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Genetic analysis of the proximal heterochromatin of chromosome-2 of Drosophila melanogaster Hilliker, Arthur James 1975

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GENETIC ANALYSIS OF THE PROXIMAL •HETEROCHROMATIN OF CHROMOSOME^ OF DROSOPHILA MELANOGASTER by ARTHUR JAMES HILLIKER B.Sc, University of British Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it • f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permission. Department of ^ Q o l o ^ The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook"Place Vancouver, Canada V6T 1W5 Date ABSTRACT The genetic function of Drosophila heterochromatin has been debated since i t s earliest description by Heitz (1933). To examine the genetic composition of the proximal region of chromosome 2 of Drosophila melanogaster, the generation of proximal deficiencies by the detachment of compound second autosomes appeared to be a promising method. Compound second autosomes were detached by gamma radiation. A fraction of the detachment products were recessive lethals owing to proximal deficiencies. Analysis of these detachment products by inter §k complementation, pseudo-dominance tests with proximal mutations and alleleism tests with known deficiencies, provided evidence for at least two l o c i between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. These data in conjunction with cytological observations further demonstrate that rolled and light are located within the proximal heterochromatin of the second chromosome. To further this analysis, lethal alleles of the largest 2L and 2R proximal deficiencies were generated, employing, as a mutagen, ethyl methane sulphonate (EMS). Analysis of the 118 EMS induced recessive lethals and visible mutations recovered provided evidence for seven l o c i in the 2L heterochromatin and six l o c i in the 2R heterochromatin, with multiple alleles being obtained for most sites. Of these l o c i , one in 2L and two in 2R f a l l near the heterochromatic-euchromatic junction of 2L and 2R respectively. None of the 113 EMS lethals behaved as a deficiency, thereby confirming that, in Drosophila, the EMS mutagenesis method of Lewis and Bacher (1968) results in true "point" mutations. A l l of the heterochromatic l o c i uncovered in this study appear to be non-repetitive cistrons. Thus functional genetic l o c i are found in heterochromatin, albeit at very low density relative to euchromatin. TABLE OF CONTENTS ABSTRACT . i TABLE OF CONTENTS i i LIST OF TABLES . v LIST OF FIGURES . . v i ACKNOWLEDGEMENT . . . • ix CHAPTER I. GENERAL INTRODUCTION 1 CHAPTER II. ANALYSIS OF DETACHMENT PRODUCTS OF COMPOUND SECOND AUTOSOMES Introduction 9 Materials and Methods 10 Mutations and chromosome rearrangements 10 Compound autosomes 10 Recovery of compound-2 detachment products .... 10 Complementation tests on lethal detachments ... 12 Cytological analysis 13 Origin of compound second autosome detachments 14 Results and Discussion 17 Detachment products of C(2L)SH3,+; C(2R)SH3,+ 18 Detachment products of C(2L)SH3,+: C(2R)VKl,Dp(2L)lt+bw 32 Detachment products of C(2L)SHl,Dp(2R)rl+/+; C S H 3 ? H ~ » ••• 39 CHAPTER III. ANALYSIS OF EMS INDUCED LETHAL ALLELES OF SECOND CtTRO^ MO SO^ E^ DEFICIENC IE S PAGE Materials and Methods .-46 Generation of lethal alleles of Df(2R)M-S2lO 46 Generation of lethal alleles of Df(2L)C' 47 Results and Discussion 48 Lethal alleles of Df(2R)M-S210 48 Lethal alleles of Df(2L)C' 73 CHAPTER IV. GENERAL DISCUSSION 89 LITERATURE CITED 95 APPENDIX I. ON THE NATURE OF POSITION-EFFECT VARIEGATION OF THE LIGHT LOCUS 100 APPENDIX II. AN IMPROVED TECHNIQUE FOR POLYTENE CHROMOSOME SQUASH PREPARATIONS IN DROSOPHILA Introduction 106 Materials and Methods 106 Results 107 Discussion 108 APPENDIX III. PREPARATION OF SOMATIC MITOSES OF DROSOPHILA AND CHROMOSOME IDENTIFICATION Chromosome preparation 110 Chromosome identification I l l APPENDIX IV. THE LOCATION OF THE ROUGHISH LOCUS 114 APPENDIX V. PRELIMINARY EXPERIMENTS ON THE EFFECTS OF HETEROZYGOSITY FOR SECOND CHROMOSOME PROXIMAL DEFICIENCIES ON MEIOSIS 118' PAGE APPENDIX VI. HETEROCHROMATIC DUPLICATIONS AND THE MEIOTIC SEGREGATION OF COMPOUND AUTOSOMES IN MALE DROSOPHILA 124 LIST OF TABLES TABLE CHAPTER II I. Recessive lethal and visible mutations used as genetic markers on compound autosomes and for complementation analysis. 11 II. Estimated frequency of putative reconstituted standard second chromosomes recovered as detachment products from compound-^ females. 15 III. Distribution of putative reconstituted standard seconds from compound-2, females treated with gamma radiation. 16 IV. The frequency of homozygous lethality associated with chromosomes recovered as detachment products from three different strains of gamma-radiation treated compound-2 females. 19 APPENDIX IV I. Exceptional progeny recovered from irradiated rh/rh virgin females crossed to C(2L)P,b;C(2R)VHKl,rl cn. 117 APPENDIX VI I. Progeny of B^Y; C(2L)P,b; C(2R)P > Px females and various compound second autosome bearing males. 127 II. Frequency of progeny nonsegregational for compound second autosomes from B^Y; C(2L)P,b; C(2R)P,px virgin females crossed to various strains of compound second autosome bearing males. 128 LIST OF FIGURES FIGURE-CHAPTER II 1. Hypothetical model depicting representative genotypes of reciprocal detachment products expected from inter-changes between C(2L) and C(2R) chromosomes that carry neither deficiencies nor duplications. 20 2. Genetic map of the centric region of chromosome 2 showing the relative positions and lengths of proximal deficiencies associated with reconstituted standard seconds generated as products of interchange between C(2L)SH3,+ and C(2R)SH3,+. 24 3. Photomicrograph of mitotic prometaphase chromosomes in a larval ganglion c e l l from a +/Dpk"2R)M-S210 heterozygote. 27 4. Hypothetical model depicting representative genotypes of reciprocal products expected from interchanges between a C(2L) that is genetically an isochromosome and a C(2R) that is heterozygous for a duplication of the most proximal segment in 2L. 33 5. Genetic map of the centric region of chromosome-2 showing the relative positions and lengths of proximal deficiencies associated with reconstituted standard seconds generated as products of interchange between C(2L)SH3,+ and C(2R)VKl,Dp(2L)lt+bw/bw. 36 FIGURE ? A G E 6. Compilation of the genetic maps given in Figures 2 and 5. 40 CHAPTER III 1. Distribution by complementation with 2R proximal deficiencies of EMS induced lethal alleles of Df(2R)M-S21Q. 50 2. Complementation map. of the Group I lethal alleles of Df(2R)M-S210. 52 3. Photograph of a third instar larva, homozygous for the Group B 2R proximal deficiency. 54 4. Complementation map of the Group II lethal alleles of . Df(2R)M-S210. 58 5. Complementation map of the Group III. lethal alleles of Df(2R)M-S21Q. 61 6. Complementation map of the Group IV lethal alleles of Df(2R)M-S210. 64 7. Complementation map of the Group V lethal alleles of Df(2R)M-S210. 67 8. Distribution by complementation with. 2L proximal deficiencies of EMS induced lethal alleles of Df(2L)C f. 74 9. Complementation map of the Group VI lethal alleles of Df(2L)C . 77 FIGURE PAGE 10. Complementation map of the Group VII lethal alleles of Df(2L)C. 79 11. Complementation map of the Group VIII lethal alleles of Df (2L)C . 82 12. Complementation map of the Group IX lethal alleles of Df (2L)C • 85 ACKNOWLEDGMENT I would like to thank Dr. David G. Holm for his support of.and interest in the present study. Thanks must also go to Dr. C. V. Finnegan for the use of his laboratory f a c i l i t i e s , especially his excellent photomicroscope, and to Mr. Brian G. MacLean for his advice in matters cytological and photographic. Finally I greatly appreciate the friendship and courtesy of those previously or presently associated with Dr. Holm's laboratory. CHAPTER I GENERAL INTRODUCTION The Biological function of heterochromatin has been debated since i t s f i r s t clear description by Heitz (1928, 1929). This debate became one of broad interest with the demonstration of the ubiquity of heterochromatin among plants and animals (Brown 1966) . Nineteenth century cytologists, in dissecting the mitotic cycle, demonstrated that after nuclear division chromosomes unravelled (decondensed) during the formation of the interphase nucleus. Thus most of the chromosomal material was condensed only for part of the c e l l cycle, namely prophase through anaphase, and in telophase began to decondense. At interphase the chromosomes could not be resolved. However, in these interphase nuclei, bodies of dark staining material were often observed. Heitz demonstrated that these darkly staining bodies corresponded to chromatin which remained condensed throughout the c e l l cycle. This permanently condensed chromatin Heitz termed "heterochromatin". In interphase nuclei the heterochromatic blocks were referred to as "chromocenters". Often chromocenters fused to form one or several large composite chromocenters. Since Heitz was of the opinion that the chromosomal genes could be active only in noncondensed chromatin, he suggested that heterochromatic regions were genetically inert. A number of cytological properties are diagnostic of heterochromatin. In most animal species heterochromatin is pericentromeric, while in plants terminal heterochromatin i s common. Further, throughout prophase heterochromatic regions are considerably denser than the remaining euchromatin. Homologous chromosomes are homologous in their sites of heterochromatin, which remain constant. Finally, hetero-chromatic regions exhibit the property of chromatid apposition during mitotic prophase. The heterochromatin described above i s "cl a s s i c a l " or "constitutive" heterochromatin (Brown 1966). A second type of heterochromatin termed "facultative" i s that associated with chromosomal inactivation through condensation of one of a pair of homologous chromosomes in the soma or of a univalent in one c e l l type as, for example, the condensation of the X-chromosome during spermatogenesis in some insects. Throughout the following text the term heterochromatin i s used s t r i c t l y in reference to constitutive heterochromatin. From their cytogenetic studies on Drosophila melanogaster Heitz (1933) and Kaufmann (1934) facili t a t e d inference as to the genetic constitution of heterochromatin. They found that the entire Y-chromosome, the proximal 1/3 to 1/2 of the .X-chromosome, and the proximal 1/4 of each of the arms of the metacentric major autosomes, chromosomes 2 and 3, were heterochromatic at prometaphase. With respect to the Y-chromosome these observations were of considerable significance as the earlier studies of Bridges (1916) had demonstrated an apparent lack of genes in the Y-chromosome. Bridges and his colleagues discovered no instance of a Y-lihked gene, and, further-, Bridges found that nullo-Y males (XO) were of perfectly normal phenotype, v i a b i l i t y and behaviour, but s t e r i l e . Thus i t appeared that the completely heterochromatic Y-chromosome, although larger than the X-chromosome, for which many genetic l o c i were known, contained no genes. However, the description of the Y-chromosome as completely hetero-chromatic and genetically inert was to be modified. Several secondary constrictions were described in the Y-chromosome (reviewed in Cooper 1959). Further, as Bridges' (1916) discovery of the s t e r i l i t y of XO males anticipated, specific f e r t i l i t y factors were subsequently revealed (reviewed in Brosseau 1960 and Hess and Meyer 1968). The numbers of genetic sites (nucleolus organizer plus f e r t i l i t y factors equalling 8) and secondary constrictions (7-9) are in close agreement, suggesting that the secondary constrictions correspond to Y-chromosome genetic l o c i . Hess and Meyer's (1968) findings in Drosophila hydei spermatogonia of lampbrush chromosome loops at the sites of Y-chromosome secondary constrictions strengthens this hypothesis. Thus, although the hetero-chromatic Y-chromosome does contain genetic l o c i , these l o c i would appear to reside at the sites of the secondary constrictions and, therefore, are not truly heterochromatic. Similarly, Heitz's and Kaufmann's (loc. cit.) cytological findings were, in the light of Heitz's speculation of the genetic inertness of heterochromatin, in agreement with the work of Muller and Painter (1932) on gene localization in the X-chromosome of Drosophila melanogaster. Muller and Painter demonstrated that few i f any gene l o c i were located in the proximal third of"the X-chromosome, precisely that region which Heitz and Kaufmann found to be heterochromatic. The recent detailed fine structure analysis of the X-chromosome proximal region by Schalet and Lefevre (1973) demonstrates that only one locus, bobbed (bb) (which was previously known to befin heterochromatin), i s definitely in the X-chromosome heterochromatin. The bobbed locus has been identified as the nucleolar organizer of the X-chromosome (Ritossa, Atwood and Spiegelman 1966) and is associated with a prominent secondary constriction. Secondary c o n s t r i c t i o n s , i n addition to the nucleolar c o n s t r i c t i o n , have been reported for the X-chromosome proximal heterochromatin (Cooper 1959) and these may correspond to further l o c i , which are as of yet undefined. L i t t l e i s known of the genetic c o n s t i t u t i o n of the heterochromatic segments of the major autosomes of Drosophila, although i t has been speculated that l i g h t (Schultz 1936) and r o l l e d (Morgan, Schultz and Curry 1940) are i n the proximal heterochromatin of chromosome-2. In addition to permanent heteropycnosis, there are other long established differences between heterochromatin and euchromatin i n Drosophila. L i t t l e or no recombination occurs i n Drosophila hetero-chromatin (Baker 1958). Further, cytophotometric data (Rudkin 1969) confirms the e a r l i e r conclusion of Heitz (1933) that heterochromatin does not r e p l i c a t e during polytene chromosome formation. Drosophila heterochromatin i n i t i a t e s DNA r e p l i c a t i o n much l a t e r i n the S phase of the c e l l cycle than euchromatin (Barigozzi et a l . 1966). In f a c t , DNA synthesis l a t e i n the S phase of the c e l l cycle i s c h a r a c t e r i s t i c of heterochromatin i n a l l species (Lima-de-Faria and Jaworska 1968). Modern biochemical and cytochemical studies have demonstrated that, as for most animal species, Drosophila heterochromatin i s enriched i n highly r e p e t i t i v e DNA (Botchan et a l . 1971; G a l l et a l . 1971; Peacock et a l . 1973). In most animal species heterochromatin i s enriched i n highly r e p e t i t i v e DNA; the Chinese hamster (Cricetulus griseus) i s the only known exception (Comings and Mattoccia 1972; A r r i g h i et a l . 1974). The r e p e t i t i v e sequences are often very short i n length, of the order of magnitude of 10 nucleotides, and thus i n a l l p r o b a b i l i t y would not be transcribed (Yunis and Yasmineh 1971) . In Drosophila melanogaster there are seven highly r e p e t i t i v e DNA sequences which make up the bulk of the c o n s t i t u t i v e heterochromatin of t h i s species (Peacock et a l . 1973,: Sederoff e_t a l . 1975), one of these sequences being an AGAAG pentamer, which i s l o c a l i z e d to the heterochromatin of the second and Y chromosomes. In assigning functions to heterochromatin, b i o l o g i s t s have been highly imaginative. The phenomenon of p o s i t i o n - e f f e c t v a r i e g a t i o n (reviewed i n Lewis 1950; Hannah 1951; Baker 1968) leads many to postulate.the existence,within heterochromatin, of regulatory genes which a f f e c t neighbouring l o c i . However, the mechanism of p o s i t i o n -e f f e c t variegation remains unclear. Others have postulated that heterochromatin plays a r o l e i n chromosome p a i r i n g and segregation during meiosis (reviewed i n Yunis and Yasmineh 1971), or that p e r i -centromeric heterochromatin protects the centromere.during nuclear d i v i s i o n (Walker 1971) . While these and many other functions have been proposed (reviewed i n Hannah 1951.; Cooper 1959;. Yunis and Yasmineh 1971), there i s l i t t l e , i f any, evidence to support any of the postulated fucntions. While the experimental findings summarized above appear to demonstrate that heterochromatin i s g e n e t i c a l l y i n e r t , a conclusion that i s today r e c e i v i n g wide acceptance, the evidence remains consistent with an hypothesis of a very low gene density i n heterochromatin r e l a t i v e to euchromatin, as no sing l e heterochromatic chromosome region has been subjected to a t r u l y thorough genetic and cytogenetic a n a l y s i s . The obvious choice of organism i n which to attempt such a complete genetic analysis of a heterochromatic region i s Drosophila melanogaster, whose genetics and, in a qualified sense, cytogenetics are more complete than any other higher organism's. In a thorough analysis of a heterochromatic chromosome segment there are a number of things one would want to accomplish. F i r s t , one would want to demonstrate the number and physical location of genes associated with that heterochromatic segment. One would want to know whether these genes are actually in or merely adjacent to heterochromatin (i.e. at the heterochromatic-euchromatic junction) and i f there is any association of these genes with secondary constrictions. In addition, one would want to investigate the nature of these l o c i in terms of whether they are repetitive or nonrepetitive sites and of their involve-ment in development. Further, one would want to investigate the effect of deletions and duplications for a l l or part of this heterochromatic segment upon meiosis, f oriit has been suggested that heterochromatin may bring homologous chromosomes together during meiosis and that centromeric heterochromatin may.protect the centromere from, breakage during the course of the meiotic division; thus predicting that heterozygosity for.partial or complete deficiencies of a centromeric heterochromatic segment may result in elevated rates of nondisjunction or chromosome loss. Finally, the thorough genetic analysis of such a heterochromatic chromosome segment should provide the material to investigate definitively whether or not recombination occurs in heterochromatin. The heterochromatic region I have chosen to analyze is the proximal region of chromosome-2 of Drosophila melanogaster. CHAPTER II ANALYSIS OF DETACHMENT PRODUCTS OF COMPOUND SECOND AUTOSOMES INTRODUCTION As an i n i t i a l approach towards examining the genetic composition of the proximal heterochromatin of chromosome-2 in Drosophila melanogaster, the detachment of compound autosomes appeared a promising method for generating proximal deficiencies. It has been well documented that detachments of compound-X chromosomes are generated by interchanges " between heterologous chromosomes (Parker 1954; Abrahamson, Herskowitz and Muller 1956; Parker and Hammond 1958; Parker and Williamson 1970). Baldwin and Suzuki (1971) , upon finding proximally located recessive lethals associated with standard thirds recovered from radiation treated compound-3-bearing females, proposed that radiation-induced detachments of compound autosomes provided a means of generating autosomal proximal deficiencies. I was cautioned, however, by the fact that Parker (1954) noted a considerable variation in lethal frequencies associated with detachments from various attached-X stocks, suggesting that some compound-X chromosomes had accumulated spontaneous lethals. He commented that "these spontaneous lethals probably accumulate near the centromere where homozygosis frequency i s low and selection relatively ineffective". Nevertheless, I found that through using homogeneous populations of compound autosomes, proximal deficiencies could be identified as products of compound-2L and compound-2R detachments. Moreover, by using the deficiencies so generated, a number of genetic l o c i have been localized to the centric heterochromatin of chromosome-2. MATERIALS AND METHODS Mutations and chromosome rearrangements: A brief description of the mutations used in this study is provided in Table I. The two multiple-break inversions employed as balancers for the reconstituted chromosomes were 1) In(2LR)bwVI; an inverted second chromosome identified by a dominant brown-variegated (bwVI) eye phenotype and 2) In(2LR)SMl.Cy; an effective balancer for the entire length of chromosome-2, marked by a dominant curly wing (Cy) phenotype. Further details on the above mutants and rearrangements are given by Lindsley and Grell (1968) . Compound autosomes: Compound-2 strains used include: 1) C(2L)SH3,+;C(2R)SH3,+, 2) C(2L)SH1,+;C(2R)SR3,+, 3) C(2L)SH3,+; C(2R)VKl,bw, 4) C(2L)VYl,b pr;C(2R)Pl,px. Each newly generated C(2L) or C(2R) chromosome is assigned an alphanumeric code and established in an independent line. The f i r s t code letter identifies the place of origin (i.e. P = Pasadena; S = Storrs; V = Vancouver). Pertinent genetic properties of the compound autosomes used in this study are described under related headings in the RESULTS AND DISCUSSION section of this chapter. Recovery of compound-2 detachment products: Compound-2 virgin females were treated with 0 (controls) or 2000 rads of gamma radiation from a 6°Co source and crossed to heterozygous In(2LR)bwVI/In(2LR)SMI,Cy males. Rare surviving progeny either with a brown variegated eye (bw^) or with a curly wing (Cy_) phenotype were scored as putative detachment products (i.e. reconstituted standard second chromosomes) and individually mated to In(2LR)bwVI/In(2LR)SMI,Cy f l i e s of the opposite sex. From each line crosses were made between F2 virgin females and males, each heterozygous Recessive lethal and visible mutations used as genetic markers on compound autosomes and for complementation analysis. Symbol b pr Bl esc 1(2)ova tvl It vl M(2)S10 M(2)S8 M(2)S4 stw ap msf pk tuf ltd on bw Map position 2-48.5 2-54.5 2-54.8 2-54.9 2-55 2-55 2-55.0 2-55.1 2^55.1 2-55.1 2.55.1-.2 2-55.1 2-55.2 2-55.2 2-55.2 2-55.5 2-56 2-57.5 2-104.5 .Chromosome arm ,2L 2L 2L 2L 2L 2L 2L 2R 2R 2R 2R 2R 2R 2R 2R 2R 2R 2R 2R .Phenotype black body purple eyes short bristles, homozygous lethal extra sex combs on male homozygous lethal dark streak on thorax yellowish-pink eyes rolled wing edges thin bristled Minute, homozygous lethal, deficient for r l Minute, homozygous lethal and with M(2)S10 and M(2)S4, deficient for stw. Minute, homozygous lethal and with M(2)S10 deficient for stw and ap yellow hairs, pale br i s t l e tips wings missing wings short, crumpled, legs short, eyes misformed anteriorly slanted costal hairs tuft of hairs between antennae and eyes yellowish-pink eyes bright red eyes, colorless o c e l l i brown eyes for the putative detachment and In(2LR)SMl,Cy. Lethal and semi-lethal detachments were identified in the F3 progeny and maintained as balanced heterozygotes over In(2LR)SMl,Cy. Following careful examination of their phenotype, fully viable homozygous detachments were discarded. Complementation tests on lethal detachments: A l l lethal detachments recovered from C(2L)SH3, + ;C(2R)SH3,+ and C(2L)SH3, + ;C(2R)VKt,bw "treated females (Experiments 1 and 2 respectively under Results) were tested for complementation in a l l possible inter se combinations and against the proximal mutations and deficiencies li s t e d in Table 1. (Lethal detachments were also tested against the recessive visible roughish, rh. However, the reported location of rh at the.base of 2L proved to be erroneous. For details see Appendix IV.) In addition, a l l lethal-bearing detachments were tested genetically for X;2, 2;3 and 2;4 translocations. Each complementation test involved 2 or 3 pairs of parents brooded for four days in shell vials containing standard Drosophila medium. Reciprocal.crosses were made in a number of tests. Progeny were screened for 20 days following the f i r s t observed eclosions. Since a l l recessive lethal detachments were balanced over In(2LR)SMl, which carried the dominant Curly (Cy_) wing marker, the criterion for complementation 1^  5^ ^^^^  A l l experiments were performed at 25^C Cytological analysis: Where described, polytene chromosomes from salivary glands and mitotic chromosomes from larval ganglia were prepared by aceto-lacto-orcein squash techniques (Appendices II and III). Photomicrographs were prepared using a Zeiss photomicroscope equipped with phase contrast optics. Origin of compound second autosome detachments: The data in Tables 2 and 3 are from a series of i n i t i a l experiments conducted to examine the source and expected recovery of reconstituted, standard second chromosomes from compound-2 female parents. Notable recoveries of reconstituted autosomes have been, obtained as apparent induced detachment products from gamma- or X-ray treated compound-2 (Bateman 1968, H i l l i k e r 1972) and compound-3 (Chovnick et a l . 1970, Baldwin and Suzuki 1971) females. However, I viewed these tests as essential to my analysis of detachment products since although spontaneous compound-autosome revertants have been reported (Leigh and Sobels 1970), their frequency of formation is unknown. For these tests I used C(2L)VY,b pr;C(2R)P,px virgin females aged 72 + 24 hours. They were divided into four groups, the f i r s t group (the control) received 0 rads, two groups received 2000 rads of gamma radiation, and the fourth group, 4500 rads. For the control and two treatment groups (2000 and 4500 rads), single females were placed in vials with two In(2LR)SMI,Cy/In(2LR)bwV1 males. For the remaining treatment group (2000 rads), two females were placed in each mating v i a l . Since only products of exceptional meiotic events w i l l be obtained from such a cross, an estimated relative productivity of these females was gained through mating approximately 10 percent of the females from each (treatment and control) group to their compound-2^  brothers. After seven days of egg laying, parents were discarded. F l progeny were counted and classified, and where necessary, F l females were tested for triploidy. Vials not yielding progeny were examined for the presence of f e r t i l i z e d eggs. The estimated frequencies of detachment were based on the following argument. Segregation of C(2L) and C(2R) during meiosis in females •. approaches 100 percent in the absence of heterologous rearrangements, whereas in males, as inferred from genetic and egg hatch studies, compound-autosome assortment is approximately random (Holm et at. 1968, Grell 1970, Holm and Chovnick 1975). Consequently, only one quarter of the f e r t i l i z e d eggs in the multiplier vials (i.e. the compound by compound crosses) are expected to produce viable progeny. In contrast, when compound-bearing females are crossed to standard males, an egg pronucleus containing a reconstituted second chromosome w i l l have, in theory, a 100 percent chance of recovery. The estimated frequencies of recoverable detachments, therefore, were calculated by dividing the number of putative reversions by the product of four times the mean number of progeny per multiplier v i a l times the number of experimental vials containing f e r t i l i z e d eggs. These results are presented (in Table II)primarily to provide a relative comparison between the frequencies of spontaneous and radiation induced events. It i s evident that the vast majority of .reconstituted autosomes recovered arose as products of radiation induced detachments. In addition, as shown in Table III the recovery of reversion products followed a Poisson distribution in the radiation experiments, thereby implying that, for the most part, each compound-compound interchange product arose independently during meiosis. )• Estimated frequency.of putative reconstituted standard second chromosomes recovered .as .detachment .-products from compound-2 females. Number of Number of Estimated Number '-Estimated Treatment progeny per ' vials total detachment .frequency of in radss multiplier containing number of products detachments v i a l ± S.E. f e r t i l i z e d zygotes recovered in percent eggs  0 27.4311.12 1683 185,000 4 0.002 2000 23.47±1.36 1104 104,000 179 0.17 2000* 32.30±0.53 935 121,000 259 0.21 4500 23.1611.22 1392 129,000 332 0.26 *Two female parents per mating v i a l TABLE III Distribution of putative reconstituted standard seconds from compound-2 females treated with gamma-radiation. Goodness:; of f i t P a 2 ) * 2000 1 Expected 938.89 152.23 12.34 0.68 0.0112 .9 Observed 940. 151. 11. 2. 2000 2 Expected 708.87 196.36 27.20 2.51 0.1239 .5 Observed 708. 196. 30. 1. 4500 1 Expected 1086.9 259.23 30.91 2.458 1.9591 .1 Observed 1103 249 37 3 Female Number Progeny per v i a l Dose ^ j. (fads)' P a r e n t s o f v ;- per v i a l vials 0 ,1 2 ..3 Classes 2 and 3 were pooled for the X2 test (df=l) . RESULTS AND DISCUSSION An Interesting parallel can be drawn between formation.and detachment of compound autosomes. Studies on the detachment of compound-X chromosomes disclose that interchange between heterologous chromatids is responsible for the separation of attached arms (Parker 1969). The interchange i s , in essence, a translocation event followed by the segregation of reciprocal products; as a consequence, thereof, one half of the translocation product is recovered. While, classically, compound chromosomes, especially in plants, were viewed as isochromosomes arising from centromeric misdivision (for example see Darlington 1939, 1940) - a view acknowledged by Bateman (1968) as a possible source of compound autosome formation in Drosophila - we have since discovered that many newly generated compound-2 chromosomes carry duplications for the most proximally known markers on the opposite arm (see Appendix IV). From this we infer that several compound autosomes are also heterozygous for proximal deficiencies. Taken together, the above observations support the notion that both the formation and the detachment of compound chromosomes arise from interchanges between heterologous chromatids and provide the frame of reference for the models offered in explaining the results that follow. In separate experiments, virgin females from three different compound-2_ lines were treated with 2000 rads of ^Co radiation and mated with VI In(2LR)bw /ln(2LR)SMl,Cy males. Each putative detachment product recovered in a f e r t i l e individual was tested for homozygous lethality (see Materials and Methods for details). The results of these tests, presented in Table IV, show a wide range in the proportion of lethal-bearing detachments recovered from the three separate lines. These differences (from approximately .25% in Experiment 2 to almost 100% in Experiment 3) w i l l derive greater significance upon relating the distribution of lethals to the distribution of the break points involved in the interchanges and to the nature of the compound autosomes from which the detachment products were obtained. Detachment products of C(2L)SH3.+:C(2R)SH3.+: Before examining the results of Experiment 1, let us consider the consequence of an interchange between a C(2L) and a C(2R) which carry neither duplications nor deficiencies for proximal genetic l o c i . Such a pair of compound autosomes is diagrammed in Figure 1. Consider that the markers a b on C(2L) and c d on C(2R) indicate the normal sequence corresponding to the standard second from which the compounds were generated. Consider, further, that deficiencies for these l o c i are recessive lethals (or possibly recessive visibles) but that these l o c i f a l l within a region for which partial trisomy has l i t t l e or no apparent effect on v i a b i l i t y . If we view the recovery of reconstituted standard seconds as. one half the products of a translocation (or interchange) between radiation induced chromatid breaks on C(2L) and C(2R) and, moreover, i f we consider that within any series breaks are randomly distributed between or adjacent to the markers a. and d on C(2L) and C(2R) respectively, a population of interchange products w i l l be heterozygous for varying degrees of segmental aneuploidy (i.e. duplications and deficiencies) in the proximal l e f t and right arms. In Figure 1 the l e f t arm of chromosome-2 is The frequency of homozygous lethality associated with chromosomes recovered as detachment products from three different strains of gamma-radiation treated compound-2 females. Total Homozygous lethals Experiment L o m P o u n c l detachments autosomes 1 C(2L)SH33+; 83 " 36 43.4 C(2R)SH33+. 2 C(2L)SH33+; 110 27 24.5 C(2R)VKl3 Dp (2L)lt+bw/bw. 3 C(2l)SEl3Dp(2R)vl~r/+; C(2R)SH33+. 60 59 98.3 FIGURE 1 ¥ Hypothetical model depicting representative genotypes of reciprocal detachment products expected from interchanges between C(2L) and C(2R) chromosomes that carry neither deficiencies nor duplications for proximal genetic l o c i . a b • + - — i -C(2L) K-2L K-2R c d H H C(2R) a b 1 2 3 Regions of Interchange 3 - 4 3 - 5 .2 - 4 1 - 5 c~d~ 4 5 6 Interchange Products a bK-2Lc d • ~ <—t-A a bK-2Rc d —H—*~T°—1— ? f ^ . ^ a b cK-2Rc d ~ * — h T H — 0 — 1 — — a bK-2Lb c d — — i — i — © — t — — < — t -aK-2Rc d - — t - j - O — i — i — -a b K-2L b a d —I 1—9 1 1 , I cK-2Rc d H O 1 h-Genotype Normal Normal Def(2R)c Dup(2R)c Dup(2L)b Def(2L)b Dup(2L)ab: Def(2R)c Def(2L)ab: Dup(2R)c divided into three regions as defined by the markers a and b, with region 3 designating, that segment of the chromosome between the most proximal locus and the centromere (K-2L) of C(2L)v The :right arm is similarly divided into three regions with 4 designating the most proximal segment. The lower portion of Figure 1 depicts representative interchange products one might anticipate when the acentric fragment from one compound fused with the centric free arm of the complementary compound autosome. When the interchange occurs between breaks in regions 3 and 4, the reciprocal products w i l l differ only in the source of the centromere (either K-2L or K-2R) and should be homozygous viable. If, however, the break is proximal in one compound autosome, for example region 3 in C(2L) and distal to a functional locus in the other (in this example compound-2R) the interchange product carrying the C(2L) centromere (K-2L) w i l l be deficient for the proximal marker in 2R, but the reciprocal product, with K-2R, w i l l carry the corresponding duplication. Similar duplication-deficiency interchange products arise from a distal break in C(2L) and a proximal break in C(2R) (e.g. regions of interchange 2-4 in Figure 1). Finally, i f the interchange occurs between induced breaks distal to functional l o c i both in C(2L) and in C(2R) (for example, regions 1 and 5 in Figure 1) the K-2L product w i l l be duplicated for the proximal l e f t arm and deficient for the proximal right, while the K-2R product w i l l carry the reciprocal combination of duplication-deficiency. In accordance with the above model, which considers compound autosomes that are isochromosomes in so far as they are diploid for a l l functional genetic l o c i , the interchange products w i l l f a l l into three separate groups: 1) homozygous viables, 2) homozygous lethals owing to a deficiency in 2L proximal and.3) homozygous lethals owing to deficiencies in 2R proximal. Interchange products deficient both for 2L and 2R l o c i w i l l not arise from compound autosome detachment where both compounds are ful l y diploid; they should only occur where at least one compound autosome is hemizygous for proximal l o c i . Furthermore, deficiencies are polar, that is they extend from the centromere distally and always include the most proximal locus. For example, in Figure 1, deficiencies in proximal 2L are either b or a b, but not a alone. The distribution of lethals (and semi-lethals) obtained in the detachment products of the G(2L)SH3,+;G(2R)SH3,+ chromosomes (Figure 2) is consistent with the predictions of the above model. Of the 83 detach-ment products recovered as f e r t i l e individuals, 28 were homozygous complete lethals and 8 were classified as semi-lethals. On the basis of inter se complementation, allelism tests with known recessive lethals and lethal deficiencies within the proximal region, and pseudodominance tests against visible markers within this region (see Table I and Figure 2), 35 of the lethals, which includes, a l l 8 semi-lethals, were localized to two main clusters, one on either side of the centromere. The remaining single lethal, which gave f u l l complementation with a l l others, probably arose from a secondary hit as i t f a l l s well outside the proximal hetero-chromatic region. It is of interest to note that the yield.of lethal detachments (less than 50 percent) and the absence of double lethals (i.e. lethals spanning the centromere) argues that neither C(2L)SH3,+ nor C(2R)SH3,+ is hemizygous for proximal l o c i . In this context i t is interesting to note that both C(2L)SH3,+ and C(2R)SH3,+ appear as isochromosomes in both polytene and somatic chromosome preparations. FIGURE 2 Genetic map of the centric region of chromosome-2^ showing the relative positions and lengths of proximal, deficiencies associated with reconstituted standard seconds generated as products of inter-change between C(2L)SH3.,+ and G(2R) SH3.+. The marker positions shown on the map are only relative and do not represent actual physical separation. Genetic markers contained in t ] represent putative positions of recessive lethals defined:by the newly generated deficiencies. Proximal heterochromatin i s indicated by XvNNW . The position of Minute (M-S2)' relative to the heterochromatic-euchromatic junction is not known. The dotted line under Group E indicates--the position, relative to It, of-the lethal associated with the 2j_3 quasi-reciprocal translocation. See text for further details. 00 i -esc(54-9) -l(2)crc(55) -tri (55) Secondary Constriction It (550) [t(2L)D] 56* ll(2UD'l ~Q Centromere ll(2R)B] r I (55-1) MS2 (551) :• stw (55-1) :- ap (55-2) - msf (552) •• pk (552) In addition to lethality tests, each detachment product was genetically tested for X;2, 2;3 and 2^ 4 translocations. Although rarely expected multiple-hit events, one such product was recovered and w i l l be described below. The nature of the proximally located lethals, depicted in Figure 2, is based on the concept that detachment products, as a function of compound interchange, w i l l carry deficiencies (and duplications) of varying lengths. Focusing attention f i r s t upon the 2R associated lethals we find that a l l 18, which represents one half of a l l lethal detachments recovered, are lethal over the proximal deficiency, Df(2R)M-S210. Deficiency M(2)S10 is defined genetically as a recessive lethal with a dominant Minute phenotype (see Lindsley and Grell 1968).and which also uncovers the more proximal marker, rolled ( r l ) ; cytologically, M(2)S10 is apparently deficient for only the most proximal three polytene chromosome bands in 2R but i t reduces the mitotic metaphase length of 2R to approximately 3/4 of i t s normal size (Morgan, Schultz and Curry 1940). It has also been reported to behave as a heterochromatic deficiency in that i t enhances position-effect variegation (Morgan et a l 1940, Hannah 1951). While I have been unable to confirm the absence of the proximal bands (in 40A) of the polytenes mitotic prometaphase preparations of M(2)S10 obtained (see Figure 3) clearly show that the proximal 2R heterochromatin is missing The detachment lethals in 2R can be subdivided into two groups. Group A mutants, of which there are 17, in addition to being lethal over Df(2R)M-S210, uncover the marker r l . The distal boundaries of these deficiencies can be defined further as they complement the Minute FIGURE 3 Photomicrograph of a stained preparation of mitotic prometaphase chromosomes in a ganglion c e l l from a +/Df(2R)M-S210 heterozygous, third-instar, male larva. The dotted arrow points to the 2R proximal heterochromatin on the + chromosome; the solid arrow points to the M(2)S10 (2R heterochromatic-deficient) chromosome. deficiencies, M(2)S4 and M(2)S8. The latter two mutants are not only lethal over M(2)S10 but, genetically, extend di s t a l l y to include the markers indicated on the map in Figure 2. The single Group B lethal, which is lethal over Group A and M(2)S10, does not uncover r l . Only i f i C(2L)SH3,+ carried a r l * duplication, which i t does not, could the Group B lethal locus (or loci) f a l l between r l and the M(2)S2 si t e . Therefore, the conclusion i s that Group B represents a deficiency that uncovers a genetic locus (or loci) proximal to r l . Turning next to the 2L associated lethals, we can assign relative map positions to 17 lethals (and semi-lethals) on the basis of inclusion within the Group C deficiencies. T,he Group C deficiencies, of which there were two, uncover light (It), the most proximal genetic marker heretofore localized on 2L. The six Group D lethals, which f a i l to complement with each other and with the Group C lethals, do not include It. Therefore, consistent with the argument presented for the positioning of Group B on the right arm, we envisage Group D to represent deficiencies in 2L that include functional genetic l o c i (or a single locus) proximal to It. The 8 semi-lethals, denoted by Group D' in Figure 2, lead us to suspect that Group D deficiencies do, in fact, uncover more than a single genetic site. The 8 semi-lethals have in common the following characteristics: 1) their v i a b i l i t y , relative to heterozygotes, i s between 5 and 15 percent; 2) those that eclose are greatly reduced in size (to approximately 1/2 the size of a normal adult) ; and 3) they eclose very late. Furthermore, a l l 8 Group D' semi-lethals manifest these same -characteristics over any of the Group D or Group C (lethal) deficiencies. While one might be tempted to interpret Group D and Group D' as representing deficiencies of varying lengths extending into a tandemly replicated series of genes, somewhat, analogous to the bobbed mutations of the X-chromosome, "I view this as unlikely for two reasons. 1) The v i a b i l i t y and phenotypic expression of a l l homozygous Group D' deficiencies are manifestly the same as heterozygous Group D'/Group C deficiencies. 2) EMS (ethylmethane sulfonate) mutagenic studies (Chapter IV) reveal at least two l o c i within Group D, one of which . •• expresses the D' mutant characteristics. I propose, therefore, that the present deficiency mapping has disclosed, at least two genetic l o c i within the heterochromatic region between It and the centromere. The single Group E recessive lethal, represented as a dotted line in Figure 2, though genetically behaving as a nonpolar deficiency, is in fact a C(2L)-3-C(2R) quasireciprocal translocation. This arose from a 3-hit event that translocated 2L, including a large block of hetero-chromatin proximal to the _2L secondary constriction, to the right arm of chromosome-3 at polytene band 92E-F of Bridges'map (see Lindsley and Grell 1968). The right arm of chromosome-3 distal to 92E-F.is appended to the centromere-bearing detachment of C(2R). While this 2^ 3 translocation is lethal over Group C deficiencies, i t is f u l l y viable over a l l Group D deficiencies. Furthermore, f l i e s heterozygous for Group E over In(2LR)bwV1, an inversion with a breakpoint in the l e f t proximal heterochromatic region, are complete lethals and Group E over It shows the classical position-effect variegation for light eyes (Schultz 1936). The rare homozygous Group E translocation progeny that emerge as adults (approximately 1.0 percent of the heterozygotes) are phenotypically It variegated. It is interesting to note that since the translocation i s not lethal over Group D deficiencies, the break in 2L must l i e proximal to the Group D lethals. Moreover, the position-effect variegation, owing to l t ^ being displaced from the centric hetero-chromatin to a euchromatic region, does not affect the more proximal l o c i sufficiently to cause lethality. (See Appendix I for further discussion of this unusual 2^3 translocation.and i t s relevance to It variegation.) Turning now to the distribution of the break points involved in the compound-compound interchanges, let us examine the following arguments. Considering the proposed, model for reconstituting standards from compound autosomes, approximately one half of the detachment products carried an acentric right arm and half carried an acentric l e f t . From the 83 detachments recovered, 18, or approximately 1/4, are deficiencies of proximal 2R and 17, again approximately 1/4, are deficiencies of proximal 2L. Considering f i r s t the 2R deficiencies, we find that 17 of the 18 lethals uncover r l but none uncover the Minute lethal, defined by the overlapping of deficiencies M(2)S10 with M(2)S4 and.M(2)S8. Furthermore, as shown above, M(2)S10 is apparently deficient for a l l proximal 2R heterochromatin. If we can correctly assume that break-points occur at random.within the heterochromatic region, we are led to.the conclusion that r l i s a gene intercalated approximately midway between the centromere and the heterochromatic-euchromatic junction. The Group B deficiency I view as revealing at least one additional locus proximal to but probably quite tightly linked to r l . For the l e f t arm I can offer a similar argument.. I have found that C(2R)VK1,Dp(2L)lt+,bw (the chromosome in Experiment 2 described below) which carries a l t ^ duplication, does not possess the prominent secondary constriction frequently observed at the euchromatic-heterochromatic junction of 2L (Kaufmann 1934). However, the prometaphase figures of the Group C (It) deficiency-chromosomes do show this prominent secondary constriction. This places It in the heterochromatic segment proximal to the constriction. The remaining Group D and Group D' deficiencies uncover genetic sites that are probably intercalated somewhere midway between lt^ and the centromere. Interestingly, the Group C deficiencies are not deficient for any proximal bands of the 2L polytene chromosome. Detachment products of C(2I/)SH3.+:Cf2RWKl.Dp(2LUt+.bw: Let us f i r s t examine (in Figure 4) the consequence of interchanges between a C(2L) that i s genetically an isochromosome and a C(2R) that carries a duplication for a proximal segment of 2L. Markers a b and c d represent the normal sequence in proximal 2L and 2R respectively. In addition, one arm of C(2R) carries a duplication for marker b intercalated between marker c and the centromere, K-2R. The designated regions 1, 2 and 3 of 2L in Figure 4 correspond to those described in Figure 1 above. However, on 2R, region 4 is homologous to region 3 on 2L; 'region 5 now designates the heterochromatic segment that normally separates the marker c and the centromere on the standard chromosome and region 6 corresponds to region 5 in Figure 1 above. The lower portion of Figure 4 depicts some of the detachment products expected following interchanges between the designated regions on C(2L) and C(2R). The predictions for the recovery of deficiency products in the previous model (Figure 1) hold true for the products expected in Figure 4, with two major differences. If the points of induced breakage FIGURE 4 Hypothetical model depicting representative genotypes of reciprocal products expected from interchanges between a C(2L) that i s genetically an isochromosome and a.C(2R) that i s heterozygous for a duplication of the most proximal segment in 2L (indicated by the marker b). a b c d H I-C(2L) K-2L K-2R' C(2R) —I 1¬1 02^3 4 * 5 ^ 6 ^ Regions of Interchange 3-4 3 - 5 3-6 2 - 5 1 - 5 Interchange Products a 6 K-2L b e d -t 1 O i I I—h¬a bK-2Rc d -i 5—r-O 1 ! — a bK-2Lc d — i — i ® . i — j — a b bK-2Rc d ^—' ^ ' o— i— i -a bK-2Ld -i 1 O i l a 6 c 6/C-#?c d H 1—j—I 1 O 1 1-a bK-2Lb c d H i—-O 1 . I 1— a bK-2Rc d " " "F 1 0 + 1  a o c <y H 1—O 1 f—T 1 1— bK2Rc d -)—o—I—I— Genotype Dup(2L)b Norma/ Normal Dup(2l)b Def(2R)c Dup(2L)b:(2R)c Dup(2L)b Normal Dup(2L)ab Def(2L)a are randomly distributed, then 1) the proportion of b (or l e f t arm) deficiencies w i l l be markedly reduced and 2) nonpolar deficiencies of proximal 2L w i l l be recovered (e.g. deficiency a, but not b, from interchanges at regions 1-5 in Figure 4) . From the analysis of detachment products in Experiment .1, the C(2L)SH3,+ chromosome was found to be an isochromosome for 2L free of a duplication for 2R genetic l o c i . The C(2R)VKl,Dp(2L)lt+,bw chromosome, in contrast, has been shown, in combination with a C(2L) homozygous for It, to carry a 2L duplicationfbrrthe lt+ gene. From this we may infer that the lt+ duplication on C(2R)VK1 includes the more proximal l o c i uncovered by the Group D deficiencies in Experiment 1. In Experiment 2, of the 1.10 compound-detachment products 27 were homozygous lethals. As shown in Figure 5, 17 of these lethals were included within the M(2)S10 deficiency of proximal 2R, while only 5 detachment lethals were uncovered by the Group C deficiencies in proximal 2Lv Of the remaining 5 homozygous lethals, one was lost prior to analysis and, through- crossover studies, the remaining four mapped well outside the proximal region. These findings are consistent with the prediction of a relative reduction in generating deficiencies in 2L. Directing attention f i r s t to the 2R associated lethals, we note that 16 of the 17 deficiencies are typically of the Group A class; that is they are lethal over M(2)S10 as well as Group A and Group-B deficiencies and uncover the marker r l . The remaining 2R deficiency (designated as Group A' in Figure 5), in addition to revealing the Group A characteristics, is lethal both with M(2)S4 and M(2)S8. However, chemical mutagenesis studies (Chapter III) reveal a locus common to M(2)S10, M(2)S4 and M(2)S8 that i s not included within the Group A' deficiency, demonstrating FIGURE 5 Genetic map of the centric region of chromosome-2 showing the relative positions and lengths of proximal deficiences associated with reconstituted standard seconds generated as products of inter-change between C(2L)SH3,+ and C(2R)VK1,Dp(2L)1t+bw/bw. 1(2)A' may be an allele of M-S2. Their separation on this map is to indicate that Df(2R)M-S210 uncovers at least one (recessive lethal) site that i s not included within the Group A' deficiency. A more detailed description i s given in the legend of Figure 2. ±l(2)crc tri -^Secondary X Constrict ion i?[i(2L)F] Q Centromere rl [l(2R)A ] MS 2 A-stw op that the Group A' deficiency does not extend to the dist a l boundary of M(2)S10. The absence of Group B deficiencies in this experiment lends support to our previous assumption of tight linkage between the Group B lethal and the r l locus. The distribution of (deficiency) lethals localized proximally in 2L clearly supports the prediction of recovering nonpolar deficiencies when C(2R) is heterozygous for a duplication of proximal 2L l o c i . The three lethals designated Group D' in this experiment were homozygous complete lethals but heterozygous combinations of these.Group D' lethals as well as any D' in combination with Group C or Group D lethals (from Experiment 1) express similar properties to the Group D* lethals in, Experiment 1. There were no Group C lethals £er se but the two Group F lethals are apparently representative of the Group C class in arising from break points distal to the It locus. The Group F deficiencies provide a further subdivision of the Group C deficiencies, obtained in Experiment 1, into a Group C'that is lethal over both Group F deficiencies and a Group C ;, whose dist a l break point must l i e between It and the Group F lethals. Since the Group C'deficiency chromosome retained the prominent secondary constriction (loc. c i t . ) , i t would appear that the Group F deficiencies uncovered an additional locus (or loci) within the heterochromatic segment distal to the It gene. Furthermore, the Group F deficiencies weakly complement the quasi-reciprocal 2jJ3 translocation (Group E lethal in Figure 2) and In(2LR)bwV1, both of which show position effect variegation for light and are lethal over Group C and C' deficiencies. This points to the possibility of an additional genetic locus (or loci) between JLt and the Group D deficiencies that i s uncovered by Group C and C* and is primarily responsible for the position-effect lethality. Further, the Group F deficiencies are not deficient for any proximal bands in the 2L polytene chromosome. Detachment, products of C(2L)SHl,DP(2R)rl+/+;C(2R)SH3,+: As a parallel to Experiment 2 (above), I examined detachment products from a C(2L) that carried a duplication for proximal 2R, including the r2* gene. Regrettably, 59 of the 60 reconstituted standard seconds recovered were homozygous lethal. With the -prior knowledge (from Experiment 1) on the genetic properties of the G(2R)SH3,+ chromosome, I viewed the high lethality rate as evidence for heterozygosity of preexisting lethals on C(2L)SH1. It is conceivable that, as a function of formation, C(2L)SH1 was heterozygous for a proximal deficiency on the same arm as the 2R(rl +) duplication and at a more distal site, but close to the centromere, a recessive lethal on the homologous arm. Since the frequency of homozygosis near the centromere i s low, this heterozygous combination of recessive lethals would remain relatively stable. Detachment products from the present experiment were not analyzed further. Nonetheless, this experiment served to emphasize the importance to this type of study for independent examination of homogeneous populations of compound autosomes. The results from both Experiments 1 and 2 are summarized in Figure 6. Cytological observations place a l l of these deficiencies within the proximal heterochromatic regions of the l e f t and right arms of chromosome-2. These regions represent approximately 25 percent of the physical length but only 0.1 percentoffthe genetic length of the entire chromosome (see Lindsley and Grell 1968) . The present method of dissecting the proximal •FIGURE 6 Compilation of the genetic maps given in Figures 2 and 5. \l(2)crc tri x Secondary Constr ict ion \ \l(2L)F] It | \I(2L)D] Centromere [K2R)B] rl [l(2R)An MS2 7 - [l(2R)MS2-10] - stw -- ap heterochromatin does not resolve completely the debate concerning the nature of the genes intercalated into this region. In particular, deletion mapping cannot provide a clear distinction between possible tandemly repeated genes and single gene l o c i . . However, i t does demonstrate that within this segment of the chromosome, where DNA sequences (Botcham et a l . 1971, Gall et a l . 1971, Peacock et a l . 1973) and cytological properties imply primarily genetic inactivity, active genetic sites do reside, albeit at low density. Moreover, the present results provide an estimate of the number of functional l o c i and their relative distribution in the heterochromatic region of chromosome-2. According to chemical mutagenesis studies each site inferred from the present deficiency analysis represents a nonrepetitive cistron. Furthermore, within.the M(2)S10 deficiency, saturation mutagenesis studies reveal only three loci in addition to the three shown by the present study, thus demonstrating a low gene density in the 2R proximal heterochromatin. Prior to this study, the markers It and r l were defined as the most proximal genetic l o c i on 2L and 2R respectively. The proximal deficiencies generated through the detachment of compound autosomes reveal these markers as functional l o c i residing within cytologically defined heterochromatic segments. The frequency with which r l and It are uncovered by deficiencies would suggest that r l is more proximal in the 2R.than is l_t in the 2L heterochromatic block. Proximal to r l , on 2R, there is at least one additional v i t a l locus defined by the Group B deficiency; and distal to r l , the lethal uncovered by the overlapping of Group A' with deficiencies M(2)S4 and M(2)S8 indicates a third heterochromatic locus. However, since M.(2)S2 is located near the heterochromatic-euchromatic junction, a region that cytologically is extremely d i f f i c u l t to resolve, T cannot discount the possibility that the distal lethal locus (uncovered by..Group A'.) f a l l s just outside the proximal heterochromatic segment. Between the centromere and It on 2L_, two functional l o c i are indicated by the differing phenotypic expressions of the Group D and Group D' deficiencies. Position-effect lethality suggests a possible third locus between Group D and It (not indicated in Figure 6). Distal to It, as defined by the Group F deficiencies, at least one additional locus is recognized as residing within the heterochromatic segment of the chromosome. Hence, on the basis of deficiency mapping I propose, as a conservative estimate, that within the proximal heterochromatin of 2R there are at least two, and possibly three, functional genetic l o c i , including r l , and within 2L at least four, including It. At the same time, I realize that less than half of the interchanges generated lethal deficiencies. Accordingly, i f I am correct in assuming a random distribution of induced breaks throughout the heterochromatic regions and an equal probability of recovering acentric l e f t and right arm detachments, 1 am led to infer genetic quiescence in the most proximal half of the centric heterochromatin of chromosome two CHAPTER III ANALYSIS OF EMS INDUCED • LETHAL ALLELES OF SECOND CHROMOSOME PROXIMAL DEFICIENCIES INTRODUCTION The previous chapter.described a study in which the proximal region of chromosome-2 was genetically dissected by employing compound second autosome-detachments. In order to supplement and further this analysis I turned next to the induction of lethal alleles of chromosome-2 proximal deficiencies through the use of the chemical mutagen, EMS (ethyl methane sulphonate). In this fashion I could determine i f l o c i , in addition to those identified by the detachment analysis, existed i n the second chromosome heterochromatin. Further, by complementing the EMS induced lethals to the deficiencies described in the previous chapter, i t was possible to demonstrate the relative map positions of the new l o c i uncovered. This chapter describes the results of the analysis of EMS induced lethal alleles of the deficiencies, Df(2L)Group. C and Df(2R)M-S210, described in Chapter II. Df(2L)Group C is the largest 2L proximal deficiency recovered by compound second autosome detachment. Df(2R)M-S210 is deficient for the 2R heterochromatin. MATERIALS AND METHODS Generation of lethal alleles.of. Df(2R)M-S210: A stock isogenic for chromosome-2 and homozygous for-.the-second chromosome dominant mutation Pin was derived. Pin/Pin (iso-2) virgin males aged 2 to 3 days (posteclosion) were fed for 24 hours on a solution of 0.025 M EMS in 1% sucrose (Lewis and Bacher 1968). The males were then removed from the treatment vessels, placed in bottles containing a standard Drosophila medium and allowed to recover for 24 hours. The treated males were then mated to- In(2LR)bwVI/ln(2LR)SMI virgin females, with, 1 treated, male and 10 virgin females per culture.. F l male progeny heterozygous for the treated paternal chromosome (Pin) and In(2LR)SMl,Cy.were single pair mated in shell vials to Df(2R)M-S210/ In(2LR)SMl virgin females. F2 cultures in which a l l progeny were of the Cy_ phenotype were scored as putative lethal alleles of Df(2R)M-S210. The presence of Pin on the treated chromosome allowed one to derive from the lethal cultures a balanced stock heterozygous for the putative lethal a l l e l e of Df(2R)M-S210 and In(2LR)SMl,Cy. Each putative lethal a l l e l e of Df(2R)M-S210 was subsequently tested for complementation with the 2R proximal deficiencies described in Chapter II. Those putative lethal alleles of Df(2R)M-S210 that proved to be lethal alleles of the M(2)S10 deficiency, rather than lethal alleles of secondary lethals accumulated by and distributed on the M(2)S10 chromosome, were complemented in a l l inter se combinations. As a l l of the proximal lethal and deficiency stocks were heterozygous for In(2LR)SMI,Cy,complementation was indicated by the presence of transheterozygote, non-Cy_ progeny, whose phenotype was carefully examined. Complementation results of key importance in determining the nature of the often complex a l l e l e complementation maps were reconfirmed. A control experiment was done for the Df(2R)M-S210 lethal a l l e l e screen; and the X-chromosome spontaneous lethal rate was assayed in the iso-2 Pin strain. A l l experiments were performed at 25°C and employed a standard, cornmeal-agar-yeast-sucrose-dextrose Drosophila medium. Generation of lethal alleles of Df(2L^Group c': EMS induced lethal alleles of Df(2L)Group C were isolated and analysed by following the same procedures employed for the recovery of EMS induced lethal alleles of Df(2R)M-S210. 2L rather than 2R proximal deficiencies were ut i l i z e d in subsequent analysis to further characterize the newly induced recessive lethal (and visible) mutations. • RESULTS. AND DISCUSSION The spontaneous X-chromosome recessive lethal rate was determined in iso-2 Pin=-males. From.10 parental males, 1618 X-chromosomes were tested of which 4 were found to bear recessive lethals. (Of the 4 lethals, 2 were derived from a single male.) This spontaneous mutation rate, 0.25%, is well within the range .'observed for most Drosophila strains (Plough 1941). Thus the iso-2 Pin stock does not appear to be a highly spontaneously mutable strain. Lethal alleles of Df (2TOM-S210: In the control experiment, no lethal alleles of Df(2R)M-S210 were recovered.in 1925 tested chromosomes. In the EMS treated series, 85 lethal alleles of Df(2R)M-S21Q were recovered in 5000 f e r t i l e cultures, each f e r t i l e culture representing an individual tested chromosome. Unlike the control, appreciable s t e r i l i t y (approximately 16%) was observed in the F l male progeny of the EMS treated iso-2 Pin males. In addition to the 85 lethal alleles of Df(2R)M-S210, 41 chromosomes were recovered which, although lethal when heterozygous with the M(2)S2 1 0 chromosome, were not lethal alleles of the M(2)S2 1 0 deficiency but, rather, were alleles of secondary lethals that the M(2)S2 1 Q chromosome had accumulated. The accumulation of lethals on permanently heterozygous second ..chromosomes has been well documented (Mukai 1964) and the M(2)S2 1 0 chromosome was constructed over 35 years ago (Morgan, Schultz and Curry 1940) . On the basis of complementation with the 35 available 2R proximal deficiencies and the recessive mutation r l , the 85 lethal alleles of Df(2R)M-S210 f e l l into five groups (Figure 1). Let us now examine the complementation maps of each of the five sites, beginning with Group I. Group I lethals were lethal in combination with the Groups B, A, A' and A" deficiencies, but they were r l + and survived when heterozygous with Df(2R)M-S2^. The complementation map of the Group I lethals i s presented in Figure 2. The Group I lethals can be divided further into two subgroups. One lethal, EMS 31, complements with a l l 4 remaining lethals. The other lethals, EMS 45-10, EMS 45-84, EMS 45-87 and EMS 45-91, provide a circular complementation map. This circular complementation map is clearly an example of i n t e r a l l e l i c complementation (reviewed in Fincham 1966). Obviously with such complex al l e l e complementation occurring at the EMS 45-10 locus i t is not possible on the basis of. the complement-ing of EMS 31 with a l l 4 other Group I lethals to assign i t to a different locus. However, observations on the phenotype of Group B deficiency homozygotes and heterozygotes with the Group I and the Groups A, A', 10 and A , and M(2)S2 deficiencies strongly imply that EMS 31 represents a locus separate from the one associated with the EMS 45-10 complex. Zygotes homozygous for the Group B deficiency or heterozygous for the Group B deficiency and a larger _2R deficiency, which encompasses the Group B deficiency, survive to the third larval instar but do not pupate. These nonpupating larvae develop extremely large melanotic masses in their haemocoel. Virtually a l l of the deficiency homozygote larvae develop these melanomas and, usually, when they die, they contain one or more very large, and numerous small, melanomas (Figure 3). Heterozygotes FIGURE 1 Distribution by complementation with 2R.proximal deficiencies of EMS-induced lethal alleles of Df(2R)M-S210. Proximal hetero-chromatin i s indicated by ^^ s^^ S^^ S^^  • See text, and Figures 2, 5, and 6 of Chapter II, for further details. o o c - o Kl Co 5 Q Centromere 5 r/ FIGURE 2 Complementation map of the Group I lethal alleles of Df(2R)M-S210. EMS 45-91 FIGURE 3 Photograph of a third-instar larvae homozygous for the Group B 2R proximal deficiency. for EMS-31 and any of the Groups A, A*, A", and B or M(2)S10 deficiencies have the Group B syndrome, being late larval lethals with the lethal larvae developing huge melanotic masses. None of the 4 lethals belonging to the EMS 45-10 complex exhibit the Group B (melanotic) syndrome. However, a l l are late larval and pupal lethals. Thus, i t would appear that the Group I lethals represent two l o c i , one represented by one lethal, EMS 31, and the other represented by four, EMS 45-10, EMS 45-84, EMS 45-87, and EMS 45-91. As both sets of Group I lethals are lethal in combination with the Group B deficiency and are r l * , i t i s clear that both l o c i are proximal to the r l locus. Their relative order i s unknown and f a i l i n g the recovery of a proximal deficiency whose distal or proximal boundary f a l l s between the two l o c i , this w i l l be resolved only by conventional genetic mapping. However, i t w i l l probably be d i f f i c u l t , i f not impossible, to separate these two sites through recombination, as indicated by the following observation. A recombination experiment between EMS 31 and r l was attempted. Virgin females heterozygous for EMS 31 and b p_r r l cn (see Table I of Chapter II for a description of these mutations) were crossed to Df(2R)M-S210/ln(2LR)SMl males. As r l is virtu a l l y lethal when hemizygous at 25°C (the r l hemizygotes have less than 1% v i a b i l i t y and the rare surviving r l hemizygote has a very severe r l phenotype) the only regular surviving progeny are In(2LR) SMI hetnerozygotes which have the Cy_ wing phenotype. A Cy^ exception could arise as a result of recombination between r l and EMS 31. However, such a recombinational event has only a 25% chance of being recovered as a Cy + exception for i t requires that the EMS 31 + r l + recombinant chromatid be selected for in a female pronucleus that i s f e r t i l i z e d by a Df(2R)M-S2 bearing sperm. As the total number of zygotes is equal to approximately twice the number of Cy_ progeny, one may estimate the recombination frequency as two times the number of recombinants divided by the number of Cy_. progeny. No recombinants between EMS 31 and r l were observed in the 13,268 progeny recovered. Clearly, as the 95% confidence limits (Stevens 1942) for the recombinational. distance between EMS 31 and r l are 0 and 0.045%, i t may well be very d i f f i c u l t to.order EMS 31 and the EMS 45-10 complex by recombination. The Group II lethals are a l l alleles of the r l locus (see Figure 4 for the complementation map). A l l 9 Group II lethals are lethal when heterozygous with the Groups A, A', and A" deficiencies but survive in 4 combination with the Group B deficiencies as well as with the M(2)S2 deficiency and the Groups I, III, IV, and V EMS lethals. Eight of the Group II lethals, EMS 43, EMS 64., EMS 34-29, EMS 45-32, EMS 45-39, EMS 45-54, EMS 45-95 and. EMS 698 are lethal when heterozygous with the original _rl mutation; one Group II lethal, however, EMS 45-^ 52, while extremely inviable when homozygous, when heterozygous with r l is associated with a f u l l y viable f l y expressing a phenotype indistinguishable from that of the original r l / r l homozygotes. Additionally, a l l 9 Group II lethals are lethal in a l l inter se combinations. Interestingly, the Group II lethals, save for EMS 45-52, when hemizygous (i.e. hetero-zygous with a r l deficiency), or in any inter se combination, are _ associated with a lethal phenotype wherein the organisms develop into third instar larvae that completely lack imaginal discs. Df(2R)M-S2 .FIGURE Complementation map of the Group II lethal alleles of 10 4 EMS 43  EMS 64  EMS 698  EMS 34-29  EMS 45-32  EMS 45-39  EMS 45-52 EMS45-54 EMS45-55 The complementation map of the Group I I I l e t h a l s i s presented i n Figure 5. The Group I I I l e t h a l s divide the previously described Group A d e f i c i e n c i e s (Chapter II) into two classes, Group A d e f i c i e n c i e s that do not include the Group I I I l e t h a l s , and Group A" d e f i c i e n c i e s that do. The 6 Group I I I l e t h a l s , EMS 34-7, EMS 45-1, EMS 45-17, EMS 45-37, EMS 45-40, and EMS 45-73 do not complement i n any i n t e r se combination and thus would d e f i n i t e l y appear to be associated with a sin g l e locus. Of the 33 compound-second autosome detachment-associated d e f i c i e n c i e s that uncovered r l , 21 include the EMS 34-7 locus. Results from compound-autosome detachment studies i n d i c a t e that a l l breaks i n C(2R) occurred proximal the heterochromatic-euchromatic junction. Since approximately one-half of the 2R heterochromatic d e f i c i e n c i e s included the marker r l , the p h y s i c a l l o c a t i o n of t h i s locus was estimated to be near the middle of the heterochromatic segment. Following t h i s same l i n e of argument, the i n c l u s i o n of the EMS 34-7 ' locus i n s l i g h t l y more than one h a l f the r l d e f i c i e n c i e s would place EMS 34-7 j u s t proximal to the d i s t a l quarter of the heterochromatic region. However, despite the appreciable p h y s i c a l distance that separates the two l o c i , roughly 1/6 to 1/4 of the 2R heterochromatic block, there i s v i r t u a l l y no recombination between r l and EMS 34-7. V i r g i n females heterozygous f or EMS 34-7.and b pr r l cn were mated to Df(2R)M-S2 1 0/ In(2LR)SMI males. From t h i s cross, no recombinant Cy + progeny were recovered i n 20,750 Cy_ progeny examined. This gives an upper 95% confidence l i m i t of 0.029% crossing over between the two heterochromatic l o c i . FIGURE 5 Complementation map of the Group III lethal alleles of Df(2R)M-S210. EMS 34-7  EMS45-1  EMS 45-17  EMS 45-37  EMS45-40 EMS 45-73 The Group III lethals, when hemizygous, usually die in the".late pupal stage with many dying while eclosing. The rare hemizygous survivors have unexpanded wings and, often, misshapen rear legs, etched tergites, and. smaller body size. The next group, the Group IV lethals, present a.rather complex complementation map (Figure 6). In addition to Df(2R)M-S210, the Group 4 IV lethals are lethal in combination with Df(2R)M—S2 and the single 4 Group A' lethal. As the Group. IV lethals f a l l within the M(2)S2 deficiency," ;•" a deficiency for which I can detect neither loss of 2R _ proximal heterochromatin in somatic chromosome preparations nor deficiency for any proximal, bands of the ^ R polytene chromosome, the locus associated with the Group IV lethals i s probably in the vi c i n i t y of the heterochromatic-euchromatic junction. Thus i t i s unclear whether the Group IV locus is within or immediately adjacent to the ZR proximal heterochromatin. The recovery of only one compound-second autosome detachment deficient for the Group IV site i s further evidence for the location of the Group IV locus near the heterochromatic-euchromatic junction. • A total of 35 recessive lethals were recovered for the Group IV site. Of these lethals, 34 f e l l into an extensive and complex inter-• a l l e l i c complementation map (Figure 6), while the remaining lethal, EMS 45-72, complemented with a l l others in this group. The complementation between alleles was surprisingly unambiguous, that i s , heterozygous combinations of different alleles were either f u l l y viable or completely inviable. Although not thoroughly studied, the lethal phases for the majority of homozygous and heterozygous combinations appeared to f a l l FIGURE 6 Complementation map of the Group IV lethal alleles of Df(2R)M-S210. (EMS 45-72, not shown in the complementation map, complements ful l y with a l l other Group IV lethals). 34-14 8*' mi 45,1 ~ ~ 885.187,788 34-2 34-8,45-4 34-3 693,34113428 34-25 into the late larval and early pupal stages of development. The exceptions to complete lethality within the Group IV lethals involve 4 combinations which were associated with partial complementation, two combinations involving EMS 34-28 and two involving EMS 45-71. In Figure 6, EMS 34-28 is shown as noncomplementary to EMS 45-16 and EMS 45-28 while, in fact, i t does complement weakly with both lethals. EMS 34-28/EMS 45-16 heterozygotes have 17% v i a b i l i t y and EMS 34-28/  EMS 45-28 heterozygotes have 8% v i a b i l i t y relative to the In(2LR)SMl, Cy heterozygotes. EMS 45-71, on the other hand, is shown in Figure 6 as complementing with EMS 34-26 and EMS 45-34, even though i t does so only weakly. EMS 45-71/EMS 34-26 heterozygotes have 10% v i a b i l i t y and EMS 45-71/  EMS 45-34 heterozygotes have 21% v i a b i l i t y . These combinations are, however, the only ones which show ambiguity with respect to complementation. Thus the Group IV lethals are associated with a locus that exhibits a relatively high rate of EMS mutability and whose lethal alleles exhibit complex i n t e r a l l e l i c complementation. The exceptional lethal in this group, EMS 45-72, may, in fact, represent an additional locus, but this is unclear. The Group V lethals are also associated with a circular complementation map (Figure 7). The Group V lethals are lethal with none of the compound-second autosome detachment 2R proximal deficiencies, but are lethal in combination with Df(2R)M-S24 and Df(2R)M-S28. Moreover, they are viable in a l l combinations with the Group IV (and Groups I, II, and III) lethals. This places the locus associated with the Group V lethals d i s t a l to that associated with the Group IV. The Group V locus, therefore, f a l l s near the 2R heterochromatic-euchromatic junction, but i t is not clear whether FIGURE 7 Complementation map of the Group V lethal alleles of Df(2R)M-S210. i t l i e s just within or just outside the 2R heterochromatic block. Complementation among the Group V lethals i s relatively straight-forward, 27 of the 30 lethals forming,a uniformly noncomplementing complex. These 27 lethals are subdivided by their complementation with 3 exceptional alleles EMS 45-8, EMS 34-20, arid EMS 45-89 which complement with most of the other alleles. Complementation is either complete or completely negative in a l l combinations of alleles save 4, 3 involving EMS 45-8 and 2 involving EMS 45-26. Although EMS 45-8 is shown in Figure 7 as noncomplementing with EMS 34-4, EMS 45-8/EMS 34-4 heterozygotes do have 8% v i a b i l i t y . Further, although EMS 45-8/EMS 45-67 heterozygotes are indicated as complementing in Figure 7, these heterozygotes have 48% v i a b i l i t y ; which is considered to be essentially f u l l , complementation. Although EMS 45-26 complements f u l l y with EMS 45-8 and EMS 34-20, the transheterozygotes have a peculiar imaginal external phenotype. EMS 45-26/EMS 45-8 heterozygotes have their wings uniformly spread out from the body at a 45° angle. EMS 45-26/EMS 34-20 heterozygotes have, in addition to the spread wing phenotype of EMS 45-26/EMS 45-8 heterozygotes, o c e l l i that are often misshapen, unpigmented, or absent. The large number of Group. V lethals recovered (a total of 30) points to a relatively high EMS mutability of the Group V locus. This is a characteristic i t shares with the immediately adjacent Group IV locus in addition to i n t e r a l l e l i c complementation. Recombinants were recovered between a Group V lethal and r l . Virgin females heterozygous for EMS 34-21 and b pr r l cn were crossed to Df (2R)M-S21(hn(2LR)SMl males. 2 recombinant Cy progeny were recovered along with 5996 Cy_ progeny. That the putative recombinant progeny were truly recombinant was verified by genetic testing. The 95% confidence limits (Stevens 1942) on the recombinational distance between r l and EMS 34-21 are 0.01 and 0.20 map units. Thus although the r l and Group V l o c i are separated by the distal half of the 2R heterochromatic block, crossing over between the two sites i s less than 1%. The cross-over distance between two markers flanking an equal extent of 2R euchromatin would be of the order of magnitude of 10 map units (Lindsley and Grell 1968). The analysis of the EMS induced lethal alleles of Df.(2R)M-S2/P has revealed the existence of 3 l o c i within the M(2)S10 deficiency in addition to the three demonstrated by the analysis of compound-second autosome detachments (Chapter II). Thus there are at least 6 l o c i within the M(2)S10 deficiency with at least 4 of these l o c i the EMS 31 locus, the EMS 45-10 locus, the r l locus and the EMS 34-7 locus, within the 2R proximal heterochromatin. The other two l o c i , the Groups IV and V l o c i , are near the 2R heterochromatic-euchromatic junction, but only one, Group V, f a l l s outside the deficiencies generated through detachment of compound-2 autosomes. Of the 85 EMS induced lethal alleles of Df(2R)M-S210, none are deficient for 2 or more l o c i ; that i s , a l l appear to be point mutations. This i s consistent with the literature on EMS mutagenesis in Drosophila. Lim and Snyder (1974), in their analysis of 85 EMS induced lethals f a l l i n g within the zeste-white region of the X-chromosome using the mutagenesis procedure of Lewis and Bacher (1968) that I have employed in my analysis of the proximal, region of chromosome-2, found none that affected more than one cistron, even though this region is very well defined genetically (Judd, Shen and Kaufman 1972). Earlier, Lim and Snyder (1968), in a cytological examination of EMS-induced.X-chromosome recessive lethals, found that none of the lethals involved deletions, inversions or other visible rearrangements. Further, EMS is unable to induce, in Drosophila females, compound autosome formation (which, like the generation of deficiencies, is an interchange event) at doses capable of inducing a rate of X-chromosome recessive lethality of approximately 2%. (W. Gibson, personal communication). It is unlikely that any of the Groups I and II lethals are deficiencies for i t i s clear from the detachment analysis that breaks are far more likely to occur distal to r l and proximal to Group I than to occur between r l and the Group. I l o c i . Thus a deficiency for r l is far more like l y to be deficient for both r l and the Group I l o c i than deficient for r l only. It is particularly unlikely that any of the Group IV or Group V lethals are deficiencies. The Group IV lethals are associated with a complementation map (Figure 6) in which no single lethal a l l e l e f a i l s to complement a l l other lethal alleles in the group, as would be expected for a deficiency. As far the Group V lethals, a deficiency in this region would be far more lik e l y to have i t s proximal break proximal to the Group IV locus than between the Group IV and V sites; and none of the Group V lethals are lethal in combination with any of the Group IV lethals. Thus, the 85 lethals recovered that f a l l within the M(2)S2 1 0 deficiency would appear to be point mutations implying that the l o c i they uncover are nonrepetitive. Moreover, the complex al l e l e complementation maps of the EMS 45-10, Group IV and Group. V l o c i provide further evidence that the genetic l o c i uncovered in 2R heterochromatin represent, single, nonrepetitive genes. From the preceding analysis of compound second autosome detachments (Chapter II) and the recovery of EMS induced lethal alleles of Df(2R)M-S210 i t i s clear that typical (i.e. nonrepetitive and viability-essential) l o c i exist within constitutive heterochromatin, although at very low density relative to euchromatin.- The estimated relative gene density of the 2R heterochromatic. block to an equal amount of 2R euchromatin is 1%, that is there is approximately one hundred times more genetic l o c i in a. block of 2R euchromatin than, there is in a block of 2R proximal heterochromatin of similar length. This estimate is based on the observation that the 2R polytene chromosome contains 1136 bands (Bridges and Bridges 1939), which would imply that a portion of 2R euchromatin equal in somatic chromosome length to the 2R heterochromatic block would be represented in the polytene chromosome by a 379 band segment. As there is substantial evidence (reviewed in Lefevre 1974) for the proposition-that each polytene chromosome band is associated with a single genetic locus (Bridges 1935), this leads one to an estimate of approximately 380 genes in. the 2R heterochromatic block. Within the 2R heterochromatin I have been able to identify 4 (and possibly. 5,if Group.IV is included) gene l o c i , thus the gene density of the 2R heterochromatin would appear:to be only in the order of magnitude of 1% that of the estimated gene density of an equal extent of 2R euchromatin. Lethal alleles of.Df(2L)Cj; EMS mutagenesis yielded 28 lethal alleles of Df (2L)C in 6467 f e r t i l e cultures, each culture representing a single tested chromosome. In addition there was approximately 20 percent s t e r i l i t y (1591 of a total of 8058 cultures) in the F l male progeny of the EMS treated iso-2 Pin males. In addition to the 28 lethal alleles of Df(2L)C, 5 alleles of It were recovered and were found to be viable over the deficiency, which, as shown in the previous chapter, i s deficient for the It locus,. A phenomenon noted in the Df(2R)M-S210 lethal a l l e l e screen was also witnessed in this series of tests. Of a total of 6490 f e r t i l e cultures in the Df(2L)C lethal a l l e l e screen, 23 had a l l Cy P i n + progeny despite the fact that the paternal genotype was Pin/ln(2LR)SMI. A possible explanation for these 23 exceptional cultures i s that the male bearing the EMS treated iso-2 Pin chromosome was mosaic for a dominant lethal mutation on the mutagenized chromosome-2, the germ line bearing the dominant lethal mutation; the soma not bearing i t . A l l of the F2 zygotes inheriting the paternal Pin chromosome (derived of course from the paternal germ line) would be heterozygous for the dominant lethal in a l l cells and, therefore, would die. These cultures were not numbered among the tested chromosomes, as I was unable to determine whether or not the paternal mutagenized Pin chromosome bore a lethal allele. of Df (2L)C . On the basis of complementation with the 2L proximal deficiencies described in Chapter II, the 28 lethal alleles of Df(2L)C f a l l into 4 groups (Figure 8), numbered VI, VII, VIII and IX to avoid confusion with the EMS lethal alleles of Df(2R)M-S210. FIGURE 8 Distribution by complementation, with 2L proximal deficiencies of EMS induced lethal alleles of Df(2L)C. Proximal heterochromatin is indicated by ^ ^ C ^ V , and the secondary constriction at the 2L heterochromatic-euchromatic junction by ( ). For further details see text and Figures 2 and 5 of Chapter II. 0 Q g 0 Q 0 0 o c o p 0 0 0 C 0 0 Q 0 Q Q< £ 2 c CD o . x x X X X X X -D D' o G r o u p E eroupEDI GrouplLT Group3ZI Let us now examine the complementation maps of each of the four groups of l e t h a l s beginning with the most proximal, Group VI (Figure 9). The Group VI l e t h a l s are l e t h a l when heterozygous f or the Groups C, C', D, and D' 2L proximal d e f i c i e n c i e s but they are l t ^ . The Group VI l e t h a l s , EMS 40-5 and EMS 56-19, were noncomplementing i n combination but complemented f u l l y when heterozygous f or a l l of the Groups VII, VIII and IX l e t h a l s . Group VI l e t h a l hemizygotes were l i k e Group D' deficiency homozygotes distinguished by infrequent adult survivors who, although of a normal external phenotype, were l a t e eclosing and greatly reduced i n s i z e (to approximately 1/2 the normal adult s i z e ) . The existence of the Group VII l e t h a l s confirms the previous inference that the Groups D and D' d e f i c i e n c i e s were g e n e t i c a l l y d i s t i n c t (Chapter II) as the Group VII l e t h a l s were l e t h a l i n combination with the Groups C, C and D d e f i c i e n c i e s but complemented f u l l y with the Group D' d e f i c i e n c i e s . C l e a r l y , the Group D d e f i c i e n c i e s have a greater d i s t a l extent than the Group D' d e f i c i e n c i e s as was previously i n f e r r e d (Chapter I I ) . Inspection of the Group VII l e t h a l complementation map (Figure 10) leads one to conclude that possibly two l o c i are associated with the Group VII l e t h a l s . EMS 56-24 complemented f u l l y with the other 3 Group VII l e t h a l s . (EMS 56-24 hemizygotes were of approximately 2% v i a b i l i t y . ) The other subgroup consists of the EMS 56-4, EMS 56-14, and EMS 56-15 l e t h a l s . The l e t h a l s of the EMS 56-4 complex were completely i n v i a b l e when hemizygous and i n a l l i n t e r se combinations of the three l e t h a l s . A semi-lethal a l l e l e of the EMS 56-4 locus was recovered, EMS 40-22, although t h i s semi-lethal i s not included i n the Group VII complementation map i l l u s t r a t e d i n Figure 10. EMS 40-22 was of approximately 20% hemi-FIGURE 9 Complementation map of the Group VI lethal alleles of Df (2L)C . EMS40-5 EMS 56-19 FIGURE 10 Complementation map of the Group VII lethal alleles of Df (2L)C . EMS 56-24 EMS 56-4 EMS 56-14 EMS 56-15 zygous v i a b i l i t y with the hemizygote progeny being l a t e eclosing. Heterozygotes for EMS 40-22 and. EMS 56-4, EMS 40-22 and EMS 56-14, and EMS 40-22.and EMS 56-15 were of 33, 26, and 35 percent v i a b i l i t y r e s p e c t i v e l y , with these heterozygous progeny being of l a t e r e c l o s i o n than t h e i r s i b s . EMS 40-22 complemented f u l l y with a l l other 2L l e t h a l s including EMS 56-24, the remaining Group VII locus. Thus i t would appear that two l o c i are associated with the Group VII l e t h a l s , one locus with EMS 56-24 and the other with the EMS 56-4 complex, but t h e i r r e l a t i v e order i s unknown. The Group VIII l e t h a l s are uncovered only by the Group C and C' d e f i c i e n c i e s and therefore define a region d i s t a l to the Groups D and D' d e f i c i e n c i e s and proximal to the Group F d e f i c i e n c i e s . Group VIII i s divided into three d i s t i n c t , nonoverlapping complementation subgroups (Figure 11), one of which corresponds to the I t locus. In ad d i t i o n to 5 v i s i b l e a l l e l e s of I t , which were hemizygous v i a b l e and of I t phenotype, 3 l e t h a l a l l e l e s of the I t locus were recovered, EMS 40-12, EMS 40-17 and EMS 56-3. These l e t h a l a l l e l e s of I t were of l i g h t phenotype when heterozygous for a nonlethal I t a l l e l e but were l e t h a l i n combination with one another. Hemizygotes for the I t l e t h a l a l l e l e s appeared to die i n the l a t e pupal stage of development. Thus, i t would appear that I t i s an e s s e n t i a l locus. The two other complementation groups within the Group VIII l e t h a l s are the EMS 40-2 complex of 3 l e t h a l s and the EMS 40-6 complex of 14 l e t h a l s . The EMS 40-2 complex consists of EMS 40-2, EMS 56-6 and EMS 56-32. ' A l l 3 l e t h a l s of the EMS 40-2 complex are I t and v i a b l e i n combination with the 25 other-Df(2L)C 1 FIGURE 11 Complementation map of the Group VIII"lethal alleles of Df (2L)C . "It" EMS 40-2 EMS 40-12 EMS 56-6 EMS 40-17 EMS 56-32  EMS 56-3 EMS 40-6  EMS 40-7  EMS 40-8  EMS 40-20  EMS 40-21  EMS 56-1  EMS 56-2  EMS 56-5  EMS 56-7  EMS 56-9  EMS 56-10  EMS 56-16 EMS 56-20 EMS 56-27 lethal a l l e l e s . The significance of the fact that 14 of the 28 EMS induced lethal alleles of Df(2L)C f a l l at this site i s unclear, although i t i s interesting to note that, unlike the two l o c i in the proximal region of 2R associated with high EMS mutability, the EMS 40-6 complex is not associated with i n t e r a l l e l i c complementation. In summation, i t would appear that the Group VIII lethals are associated with three l o c i , one corresponding to the It' locus, for which in addition to 3 lethal alleles 5 hemizygous viable visible alleles were recovered; another, to the EMS 40-2 complex for which 3 lethal alleles were obtained; and the third locus, to the EMS 40-6 complex which represented 14 lethal a l l e l e s . The relative order of these three l o c i remains unknown. The most distal 2L lethals are those associated with Group IX. The 2 Group IX lethals, EMS 40-18 and EMS 56-8, although lethal when heterozygous for the Group C' deficiency, are f u l l y viable in combination with the Group C deficiency and are l t \ Both lethals are, however, lethal in combination with the Group F deficiencies; arid EMS 40-18/ EMS 56-8 heterozygotes are inviable, thus the two lethals are a l l e l i c . Hemizygotes for EMS 40-18 and EMS 56-8 are associated with a rather interesting lethal phenotype. These hemizygotes pupate but subsequently undergo complete autolysis with no adult structures present and larval tissues being reduced to an oily mass at the bottom of the pupa case. Thus, the Group IX lethals are associated with a single locus, and when hemizygous exhibit a rather dramatic tissue degradation following pupation. The analysis of the EMS induced lethal alleles of Df(2L)C has revealed 7 l o c i within the deficiency of 3 in addition to the 4 inferred FIGURE 12 Complementation map of the Group IX lethal alleles of Df (2L)C . EMS 40-18 EMS 56-8 from the compound-second autosome detachment analysis (Chapter II). Multiple lethal alleles were recovered for each locus save one (the site inferred from the existence of the EMS 56-24 lethal) with 2 l o c i associated with 2 lethal alleles, 3.loci with 3, and 1 locus (the EMS 40-6 complex) with 14 lethal a l l e l e s . Cytological observations place a l l 7 of these l o c i in the 2L heterochromatin. Df(2L)C i s not deficient for any proximal bands in the euchromatic 2L polytene chromosome nor is i t deficient for the secondary constriction at the 2L heterochromatic-euchromatic junction. In addition the C(2R)VK1,Dp(2L)lt+bw chromosome, which, as the recovery of the Group F deficiencies demonstrated (see text and Figure 4 of Chapter II), carries a duplication of the 6 l o c i associated with the Groups VI, VII and VIII lethals, i s not duplicated for any portion of the secondary constriction at the 2L heterochromatic-euchromatic junction. Further, although the Group F deficiencies on the basis of. their Minute phenotype would appear to have a greater distal extent than the non-Minute Df (2L)C , neither of the' 2 Group F deficiencies is deficient for proximal bands in the 2L polytene chromosome and neither of them lacks the 2L secondary constriction. The 4 l o c i associated with the Groups VIII and. IX lethals may l i e in the immediate vi c i n i t y of the 2L heterochromatic-euchromatic junction as only 1 deficiency deficient for the Group IX lethal locus (the group C deficiency) and 2 deficiencies deficient for the Group VIII lethal l o c i (the Groups C and C deficiencies) were obtained among the C(2L)SH3,+;C(2R)SH3,+ detachments. However, 6 detachments (the Group D deficiencies) deficient for the two l o c i associated with the Group VII lethals were recovered among the C(2L)SH3,+;C(2R)SH3,+ detachments indicating that these l o c i and the more proximal locus associated with the Group VI lethals, for which 14 C(2R)SH3+;C(2R)SH3,+ detachments were deficient, (the Groups. D and D' deficiencies) are well within the 2L proximal heterochromatin. Consistent with the findings for EMS induced lethal alleles of Df(2R)MS-10, none of the lethal alleles of Df(2L)C induced, recessive lethals associated with the proximal region of chromosome-^ appear to be deficiencies, i t seems evident that, when one employs the mutagenesis procedure of Lewis and Bacher (1968), EMS behaves as a point mutagen in Drosophila heterochromatin, as has previously been demonstrated in Drosophila euchromatin (Lim and Snyder 1974). CHAPTER IV GENERAL DISCUSSION The preceding sections orf>this thesis describe the genetic dissection of the proximal region of chromosome-2; of Drosophila/melanogaster through the detachment of compound-second autosomes (Chapter II) and the induction, with ethyl methane sulphonate (EMS), of recessive lethals a l l e l i c to the 2L and 2R proximal deficiencies associated with' the detachment products. In combination with cytological analyses of the compound autosomes employed and proximal deficiencies obtained, these studies have demonstrat-ed that genetic l o c i are situated within the constitutive heterochromatin of the second chromosome. Further, these l o c i are not associated with secondary constrictions as observed for the bobbed locus in the X-chromosome heterochromatin and, probably, for the f e r t i l i t y factors in the Y-chromosome. These heterochromatic l o c i appear to be nonrepetitive genes, i.e. only one copy of the structural gene is present at each locus. Lethal alleles of the chromosome-2 heterochromatic l o c i uncovered in this study have late larval and pupal lethal phases, an observation of possible significance. Finally, although genes have been demonstrated in chromosome-2 proximal heterochromatin, the gene, density of this region i s very low relative to that estimated for the second chromosome euchromatin (approximately 1%). The observation of a low gene density in chromosome-2 heterochromatin is perfectly consistent with results,previously cited, that<ha\e hetetofore been interpreted as conclusive evidence of the complete genetic inactivity of heterochromatin (e.g. Yunis and Yasmineh 1971). The assignment of a few genetic l o c i to a heterochromatic chromosome segment is not of course inconsistent with the assignment of the bulk of the DNA of the hetero-chromatic segment to short, highly repetitive base sequences. Further the lack of DNA-RNA hybridization of chromocentral DNA with nuclear (and cytoplasmic) RNA merely demonstrates the apparent genetic inactivity of heterochromatin relative to euchromatin. A few gene l o c i within a large heterochromatic chromosome segment can not be demonstrated bio-chemically, only genetically, as has been accomplished here for the Drosophila second chromosome heterochromatin. While one may find i t d i f f i c u l t to conceive how transcription of RNA may occur at sites intercalated in the condensed chromocentral material, i t is evident that DNA replication, a process fundamentally similar to RNA transcription, does occur within heterochromatin. Moreover, i t is conceivable that the folding of the heterochromatic material to form the chromocenter may have a pattern of organization such that functional genetic l o c i inter-calated in heterochromatin are positioned on the exterior of the chromo-center. (The significance of the location of those l o c i intercalated in the chromosome-2 heterochromatin and those associated with hetero-chromatic-euchromatic junctions i s unclear, although the observation that the lethal phases of the lethal alleles of these l o c i are late larval and pupal i s , perhaps, instructive ) The analyses of the EMS-induced lethal alleles of 2L and 2R proximal deficiencies have demonstrated that EMS behaves as a point mutagen, i.e. EMS does not induce deletions. Lim and Snyder (1974) have arrived at foe same conclusion from their analysis of EMS-induced recessive lethals f a l l i n g within a small segment of X-chromosome euchromatin. Thus, EMS appears to be a nonradiomimetic mutagen in that i t does not induce deletions (Chapter III; Lim and Snyder 1974) or other inter-changes (Lim and Snyder 1968; W. Gibson unpublished) in Drosophila heterochromatin.or euchromatin. EMS may well operate, therefore, by a base substitution mechanism resulting in the .alteration,of a single nucleotide, in.the nucleotide sequence of.a gene. In addition to confirming that EMS does not generate chromosomal aberrations or interchanges, the analysis of the EMS induced.chromosome-2 proximal lethals has provided a caveat for the general procedure of defining the genetic constitution of.a chromosome segment through the analysis of mutagen,induced recessive lethals. This caveat is that.the phenomenon of in t o r a l l e l i c complementation may lead one to overestimate the number of genetic l o c i within a chromosome segment under investigation. This possibility i s illustrated by the complementa-tion maps of lethal alleles of l o c i within Df(2R)M-S210. For example, i f any one lethal was missing from the EMS 45-10 complex of the Group I lethals (Figure 2 of Chapter III), then the complementation map would resemble that of three overlapping deficiencies. This would imply that at least three genetic l o c i were associated with the EMS 45-10 complex. Another example is that of the Group V lethals (Figure 7 of Chapter III). Were i t not for the existence of EMS 34-4, the complementation of EMS 45-89 with the remaining Group V lethals would suggest that i t represented a lethal a l l e l e of a second locus. Only when adjacent complementing recessive lethals are separable by a definite deficiency can i t be said to be conclusively demonstrated that they represent lethal alleles of separate l o c i . Furthermore, the results of this study imply that, in Drosophila, when the complementation map of adjacent EMS-induced recessive lethals resembles that of overlapping deficiencies one i s , in reality, observing inte'riallelie complementation among lethal, alleles of a single locus.' In addition to the genetic analysis of the proximal heterochromatin in chromosome-2, the effects of various heterochromatic deficiencies and duplications on meiotic segregation, chromosome loss, and.recombination were assayed. Heterozygotes for Df(2 ) M-S2 1 0, a deficiency for.the 2R heterochromatic block, were tested for elevated rates of chromosome-2^ nondisjunction and chromosome loss (Appendix V). Virtually no spontaneous nondisjunction of chromosome loss for the second chromosome were observed in males or females heterozygous for the M(2)S10 deficiency. Thus the removal of the proximal heterochromatin to the right of the centromere of chromosome-2 does not result in a meiotically-unstable chromosome. In preliminary experiments (Hilliker, unpublished), however, i t was found that Df(2R)MtS2 1° heterozygosity results in a marked reduction in recombination for the right arm of chromosome-2. The nature and significance of this reduction awaits further experimental analysis. The Group C' deficiency was assayed for heterozygous nondisjunction and chromosome loss. Df(2L)C', in addition to being deficient for much of the 2L proximal heterochromatin, i s , on the basis of genetic and cytological evidence, duplicated for a large segment of the _2_R hetero-chromatic block. Heterozygosity for Df(2L)C i s not, however, associated with nondisjunction (or chromosome loss) of the second chromosome (Appendix V). Further, compound-second, autosomes bearing duplications of the proximal heterochromatin of thVother chromosome arm were assayed in males for increased meiotic segregation from the complementary compound. In his analysis of the meiotic segregation of several combinations of compound-second autosomes in males, Holm (19.69) . demonstrated an apparent random assortment of C(2L) and C(2R), with nearly, equal, frequencies of C(2L) ;. C(2R) ; nullo-2; and diplo-2^ sperm being produced. In my analysis of the meiotic segregation, in males, of compound autosomes bearing duplications of proximal heterochromatin of the opposite arm I witnessed no increase in the recovery of segregant classes (C(2L);;and.C(2R) sperm), indicating that these heterochromatic duplications had no effect on the segregation of compound autosomes (Appendix VI). Finally, this study has provided material for examining .the nature of recombination within heterochromatin. Lethal alleles have been - . generated for l o c i intercalated in the chromosome-2 heterochromatin; recombinants between these lethals can.be easily detected and. genetically confirmed; and only large-scale recombination experiments remain to determine whether or not recombination, and/or the related phenomenon of gene conversion (Chovnick, Ballantyne and Holm 1971), occurs within Drosophila heterochromatin. In conclusion, while i t i s clear from this study that heterochromatin is not genetically inert, i t i s evident that gene density is very low relative to euchromatin. Perhaps, as an analysis of the phenomenon of lt-variegation suggests (Appendix I), the genetically inactive regions serve to provide a necessary environment for the normal expression of genes intercalated in or adjacent to heterochromatic regions. However, although heterochromatin is not completely devoid of genes, the principal functions, of heterochromatin, i f any, remain undefined. LITERATURE CITED Abrahamson, S., I.H. Herskowitz and H. J. Muller, 1956 Identification of half-translocations,produced by X-rays in detaching attached-X chromosomes of Drosophila melanogaster. Genetics 41: 410-419. Arrighi, F. E., T. C. Hsu, S. Pathak and H. Sawada,,1974 The sex chromosomes of the Chinese hamster: constitutive heterochromatin deficient in repetitive DNA sequences. Cytogenet. Cell Genet. 13: 268-274. Baker, W. K., 1958 Crossing-over in heterochromatin. American Naturalist 92: 59-60. Baker, W. K., 1968 Position-effect variegation. Advances in Genetics 14: 133-169. Baldwin, M. C. and D. T. Suzuki, 1971 A screening procedure for the detection of putative deletions in proximal heterochromatin. Mutation Research 11:. 203-213. Barigozzi, C , S. Dolfini, M. Fraecaro, G. Rezzonico Raimondi and L. Tiepolo, 1966 In vitro study of the DNA replication patterns of somatic chronos^ToTBrosophlla melanogaster. Exp.. Cell Res. 43: 231-234. Bateman, A. J., 1968 Nondisjunction and isochromosomes from irradiation of chromosome 2 in Drosophila. In: Effects of radiation on meiotic  systems. Vienna: International Atomic Energy Agency. Botcham, M., R. Kram, C. W. Schmid and J. E. Hearst, 1971 Isolation and chromosomal location of highly repeated DNA sequences in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S. 68: 1125-1129. Bridges, C. B., 1916 Non-disjunction as proof of the chromosomal, theory of heredity. Genetics 1,: 1-52, 107-163. Bridges, C. B., 1935 Salivary chromosome maps. J. Heredity 26: 60-64. Brosseau, G. E. Jr., 1960 Genetic analysis of the male f e r t i l i t y factors on the Y-chromosome of Drosophila melanogaster. Genetics 45: 257-274. Brown, S. W., 1966 Heterochromatin. Science 151: 417-425. Chovnick, A., G. H. Ballantyne, D. L-. B a i l l i e and D. G.. Holm, 1970 Gene conversion in higher organisms: half-tetrad analysis of recombination within the rosy cistron of Drosophila melanogaster. Genetics 69: 179-209. Comings, D. E. and E. Mattoccia, 1972 DNA of mammalian and avian heterochromatin. Exp. Cell.Res. 71: 113-131. Cooper, K. W., 1959 Cytogenetic analysis of major heterochromatic elements (especially Xh and Y) in Drosophila melanogaster and the theory of "heterochromatin". Chromosoma 10: ,535-588. Darlington, C. D., 1939 Misdivision and. the genetics of the centromere. J. Genet. 37: 341-364. Darlington, C. D., 1940 The origin of isochromosomes. J. Genet. 39: 351-361. Demerec, M. and B. P. Kaufmann, 1969 Drosophila guide. 8th ed. Washington: Carnegie Institute. Fincham, J. R. S., 1966 Genetic complementation. New York: W. A. Benjamin, Inc. Gall, J. G., E. H. Cohen and M. L. Polan, 1971 Repetitive DNA sequences in Drosophila. Chromosoma 33: 319-344. Grell, E. H., 1970 Distributive pairing: mechanism for segregation of compound autosomal chromosomes in oocytes of Drosophila  melanogaster. Genetics 65* 65-74. Hannah, A., 1951 Localization and function of heterochromatin in Drosophila melanogaster. Advances in Genetics 4_: 87-125. Harrisson, C. M. H., 1971 The arrangement of chromatin in the interphase nucleus with reference to c e l l differentiation and repression in higher organisms. Tissue and Cell 3: 523-550. Heitz, E., 1928 Heterochromatin der Moose I. Jb. wiss Bot. 69: 762-818. Heitz, E., 1929 Heterochromatin, Chromozentren, Chromomenen. Ber.dtsch. bot. Ges. 47: 274-284. Heitz, E., 1933 Die somatische Heteropyknose bei Drosophila melanogaster und ihre genetische Bedeutung. Z. Zellforsch 20: 237-287. Hess, 0. and G. F. Meyer, 1968 Genetic activities of the Y chromosome in Drosophila during spermatogenesis. Advances in Genetics 14: 171-228. Hessler, A. Y., 1958 V-type position effects at the light locus in Drosophila melanogaster. Genetics « : 395-403. Hi l l i k e r , A. J., 1972 Deficiency mapping of,the proximal region of chromosome 2 of Drosophila melanogaster. Can. J. Genet. Cytol. 14: 729 (abstract). Holm,.D. G., 1969 The meiotic behavior of compound, autosomes in Drosophila melanogaster. Ph.D. Thesis, University of•Connecticut. Holm, D. G., M. C. Baldwin, P. Duck and A. Chovnick, 1969 The use of compound autosomes to, determine the relative centromeric position of chromosome three. Drosophila Information Service 44: 112. Holm, D. G. and A. Chovnick, 1975 Compound autosomes in Drosophila melanogaster: The meiotic behaviour of compound thirds. Genetics (in press). Holm, D. G., M. Deland and A. Chovnick, 1967 Meiotic segregation of C(3L) and C(3R) chromosomes in Drosophila melanogaster. Genetics 56: 565-566 (abstract). Judd, B. H., M. W. Shen and T. C. Kaufman, 1972. The anatomy and function of a segment of the X chromosome of Drosophila melanogaster. Genetics 71: 139-156. Kaufmann, B. P., 1934 Somatic mitoses of Drosophila melanogaster. J. Morphology 56: 125-155. Lefevre, G. Jr., 1974 The relationship between genes and polytene chromosome bands. Annual Review of Genetics 8-: 51-62. Leigh, B. and F. H. Sobels, 1970 Induction by X-rays of isochromosomes in the germ cells of Drosophila melanogaster males: Evidence for nuclear selection in embryogenesis. Mutation Research 10: 475-487. Lewis, E. B., 1950 The phenomenon of position effect. Advances in Genetics 3: 72-115. Lewis, E. B. and F. Bacher, 1968 Method of feeding ethyl, methane sulphonate (EMS) to Drosophila males. Drosophila Information Service 4j3: 193. Lim, J. K. and L. A. Snyder, 1868 The mutagenic effects of two mono-functional alkylating chemicals on mature spermatozoa of Drosophila. Mutation Research 6: 129-137. Lim, J. K. and L. A. Snyder, 1974 Cytogenetic and complementation analyses of recessive lethal mutations induced in the X chromosome of^Drosophila by three alkylating agents. Genetical Research 24: Lima-de-Faria, A. and H. Jaworska, 1968 Late DNA synthesis in hetero-chromatin. Nature 217: 138-142. Lindsley, D. L. and E. H. Grell, 1968 Genetic variations of Drosophila  melanogaster. Carnegie Institute of Washington Publ. No. 627. Moore, C. M. , 1971 Nonhomologous pairing in oogonia and ganglia of Drosophila melanogaster. Genetica 42: 445-456. Morgan, T. H., C. B. Bridges and J. Schultz., 1932 The constitution of the germinal material in relation to heredity.. Carnegie Inst. Wash. Ybk. 31: 303-307. Morgan, T. H., C. B. Bridges and J. Schultz, 1935 Constitution of the germinal material in relation to heredity. Carnegie Inst. Wash. Ybk. 34: 284-291. Morgan, T. H. and J. Schultz, 1942. Investigations on the constitution of the germinal material in relation to heredity. Carnegie Inst. Wash. Ybk. 41: 242-245. Morgan, T. H., J. Schultz and V. Curry, 1940 Investigations on the constitution of the germinal material in relation to heredity. Carnegie Inst. Wash. Ybk. 39: 251-255. Morgan, T. H., J. Schultz and V. Curry, 1941 Investigations on the constitution of the germinal material in relation to heredity. Carnegie Inst. Wash. Ybk. 40: 282-287. Muller, H. J. and T. Painter, 1932 The differentiation of the sex chromosomes of Drosophila into genetically active and inert regions. Mu. Indukt. Abstamm. - Vererb. Lehre 62: 316-365. Mukai, T., 1964 The genetic structure of natural populations of Drosophila melanogaster. I. Spontaneous mutation rate of polygenes controlling v i a b i l i t y . Genetics 50: 1-19. Parker, D. R., 1954 Radiation induced exchanges in Drosophila females. Proc. Natl. Acad. Sci. U . S . ^ j ; 795-800. Parker, D. R. 1969 Heterologous interchange at meiosis in. Drosophila. II. Some disjunctional consequences of interchange. Mutation Research ]_: 393-407. Parker, D. R. and A. E. Hammond, 1958 The production of translocations in Drosophila oocytes. Genetics 43: 92-100. Parker, D. R. and J. H. Williamson, 1970 Heterologous interchange at meiosis in Drosophila. III. Interchange-mediated nondisjunction. Mutation Research 273-286. Peacock, W. J., D. Brutlag, E. Goldring, R. Appels, C. W. Hinton and D.~.L. Lindsley, 1973 The organization of highly repeated DNA sequences in Drosophila melanogaster chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38: 405-416. Plough, H. H., 1941 Spontaneous mutability in Drosophila. Cold Spring Harbor Symp. Quant. Biol. 9: 127-137. Ritossa, F. M., K. C. Atwood and S. Spiegelman, 1966 A molecular explanation of the bobbed mutants of Drosophila . as .partial deficiencies of ribosomal DNA.. Genetics .54.: 819-834. Rudkin, G. T., 1969, Nonreplicating DNA in Drosophila. Genetics (Suppl.) 61: 227-238 Schalet, A. and G. Lefevre Jr., 1973 The localization of "ordinary" sex-linked genes in section 20 of the polytene X chromosome of Drosophila melanogaster. Chromosoma 44: 183-202. Schultz, J., 1936 Variegation in Drosophila and the inert chromosome regions. Proc. Natl. Acad. Sci. U.S. 22:, 27-33. Schultz, J. 1941 The function of heterochromatin. Proc. 7th Intern. Cong.,, Genet. 257-262. Schultz, J. and T. Dobzhansky, 1934 The relation of a dominant eye color in D. melanogaster to the associated chromosome rearrangement. Genetics 19: 344-364. Sederoff, R., L. Lowenstein and H. C. Birnboim, 1975 Polypyrimidine segments in Drosophila melanogaster DNA: II chromosome location and nucleotide sequence, (submitted for publication.) Sharma, A. K. and A. Sharma, 1972 Chromosome t e c h n i q u e s 2 n d ed. London: Butterworths. Stevens, W. L., 1942 Accuracy of mutation rates. J. Genet. 43: 301¬307. Vosa, C. G., 1961 A modified acetic-orcein method for pollen mother cells . Caryologia 14: 107-110. Walker, P. M. B., 1971 Origin of Satellite DNA. Nature 229: 306¬308. Yunis, J. J. and W. G. Yasmineh, 1971 Heterochromatin, sa t e l l i t e DNA and c e l l function. Science 174: 1200-1209. APPENDIX I ON THE NATURE. OF POSITION-EFFECT VARIEGATION OF THE LIGHT LOCUS The light locus of Drosophila melanogaster i s located near the heterochromatic-euchromatic junction of the l e f t arm of chromosome-2 (Schultz 1936; Chapter II). Position-effect variegation of this locus was f i r s t investigated by Schultz and his collaborators ;(Schultz and Dobzhansky 1934; Morgan, Bridges and Schultz 1932, 1935; Morgan, Schultz and Curry 1941; Morgan and Schultz 1942; Schultz 1936, 1941). Light variegation was readily induced by X-rays and was found to be associated with chromosomal rearrangements involving one breakpoint in the proximal region of 2L and the other in the euchromatin of the X, second, or third chromosome?. Hessler (1958) using X-radiation induced a number of light-variegating chromosomal rearrangements and examined the breakpoints in the salivary gland and somatic chromosomes. From this cytological analysis i t was clear that in most light-variegating chromosomal rearrangements one breakpoint was in the distal, euchromatin of the X—chromosome or an autosome, and the second breakpoint proximal to the 2L heterochromatic-euchromatic junction. Hessler interpreted these data to imply that the light locus was located in or next to the proximal heterochromatin of 2L and that the removal of this locus from heterochromatin to distal euchromatin, i.e. euchromatin far removed from a centromere, resulted in. variegation. However, as Hessler recognized, i t could not be determined whether the light locus was actually displaced. The breaks may have been dist a l to the light locus, the juxtaposition of previously distal euchromatin with the basally located light locus resulting in variegation. The fortuitous construction of a 2jJ3 translocation from a compound second autosome bearing female enabled me to. obtain a light-variegating rearrangement in which i t could be conclusively, demonstrated.that the light locus was transposed (i.e. distal.to the 2L breakpoint) .. This 2^3 translocation was constructed by the irradiation of C(2L)SH3,rh;G(2R)SH3,+ females and is referred to as the Group E lethal in Chapter II. As described in Chapter II, this lethal arose from a 3 hit event that translocated 2L, including a large block of heterochromatin proximal to the 2L secondary constriction to the right arm of chromosome-3 at the 92E-F region of Bridges' polytene chromosome map (see Lindsley and Grell 1968). The right arm of. chromosome-^ distal to 92E-F is appended to the centromere-bearing detachment of C(2R) (the, break in C(2R)SH3,+ was in the proximal, heterochromatin). The centric 2L and acentric 2R fragments, which together represent the reciprocal product of the translocation were lost. Since C(2R)SH3,+ does not. carry a l t ^ duplication, one may follow with certainty the It locus, as the translocation derived i t s 2L from C(2L)SH3,+, the centromere^bearing fragment of which was lost during the construction of this 2^3 quasi-reciprocal translocation. The Group E translocation proved to be very weakly homozygous viable and progeny homozygous for the translocation or heterozygous for the translocation and It had a light-variegated phenotype. In addition, the Group E translocation was lethal in combination with the 2 Group C It deficiencies (see Chapter II). Further, upon careful examination of the somatic chromosomes i t was found that virtually, the whole of 2L including the 2L heterochromatic block was translocated to the distal euchromatin of 3R. From the above observations i t is clear that i t i s the displacement of the light locus to distal euchromatin, rather than the displacement of previously distal euchromatin into.a position in the vic i n i t y of the light locus,,.that resulted in light-variegation. However, i t would appear, that, i t is not the removal of light from heterochromatin and i t s juxtaposition with euchromatin that is responsible for light variegation, but rather the removal, from the centromere of the heterochromatic block with which I t . i s associated. That i s , in order for the light locus to function normally, the hetero-chromatic block with which i t i s associated must be near a centromere. Thus, although the heterochromatin immediately adjacent to the It locus may remain undisturbed by. a particular chromosomal rearrangement, light variegation w i l l result i f the 2L heterochromatic block has been displaced far from a centromere. Recent studies indicate that the centromeric heterochromatin of eukaryotes i s bound during interphase to the nuclear membrane (see Harrisson 1971). Thus genes associated with centromeric heterochromatin may have a somewhat defined environment. It may be that the centromere migrates to the nuclear membrane when the nuclear membrane reforms subsequent to the i n i t i a t i o n of telophase. Heterochromatin associated with the centromere may then bind to the nuclear membrane. However, i f by a chromosomal rearrangement, heterochromatin i s moved some.distance from the centromere, the probability of binding to the nuclear membrane may be reduced. That i s , the further heterochromatin i s removed from the centromere, the less the probability of it's binding to the nuclear membrane. It is postulated, that the association of the 2L heterochromatic block with the nuclear membrane is necessary for the normal function of the light locus and that the inhibition of this association by translocating It and-2L heterochromatin to dist a l euchromatin results in light variegation. In conclusion, light variegation results from the displacement of the light locus and i t s associated heterochromatic block to a position well removed from a centromere. It is speculated that the physiological basis of light variegation may l i e in the effect that the binding of constitutive heterochromatin to the nuclear membrane:has on the functioning of individual heterochromatic l o c i . APPENDIX II AN IMPROVED. TECHNIQUE FOR POLYTENE CHROMOSOME SQUASH PREPARATIONS IN DROSOPHILA INTRODUCTION In the past, obtaining consistently good polytene chromosome preparations from the larval salivary glands of.. Drosophila. melanogaster has required optimal conditions - f u l l y mature larvae (prepupae) raised in uncrowded cultures at low temperature (Demerec and Kaufmann 1969) . By a single mechanical modification of the standard squash technique I have been able to obtain routinely superior preparations under less than optimal conditions. .. Moreover, this modification, allows one easily to obtain good preparations of complex chromosomal rearrangements, heretofore rather d i f f i c u l t to achieve even.under optimal, conditions. MATERIALS AND METHODS 1. Dissect third instar Drosophila larvae in. a large drop of 50% acetic acid on a depression slide. (Dissection in 45% acetic acid was suggested by T. Kaufman. One may also dissect in Drosophila Ringers' but dilute acetic acid gives better preparations.) 2. Transfer the salivary glands to a. drop of 2% aceto-lacto-orcein (Vosa 1961) on a siliconized slide. 3. Place the coverslip on the drop of stain in which the salivary glands are immersed. Use 18 or 22 mm^  coverslips. 4. Tap the coverslip gently 10 or more times directly over the glands with the blunt end of a pair of forceps. This is the innovative step and the most important in the procedure. Although one taps gently, the glands spread out considerably. The exact strength of the taps w i l l be best learned by experience. The coverslip moves laterally somewhat but this movement should be minimal. 5. Squash the glands with the thumb, applying firm, hut not excessive pressure through paper towelling directly.on the coverslip and over the glands. Do not move the coverslip laterally.while squashing. Seal the preparation with clear n a i l polish or wax. 6. Observe with phase contrast optics. RESULTS Well spread polytene chromosomes are obtained routinely under less than optimal conditions. One may virtually disregard temperature, and overcrowding and precise larval age are of less concern.. One can rely on several good spreads from each preparation, with minimal chromosome breakage and distortion. With complex chromosome rearrangements the results are particularly pleasing. Normally, even under optimal conditions, good polytene chromosome spreads of such rearrangements are d i f f i c u l t to obtain. One may resort to extreme methods, such as squashing with excessive force or pressing down ontthe coverslip directly over the salivary glands with the eraser end of a pencil, but these techniques are inefficient and often result in lateral movement of the coverslip and broken or distorted chromosomes that usually are not well spread. By the new method reported herein, however, one may easily obtain good spreads of complex rearrangements without chromosome breakage or distortion. DISCUSSION In the unmodified squash technique nuclei were ruptured and chromosomes were spread and fixed between the slide and coverslip simultaneously. The taps probably "shock the nuclei and chromosomes, bursting the nuclei and forcing the chromosomes to disperse, giving them time to do so before fixing them between the coverslip and slide. Once the chromosomes are fixed between the coverslip and slide, additional squashing is relatively ineffective in promoting further chromosome dispersal. APPENDIX III PREPARATION OF SOMATIC MITOSES OF DROSOPHILA . AND CHROMOSOME IDENTIFICATION Chromosome, preparation (modified from Moore 1971) 1. Select an active mature third-instar larvae.and dissect out the brain in Drosophila Ringers' (0.75% NaCl). 2. Place the brain in 1% Na Citrate for 30 to 90 seconds. 3. Place the tissue in a drop of 2% aceto-lacto-orcein (Vosa 1961) on a siliconized.slide and allow i t to f i x and stain for 5 minutes at room temperature. Prefixation in any of acetic-methanol (various proportions); and Farmer's; Navashin's; Carnoy's; Flemming's; and Benda's fixatives (Sharma and Sharma 1972), I found to be of l i t t l e value save for Benda's fixative i f one desires to observe anaphase chromosomes. 4. Drop a coverslip onto the drop of stain. 5. Squash with the thumb, applying firm pressure through paper towelling directly on the coverslip and over the tissue. . Do not move the coverslip laterally while squashing. 6. Seal the edges of the coverslip with clear n a i l polish or wax. 7. Observe the preparation 24 to 48 hours later with phase-contrast optics. Chromosome.Identification This account i s based largely on my own observations. Valuable published descriptions of-Drosophila melanogaster somatic chromosome morphology include Kaufmann (1934) and Cooper (1959). Drosophila melanogaster has four pairs of chromosomes, one pair of which i s heteromorphic in the male (the X/Y bivalent). The X-ehromosome is acrocentric; the Y-chromosome, submetacentric; the second and.third chromosomes are metacentric; and the tiny fourth chromosome is probably acrocentric. The proximal third of the X-chromosome is heterochromatic. This heterochromatic region is often subdivided into two equal blocks by a prominent secondary constriction, viz. the NO (nucleolar organizer). Much less frequently, other, smaller constrictions are observed which further subdivide the X-chromosome heterochromatin (see Cooper 1959). The Y-chromosome is completely heterochromatic. A NO is present on the short arm (Y^) . Six other Y-chromosome secondary constrictions have been observed (Cooper 1959)/two of which, on the long arm (Y_b , I have frequently observed. The major autosomes, chromosomes 2 and _3, are of roughly equal size, the third chromosome being somewhat longer. In overall length the major autosomes are roughly twice the size of the X-chromosome. The centromeres of these chromosomes are bounded on each side by heterochromatin. The proximal quarter (roughly) of each autosomal arm is heterochromatic. In favorable preparations i t can be observed that while the 3L and 3R heterochromatic blocks are of approximately equal mass, the 2R hetero-chromatic block i s somewhat larger than that of 2L. Further, the hetero-chromatic-euchromatic junction of 2L is the site of a very frequently observed secondary constriction (Kaufmann 1934).. This 2L secondary constriction is as prominent as that associated, with the X-chromosome NO and may be considered a very important landmark. Much less frequently, secondary constrictions are observed at the heterochromatic-euchromatic junctions of the other autosomal arms, although never at the hetero-chromatic-euchromatic junction of the X-chromosome. I have observed in early prophase nuclei indications of secondary constrictions within the autosomal heterochromatic blocks. However, these constrictions are not prominent and are not observed at late prophase. The distribution of heterochromatin on,the small fourth chromosome is highly debatable. Constrictions are also observed occasionally within the=:euchromatin; although this i s controversial. Often I have observed what appears to be a constriction in the distal third of the X-chromosome euchromatin. The l e f t and right arms of chromosome three can be differentiated easily only during early prophase, in which the greater length of 3R i s , in favourable preparations, clearly observable. Chromosome morphology is more distinct in early than in late prophase nuclei. In early prophase nuclei the chromatids remain in apposition, that is each chromosome is not separated into two chromatids. The heterochromatic blocks are easily separable from euchromatin on the basis of their much higher degree of condensation. It i s in early prophase nuclei that secondary constrictions may be most profitably studied. However, given the lack of condensation of the euchromatic regions, the chromosomes are less distinct, longer, and overlap each other considerably. By late prophase the euchromatic portions of. the chromosomes have separated into chromatids. The heterochromatic-.regions, however, remain together and this property of chromatid apposition is characteristic, in fact diagnostic, of heterochromatin in late prophase. Although the heterochromatic. regions may be resolved into chromatids precociously (usually by energetic squashing'.), ordinarily they do not separate into chromatids until the onset of anaphase. By late prophase the euchromatin has considerably.condensed and the differential staining intensity between i t and heterochromatin is much less marked. Prominent secondary constrictions are less pronounced and smaller constrictions usually are no longer distinguishable. Late prophase chromosomes are more easily separated by squashing than those of early prophase and are more distinct. For routine karyotype analysis late prophase figures are most suitable. However, for detailed morphological analysis early prophase nuclei must be studied. APPENDIX IV THE LOCATION OF THE ROUGHISH LOCUS The roughish (rh) locus, the mutant recessive a l l e l e of which i s associated.with a rough eyed phenotype, i s reported to map at 54.7 on the second chromosome of Drosophila melanogaster (Lindsley and G r e l l 1968). This places rh i n the proximal region of 2L, d i s t a l t o . l t , t r i , l ( 2 ) c r c , esc, and B l . (For a d e s c r i p t i o n of these and.other genetic markers discussSd i n t h i s section see Table I of Chapter II.) As rh i s a proximal, gene, the rh mutant a l l e l e was tested against the proximal d e f i c i e n c i e s described i n Chapter II for pseudodominance i n order to asc e r t a i n whether the rh locus f e l l within theheterochromatic regions defined by these d e f i c i e n c i e s . However, none of the proximal d e f i c i e n c i e s uncovered the rh locus. Coincident with the t e s t i n g of chromosome-^'\proximaT mutations with proximal d e f i c i e n c i e s , attempts were made to asc e r t a i n the accuracy of assignment of these l o c i to chromosome arms. Deciding on which side of the centromere a proximal locus l i e s i s d i f f i c u l t to accomplish by conventional genetic mapping. A superior method of determining on which arm of an autosome a proximal locus i s situated i s to construct compound autosomes f o r the chromosome i n question from s t r a i n s homozygous for a mutant a l l e l e of the proximal gene (Holm, Baldwin, Duck and Chovnick 1969). I f the gene's mutant phenotype i s associated withfthe newly induced compound l e f t autosome, then the locus i s to the l e f t of the centromere. S i m i l a r l y , i f the mutant phenotype i s associated with newly induced compound ri g h t autosomes, then the gene i s i n the r i g h t arm. By t h i s method D. Holm has confirmed the assignment of I t and H. Harger, the assignment of. B l to 2L; and D. Holm has confirmed the assignment of r l and pk, J . Gavin, the assignment of stw, W. Gibson the assignment of ap, and A H i l l i k e r , the assignment of M(2)S2, tuf, and l t d to 2R. However, I found that rh,which had been tentatively assigned to 2L (Lindsley and Grell 1968) was, in fact, actually located on 2R. From the irradiation (4250 rads) of 355 rh/rh virgin females which were subsequently single pair mated to C(2L)P,b C(2R)VHkl,rlcn males and brooded for 7 days, 44 exceptional F l progeny were obtained (Table I ) . Of these exceptional progeny, 2 were matroclinous; 33, patroclinous; and 9 represented newly induced compound autosomes. The rh phenotype was associated with a l l newly induced C(2R) but none of the 5 newly induced C(2L) chromosomes, thereby demonstrating that rh is on the right arm of chromosome two and not the l e f t as previously reported. (Note that a l l 5 of the newly induced C(2L)'s gave a r l + phenotype in combination with C(2R)VHkl,rlcn. Many C(2L) chromosomes, synthesized from a variety of strains, have been found (by W. Gibson, T. Yeomans, D. Holm, and myself) to carry r l + duplications of 2R.) . TABLE I Exceptional progeny recovered from irradiated vh/rh virgin females crossed to C(2L)P,b C(2R)VHKlJrlcnr:males: Phenotype Number recovered Phenomenon rh (matroclinous) Nondisjunction b; vl; on (patroclinous) 33 ion or Nondisjunction Chromosome loss on Newly induced C(2L) bjrh Newly induced C(2R) APPENDIX V PRELIMINARY EXPERIMENTS ON THE EFFECTS OF HETEROZYGOSITY FOR SECOND CHROMOSOME PROXIMAL DEFICIENCIES ON MEIOSIS It has been suggested (Walker 1971) that centromeric heterochromatin may promote the i n i t i a t i o n of meiotic pairing of homologous chromosomes and, further, protect the centromere from the "rigours of meiosis" -presumably the terminalisation of chiasmata and subsequent reductional segregation of homologous dyads. This hypothesis suggested - the following experiments, namely assaying the effects of chromosome-2 proximal deficiencies on second chromosome nondisjunction and loss. Walker's theory (loc. cit.) would suggest that heterozygosity for second chromosome heterochromatic deficiencies w i l l result in appreciable second chromosome nondisjunction and chromosome loss. The proximal deficiencies studied were Df(2R)M-S210, which, as previously described (Chapter II) is deficient for the 2R :heterochromatic block and Df(2L)C' which is undoubtedly deficient for much of the 2L proximal heterochromatin (Chapter II), although cytological observations imply that i t must therefore be duplicated for much of the 2R heterochromatin Dff2R->MS-in: Virgin females heterozygous for Df(2R)M-S210 and b pr cn (for a description of these mutations see Table I of Chapter II) were crossed, singly in vi a l s , to C(2L)VHl,lt; C(2R)P,px males and brooded for six days. As compound-second autosome bearing males produce nearly equal frequencies of the four classes of C(2L) ; C(2R); nullo-2; and diplo-2 sperm (Holm 1969), nullo-2 and diplo-2 female gametes, the C OT1S 6 (J U 6T1C 6 of second chromosome nondisjunction or chromosome loss, may be recovered as viable zygotes with 25% efficiency. Thus by the use of multipliers to estimate the total number of f e r t i l i z e d eggs (see MATERIALS AND METHODS) section of Chapter II), one may assay second chromosome loss and non-disjunction in Drosophila females by mating them with compound second autosome bearing males. From an estimated 4844 f e r t i l i z e d eggs, no nondisjunctional progeny were recovered. Thus neither chromosome loss nor nondisjunction of chromosome two is associated with heterozygosity for Df(2R)M-S21Q. The absence of chromosome loss is a partial refutation of Walker's (loc. cit.) theory of the protection of centromeres by flanking heterochromatin. No newly induced isochromosome bearing exceptions (a possible consequence of chromosome "breakage") or patroclinous progeny were observed. Clearly the M(2)S10 chromosome is stable despite the absence of the 2R hetero-chromatic block. The upper 95% confidence limit of second chromosome loss, and therefore chromosome instability is 0.25%. (Nor did M(2)S10/b pr cn heterozygotes prove particularly sensitive to radiation-induced non-disjunction. From mating Df(2R)M-S210/b pr cn females irradiated with 2000 rads of gamma radiation to C(2L)VHl,lt;C(2R)P,px males only 1 matroclinous progeny was observed in g gg g although 33 patriclinous progeny and 15 newly induced compound second autosome bearing exceptional progeny were recovered ) Second chromosome nondisjunction, and loss, was also assayed in males heterozygous for Df(2R)M-S210 and In(2LR)SMl. Df(2R)M-S210/ln(2LR)SMI males were crossed to BSY;C(2L)P,b;C(2R)P,px virgin females, singly in vials and brooded for 9 days. B^ Y i s a Y-chromosome carrying a small duplication of the X-chromosome including a dominant al l e l e of the Bar (B) locus (see Lindsley and Grell 1968 for further details). S B Y;C(2L)P,b;C(2R)P,px females produce 40% second chromosome non-segregational progeny when crossed to compound second autosome bearing males. Thus 20% of the female gametes are diplo-2 and.20% are nullo-2 (see Appendix. VI). Consequently, nondisjunction for chromosome 2 s may be assayed in males by mating them to B Y;C(2L)P.,b;C(2R)P,px. females. In order to estimate the number of nondisjunctional.female gametes produced per female per v i a l , a sample of BSY;C(2L)P,b:C(2R)P,px virgin females were crossed to C(2L)SH3,+;C(2R)SH3,+ males. As these males produce approximately 25% diplo-2 and 25% nullo-2 sperm, the nondisjunctional progeny represent 1/4 of the nondisjunctional female gametes. Thus,- the* number of nondis junctional female gametes per experimental v i a l may be estimated as 4 times the number of non-disjunctional progeny per multiplier v i a l . However, in the cross to the i n Df(2R)M-S2 /In(2LR)SMl males a nondis junctional (diplo-2 or nullo-2') sperm f e r t i l i z i n g a nondisjunctional female gamete has a 50% chance of resulting in a viable, diplo-2 zygote. Thus chromosome-2 nondisjunction per experimental v i a l may be estimated as 2 times the number^of exceptional (i.e. nondisjunctional) progeny per experimental v i a l divided by 4 times the number of non-dis junctional progeny per multiplier v i a l . In the experimental series, an estimated 8810 nondisjunctional female gametes resulted in no diploid nondisjunctional progeny. (The only exceptional progeny recovered were one tr i p l o i d female and 2 intersexes.) Thus a 95%.upper.confidence limit on chromosome-2 nondisjunction in Df(2R)M-S210/ln(2LR)SMI heterozygotes of 0.068% is established. With chromosome-2 nondisjunction and chromosome loss less than 0.1% in males and 0.3% in females heterozygous for Df(2R)MS-10, the loss of the heterochromatic block to the right of the centromere of chromosome-2. clearly does not result in any meiotic instability of the second chromosome. Df (2L)C Virgin females heterozygous for .Df(2L)C and b pr cn were mated to C(2L)VHl,lt;C(2R)P,px males and brooded for 6 days. In an estimated 31,200 f e r t i l i z e d eggs, 2 matroclinous and 1 patroclinous progeny were recovered. The frequency of spontaneous second chromosome nondisjunction in Df(2L)C heterozygotes i s 4x3/31,200 or 0.038% with 95% confidence limits of 0.008% and 0.112% (Stevens 1942), well within the range observed for Drosophila females homozygous for normal second chromosomes (W. Gibson, personal communication). Although the genetic evidence (Chapters II and III) strongly argued that Df(2L)C was deficient for much of the 2L heterochromatin, upon cytological examination of the Df(2L)C somatic chromosome I found a substantial block of heterochromatin to the l e f t of the centromere. This could be explained by the following hypothesis. In the construction o f Df(2L)C from the detachment of C(2L)SH3,+;C(2R)SH3,+ the acentric 2L fragment was generated by a break in the distal 2L heterochromatin (that this break was proximal to the secondary constriction at the 2L heterochromatic-euchromatic junction was clear, as Df(2L)C was not deficient for this constriction) with the centric 2R fragment being generated by a break in the distal heterochromatin of 2R resulting in a centric 2R fragment dujsiicai&S.: f o r m u c h o f ; t h e 2R heterochromatin including the r l * locus. Df(2L)C therefore would be a 2L proximal deficiency but a 2R proximal duplication with a r l + locus on each side of the centromere. In order to test the hypothesis I constructed.with radiation non-sister 2L compound autosomes (compound autosomes with one 2L chromatid from one second chromosome and the other.2L chromatid from i t s homologue)'. from females heterozygous for Df (2L)C and b pr cn. If Df(2L)C carries a r j * duplication in the l e f t arm them compound l e f t autosomes deriving one. arm from the Df(2L)C chromosome should more frequently carry r l * duplications of 2R than do compound lefts derived from normal second chromosomes. Of 21 nonsister compound l e f t autosomes, derived from Df(2L)C'/b pr cn heterozygotes, 17 were r l * whereas Yeomans (1972) found only 10 of 21 compound l e f t second chromosomes derived from It stw3/b pr cn heterozygous females were r l * and W. Gibson (unpublished), upon examining 39 compound l e f t second autosomes derived from females of a number of different heterozygous genotypes, found that 25 carried a duplication r l * . Thus Df(2L)C would appear to be duplicated for r l * and, therefore, much of the 2R heterochromatin. This conclusion i s also supported by biochemical evidence. Sederof et a l . (1975) have found that the highly repeated polypyrimidine sequence TCTTC is localized to the Y-chromosome and. the 2R heterochromatin. J. Stone (personal communication) has found that the nuclear DNA of Df(2L)C heterozygotes has a larger fraction of the TCTTC repeated pentamer than does the nuclear DNA of wild type Drosophila. APPENDIX VI HETEROCHROMATIC DUPLICATIONS AND THE MEIOTIC SEGREGATION OF COMPOUND AUTOSOMES IN MALE DROSOPHILA Holm (1969) demonstrated that in the several strains he examined compound second autosomes segregated randomly during male meiosis producing equal frequencies of C(2L); C(2R>; diplo-2 ..(C(2L) ;C(2R)) and nullo-2 (neither C(2L) nor C(2R)) bearing sperm. In female.Drosophila, however, Holm found that the C(2L) chromosome regularly segregates from the C(2R) chromosome. In order to examine the role, i f any, of heterochromatic homology in meiotic pairing in males, the segregation of compound autosomes bearing duplications for heterochromatic material of the complementary compound autosomes was assayed. Segregation was assayed by crossing males of the selected compound-second autosome bearing strains to differentially marked compound-second autosome bearing females possessing a Y-chromosome. These B^Y,; C(2L)P,b; C(2R)P,px females give, as f i r s t demonstrated by E. H. Grell (1970)for B^Y; C(2L); C(2R) bearing females in general, a high frequency of compound-second autosome nonsegregation. Female gametes nonsegregational for the compound second autosomes w i l l result in a viable zygote only i f f e r t i l i z e d by a sperm nonsegregational for the paternal compound-second autosomes. Thus a strain in which compound-second autosomes partially segregate in males when crossed to BSY; C(2L)P,b; C(2R)P,px females w i l l give a lower frequency of progeny completely matroclinous or patroclinous for the two compound-second autosomes than w i l l a strain in which C(2L) andilC(2R) segregate at random in the male. To examine the effect, i f any, of heterochromatic homology on compound-second autosome meiotic segregation in males, males of the C(2L)SH3+;C(2R)SH3+ strain (a strain in which nearly equal frequencies " of C(2L); C(2R); diplo-J2; and nullo-2 sperm are produced) and of several-other strains in which one or both compound autosomes bore heterochromatic duplications of the other arm were crossed singly in shell vials to B Y;C(2L)P,b;C(2R)P,px virgin females. The results are presented in Table I and in summary form in Table II. Since there is no significant reduction in the frequency of non-segregation in those crosses involving males carrying C(2L) and C(2R) chromosomes with measurable heterochromatic homology (Table II), i t i s apparent that heterochromatic. homology is not a major factor in regulating segregation in males of.Drosophila melanogaster. CM TABLE I Progeny of BSY; C(2L)P,b: C(2L)P,b; C(2R)P,px females and various compound second autosome bearing males. Chromosome from mother BSI BSY BSY- . • BSY C(2L)P,b; C(2L)P3b; C(2R)PJPX C(2R)P3px E(2R)F>P* •C(2R)P,px C(2L)P3b C(2L)P3b • 0 Total Male genotype + C(2L)SHZ3 + C(2R)SH3,+ C(2L)SHl3Dp(2R)rl> C(2R)SH1J + C(2L)SHl,Dp(2R)vl C(2R)SH3J + C(2L)SK3,+ C(2R)VK23Dp(2L)lt+,bw C(2L)VHl3Up(2R)rl+,lt C(2R)VK2,Dp(2L)lt+,bw 462 567 57 127 122 300 395 39 153 127 273 204 279 247 5 1771 365 286 383 447". 4 2448 46 102 31 47 44 0 264 146 122 174 21$ 3 940 80 146' 163 1 742 TABLE I I . Frequency of progeny nonsegregational.for compound-second autosomes from BSI; C(2L)P3b; C(2R)P3px v i r g i n females crossed to various s t r a i n s of compound-second autosome bearing males. C(2L) C(2R) Percent nonsegregational progeny N SH3+ SH3+ 40.4 1771 SE1+ SH1+ 41.6 2448 SH1+* SH3+ 38.3 264 SH3+ VK2bw** 36.7 940 VHllt VKSba** 38.7 742 * Bears a vl+ d u p l i c a t i o n of 2R. Bears a l t + d u p l i c a t i o n of 2L. 

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