Dataset Information

A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells.

ABSTRACT: Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 15-100 bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary-phase cells. Alterations within minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy, and several types of cancer. However, little is known about the factors preventing minisatellite alterations. Previously, our laboratory developed a color segregation assay in which a minisatellite was inserted into the ADE2 gene in the yeast Saccharomyces cerevisiae to monitor alteration events. We demonstrated that minisatellite alterations that occur in stationary-phase cells give rise to a specific colony morphology phenotype known as blebbing. Here, we performed a modified version of the synthetic genetic array analysis to screen for mutants that produce a blebbing phenotype. Screens were conducted using two distinctly different minisatellite tracts: the ade2-min3 construct consisting of three identical 20-bp repeats, and the ade2-h7.5 construct, consisting of seven-and-a-half 28-bp variable repeats. Mutations in 102 and 157 genes affect the stability of the ade2-min3 and ade2-h7.5 alleles, respectively. Only seven hits overlapped both screens, indicating that different factors regulate repeat stability depending upon minisatellite size and composition. Importantly, we demonstrate that mismatch repair influences the stability of the ade2-h7.5 allele, indicating that this type of DNA repair stabilizes complex minisatellites in stationary phase cells. Our work provides insight into the factors regulating minisatellite stability.

SUBMITTER: Alver B

PROVIDER: S-EPMC3618361 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells.

Alver Bonnie B Jauert Peter A PA Brosnan Laura L O'Hehir Melissa M VanderSluis Benjamin B Myers Chad L CL Kirkpatrick David T DT

G3 (Bethesda, Md.) 20130409 4

Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 15-100 bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary-phase cells. Alterations within minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy, and several types of cancer. However, little is known about the facto ...[more]

PMID: 23550123

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Project description:The screen for genes involved in the spontaneous loss of heterozygosity mutagenesis (SLM) in diploid cells was done on the collections of deletion strains of Saccharomyces cerevisiae created by the Saccharomyces Genome Deletion Project (http://www-sequence.stanford.edu/group/yeast_deletion_project/). Screens were performed on the pools of clones. Barcode microarrays were used to detect the increased SLM frequency leading to relative increase in abundance of deletion clone in a pool. Collections used were: Homozygous Diploid (HD) and Essential (E) mixed to create single HDE pool. Three mutagenesis markers were used: (1) naturally existing mating type locus, and two newly introduced markers, (2) CAN1/can1delta, (3) URA3/ura3delta. 1. To detect the increased SLM frequency at the mating type locus HDE pool was crossed to MATa or MATalpha strain. Deletion clones that gained mating competence due to loss of heterozygosity (LOH) at the mating type locus gave rise to clones with the ability to grow on minimal medium. The relative abundance of these clones was compared to the abundance in the original HDE pool. 2. To detect the increased SLM frequency at the CAN1/can1delta locus the derivative HDE pool was created. Every cell of this pool had one of two copies of CAN1 gene deleted. The increased SLM frequency led to increased relative abundance of respective deletion clone under canavanine selection and could be estimated by comparing it to the abundance when pool was grown without selection. 3. To detect the increased SLM frequency at the URA3/ura3delta locus the derivative HDE pool was created. Every cell of this pool had one of two copies of URA3 gene deleted. The increased SLM frequency led to increased relative abundance of respective deletion clone under 5M-^R-fluoroorotic acid selection and could be estimated by comparing it to the abundance when pool was grown without selection. 4. For screens of SLM at CAN1 and URA3 markers the resistance control experiments were performed to detect genes whose deletion is sufficient to enable yeast cells with functional CAN1 or URA3 to grow in the presence of canavanine or 5M-^R- fluoroorotic acid respectively. 5. To include in the analysis slow-growing deletion clones control hybridizations were performed to detect genes whose deletion extends the doubling time of yeast cells.

Dataset Information

A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells.

Publications

A Whole Genome Screen for Minisatellite Stability Genes in Stationary-Phase Yeast Cells.

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