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.

Evolutionary history and mechanism of the Drosophila cecropin gene family.

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.

Evolution of the mitochondrial ATPase 6 gene in Drosophila: unusually high level of polymorphism in D. melanogaster.

We have determined 1990 bp mitochondrial DNA sequence which extends from 3' end of the cytochrome oxidase subunit I (COI) gene to 5' end of the COIII gene from two sibling species of Drosophila, D. simulans and D. mauritiana. Analyses of the sequences and part of the NADH dehydrogenase subunit 2 gene and the COI gene together with those from D. melanogaster and D. yakuba revealed that amino-acid substitution rate of the ATPase 6 gene seems to be higher in some strains of D. melanogaster than in the other species. High level of amino-acid polymorphism in this gene was observed in D. melanogaster. Synonymous substitution rate is relatively constant in all the genes examined, suggesting that mutation rate is not higher in the ATPase 6 gene of D. melanogaster. The amino-acid substitutions found specifically in D. melanogaster are at the sites which are not conserved among mammals, yeast and E. coli. These sites of the ATPase 6 gene might lose the selective constraint in D. melanogaster, and the amino-acid substitutions can be explained by neutral mutations and random genetic drift.

Temperature-dependent selection in the transmission of mitochondrial DNA in Drosophila.

We previously reported a selective mode of mitochondrial DNA (mtDNA) transmission in mtDNA heteroplasmy that was induced artificially in Drosophila melanogaster; the transmission bias appeared to depend on the particular temperature at which heteroplasmic lines were maintained. Here we report investigations of the temperature-dependent mode of mtDNA transmission in heteroplasmic lines for intra- and interspecific combinations maintained separately at 22.5 degrees C, 25 degrees C and 29 degrees C for 20 generations. We have examined a selection model for mitochondrial transmission, similar to genetic selection in haploid organisms. Changes in the relative proportions of two types of mtDNA fit the expectations from the model well. The intensity of selection estimated as a selection coefficient depends on temperature. Temperature-sensitive processes thus appear to be involved in the transmission and maintenance of mitochondria.

Hybrid dysgenesis in natural populations of Drosophila melanogaster in Japan. III. The P-M system in and around Japan.

The P-M system of hybrid dysgenesis in Drosophila melanogaster was investigated on the basis of gonadal dysgenesis, using 1,590 strains from 28 natural populations in Japan, and 20 populations from Southeast Asia, the Pacific area and Africa. Strong P strains were found sporadically in several populations in Japan. Few strong M strains were observed. Q strains were present at a high frequency in most populations. Thus, most populations in these areas were regarded as Q populations. The distribution of the P element and the evolution of P, Q and M populations are also discussed.

Further observation of paternal transmission of Drosophila mitochondrial DNA by PCR selective amplification method.

By designing 3' ends of primers in PCR (polymerase chain reaction), a specific DNA fragment was selectively amplified in the presence of a 10(3)-fold excess of highly homologous (sequence difference ca. 2%) opponent DNA. This technique was applied in detecting paternal leakage of mitochondrial DNA (mtDNA) in intraspecific crosses of Drosophila simulans and interspecific crosses of Drosophila simulans and Drosophila mauritiana. The mtDNA types of their progeny were analysed by selective amplification of the paternal mtDNA fragment possessing a polymorphic restriction site and detecting its cleaved fragments. Paternal mtDNA was detected in the progeny of 14 out of 16 crosses. The present result indicates small but frequent inheritance of sperm mtDNA in Drosophila, which is supportive to our previous finding.

Mitochondrial DNA heteroplasmy maintained in natural populations of Drosophila simulans in Réunion.

Mitochondrial DNA (mtDNA) variation in Drosophila simulans was studied to determine whether the cytoplasmic state of mtDNA heteroplasmy persists in natural populations in Réunion. For this purpose, 172 isofemale lines, newly collected from two local populations, were examined, among which three types of mtDNA (siII, siIII and siIII') were found, based on the Hpa II restriction pattern. Ten of the lines were heteroplasmic for a combination of siII and siIII, as determined by autoradiography. The same type of heteroplasmy had been noted in one of the two local populations 8 years before (Satta et al. 1988). The present results suggest that the heteroplasmic state occurs recurrently in natural populations of D. simulans in Réunion.

Selective transmission of mitochondrial DNA in heteroplasmic lines for intra- and interspecific combinations in Drosophila melanogaster.

The transmission of mitochondrial DNA (mtDNA) was investigated in the heteroplasmic lines of Drosophila melanogaster at 19 degrees C and at 25 degrees C. The selective transmission of one type of mtDNA was dependent on the temperature at which the lines were maintained. In heteroplasmic lines for an intraspecific combination induced by germ-plasm transplantation using D. melanogaster as a germ-plasm donor, the proportion of donor mtDNA decreased in four out of five lines examined, the decreasing rate of which being greater at 25 degrees C than at 19 degrees C. Donor mtDNA was lost by the 20th generation at 25 degrees C. For an interspecific combination using D. mauritiana as a germ-plasm donor, the proportion of donor mtDNA increased and endogenous mtDNA was replaced with donor mtDNA at 25 degrees C. But donor mtDNA was almost lost at 19 degrees C by the 14th generation in all four lines examined. Possible mechanisms involved in the temperature-dependent modes of mtDNA transmission are discussed.

Incomplete maternal transmission of mitochondrial DNA in Drosophila.

The possibility of incomplete maternal transmission of mitochondrial DNA (mtDNA) in Drosophila, previously suggested by the presence of heteroplasmy, was examined by intra- and interspecific backcrosses of Drosophila simulans and its closest relative, Drosophila mauritiana. mtDNAs of offspring in these crosses were characterized by Southern hybridization with two alpha-32P-labeled probes that are specific to paternal mtDNAs. This method could detect as little as 0.03% paternal mtDNA, if present, in a sample. Among 331 lines that had been backcrossed for ten generations, four lines from the interspecific cross D. simulans (female) x D. mauritiana (male) showed clear evidence for paternal leakage of mtDNA. In three of these the maternal type was completely replaced while the fourth was heteroplasmic. Since in this experiment the total number of fertilization is known to be 331 x 10 = 3310, the proportion of paternal mtDNA per fertilization was estimated as about 0.1%. The mechanisms and evolutionary significance for paternal leakage are discussed in light of this finding.

Differences in the modes of transmission of foreign mitochondrial DNA in heteroplasmic lines for intra- and interspecific combinations in Drosophila melanogaster.

The transmission of foreign mitochondrial DNA (mtDNA) was investigated in heteroplasmic lines of Drosophila melanogaster constructed by germ-plasm transplantation and maintained at 19 degrees C. When D. melanogaster was used as a germ-plasm donor, donor mtDNA was retained in all four heteroplasmic lines examined. Individual females were found to be heteroplasmic at the 17th and 18th generations. Donor mtDNA derived from D. mauritiana was found to have decreased in all four heteroplasmic lines examined. It could no longer be found after the 16th generation. This difference in the modes of transmission of donor mtDNA in intra- and interspecific combinations of heteroplasmy indicates that there may be certain species-specific functions which propagate and transmit endogenous mtDNA under the nuclear genome of D. melanogaster.

Complete replacement of mitochondrial DNA in Drosophila.

The introduction of foreign mitochondria or mitochondrial DNA into a cell is a useful technique for clarifying the molecular mechanisms responsible for the maintenance of mitochondria. Novel combinations of mitochondrial and nuclear genomes have been studied in mammalian cells in culture and in yeast. In Drosophila, we have recently constructed heteroplasmic flies possessing both endogenous mitochondrial DNA and foreign mitochondrial DNA by intra- and interspecific transplantation of germ plasm. During the maintenance of these heteroplasmic lines, flies of D. melanogaster are produced that no longer possess their own mitochondrial DNA but retain the foreign mitochondrial DNA from D. mauritiana. . These flies are fertile and the foreign mitochondrial DNA is stably maintained in their offspring. Here we report the complete replacement of endogenous mitochondrial DNA with that from another multicellular species. Molecular and genetic analysis of this replacement in Drosophila should provide new insight into the functional interaction between nuclear and organelle genomes.

Induction of mitochondrial DNA heteroplasmy by intra- and interspecific transplantation of germ plasm in Drosophila.

A new experimental system for inducing mitochondrial DNA heteroplasmy in Drosophila was developed. By transplanting the germ plasm of Drosophila melanogaster and Drosophila mauritiana into the posterior pole of the recipient eggs of D. melanogaster, it was possible to introduce foreign mitochondria into the recipient female germline. Heteroplasmic individuals containing both donor and recipient mtDNA were obtained in intra- and interspecific combinations at similar frequencies. The proportion of donor-derived mtDNA in the heteroplasmic individuals varied considerably from individual to individual irrespective of the donor species used. No significant decrease in or elimination of donor mtDNA was observed, and the heteroplasmic state in female germlines persisted for several generations. The present system should serve very much to promote the study and clarification of the transmission genetics of mtDNA in insects.

Analysis of nucleotide substitutions of mitochondrial DNAs in Drosophila melanogaster and its sibling species.

To study the rate and pattern of nucleotide substitution in mitochondrial DNA (mtDNA), we cloned and sequenced a 975-bp segment of mtDNA from Drosophila melanogaster, D. simulans, and D. mauritiana containing the genes for three transfer RNAs and parts of two protein-coding genes, ND2 and COI. Statistical analysis of synonymous substitutions revealed a predominance of transitions over transversions among the three species, a finding differing from previous results obtained from a comparison of D. melanogaster and D. yakuba. The number of transitions observed was nearly the same for each species comparison, including D. yakuba, despite the differences in divergence times. However, transversions seemed to increase steadily with increasing divergence time. By contrast, nonsynonymous substitutions in the ND2 gene showed a predominance of transversions over transitions. Most transversions were between A and T and seemed to be due to some kind of mutational bias to which the A + T-rich mtDNA of Drosophila species may be subject. The overall rate of nucleotide substitution in Drosophila mtDNA appears to be slightly faster (approximately 1.4 times) than that of the Adh gene. This contrasts with the result obtained for mammals, in which the mtDNA evolves approximately 10 times faster than single-copy nuclear DNA. We have also shown that the start codon of the COI gene is GTGA in D. simulans and GTAA in D. mauritiana. These codons are different from that of D. melanogaster (ATAA).

The Genetic Structure of Natural Populations of DROSOPHILA MELANOGASTER. Xv. Nature of Developmental Homeostasis for Viability.

Developmental homeostasis of relative viability was examined for homozygotes and heterozygotes using second chromosomes from two populations of Drosophila melanogaster. One was a chromosome population in which spontaneous mutations were allowed to accumulate since it was begun with a single near-normal second chromosome. The second was a natural population approximately at equilibrium. For the estimation of relative viability, the Cy method was employed (Wallace 1956), and environmental variance between simultaneously replicated cultures was used as the index of developmental homeostasis. A new method was used in the estimation of sampling variance for relative viability that was employed for the calculation of environmental variance (error variance between simultaneously replicated cultures - sampling variance). The following findings were obtained.: (1) The difference in environmental variance between homozygotes and heterozygotes could not be seen when a chromosome population with variation due to new mutations was tested. (2) When a chromosome group isolated from an approximate equilibrium population was examined, heterozygotes manifested a smaller environmental variance than the homozygotes if their relative viabilities were approximately the same. (3) There was a slight negative correlation between viability and environmental variance, although opposite results were found when the viabilities of individuals were high, especially when overdominance (coupling overdominance, Mukai 1969 a, b) was manifest. On the basis of these findings, it was concluded that developmental homeostasis was a product of natural selection, and its mechanism was discussed.

Mutation rate and dominance of genes affecting viability in Drosophila melanogaster.

Spontaneous mutations were allowed to accumulate in a second chromosome that was transmitted only through heterozygous males for 40 generations. At 10-generation intervals the chromosomes were assayed for homozygous effects of the accumulated mutants. From the regression of homozygous viability on the number of generations of mutant accumulation and from the increase in genetic variance between replicate chromosomes it is possible to estimate the mutation rate and average effect of the individual mutants. Lethal mutations arose at a rate of 0.0060 per chromosome per generation. The mutants having small effects on viability are estimated to arise with a frequency at least 10 times as high as lethals, more likely 20 times as high, and possibly many more times as high if there is a large class of very nearly neutral mutations.-The dominance of such mutants was measured for chromosomes extracted from a natural population. This was determined from the regression of heterozygous viability on that of the sum of the two constituent homozygotes. The average dominance for minor viability genes in an equilibrium population was estimated to be 0.21. This is lower than the value for new mutants, as expected since those with the greatest heterozygous effect are most quickly eliminated from the population. That these mutants have a disproportionately large heterozygous effect on total fitness (as well as on the viability component thereof) is shown by the low ratio of the genetic load in equilibrium homozygotes to that of new mutant homozygotes.

Linkage disequilibrium in a local population of Drosophila melanogaster.

461 second chromosomes of Drosophila melanogaster were extracted from a Raleigh, N.C. population and four enzymes controlled by the genes located in this chromosome (alcohol dehydrogenase (EC 1.1.1.1.), malate dehydrogenase-1 (EC 1.1.1.37), glycerol-3-phosphate dehydrogenase-1 (EC 1.1.1.8), and alpha-amylase (EC 3.2.1.1), were assayed electrophoretically and cytologically (salivary-gland chromosomes). Linkage disequilibrium could not be detected among any pair of isozyme genes, except in one case that is best explained as due to a chance error in estimation. Some disequilibria were detected, however, between isozyme genes and polymorphic inversions. The relative viabilities of homozygous and heterozygous combinations of these chromosomes were estimated with respect to the alcohol dehydrogenase alleles and the glycerol-3-phosphate dehydrogenase alleles; no significant difference could be detected. The role of epistasis in natural populations is discussed on the basis of these results.