Search

The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4 allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression of SIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner.

Abstract: Specific mutations in nuclear MGI genes encoding the α, β and γ 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.

Purified mitochondria from the petite positive yeast Torulopsis glabrata contain a circular deoxyribonucleic acid (DNA) with a length of 6 mum and a buoyant density of 1.686 g/cm3. This DNA is absent from ethidium bromide induced respiratory-deficient mutants.

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.

Summary Threeori elements (ori 2,ori 5, andori 7) have been sequenced inSaccharomyces cerevisiae strain Dip 2 and compared to the equivalentori elements of a second strain (B). Bothori 2 andori 5 exhibit 98% base matching between strains Dip 2 and B. In contrast, the thirdori element (ori 7) exhibits extensive sequence rearrangements whereby a segment located downstream in the consensus strain occurs within theori structure in Dip 2. This represents a novel polymorphic form of the yeast mitochondrial genome.

Summary A spontaneously arising mitochondrial DNA (mtDNA) variant ofSaccharomyces cerevisiae has been formed by two exta copies of a 14-bp sequence (TTAATTAAATTATC) being added to a tandem repeat of this unit. Similar polymorphisms in tandemly repeated sequences have been found in a comparison between mtDNAs from our strain and others. In 5850 bp of intergenic mtDNA squence, polymorphisms in tandemly repeated sequences of three or more base pairs occur approximately every 400–500 bp whereas differences in 1–2 bp occur approximately every 60 bp. Some polymorphisms are associated wit optional G+C-rich sequences (GC clusters). Two such optional GC clusters and one A+T repeat polymorphism have been discovered in the tRNA synthesis locus. In addition, the variable presence of large open reading frames are documented and mechanisms for generating intergenic sequence diversity inS. cerevisiae mtDNA are discussed.

Summary Mapping of sequences specifying the large and small ribosomal RNAs and six polypeptides in the circular 23.7 kbp mitochondrial DNA ofSaccharomyces 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 fromTorulopsis 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 theEscherichia 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 ofS. exiguus andT. glabrata.

Summary Analysis of gene order and orientation in the circular 18.9 kbp mitochondrial DNA molecule ofTorulopsis 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 andSaccharomyces exiguus mtDNA (see accompanying paper).

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.

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.

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 preveailing 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, 1.5a, and 1.5b 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 cox1.5a and K1 cox1.4/Sc cox1.5b 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%.

The role of specialized regions of insect rectal papillae in the regulation of water and ion uptake is well documented. Although the apparatus for active uptake of water or ions is located in various cell membranes, the absorbed molecules must first pass through the cuticle which lines the rectal epithelium. Most cuticle (e.g. abdominal) has been shown to be permeable only to molecules soluble in wax, and to be impermeable to water and ions. Obviously if such cuticle lined the rectum, absorption of water and ions would be severely restricted. The present freeze-fracture and lanthanum tracer study was undertaken to investigate in more detail both the morphological features of the rectal papillae cuticle which could be responsible for its anomalous permeability and the various cell membranes involved in this transport. It has been suggested from permeability studies that the anomalous permeability of rectal papillae cuticle could be due to the lack of a complete wax layer over the surface of the rectal cuticle. The present study strongly supports this suggestion. Thus, the freeze-fracture micrographs have shown that a surface layer of the cuticle reacts during fracturing like a lipid bilayer. However, in rectal papilla cuticle this surface bilayer is interrupted at each epicuticular depression by areas of different fracturing behaviour. These discontinuities in the surface bilayer probably allow the rectal contents to contact directly the true cuticular matrix. They could, therefore, explain the ease with which water and ions penetrate the rectal cuticle and so gain access to the underlying epithelial cells. Although similar discontinuities are present on some of the rectal cuticle surface external to the rectal papillae, they appear to be filled in by plugs of lipid-like material. The lateral plasma membranes of the rectal papillae cells are generally considered to be the main site of active transport. The present lanthanum tracer and freeze-fracture study has shown that the lateral plasma membranes contain 3 distinct differentiations. Septate junctions are present at the apical and basal surfaces of the epithelial layer; a further membrane differentiation is found adjacent to the septate junctions; and thirdly, an array of short, variable length, non-anastomosing linear structures covers most of the lateral plasma membrane surface. These latter structures, unlike known types of cell junctions do not show equivalent arrays in apposing membranes even when the lateral plasma membranes of adjacent cells are closely apposed. The possible function of these structures is discussed.

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.

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 hybridzing 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. custersd 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.

Abstract: 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 α-, β- and γ-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPδ gene encoding the δ-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 ATPδ from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPδ gene, like strains with disruptions of α-, β- and γ-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 ATPδ in the presence of some mgi alleles does not abolish the Mgi– phenotype. By contrast, elimination of ATPδ in other mgi strains removes resistance to ethidium bromide and ρ 0 mutants are not formed. Hence the ATPδ subunit of F1-ATPase, while not mandatory for a Mgi– phenotype, aids some mgi alleles in suppressing ρ 0 lethality.

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.

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms.

Mucor genevensiswas grown in both glucose-limited and glucose-excess continuous cultures over a range of dissolved oxygen concentrations (<0.1 to 25 μM) to determine the effects of glucose and the influence of metabolic mode (fermentative versus oxidative) on dimorphic transformations in this organism. The extent of differentiation between yeast and mycelial phases has been correlated with physiological and biochemical parameters of the cultures. Under glucose limitation, oxidative metabolism increased as the dissolved oxygen concentration increased, and this paralleled the increase in the proportion of the mycelial phase in the cultures. Filamentous growth and oxidative metabolism were both inhibited by glucose even though mitochondrial development was only slightly repressed. However, the presence of chloramphenicol in glucose-limited aerobic cultures inhibited mitochondrial respiratory development but did not induce yeast-like growth, indicating that oxidative metabolism is not essential for mycelial development. Once mycelial cultures had been established under aerobic, glucose-limited conditions, subsequent reversal to anaerobic conditions or treatment with chloramphenicol caused only a limited reversal (<35%) to the yeast-like form. Glucose, however, induced a complete reversion to yeast-like form. It is concluded that glucose is the most important single culture factor determining the morphological status of M. genevensis; mitochondrial development and the functional oxidative capacities of the cell appear to be less important factors in the differentiation process.

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/cm3, which is characteristic of mitochondrial DNA, but the major component has a buoyant density of 1.701 g/cm3. 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/cm3, 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/cm3and the lower band DNA of buoyant density 1.684 g/cm3. 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/cm3may be located in peroxisomes.

Circular DNA has been investigated in the water mold Saprolegnia. This organism was shown to contain two satellite DNA components at 1.685 and 1.707 g/cm3 in addition to main band DNA at a buoyant density of 1.717 g/cm3. The component banding at a density of 1.685 g/cm3 was found to occur as closed circles of length 14 µm in the relaxed form. On the basis of sucrose gradient studies this DNA is thought to be mitochondrial in origin. No other class of circular molecules was observed.

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, a3, b, and c1 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.

Growth of Mucor genevensis, a facultatively anaerobic dimorphic mold, in high concentrations of chloramphenicol (4 mg/ml) leads to increased numbers of yeast-like cells and small club-like mycelial forms. This change in morphology is accompanied by a threefold increase in the mass doubling time, the loss of cyanide-sensitive respiration, and the development of cyanide-insensitive respiration. Associated with these changes is the absence of cytochromes aa3and band the inability of the organism to utilize ethanol; in addition, mitochondria appear more numerous and have less internal membrane. A further inhibitory action of the antibiotic, other than eliminating functional mitochondria, appears likely since microaerobic cultures which lack respiratory ability have twice the mass doubling time in the presence of the drug. Although a small inhibition of amino acid incorporation by cytoplasmic ribosomes is found with a high chloramphenicol concentration, it is insufficient to account for the effect on growth of the microaerobic culture. The nature of this additional effect of chloramphenicol remains to be determined, but it has been shown that increasing the glucose concentration can partially reverse this action of the antibiotic. The effect of the drug on the morphology of the organism is not as dramatic as that of phenethyl alcohol in producing yeast-like forms. However, in view of the action of chloramphenicol in eliminating functional mitochondria in M. genevensisthe suggestion that phenethyl alcohol exerts its effect in promoting yeast-like morphology by uncoupling oxidative phosphorylation should be re-examined.

Fatty acid and sterol content and composition were determined for the dimorphic mold, Mucor genevensis, grown under a variety of experimental conditions. Fatty acids account for 6 to 9% of the dry weight of aerobically grown mycelium, and 70 to 80% of these are unsaturated. The organism contains γ-linolenic acid which is characteristic for Phycomycetes, and in sporangiospores this compound represents 40% of the total fatty acids. Of the sterols found in mycelium, 80% is ergosterol, and stigmasterol was positively identified as one of the minor components. In anaerobically grown yeastlike cells, sterol content is less than 10% of the level found in aerobically grown cells, and fatty acids amount to less than 2% of the dry weight. These fatty acids are predominantly short chain and less than 10% are unsaturated. Yeastlike cells obtained under aerobic conditions by growth in the presence of phenethyl alcohol have fatty acid and sterol compositions characteristic of aerobically grown mycelium. It is concluded that the dimorphology of the organism is not directly related to lipid composition.

The addition of nitrate to cultures of Spirillum itersoniiincubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 × g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome cwas increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome crose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome cin response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome cdoes not require the presence of a functional nitrate reductase.

Mitomycin C and ultraviolet light were found to induce the formation of microcysts in Spirillum itersonii. These forms, as well as spontaneously occurring microcysts in this species, were found to contain phage tail parts, rhapidosomes, and a granular substance not seen in normal cells. It is suggested that microcysts are formed as the result of the induction of a defective phage. The production of phage lysozyme within the cell could lead to the formation of spherical forms as the cells lose their structural mucopeptide layer. Complete virus particles were not seen, nor was any biological activity demonstrated when the induced cultures were tested against two other strains of S. itersonii. The other strains of this bacterium also formed microcysts and phage tail parts when induced with mitomycin. Attempts to isolate an organism lacking the defective phage have been unsuccessful.

Spirillum itersoniicontains b- and c-type cytochromes as well as a carbon monoxide-binding pigment of the cytochrome otype. Synthesis of cytochromes band cis increased by about two- and fourfold, respectively, when cells are transferred from high to low aeration. The increased concentration of cytochrome is not accompanied by an increase in the respiration rate of the cells. Both cytochrome band cytochrome care located in the particulate fraction of cells grown under high or low aeration, and both pigments are fully reducible by succinate. No evidence was found for the accumulation of the protein component of either cytochrome when synthesis of the prosthetic group was limited by iron deficiency, nor did heme or precursors accumulate when protein synthesis was prevented. It was therefore concluded that the formation of the heme prosthetic group is closely integrated with the synthesis of the protein moiety. δ-Aminolevulinate synthase was detected in extracts of the organism. Its activity was correlated with cytochrome synthesis; it was reduced by high aeration and increased under low aeration. The synthase was inhibited by hemin at concentrations of 10 μmor higher. The observations are consistent with a central role for the heme prosthetic group in the regulation of cytochrome synthesis.

Respiration and mitochondria in Mucor genevensis, a facultatively anaerobic dimorphic mold, have been studied in aerobically and anaerobically grown cells and in anaerobically grown cells adapting to aerobic conditions. Respiration in hyphae continues at a high level during aerobic growth but drops rapidly on exhaustion of glucose. In anaerobically grown yeastlike cells, containing no recognizable aerobic cytochromes, a small cyanide-insensitive respiration occurs. Mitochondria with well defined cristae are visible in negative contrast after KMnO4fixation of stringently anaerobic cells containing low amounts of fatty acid of which 10% or less are unsaturated. On aeration of anaerobically grown cells, respiratory capacity and cytochromes develop rapidly, even in the presence of 10% glucose, indicating that glucose does not repress development of respiration. However, mycelium formation by adapting yeastlike cells is repressed by high glucose concentration. In adapting cells, apparent changes in mitochondrial ultrastructure appear to be more related to changes in fixation properties of cells than to changes in the structure of mitochondria.

CYTOPLASMICALLY inherited respiratory deficient mutants termed petites, were first described in baker's yeast over 20 years ago1. Since then in laboratory studies several species of yeast have been shown to give rise to respiratory deficient mutants2,3. However, although some respiratory deficient (obligatory fermentative) yeasts have been found in nature3, it is not clear whether these species are the result of cytoplasmic (petite) or chromosomal lesions4because no suitable genetic tests are available. Nevertheless it is known that petitemutants of both Saccharomyces cerevisiae5and Tornlopsis glabrata6have large deletions from their mitochrondrial DNA with each independent isolate having a unique circular DNA size profile which can be regarded as a ‘fingerprint’. By contrast chromosomal or segregational mutants have intact and functional mitochondrial DNA, as shown by genetic tests3,7,8. Therefore it seemed possible that the type of lesion in naturally occurring respiratory deficient species could be determined by circular DNA profile analysis. Using this method, we show here that three independently isolated strains of Candida slooffiiresemble petitemutants.

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.

Sialography is the best way of assessing disease of the duct system of the parotid and submandibular glands. However, it can often be difficult to cannulate the orifices of Stenson's and Wharton's ducts with the available range of plastic and metal instruments.A 24 G Jelco Teflon intravenous cannula (Critikon Ltd) offers practical benefits whilst costing less than 5% of one commercially produced alternative in use in the department.