Sadao I. Chigusa

A trace amine, tyramine, functions as a neuromodulator in Drosophila melanogaster.

The tyramine receptor (TyrR) is a G protein-coupled receptor for trace amines, cloned in Drosophila melanogaster, and claimed to be either an octopamine receptor or a tyramine receptor. We previously reported that in the larval neuromuscular junctions, the modulatory effect on the excitatory junction potentials of tyramine is distinctly different from that of octopamine. The effect of tyramine but not of octopamine was selectively abolished in the TyrR mutant hono, suggesting that this gene encodes a receptor for tyramine, and not for octopamine. We examined whether there was a gene-dosage effect of this tyramine modulation using combinations of hono, deficiency (Df) and wild-type alleles. The tyramine effect was observed in hono heterozygotes (+/hono), which showed intermediate levels of response, but was not seen in +/Df or hono/Df hemizygotes. While these further suggest that tyramine is the true ligand, it is possible that the gene-dosage effect is only evident above some threshold of gene expression levels. Immunohistochemical staining using an anti-tyramine antibody identified tyramine-containing neurons in the larval central nervous system, some of which were distinct from the octopamine-containing neurons. Taken together, these results strongly suggest that tyramine functions as a neuromodulator.

Dubious maternal inheritance of mitochondrial DNA in D. simulans and evolution of D. mauritiana.

Within-line heterogeneity has been found in the mitochondrial DNA (mtDNA) in two isofemale lines of D. simulans . The co-existing types, S and M, were typical of the mtDNA in D. simulans and in D. mauritiana , respectively, their nucleotide divergence per site being ca. 2·1%. Segregation analysis confirmed that some individuals in these lines were heteroplasmic and suggested incomplete maternal inheritance of mtDNA in Drosophila . Examination of other lines of D. simulans revealed that the M type of D. mauritiana occurs at 71% in Reunion, 38% in Madagascar and 0% in Kenya. This finding and interspecific sequence comparisons of both M types indicate that D. mauritiana diverged from D. simulans probably less than 240000 years ago.

Diversification of olfactory receptor genes in the Japanese medaka fish, Oryzias latipes

Vertebrate olfactory receptors (OR) exists as the largest multigene family, scattered throughout the genome in clusters. Studies have shown that different animals possess remarkably diverse set of OR genes to recognize diverse odor molecules. In order to examine the evolutionary process of OR diversification, we examined three OR gene subfamilies from Japanese medaka fish (seven lines sampled from four populations). For each subfamily, the sequences of ancestral genes were inferred based on distance method. Examination of d(N)/d(S) ratios for each branch of phylogenetic trees suggested that purifying selection is the major force of evolution in medaka OR genes. However, for the mfOR1 and mfOR2 paralogous gene pairs, a nonrandom distribution of fixed amino acid changes and the d(N)>d(S) in a branch suggested that diversifying selection occurred after gene duplication. The fixed amino acid changes were observed in the third, fifth and sixth transmembrane domains, which has been predicted to serve as a ligand-binding pocket in a structural model. Compatibility test suggested that interlocus recombinations involving the fourth transmembrane domain occurred between the mfOR1 and mfOR2 gene pairs. The pattern of nucleotide substitutions in other OR genes agrees with the hypothesis that a limited number of amino acid residues are involved in odorant binding. Such comparative analyses of paralogous OR genes should provide bases for understanding the evolution, the structure, and the functional specificity of OR genes.

Evolutionary history and mechanism of the Drosophila Cecropin gene family

Abstract Upon bacterial infection, insects secrete a set of synthesized antibacterial proteins into the hemolymph and initiate synergistic destruction of invaders. Cecropin is one such antibacterial protein which is also found in vertebrates. To study the evolutionary history and mechanism of the Cecropin gene family, we determined DNA sequences of one isogenic In(3R)C and six isofemale lines of Drosophila melanogaster as well as one line of D. simulans and of D. yakuba. The phylogenetic analysis of these sequences together with those published for D. virilis and Sarcophaga peregrina reveals frequent gene re-organization. It was also found that silent nucleotide differences within D. melanogaster are quite heterogeneous across the gene region of approximately 3 kilobases and the extent of polymorphism is unusually usually high. These data suggest that the Cecropin gene region of D. melanogaster underwent intragenic recombination as well as introgression from a closely related sibling species, D. simulans.

Evolutionary analysis of putative olfactory receptor genes of medaka fish, Oryzias latipes

To obtain an understanding of the origin, diversification and genomic organization of vertebrate olfactory receptor genes, we have newly cloned and characterized putative olfactory receptor genes, mfOR1, mfOR2, mfOR3 and mfOR4 from the genomic DNA of medaka fish (Oryzias latipes). The four sequences contained features commonly seen in known olfactory receptor genes and were phylogenetically most closely related to those of catfish and zebrafish. Among them, mfOR1 and mfOR2 showed the highest amino acid (aa) similarity (93%) and defined a novel olfactory receptor gene family that is most divergent among all other vertebrate olfactory receptor genes. Southern hybridization analyses suggested that mfOR1 and mfOR2 are tightly linked to each other (within 24kb), although suitable marker genes were not available to locate their linkage group. Unlike observation in catfish olfactory receptor sequences, nucleotide (nt) substitutions between the two sequences did not show any evidence of positive natural selection. mfOR3 and mfOR4, however, showed a much lower aa similarity (26%) and were both mapped to a region in the medaka linkage group XX. After including these medaka fish sequences, olfactory receptors of terrestrial and aquatic animals formed significantly different clusters in the phylogenetic tree. Although the member genes of each olfactory receptor gene subfamily are less in fish than that in mammals, fish seem to have maintained more diverse olfactory receptor gene families. Our finding of a novel olfactory receptor gene family in medaka fish may provide a step towards understanding the emergence of the olfactory receptor gene in vertebrates.

Rapid evolution of the male-specific antibacterial protein andropin gene in Drosophila.

Abstract.Andropin, which encodes an antibacterial protein, is closely linked to the Cecropin gene cluster of D. melanogaster. Andropin and Cecropins are considered to have originated from one common ancestor. However, the expression pattern of Andropin is distinct from that of Cecropins, being restricted to the adult male ejaculatory duct. To elucidate the evolutionary process of Andropin, we have sequenced Andropin genes from D. melanogaster and its closely related species. In D. melanogaster, the nucleotide diversity of Andropin is remarkably low compared to that of Cecropin. In contrast, nonsynonymous substitutions of Andropin are conspicuously frequent between species. From genomic Southern analysis, Andropin-like genes are present in at least the melanogaster species subgroup. The series of present results suggests that Andropin was born in the course of constructing the Drosophila Cecropin gene family and then started to evolve rapidly, in contrast to Cecropins.

Hybrid dysgenesis in natural populations of Drosophila melanogaster in Japan. II: Strains which cannot induce P-M dysgenesis may completely suppress functional P element activity

Many inbred and isofemale lines derived from wild populations of Drosophila melanogaster were tested for gonadal dysgenic sterility, male recombination and sn w secondary mutation. Among them, we have found strains whose dysgenic offspring show negligible sterility, and undetectable male recombination and sn w mutation. They can be considered to be neutral strains in the strict sense. Such neutral strains appear to carry only defective P elements in their genomes. Taking the observations of Karess & Rubin (1984) into account, it is suggested that some defective P elements retain the function necessary for P cytotype. Cytotype determination mechanisms are discussed.

Molecular Population Genetics of Olfactory Systems in Drosophila melanogaster Complex: Cloning of Putative Olfactory Receptor Genes

A large number of different odorant molecules are discriminated by the olfactory system. The putative olfactory receptor genes of the rat [1], catfish [2], and a few other organisms have recently been cloned. Interestingly, the following sequence analyses suggest that the genetic variation of these gene subfamilies is maintained by a certain type of Darwinian selection [2,3]. Fruit flies seem to have a relatively simple, olfactory system and can be studied by molecular, genetic and behavioral methods [4]. However, no nucleotide sequence data of any Drosophila olfactory receptor gene has yet been reported. Drosophila should be very useful as material for studies of molecular population genetics and molecular evolution. To elucidate the genetic basis of olfaction and the mechanisms that maintain genetic diversity, we have begun to clone the olfactory receptor genes of Drosophila, based on three kinds of methods; the polymerase chain reaction (PCR), cDNA library screening, and the enhancer trap assay: 1. In PCR amplification we have constructed six series of PCR primers, corresponding to the highly conserved regions between the putative olfactory receptor genes of the rat [1] and the catfish [2]. Many PCR products were amplified and sequenced, but unfortunately, we found no clone showing significant homology to the olfactory receptor genes published. At present, we are examining other DNA products, using the primers 5′ CGGAGCTCGA (CT) (AC)GITA(CT)GTIGCIAT(ACT)TG and 3′ GCGGATCCTA(AGT)AT(AG)AAIGG(AG) TTIA(AG)CAT. 2. cDNA library screening plays an important role in our experiment. We are constructing libraries from the whole body and the head and we have prepared a series of oligonucleotides for the use of library screening. After performing Southern blot hybridization experiments, we found two appropriate oligonucleotides as a probe. 3. We are planning to isolate genome and cDNA clones by means of the enhancer trap assay. Following jump starter methods for the construction of insertional mutant lines with the P-lwB vector, we (R.U. and D.Y.) have obtained nearly 1000 homozygous fertile lines which may contain a single P-1wB insert in the second or third chromosome. After staining for β-galactosidase expression, we carefully observed the third antennal segment and maxillary palpus, since these organs are believed to have olfactory function [5]. Thus far, from this assay, we have found several lines with interesting staining patterns. We will be continuing with all the experiments described above. After we have obtained the DNA sequence data, which we should have in the near future, we will discuss Drosophila olfactory systems from the molecular evolutionary viewpoint.