Etsuko T. Matsuura

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.

GENETIC ANALYSIS OF TEMPERATURE-DEPENDENT TRANSMISSION OF MITOCHONDRIAL DNA IN DROSOPHILA

In artificially induced mitochondrial DNA (mtDNA) heteroplasmy in Drosophila, the effects of chromosome substitution on temperature-dependent selection in mtDNA transmission were investigated. Using two strains of D. melanogaster, bw;e11 and y;bw;st, which showed a different temperature dependency in mtDNA transmission, chromosomes were substituted reciprocally, and mtDNA of D. mauritiana was introduced into each newly constructed strain. For each heteroplasmy, the transmission of mtDNA was examined at 25°C and 19°C. When either the second or the third chromosome of the y;bw;st strain was substituted with that of the bw;e11 strain, the temperature-dependent selection in mtDNA transmission was altered. The selection was not changed when either the second or the third chromosome of the bw;e11 strain was substituted with that of the y;bw;st strain, or even when both the second and the third chromosomes of the bw;e11 strain were substituted with those of the y;bw;st strain. These results suggest that the temperature-dependent selection in mtDNA transmission is co-operatively regulated by gene products that are encoded by the X, second and third chromosomes.

Evolution of the A+T-rich region of mitochondrial DNA in the melanogaster species subgroup of Drosophila.

We determined the nucleotide sequences of two regions in the A+T-rich region of mitochondrial DNA (mtDNA) in the siI and siII types of D. simulans, the maII type of D. mauritiana, and D. sechellia. The sequences were aligned with those of the corresponding regions of siIII of D. simulans and maI of D. mauritiana, D. melanogaster, and D. yakuba. The type I and type II elements and the T-stretches were detected in all eight of the mtDNA types compared, indicating that the three elements are essential in the A+T-rich region of this species subgroup. The alignment revealed several short repetitive sequences and relatively large deletions in the central portions of the region. In the highly conserved sequence elements in the type II elements, the substitution rates were not uniform among lineages and acceleration in the substitution rate might have been due to loss of functional constraint in the stem–loop-forming sequences predicted in the type II elements. Patterns of nucleotide substitutions observed in the A+T-rich region were further compared with those in the coding regions and in the intergenic regions of mtDNA. Substitutions between A and T were particularly repressed in the highly conserved sequence elements and in the intergenic regions compared with those in the A+T-rich region excluding the highly conserved sequence elements and in the fourfold degenerate sites in the coding regions. The functional and structural characteristics of the A+T-rich region that might be involved in this substitutional bias are discussed.

Effects of overexpression of mitochondrial transcription factor A on lifespan and oxidative stress response in Drosophila melanogaster

Mitochondrial transcription factor A (TFAM) plays a role in the maintenance of mitochondrial DNA (mtDNA) by packaging mtDNA, forming the mitochondrial nucleoid. There have been many reports about a function of TFAM at the cellular level, but only a few studies have been done in individual organisms. Here we examined the effects of TFAM on the Drosophila lifespan and oxidative stress response, by overexpressing TFAM using the GAL4/UAS system. Under standard conditions, the lifespan of TFAM-overexpressing flies was shorter than that of the control flies. However, the lifespan of TFAM-overexpressing flies was longer when they were treated with 1% H(2)O(2). These results suggest that even though excess TFAM has a negative influence on lifespan, it has a defensive function under strong oxidative stress. In the TFAM-overexpressing flies, no significant changes in mtDNA copy number or mtDNA transcription were observed. However, the results of a total antioxidant activity assay suggest the possibility that TFAM is involved in the elimination of oxidative stress. The present results clearly show the effects of TFAM overexpression on the lifespan of Drosophila under both standard conditions and oxidative stress conditions, and our findings contribute to the understanding of the physiological mechanisms involving TFAM in mitochondria.

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.

Age-related changes in the activities of respiratory chain complexes and mitochondrial morphology in Drosophila.

Using Drosophila melanogaster, we examined changes in the activities of some of the respiratory enzyme complexes with age. The age-related decreases of enzyme activities were observed especially in complex I. We also examined changes in the ultrastructure of mitochondria in the flight muscles of thoraces. The results indicated that the mitochondrial size varied more widely in aged flies than in young ones, in addition to the slight increase in the average size with age. These changes had already appeared before the survival began to decrease, clearly indicating that the accumulation of such changes seriously damages mitochondrial function.

Mitochondria-mediated transformation of Drosophila.

Publisher Summary This chapter describes the methods for achieving heteroplasmy and the experimental conditions for replacing mtDNA. It also discusses the usefulness of mitochondria-mediated transformation for the further study of mitochondrial genetics. The relationship between mitochondrial and nuclear genomes can be studied by the introduction of foreign mitochondrial genomes into a cell or an organism. Drosophila, especially Drosophila melanogaster, is one of the most well studied organisms in genetics, and a variety of classic genetic and molecular biological methods have been developed to study and manipulate the Drosophila genome. Many species of Drosophila, from various taxonomic groups, are available and can be easily grown under laboratory conditions. Studies on mitochondria and mtDNA using Drosophila have been widely conducted, ranging from the molecular analysis of mtDNA itself to evolutionary and population studies. In Drosophila, it is even possible to isolate the mtDNA from a single individual.

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.