DCg1 αIV collagen chain of Drosophila melanogaster is synthesized during embryonic organogenesis by mesenchymal cells and is deposited in muscle basement membranes

We report the purification and characterization of three sequence-specific polyclonal antibodies raised against specific portions of the Drosophila αIV collagen chain produced from the gene DCg1. These antibodies were used for immunolocalization experiments on tissue sections from embryonic organogenesis stages (13–17) and first larval stages. This analysis was paralleled by in situ hybridization experiments with a labeled fragment of the gene DCg1. We demonstrated that, by late embryogenesis, the DCg1 αIV chain was synthesized by individual mesoblasts and deposited in basement membranes of skeletal and visceral muscles. These sites of αIV collagen deposition were the same, by first and second instars, but the protein was then synthesized by fat body cells. Our results were reminiscent of those obtained for vertebrate in vitro myogenesis, they suggested, moreover, a tissue-specific composition of basement membranes in Drosophila melanogaster.     

Duplicative and conservative transpositions of larval serum protein 1 genes in the genus Drosophila

Interspecific comparative molecular analyses of transposed genes and their flanking regions can help to elucidate the time, direction, and mechanism of gene transposition. In the Drosophila melanogaster genome, three Larval serum protein 1 (Lsp1) genes (alpha, beta and gamma) are present and each of them is located on a different chromosome, suggesting multiple transposition events. We have characterized the molecular organization of Lsp1 genes in D. buzzatii, a species of the Drosophila subgenus and in D. pseudoobscura, a species of the Sophophora subgenus. Our results show that only two Lsp1 genes (beta and gamma) exist in these two species. The same chromosomal localization and genomic organization, different from that of D. melanogaster, is found in both species for the Lsp1beta and Lsp1gamma genes. Overall, at least two duplicative and two conservative transpositions are necessary to explain the present chromosomal distribution of Lsp1 genes in the three Drosophila species. Clear evidence for implication of snRNA genes in the transposition of Lsp1beta in Drosophila has been found. We suggest that an ectopic exchange between highly similar snRNA sequences was responsible for the transposition of this gene. We have also identified the putative cis-acting regulatory regions of these genes, which seemingly transposed along with the coding sequences.     

Annotation and expression profiling of apoptosis-related genes in the yellow fever mosquito, Aedes aegypti

Apoptosis has been extensively studied in Drosophila by both biochemical and genetic approaches, but there is a lack of knowledge about the mechanisms of apoptosis regulation in other insects. In mosquitoes, apoptosis occurs during Plasmodium and arbovirus infection in the midgut, suggesting that apoptosis plays a role in mosquito innate immunity. We searched the Aedes aegypti genome for apoptosis-related genes using Drosophila and Anopheles gambiae protein sequences as queries. In this study we have identified eleven caspases, three inhibitor of apoptosis (IAP) proteins, a previously unreported IAP antagonist, and orthologs of Drosophila Ark, Dnr1, and BG4 (also called dFadd). While most of these genes have been previously annotated, we have improved the annotation of several of them, and we also report the discovery of four previously unannotated apoptosis-related genes. We examined the developmental expression profile of these genes in Ae. aegypti larvae, pupae and adults, and we also studied the function of a novel IAP antagonist, IMP. Expression of IMP in mosquito cells caused apoptosis, indicating that it is a functional pro-death protein. Further characterization of these genes will help elucidate the molecular mechanisms of apoptosis regulation in Ae. aegypti.     

Origin and evolution of the regulatory gene male-specific lethal-3

Dosage compensation in Drosophila is mediated by genes known as "male-specific lethals" (msls). Several msls, including male-specific lethal-3 (msl-3), encode proteins of unknown function. We cloned the Drosophila virilis msl-3 gene. Using the information provided by the sequences of the Drosophila melanogaster and D. virilis genes, we found that sequences of other species can be aligned along their entire lengths with msl-3. Among them, there are genes in yeasts (the Schizosaccharomyces pombe Alp13 gene, as well as a putative Alp13 homolog, found in Saccharomyces cerevisae) and in mammals (MRG15 and MSL3L1 and their relatives) plus uncharacterized sequences of the nematode Caenorhabditis elegans and the plants Arabidopsis thaliana, Lycopersicon esculentum, and Zea mays. A second Drosophila gene of this family has also been found. It is thus likely that msl-3-like genes are present in all eukaryotes. Phylogenetic analyses suggest that msl-3 is orthologous to the mammalian MSL3L1 genes, while the second Drosophila melanogaster gene (which we have called Dm MRG15) is orthologous to mammalian MRG15. These analyses also suggest that the msl-3/MRG15 duplication occurred after the fungus/animal split, while an independent duplication occurred in plants. The proteins encoded by these genes have similar structures, including a putative chromodomain close to their N-terminal end and a putative leucine zipper at their C-terminus. The possible functional roles of these proteins are discussed.     

Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequences removed by deletion Df(1)faswb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5' and 3' nontranslated gene regions are located. These results suggest that Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs.

We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus of Drosophila melanogaster. The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2). The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes. Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome. Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases. The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases. Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines.     

A uniquely conserved regulatory signal is found around the translation initiation site in three different collagen genes

We have determined the nucleotide sequence of the 5' untranslated region and the sequence encoding the signal peptide for mRNAs of the chick alpha 1 type I and alpha 1 type III collagen. These sequences were obtained by synthesizing the corresponding cDNAs using as primers either a synthetic oligonucleotide to prime alpha 1 type I cDNA or a DNA fragment isolated from a genomic clone coding for alpha 1 type III collagen to prime the cognate cDNA. Both primers were selected so that the resulting cDNAs would be short and would contain sequence information for the 5' untranslated region and the signal peptide of the proteins. The nucleotide sequences of these cDNAs were compared with the corresponding sequence of alpha 2 type I collagen. In each mRNA the 5' untranslated segment is approximately 130 nucleotides and contains two or more AUG triplets preceding the AUG which serves as a translation initiation codon. A sequence of about 50 nucleotides surrounding the translation initiation codon is remarkably conserved in all three mRNAs, whereas the sequences preceding and following this segment diverge markedly. This homologous sequence contains an almost identical inverted repeat sequence which could form a stable stem-loop structure. The initiation codon and the AUG which precedes it are found at the same place within this symmetrical sequence and the distance between them is invariant. The rest of the conserved sequence shows a less perfect symmetry. This conserved sequence has not been found in other genes. Our data suggest that these three and perhaps other collagen genes contain an identical regulatory signal that may play a role in determining the level of expression of these genes by modulating translational efficiency.     

Chorion cDNA clones of D. melanogaster and their use in studies of sequence homology and chromosomal location of chorion genes

Clones corresponding to two distinct A1 and A2 chorion genes have been isolated from a cDNA library in Drosophila melanogaster and characterized by hybrid-selected translation and blotting-hybridization analysis. These sequences detectably cross hybridize, thus indicating that at least some chorion genes in the fruit fly are homologous. According to in situ hybridization results, the A1 and A2 genes are not linked (mapping in regions 66D 10-12 and 54C-D of the third and second chromosomes, respectively). In conjunction with other evidence, these results suggest that in Drosophila, clustering of chorion genes may be limited to genes which are expressed in parallel during development.