Michael S. Esposito

Diploid yeast cells yield homozygous spontaneous mutations

A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1–12/leu1–12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1–12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1–12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate postreplicational chromatid breakage and exchange near the site of leu1–12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

Simultaneous detection of changes in chromosome number, gene conversion and intergenic recombination during mitosis of Saccharomyces cerevisiae: spontaneous and ultraviolet light induced events

SummaryWe have employed a hyperhaploid strain of Saccharomyces cerevisiae disomic for chromosome VII to monitor spontaneous and ultraviolet light induced restitution of haploidy (chromosomal loss and/or nondisjunction), mitotic gene conversion and mitotic intergenic recombination. The disomic chromosomal pair incorporates six heterozygous markers, including cyh2r, distributed on both sides of the centromere. Cycloheximide resistant segregants of spontaneous origin were analyzed to calculate the spontaneous mitotic rates of restitution of haploidy, intergenic recombination and gene conversion that result in expression of the cyh2r mutation. Restitution of haploidy was found to be the most common source of spontaneously arising cycloheximide resistant segregants. In contrast, those induced by ultraviolet light resulted most frequently from gene conversion of CYH2s to cyh2r. The chromosome VII hyperhaploid system provides a sensitive method to detect the aneugenic and recombinagenic effects of suspect chemical and physical agents.

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.

The genetics of complex diseases.

Genetic factors influence virtually every human disorder, determining disease susceptibility or resistance and interactions with environmental factors. Our recent successes in the genetic mapping and identification of the molecular basis of mendelian traits have been remarkable. Now, attention is rapidly shifting to more-complex, and more-prevalent, genetic disorders and traits that involve multiple genes and environmental effects, such as cardiovascular disease, diabetes, rheumatoid arthritis and schizophrenia. Rather than being due to specific and relatively rare mutations, complex diseases and traits result principally from genetic variation that is relatively common in the general population. Unfortunately, despite extensive efforts by many groups, only a few genetic regions and genes involved in complex diseases have been identified. Completion of the human genome sequence will be a seminal accomplishment, but it will not provide an immediate solution to the genetics of complex traits.

Recombinators, recombinases and recombination genes of yeasts

Genetic recombination in the nuclear, organellar, and plasmid genomes of eukaryotic organisms occurs during mitosis and meiosis. Mitotic recombination is an important mechanism for the repair of DNA damaged by ultraviolet light, ionizing radiation, and chemical agents (Friedberg et al. 1991). Moreover, nuclear chromosomal mitotic recombination is involved in a number of basic cellular processes seen in a wide variety of eukaryotic systems including: cell-type differentiation of homothallic budding yeast (Saccharomyces cerevisiae) (Strathern et al. 1982) and fission yeast (Schizosaccharomyces pombe) (Egel et al. 1984); transpositions of P-elements in Drosophila melanogaster (Gloor et al. 1991); mammalian and human site-specific V-D-J gene rearrangements that give rise to immunoglobulin and T-cell receptor diversity (Oettinger et al. 1990; Kallenbach and Rougeon 1992); and loss of heterozygosity implicated in carcinogenesis (Solomon et al. 1991). Meiotic recombination plays a key role in ensuring accurate chromosome segregation at the first meiotic division (the reductional division). Additionally, meiotic interchromosomal and intrachromosomal recombination of homologous DNA sequences between members of multigene families, such as the mammalian major histocompatibility complex, MHC, (Kuhner et al. 1991) and the isoaccepting multiple tRNA genes of fission yeast (Amstutz et al. 1985), contributes to allelic diversity and concerted evolution of members of multigene families. These recombinational events and conventional allelic meiotic gene conversion, crossing over, and independent assortment, produce an abundance of gametic genotypes.

Erythromycin and cycloheximide sensitivities of protein and RNA Synthesis in sporulating cells of Saccharomyces cerevisiae: Environmentally induced modifications controlled by chromosomal and mitochondrial genes.

SummaryThe purpose of the experiments reported below was to examine the response in sporulation medium of the three diploid cell types MATα MATα, MATα MATα (asporogenic diploids) and MATα MATα (sporogenic diploid) to erythromycin, a specific inhibitor of mitochondrial protein synthesis (MPS) in vegetative cultures, and cycloheximide, a specific inhibitor of cytosol protein synthesis (CPS) in vegetative cultures. When MATα MATα diploids are transferred to sporulation medium a significant fraction of total protein synthesis (CPS + MPS) becomes sensitive to erythromycin in contrast to the behavior of MATa MATa and MATα MATα diploids in which the resistance of CPS to erythromycin is maintained. The decompartmentalization of erythromycin sensitivity is thus cell type specific. Erythromycin stimulates total RNA synthesis of MATα MATα cells in sporulation medium but not of MATα MATα and MATα MATα cells. Cycloheximide inhibits protein synthesis and stimulates RNA synthesis in all three diploid cell types. An erythromycin resistant mutant, shown to be due to a mutation of the mitochondrial genome, exhibited only partial resistance of CPS to erythromycin in sporulation medium in the background of the MATα MATα mating type genotype. Total RNA synthesis in this mutant was not stimulated. The results reported indicate that mitochondrial functions during sporulation are not restricted to those involving respiratory metabolism.

Nonrandomly-associated forward mutation and mitotic recombination yield yeast diploids homozygous for recessive mutations.

We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L-α-aminoadipate as a nitrogen source (α-Aa+) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. α-Aa- diploid cells yield α-Aa+ mutants at a rate of 7.8±3.6×10-9. As in haploid strains, approximately 97% (30/31) of α-Aa+ mutants are spontaneous lys2-x recessive mutations. α-Aa+ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2±0.4×10-6. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3±0.6×10-3. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarly of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval. The methods described can be used to simultaneously monitor the effects of yeast gene mutations and carcinogens on the principal parameters affecting the genomic stability of diploid mitotic cells: mutation, gene conversion, intergenic recombination, and chromosomal loss or rearrangement.

Conditional hyporecombination mutants of three REC genes of Saccharomyces cerevisiae

SummaryWe have isolated and characterized three conditional hyporecombination mutants, rec1-1, rec3-1 and rec4-1, that define three REC genes of Saccharomyces cerevisiae required for spontaneous general mitotic interchromosomal recombination. Each MATa/MATα rec/rec diploid is deficient in mitotic single site gene conversion, intragenic recombination, intergenic recombination and sporulation at the restrictive temperature (36°C). The rec1-1 mutation also confers conditional enhanced sensitivity to the killing effects of X-rays. The rec1-1 and rec3-1 mutations have been mapped to chromosome VII. The rec1-1, rec3-1 and rec4-1 mutations exhibit complementation at 36°C for both mitotic recombination and sporulation.

The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability, recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation

SummaryThe recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MATα n+1 chromosome VII disomic strain LBW (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MATα n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATaIMaTα ree46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MATα rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intergenic recombination resulting in prototrophy. When compared to MATα rec46-1 n+1 disomics however, MATa/MATα rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATaIMATα rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).