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.

Nucleotide sequence variation is frequent in the mitochondrial DNA displacement loop region of individual human tumor cells.

The mitochondrial DNA (mtDNA) displacement loop (D-loop) regions of 76 various tumor cell lines were examined to investigate the existence of a specific relationship between a somatic mtDNA sequence and initiation and/or progression of a tumor. Based on molecular cloning-sequencing analysis, a nucleotide sequence in the D-loop region in each cell line was found to be homoplasmic. Several site-specific nucleotide variations were found in stomach and liver tumor cell lines more frequently than those in other tumor cell lines. Subsequently, 20 pairs of noncancerous and cancerous parts from stomach and liver tumor tissues were examined. In the liver tumor tissue, 80% of the noncancerous parts exhibited slightly higher heterogeneity than the corresponding cancerous parts. Several site-specific nucleotide variations found in 76 tumor cell lines were also detected in noncancerous or cancerous parts of stomach and liver tumor tissues. However, it remains unclear why the mtDNA D-loop sequence is homoplasmic in each tumor cell line. The data indicate that mtDNA exhibits heterogeneity even in the noncancerous part and a slight decrease in heterogeneity during tumorigenesis and/or tumor progression. Homoplasmy of the mtDNA population in the tumor cell line would be acquired in the cloning process of establishing a cell line. Site-specific nucleotide substitutions might not be directly involved in the tumorigenesis process.

Analysis of paternal transmission of mitochondrial DNA in Drosophila.

It has previously been shown that paternal mitochondrial DNA (mtDNA) can be detected in later generations in Drosophila. To further analyze the paternal transmission of mtDNA, the progeny of two intraspecific and three interspecific crosses were examined in the frequency of the paternal transmission of mtDNA, using closely related species of the melanogaster species subgroup. Types of mtDNA in the progeny of the individual backcrosses of F(1) females were analyzed by selective amplification of paternal mtDNA. More than 100 F(1) females were examined for each backcross. The same type of mtDNA as that of the paternal mtDNA was detected in approximately 20-60% of the backcrosses. The present results indicate that paternal leakage occurs in the intraspecific crosses as well as in the interspecific crosses in Drosophila.

Complete nucleotide sequence of the A+T-rich region of Drosophila mauritiana mitochondrial DNA.

We determined the complete nucleotide sequence of the A+T-rich region of the maII type of mtDNA in D. mauritiana. The nucleotide sequence was found to contain 3,206 bp. Three types of conserved element, i.e., type I element, type II element, and T-stretch, were included in this sequence, as reported for D. melanogaster. Comparison between the two species revealed that the type I elements were less conserved than the type II elements. However, each of these type I elements contained a G-stretch within a loop of a putative stem-loop-forming sequence, which has also been observed in D. melanogaster. Moreover, in both type I and type II repeat arrays, the elements closest to the T-stretch diverged the most, due to nucleotide substitution and/or the insertion of short repeats. Sequence comparison of the two complete sequences of the A+T-rich region of D. melanogaster and the maII type of D. mauritiana, as well as comparison of partial sequences in other types of mtDNA within the melanogaster complex, suggested that the A+T-rich region in this complex has been maintained by concerted evolution after the duplication of two types of element, i.e., type I and type II.

Involvement of Drosophila Sir2-like genes in the regulation of life span.

In Drosophila melanogaster, the Sir2 gene and four Sir2-like genes have been found to be homologous to yeast SIR2 genes. To examine whether the fly Sir2, CG5216, and two Sir2-like genes, CG5085 and CG6284, affect life span, we suppressed their expression using RNAi. Decreased expression of the Sir2 and Sir2-like genes in all cells caused lethality during development. Suppression of the Sir2 in neurons and ubiquitous silencing of the Sir2-like genes shortened life spans. The effects were severer at 28 degrees C than at 25 degrees C. These results suggest that Sir2-like genes as well as Sir2 are involved in the regulation of life span in Drosophila.

Detection of deletions flanked by short direct repeats in mitochondrial DNA of aging Drosophila.

Cumulative damage due to reactive oxygen species (ROS) in mitochondria, especially in mitochondrial DNA (mtDNA), would result in a decrease in mitochondrial respiratory function and contributes to the age-related decline in the physiological functioning of organisms. Previously, we reported the tissue-specific accumulation of deleted mtDNA with age in Drosophila melanogaster. In the present study, to understand the mechanism by which mtDNA deletion is generated with age, nucleotide sequences of deleted mtDNA were determined. Consequently, 33 different sequences each containing a deletion were obtained from flies that were more than 55-day-old. Most of the deletions were found to be flanked by short direct repeats. The present results, together with those from other animals, suggest that there is a common mechanism generating mtDNA deletions through direct repeats.

Accumulation of deleted mitochondrial DNA in aging Drosophila melanogaster.

Cumulative damage in mitochondria by reactive oxygen species is thought to result in a decrease in mitochondrial respiratory function and to contribute to the age-related decline in the physiological function of organisms. The mitochondrial genome is also subjected to damage with age through deletions. The accumulation of deleted mitochondrial DNA (mtDNA) has been observed in various animals, but still remains unclear in insects. We examined the accumulation of deleted mtDNA in D. melanogaster at various ages from larvae to 65-day-old adults. When DNA extracted from whole bodies was examined by PCR and Southern hybridization, the age-related accumulation of deletions was not clear. However, when the accumulation of deleted mtDNA with age was examined separately in three parts of the body (head, thorax and abdomen), deleted mtDNA signals were detected more frequently in the thorax and the accumulation was age-dependent. Three of the deleted mtDNA were cloned, and the breakpoints of the deletions were identified. These results strongly suggest that deleted mtDNA accumulates in Drosophila with age in a tissue-specific manner.

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.

Comparative proteomic analyses of macular and peripheral retina of cynomolgus monkeys (Macaca fascicularis).

The central region of the primate retina is called the macula. The fovea is located at the center of the macula, where the photoreceptors are concentrated to create a neural network adapted for high visual acuity. Damage to the fovea, e.g., by macular dystrophies and age-related macular degeneration, can reduce central visual acuity. The molecular mechanisms leading to these diseases are most likely dependent on the proteins in the macula which differ from those in the peripheral retina in expression level. To investigate whether the distribution of proteins in the macula is different from the peripheral retina, proteomic analyses of tissues from these two regions of cynomolgus monkeys were compared. Two-dimensional gel electrophoresis and mass spectrometry identified 26 proteins that were present only in the macular gel spots. The expression levels of five proteins, cone photoreceptor specific arrestin-C, gamma-synuclein, epidermal fatty acid binding protein, tropomyosin 1alpha chain, and heterogeneous nuclear ribonucleoproteins A2/B1, were significantly higher in the macula than in the peripheral retina. Immunostaining of macula sections by antibodies to each identified protein revealed unique localization in the retina, retinal pigment epithelial cells and the choroidal layer. Some of these proteins were located in cells with higher densities in the macula. We suggest that it will be important to study these proteins to determine their contribution to the pathogenesis and progression of macula diseases.

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.