The term, heterochromatin, refers to those portions of a chromosome which stain very darkly early in the division cycle at a time when most of the chromosomal material stains very lightly. After the discovery of the mutagenic effects of X-rays, large numbers of mutants were recovered in Drosophila. These mutants were genetically mapped but none of them seemed to be located in the heterochromatic regions of the chromosomes. This observation led to the hypothesis that heterochromatin was genetically inert. Since then the problem of the function of heterochromatin has engaged the attention of many different researchers. One of the few mutants known to reside in the proximal heterochromatin of the X chromosome is bobbed. Ritossa and Spiegelman (1965) have presented evidence that bobbed is the nucleolus organizer, the site of ribosomal RKA synthesis. Their results suggest that this is a highly redundant region of the chromosome. If heterochromatin in general is greatly redundant, this would offer one explanation for the apparent lack of genetic loci. A mutant phenotype might result only after large blocks of the chromosome have been deleted. A scheme involving the use of attached-autosomes to select for those third chromosomes which have sustained breaks in the proximal heterochromatin was devised. In general, virgin females were irradiated and attached-third chromosomes were selected for by mating these females to attached-third males. Female progeny carrying these newly synthesized attached-autosomes are, in turn, irradiated and newly reconstituted chromosomes were selected for by mating the females to males carrying normal third chromosomes. Thus, each newly reconstituted chromosome has sustained a number of breaks at different positions in the proximal heterochromatin. Using this procedure, we recovered sixty-six homozygous lethal chromosomes. Fifty-three of these sixty-six lethals may be placed in one of the six complementation groups, two of which appear to be multisite deletions. In addition, these fifty-three lethals appear to be located, by the results of genetic mapping, in the region spanning the centromere. These two pieces of evidence suggest that the method used here does, indeed, select for deletions in the proximal heterochromatin. / Science, Faculty of / Zoology, Department of / Graduate

The monofunctional ethylating agent EMS has been found to induce ts lethal mutations in Drosophila melanoqaster. Temperature sensitive lethals can be induced on both the X and the second chromosomes. The frequencies of ts lethal induction with respect to the estimated number of "single hit" lethal mutations are not significantly different (for the X chromosome 12.5% and for the second, 10.9%). The ts lethals from each chromosome have similar visibilities at high and low temperatures. As with the ts lethals on the X chromosome, Suzuki et al, 1967) those on the second behaved in a manner compatible with the suggestion that Drosophila ts lethals are similar to those found in micro-organisms. These mutants, particularly dominant ts lethals, may provide powerful tools for Drosophila genetic investigation. / Science, Faculty of / Zoology, Department of / Graduate

The experiments reported in the present dissertation were undertaken to obtain further evidence for the possible roles of protein, RNA and DNA macromolecules in radiation-mutagenesis in Drosophila melanogaster. Several antibiotics were tested for their modifying effects on the frequency of radiation-induced sex-linked recessive lethals. Pre-radiation treatment with actinomycin D significantly reduces the frequency of induced mutations in germ cell stages assumed to include spermatids and spermatocytes. These results are consistent with the hypothesis of a role of proteins in the stabilization (repair) of radiation-induced premutational lesions. Puromycin, a specific inhibitor of protein synthesis is ineffective in the modification of induced mutation frequencies in Drosophila melanogaster. Mitomycin C, is itself a potent mutagen in all germ cell stages, peak mutagenicity occurring in spermatid stages. In combination with ɣ-rays, mitomycin C shows an overall additivity of effect. Mutation frequencies due to mitomycin C are not altered by pre- or concurrent treatment with actinomycin D. This may indicate a different mechanism for mutagenesis by mitomycin C and radiation. / Science, Faculty of / Zoology, Department of / Graduate

Polyhomeotic (ph) is a developmentally important gene in Drosophila melanogaster which has been genetically characterized and recently cloned. ph is genetically and molecularly complex and has a strong maternal effect. Analysis of null or amorphic alleles reveal phenotypic effects that include embryonic lethality, cell death of the ventral epithelium, homeotic transformations, and alteration in the pattern of axon pathways. Two independent point mutations are required to produce a ph null allele. I have shown that the ph locus contains two, large, highly conserved, tandem repeats that are both transcribed. I have identified transcripts that are altered in ph mutants and that are developmentally regulated. Fourteen cDNA's have been isolated, and mapped. Northern and Southern blot analysis, and comparisons between cDNA and genomic restriction maps shows that the cDNAs represent at least 4 different transcripts that include distinct products of both repeats as well as non-repeated sequence. Both the genetic behavior and molecular organization of the ph locus are unique in Drosophila. / Science, Faculty of / Zoology, Department of / Graduate

Multigene families have posed an acute problem for evolutionary biologists ever since the revelation that many families exhibit unexpected sequence homogeneity within and between individuals of a species. A family that is shared between several species, in contrast, often reveals substantial heterogeneity between them. This cohesive and species-specific pattern of variation, which disengages from the classical mode of random genetic drift and selection, has been formally described as Molecular Drive (Dover, 1982). Based on initial observations (Cribbs 1982), the tRNA₄Ser and tRUA₇Ser genes on the X-chromosome of Drosophila melaaogaster also showed intriguing characteristics reminiscent of Molecular Drive. However, in this unusual case, the coevolution process would not only encompass the individuals within a family, but would also ensnare members from a different family. This thesis is an in depth study on the concerted evolution of both gene families and provides evidence consistent with the view that they are undergoing Molecular Drive. Eight tRNA₄,₇Ser genes have been cloned from bands 12DE on the X-chromosome of D metanogaster by molecular walking. There are two tRNA₄Ser and two tRNA₇Ser genes that contain sequences expected from their known tRNAs (Cribbs et. al., 1987a). Of the 86 nucleotides, they only differ from each other at positions 16, 34 and 77 (non-standard numbering, see Sprinzl et al., 1987). The difference at position 34 corresponds to the anticodon and accounts for their difference in codon recognition. These genes have been designated as either 444 or 777 genes, based solely on the three diagnostic differences. However, there is also a single 474 and two 774 genes, which are recombinant structures of the bona fide genes. The remaining gene, 444*, has the three nucleotides diagnostic of tRNA₄Ser but contains a mutation at the tip of the extra arm. Thus collectively, the entire caste of tRNA₄,₇Ser genes at 12DE forms a graded series of transitional states, bridging the narrow sequence variability between true tRNA4Ser and tRNA7Ser. Flanking sequences of these hybrid and the 444* genes show segmental homologies related to both the 444 and 777 genes within the cluster, again a strong indication that both gene types are undergoing concerted evolution. Examination of selected genes from two distantly related sibling species, D, erecta and D. yakuba, shows their equivalent flanking sequences have diverged from those of melanogaster. As expected, the base changes in these species, often occurring as clusters, are also non-random and appear to have been propagated to certain respective members to maintain a species-specific and cohesive pattern of variation consistent with Molecular Drive. One possible mode of spreading sequence variation and creating the hybrid genes in the process could involve an initial stage of asymmetric pairing between 444 and 777 DNA. To examine this possibility, a tRNAArg gene cluster also from 12DE was conveniently exploited as independent "monitors". This family shows fluctuations in the number of genes among the different species and strains (Newton, unpublished), which could also be explained by asymmetric pairing of DNA followed by unequal exchange. Thus, even though the tRNAArg and tRNA₄,₇Ser genes have embarked on different evolutionary pathways, both phenomena may be explained by their common susceptibility to local asymmetric pairing of DNA. / Science, Faculty of / Zoology, Department of / Graduate

The genetic function of Drosophila heterochromatin has been debated since its 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 se complementation, pseudo-dominance tests with proximal mutations and alleleism tests with known deficiencies, provided evidence for at least two loci 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 loci in the 2L heterochromatin and six loci in the 2R heterochromatin, with multiple alleles being obtained for most sites. Of these loci, one in 2L and two in 2R fall 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. All of the heterochromatic loci uncovered in this study appear to be non-repetitive cistrons. Thus functional genetic loci are found in heterochromatin, albeit at very low density relative to euchromatin. / Science, Faculty of / Zoology, Department of / Graduate

Testes and paragonial glands of Drosophila melanogaster wild type males were labelled in vitro using ³⁵S-methionine, and the proteins synthesized were analysed by 2-dimensional gel electrophoresis (O'Farrell, 1975). Testes and paragonial glands were also labelled in vivo by feeding male or larvae on ³⁵S-labelled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. ³H-proline was also used to label testes, and no significant differences from the pattern obtained with ³⁵S-methionme were found. Different laboratory stocks were analyzed to examine the degree of genetic heterozygosity of testicular proteins. The variation between patterns was very low, facilitating subsequent studies in which flies of defined genetic constitution, but with different genetic backgrounds, were compared. Testes and paragonial glands from X/0 and X/Y/Y males were labelled in vitro with ³⁵S-methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Non-equilibrium pH gradient 2 dimensional gels (O'Farrell et al., 1977) were also run on testis proteins from X/0, X/Y and X/Y/Y males. These gels will resolve basic as well as acidic proteins and once again no differences attributable to the Y chromosome were seen. Pure sperm was manually dissected from in vivo labelled males and the proteins analyzed. Ninety-two proteins were detected, and all were synthesized in comparable amounts by X/0, X/Y and X/Y/Y males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational factors, or regulatory proteins only present in very small amounts in the adult testes. ³⁵S labelled males were also mated to unlabel led females, and the proteins of the transferred sperm were analyzed by 2DPAGE. The contributions of the testes and paragonial gland to the ejaculate were determined. Testes at various stages of development were also cultured in vitro in ³⁵S-methiomne containing media. A profile of the proteins synthesized during development revealed that the spectrum of proteins synthesized at different stages between third instar larvae and the imago were remarkably similar, despite the morphological changes taking place in the organ. Sperm proteins were localized on the patterns, and the quantitative changes occurring during this period were examined. The basic proteins of the testis were studied in an attempt to biochemically identify a Drosophila protamine. Sperm was isolated by dissection, and the acid soluble proteins were separated on a 15% modified Laemmli SDS gel. No unusually small basic protein was seen upon staining, but a protein was present which comigrated with trout histone H4. This suggests that D. melanogaster males may retain somatic histones in the nucleus during the condensation of the sperm head. Testes were labelled in vitro with ³H-arginine and the basic proteins were analyzed on a 15% modified Laemmli SDS gel. The gel was autoradiographed and a prominent doublet was seen at the front of the gel, suggesting that a small highly basic protein is synthesized in the testis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In order to study and compare spontaneous and radiation induced recombination frequencies of the euchromatic and heterochromatic regions of chromosome 2 Drosophila melanogaster females were exposed to 0, 2, 3, 4, or 5 krads of gamma radiation. Exchange was measured in the b-Bl, Bl-It, lt-rl, rl-pk, and pk-cn intervals. The lt-rl interval defines a wholly heterochromatic segment. Analysis of the recombination data demonstrates that spontaneous recombination occurs in the heterochromatic interval at a frequency of approximately 0.1% and the frequency of induced recombination is primarily increased in the heterochromatic interval. Moreover, the frequency of recombination in the heterochromatic interval is correlated with the dose of radiation. There are slight or no increases in the recombination frequencies of the euchromatic segments and the regions containing the heterochromatic-euchromatic boundaries show responses intermediate to the heterochromatic and euchromatic regions. Testing of the recombinant chromosomes indicates that 22% of them are associated with recessive lethals. The association is greater in eggs laid the first four days after radiation treatment than in thoselaid five-eleven days after the radiation treatment. It is postulated that induced recombination can occur via symmetrical as well as asymmetrical interchange. The interchromosomal effect of chromosome 3_, heterozygous for In(3LR)DcxF, on recombination in chromosome 1_ has been studied. The results show that its effect is not significant in the heterochromatic region. Thus, the alterations in the recombination frequencies owing to radiation treatment appear to be independent of those owing to the interchromosomal effect. Recombination was also measured in the presence of the heterochromatic deficiency - Df(2R)MS2¹º. The results indicate that the frequency of recombination is decreased in the chromosome arm containing the deficiency and in the heterochromatic interval of the left arm. The euchromatic regions of the opposite arm show a slight increase in recombination. A higher number of multiple crossover progeny are recovered than would be expected according to map distances in the presence of the heterochromatic deficiency, Df(2R)MS2¹º, the heterozygous inversion, In(3LR)DcxF, and for double crossovers involving the heterochromatic region, but only at high doses of radiation. / Science, Faculty of / Zoology, Department of / Graduate

Two types of sex-linked recessive mutations in Drosophila melanogaster have been investigated in the present study. The first type includes 5 different mutations which exhibit a stress-sensitive (ses) phenotype. Flies of all five mutant stocks become paralyzed when their containers are lightly tapped; wild type flies are unaffected by the same treatment. The five mutations form three complementation groups (cistrons). Flies mosaic for mutant and non-mutant tissue were studied to determine the foci of their action in embryos by fate mapping. These studies suggest that the mutation ses D² has 6 foci in the presumptive nervous system of the blastoderm. Each focus corresponds to a site in the thoracic ganglion which controls the movement of one leg. The focus for ses E¹ mutation is rather diffuse and occupies a larger area in the thoracic nervous system of the blastoderm fate map. Focus mapping studies with the ses B¹ mutation were inconclusive because of the highly variable expressions of the adult behavioural phenotype in mosaic individuals. Developmental studies, involving temperature shifts from 22°C to 29°C (permissive to restrictive temperatures) revealed that the ses E¹ mutation has 2 temperature-sensitive periods (TSPs) for lethality during its development, one in the late 2nd larval instar stage and the other in the late pupal stage. In addition to developmental TSPs of ses B² at the embryonic, 1st larval instar and pre-pupal stage, temperature-shift studies also revealed a ts maternal- effect lethal for ses B². The second type of mutation studied has a temperature-sensitive (ts) phenotype of adult death induced by shifting up to the restrictive temperature. The add Atsl flies have normal behaviour and longevity at 22°C but die within 24 hours after shift-up to 29°C. In contrast, the ses E¹ flies are less active and require 168 hours at 29°C to induce death. Both mutations studied have a 3rd larval instar-pupal TSP with add Atsl and ses E¹ also having an additional TSP at the embryonic and 1st larval instar-2nd larval instar stage respectively. Fate mapping studies suggest that the adult lethal phenotype of add Atsl is caused by a lesoon in tissues derived from the mesodermal cells of the blastoderm, and for ses E¹ the lesion is in the neural cells. / Science, Faculty of / Zoology, Department of / Graduate

There has recently been renewed interest concerning the location of the major loci responsible for the Segregation Distortion phenomenon in Drosophila melanogaster. Hartl (1974) has shown that two major sites are involved: Sd and Rsp. Rsp confers insensitivity to SD chromosomes, while Sd is considered to be the major locus that initiates distortion, Sd is located to the left of Rsp and both are located between Tft and cn. Ganetzky (1977) has extended these findings by showing that just distal to pr there is a locus that, if deleted on a SD chromosome, eliminates distortion and he argues that this is the Sd site. Ganetzky (1977) also uncovered another important locus, in or near the heterochromatin of 2L, that, if deleted from a SD chromosome, greatly reduces the ability of that chromosome to distort and he argued that this site is an enhancer of SD, E(SD). Ganetzky (1977) , also suggests that Rsp might be located very close to the centromere in the proximal heterochromatin of 2R. The results presented here demonstrate the presence of an important component of SD located within the proximal heterochromatin of 2L. These results also show that there is another important site located just distal to pr. However, when this site is removed by recombination from a SD chromosome, a certain level of residual distortion remains. It is argued that the site that Ganetzky (1977) called E(SD) is likely responsible for this residual distortion in the absence of the site just distal to pr. Thus the site near pr is called Sd₁ and the site near 1t is called Sd₂,. Loss of either site results in a large reduction, but not complete elimination, of the distorting ability of a SD chromosome. Other data are presented that, on the whole, agree with Ganetzky's (1977) proposal that Rsp is located in the centromeric heterochromatin of 2R, very close to the centromere. Miklos and Smith-White (1971) have suggested that k (the segregation ratio observed from a given mating) is a deceptive measure of the degree of distortion and they have proposed another method of measuring distortion based on their model of sperm dysfunction. Some of the weak assumptions of this model are discussed and a simpler alternative is presented. The alternative model assumes that the potential segregation ratios of a population of SD males follow a truncated normal distribution. Data are presented that are not necessarily inconsistent with this assumption. The same data show that it is likely that certain SJJ chromosomes differ in their susceptibility to modifiers of It is concluded that at present k provides the clearest measure of distortion. / Science, Faculty of / Zoology, Department of / Graduate