Differential effects of nalidixate on the cell growth of respiratory competent strains and cytoplasmic petite mutants of Saccharomyces cerevisiae

Summary Sodium nalidixate inhibited the cell growth and division of several respiratory competent strains of Saccharomyces cerevisiae. A number of cytoplasmic petite strains (both spontaneous and induced by ethidium bromide) were shown to be more resistant to sodium nalidixate than the wild-type strains from which they were derived. There was considerable variation in sensitivity of different petites derived from the same wild-type. Usually petite strains which were induced by ethidium bromide were more resistant than spontaneously arising petites. The susceptibility of a wild-type strain to nalidixate was found to be least when the mitochondrial respiratory system was maximally repressed. It was also noted that sodium nalidixate (100 g/ml) induced petite mutants.Dr. Carnevali is a Senior Research Worker of the Centro di Studio per gli Acidi Nucleici of the National Research Council of Rome and is on leave of absence at the above address     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.     

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae

The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85 779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.     

Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.     

Saccharomyces cerevisiae mutants defective in the maturation of ribosomal RNA

Summary Slow-growing mutants were isolated after mutagenesis of the osmotic-sensitive strain Saccharomyces cerevisiae VY1160. The isolated mutants in rich media have generation times from 300 to 400 min at 30°C. Studies on the biosynthesis of rRNA^x have shown, that the processing of 37S pre-rRNA in 6 of the slow-growing mutants occurs 3 to 4 times slower than in the parental strain. These mutants with decreased rate of rRNA maturation are of two different types. In some of them the processing of both 37S and 27S pre-rRNA is slowed down, while the mutants from the second group are acharacterized by a specific inhibition of the step 27S pre-rRNA25S rRNA. Experiments in which the synthesis of macromolecules was studied, have shown that in the mutants and in the parental strain, RNA and proteins are synthesized at comparable rates. Preliminary results suggest that the decreased rate of rRNA processing in three of the isolated mutants might be due to an insufficient function of the enzymes involved in the maturation of rRNA.Abbreviations rRNA ribosomal RNA - pre-rRNA precursor to ribosomal RNA     

Denaturation mapping of the ribosomal DNA of Saccharomyces cerevisiae

Summary The thermal melting profile of purified Saccharomyces cerevisiae ribosomal DNA (rDNA) is biphasic indicating considerable intramolecular heterogeneity in base composition. The first phase of the transition, about 20% of the total hyperchromic shift, has a T_m of 80.6°C and the second phase has a T_m of 87.3°C, corresponding to GC contents of 28 and 44%, respectively. The T_m of the nonribosomal nuclear DNA, called DNA, is 85.7°C. This heterogeneity in GC distribution in the rDNA is also reflected in its denaturation map. A denaturation map of the 5.6×10⁶ dalton rDNA SmaI restriction fragment, which represents monomer units of the rDNA, shows that specific regions of the repeating unit denature more readily than the remainder and apparently have a significantly higher AT content. By aligning the rDNA denaturation map with the restriction endonuclease map, we have been able to determine that the AT-rich segments are localized in the transcribed and nontranscribed spacer regions of the rDNA repeating unit. Buoyant density determinations of individual rDNA restriction fragments corroborate the locations of AT-rich regions.A denaturation map of the tandem repeating units in higher molecular weight rDNA has also been constructed and compared with the map of the SmaI fragment. The results show that the repeating units are uniform in size, that they are not separated by large heterogeneous regions, and that they are arranged in head-to-tail array.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

Circular mitochondrial DNA molecules from petite mutants of Saccharomyces cerevisiae: resolution by polyacrylamide gel electrophoresis

Mitochondrial DNA (mtDNA) from petite strain K45 ofSaccharomyces cerevisiae contains about 7% circular DNA molecules which comprise a simple oligomeric series based on a monomeric size of 1.7 kilobase pairs. Electrophoresis of K45 mtDNA on a polyacrylamide-agarose slab gel fractionates the mtDNA into a major band (containing linear DNA) and several faster running minor bands each containing particular size class of circular DNA molecules. From study of mtDNA from K45 and two other simple petites it was found that the mobility of circles is inversely proportional to the logarithm of the circle size. Polyacrylamide gel electrophoresis thus permits the separation of circular mtDNA from the linear mtDNA of simple petites, and physically resolves circles of different size from one another.     

Transfer RNA genes in the mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Hybridization saturation analyses of mitochondrial DNA from 11 petite clones genetically characterized with respect to chloramphenicol and erythromycin resistance markers, have been carried out with 11 individual mitochrondrial transfer RNAs. Mitochondrial tRNA cistrons were lost, retained, or amplified in different petite strains. In some cases hybridization levels corrected for kinetic complexity of the mtDNA³ were two- to threefold greater than that for grande mtDNA indicating selective amplification, or increased number of copies, of the segment of mtDNA containing that tRNA cistron. Hybridization levels corrected for reduced kinetic complexity of petite mtDNAs in many cases were only 1 to 10% of that for grande mtDNA suggesting a low level of intracellular molecular heterogeneity of mtDNA with respect to tRNA cistrons. Some petite clones that retained tRNA genes continued to transcribe mitochondrial tRNAs, since tRNA isolated from these strains could be aminoacylated with Escherichia, coli synthetases and hybridized with mtDNA. Hybridization data allow us to order several of the tRNA cistrons on the mitochondrial genome with respect to the chloramphenicol and erythromycin antibiotic resistance markers.     

Antimutators of mitochondrial and nuclear DNA in Saccharomyces cerevisiae. Relationship with gamma-ray sensitivity

Summary In Saccharomyces cerevisiae ten antimutator mutants have been isolated. The spontaneous occurrence of mitochondrial mutants resistant to erythromycin, oligomycin, and diuron is decreased 2-60-fold in these strains. The rate of forward and reverse spontaneous mutations of the nuclear genome is also reduced. The meiotic progenies arising from the crosses of seven mutants (LB₁, LB₂, LB₄, LB₅, LB₆, LB₇, LB₁₀) with an isogenic parental strain exhibit 2:2 segregations and therefore are the result of mutations in a single nuclear gene. The six mutants LB₁, LB₂, LB₄, LB₆, LB₇, LB₁₀ are semidominant and determine six complementation groups. The mutant LB₅ is dominant and therefore cannot be assigned to any complementation group. The mutants. LB₁, LB₄ and LB₁₀ are gamma-ray sensitive and, by tetrad analysis, it has been shown that gamma-ray sensitivity and spontaneous antimutability are the result of a single nuclear gene mutation. The other three mutants LB₃, LB₈ and LB₉ exhibit complex tetrad segregations typical of cytoplasmic inheritance and do not complement each other. However, although the mutations are semidominant, it has not been possible to detect any antimutator cytoductant among some 500 cytoductants carrying the kar1-1 nucleus. These results suggest that either several nuclear genes are involved in the expression of the antimutator phenotype or that the antimutator gene is located on nonchromosomal elements of the nucleus. The present study leads to the conclusion that a large number of nuclear genes are able to control simultaneously the spontaneous mutation rate of nuclear and mitochondrial genes. Since out of the ten antimutator mutants, three are also deficient in the repair of gamma-ray damage, it is also concluded that spontaneous and gamma-ray-induced lesions of DNA can be repaired by the same error-free process.     

Mapping of the mitochondrial 16S ribosomal RNA gene and its expression in the cytoplasmic petite mutants ofSaccharomyces cerevisiae

The 16S ribosomal RNA gene of yeast mitochondria was titrated in various cytoplasmic petite mutants by DNA-RNA hybridization. The gene was located close to the prolyl transfer RNA gene. The properties of the rho^? strains suggest that the gene order would be: - P_I - 16S - prolyl tRNA - valyl tRNA - (tRNAs) - R_I - R_III -; the 23S ribosomal gene is far from the 16S one. Several petite mutants were found which have retained, in addition to many transfer RNA genes, both of the 23S and 16S ribosomal RNA genes. The two genes seem to be transcribed in these mutants.     

Mitochondrial DNA synthesis during the cell cycle of Saccharomyces cerevisiae

Logarithmically growing cultures of Saccharomyces cerevisiae were separated into fractions of increasing average age by zonal centrifugation. Nuclear and mitochondrial DNA labeled for both long and short periods were examined at different times in the cell cycle following isolation of the DNA on CsCl gradients. The data thus obtained were used to determine the relative times of replication. In S. cerevisiae the synthesis of nuclear and mitochondrial DNA was found to occur at the same time in the cell cycle. Some data indicate that mitochondrial DNA synthesis was completed before nuclear DNA synthesis.     

The size of mitochondrial DNA from a cytoplasmic petite mutant of Saccharomyces cerevisiae

Summary Mitochondrial DNA has been isolated from a cytoplasmic petite mutant of Saccharomyces cerevisiae which has retained only about 2% of the mitochondrial wild type genome. The denatured DNA was analyzed by agarose gel electrophoresis and a homogeneous, single band of DNA was found. Petite and wild type mitochondrial DNAs exhibited similar gel electrophoretic mobilities. Using denatured DNA from the E. coli phages T4 and T3 for comparison a molecular weight of 55×10⁶ daltons has been calculated for the double-stranded petite mitochondrial DNA. On the basis of this observation most of the mitochondrial DNA of this petite mutant appeared to consist of a polymer of about 50 repeats to account for a size similar to that of the wild type molecule. Thus a regulatory mechanism might exist which keeps constant the physical size of the mitochondrial DNA molecule in spite of the elimination of large fractions of the wild type genome.Dedicated to Dr. Dr. h. c. Peter Michaelis on the occasion of his 75th birthday     

Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae

In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast. For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene. A 400 by deletion in plasmid DNA had no effect on recombinational plasmid repair. However, a 400 by deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process. We have also studied the competition between recombination and ligation in the repair of linearized plasmids. Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA. This situation results in a decreased probability of non-recombinational (i.e. ligation) repair of linearized plasmid DNA.     

A simple method for the isolation and characterization of thymidylate uptaking mutants in Saccharomyces cerevisiae

Summary The mutant tmp1–10 ^ts which confers thermosensitive auxotrophy for thymidylate is employed for the selection of 5-dTMP uptaking mutants. At the nonpermissive temperature yeast cells phenotypically wild type for thymidylate uptake can grow for only 3 to 4 generations in the presence of 10^–2 M 5-dTMP. Thymidylate utilizing mutants (tum mutants) were isolated which can grow in the presence of 12 to 24 g 5-dTMP/ml. Genetical analysis revealed one of these mutant strains to be a double mutant, tuml tum2. For normal growth haploid thymidylate auxotrophic strains require approximately 360 g 5-dTMP/ml when tum1 and 24 g 5-dTMP when tum2 is present, respectively. Cells prototrophic for thymidylate (TMP) harbouring tum1 tum2 will also take up 5-dTMP and incorporate it specifically into their DNA. Thymidylate utilization in such strains is independent of functional mitochondria, as similar incorporation of labelled 5-dTMP is found in isogenic strains with rho ⁺, rho ^– and rho ⁰ status. Optimal stimulation of the 5-dTMP uptaking principle in haploid TMP strains is found at 4 g 5-dTMP/ml when tum1 and tum2 are present.     

A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae

Summary A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura5 mutation affecting the activity of orotidine-5-phosphate carboxy-lyase (EC 4.1.1.23). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 2.4.2.10). The orotidine-5-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridises to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120.000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5-phosphate carboxy-lyase and orotate phosphoribosyl transferase.Abbreviations bp basepair - kb kilobasepair - MOPS Morpholino propane sulfonic acid