small wing encodes a phospholipase C-(gamma) that acts as a negative regulator of R7 development in Drosophila.

Phospholipase C-(gamma) (PLC-(gamma)) is activated in many cell types following growth factor stimulation. Our understanding of the role of PLC-(gamma) in cell growth and differentiation has been severely limited by the dearth of mutations in any organism. In this study, we show that the Drosophila gene small wing (sl), identified by Bridges in 1915, encodes a PLC-(gamma). Mutations of sl result in extra R7 photoreceptors in the compound eye, consistent with overactivation of the receptor tyrosine kinase pathways that control R7 development. The data presented here provide the first genetic evidence that PLC-(gamma) is involved in Ras-mediated signaling and indicate that PLC-(gamma) acts as a negative regulator in such pathways in Drosophila.

Conserved alternative splicing patterns and splicing signals in the Drosophila sodium channel gene para.

We cloned genomic DNA corresponding to the Drosophila virilis homologue of para, a gene encoding a sodium channel alpha-subunit, and obtained many partial cDNA clones from embryos and adults. Para protein has been well conserved, and the optional elements at six different sites of alternative splicing in D. melanogaster are present in D. virilis, in addition to one new optional exon. Among 31 different splice-types observed in D. virilis, the stage-specific pattern of alternative splicing seen in D. melanogaster is also conserved. Comparison of genomic DNA sequence revealed three aspects that vary between alternatively and constitutively used exon sequences. Sixteen short blocks (10-75 bp), the only recognizably conserved intron sequence, were disproportionately associated with alternatively used splice sites. Silent site substitutions were found much less frequently in alternative than constitutive exon elements, and the degree of match to the Drosophila splice site consensus tended to be lower at less frequently selected alternative splice junctions. This study shows that the developmentally regulated variability of para products is highly conserved and therefore likely to be of functional significance and suggests that a variety of different sequence-dependent mechanisms may regulate this pattern of alternative splicing.

Developmentally regulated alternative splicing generates a complex array of Drosophila para sodium channel isoforms.

The para locus encodes the predominant class of sodium channels expressed in Drosophila neurons. Previous sequence analysis of para cDNAs indicated the occurrence of alternative splicing at several sites within the open reading frame. Here we report a detailed analysis of this alternative splicing and its regulation during development. We have used a combination of RNA-PCR and sequence analysis to examine a 1.7 kilobase region of the para mRNA that encompasses the previously reported sites of alternative splicing. Five sites of alternative splicing were identified; 48 different splice variants could be generated by the differential exon usage observed. The number of splice forms and their relative frequency in vivo were characterized in RNA samples of both embryos and adults. The range of splice types was found to be much more diverse in adults than in embryos; of a total of 19 different combinations of alternative exons, 11 splice types were found in embryos and 18 in adults. Usage of some individual alternative exons changed during development; a newly identified exon, which is found in one of two forms either 24 or 30 base pairs long, was present in about 85% of para transcripts from embryos but only 7% of those in adults. These data suggest that a wide variety of subtly distinct Na channel isoforms are present in Drosophila, and that these may provide a range of voltage-gated sodium channel functions. Although multiple sodium channel genes have already been described in both Drosophila and mammalian systems, this study provides a clear indication that sodium channel variability may be much greater than previously thought.

Length polymorphism in the threonine-glycine-encoding repeat region of the period gene in Drosophila.

Single-fly polymerase chain reaction amplification and direct DNA sequencing revealed high levels of length polymorphism in the threonine-glycine encoding repeat region of the period (per) gene in natural populations of Drosophila melanogaster. DNA comparison of two alleles of identical lengths gave a high number of synonymous substitutions suggesting an ancient time of separation. However detailed examination of the sequences of different Thr-Gly length variants indicated that this divergence could be understood in terms of four deletion/insertion events. In Drosophila pseudoobscura a length polymorphism is observed in a five-amino acid degenerate repeat, which corresponds to melanogaster's Thr-Gly domain. In spite of the differences between D. melanogaster and D. pseudoobscura in the amino acid sequence of the repeats, the predicted secondary structures suggest evolutionary and mechanistic constraints on the per protein of these two species.

Molecular evolution in the Drosophila yakuba period locus.

Two nuclear genes from Drosophila yakuba were cloned, the orthologue of the Drosophila melanogaster period (per) clock gene and the orthologue of an unnamed D. melanogaster gene adjacent to per, which encodes a 0.9-kb RNA transcript. The DNA and presumed protein sequences of both genes are presented and compared with their orthologues in D. melanogaster. Consistent with the per orthologues described in other Drosophila species, some parts of the per gene have accumulated nonsynonymous substitutions at a much higher rate than others. This contrasts markedly with the evenly distributed amino acid replacements observed in the protein encoded by the adjacent gene. The level of synonymous and nonsynonymous substitutions between D. yakuba and D. melanogaster per were compared in small subsections across the gene. The results suggest that the divergence observed in the less well-conserved regions of the per protein is principally due to reduced selective constraint, although the limitations of the method used prevent positive selection acting upon a small proportion of sites being ruled out. The level of silent substitution observed in both of these nuclear genes is very similar to the level of silent substitution previously reported between D. melanogaster and D. yakuba mitochondrial genes, confirming several indirect studies, which have suggested that, in contrast to the case in mammals, silent sites are evolving at similar rates in mitochondrial and nuclear genes of Drosophila.

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution.

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma-eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.