Gene conversions within the Cup1r region from heterologous crosses in Saccharomyces cerevisiae.

Meiotic recombination among unselected tetrads was analyzed genetically and physically in a heterologous cross where one parent carried six copies of a 1.6 kb CUP1 repeat while the other parent carried seven copies of a 1.1 kb repeat. In the heterologous cross, 140 unselected, complete tetrads were subjected to Southern analysis and 20% exhibited meiotic copy number alterations at the CUP1 locus. Most events, more than 75%, involved only a single spore of a tetrad, and were largely intrachromosomal or sister chromatid events. However, some conversions and associated crossovers between homologs were also observed. We propose that the high level of heterologies interferes with homologous exchanges and leads to an increase in intrachromosomal events.

The detection of mitotic and meiotic chromosome gain in the yeast Saccharomyces cerevisiae: effects of methyl benzimidazol-2-yl carbamate, methyl methanesulfonate, ethyl methanesulfonate, dimethyl sulfoxide, propionitrile and cyclophosphamide monohydrate.

The diploid yeast strain BR1669 was used to study induction of mitotic and meiotic chromosome gain by selected chemical agents. The test relies on a gene dosage selection system in which hyperploidy is detected by the simultaneous increase in copy number of two alleles residing on the right arm of chromosome VIII: arg4-8 and cup1S (Rockmill and Fogel. 1988; Whittaker et al., 1988). Methyl methanesulfonate (MMS) induced mitotic, but not meiotic, chromosome gain. Methyl benzimidazol-2-yl carbamate (MBC) and ethyl methanesulfonate (EMS) induced both mitotic and meiotic chromosome gain. Propionitrile, a polar aprotic solvent, induced only mitotic chromosome gain; a reliable response was only achieved by overnight incubation of treated cultures at 0 degrees C. MBC is postulated to act by binding directly to tubulin. The requirement for low-temperature incubation suggests that propionitrile also induces aneuploidy by perturbation of microtubular dynamics. The alkylating agents MMS and EMS probably induce recombination which might in turn perturb chromosome segregation. Cyclophosphamide monohydrate and dimethyl sulfoxide (DMSO) failed to induce mitotic or meiotic chromosome gain.

Unequal crossing-over and gene conversion at the amplified CUP1 locus of yeast.

Meiotic recombination was analyzed between two twelve-copy arrays of a gene amplification at the CUP1 locus of Saccharomyces cerevisiae. Utilizing Southern analysis to identify spores with non-parental repeat arrays, we find that approximately 11% of a sample with 202 unselected tetrads possess at least one nonparental spore array. Both reciprocal and non-reciprocal changes are observed. The data suggest a model in which frequent mispairing among identical copies of the 2.0 kb repeat unit leads to the formation of unpaired loops containing integral numbers of repeat units. In this model, conversions involving the loops lead to non-reciprocal changes in arrays: about half are associated with reciprocal exchange, and net increases in repeat unit numbers occur about as frequently as net decreases. Thus, the known properties of gene conversion can account for all the segregations we observe.

The detection of mitotic and meiotic aneuploidy in yeast using a gene dosage selection system.

A system is described in which spontaneous and chemically-induced mitotic and meiotic hyperploidy can be assayed in the same diploid culture of Saccharomyces cerevisiae. Monitoring gene dosage changes at two loci on chromosome VIII, the test utilizes a leaky temperature-sensitive allele arg4-8 and low level copper resistance conferred by the single copy allele cup1s. An extra chromosome VIII provides simultaneous increased dosage for both genes, resulting in colonies that are both prototrophic for arginine at 30 degrees C and copper resistant. During mitotic cell divisions in diploids, spontaneous chromosome VIII hyperploids (trisomes and tetrasomes) occur at a frequency of 6.4 x 10(-6) per viable cell. Among ascospores, the spontaneous chromosome VIII disome frequency is 5.5 x 10(-6) per viable spore. The tubulin-binding reagent methyl benzimidazol-2-yl carbamate (MBC) elicits enhanced levels of mitotic and meiotic aneuploidy relative to control levels. The system represents a novel model for examining chromosome behavior during mitosis and meiosis and provides a sensitive and quantifiable procedure for examining chemically induced aneuploidy.

The molecular genetics of copper resistance in Saccharomyces cerevisiae--a paradigm for non-conventional yeasts.

After a short introduction on the historical background of the development of Saccharomyces cerevisiae as a model eukaryote, a review is given on the present state of genetics and molecular biology of copper resistance in S. cerevisiae. The gene CUP1 encodes a protein (copper metallothionein) of a molecular weight of 6570 dalton. The synthesis of this copper chelatin is induced by copper and is regulated at the level of transcription. Copper resistance (CUPr) is correlated with amplification of CUP1 and resulted in a higher copy number of this gene on chromosome VIII. Spontaneous meiotic alterations of the gene copy number have been studied.

Gene conversion, unequal crossing-over and mispairing at a non-tandem duplication during meiosis of Saccharomyces cerevisiae.

We have developed a novel system to examine conversion, exchange and mispairing involving a non-tandem duplication of the ade8 locus in yeast by monitoring the segregation of heterozygous markers between the duplicated sequence. Plasmid Yrp17 carries the yeast selectable markers URA3+ and TRP1+. Yrp17 derivatives with a 4 kb insert carrying ade8-18 were used to clone the mutations trp1-1 and ura3-1 by gap repair. Integrants of the resulting plasmids at the Ade8 locus were crossed to yield diploid hybrids with a non-tandem duplication of Ade8 and heterozygosity for the plasmid markers between the duplicated sequences. 1192 complete, unselected asci were analyzed and 270 exhibiting recombination of the markers contributed by the plasmid were analyzed by Southern transfers to detect changes in plasmid sequences. Twenty-seven tetrads had unequal homologous exchanges and five had unequal sister-chromatid exchanges. Seven tetrads carry an additional copy of the integrated plasmid and ten are missing one. We propose that these two classes represent conversions of the entire 11 kb plasmid, which occur after misalignment and formation of an unpaired loop. Mispairing is a frequent event, and occurs in approximately fifty percent of all meioses. The system described provides a means to determine the meiotic rules of conversion, exchange and pairing for duplicated DNA sequences.

Synaptic relations in meiotic gene conversion at the iterated CUP1r locus of S. cerevisiae.

This study concerns a comparative molecular analysis of copy number changes in two hybrids that differ in the extent of homologies at the CUP1r locus. Hybrid JW1020 is a diploid wherein each parent contributed nine identical, tandemly arrayed 2.0 kb repeat units. Genomic DNA was isolated from each of the spore colonies in a sample of 200 unselected tetrads. About 15% displayed copy number changes, i.e., increases or decreases of one or more complete units. Changes on a per tetrad basis occurred as often in a single spore colony as changes in each of two spores. Such double changes are rarely reciprocal in character. To account for the observed qualitative and quantitative copy number shifts, we propose a molecular recombination model that posits partial, incomplete synaptic pairing and gene conversion of the unpaired regions with or without associated crossing over. A second contrasting study centers on the copy number alterations and recombinational events uncovered in a molecular analysis of 50 unselected tetrads generated by hybrid EB8. Unlike the hybrid JW1020, the EB8 diploid strain carried a six copy tandem array of 1.1 kb units at the CUP1r locus in one parental homologue and a five copy array of 1.6 kb units at the corresponding chromosome VIII locus. These natural alleles were recovered from industrial yeast strains by conventional genetic procedures and characterized by restrictional analysis. Twelve tetrads exhibit evidence for several different types of recombination events. However,ordinary crossover exchanges are conspicuously absent. We suppose that the repetitious nonhomologies generate DNA configurations sufficient to disrupt the effective synapsis over the entire locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Meiotic recombination and sporulation in repair-deficient strains of yeast.

A genetic system designed to monitor recombination and sporulation in various repair-deficient yeast strains was constructed. Variously heterozygous at seven or eight sites distributed across the genome, the system facilitated sensitive detection of changes in frequency or pattern of meiotic recombination. Ten rad mutants sensitive primarily to UV-irradiation and without terminal blocks in the sporulation process were studied. Seven were defective in excision repair (rad1, rad2, rad3, rad4, rad10, rad14 and rad16), and three were defective in mutagenic repair (rad5, rad9 and rad18). Individually, each mutant displayed behavior consistent with an orthodox meiosis including a wild-type meiotic recombination profile with respect to gene conversion, PMS and intergenic map distances. Accordingly, we conclude that these mutants are without major effect on meiotic heteroduplex formation or correction. However, certain combinations of excision-defective mutants with rad18 exhibited marked ascosporal inviability. Tetraploids homozygous for rad1 and rad18 produce a large proportion of diploid spores containing a recessive lethal.

Mitotic recombination in yeast: isolation and characterization of mutants with enhanced spontaneous mitotic gene conversion rates.

Semi-dominant mutants displaying greatly elevated (up to 200-fold above control) levels of spontaneous mitotic recombination have been isolated in a disomic haploid strain of yeast heteroallelic at the arg4 locus. They are designated by the symbol MIC. The mutants variously exhibit associated sensitivity to UV and ionizing radiation and to methyl methanesulfonate, enhanced UV-induced mitotic recombination, and enhanced spontaneous forward mutation rates. Possible enzyme defects and involvement in repair and editing of DNA are discussed. The mutants are expected to simplify the analysis of recombination pathways in yeast.

Polymethylpolysaccharide synthesis in an ethionine-resistant mutant of Mycobacterium smegmatis.

Mutants of Mycobacterium smegmatis were selected for resistance to ethionine in an effort to obtain methylation-defective strains that were altered in their ability to make methylmannose polysaccharides (MMP) or methylglucose lipopolysaccharides. Two methods were developed for the detection of MMP in cell extracts to aid in the screening for potential mutants, one a qualitative procedure based on iodine binding by the sample after paper chromatography and the other a quantitative procedure based on fluorimetric titration of the MMP with parinaric acid. An ethionine-resistant mutant was obtained that contained only about 25% of the normal level of S-adenosylmethionine and 10% of the normal level of methionine adenosyltransferase (adenosine 5'-triphosphate:L-methionine S-adenosyltransferase, EC 2.5.1.6) activity. When grown in the presence of 0.1% ethionine, the mutant cells contained about 50% of the wild-type levels of methylglucose lipopolysaccharides but only about 7% of the normal level of MMP (wild-type cells contain about 0.14 mM MMP and the mutant contains about 0.01 mM MMP). The amount of fatty acid synthesis in the ethionine-resistant mutant grown in the presence of ethionine was not dramatically altered although the mutant accumulated more short-chain and less long-chain unsaturated fatty acids than the wild-type cells.