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Kluyveromyces lactis is a petite-negative yeast that does not form viable mitochondrial genome-deletion mutants (petites) when treated with DNA-targeting drugs. Loss of mtDNA is lethal for this yeast but mutations at three loci termed MGI, for mitochondrial genome integrity, can suppress this lethality. The three loci encode the alpha-, beta- and gamma-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPdelta gene encoding the delta-subunit of F1-ATPase. The deduced protein contains 158 amino acids showing 72% identity to the protein from Saccharomyces cerevisiae and a putative mitochondrial targeting sequence of 23 amino acids. Disruption of the gene causes cells to become respiratory deficient while the introduction of ATPdelta from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPdelta gene, like strains with disruptions of alpha-, beta- and gamma-F1-subunits, do not produce petite mutants when treated with ethidium bromide. However, unlike strains with disruptions in the three largest F1-subunits, disruption of ATPdelta in the presence of some mgi alleles does not abolish the Mgi- phenotype. By contrast, elimination of ATPdelta in other mgi strains removes resistance to ethidium bromide and rho0 mutants are not formed. Hence the ATPdelta subunit of F1-ATPase, while not mandatory for a Mgi- phenotype, aids some mgi alleles in suppressing rho0 lethality.

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the "gain-of-function" phenotype found in mgi1 point mutants. The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations.

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondrial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeast Kluyveromyces lactis, specific mutations in the nuclear genes MG12 and MG15 encoding the alpha- and gamma-subunits of the mitochondrial F1-ATPase, allow mtDNA to be lost. In this study we show that wild-type K. lactis does not survive in the absence of its mitochondrial genome and that the function of mgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing a mgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned the K. lactis homologue of the Saccharomyces cerevisiae mitochondrial genome maintenance gene MGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption of MGM101 is not lethal in a haploid mgi strain with a specific mutation in a subunit of the mitochondrial F1-ATPase. These observations suggest that mtDNA in K. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of Fo.

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.

Mapping the 23-kb circular mitochondrial DNA from the yeast Kluyveromyces thermotolerans has shown that only one change occurs in the gene order in comparison to the 19-kb mtDNA of Candida (Torulopsis) glabrata. Sequence analysis of the mitochondrially encoded cytochrome oxidase subunit 2 gene reveals that despite their conserved gene order, the two small genomes are more distantly related than larger mtDNA molecules with multiple rearrangements. This result supports a previous observation that larger mitochondrial genomes are more prone to rearrange than smaller forms and suggests that the architecture of the two small molecules is likely to represent the structure of an ancestor.

Mitochondrial genomes from yeasts in the Dekkera/Brettanomyces/Eeniella group vary in size from 28 to 101 kb. Mapping of genes has shown that the three smallest genomes, of 28-42 kb, have the same gene order, whereas the three larger mitochondrial DNAs of 57-101 kb are rearranged relative to the smaller molecules and between themselves. To examine the relationships between these genomes, a phylogenetic tree has been constructed by sequence comparison of the mitochondrial-encoded cytochrome oxidase subunit gene (COX2) from the six species. Contrary to expectation, the tree shows that the larger rearranged genomes are more closely related than the smaller mtDNAs. This result indicates that the gene order of the smaller mtDNAs (28-42 kb) is ancestral and that larger mtDNA molecules (57-101 kb) are more prone to rearrangement than smaller forms.

By employing pulsed-field gel electrophoresis we have determined the size of the rDNA cluster in wild-type yeast strains representing genera of Candida, Kluyveromyces, Pachysolen, Schizosaccharomyces and Torulaspora. Although the genome size of the examined species is similar (12.3-13.9 Mb), at least a four-fold variation has been observed between the lowest amount of rDNA repeats in P. tannophilus (28) and the highest in C. glabrata and S. poombe (> 115). In two species the rDNA cluster is represented by two loci, residing either in one (S. pombe) or two chromosomes (C. glabrata).

Migratory behaviour of mitochondrial DNA (mtDNA) from fungal species belonging to Plectomycetes, Loculoascomycetes and the zoosporic moulds, Oomycetes, has been studied by Pulsed Field Gel Electrophoresis (PFGE). Electrophoretic profiles demonstrate that long, linear molecules of a heterogenous size are the prevailing in-vivo form of organelle DNA in all examined species. These profiles are consistent with the presence of the rolling-circle mode of mtDNA replication that occurs in all branches of true fungi and zoosporic moulds.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.

By employing pulsed field gel electrophoresis we find that slow growing strains of Kluyveromyces lactis have only 43%-55% of the wild-type level of ribosomal DNA (rDNA) repeats. When subjected to prolonged vegetative growth these strains can increase both the number of rDNA repeats and their growth rate.

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and cox2-cox1. In addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K. lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K. lactis is closely related to that of Saccharomyces cerevisiae.

Mapping of sequences specifying the large and small ribosomal RNAs and six polypeptides in the circular 23.7 kbp mitochondrial DNA of Saccharomyces exiguus has shown that these genes have the same orientation and that a 5 gene cluster is common to this DNA and the 18.9 kbp mtDNA from Torulopsis glabrata. Included in the preserved region are juxtaposed sequences specifying ATPase subunits 6 and 9 which have the same order and orientation as analogous genes in the Escherichia coli unc operon. The above data, together with knowledge that these two sequences are dispersed in larger yeast mtDNAs, leads us to suggest that larger forms are derived from a smaller ancestral molecule that would have had some resemblance to the mtDNAs of S. exiguus and T. glabrata.

Sequences hybridizing to mitochondrial DNA probes from Saccharomyces cerevisiae have been mapped in six mitochondrial genomes from the Dekkera/Brettanomyces yeasts and in mtDNA from the closely related Eeniella nana. Sequence order for the 34.5 kbp mtDNA of E. nana is identical to that for mtDNAs from B. custersianus (28.5 kbp) and B. naardenensis (41.7 kbp) thereby suggesting that the former yeast is affiliated with the latter two species. A closer relationship is suggested for D. intermedia and D. bruxellensis as mtDNAs from these yeasts, 73.2 and 85.0 kbp respectively, have the same sequence order and mostly common restriction endonuclease sites. Differences between the two molecules are reminiscent of those found in mtDNA polymorphisms of S. cerevisiae strains thereby suggesting that the two Dekkera yeasts are variants of a single species. An unusual feature of the Dekkera species mtDNA is an inversion of the cytochrome b hybridizable region relative to the LrRNA sequence. Likewise mtDNA from B. anomalus (57.7 kbp) has an inversion of the cytochrome oxidase subunit 1 sequence with respect to the LrRNA sequence. By contrast the largest mtDNA (101.1 kbp) from B. custersii has the cytochrome b and LrRNA sequences in the same orientation. In addition hybridizable regions in this mtDNA are found in three clusters that are separated by several thousand base pairs of sequence deficient in restriction endonuclease sites. This observation together with the low guanine and cytosine content of the mtDNA suggests that the regions separating the sequence clusters are mostly adenine and thymine residues.

Analysis of gene order and orientation in the circular 18.9 kbp mitochondrial DNA molecule of Torulopsis glabrata has shown that the eight large genic sequences have the same orientation and that a five gene cluster which runs--cytochrome b, cytochrome oxidase subunit 1, ATPase subunits 6 and 9 and cytochrome oxidase subunit 2--is common to this DNA and Saccharomyces exiguus mtDNA (see accompanying paper).

Intra-species fusion products of Saccharomyces cerevisiae, Saccharomyces unisporus and Torulopsis glabrata have been isolated following polyethylene glycol-induced fusion of protoplasts and selection for prototrophic colonies. Staining with lomofungin showed that all fusion products were uninucleate. Measurement of DNA content mostly gave values between haploid and diploid levels indicating that the majority of fusion products were aneuploid, Nevertheless fusion products of S. cerevisiae and S. unisporus were, as expected, more resistant to X-irradiation than their haploid parents. By contrast, the X-ray dose-response curve of all T. glabrata fusion products was indistinguishable from their progenitors despite the fact that mitotic segregants could be recovered amongst the survivors to X-rays. A possible explanation for the behaviour towards X-rays of T. glabrata fusion products is that this species lacks a DNA repair pathway involving recombination between homologous chromosomes. We conclude from this study that the shape of the X-ray dose-response curve should not be taken to indicate the ploidy of new yeast isolates without supporting data.