. Reuter (1988). Cytogenetic analysis of the echnoid (ed), dumpy (dp) and clot (cl) region i n Drosophila melanogaster. Genet. Res., Camb. 51, 197-208. Tabor, S., and C.C. Richardson (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl . Acad. S c i . USA 84, 4767-4771. Tartof, K.D., C. Bishop, M. Jones, C.A. Hobbs, and J . Locke (1989). Towards an understanding of p o s i t i o n e f f e c t variegation. Dev. Genet. 10, 162-176. Tartof, K.D., C. Hobbs, and M. Jones (1984). A s t r u c t u r a l basis f o r variegating p o s i t i o n e f f e c t s . C e l l 37, 869-878. Wakimoto, B.T., and M.G. Hearn (1990). The e f f e c t s of chromosome rearrangements on the expression of heterochromatic genes i n chromosome 2L of Drosophila melanogaster. Genetics 125, 141-154. Whit f i e l d , J.M., G.F. Gonzalez, E. Sanchez-Herrero, and D.M. Glover (1989). Transcripts of one of the two Drosophila c y c l i n genes become l o c a l i z e d i n pole c e l l s during embryogenesis. Nature 338, 337-340. Whiting, J.H. J r . , J.L. Farmer, and D.E. J e f f e r y (1987). Improved in situ hybridization and detection of b i o t i n - l a b e l e d D. melanogaster DNA probes hybridized to D. v i r i l i s s a l i v a r y gland chromosomes. Drosophila Inform. Ser. 66, 170-171. 230 Wood, W.B. (1988). The nematode Caenorhabditis elegans. p p . l -667. Cold Spring Harbor Laboratory. Workman, J.L., and R.G. Roeder (1987). Binding of t r a n s c r i p t i o n factor TFIIID to the major l a t e promoter during in vitro nucleosome assembly potentiates subsequent i n i t i a t i o n by RNA polymerase I I . C e l l 51, 613-622. Wustmann, G., J . Szidonya, H. Taubert, and G. Reuter (1989). The genetics of p o s i t i o n - e f f e c t variegation modifying l o c i i n Drosophila melanogaster. Mol. Gen. Genet. 217, 520-527. Yoon, J.S., R.H. Richardson, and M.R. Wheeler (1973). A technique f o r improving s a l i v a r y chromosome preparations. Experientia 29, 639-641. Zhang, P., and R.S. Hawley (1990). The genetic analysis of d i s t r i b u t i v e segregation i n Drosophila melanogaster. I I . Further genetic analysis of the nod locus. Genetics 125, 115-127 Zink, B., and R. Paro (1989). In vivo binding pattern of a trans-regulator of homeotic genes i n Drosophila melanogaster. Nature 337, 468-471. Zuckerkandl, E. (1974). Recherches sur l e s propriétés et l'activité biologique de l a chromatine. Biochimie 56, 937-954. 231 

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