Rates of movement of transposable elements inDrosophila melanogaster

Mobilization rates of nine families of transposable elements (P, hobo, FB, gypsy, 412, copia, blood, 297, andjockey) were estimated by using 182 lines. Lines were started from a completely isogenic population ofDrosophila melanogaster, carrying the markersepia as an indicator of possible contamination, and have been accumulating spontaneous mutations independently for 80 generations of brother-sister (or two double-first-cousin) matings. Transposable element movements have been analyzed in complete genomes by the Southern technique. Mobilization was a rare event, with an average rate of 10^–5 per site per generation. The most active element wasFB. In contrast, the retroelementsgypsy andblood did not move at all. Most changes in restriction patterns were consistent with rearrangements rather than with true transposition. The euchromatic or heterochromatic location of elements was tested by comparing insertion patterns from adults and salivary glands. Certain putative rearrangements involved heterochromatic copies of the retroelements412, copia or297. Clustering of movement across families was observed, suggesting that movement of different families may be non-independent. An association between modified insertion patterns and mutant effects on quantitative traits shows that spontaneous transposition events cause continuous variation.     

RNA干扰技术在果蝇中的应用

RNA干扰是双链RNA特异诱导的转录后期基因沉默.该技术随着不断完善而越来越被广泛地运用于果蝇的功能基因组研究上,双链RNA已经成为果蝇中功能基因的一个十分有效的抑制子,势必使RNA干扰技术成为研究果蝇体内基因功能的强有力的反向遗传学研究技术.     

Molybdenum hydroxylases in Drosophila

Summary The molybdenum hydroxylases are a ubiquitous class of enzymes which contain molybdenum in association with a low molecular weight cofactor. Genetic evidence suggests that the Drosophila loci, ma-1, cin and lxd are concerned with this cofactor because mutants for any one of these loci simultaneously interrupt activity for two molybdenum hydroxylases, XDH and A0. A third enzyme activity, P0, is also absent in each of the three mutants but evidence classifying P0 as a molybdoenzyme has been lacking. This study utilizes the known tungsten sensitivity of molybdoenzymes to demonstrate directly that pyridoxal oxidase is also a molybdoenzyme. The low molecular weight molybdenum cofactor is found to be severely reduced in extracts of the lxd and cin mutants but ma-1 mutants have high levels of cofactor. A partially purified preparation of XDH crossreacting material from ma-1 was also shown to contain the molybdenum cofactor. These results, considered with data from other workers are taken to indicate that the functions of all three of the loci examined could be concerned with some aspect of cofactor biosynthesis.This work was supported by PHS grant GM 23736 to V. Finnerty     

Molybdenum hydroxylases in Drosophila

Summary A reliable method for visualizing the Drosophila enzyme pyridoxal oxidase in polyacrylamide gels is described. Antiserum to pyridoxal oxidase has been produced and used in quantitative immunoelectrophoresis to determine the relative amounts of pyridoxal oxidase cross reacting material (CRM) in several mutants including lpo, lxd, ma-l and cin. The lpo variant did not have CRM for PO, thus further supporting the idea that it represents a structural gene for pyridoxal oxidase in Drosophila. CRM for PO was found in ma-l and lxd indicating that their effects upon the enzyme are probably post-translational. No CRM for PO could be found in the cin mutants.This work was supported by PHS grant GM 23736 to V. Finnerty     

Postembryonic Hematopoiesis in Drosophila

We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte.     

The Drosophila genome

The past year has been a spectacular one for Drosophila research. The sequencing and annotation of the Drosophila melanogaster genome has allowed a comprehensive analysis of the first three eukaryotes to be sequenced—yeast, worm and fly—including an analysis of the fly's influences as a model for the study of human disease. This year has also seen the initiation of a full-length cDNA sequencing project and the first analysis of Drosophila development using high-density DNA microarrays containing several thousand Drosophila genes. For the first time homologous recombination has been demonstrated in flies and targeted gene disruptions may not be far off.     

The ribosomes of Drosophila

Summary One- and two-dimensional gel electrophoresis was employed to characterize and compare ribosomal subunit proteins from wild-type D. melanogaster and several mutants, including suppressor-offorked and four X-linked cold sensitive lethals. The sibling species D. simulans was also studied. We found the electropherogram patterns indistinguishable in all cases.This work was supported by grants from the National Institutes of Health (GM 18910, GM 22866) and the National Science Foundation (BM S75-11890) to Edward Berger     

The ribosomes of Drosophila

Summary The assembly of proteins and RNA into mature ribosomal subunits has been studied in Drosophila cell cultures by pulse-chase experiments. Pulse labeled rRNA has a transit time of 3 h, while the transfer of ribosomal protein occurs completely within 30 min. Inhibition of protein synthesis by cycloheximide results in an almost immediate cessation of ribosome assembly, a result which indicates that no large pool of free ribosomal proteins exists in the cell. Substituting pre-ribosomal RNA with the analogue 5-fluorouridine (5-FU) results in a cessation of ribosome maturation. Under these conditions at least three large subunit proteins continue to accumulate on pre-existing cytoplasmic subunits, indicating an exchange. A portion of ribosomal subunit proteins synthesized in the presence of 5-FU can be recovered in cytoplasmic subunits once the effect of 5-FU has been reversed. This is most easily interpreted in terms of their stabilization on substituted pre-rRNA within the nucleolus, and subsequent utilization on unsubstituted RNA.Work supported by a grant from the NIH (GM 22866)     

A genetic screen for elements of the network that regulates neurogenesis in Drosophila

The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute. Their activity defines proneural cell clusters in the wing imaginal disc. Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles. Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell. This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster. Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis. This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles. In this way, about 100 000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate. In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers. Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis. Received: 20 September 1997 / Accepted: 8 October 1997     

Interstrain crosses enhance excision of Tc1 transposable elements in Caenorhabditis elegans

Summary We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3–5 exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su⁺ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su⁺ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su⁺ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photoproducts can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity. We demonstrate that the transformant shows much higher UV sensitivity than recA1207 carrying the vector plasmid pBR325, indicating that translesion synthesis significantly contributes to DNA repair capacity of cells deficient in recombination.     

Non-random association of transposable elements with duplicated genomic blocks in Arabidopsis thaliana

The genome of Arabidopsis thaliana is known to contain numerous open reading frames apparently encoding transposases. In order to test the hypothesis that transposable elements have played a role in segmental duplication in this species, we compared the distribution of transposable elements with that of genomic windows that shared gene families to a greater extent than expected by chance. Phylogenetic analyses indicated that duplication of these segments occurred after the monocot-dicot divergence and probably after the eurosid I–eurosid II divergence. Known transposable elements were found to occur in putatively duplicated segments to a far greater extent than expected on the basis of their genome-wide distribution, suggesting that transposition may have played a role in segmental duplication in this species.