Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en²/en³ genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.     

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.     

Regeneration following duplication in imaginal wing disc fragments of Drosophila melanogaster

Fragments from the imaginal wing disc of Drosophila melanogaster were cultured in vivo for periods up to 28 days. One type of edge fragment first duplicated and then ceased to grow, but others often continued to grow following initial duplication and regenerated structures characteristic of other areas of the disc. After 28 days of culture, about 50% of fragments from the presumptive ventral hinge region of the disc grew extensively and produced regenerated as well as original structures. The regenerated structures in some implants were produced at the line of mirror-image symmetry. Regeneration was associated with fragment growth and in many cases was accompanied by loss of duplicate structures. Fragments which were only duplicated after the culture period could in some cases be stimulated to grow by additional culture in fresh hosts, but the results of coculturing two fragments in each host show that culture conditions alone do not control growth and regulation in the fragments. The large, normally regenerating fragment, complementary to the ventral fragment, did not appear to grow following regeneration and only occasionally produced supernumerary structures during prolonged culture. Intact wing discs cultured under similar conditions never produced supernumerary structures. Our results suggest that a duplicated pattern is less stable than a complete, regenerated pattern, which in turn is less stable than an intact disc. We propose that the growth of duplicated disc fragments is stimulated by polarity reversals present at lines of mirror-image symmetry.     

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.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

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.     

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.     

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.     

The behavior during the initial phase of in vitro aggregation of dissociated imaginal disc cells from Drosophila melanogaster

Aspects of the initial phase of aggregation in vitro of dissociated imaginal disc cells from Drosophila melanogaster are described. Using the methods described certain interdisc differences in the percent decrease in single cell units during the first hour of aggregation can be demonstrated. In addition it is shown that prospective notum cells isolated from the dorsal mesothoracic disc show less of a decrease than do prospective wing cells. This difference shows up in a variety of different wild-type stocks and in several mutant stocks as well. Prospective notum cells from the mutation fu59 show only a limited ability to adhere to one another, while the percentage of single cell units from prospective wing blade cells from r9 larvae grown on pyrimidine-poor medium decrease less compared to cells from the same stock grown on RNA-supplemented medium. There is a significantly greater decrease in the percent single cell units in cultures of prospective eye cells than in cultures of prospective antennal cells. Furthermore, cells from the antennal disc of larvae bearing the homeotic mutation ssa show a significantly lower decrease in single cell units when grown at restrictive temperatures. In contrast, antennal disc cells from the homeotic mutation ophthalmoptera; eyeless Dominant, a mutation which affects the eye disc, are unaffected, while cells from the eye disc are slightly less able to reassociate with one another.     

Loss of centrioles and polyploidization in follicle cells of Drosophila melanogaster

Centrioles of the nurse cells of Drosophila have been shown to move into the oocyte prior to polyploidization of the nurse cells. In order to determine whether or not centriolar loss always occurs in polyploid insect cells, the follicular epithelium of the Drosophila ovary was studied. The DNA content of the cells was determined by cytophotometry of Feulgen-stained squash preparations. The first two endomitotic replications occur at stage 7 and 8. Two additional replications occur prior to stage 11, but the DNA content appears to be under-replicated. Centrioles are found in follicle cells until stage 10 at which time they are no longer present. At the inception of polyploidization the centrioles are no longer closely associated with each other or the nuclear envelope. Instead, they are located adjacent to the plasma membrane at the basal surface. These results closely parallel the previous results found for the nurse cells. Hence, it may be a general observation that centrioles are gradually lost in polyploid insect cells.     

Autonomous differentiation of the tumorous-head phenotype in Drosophila melanogaster

Summary Transformed areas derived from mature imaginal eye discs of the tumorous-head (tuh) mutant of Drosophila melanogaster were transplanted either into larval hosts (metamorphosis test) or into adult females (long-term in vivo culture). These disc fragments showed characteristic morphologic and enzymatic (aldehyde oxidase (aldox) positive) differences in comparison to a similar region in wildtype eye discs.The tissues derived from the central portion of the tuh eye disc which would normally give rise to eye facets transformed predominantly into homoeotic structures of the abdominal region of the fly. Posterior abdominal tergites arose in 88% of the transplants, of which 7% also possessed genital tissue. In addition, 10% showed duplicated vibrissae with no accompanying homoeotic alteration and 2% differentiated into unidentifiable structures.Our preliminary results from long-term cultures have shown the capacity of the tuh transformed area to grow in vivo and to maintain its differentiation potential. This kind of approach therefore provides an opportunity to follow transdetermination properties of a homoeotically altered tissue.In the present study we demonstrate that during larval life, the presumptive region of the tuh transformed area can be removed from the surrounding unaffected eye disc tissue. From the autonomous differentiation of the tuh phenotype we conclude that the homoeotic change is cell-intrinsically expressed and that the aldox positive areas in the tuh eye discs signal an altered state of determination. Leave of absence: Department of Biological Sciences, Florida Technological University, Orlando, Florida 32816, USA     

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.     

The action of the Notch locus in Drosophila melanogaster

Summary It is shown that the Notch⁸ deficiency in Drosophila melanogaster affects a number of enzyme activities localized in the mitochondria, such as NADH oxidase (activity of the complete respiratory chain), NADH dehydrogenase (the first step in the respiratory chain before transfer to ubiquinone), Succinate dehydrogenase and -Glycerophosphate dehydrogenase. The experiments reported here do not exclude the possibility of involvement of other genes in the deficiency. The effect of duplications of the Notch locus on NADH oxidase and NADH dehydrogenase suggest that this locus determines the enzyme activities.The dosage effects of the Notch locus on activity suggest that this locus contains the structural genes for these enzymes.     

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.     

A further characterization of the cinnamon gene in Drosophila melanogaster

Summary Two mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC.2.7.1.40) are described. The mutations are recessive, segregate 2⁺:2^- in tetrads and do not complement each other. Single-step spontaneous revertants, isolated on glucose plates, get back pyruvate kinase activity. The enzymes from various revertants display a wide spectrum of specific activity, thermolability and altered affinity for ligands such as P-enol pyruvate, ADP and fructose 1,6-diphosphate. The mutants produce materials crossreacting to the rabbit antibody raised against purified pyruvate kinase from the wild type yeast. These mutations thus define the structural gene of pyruvate kinase.The mutations map on the leaft arm of chromosome I and form a single complementation group with five other pyruvate kinase mutations in the pyk1 gene that was earlier suggested to be a regulatory locus controlling the synthesis of this enzyme. A comparative study of these mutants has been made with the structural mutants described here.