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. 2008 Dec;180(4):1799-808.
doi: 10.1534/genetics.108.087999. Epub 2008 Oct 1.

Chromosome-scale genetic mapping using a set of 16 conditionally stable Saccharomyces cerevisiae chromosomes

Robert J D Reid  1 , Ivana Sunjevaric, Warren P Voth, Samantha Ciccone, Wendy Du, Aileen E Olsen, David J Stillman, Rodney Rothstein
Affiliations

Chromosome-scale genetic mapping using a set of 16 conditionally stable Saccharomyces cerevisiae chromosomes

Robert J D Reid et al. Genetics. 2008 Dec.

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.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
Construction of pCEN-UG plasmids and conditional centromere strains. (A) The pCEN-UG plasmids were constructed by amplifying centromere-proximal DNA (thick solid offset lines), including the CEN locus (solid circle) and cloning into plasmid pKlUGAL-NotI. This cloning generates a CEN-proximal region that is interrupted by the K. lactis URA3 gene and a GAL1 promoter. The CEN disruption fragment is liberated from the plasmid by digestion with NotI. AMP-R indicates the beta lactamase gene from the pUK21 plasmid backbone. (B) The CEN14 insertion DNA was made by PCR amplification of centromere-proximal sequences using primers indicated by the open arrows. (C) The promoter-marker cassette from A was amplified using primers indicated by the open arrows. The PCR products from B and C contain sequence tags on the primers that are reverse and complementary so that the resulting DNAs could be fused in a second round of PCR (open circle in B fuses to open circle in C and open diamond in B fuses to open diamond in C). (D) Recombination between the marker segments and genomic integration (denoted by x's) replaces the native centromere with a conditional centromere.
F<sc>igure</sc> 2.—
Figure 2.—
Induction of chromosome-14 loss induces LOH for multiple alleles. (A) The conditional CEN strain W3617-1B was mated to strain K393-27C to produce a diploid with the following genotype: MATa/α CEN14/cen14∷K lactis-URA3 ura3-1/ura3-1 MET17/met17 met4/MET4 pet8/PET8 his2/HIS2 HIS3/his3-11,15 lys1-1/LYS1 LYS2/lys2. The resulting diploid contained heterozygous markers for PET8 and MET4 on chromosome 14 drawn approximately to scale. (B) The diploid strain was grown on galactose-containing medium and then 5-FOA medium to induce LOH. The diploid parent and subsequent LOH strain (LOH14) were streaked onto the indicated media to check for viability. Failure to grow signifies loss of URA3, PET8, and MET4.
F<sc>igure</sc> 3.—
Figure 3.—
Mapping an unknown camptothecin-sensitive mutant. An unknown gene (cptS) in the MATα library his5Δ∷KanMX strain results in camptothecin sensitivity. The cptS gene was separated from the his5Δ allele by backcross. The resulting mutant strain and a wild-type control were subsequently crossed to the 16 CEN-conditional strains, chromosome loss was induced, and the resulting LOH strains were grown, diluted, and spotted on plates with or without 0 or 5 μg/ml camptothecin. The LOH2 strain crossed to the cptS gene fails to grow in the presence of 5 μg/ml of camptothecin.
F<sc>igure</sc> 4.—
Figure 4.—
Complementation analysis of a camptothecin-sensitive gene on chromosome 2. The cptS mutant strain was crossed to three strains with deletions on chromosome 2. The resulting diploids (top three strains) and the parent haploid strains (bottom four) were spotted onto plates with 0 or 5 μg/ml camptothecin to assay drug sensitivity. Failure to complement shows that cptS is allelic to mms4.
F<sc>igure</sc> 5.—
Figure 5.—
LOH method to verify camptothecin sensitivity. Four yeast strains with gene deletions on chromosome 8 were identified in a screen for sensitivity to expression of the camptothecin mimetic top1-T722A. These strains were crossed to a CEN8-conditional strain that was transformed with plasmid-borne TOP1 expressed from the CUP1 promoter. The diploids and subsequent LOH8 strains were grown and diluted for spot assays on media with 0 or 5 μg/ml camptothecin (CPT) and 0 or 50 μm copper to induce TOP1 expression and increase camptothecin sensitivity. Only the dia4 disruption shows CPT resistance after LOH8 indicating that the sensitivity of the haploid strain is not due to dia4.
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