Academic journal article Genetics

Chromosome-Scale Genetic Mapping Using a Set of 16 Conditionally Stable Saccharomyces Cerevisiae Chromosomes

Academic journal article Genetics

Chromosome-Scale Genetic Mapping Using a Set of 16 Conditionally Stable Saccharomyces Cerevisiae Chromosomes

Article excerpt

ABSTRACT

We have created a resource to rapidly map genetic traits to specific chromosomes in yeast. This mapping is done using a set of 16 yeast strains each containing a different chromosome with a conditionally functional centromere. Conditional centromere function is achieved by integration of a GAL1 promoter in cis to centromere sequences. We show that the 16 yeast chromosomes can be individually lost in diploid strains, which become hemizygous for the destabilized chromosome. Interestingly, most 2n - 1 strains endoduplicate and become 2n. We also demonstrate how chromosome loss in this set of strains can be used to map both recessive and dominant markers to specific chromosomes. In addition, we show that this method can be used to rapidly validate gene assignments from screens of strain libraries such as the yeast gene disruption collection.

THE centromere is the chromosomal element that ensures faithful segregation of duplicated chromosomes to both the mother and daughter during cell division. The yeast centromere is composed of an ~125-bp sequence that can be subdivided into three functional elements present at all yeast centromeres (Fitzgerald-Hayes et al. 1982; Panzeri et al. 1985). These conserved DNA elements are binding sites for the centromere binding proteins Cbf1, Cse4, Mif2, and the CBF3 complex (Ndc10, Ctf13, Cep3, and Skp1) that together make up the inner kinetochore (reviewed in Cheeseman et al. 2002). The inner kinetochore associates with the central and outer kinetochore proteins to establish a link between the centromere of every chromosome and the mitotic spindle apparatus to ensure proper segregation during mitosis (Cheeseman et al. 2002).

Early experiments designed to study centromeric DNA showed that chromosomal insertion of a strong promoter adjacent to CEN DNA abrogated centromere function (Panzeri et al. 1984). This led to the construction of conditional centromeres by cloning strong regulatable promoters such as GAL1 adjacent to CEN sequences to allow controlled inactivation (Chlebowicz-Sledziewska and Sledziewski 1985; Hill and Bloom 1987). Conditional centromeres have been used to alter the stability of plasmids, YACs and whole chromosomes (Chlebowicz-Sledziewska and Sledziewski 1985;Hill and Bloom 1987; Smith et al. 1990). In a plasmid context, conditional centromeres can be used to amplify copy number since inactivating a CEN sequence relieves copy number control causing plasmid number to increase in the mother cell. At the same time, inactivation of the CEN sequence results in a higher frequency of plasmid loss, which can be selected with a counter-selectable marker. Likewise, when conditional centromeres are placed in a chromosomal context, chromosome loss can be induced to generate 2n - 1 diploids by counterselecting a marker on the conditional chromosome (Hill and Bloom 1987).

We took advantage of the Hill and Bloom (1987) observation to produce a set of 16 centromere-conditional, counterselectable chromosomes in both MATa and MATα strains. Here we show that these strains produce chromosome specific 2n - 1 monosomy and concomitant loss of heterozygosity (LOH).We show that chromosomespecific LOH can be used to map both recessive and dominant mutations using different readouts of the induced homozygous phenotype. This procedure is useful to map any unknown gene to a specific chromosome, which in turn accelerates the refinement of its genetic location. Finally, we show that this same approach can be used to verify that the phenotype of a haploid deletion library strain is indeed due to the "advertised" gene disruption.

MATERIALS AND METHODS

Reagents and yeast media: G418 was purchased from MediaTech (Herndon, VA). Five (5)-fluoroorotic acid (5-FOA) was purchased from American Bioanalytical (Natick, MA). Taq DNA polymerase was purchased from Continental Laboratory Products (San Diego).

Yeast extract-peptone-dextrose (YPD) medium, synthetic complete (SC) medium, synthetic dropout (e. …

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