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.     

Clonal analysis of primordial disc cells in the early embryo of Drosophila melanogaster

Single cells were marked by X-ray-induced somatic recombination at the blastoderm stage (3 hr) and at 7 and 10 hr of development, when the embryos are relatively insensitive to radiation damage. The resulting clones of genetically marked cells were analyzed on the adult cuticle. For the three stages examined there is a decrease in clone size with developmental age indicating proliferation of the primordial disc cells which give rise to the adult cuticular structures. The extent of the clones has been used to study the specificity of determination in the primordial disc cells at the time of induction. A significant fraction of the clones induced at all three stages extend from the antenna into the head. In the thorax, some of the clones induced at 3 hr extend from the second leg into the wing, thus indicating that these cells are not yet disc-specifically determined. No clones, however, were found extending from the first leg into the second or from the second into the third, and by 7 hr all thoracic clones were restricted to a single disc. The results provide evidence that determination at the blastoderm stage is not yet disc-specific.     

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.     

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.     

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.     

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.     

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     

Compartments in the wing of Drosophila: A study of the engrailed gene

It has recently been suggested that the wildtype alleles of homeotic genes are responsible for controlling the development of compartments. Because the mutation engrailed gives the posterior wing compartment anterior characteristics, it can be regarded as such a homeotic gene. Our experiments confirm the role of the engrailed gene in development of the posterior wing compartment, results which strongly support and extend the compartment hypothesis.Clonal analysis reveals that the state of the engrailed gene is immaterial to the entire anterior compartment, and crucial to the normal development of the posterior compartment, where it controls the pattern of veins and bristles. The presence of a straight and precisely positioned compartment border is dependent on the activity of the engrailed gene until late in development. We suggest that this is due to the gene's effects on cell affinities of the posterior compartment.The engrailed mutation increases the size and changes the shape of the posterior compartment. engrailed clones cause local wing enlargement only if they are dorsal and include the posterior margin of the wing. Wildtype cells outside the clone contribute to this change of shape. This result suggests that the postero-dorsal margin is primarily responsible for the control of shape, and that the ventral compartment is, to some extent, modeled on the dorsal.     

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.     

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.     

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.     

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.