Seymour Fogel

Mechanisms of Meiotic Gene Conversion, or “Wanderings on a Foreign Strand”

INTRODUCTION Over the last decade and a half, studies in organisms amenable to tetrad (or octad) analysis have provided a reasonably complete descriptive inventory of intragenic recombination (Fogel et al. 1979; Nicolas 1979; Rossignol et al. 1979; Sang and Whitehouse 1979). This paper aims to provide an overview of the relevant data. At the same time it attempts a synthesis of the new information in the expectation that a scaffold will emerge from which testable molecular hypotheses concerning generalized and intragenic recombination can be constructed. A comprehensive review of the literature concerning meiotic recombination will not be attempted. Much of the data have been cogently summarized in texts by Esser and Kunnen (1967), Whitehouse (1969), Grell (1974), Kushev (1974), Catcheside (1977), Fincham et al. (1979), Stahl (1979b) and in several critical reviews by Emerson (1969), Mortimer and Hawthorne (1969), Fogel and Mortimer (1971), Radding (1973), Hotchkiss (1974), Hastings (1975), Esposito and Esposito (1977), Pukkila (1977), Resnick (1979), Stahl (1979a), and Petes (1980b). Here, we approach the problem of intragenic meiotic recombination or recombinagenic events within short DNA segments by considering four questions. What is the phenomenon of gene conversion? What are the methods that can be employed to study this phenomenon? What are the salient features of the conversion process? Can methodologies of recombinant DNA technology be adapted to advance the analysis of intragenic recombination from the genetic level to the molecular level? We may begin by asking, What is meiotic gene conversion? In organisms subject...

Gene amplification in yeast: CUP1 copy number regulates copper resistance

The CUP1 locus in yeast confers resistance to copper toxicity. We determined the molecular basis for copper resistance in three yeast strains, with differing degrees of resistance. Increased resistance to copper is associated with overproduction of a low molecular weight copper-binding protein, copper-chelatin. Increased chelatin synthesis results from amplification of the CUP1r gene and increased synthesis of the copper inducible mRNA. The copper resistance level of a given strain correlates directly with the gene copy number.Strains containing one copy and ten tandemly iterated copies of the CUP1 gene were studied. From the latter, a haploid strain with enhanced resistance was isolated following several selection cycles at elevated copper concentrations. This strain was disomic for chromosome VIII, the chromosome containing the CUP1 locus. The disomic chromosomes exhibit differential CUP1 gene amplification: 11 and 14 tandemly organized repeat units are found in the respective chromosome VIII homologues. We propose that the molecular mechanisms of gene amplification involve unequal sister chromatid exchange and intrachromosomal gene conversion, as well as disomy.

A system selective for yeast mutants deficient in meiotic recombination

SummaryAn experimental design and rationale for detecting and recovering Saccharomyces cerevisiae mutants specifically blocked in meiotic gene conversion is presented. The system utilizes an otherwise haploid strain disomic (n+1) for chromosome III which is simultaneously heterozygous for the mating-type locus and heteroallelic at leu2. The former is an essential requirement for inducing meiotic development; i.e., DNA replication and sporulation upon transfer to acetate media, while the latter provides a convenient signal for assaying recombination at the intragenic level. Of 940 clones screened qualitatively after mutagenesis with ethyl methanesulfonate, 91 presumptive mutants were isolated. These are classed arbitrarily into four groups according to the reduction in interallelic recombination observed in quantitative tests.

Unequal crossing-over and gene conversion at the amplifiedCUP1 locus of yeast

SummaryMeiotic recombination was analyzed between two twelve-copy arrays of a gene amplification at theCUP1 locus ofSaccharomyces 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.

Multicopy CUP1 plasmids enhance cadmium and copper resistance levels in yeast

SummaryA 3.3 kb fragment of yeast genomic DNA was isolated by screening a genomic library constructed in the high copy number 2 micron plasmid YEp351 vector for clones capable of enhancing the degree of resistance of Saccharomyces cerevisiae strain MW3070-8B to cadmium. The insert contained two complete copies of the CUP1 gene open reading frame (183 bp), including the upstream promoter sequences (450 bp) with two conserved metal responsive cis-acting elements. Northern analysis showed that addition of cadmium (0.02 μM) or copper (50 μM) to overnight liquid cultures of yeast induced expression of CUP1 transcripts from both chromosomal and plasmid-borne gene copies. The cloned 3.3 kb DNA in a high copy number plasmid restored copper resistance to the sensitive strain LS70-313Δ, deleted for the CUP1 gene (cup1Δ), but failed to restore cadmium resistance. Thus, CUP1 gene expression in yeast appears to be influenced differently by cadmium and copper ions. Resistance to heavy metal poisoning resulted from enhanced gene product levels attributable to amplification of the CUP1 gene as well as to increased transcriptions. Two distinct gene product levels mediate cadmium and copper resistance; a higher gene product level was required to confer cadmium resistance.

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

SummaryA 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° C and copper resistant. During mitotic cell divisions in diploids, spontaneous chromosome VIII hyperploids (trisomes and tetrasomes) occur at a frequency of 6.4×10-6 per viable cell. Among ascospores, the spontaneous chromosome VIII disome frequency is 5.5×10-6 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.

Fidelity of meiotic gene conversion in yeast.

SummaryGene conversion was studied in a sample of 3869 unselected meiotic tetrads obtained from three diploids, respectively; heterozygous for a single ochre mutant, heteroallelic for a pair of ochre alleles, and heterozygous for an ochre specific suppressor. Although the genetic system were sufficiently sensitive to detect single base changes at the mutant codon level, none were found among 36 conversions (1+:3m) of the ochre mutants and 153 conversions (3S:1+) of the suppressor locus. These findings lead to the conclusion that the informational transfer in gene conversion occurred with complete fidelity. Gene conversion conserved and did not generate new genetic information. The error level of conversion was estimated as less than 10-2/N.P.

Recombinational repair of diverged DNAs : a study of homoeologous chromosomes and mammalian YACs in yeast

SummaryRecombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms+ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.

Mutations affecting meiotic gene conversion in yeast.

SummaryMeiotic development and genetic recombination, including gene conversion, occur when cells of Saccharomyces cerevisiae are exposed to nitrogen-limited acetate sporulation medium. Mutations blocking acetate-induced conversion as detected by prototroph formation at the heteroallelic leu2 locus were isolated in a previous study (Roth and Fogel, 1971) in a marked disomic (n+1) haploid strain. Five mutant strains were further characterized. Diploids homozygous for each mutation were constructed and analyzed for intragenic recombination, premeiotic DNA replication, and ascospore formation. Although prototroph formation was blocked in all five diploids, only three, which permitted the initiation and completion of premeiotic DNA replication, could be considered specifically deficient in meiotic gene conversion as assayed by the production of wild type recombinants. From tetrad analysis and complementation tests, the mutations were identified as recessive and nonallelic. To denote the conversionless phenotype, the loci marked by the mutations have been designated con1, con2, con3.

Saturated fatty acid mutants in yeast

SummaryThirty-nine saturated fatty acid requiring mutants were isolated after treatment with ethyl methanesulfonate (EMS). These mutants have been assigned to three major complementation classes and from tetrad analysis appear to represent three unlinked genes: at least two loci are not linked to any centromere. Two major complementation categories are delineated by two non-allelic mutnats. Though fully complementary to each other, each fails to complement any mutant in several different complementation subgroups. Characterized by their response to external suppressors, these mutants represent nonsense mutations. One mutant is of the amber variety and the other is ochre. Tetrad analysis of diverse hybrids involving the saturated fatty acid auxotrophs revealed cryptic aneuploidy for chromosomes III, VIII and the as yet unidentified chromosomes that carry the above ochre and amber mutants. Clearly, this represents a minimal estimate. A more precise evaluation requires inclusion of sufficient genetic markers adequate to monitor the entire yeast genome. Gene dosage alterations due to aneuploidy had no apparent effect on the efficiency of interallelic complementation. The implications of these findings on the regulation and assembly of fatty acid synthetase are discussed. A working hypothesis concerning the relation between the effect of the primary lesion and nondisjunction is presented.