Genetic instability in Drosophila melanogaster

Summary An unstable long tandem duplication which includes the white locus twice, marked with w ^sp in the left and w ^17G in the right locus, when kept in males has been found to produce red-eyed sons which have lost the long duplication and with it the w ^sp and w ^17G mutants. Such exceptions were produced also when w ^17G had been exchanged for w ^a.Stocks originating from these exceptions are unstable, producing: 1) zeste males, also unstable, 2) w ^- deletions, stable, 3) transpositions of the white locus to sites in other chromosomes.The instability is interpreted as the effect of an IS element, within or adjacent to the white locus, which is supposed to retain a duplication of the proximal zeste interacting part of this locus. According to the orientation of the IS element the duplicated part can be active or inactive, giving a zeste or red eye phenotype.The frequency of exceptional offspring after X-ray treatment of the red and zeste unstable stocks have been compared to stable stocks with corresponding genotypes.     

Male recombination with single and homologous P elements in Drosophila melanogaster

It has previously been shown that, in the presence of a source of P element transposase, male recombination in Drosophila melanogaster is induced at a rate of about 1% in the region of a single P[CaSpeR] element. This paper shows that recombinant chromosomes retain unaltered P[CaSpeR] elements at the original site in a high proportion of cases. This result is incompatible with a simple model in which recombination occurs by resolution of a Holliday junction following P element excision and repair. It has also previously been shown that homozygous regions containing a P element produce male recombination levels of 10–20%, an order of magnitude higher than that given by a single element. This paper shows that reciprocal recombinant chromosomes retaining P[CaSpeR] elements can be combined to produce similarly high levels of recombination. This result potentially allows for recombinant chromosomes from homologous recombination to be analysed at the molecular level in the region of the inserted element.     

Expression and localization of clathrin heavy chain in Drosophila melanogaster

Clathrin-coated vesicles mediate cellular endocytosis of nutrients and molecules that are involved in a variety of biological processes. Basic components of the vesicle coat are clathrin heavy chain (Chc) and clathrin light chain molecules. In Drosophila melanogaster the chc gene function has been analyzed in a number of previous studies mainly using genetic approaches. However, the chc mRNA and protein expression patterns have not been studied systematically. We have generated an antibody that specifically recognizes Chc and we have analyzed chc RNA and protein expression patterns throughout embryonic and larval stages. We found that chc mRNA and protein are highly expressed from early stages of embryogenesis onwards, consistent with genetic studies predicting a maternal contribution of the gene function. During subsequent stages mRNA and protein are co-expressed in all embryonic cells; however we found an up-regulation in specific tissues including the gut, the salivary glands, tracheal system and the epidermis. In addition the central nervous system and the nephrocyte-like garland cells show strong Chc expression at late embryogenesis. In larvae Chc is highly expressed in garland cells, imaginal discs, fat body, salivary glands and the ring gland. Subcellularly, we found Chc protein in a vesicle-like pattern within the cytoplasm and at the plasma membrane. Co-labeling studies show that Chc is partially in contact with the trans-Golgi network and co-localizes with markers for early endocytosis. Together, the antibody may serve as a new tool to study the function of Chc in clathrin-dependent cellular processes, such as endocytosis.     

Nucleotide metabolism variability in Drosophila melanogaster

Summary We have studied the metabolic variability within different wild-type strains of Drosophila melanogaster for resistance to antimetabolites (aminopterin, 8-azaguanine), the target enzymatic activities (dihydrofolate reductase, hypoxanthine guanine phosphoribosyltransferase) and capacity to survive on minimal medium with or without exogenous bases or nucleosides (thymidine, hypoxanthine). No correlation was found between dihydrofolate reductase activity and resistance to aminopterin. The results indicated the importance of salvage pathways in the resistance mechanisms in Drosophila.     

Behavioral mutants of Drosophila melanogaster

Summary Sex-linked behavioral mutants were induced in Drosophila melanogaster with ethyl methanesulfonate (EMS) and isolated by direct visual observation of abnormal phenotypes. The four behavioral phenotypes used were flight-reduction, hyperactivity, hypoactivity and stress-sensitivity, and are easily discernable in either single or small populations of mutant flies. In one screen, forty-two behavioral mutants were recovered from strains derived from 800 mutagen-treated X chromosomes. In a second screen, 139 behavioral mutants were obtained from 2369 X chromosomes. The high rate at which behavioral mutants were recovered in the second screen, when compared to new visibles (28) and new temperature-sensitive lethals (124), suggests that the isolation of behavioral mutations on the autosomes of Drosophila and in the genomes of larger insects should be practical.This research was supported by National Research Council of Canada grant A-1764 to D.T.S.     

Dopa decarboxylase from Drosophila melanogaster

Summary Dopa decarboxylase (EC 4.1.1.26) has been purified to near homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 113,000 measured by sucrose gradient sedimentation and 102,000 measured by variable porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions revealed the enzyme consists of two subunits of molecular weight 54,000. The affinity of the enzyme for L-dopa is 30-fold greater than for L-tyrosine. Activity is strongly inhibited by heavy metal ions and the sulfhydryl reagent N-ethylmaleimide. N-acetyl dopamine acts as a competitive inhibitor of the enzyme.Antibodies were elicited against the purified enzyme and measurements of the amount of cross-reacting material (CRM) in two groups of mutants were made. The first group comprised the recessive lethal mutants l(2)amd. Heterozygous mutant stocks are hypersensitive to -methyl dopa, an inhibitor of dopa decarboxylase. These stocks were found to have nearly normal amounts of CRM and enzyme activity.A second group of recessive lethal mutants, characterized by lower levels of dopa decarboxylase, was also analysed. These mutants, designated l(2) Ddc, as heterozygotes exhibited CRM levels between 25 and 75% of normal. Although they are alleles at a single locus, they were classifiable into three distinct groups whose properties readily could be ascribed to a homodimeric structure of the enzyme. This structure would also account for the pattern of intracistronic complementation exhibited by the mutants. Finally, the severity of the mutant defects, as judged by our measurements of CRM and activity, closely parallels that deduced from their complementation pattern. We conclude that these mutations are lesions in the structural gene for dopa decarboxylase.     

High levels of recombination induced by homologous P elements in Drosophila melanogaster

Summary P element transposons in Drosophila melanogaster are capable of mobilizing incomplete P elements elsewhere in the genome, and of inducing recombination. This recombination is usually only of the order of 1% or less. We show that two P elements, located at exactly homologous sites, induce levels of recombination of 20% or higher. The recombination appears to be exact, as determined by the lack of phenotypic effects in recombinant products and the lack of size changes detectable by Southern hybridization. Female recombination is increased, but to a lesser extent than male recombination. Somatic recombination levels are also elevated. Alternative explanations for the high recombination levels are given in terms of the consequences of repair of an excision site and in terms of recombination as part of the replicative transposition process.     

Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster

There is a rich diversity of paucimannose N-glycans in worms and flies, and these may play a role in the survival of these organisms. Although paucimannose N-glycans are not expressed in vertebrates, complex N-glycans may take over some of the functions of paucimannose N-glycans. Identification of the target proteins of β-1,2-N-acetylglucosaminyltransferase I (GnTI) in worms and flies and elucidation of their functions may thus lead to a better understanding of the role of GnTI-dependent glycoproteins in the survival/longevity of both invertebrates and vertebrates.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

Carnitine suppression of position-effect variegation in Drosophila melanogaster

Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone deacetylase and is capable of suppressing position-effect variegation in Drosophila melanogaster, we tested a further possible function of carnitine in the nucleus, using an assay for the suppression of position-effect variegation. We tested three physiological forms of carnitine (l-carnitine, l-propionylcarnitine, l-acetylcarnitine) for the ability to suppress two different chromosomal rearrangements, inducing variegation of the white ⁺ and brown ⁺ genes. The results show that the carnitine derivatives are capable of suppressing the position-effect variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2)1 ⁰¹, a mutation that induces hyperacetylation of histones, whilst hyperacetylated histories accumulated in both the nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the chromatin level.     

Movement of Drosophila melanogaster transposable elements other than P elements in a P-M hybrid dysgenic cross

Summary The Drosophila melanogaster mobile DNA sequences P factors and P elements transpose at elevated rates when P strain males are mated to M strain females in a hybrid dysgenic cross (Engels 1983). Isofemale lines derived from such a cross were analysed by in situ hybridisation using cloned copies of the transposable elements copia, 412 and F. It was found that lines derived from dysgenic crosses showed a statistically significant number of new sites for these elements when compared to a non-dysgenic control cross. This result suggests a functional coupling of copia, 412 and F transposition and some component present in the P-M dysgenic system.     

The genetics of dopa decarboxylase in Drosophila melanogaster

Summary Data on 46 mutations in the structural gene, Ddc. for dopa decarboxylase and 33 mutations in the methyl dopa hypersensitive gene, 1(2)amd, in Drosophila melanogaster are presented including information on their isolation, their effects on DDC activity, and their sensitivity to dietary methyl dopa. Intragenic complementation of both loci is documented, the effects of heteroallelic complementing heterozygosity on DDC activity, in vitro thermolability of DDC, and on temperature sensitive viability are presented. Data are marshalled to support rejection of the hypothesis that Ddc mutations and 1(2)amd, mutations are lesions in a single gene.     

Constitutive heterochromatin in early embryogenesis of Drosophila melanogaster

Summary The formation of constitutive heterochromatin was studied during the embryonic development of Drosophila melanogaster, using the C-banding technique. During embryonic cleavage, C-banded material is not seen in mitotic chromosomes; the differentiation between euchromatin and heterochromatin only occurs at blastoderm. This event correlates with the establishment of position-effect variegation.     

Simultaneous transposition of different mobile elements: Relation to multiple mutagenesis in Drosophila melanogaster

Summary Several different transposition events occur simultaneously in one and the same germ cell, as we have found by analyzing different genetic systems in Drosophila melanogaster. (i) In unstable ct ^MR2 strains, stable reversions to ct ⁺ and changes in the type of ct mutation, which depend on an excision or transposition of the mobile element mdg4 (Gerasimova 1981; Gerasimova et al. 1984), are frequently accompanied by the appearance of novel mutations in different loci of the X chromosome. Some of these (sn, w, g) seem to be induced by the P-element and copia. (ii) A stable ct ^MR2 reversion to the wild type frequently coexists with an insertion of one to five copies of the P-element in the X-chromosome. Thus, the number of independent transposition events registered by genetic analysis and in situ hybridization may be as great as six. (iii) In two strains with double unstable mutations (cm, ct, and ct, r), double reversions to the wild type occurred at a high rate (80%–97% of total revertants). They frequently coexisted with novel strain-specific mutations. (iv) The stable strain ct ⁶ g²is destabilized by crossing with the MRh12/Cy strain (which contains a number of P-element copies). Both mutations begin to revert to the wild type. Of the revertants 50% have double reversions. Our experiments revealed a high specificity of insertion sites depending on the nature of transposon and the strain genotype. A possible role played by the burst of transposition in the evolution and possible mechanisms of transposition specificity are discussed.