Asymmetric gene conversion at inserted segments on yeast mitochondrial DNA.

Different molecular weight forms of the protein product of the yeast mitochondrial gene var 1 are shown at arise by a process of asymmetric gene conversion. These different forms can be accounted by two DNA segments, 36 and 57 base pairs long, present in one allelic form of the var 1 structural gene, which can be inserted independently and at different frequencies into other var 1 alleles.     

Rearranged mitochondrial genes in the yeast nuclear genome

We have found a contiguous DNA sequence in the yeast nuclear genome with extensive homology to non-contiguous yeast mitochondrial DNA sequences. Closely linked to this nuclear sequence in some, but not all, yeast strains is a tandem pair of transposable (Ty) elements. Certain features of the content and organization of this nuclear DNA sequence suggest that it may have originated from petite mitochondrial DNA which integrated into the nuclear genome.     

Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies

In vitro studies of muscle mitochondrial metabolism in patients with mitochondrial myopathy have identified a variety of functional defects of the mitochondrial respiratory chain, predominantly affecting complex I (NADH-CoQ reductase) or complex III (ubiquinol-cytochrome c reductase) in adult cases. These two enzymes consist of approximately 36 subunits, eight of which are encoded by mitochondrial DNA (mtDNA). The increased incidence of maternal, as opposed to paternal, transmission in familial mitochondrial myopathy suggests that these disorders may be caused by mutations of mtDNA. Multiple restriction endonuclease analysis of leukocyte mtDNA from patients with the disease, and their relatives, showed no differences in cleavage patterns between affected and unaffected individuals in any single maternal line. When muscle mtDNA was studied, nine of 25 patients were found to have two populations of muscle mtDNA, one of which had deletions of up to 7 kilobases in length. These observations demonstrate that mtDNA heteroplasmy can occur in man and that human disease may be associated with defects of the mitochondrial genome.     

DNA sequences of telomeres maintained in yeast

Telomeres, the ends of eukaryotic chromosomes, have long been recognized as specialized structures. Their stability compared with broken ends of chromosomes suggested that they have properties which protect them from fusion, degradation or recombination. Furthermore, a linear DNA molecule such as that of a eukaryotic chromosome must have a structure at its ends which allows its complete replication, as no known DNA polymerase can initiate synthesis without a primer. At the ends of the relatively short, multi-copy linear DNA molecules found naturally in the nuclei of several lower eukaryotes, there are simple tandemly repeated sequences with, in the cases analysed, a specific array of single-strand breaks, on both DNA strands, in the distal portion of the block of repeats. In general, however, direct analysis of chromosomal termini presents problems because of their very low abundance in nuclei. To circumvent this problem, we have previously cloned a chromosomal telomere of the yeast Saccharomyces cerevisiae on a linear DNA vector molecule. Here we show that yeast chromosomal telomeres terminate in a DNA sequence consisting of tandem irregular repeats of the general form C1-3A. The same repeat units are added to the ends of Tetrahymena telomeres, in an apparently non-template-directed manner, during their replication on linear plasmids in yeast. Such DNA addition may have a fundamental role in telomere replication.     

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.     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

Paternal inheritance of mitochondrial DNA in mice.

For nearly 20 years it has been assumed on the basis of low-resolution experiments that mitochondrial (mt)DNA, in contrast to the genes in the nucleus, has an exclusively maternal mode of inheritance in animals. Using the polymerase chain reaction, paternally inherited mtDNA molecules have now been detected in mice at a frequency of 10(-4), relative to the maternal contributions. These mice were hybrids between two inbred strains (C57BL/6J and Mus spretus) whose mtDNAs can be distinguished easily. This new mode of inheritance provides a mechanism for generating heteroplasmy and may explain mitochondrial disorders exhibiting biparental transmission.     

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.     

Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast

Although single heterozygous markers in yeast usually segregate during meiosis in a 2:2 ratio, abberant 3:1 segregations occur quite frequently as a result of gene-conversion events. A second type of aberrant segregation, post-meiotic segregation, results from the segregation of two genotypes from a single haploid spore; in yeast such events are detected as sectored spore colonies and usually occur rarely. Post-meiotic segregation is thought to result from the replication of heteroduplex DNA formed during meiotic recombination. We report here that if the heteroduplex includes a palindromic insertion sequence, a high frequency of post-meiotic segregation results. This suggests that palindromic insertions are poorly repaired, which may be the result of hairpin-loop formation that affects the efficiency of repair of heteroduplex DNA.     

Premature ageing in mice expressing defective mitochondrial DNA polymerase

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.