Alleles of the var1 locus on yeast mitochondrial DNA specify the size of var1 ribosomal protein. We report the nucleotide sequence of a var1 allele that determines the smallest var1 protein. It contains an open reading frame of 396 codons, which we identify as the structural gene for var1 protein. The var1 protein specified by this allele has an amino acid composition in close agreement with that predicted by the DNA sequence. The var1 coding region is highly unusual: it is 89.6% AT and contains a 46 bp GC-rich palindromic cluster that accounts for 38% of the total GC residues. Our results strongly suggest that like mammalian mitochondria but unlike those from Neurospora, yeast mitochondria use AUA as a methionine codon. Comparison with the sequence of a var1 allele specifying a larger protein suggests that some size polymorphism of var1 protein results from in-frame insertions of a variable number of AAT (Asn) codons.

A procedure is presented for the rapid isolation of mitochondrial DNA (mtDNA) in high yield from Saccharomyces cerevisiae. Yeast cells, which may be grown to late stationary phase, are broken by a combination of enzymatic and mechanical means; mtDNA is then isolated from a crude mitochondrial lysate by a single cycle of bisbenzimide-CsCl buoyant density centrifugation. mtDNA so isolated is at least 99.5% pure, and has a mean duplex molecular weight of 24.5 . 10(6). In addition to mtDNA and bulk nuclear DNA, several other yeast nucleic acid species, identified as ribosomal DNA and a mixture of duplex RNAs, form discrete bands in these gradients.

We have investigated mtDNAorganization in the o6mycetous water mold; Achlya, and report here that this primitive organism contains a circular mitochondrial genome of 49.8 kilobase, pairs. Extensive restriction endonuclease analysis indicates that a significant portion of the genome is present as an inverted repeat. Of 52 restriction sites for 14 enzymes. thus far mapped, 28 sites cluster in two 9.6-kilobase-pair regions; within these regions, the sequence of sites is inverted but the spacing between analogous sites is identical. The repeat arms have a maximum length of 12.1 kilobase pairs and are separated by 4.64.4 and 21.0-22.3 kilobase pairs of unique sequences. Transfer hybridization experiments show that genes for both the large and the small rRNAs are contained within each repeat. Restriction endonuclease analysis shows that the unique regions between the inverted repeats are present in both possible orientations with respect to each other and in approximately equal proportions. These orientational, or "flip-flop," isomers of the unique regions are postulated to occur by intramolecular homologous recombination between the repeated regions.Mitochondrial genomes, while functionally similar, present significant contrasts in gene structure and organization. Vertebrate mitochondrial genomes are circular with a length of about 16.4 kilobase pairs (kb) and a uniform gene order. However, ascomycetous fungi such as yeasts and Neurospora contain mtDNAs that vary in size, form, and organization and are up to five times larger (reviewed in refs. 1-3).Studies ofmitochondrial genes and gene organization among a small number of lower eukaryotes show a complex picture, with surprising differences among organisms. Some ofthe most striking contrasts are seen on comparing Saccharomyces cerevisiae with the related ascomycete Neurospora crassa. It has been known for several years that the ATPase subunit 9 gene is found on mtDNA in yeast (4) and on nuclear DNA in Neurospora (5). Recently, it has become clear that these organisms also use different mitochondrial genetic codes:-yeast uses the CUN series of codons (where N is any ribonucleotide) to represent threonine (6) and, like mammalian mitochondria (7, 8), uses AUA to code for methionine (9), while Neurospora uses CUN and AUA for leucine and isoleucine, respectively (10).Consequently, we have started to examine the mtDNA of other lower eukaryotes; here, we report the restriction map of mtDNA from the primitive oomycetous water mold Achlya ambisexualis. We find the unusual mitochondrial feature ofa large inverted repeat containing both rRNA genes. The population of molecules, moreover, contains two isomers that differ in the orientation of the unique regions with respect to each other. MATERIALS AND METHODSGrowth of Achlya. A. ambisexualis E-87 was obtained from W. Timberlake (University of California, Davis). Growth in PYG medium (11) was carried out at 300C on a Gyrotory shaker at 200 rpm from a spore inoculum (12). Mycelia were harvested in late logarithmicphase...

We constructed restriction-site and gene maps for mitochondrial DNAs from seven isolates of five species of Suillus (Boletaceae, Basidiomycotina). Each mitochondrial genome exists as a single circular chromosome, ranging in size from 36 to 121 kb. Comparisons within species and between two closely related species revealed that insertions and deletions are the major form of genome change, whereas most restriction sites are conserved. Among more distantly related species, size and restriction-site differences were too great to allow precise alignments of maps, but small clusters of putatively homologous restriction sites were found. Two mitochondrial gene orders exist in the five species. These orders differ only by the relative positions of the genes for ATPase subunit 9 and the small ribosomal RNA and are interconvertible by a single transposition. One of the two gene arrangements is shared by four species whose mitochondrial DNAs span the entire size range of 36 to 121 kb. The conservation of gene order in molecules that vary over three-fold in size and share few restriction sites demonstrates a low frequency of rearrangements relative to insertions, deletions, and base substitutions.