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TIM9 has been identified as an additional novel gene required for the petite-positive phenotype in Saccharomyces cerevisiae. tim9-1 was obtained through a screen for respiratory-deficient strains that are unable to survive in the absence of mitochondrial DNA. A point mutation found in the tim9-1 coding region converts codon 71 from Gly to Arg. Examination of genes encoding other Tim components indicated that the temperature-conditional alleles of essential genes for the viability of S. cerevisiae, TIM9, TIM10 and TIM12, are required for petite survival, while deletion of TIM8 and TIM13 has no notable effect on petite cell viability. Northern hybridization results suggested that the Spt7 transcription factor is strictly involved in transcription of TIM9 and that the synergistic lethality of tim9-1/spt7Delta dual mutations is due to the deficiency of TIM9 transcription together with defective function of the tim9-1 protein.

The nature of mutations affecting several cytochrome-deficient mutants of Debaryomyces (Schwanniomyces) occidentalis has been characterized. The DR12 mutant, which is deficient in cytochrome b, and the B10Mn mutant, which is deficient in cytochromes b and a, a3, are deleted in the mitochondrial CYB and COX1 genes respectively. The B10 strain, which is partially deficient in cytochrome b, has no detectable change in its mitochondrial DNA and possibly carries nuclear lesion(s). These three mutants, unlike the rho(-) and rho degrees "petite" mutants of Saccharomyces cerevisiae, can still grow on non-fermentable substrates, due to the development of a salicylhydroxamic acid (SHAM)-sensitive alternative pathway linked to phosphorylation at site 1. A gly(-) mutant lacking mtDNA and respiratory capacity has been isolated. For the first time, it is demonstrated that mtDNA can be altered or even lost without lethal consequence in D. occidentalis, although this yeast was classified as a petite-negative species.

The Kluyveromyces lactis nuclear gene, MRP-L23, encodes a polypeptide of 155 amino acids that shares 70% and 43% identity to the ribosomal proteins L23 and L13 of Saccharomyces cerevisiae and Escherichia coli. The deduced protein, designated K1L23, is a likely component of the large subunit of mitochondrial ribosomes as it can complement the respiratory deficient phenotype of a S. cerevisiae mrp-L23 mutant. As in S. cerevisiae, KlMRP-L23 is essential for respiratory growth of K. lactis because disruption of the gene in a "petite-positive" strain carrying a rho o-lethality suppressor atp mutation rendered cells unable to grow on a nonfermentable carbon source. However, in contrast to S. cerevisiae, disruption of MRP-L23 in wild type K. lactis is lethal. Meiotic segregants of K. lactis with a disrupted MRP-L23 allele form microcolonies with cell numbers varying from 32 to 300. These data clearly indicate an essential role of mitochondrial protein synthesis for viability of the petite-negative yeast K. lactis.

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.

Specific mutations in nuclear MGI genes encoding the alpha, beta and gamma subunits of the mitochondrial inner membrane F1-ATPase complex allow mitochondrial DNA (mtDNA) to be lost from K. lactis. In the absence of a mutation in any of these three nuclear genes, loss of mtDNA is lethal. These results imply that mtDNA encodes a gene that is essential. Likely candidates for such an essential role are the ATP6, 8 and 9 genes coding for proteins of the ATP synthase-F0 component. The present study removes ATP9 from contention as a vital mitochondrial gene because in a respiratory deficient mutant, Gly- 3. 9, lacking a nuclear mgi mutation, we have found that a rearrangement in mtDNA has deleted 22 amino acids from the carboxy terminus of the 75 amino-acid subunit-9 protein. Rearrangement in mtDNA has occurred by recombination at a 23-bp repeated sequence in the introns of the ATP9 and large ribosomal RNA (LSU) subunit genes. These two introns, of 394 (ATP9) and 410 (LSU) nucleotides, both belong to group 1.

The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

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.

Base substitutions have been compared in two mitochondrial and two nuclear genes from three yeasts and three mammals. In yeasts, the two mitochondrial genes, cytochrome oxidase subunit 2 (COX2) and apocytochrome b (CYB), have fewer changes on a percentage basis than the nuclear-encoded cytochrome c (CYC) gene. By contrast, in mammals, the same mitochondrial genes have more mutations than CYC on a percentage basis. Sequence comparisons of the nuclear small-subunit ribosomal RNA (nSSU) gene shows that there are more substitutions per unit length in the three yeasts than in the three mammals. This result suggests that although the yeasts are more distantly related than the mammals, their mitochondrial genes have accumulated fewer changes.

The cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8,826 bp and contains five exons (termed E1-E5) totalling 1,602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1-1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5 a, and 1.5 b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5 a and K1 cox1.4/Sc cox1.5 b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.

Mitochondrial intron content is variable in the yeast Kluyveromyces lactis. Strains can be divided into three classes depending on the structure of the cytochrome oxidase subunit 1 (COX1) gene: (1) those containing intron K1 cox1.1, (2) those containing K1 cox1.2, 3 and 4 and, (3) those that contain all four introns. In addition, strains belonging to the first class (designated Type B strains), have an altered mitochondrial gene order relative to strains from classes (2) and (3) (Type A, Hardy et al. 1989). Crossing experiments reveal that K1 cox1.1 (a group II intron) transfers at high frequency (89%) to mitochondrial genomes lacking this intron. By contrast, the mobility of the remaining introns (all group I) is of the order of 7%.

Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble rho 0 mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit-) or nuclear lesions (pet-) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (rho-) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G + C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A + T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.

Large and small rRNAs have been isolated from mitochondria of the yeast Torulopsis glabrata and have been shown to have lengths of 2,700 bases and 1,400 bases respectively. Construction of a restriction endonuclease site map of mitochondria) DNA has enabled us to position these rRNAs by hybridization of labelled RNA to DNA fragments transferred to nitrocellulose. The large and small mt rRNA genes are separated by a minimum of 1,820 by and a maximum of 2,765 by on the 18,870 by mitochondria1 genome. tRNA genes map within this separating sequence but they are also located distal to both rRNA genes. The implication of these results to the structural relationships of mitochondrial DNAs from yeasts is discussed.

Results from the theory of random walks applied to the random excision hypothesis for production of petite mutants in yeast suggest that frequency of excision should increase as a linear function of mitochondrial DNA length (see appendix). For a series of petite positive yeasts we have determined the spontaneous petite frequency (ranging from about 0.003% to 9%) and length of mtDNA (ranging from about 19 Kbp to c. 108 Kbp) and found that, while the frequency of petite mutants does generally increase with mtDNA length, the relationship is far from linear. Although these results are inconclusive concerning the random excision hypothesis they do indicate that amongst related yeasts other factors have a greater influence than mtDNA length in determining the frequency of petite mutants.

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.

By employing Pulsed Field Gel Electrophoresis we have shown that the chromosomal DNA pattern of a petite-positive strain of Kluyveromyces lactis, designated KF4, differs from the karyotype of the reference strain. Digestion with SfiI and hybridization with an rDNA probe, demonstrate that a novel chromosomal band in KF4 results from a deletion of 35-40 rDNA cistrons from the rDNA cluster. The significance of this finding to possible alterations in cell physiology and the ability to generate "petite" mutants is discussed.

Restriction endonuclease digestion of mitocondrial DNAs from the nine Dekkera/Brettanomyces yeasts have revealed that three separate pairs of species, namely D. bruxellensis/B. lambicus; B. abstinens/B. custersii and B. anomalus/B. clausenii have identical genomes. This observation suggests that such analysis of mtDNA could be an important procedure for yeast taxonomy. Sizes of mtDNAs showed a graded range from the 28 kbp molecule in B. custersianus to the 100 kbp molecule in B. custersii. Furthermore, although the mtDNAs from D. intermedia (72 kbp) and D. bruxellensis (82 kbp) differ in size by 10 kbp the restriction enzyme fragmentation patterns are generally similar. The differences are reminiscent of mtDNA polymorphisms found in strains of Saccharomyces cervisiae which result from insertions or deletions, chiefly within genic sequences. By analogy, the two Dekkera species may, on further analysis, be revealed as variants of a single species.