Analysis of a fushi tarazu autoregulatory element: multiple sequence elements contribute to enhancer activity.

Regulatory sequences or factors involved in the regulation of target genes of Drosophila homeodomain proteins are largely unknown. Here, we identify sequence elements that are involved in the function of the fushi tarazu (ftz) autoregulatory element AE, a direct in vivo target of the homeodomain protein ftz. A systematic deletion analysis of AE in transgenic embryos defines multiple elements that are redundantly involved in enhancer activity. Sequences juxtaposed to ftz binding sites are not strictly required for enhancer function. Several sequence motifs are conserved in other developmentally regulated genes of Drosophila melanogaster and in the AE homologue of Drosophila virilis. The D. virilis AE is functional in D. melanogaster. The sequence motifs identified here are candidate elements contributing to the target specificity of the homeodomain protein ftz.     

Hsp70 duplication in the Drosophila melanogaster species group: how and when did two become five?

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.     

DNA Sequence Evolution of the Amylase Multigene Family in Drosophila Pseudoobscura

The alpha-Amylase locus in Drosophila pseudoobscura is a multigene family of one, two or three copies on the third chromosome. The nucleotide sequences of the three Amylase genes from a single chromosome of D. pseudoobscura are presented. The three Amylase genes differ at about 0.5% of their nucleotides. Each gene has a putative intron of 71 (Amy1) or 81 (Amy2 and Amy3) bp. In contrast, Drosophila melanogaster Amylase genes do not have an intron. The functional Amy1 gene of D. pseudoobscura differs from the Amy-p1 gene of D. melanogaster at an estimated 13.3% of the 1482 nucleotides in the coding region. The estimated rate of synonymous substitutions is 0.398 +/- 0.043, and the estimated rate of nonsynonymous substitutions is 0.068 +/- 0.008. From the sequence data we infer that Amy2 and Amy3 are more closely related to each other than either is to Amy1. From the pattern of nucleotide substitutions we reason that there is selection against synonymous substitutions within the Amy1 sequence; that there is selection against nonsynonymous substitutions within the Amy2 sequence, or that Amy2 has recently undergone a gene conversion with Amy1; and that Amy3 is nonfunctional and subject to random genetic drift.     

The Evolution of Duplicate Glyceraldehyde-3-Phosphate Dehydrogenase Genes in Drosophila

In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat shock and ecdysterone activation of the Drosophila melanogaster hsp23 gene; a sequence element implied in developmental regulation.

The regulation of the Drosophila melanogaster hsp23 gene by heat shock and ecdysterone has been analysed by measuring activities of hsp--Escherichia coli beta-galactosidase hybrid genes in transfected hormone-sensitive D. melanogaster cells. Mutation analysis identified multiple, distinct promoter elements. A sequence element, which also occurs in the promoters of several other developmentally regulated Drosophila genes, is present in regions of the hsp23 promoter that are essential for its ecdysterone, but not its heat-regulated activity; this element may represent a binding site for an ecdysterone--receptor complex. Mutant promoters that can be activated only by heat shock or by hormone have been constructed. Thus the two types of regulation of the hsp23 gene can function independently of each other.     

Drosophila melanogaster Parkin ubiquitinates peanut and septin1 as an E3 ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR-JP), a common familial form of Parkinson's disease, is caused by mutations of human Parkin. To deepen the understanding of Parkin biology in an in vivo model of Drosophila, we attempted to characterize the function of Drosophila melanogaster Parkin and found that D. melanogaster Parkin exhibited UbcH8-dependent E3 ubiquitin-protein ligase activity. Using E2 binding and in vitro ubiquitination assays, UbcH8 preferentially was found to bind to Parkin mutants harboring functional RING1 domains, but failed to bind to mutants harboring point mutants with complete loss of function. This inability of UbcH8 binding to such mutants was accompanied by abrogation of an E3 ligase activity, indicating that D. melanogaster Parkin as an E3 ligase interacts with UbcH8 through its RING1 domain. An in vivo ubiquitination assay revealed that D. melanogaster Parkin existed in ubiquitinated form in vivo. Moreover, peanut and septin1, D. melanogaster septin proteins, were also ubiquitinated by D. melanogaster Parkin. Co-immunoprecipitation with membrane protein Syntaxin indicated direct binding of septin proteins to syntaxin, implicating their relevance in the exocytosis of dopamine in cells. Western blot analysis and DNA fragmentation indicated that the rate and efficiency of p53-dependent apoptosis were significantly higher in the presence of dopamine than without the septin proteins. Therefore, our findings in the present study demonstrate that Parkin possibly influences septin protein effects on p53-mediated apoptosis, helping to extend the utility of Drosophila as a model system for the study of neurodegeneration.     

Evolution of the Adh locus in the Drosophila willistoni group: the loss of an intron, and shift in codon usage

We report here the DNA sequence of the alcohol dehydrogenase gene (Adh) cloned from Drosophila willistoni. The three major findings are as follows: (1) Relative to all other Adh genes known from Drosophila, D. willistoni Adh has the last intron precisely deleted; PCR directly from total genomic DNA indicates that the deletion exists in all members of the willistoni group but not in any other group, including the closely related saltans group. Otherwise the structure and predicted protein are very similar to those of other species. (2) There is a significant shift in codon usage, especially compared with that in D. melanogaster Adh. The most striking shift is from C to U in the wobble position (both third and first position). Unlike the codon-usage-bias pattern typical of highly biased genes in D. melanogaster, including Adh, D. willistoni has nearly 50% G + C in the third position. (3) The phylogenetic information provided by this new sequence is in agreement with almost all other molecular and morphological data, in placing the obscura group closer to the melanogaster group, with the willistoni group farther distant but still clearly within the subgenus Sophophora.      

Evidence for redundancy but not trans factor-cis element coevolution in the regulation of Drosophila Yp genes.

In Drosophila melanogaster and the endemic Hawaiian species D. grimshawi three Yolk protein (Yp) genes are expressed in a similar sex- and tissue-specific pattern. In contrast, DNA sequence comparisons of promoter/enhancer regions show low levels of similarity. We tested the functional significance of these observations by transforming D. melanogaster with the genomic region that includes the divergently transcribed D. grimshawi DgYp1 and DgYp2 genes; we found that the introduced genes were expressed in female fat body and in ovaries but not in males. Moreover, we found D. grimshawi proteins in the hemolymph and accumulating in ovaries. Using reporter constructs we showed that the intergenic region from D. grimshawi was sufficient to drive accurate expression, but some low level of ectopic expression was seen in males. Transforming D. melanogaster with constructs bearing deletions within the D. grimshawi intergenic region revealed only subtle effects in the overall level of expression, suggesting a high level of redundancy. Testing mutants in the sex-specific regulator doublesex revealed that it is capable of repressing the DgYp genes in males. Together, these data show that D. melanogaster trans-acting factors can regulate the in vivo pattern of DgYp expression and support the notion of a redundant and complex system of cis-acting elements.     

Novel odorant-binding proteins expressed in the taste tissue of the fly

A taste tissue cDNA library of the fleshfly Boettcherisca peregrina was screened with a subtracted cDNA probe enriched with taste-receptor-tissue-specific cDNA. Seven genes were identified with sequence similarity to insect odorant-binding protein (OBP) genes. The predicted amino acid sequences of the genes contain the putative signal peptide sequence at the N-terminal and most of them conserve the six cysteines common to known insect OBPs. These genes show a high degree of sequence divergence with approximately 20% amino acid identity. The most striking feature was that all seven of these genes are expressed mainly in the taste tissues, such as the labellum and tarsus, unlike the known insect OBP genes expressed in olfactory tissue. The predicted amino acid sequences had the highest degree of sequence similarity to the Drosophila melanogaster OBPs named pheromone binding protein-related proteins (PBPRPs). These gene products are here referred to as gustatory PBP-related proteins (GPBPRPs) 1-7. Homologous GPBPRP genes were found also in D. melanogaster by database search and are shown to be expressed in Drosophila taste tissues.