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Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy beta-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 +/- 920, significantly higher than that in skeletal muscle, 3650 +/- 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.

The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex. Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector. These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene. The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane. In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane. Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long. In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species. The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.

A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.

A series of yeast mitochondrial mit- mutants with defects in the oli2 gene, coding for subunit 6 of the mitochondrial ATPase complex, has been analyzed at the DNA sequence level. Fifteen of sixteen primary mit- mutants were shown to contain frameshift or nonsense mutations predicting truncated subunit 6 polypeptides, in various strains ranging from about 20% to 95% of the wild-type length of 259 amino acids. In only one strain could the defect in subunit 6 function be assigned to amino acid substitution in an otherwise full-length subunit 6. Many mutants carried multiple base substitutions or insertions/deletions, presumably arising from the manganese chloride mutagenesis treatment. Revertants from three of the mit- mutants were analyzed: all contained full-length subunit 6 proteins with one or more amino acid substitutions. The preponderance of truncated proteins as opposed to substituted full-length proteins in oli2 mit- mutants is suggested to reflect the ability of subunit 6 to accommodate amino acid substitutions at many locations, with little or no change in its functional properties in the membrane FO-sector of the ATPase complex.

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.

The mutation in the temperature-conditional mit- mutant h56, mapped previously to the var1 gene region of Saccharomyces cerevisiae mitochondrial DNA, results in a specific inhibition of var1 protein synthesis in cells incubated at the non-permissive temperature, 36 degrees C (1). We have now characterized the mutation present in mutant h56 by DNA sequencing and found it to be an A to T transversion located 109 nucleotides upstream of the var1 reading frame. Two spontaneous revertants of mutant h56 restore the parental strain sequence at residue -109, confirming that this single base change within the 5'-untranslated region of the var1 mRNA is responsible for defective synthesis of the var1 protein. A comparison of var1 transcripts in the parental and mutant strains has shown that the mutation specifically blocks formation of var1 mRNA at 36 degrees C and leads to accumulation of precursor transcripts. Expression of the oli1 gene, co-transcribed with the var1 gene in primary transcripts, is not affected. It is concluded that the mutation in mutant h56 alters the secondary structure of the precursor RNA, inhibiting an endonucleolytic cleavage required to generate the 5' end of var1 mRNA.