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Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.

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.

Fifty years ago it was reported that baker's yeast, Saccharomyces cerevisiae, can form "petite colonie" mutants when treated with the DNA-targeting drug acriflavin. To mark the jubilee of studies on cytoplasmic inheritance, a review of the early work will be presented together with some observations on current developments. The primary emphasis is to address the questions of how loss of mtDNA leads to lethality (rho 0-lethality) in petite-negative yeasts and how S. cerevisiae tolerates elimination of mtDNA. Recent investigation have revealed that rho 0-lethality can be suppressed by specific mutations in the alpha, beta, and gamma subunits of the mitochondrial F1-ATPase of the petite-negative yeast Kluyveromyces lactis and by the nuclear ptp alleles in Schizosaccharomyces pombe. In contrast, inactivation of genes coding for F1-ATPase alpha and beta subunits and disruption of AAC2, PGS1/PEL1, and YME1 genes in S. cerevisiae convert this petite-positive yeast into a petite-negative form. Studies on nuclear genes affecting dependence on mtDNA have provided important insight into the functions provided by the mitochondrial genome and the maintenance of structural and functional integrity of the mitochondrial inner membrane.

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (rho-) and can survive complete loss of the organellar genome (rho(o)), the genetic factor(s) that permit(s) survival of rho- and rho(o) mutants remain(s) unknown. In this report we show that a function associated with the F1-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the alpha or beta subunit, but not the gamma, delta, or epsilon subunit of F1, renders cells petite-negative. The F1 complex, or a subcomplex composed of the alpha and beta subunits only, is essential for survival of rho(o) cells and those impaired in electron transport. The activity of F1 that suppresses rho(o) lethality is independent of the membrane Fo complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F1 subunits to suppress rho(o) lethality has been achieved by simultaneous expression of S. cerevisiae F1 alpha and gamma subunit genes in Kluyveromyces lactis - which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of rho- or rho(o) mutations in S. cerevisiae.

The nucleotide sequences of two segments of 6,737 ntp and 258 nto of the 18.4-kb circular mitochondrial (mt) DNA molecule of the soft coral Sarcophyton glaucum (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) have been determined. The larger segment contains the 3' 191 ntp of the gene for subunit 1 of the respiratory chain NADH dehydrogenase (ND1), complete genes for cytochrome b (Cyt b), ND6, ND3, ND4L, and a bacterial MutS homologue (MSH), and the 5' terminal 1,124 ntp of the gene for the large subunit rRNA (1-rRNA). These genes are arranged in the order given and all are transcribed from the same strand of the molecule. The smaller segment contains the 3' terminal 134 ntp of the ND4 gene and a complete tRNA(f-Met) gene, and these genes are transcribed in opposite directions. As in the hexacorallian anthozoan, Metridium senile, the mt-genetic code of S. glaucum is near standard: that is, in contrast to the situation in mt-genetic codes of other invertebrate phyla, AGA and AGG specify arginine, and ATA specifies isoleucine. However, as appears to be universal for metazoan mt-genetic codes, TGA specifies tryptophan rather than termination. Also, as in M. senile the mt-tRNA(f-Met) gene has primary and secondary structural features resembling those of Escherichia coli initiator tRNA, including standard dihydrouridine and T psi C loop sequences, and a mismatched nucleotide pair at the top of the amino-acyl stem. The presence of a mutS gene homologue, which has not been reported to occur in any other known mtDNA, suggests that there is mismatch repair activity in S. glaucum mitochondria. In support of this, phylogenetic analysis of MutS family protein sequences indicates that the S. glaucum mtMSH protein is more closely related to the nuclear DNA-encoded mitochondrial mismatch repair protein (MSH1) of the yeast Saccharomyces cerevisiae than to eukaryotic homologues involved in nuclear function, or to bacterial homologues. Regarding the possible origin of the S. glaucum mtMSH gene, the phylogenetic analysis results, together with comparative base composition considerations, and the absence of an MSH gene in any other known mtDNA best support the hypothesis that S. glaucum mtDNA acquired the mtMSH gene from nuclear DNA early in the evolution of octocorals. The presence of mismatch repair activity in S. glaucum mitochondria might be expected to influence the rate of evolution of this organism's mtDNA.

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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.

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.

We have shown previously that mutations in nuclear genes, termed MGI, for mitochondrial genome integrity, can convert the petite-negative yeast Kluyveromyces lactis into a petite-positive form with the ability to produce mitochondrial genome deletion mutants (Chen and Clark-Walker, Genetics, 133, 517-525, 1993). Here we describe that two genes, MGI2 and MGI5, encode the alpha- and gamma-subunits of mitochondrial F1-ATPase. Specific mutations, Phe443-->Ser and Ala333-->Val in MGI2, and Thr275-->Ala in MGI5, confer on cells the ability to produce petite mutants spontaneously with deletions in mitochondrial (mt) DNA and the capacity to lose their mitochondrial genomes upon treatment with ethidium bromide. Structural integrity of the F1 complex seems to be needed for expression of the Mgi- phenotype as null mutations in MGI2 and MGI5 remove the ability to form mtDNA deletions. It is suggested that mgi mutations allow petites to survive because an aberrant F1 complex prevents collapse of the mitochondrial inner membrane potential that normally occurs on loss of mtDNA-encoded F0 subunits.

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.

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A nuclear mutation, mgm101, results in temperature sensitive loss of mitochondrial DNA (mtDNA) in the yeast Saccharomyces cerevisiae. The corresponding gene, MGM101, was isolated from a genomic DNA library by complementation. Sequence analysis shows that MGM101 encodes a positively charged protein of 269 amino acids with a calculated molecular weight of 30 kDa. This analysis also reveals that MGM101 is adjacent to the ribosomal protein gene RPS7A on chromosome X and hybridization indicates it occurs in single copy. Creation of a null mutant by targeted disruption showed that the gene has no essential cellular function, aside from its participation in mitochondrial genome maintenance. As no counterpart has been identified in databases it is a novel protein whose role has yet to be determined. Expression of MGM101 is low on glucose medium but on galactose there is a two-fold increase in the level of the transcript.

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.

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.

Shuttle plasmids, pE1.CUP1B and pE1.CUP1E of 10.6 kb, have been constructed between the metallothionein-encoding CUP1 gene of Saccharomyces cerevisiae and a vector capable of replication in Kluyveromyces lactis. Introduction of these plasmids into K. lactis confers resistance to copper as well as to cadmium and silver. Resistance to these latter metal ions, in the absence of induction by copper, suggested that the CUP1 gene is constitutively expressed in the foreign background. Introduction of the lacZ reporter gene from Escherichia coli into a cloning site downstream from the CUP1 promoter showed that expression of this gene is constitutive in K. lactis but in S. cerevisiae induction by copper is necessary. Sequences upstream from the CUP1 promoter are involved in the constitutive expression since deletion of 91 nucleotides from this region abolishes metal resistance. It is suggested that a K. lactis protein, normally involved in activating transcription of the resident CUP1 gene in the presence of copper, can promote transcription in the absence of metal ion by binding to the upstream activation sequence of the introduced CUP1 gene.

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.

The antral-duodenal contractile relationship was studied in control, after parietal cell vagotomy and truncal vagotomy conditions using extraluminal strain gage transducers. All conditions were investigated under interdigestive and digestive states and after insulin, bethanechol and histamine. After parietal cell vagotomy, there was minimal alteration of the antral-duodenal relationship in both the interdigestive and digestive states. The number and amplitude of contractions on both the antrum and duodenum (as reflected by a motility index) were not changed from control by the various stimulants. The one exception was that histamine markedly stimulated the duodenal contractile activity. In the truncal vagotomy condition, there was a total disruption of the antral-duodenal relationship in the interdigestive and digestive states. There was a significant decrease in the number and amplitude of contractions occurring on the antrum during the interdigestive and after insulin stimulation. Food was ineffective in stimulating the antrum in 2 of 3 dogs. In contrast, motor activity of the duodenum was minimally influenced by truncal vagotomy. In conclusion, parietal cell vagotomy has minimal disruptive effects on the antralduodenal relationship while truncal vagotomy reduces antral contractile activity.

A revertant (SPR1) from a high-frequency petite strain of Saccharomyces cerevisiae has been shown by mapping and sequence analysis to have a rearranged mitochondrial genome. In vivo rearrangement has occurred through a subgenomic-recombination pathway involving the initial formation of subgenomic molecules in nascent petite mutants, recombination between these molecules to form an intermediate with direct repeats, and subsequent excision of the resident or symposed duplication to yield a molecule with three novel junctions and a changed gene order. Sequencing of the novel junctions shows that intramolecular recombination in each case occurs by means of G + C-rich short direct repeats of 40-51 base pairs. Mapping and sequence analysis also reveal that the SPR1 mitochondrial genome lacks three sectors of the wild-type molecule of 4.4, 1.7, and 0.5 kilobases. Each of these sectors occurs in nontemplate, base-biased DNA, that is over 90% A + T. Absence of these sectors together with a rearranged gene order does not appear to affect the phenotype of SPR1, as colony morphology and growth rate on a number of different substrates are not detectably different from the wild type. Lack of phenotypic change suggests that mitochondrial gene expression has not been noticeably disrupted in SPR1 despite deletion of the consensus nonomer promoter upstream from the glutamic acid tRNA gene. Dispensability of DNA sectors and the presence of recombinogenic short, direct repeats are mandatory features of the subgenomic-recombination pathway for creating rearrangements in baker's yeast mtDNA. It is proposed that, in other organisms, organelle genomes containing these elements may undergo rearrangement by the same steps.

Determination of sequences from the nine regions separating the large genes in the 19-kbp mitochondrial DNA from Torulopsis glabrata has led to the identification of 23 tRNA genes and to the recognition of two types of short repeated sequence implicated in mitochondrial genome expression. The two short repeated sequences are a nonanucleotide motif, 5'-TATAAGTAA-3' and a dodecanucleotide motif, 5'-TATAATATTCTT-3'. By RNA sequence determination it has been found that primary transcripts of the small and large rRNAs commence at the 3' penultimate adenine of the nonanucleotide sequence. This motif has also been found in the DNA sequence upstream from f-methionine, phenylalanine, leucine, tyrosine and glycine tRNAs, cytochrome oxidase subunit 2 and ATPase subunit 9. The dodecanucleotide sequence is found at least once in each of the nine regions between the large genes. Determination of the 3' ends of the small and large rRNAs has shown their location to be 8 and 23 nucleotides downstream from the dodecanucleotide sequence. This motif is thought to be involved in signalling processing of polycistronic transcripts. Such transcripts are invoked to account for the production of mRNAs for cytochrome b, cytochrome oxidase subunits 1 and 3, and the joint mRNA for ATPase subunits 8 and 6 genes that lack an adjacent upstream nonanucleotide transcription initiation signal sequence. Processing of polycistronic transcripts at tRNA sequences is also implicated in the formation of mature mRNAs. From the position of tRNA genes relative to the nonanucleotide motif it appears that clusters of these genes are co-transcribed with downstream sequences for cytochrome oxidase subunits 1 and 3.

Seasonal changes in the content of Ca and Mg in leaf blades collected from mature kiwifruit [Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson var. deliciosa] vines in a high-producing orchard were measured by a sequential fractionation procedure (14 M acetic acid, 0.25 M HC1 and residual). Total Ca and Mg concentrations decreased within the first 4 weeks of growth, but increased linearly thereafter to be 1322 and 197 μmol g -1 dry weight, respectively, by leaf fall. Total quantities of Ca and Mg accumulated at this time were approximately 2500 and 380 μmol per blade. Calcium oxalate, dissolved in the HC1 extract, was the predominant fraction at every harvest, accounting for up to 79 % of the total Ca content shortly after leaf emergence, and decreasing to 42 % at leaf fall. The quantity of physiologically active Ca (acetic acid-soluble) ranged from a minimum of 15 % (8 weeks after emergence) to a maximum of 41 % at the end of the season. Forms of Ca remaining in the solid residue after extraction made only a minor contribution (3 to 21 %) to the total content at any time during the season. In contrast, however, 89 to 96% of the total Mg was extracted by acetic acid at every harvest, with the remainder dissolved by HC1. Scanning electron microscopy and X-ray microanalysis of the lamina of mature leaves indicated the presence of two crystalline, Ca-rich products which differed in their morphology, location and distribution. Large styloids (120 to 270 μm in length) were concentrated in idioblasts adjacent to vascular tissue, while bundles of four-sided raphides, containing wedge-shaped apices, were located predominantly between the major transport vessels of the vascular tissue. X-ray diffraction analysis of crystalline isolates confirmed that both morphological forms are calcium oxalate monohydrate (whewellite).

Unique transcripts for cytochrome b, ATPase subunits 6 and 9, cytochrome oxidase subunits 2 and 3 and S and L rRNA have been mapped by the S1 protection technique to the circular 19-kbp mitochondrial DNA (mtDNA) of the yeast Torulopsis glabrata. In contrast, a number of transcripts have been detected for the mosaic cytochrome oxidase subunit 1 gene with the largest being approximately 5000 nucleotides and the mature message having a length of 1760 nucleotides. Despite the presence in T. glabrata mtDNA of a sequence that hybridizes to the variant 1 gene of Saccharomyces cerevisiae mtDNA we have not detected a transcript of this region. Neither have we detected co-transcripts of adjacent genes in RNA from either glucose-repressed or derepressed cells. However, by comparison of RNA species from the two growth conditions, we have found that the ATPase subunit 6 transcript is lower in amount relative to other species in preparations from glucose-repressed cells. This information, together with the observation of separate transcripts and the knowledge that there are several species of mitochondrial RNA which can be capped by the guanylyl transferase catalysed addition of GMP, suggests that each of the genes investigated in the present study is separately transcribed.

Fifty-seven patients presenting with virologically confirmed first attacks of genital herpes were assessed for risk factors for time to the first recurrence. These factors included demographic details, personality traits, recent life events, herpes simplex biotype, frequency of orogenital intercourse and psychiatric illness as measured by the General Health Questionnaire (GHQ). This and our similar previous study (58 patients) were analysed separately and combined using a novel statistical technique (Cox's proportional hazards model). In the previous study there was a significant association only for GHQ score (p less than 0.02). For the present study age was the only statistical significant factor (the younger the patient the more likely a recurrence sooner (p less than 0.05), although there was a trend for high GHQ scorers to have recurrences sooner than low scorers. Data of the two studies combined showed that the only significant association with time to recurrence was the GHQ (p less than 0.02). These data support the notion that there is an association between psychiatric illness and recurrence of genital herpes.

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.

Complementation has been observed in cytoplasmic respiratory deficient yeast cells (petites) to yield respiratory competent diploids. This successful demonstration depended on the use of spontaneous petites of recent origin and on crosses involving all possible apirwise combinations between the many different petite isolates of opposite mating type. The possibility of deletion of a single unique region of yeast mitochondrial DNA as the initial lesion in petite formation has been eliminated by using strains isogenic for their mitochondrial DNA.

We have shown that 2.8 and 3.1 micron circular DNA molecules, previously reported to be present in Saccharomyces cerevisiae and Torulopsis glabrata respectively, contain sequences hybridizing to cytoplasmic ribosomal RNAs. In S. cerevisiae the 2.8 micron circular DNA appears to be identical to the rDNA repeating unit from nuclear DNA, both in length (approximately 9000 base pairs) and in the location of the 25, 18 and 5.8S rRNA sequences on the large HindIII fragment (6500 bp) and the presence of the 5S rRNA sequence on the small HindIII fragment. The 3.1 micron molecule from T. glabrata is approximately 2000 base pairs longer than the S. cerevisiae molecule and in addition, one of the HindIII sites lies within the region hybridizing to 25, 18 and 5.8S rRNAs. In S. cerevisiae the 4-5 copies of the 2.8 micron circular DNA molecules per cell, which have an extra-nuclear location, do not appear to be essential for cell viability as in one strain they were undetectable.

Breakage and fractionation of respiratory competent yeast in the presence of ethidium bromide, and subsequent centrifugation of a detergent lysate of the mitochondrial fraction by the dye-buoyant-density technique, results in the isolation of closed-circular DNA. After removal of bound dye, this DNA has two components when analyzed by equilibrium buoyant density in the analytical ultracentrifuge. A minor component has a buoyant density of 1.684 g/cm(3), which is characteristic of mitochondrial DNA, but the major component has a buoyant density of 1.701 g/cm(3). This species of DNA is also present in yeast that have been mutagenized to respiratory deficiency in the presence of the highest concentration of ethidium bromide compatible with cell growth. The closed-circular DNA of buoyant density 1.701 g/cm(3), and free of linear DNA, is associated with the sole particulate band obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-deficient cells. Two particulate bands are obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-competent cells, the upper band containing DNA of buoyant density 1.701 g/cm(3) and the lower band DNA of buoyant density 1.684 g/cm(3). The suggestion is advanced, in view of the reputed sedimentation behaviour of yeast peroxisomes, that the closed-circular DNA of buoyant density 1.701 g/cm(3) may be located in peroxisomes.

The effects of chloramphenicol on S. cerevisiae and on a cytoplasmic respiratory-deficient mutant derived from the same strain are compared. In the normal yeast, high concentrations of chloramphenicol in the growth medium completely inhibit the formation of cytochromes a, a(3), b, and c(1) and partially inhibit succinate dehydrogenase formation, whereas they do not affect cytochrome c synthesis. This has been correlated with the marked reduction of mitochondrial cristae formation in the presence of the drug. In glucose-repressed normal yeast, chloramphenicol has little effect on the formation of outer mitochondrial membrane, or on the synthesis of malate dehydrogenase and fumarase. However, both these enzymes, as well as the number of mitochondrial profiles, are markedly decreased when glucose de-repressed yeast is grown in the presence of chloramphenicol. The antibiotic did not appear to affect the cytoplasmic respiratory-deficient mutant. The results have been interpreted to indicate that chloramphenicol inhibits the protein-synthesizing system characteristic of the mitochondria. Since the drug does not prevent the formation of cytochrome c, of several readily solubilized mitochondrial enzymes, or of outer mitochondrial membrane, it is suggested that these are synthesized by nonmitochondrial systems.