Selection and analysis of spontaneous reciprocal mitotic cross-overs in Saccharomyces cerevisiae

Abstract

We developed a system that allows the selection of the reciprocal products resulting from spontaneous mitotic cross-overs in the yeast Saccharomyces cerevisiae. A number of other types of genetic events, including chromosome loss, can be monitored with this system. For a 120-kb chromosome interval on chromosome V (CEN5-CAN1), the rate of mitotic cross-overs was 4x10(-5) per division, a rate approximately 25,000-fold lower than the meiotic rate of cross-overs. We found no suppression of mitotic cross-overs near the centromere of chromosome V, unlike the suppression observed for meiotic exchanges. The rate of reciprocal cross-overs was substantially (38-fold) elevated by treatment of cells with hydroxyurea, a drug that reduces nucleotide pools and slows DNA replication.

Fig. 1.

Two systems for the detection of mitotic recombination and chromosome loss in diploid yeast cells. (A) One commonly used system for detection of mitotic recombination events uses a diploid that is heterozygous for mutations in the can1 and hom3 loci. The starting diploid strain is Can^S and Met⁺. The depicted strain is also homozygous for the ade2-1 mutation that results in cells that are Ade⁻ and form red colonies. Cells are transferred to plates containing Can, and any Can^R derivatives are tested for their ability to grow in the absence of methionine. Met⁻ cells represent chromosome loss events, and Met⁺ cells are assumed to represent mitotic cross-overs. Note that the CAN1/CAN1 product cannot be selected by this system. (B) Selection of both products of an RCO in the diploid MAB6. The starting diploid is phenotypically Can^S Gen^R Hyg^R His⁺ Leu⁺ Ade^+/− and forms white colonies. An RCO between the centromere and the CAN1 locus will result in a Can^R colony with one red and one white sector, resulting from the growth of two Can^R cells, one with the genotype can-100/can1-100 and one with the genotype SUP4-o/SUP4-o. Hyg^S, hygromycin-sensitive; Gen^S, geneticin-sensitive.

Fig. 2.

Phenotypic classes of unsectored Can^R colonies (derived from MAB6) resulting from nonreciprocal mitotic recombination or chromosome loss events. (A) Class 1: A BIR event initiated in the SUP4-o-containing chromosome will give rise to one Can^R cell and one Can^S cell. It is also possible that class 1 events could reflect an RCO that occurred in the culture, before the plating of the cells on medium containing Can. (B) Class 2: These BIR events are comparable to class 1, except that the event initiates by breakage of the can1-100-containing homologue. (C) Class 3: A local gene conversion (unassociated with a cross-over) in which SUP4-o is converted to the can1-100 allele will produce a Can^R Hyg^R Gen^R Leu⁺ His⁺ Ade⁻ red colony. The same phenotype can be produced by a new mutation within SUP4-o. These two possibilities can be distinguished by PCR (as described in Supporting Text). (D) Class 4: This class is similar to class 3 except can1-100 is converted to SUP4-o. (E) Class 5: This class results from loss of the chromosome containing SUP4-o by nondisjunction. (F) Class 6: This class results from loss of the chromosome containing can1-100.

Fig. 3.

Photographs of the different classes of Can^R colonies. Cells of the MAB6 strain were allowed to form colonies on Can-containing medium and were then replica-plated to five different types of diagnostic media; those containing hygromycin or geneticin and those lacking adenine, histidine, or leucine. The colony marked “sector” reflects an RCO, and the numbers represent class 1–6 colonies. The sizes of colonies in the photograph are about the same as the sizes on the plates.

Fig. 4.

Comparisons of rates of mitotic recombination and chromosome loss and rates of mitotic and meiotic recombination. (A) Rates of mitotic recombination and chromosome loss in MAB6 (MATa/MATα), MAB35 (MATa/matαΔ), and MAB38 (mataΔ/MATα). In addition, we examined MAB6 pregrown in medium containing 100 mM HU. Error bars indicate 95% confidence limits. (B) Comparisons of the physical and genetic distances for two intervals on chromosome V, CEN5-URA3 and URA3-CAN1. For each interval, the data are shown as a percentage of the distance between CEN5 and CAN1. The physical distances for CEN5-URA3 and URA3-CAN1 are 36 and 84 kb, respectively. The meiotic recombination distances (95% confidence limits shown in parentheses) for these same two intervals are 8 cM (7.5–8.4 cM) and 42 cM (41–44 cM) for >1,500 tetrads in the Saccharomyces Genome Database (SGD), and 7 cM (5–9 cM) and 41 cM (37–45 cM) based on 360 tetrads in our study. The meiotic data shown use the SGD data. The mitotic distances were derived from our analysis of sectors in MAB13 as described in Results. The 95% confidence limits are indicated for the meiotic and mitotic intervals.