Sensitivity to ethidium bromide during meiosis in Saccharomyces cerevisiae

Summary Ethidium bromide was found to inhibit nuclear and mitochondrial DNA synthesis during meiosis which resulted in the inhibition of meiotic gene conversion and sporulation and was also lethal. Protection from the effects of ethidium bromide on meiotic gene conversion and survival was found to coincide with DNA synthesis, but it is possible that protection from sporulation inhibition occurs only later in meiosis.     

Meiotic differentiation during colony maturation in Saccharomyces cerevisiae

As yeast colonies ceased growth, cells at the edge of these colonies transited from the cell division cycle into meiosis at high efficiency. This transition occurred remarkably synchronously and only at late stages of colony maturation. The transition occurred on medium containing acetate or low concentrations of glucose, but not on medium containing high glucose. The repression by high glucose was overcome when IME1 was overexpressed from a plasmid. Experiments with different growth media imply that meiosis in colonies is triggered by changes in the nutrient environment as colonies mature. HAP2 is required to sporulate in any carbon source, whereas GRR1 is required for glucose repression of sporulation. CLN3 is required to repress meiosis in colonies but not in liquid cultures, indicating that the regulators that mediate the transition to meiosis in colonies are not identical to the regulators that mediate this transition in liquid cultures.     

Mitotic hyperploidy for chromosomes VIII and III in Saccharomyces cerevisiae

Summary The arg4–8 and cup1 ^s markers comprise a copy-number-dependent signal device in the yeast Saccharomyces cerevisiae. These alleles permit reliable discrimination between euploid and disomic haploids as well as between euploid and trisomic diploids. To investigate and compare inherent inter-chromosomal differences as regards propensity for hyperploidy, we transplaced arg4–8 and cup1 ^s by deleting them from chromosome VIII and then re-introducing them at the leu2 locus on chromosome III. The rate of chromosome gain was significantly greater for the chromosome III construct compared to the native chromosome VIII, in both diploid and haploid strains. In addition, more coincident aneuploidy for other chromosomes was found among chromosome VIII hyperploids compared to chromosome III hyperploids.     

Time-dependent mitotic recombination in Saccharomyces cerevisiae

The time-dependent appearance of prototrophic recombinants between heterologously located artificial repeats has been studied in Saccharomyces cerevisiae. While initial prototrophic colony numbers from independent cultures were highly variable, additional recombinants were found to arise daily at roughly constant rates irrespective of culture. These late-appearing recombinants could be accounted for neither by detectable growth on the selective media nor by delayed appearance of recombinants present at the time of selective plating. Significantly, at no time did the distributions of recombinants fully match those expected according to the Luria-Delbruck model and, in fact, after the first day, the distributions much more closely approximated a Poisson distribution. Prototrophic recombinants accumulated not only on the relevant selective medium, but also on media unrelated to the acquired prototrophy.     

Mitotic versus meiotic recombination in Saccharomyces cerevisiae

Summary As part of a comparative analysis of spontaneous mitotic and meiotic recombination we have compared the mitotic and meiotic maps of the wild type and yeast hybrids homozygous for reml-l, a mitosis-specific hyper-rec mutation (Golin and Esposito, 1977; Golin, 1979). In wild type yeast strains recombination in centromere proximal intervals occurs relatively more frequently in mitosis than in meiosis. In reml-1/rem1-1 hybrids the distribution of mitotic exchange events is more similar to the distribution observed in meiosis.     

Thymineless recombination in Saccharomyces cerevisiae is independent of the ability to undergo meiosis

Summary Thymine nucleotide starvation is recombinagenic in Saccharomyces cerevisiae and induces formation of the nuclear dense body, a structure characteristic of yeast cells in meiosis. Conceivably, thymineless recombination in yeast, presumed to be mitotic, might be meiotic in nature. We have tested this hypothesis and have found that thymineless recombination can be induced in strains incapable of meiotic exchange.     

Repair of 2 um Plasmid DNA in Saccharomyces cerevisiae

Summary We have developed a system for assaying pyrimidine dimers in the 2 m DNA plasmid of Saccharomyces cerevisiae, using Micrococcus luteus UV endonuclease to nick dimer-containing plasmid molecules and measuring percentages of nicked and covalently closed circles on agarose gels. UV-irradiation induced dimers in plasmid DNA, in vivo, at the same rate as in chromosomal DNA. After a dose of 20 Joules·m^–2, approximately 86% of plasmid molecules had. at least one dimer. After 3 h incubation under normal growth conditions only 4% still retained dimers in a wild-type strain. In a rad1 (excision-defective) mutant 81% of plasmid molecules still had dimers after 3 h, suggesting that excision repair operates to remove dimers from plasmid DNA in wild-type yeast. Dimers can be removed from 2 ,um DNA in a rad1 mutant by photoreactivation.     

Instability of Saccharomyces cerevisiae heterokaryons

Summary We have constructed heterokaryons of Saccharomyces cerevisiae by crossing kar1 — mutants incapable of nuclear fusion. Approximately 1% of the total zygotes from kar1 — crosses can form heterokaryotic clones. These are very small as compared to diploid colonies, and are composed mainly of a mixture of both types of heteroplasmons (cells which contain the cytoplasmic components of both parents, but the nuclear genotype of only one of them). This fact indicates that heterokaryons are unstable.This instability is already observed by pedigree analysis in the first zygotic divisions. We suggest that missegregation is the main factor in heterokaryon instability and results from an unequal nuclear transmission, which occurs when one of the mother nuclei divides and, although viable, does not migrate to the daughter bud. However, the proportion of inviable zygotes and buds found in the pedigree analysis, as well as the recovery of only one type of heteroplasmon, indicates the complete loss of one of the parental nuclei. Consequently nuclear inactivation is suggested as the second reason for heterokaryon instability.     

UV-inducible proteins in Saccharomyces cerevisiae

Summary Two UV-inducible proteins have been detected in the yeast, Saccharomyces cerevisiae. The proteins have molecular weights of 78,000 Daltons and 23,000 Daltons. This induction is specific for UV-irradiation as exposure to X-rays, mitomycin C and heat shock does not result in the synthesis of the proteins. The larger (78 kD) protein is induced in various rad strains and in a ° cir° strain. Attempts are being made to isolate the genes coding for these inducible proteins.     

Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is characterized by asymmetric cell division and the asymmetric inheritance of spindle components during normal vegetative growth and during certain specialized cell divisions. There has been a longstanding interest in the possibility that yeast chromosomes segregate non-randomly during mitosis and that some of the differences between mother and daughter cells could be explained by selective chromatid segregation. This review traces the history of the experiments to determine if there is biased chromatid segregation in yeast. The special aspects of spindle morphogenesis and behavior in yeast that could accommodate a mechanism for biased segregation are discussed. Finally, a recent experiment demonstrated that yeast chromatids segregate randomly without mother–daughter bias in a common laboratory strain grown under routine laboratory conditions.     

Nature of abortive transformation in Saccharomyces cerevisiae

Disruption mutagenesis by homologous recombination in Saccharomyces cerevisiae is carried out by transforming-DNA fragments containing the target gene disrupted by a selectable marker. A large number of transient (abortive) transformants are often formed that may hinder the isolation of integrants containing the gene disruption. We show that abortive transformants result from re-circularization of the linear transforming-DNA in vivo. Their number was greatly reduced when the cut DNA could not readily re-ligate, either by digestions that gave non-compatible or blunt ends, or by de-phosphorylation. In addition, true integrants could be readily distinguished from abortive transformants through replica plating onto selective media. Enhanced disruption-mutagenesis was also observed when non-compatible ends were generated in an ARS-containing insertion vector.     

A new killer toxin produced by Saccharomyces cerevisiae

A wine-making Saccharomyces cerevisiae yeast strain isolated in our laboratory produces two different killer toxins, each one encoded by one dsRNA plasmid. One toxin has the same specificity as the one produced by strain M437 described by Naumov, but the dsRNA plasmid which encodes it migrates slightly faster in poly acrylamide gel electrophoresis. The other toxin has not been previously described, and is encoded by a dsRNA fraction which migrates at a lower rate than the × fraction of M437. These two dsRNA plasmids can be maintained separately in different yeast strains.     

Interspecific protoplast fusion between the yeasts Saccharomyces cerevisiae and Saccharomyces fermentati

Summary Protoplasts of Saccharomyces cerevisiae his1 trp2 resistant to acriflavine and able to ferment galactose and of Saccharomyces fennentati arg resistant to DL-p-fluorophenylalanine and able to ferment lactose were fused. As a result of fusion two types of prototrophic hybrids were obtained. Type 1 hybrids were able to grow on medium with galactose or lactose as sole carbon source and were sensitive to acriflavine and resistant to DL-p-pfluorophenylalanine. Type 2 hybrids were able to grow on medium with galactose as sole carbon source and were resistant to acriflavine and sensitive to DL-p-fluorophenylalanine.     

Heterologous expression of human 5-aminolevulinate dehydratase in Saccharomyces cerevisiae

Summary A cDNA coding for human 5-aminolevulinate dehydratase was placed in a yeast expression vector under the control of the GAL10 promoter. The resulting multicopy plasmid was then used to transform a yeast mutant which contains a defective hem2 gene coding for 5-aminolevulinate dehydratase. Expression of the human cDNA was shown in four ways: (1) restoration of normal growth on glycerol/galactose as primary carbon source, (2) decrease in intracellular 5-aminolevulinic acid concentration, (3) restoration of cytochrome biosynthesis and (4) direct, in situ assay of 5-aminolevulinic acid dehydratase. Curing transformed cells of plasmid restored the hem2 mutant phenotype. This heterologous system could be used to produce large quantities of human 5-aminolevulinic acid dehydratase for physical and biochemical studies.     

Plasmid associations with residual nuclear structures in Saccharomyces cerevisiae

Summary Acentric yeast plasmids are mitotically unstable, apparently because they cannot freely diffuse after replicating and therefore are not included in the daughter nucleus. This behavior could result if plasmids remain attached to structural elements of the nucleus after replicating. Since DNA replication is believed to take place on the nuclear matrix, we tested whether there was a correlation between the mitotic stability of a given plasmid and the extent to which it was found associated with residual nuclear structures. Residual nuclei were prepared from yeast nuclei by extraction with either high salt, 2 M NaCl, or low salt, 10 mM lithium diiodosalicylate (LIS). Hybridization analysis was used to estimate the fraction of plasmid molecules remaining after nuclei were extracted. We examined the extent of matrix association of three ARSI plasmids, Trpl-RI circle (1.45 kb), YRp7 (5.7 kb) and pXBAT (45.1 kb) with mitotic loss rates ranging from 3–25%. In addition we examined the matrix binding of the endogenous 2 m plasmid and the 2 m-derived YEp 13 which is relatively stable in the presence of 2 m and less stable in cir° strains. Among the ARS1 plasmids we observed a negative correlation between stability and matrix association, consistent with models in which binding to the nuclear matrix prevents passive segregation of ARS1 plasmid molecules. No such correlation was observed among the 2 n plasmids. Among all plasmids examined there is a positive correlation between size and matrix association.     

Newly synthesised DNA of high molecular weight in the yeast Saccharomyces cerevisiae

Summary Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster.     

Reversible pseudohyphal growth in haploid Saccharomyces cerevisiae is an aerobic process

Pseudohyphal growth in Saccharomyces cerevisiae has been postulated to be an adaptation to foraging for nitrogen during nitrogen starvation. This process was described as a strictly diploid phenomenon which did not occur in haploid yeast cells and was under the genetic control of both the mating-type locus and a group of five genes, the BUD genes, regulating bud formation. We have also observed a dimorphic growth pattern in yeast growing on various nitrogen-limiting synthetic media. However, and in contrast to a previous report, we find that pseudohyphal growth is not precluded in haploid cells. We demonstrate that haploid pseudohyphal growth is strictly oxygen-dependent and is rapidly reversible, defining pseudohyphal growth as a reversible developmental pathway in yeast.     

Uptake of DNA by fragile mutants of Saccharomyces cerevisiae

Summary When Saccharomyces cerevisiae SY15 rho° mutant cells grown in media stabilized with 10% sorbitol were suspended in 2% sorbitol solutions, 60–70% of the population did not lyse and became permeable to native high molecular weight DNA. Maximal incorporation of DNA to DNase resistant state was measured after 60 min of incubation in presence of 5 g/ml DNA and 10 mM CaCl₂. These results suggest that the fragile mutants might be tested as hosts for transformation of whole yeast cells.     

Meiotic segregation in familial reciprocal translocation t(8q;22q)

We studied the chromosomes of a mentally retarded boy with minor anomalies and of his parents using a G-band stained high-resolution chromosome method. This documented dup (8q24.1 → 8qter) and dup(22pter → 22q11.2) in the boy due to a maternal balanced reciprocal translocation of chromosomes 8 and 22 and 3:1 disjunction during meiosis I. The karyotype of the boy is 47, XY, + der(22) (22pter → 22q11.2::8q24.1 → 8qter). The der(22) was involved in satellite associations and stained positively with AgNO₃ in mother and child. The case is compared to similar cases in the literature and the function of the small acrocentric marker chromosome during meiosis is discussed.     

Functional co-operation between the nuclei of Saccharomyces cerevisiae and mitochondria from other yeast species

We elaborated a simple method that allows the transfer of mitochondria from collection yeasts to Saccharomyces cerevisiae. Protoplasts prepared from different yeasts were fused to the protoplasts of the ade2-1, ura3-52, kar1-1,ρ⁰ strain of S. cerevisiae and were selected for respiring cybrids on plates containing 5-fluoroorotic acid and a non-fermentable carbon source. The identity of putative cybrids was assessed by restriction analysis of mitochondrial DNA, pulse field electrophoresis and tetrad analysis. In the comprehensive screening, only mitochondrial genomes from synonymous species (S. italicus, S. oviformis, S. capensis and S. chevalieri) exhibited complete compatibility with S. cerevisiae nuclei. The closely related S. douglasii mitochondrial genome could also partially restore respiration-deficiency in ρ⁰ S. cerevisiae, whereas mitochondrial genomes from phylogenetically less related species could not. Received: 17 April 2000 / Accepted: 20 June 2000