The hemoglobin genes of Drosophila

We recently reported the unprecedented occurrence of a hemoglobin gene (glob1) in the fruitfly Drosophila melanogaster. Here we investigate the structure and evolution of the glob1 gene in other Drosophila species. We cloned and sequenced glob1 genes and cDNA from D. pseudoobscura and D. virilis, and identified the glob1 gene sequences of D. simulans, D. yakuba, D. erecta, D. ananassae, D. mojavensis and D. grimshawi in the databases. Gene structure (introns in helix positions D7.0 and G7.0), gene synteny and sequence of glob1 are highly conserved, with high ds/dn ratios indicating strong purifying selection. The data suggest an important role of the glob1 protein in Drosophila, which may be the control of oxygen flow from the tracheal system. Furthermore, we identified two additional globin genes (glob2 and glob3) in the Drosophilidae. Although the sequences are highly derived, the amino acids required for heme- and oxygen-binding are conserved. In contrast to other known insect globin, the glob2 and glob3 genes harbour both globin-typical introns at positions B12.2 and G7.0. Both genes are conserved in various drosophilid species, but only expression of glob2 could be demonstrated by western blotting and RT-PCR. Phylogenetic analyses show that the clade leading to glob2 and glob3, which are sistergroups, diverged first in the evolution of dipteran globins. glob1 is closely related to the intracellular hemoglobin of the botfly Gasterophilus intestinalis, and the extracellular hemoglobins from the chironomid midges derive from this clade.     

Evolution of protein-coding genes in Drosophila

Several contributing factors have been implicated in evolutionary rate heterogeneity among proteins, but their evolutionary mechanisms remain poorly characterized. The recently sequenced 12 Drosophila genomes provide a unique opportunity to shed light on these unresolved issues. Here, we focus on the role of natural selection in shaping evolutionary rates. We use the Drosophila genomic data to distinguish between factors that increase the strength of purifying selection on proteins and factors that affect the amount of positive selection experienced by proteins. We confirm the importance of translational selection in shaping protein evolution in Drosophila and show that factors such as tissue bias in expression, gene essentiality, intron number, and recombination rate also contribute to evolutionary rate variation among proteins.     

In vitro transcription of Drosophila ribosomal genes using Drosophila RNA polymerases

Drosophila RNA polymerases I &; II were used to transcribe a recombinant bacterial plasmid containing one copy of Drosophila ribosomal DNA. Both supercoiled and relaxed, closed circular plasmids were used. With Mg⁺² as the divalent cation, enzyme I is much more active on both forms of the plasmid; the relaxed form in particular supports almost no RNA synthesis by enzyme II. When Mn⁺² is present, differences in template efficiencies are minimal. The differences observed in the absence of Mn⁺² seem to depend only on different preferences for the physical state of the template and not on recognition of specific promotor sequences, since enzyme I shows no strand selection when transcribing these plasmids.     

Drosophila melanogaster U1 snRNA genes

We have isolated and characterized a recombinant which contains a Drosophila melanogaster U1 small nuclear RNA (snRNA) gene colinear with the published snRNA sequence. Southern hybridizations of the fly genomic DNA, using as probe a plasmid containing only the coding region of the gene, shows that the fly contains at most three or four genes and very few related sequences for the small nuclear U1 RNA. These genes were localized by in situ hybridization at different chromosomal loci and show no spatial relationship to the U2 snRNA genes.     

cGMP-dependent protein kinase genes in Drosophila

Two Drosophila genes encoding products related to cGMP-dependent protein kinase have been isolated by cross-hybridization to a Drosophila cAMP-dependent protein kinase catalytic subunit gene. Both genes encode products with putative cGMP binding and kinase domains on the same polypeptide chain, as found for the prototypical bovine lung cGMP-dependent protein kinase. The deduced product of one gene (DG1; cytological position, 21D) is 14% larger than the bovine enzyme and differs substantially in sequence at the amino terminus, the region responsible in the bovine enzyme for dimerization. The second gene (DG2; cytological position, 24A) is transcribed into three major RNA species of different size. The largest (DG2; T1) and smallest (DG2;T3) RNAs encode overlapping polypeptides of similar sequence to the whole length of bovine lung cGMP-dependent protein kinase. The translation product of the third major RNA (DG2;T2) lacks sequences similar to those that constitute the dimerization and kinase inhibitory domains of the bovine enzyme. The percentage amino acid identity between DG1 or DG2 and bovine lung cGMP-dependent protein kinase is 55 and 64%, respectively. A common progenitor of the two cGMP-dependent protein kinase genes, DG1 and DG2, is strongly suggested by the conserved positions of introns in these genes.     

Shaw potassium channel genes in Drosophila

Drosophila Shaw encodes a voltage-insensitive, slowly activating, noninactivating K(+) current. The functional and developmental roles of this channel are unknown. In this study, we use a dominant transgenic strategy to investigate Shaw function and describe a second member of the Shaw family, Shawl. In situ hybridization showed that the two Shaw family genes, Shaw and Shawl, have largely nonoverlapping expression patterns in embryos. Shaw is expressed mainly in excitable cells of the CNS and PNS of late embryos. Shawl is expressed in many nonexcitable cell types: ubiquitously in embryos until the germband extends, then transiently in the developing CNS and PNS, becoming restricted to progressively smaller subsets of the CNS. Ectopic full-length and truncated Shaw localize differently within neurons, and produce uneclosed small pupae and adults with unfurled wings and softened cuticle. This phenotype was mapped to the crustacean cardioactive peptide (CCAP)-neuropeptide circuit. Widespread expression of Shaw in the nervous system results in a reduction in body mass, ether-induced shaking, and lethality. Expression of full-length Shaw had more extreme phenotypic consequences and caused earlier lethality than expression of truncated Shaw in a given GAL4 pattern. Whole cell recordings from ventral ganglion motor neurons expressing the truncated Shaw protein suggest that a major role of Shaw channels in these cells is to contribute to the resting potential.     

Recent origins of sperm genes in Drosophila

Newly created genes often acquire testis-specific or enhanced expression but neither the mechanisms responsible for this specificity nor the functional consequences of these evolutionary processes are well understood. Genomic analyses of the Drosophila melanogaster sperm proteome has identified 2 recently evolved gene families on the melanogaster lineage and 4 genes created by retrotransposition during the evolution of the melanogaster group that encode novel sperm components. The expanded Mst35B (protamine) and tektin gene families are the result of tandem duplication events with all family members displaying testis-specific expression. The Mst35B family encodes rapidly evolving protamines that display a robust signature of positive selection within the DNA-binding high-mobility group box consistent with functional diversification in genome repackaging during sperm nuclear remodeling. The Mst35B paralogs also reside in a significant regional cluster of testis-overexpressed genes. Tektins, known components of the axoneme, are encoded by 3 nearly identical X-linked genes, a finding consistent with very recent gene family expansion. In addition to localized duplication events, the evolution of the sperm proteome has also been driven by recent retrotransposition events resulting in Cdlc2, CG13340, Vha36, and CG4706. Cdlc2, CG13340, and Vha36 all display high levels of overexpression in the testis, and Cdlc2 and CG13340 reside within testis-overexpressed gene clusters. Thus, gene creation is a dynamic force in the evolution of sperm composition and possibly function, which further suggests that acquisition of molecular functionality in sperm may be an influential pathway in the fixation of new genes.     

Organization and expression of Drosophila tropomyosin genes

It has been shown (Jockusch &; Isenberg, 1981) that vinculin (130K protein) binds to actin and induces actin filaments to form bundles even at low ionic strength. Here, we present structural details on the vinculin molecule itself and on its interaction with actin. In negatively stained preparations, vinculin appeared as a globular protein with an average diameter of 85 Å. The ability of vinculin to form actin filament bundles was confirmed using shadowing techniques and gel analysis of sedimented material. Analysis of vinculin-induced paracrystals by optical diffraction and computer processing revealed their structural similarity to Mg-induced paracrystals. The lateral position of vinculin on surface-exposed actin filaments of such paracrystals was demonstrated directly in electron micrographs and indirectly by labelling vinculin with ferritin-coupled anti-vinculin F(ab′) fragments. Polymerization of actin in the presence of vinculin-coated polystyrene beads did not result in an “end-on” binding of filaments to the beads. Rather, actin bundles were laterally associated with the whole surface of the beads, from where they radiated in a star-like pattern. The growth of actin filaments onto myosin subfragment-I decorated, vinculin-incubated. fixed filament fragments was not inhibited, as was shown directly by electron microscopy and monitored viscometrically in a nucleation assay. These results suggest that in vivo at the site of an adhesion plaque vinculin may link actin filaments together into a suitable configuration to interact with the plasma membrane.     

Molecular evolution of sex-biased genes in Drosophila

Studies of morphology, interspecific hybridization, protein/DNA sequences, and levels of gene expression have suggested that sex-related characters (particularly those involved in male reproduction) evolve rapidly relative to non-sex-related characters. Here we report a general comparison of evolutionary rates of sex-biased genes using data from cDNA microarray experiments and comparative genomic studies of Drosophila. Comparisons of nonsynonymous/synonymous substitution rates (d(N)/d(S)) between species of the D. melanogaster subgroup revealed that genes with male-biased expression had significantly faster rates of evolution than genes with female-biased or unbiased expression. The difference was caused primarily by a higher d(N) in the male-biased genes. The same pattern was observed for comparisons among more distantly related species. In comparisons between D. melanogaster and D. pseudoobscura, genes with highly biased male expression were significantly more divergent than genes with highly biased female expression. In many cases, orthologs of D. melanogaster male-biased genes could not be identified in D. pseudoobscura through a Blast search. In contrast to the male-biased genes, there was no clear evidence for accelerated rates of evolution in female-biased genes, and most comparisons indicated a reduced rate of evolution in female-biased genes relative to unbiased genes. Male-biased genes did not show an increased ratio of nonsynonymous/synonymous polymorphism within D. melanogaster, and comparisons of polymorphism/divergence ratios suggest that the rapid evolution of male-biased genes is caused by positive selection.     

Molecular evolution of Drosophila odorant receptor genes

A total of 752 odorant receptor (Or) genes, including pseudogenes, were identified in 11 Drosophila species and named after their orthologs in Drosophila melanogaster. The 813 Or genes, including 61 from D. melanogaster, were classified into 59 orthologous groups that are well supported by gene phylogeny. By reconciling with the gene family phylogeny, we estimated the number of gene duplication/loss events and intron gain/loss events in the species phylogeny. We found that these events are particularly frequent in Drosophila grimshawi, Drosophila willistoni, and obscura group. More than half of the duplicated genes stay as tandem arrays, whose size range from 2 to 8. These genes vary in sequence and some likely underwent positive selection, indicating that the gene duplication was important for flies to acquire new olfactory functions. We hypothesize that Or genes conferred the basic olfactory repertoire to ancestral flies before the speciation of the Drosophila and Sophophora subgenera about 40 Mya. This repertoire has been largely maintained in the current species, whereas lineage-specific gene duplication seems to have led to additional specialization in some species in response to specific ecological conditions.     

Transfer RNA genes of Drosophila melanogaster.

Three recombinant plasmids containing randomly sheared genomic D. melanogaster tRNAs have been identified and characterized in detail. One of these, the plasmid 14C4, has a D. melanogaster (Dm) DNA segment of 18 kb, and has three tRNA2Arg and two tRNAAsN genes. The second plasmid, 38B10, has tRNAHis genes, while the third plasmid, 63H5, contains coding sequences for tRNA2Asp. The Dm DNA segments in each recombinant plasmid are derived from unique cytogenetic loci. 14C4 is from 84 F, 38B10 is from 48 F and 63H5 is from 70 A.     

Functional interactions of neurogenic genes of Drosophila melanogaster

The neurogenic genes of Drosophila melanogaster are involved in the decision of ectodermal cells to take on a neural or an epidermal fate. We present evidence in support of the notion that six of the neurogenic genes are functionally related. We studied the phenotype of embryos lacking one of the neurogenic genes in the presence of an increased dosage of the wild-type allele of another neurogenic gene. Our analysis also included the Hairless locus, whose function is related to that of the neurogenic genes, as well as to many other genes. The effects observed were asymmetric in that triploidy for a given gene modified the phenotype of loss of the function of another gene, but triploidy of the latter gene did not modify the phenotype of loss of the function of the former gene. These asymmetries allowed us to establish a polarity of gene interactions, as well as to order the genes according to the assumed ability of some of them to modify the activity of others. In this sequence, almondex is the first link and Enhancer of split the last one. Our evidence suggests that the function of big brain is independent of the function of the other six. The consequences of this arrangement for the commitment of ectodermal cells are discussed.     

Drosophila segmentation genes and blastoderm cell identities

The formation of the segmentation pattern in Drosophila embryos provides an excellent model for investigating the process of pattern formation in multicellular organisms. Several genes required in an embryo for normal segmentation have been analyzed by classical and molecular genetic and morphological techniques. A detailed consideration of these results suggests that these segmentation genes are combinatorially involved in translating the positional identities of individual cells at an early stage in Drosophila development.