The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

Genetic and biochemical studies on glutamate-pyruvate transaminase from Drosophila melanogaster

We have used electrophoretic variants of glutamate-pyruvate transaminase (GPT, E.C. 2.6.1.2) in Drosophila melanogaster to genetically map the structural gene to position 42.6 on the X chromosome. By pseudodominance tests over several deficiencies we have localized it cytogenetically to the interval 11Fl-2 to 12Al-2. The sedimentation constant (s _20,w) of the native enzyme was determined in sucrose density gradients to be 5.9 and the native molecular weight approximately 87,000. The similarity in physical properties to mammalian enzymes suggests that the enzyme may also be dimeric in D. melanogaster.     

Analysis of the phenotypes exhibited by rudimentary-like mutants of Drosophila melanogaster

Flies mutant for one or both of the last two enzymes of de novo pyrimidine biosynthesis express a number of phenotypes that are also expressed by mutants of the first four pathway enzymes (r and Dhod-null mutants). However, r-1 flies also express two phenotypes, mottled eyes and poor viability, that are not usually expressed by r and Dhod-null flies. Chemical determinations show that orotic acid, a substrate for the fifth pathway enzyme, accumulates in r-1 individuals but not in r and wild-type individuals. Moreover, flies simultaneously mutant for r and r-1 do not express the mottled-eye phenotype, showing that r is epistatic to r-1 for this r-1-specific phenotype. When genotypically wild-type flies are cultured on a medium containing 6-azauracil, the base of a potent inhibitor of the last enzyme of de novo pyrimidine biosynthesis, phenocopies are obtained that include the mottled-eye as well as the wing phenotypes of r-1 flies. These results support hypotheses that the phenotypes common to r, Dhod-null, and r-1 flies are consequences of uridylic acid deficiency, whereas the r-1-specific phenotypes result from orotic acid accumulation in flies lacking either or both of the last two enzymes of de novo pyrimidine biosynthesis.This research was supported by NSF Research Grant PCM 78-14164, an NSF predoctoral fellowship award to T. Conner, and an NIH research career development award to J. Rawls.     

Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans

We have analysed the viability of cellular clones induced by mitotic recombination in Drosophila melanogaster/D. simulans hybrid females during larval growth. These clones contain a portion of either melanogaster or simulans genomes in homozygosity. Analysis has been carried out for the X and the second chromosomes, as well as for the 3L chromosome arm. Clones were not found in certain structures, and in others they appeared in a very low frequency. Only in abdominal tergites was a significant number of clones observed, although their frequency was lower than in melanogaster abdomens. The bigger the portion of the genome that is homozygous, the less viable is the recombinant melano-gaster/simulans hybrid clone. The few clones that appeared may represent cases in which mitotic recombination took place in distal chromosome intervals, so that the clones contained a small portion of either melanogaster or simulans chromosomes in homozygosity. Moreover, Lhr, a gene of D. simulans that suppresses the lethality of male and female melanogaster/simulans hybrids, does not suppress the lethality of the recombinant melanogaster/simulans clones. Thus, it appears that there is not just a single gene, but at least one per tested chromosome arm (and maybe more) that cause hybrid lethality. Therefore, the two species, D. melanogaster and D. simulans, have diverged to such a degree that the absence of part of the genome of one species cannot be substituted by the corresponding part of the genome of the other, probably due to the formation of co-adapted gene complexes in both species following their divergent evolution after speciation. The disruption of those coadapted gene complexes would cause the lethality of the recombinant hybrid clones.     

Genetic Variants Affecting Phenoloxidase Activity in Drosophila melanogaster

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A ₁ and A ₃ , in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.     

Identification of new immune induced molecules in the haemolymph of Drosophila melanogaster by 2D-nanoLC MS/MS

Antimicrobial peptides (AMPs) play an important role in the innate immunity of insects. In Drosophila 17 additional immune induced molecules (DIMs) were found in the haemolymph of adult flies upon septic injury. Previous studies using MALDI mass spectrometry combined with Edman degradation, detected AMPs and DIMs of a predominantly large size. By means of 2D-nanoLC ESI MS/MS, 43 DIMs were identified in this study from the haemolymph of Drosophila third instar larvae 12h after challenge with a mixture of Micrococcus luteus and Escherichia coli. Most peptides were derived from known AMP or DIM precursors, but only four peptides were purified and identified before. The majority of the peptides that we detected were smaller in size. Interestingly, two previously unknown peptide precursors were found and hereby related to immune defense. These include CG7738 and CG32185. Many of the identified peptides are post-translationally modified by an N-terminal pyroglutamic acid and/or a C-terminal amide. Haemolymph of control larvae was treated in the same way and revealed only one peptide.     

Further studies on the ring canal system of the ovarian cystocytes of Drosophila melanogaster

Summary An ultrastructural study was made of the ring canal system which connects the sister ovarian cystocytes that arise in the germaria of wild type Drosophila melanogaster females. It was discovered that during an oogonial mitosis both chromosomes and spindle are enclosed by a multilayered, perforated membrane system derived (at least in part) from the nuclear envelope. The cytokinesis of stem line oogonia takes place through the formation of a cleavage furrow. A second method of formation of plasma membrane is found in the case of cystocytes. It involves the production along the plane of division of a plaque of interconnected vesicles and tubules and later the coalescence of nearby tubules to form continuous sheets of membrane which segregate the cytoplasms of the sister cells. However, these remain connected by a canal which is enclosed by a ring-shaped rim that is completed prior to the plasma membrane to which the rim is subsequently attached. It is postulated that the rim represents a transformed midbody. As development proceeds the canal becomes wider, its rim becomes thicker, and the inner circumference of the rim becomes coated with a thick deposit having different cytochemical properties than the rim itself. Cystocyte divisions produce sister cells which differ in that one receives all previously formed canals; the other none. In the case of the last division (and perhaps in earlier ones as well) the sister cell receiving all previously formed canals also receives more cytoplasm than its sister. As the cells of the cluster grow, the canals remain close together. This finding suggests that when new plasma membrane is synthesized, it is added in areas remote from the canals. An investigation of the positioning in three dimensions of the fifteen canals of a newly formed, 16 cellcluster suggests that the spindles produced at one division are never parallel to those formed at the subsequent division. This continual shifting of the axes of the spindles at consecutive divisions presumably results in the branching chains of cells which characterize a cystocyte cluster. The possession of a unique pattern of cortical structures by two cystocytes is accompanied by the nuclear synthesis of synaptonemal complexes. The other fourteen cystocytes differentiate into nurse cells. In the most posterior portion of the germarium one of the two potential oocytes switches to the nurse cell developmental pathway. This switched off oocyte and the definitive oocyte grow at rates which differ greatly and are correlated to the amount of contact between their surfaces and certain follicle cells. As development proceeds centrioles accumulate in the oocyte, and most of these are thought to have been carried from the nurse cells into the oocyte in the nutrient stream.The authors are grateful to Richard Z. Belch and James E. Bradof for their conscientious assistance and to E. John Pfiffner for preparation of the inked drawings and construction of the Polyform models. This research was supported by the National Science Foundation grant GB7457.     

Differential characterization of two leucine aminopeptidases in Drosophila melanogaster

Two leucine aminopeptidases from Drosophila melanogaster larvae have been partially purified. The LAP A and D enzymes have similar biochemical characteristics including molecular weights of 280,000 daltons, Michaelis-Menten constants of 0.05 mM, associations with metal cofactors, and specificities toward natural and chromogenic substrates. They differ in their pH optima and spatial distributions. If the closely linked genes that code for these enzymes have resulted from a tandem gene duplication event, it is suggested that there has been subsequent evolutionary divergence. This would provide Drosophila larvae with two related, but functionally distinct enzymes.Virginia K. Walker was supported by an NRC Predoctoral Scholarship and a Killam Merit Scholarship.     

Naturally occurring quantitative variants of acid phosphatase-1 in Drosophila melanogaster

We have examined 111 wild Drosophila melanogaster lines for cis-acting quantitative variants of the Acph-1 gene, which codes for acid phosphatase-1 (ACPH). Three variants with obvious, reproducible phenotypes were isolated. All variants acted equally on all tissues and developmental stages examined. No recombinants were detected between one quantitative variant and the site determining the electrophoretic mobility of Acph-1 among 3885 flies examined. Several enzymatic properties of the variant enzymes were tested, including the K _m values for two substrates, inhibition by three different inhibitors, and thermal stability; the variant enzymes behaved identically to the wild-type enzyme in all cases. Immunological titration experiments showed that the variant enzymes had the same enzyme activity per molecule of ACPH as the wild-type enzyme. These results suggest that the quantitative variants we have identified are altered in the regulatory portion of Acph-1 so as to produce altered numbers of normal ACPH molecules.This work was supported by NIH Grant 21548. MAJ was supported by NIH Predoctoral Training Grant GM07413.     

High temperature initiates changes in Wolbachia ultrastructure in ovaries and early embryos of Drosophila melanogaster

Electron microscopic analysis of Drosophila melanogaster (w1118) ovarian cells has shown that stressful heat treatment of flies causes the appearance of electron-dense granules and large lysosomes in the cytoplasm of ovarian cells. These changes are not due to the presence of the endosymbiotic bacteria Wolbachia, as these changes were observed in both infected and uninfected flies. Essential envelope disturbances and other structural alterations have been revealed in the bacteria present in the ovarian cell cytoplasm of the flies. Some of the fly embryos died after heat shock; however, the bacteria retain their typical morphology in survived embryos. Endosymbionts did not change their localization in ovarian cells and in early fly embryos; they closely interacted with mitochondria and endoplasmic reticulum after the heat-shock treatment of flies. The performed study has shown that the high temperature affects both the host and the endosymbiont, but does not change the character of their structural interaction. Original Russian Text M.V. Zhukova, D.A. Voronin, E.V. Kiseleva, 2008, published in Tsitologiya, vol. 50, No. 5, 2008.     

Enzyme polymorphism in Drosophila melanogaster populations in Iraq

Electrophoretic studies of the degree and pattern of polymorphism at two third-chromosome loci, esterase-6 (Est-6) and phosphoglucomutase (PGM), were carried out in three Drosophila melanogaster populations collected from different localities in Iraq: Mosul, Tuwaitha, and Basrah. The results show that only the Tuwaitha population was polymorphic for both loci; the other two populations were polymorphic for Est-6 and monomorphic for PGM. The allele frequency changes at both loci were followed for 20 generations in an experimental cage derived from the Tuwaitha population; it was found that there is a deviation from Hardy-Weinberg equilibrium at both loci toward the homozygote.     

The pattern of proliferation of the neuroblasts in the wild-type embryo of Drosophila melanogaster

Summary The pattern of neuroblast divisions was studied in thoracic and abdominal neuromeres of wild-type Drosophila melanogaster embryos stained with a monoclonal antibody directed against a chromatin-associated antigen. Since fixed material was used, our conclusions are based upon the statistical evaluation of a large number of accurately staged embryos, covering the stages between the formation of the cephalic furrow up to shortened germ band. Our observations point to a rather stereotypic pattern of proliferation, consisting of several parasynchronous cycles of division. The data suggest that all SI neuroblasts divide at least eight times, all SII neuroblasts six or seven times and all SIII neuroblasts at least five times. This conclusion is based on the mapping of mitotic neuroblasts and is supported by the progressive reduction of the neuroblast volume and by the results of cell countings performed on embryos of increasing age. No conclusive evidence was obtained concerning the fate of the neuroblasts after their last mitosis, i.e. it cannot be decided whether the neuroblasts degenerate or become incorporated as inconspicuous cells in the larval ventral cord. The duration of the cycles of division of the neuroblasts was found to be 40–50 min each, while in the case of ganglion mother cells about 100 min are required to complete one cell cycle.     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Biochemical properties of esterase 6 in Drosophila melanogaster

Biochemical properties of esterase 6 in Drosophila melanogaster were investigated using partially purified preparations from three genotypes, 1/1, 1/2, and 2/2. The molecular weight of the enzyme is estimated to be about 90,000, and treatment with sodium dodecylsulfate cleaves the enzyme into four units with a molecular weight of about 22,000. The activity toward 28 naturally occurring esters was assayed and shown to vary considerably with substrate, the 1/1 preparation having in general higher activity than 1/2 and 2/2, which were very similar. Heat sensitivity, the effect of metal ions, and the effects of the presence or absence of an end product were also studied. The differences demonstrated between allozymes would allow considerable scope, under appropriate conditions, for differential selection to operate between genotypes.Supported in part by an SRC Research Studentship (N.D.D.).     

LTR Retrotransposon-Gene Associations in Drosophila melanogaster

Thirty-three percent (228/682) of all long terminal repeat (LTR) retrotransposon sequences (LRSs) present in the sequenced Drosophila melanogaster genome were found to be located in or within 1000 bp of a gene. Recently inserted LTR retrotransposons are significantly more likely to be located in or within genes than are older, fragmented LTR retrotransposon sequences, indicating that most LRS-gene associations are selected against over evolutionary time. LRSs associated with conserved genes (homologenes) are especially prone to negative selection. In contrast, fragmented LRSs that have persisted in the genome over long spans of evolutionary time are preferentially associated with genes involved in signal transduction and other newly evolved functions. Reviewing Editor: Dr. Juergen Brosius     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

DNA-dependent RNA polymerases from Drosophila melanogaster adults: Isolation and partial characterization

In preparation for the isolation and biochemical characterization of putative RNA polymerase mutants, DNA-dependent RNA polymerases of Drosophila melanogaster adults were isolated and partially characterized. Approximately 70% of the female adult RNA polymerase is located in ovaries. Multiple forms of ovarian RNA polymerases I and II are separable by DEAE-Sephadex chromatography. The two forms of RNA polymerase II differ in ammonium sulfate optima. RNA polymerase IIA is more active with double-stranded DNA as template, whereas RNA polymerase IIB transcribes single-stranded DNA most efficiently. Rechromatography of RNA polymerase IIA on DEAE-Sephadex results in the loss of ability of this form to transcribed double-stranded DNA most efficiently. Ovariectomized carcasses have two forms of RNA polymerase I and one form of RNA polymerase II and each transcribes single-stranded DNA most efficiently. As judged by gel filtration chromatography, female adult extracts have forms of RNA polymerase II that differ in molecular weight and template preference.Supported by Grants GM23456 from the NIH and 11259 from the City University Research Foundation.     

Phenol oxidase activity and the Lozenge locus of Drosophila melanogaster

We have found that the phenol oxidase activity in 50-hr Drosophila melanogaster pupae is much greater than that of adult flies. The mutants lz and lz ^g have all of the phenol oxidase components present in wild type, whereas the mutant tyr-1 has all of the wild-type components but the activity of each component is greatly reduced in comparison with wild-type activity. The newly discovered lozenge allele, lz ^rfg, lacks all phenol oxidase activity.Predoctoral fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated for the U.S. Atomic Energy Commission by Union Carbide Corporation.