A strategy for constructing aneuploid yeast strains by transient nondisjunction of a target chromosome

Abstract

Background: Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In Saccharomyces cerevisiae, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication.

Results: The centromere of each chromosome was replaced by a centromere that can be blocked by growth in galactose, and ura3::HIS3, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH) confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes that are known to be deleterious when overexpressed, including the beta-tubulin gene TUB2. To test whether a tubulin stoichiometry imbalance is necessary for the apparent lethality caused by an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene TUB1, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes.

Conclusion: This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate that a tubulin imbalance is necessary for disomic VI lethality, but it may not be the only dosage-dependent effect.

Figure 1

Strategy for modifying and duplicating target chromosomes. (A) PCR amplifies P_GAL1-CEN3 URA3 from the plasmid pGALCEN-JC3-13 [13] and, upon transformation into yeast, replaces the target centromere by homologous recombination. (B) The HIS3 plasmid pKA52 is integrated into URA3 adjacent to the conditional centromere, disrupting the URA3 open reading frame and generating direct repeats (shaded). At an approximate frequency of 10^-4, HIS3 is lost by homologous recombination between the direct repeats, regenerating a functional URA3 gene (reverse arrow). The recipient strain carries the deletion alleles ura3Δ0 and his3-Δ200. (C) A haploid carrying a modified chromosome from (B) is grown in galactose for one cell division, generating N+1 and N-1 cells by nondisjunction. Since the ura3::HIS3 marker is present in two copies, cells with URA3 and HIS3 can be produced by HIS3 excision and are identified as Ura⁺His⁺ papillae on selective medium.

Figure 2

Papillation pattern in strains that contain one or two copies of the duplication marker ura3::HIS3. Ura^-His⁺ strains were grown overnight in YPD rich medium. 10⁶ and 10⁴ cells from each culture were spotted to plates containing selective media lacking uracil (-ura) or uracil and histidine (-ura, -his). Papillae were scored after 3 days. The strains shown are KAY579 (haploid), KAY626 (hemizygous diploid), and KAY625 (homozygous diploid).

Figure 3

Frequency of appearance of Ura⁺His⁺ papillae in strains carrying a modified chromosome. Haploid strains were grown to log phase in raffinose-containing medium, exposed to galactose (dark bars) or not (light bars), then plated to glucose-containing medium lacking uracil and histidine. Papillae were scored after 3 days. Bars represent the mean frequencies ± standard deviations from at least 2 independent trials. (A) Strains carrying modified chromosome III were KAY418 and KAY419; modified IV, KAY614 and KAY619; modified VI, KAY539 and KAY568. (B) Strains carrying modified chromosome VI were KAY591 and KAY628 that harboured vectors pRS425 (2-micron) or pRS315 (CEN), or TUB1 plasmids pRB327 (2-micron) or pKA55 (CEN). The frequencies in strains treated with no galactose (light bars) are not statistically different from each other, with one exception. Strains with modified VI that carry pTUB1 plasmids exhibited higher spontaneous frequencies than did VI strains without pTUB1 (Tukey-Kramer test, p < 0.05).

Figure 4

Delayed selection strategy to identify chromosome IV disomes. (A) Cells were grown approximately one doubling in the presence of galactose to induce nondisjunction, diluted and plated to YPD rich medium to allow all viable cells to form colonies. If any colonies are clones of stable disomes, they should produce Ura⁺His⁺ papillae at high frequency when replica-plated to selective medium. Spontaneous duplications that occur after colony formation on YPD should appear as isolated papillae on selective medium. (B) Photographs of replica plates after 3 days. Medium lacks uracil and histidine. Triangles indicate isolated papillae appearing on non-growing "ghost colonies." Arrows indicate single colonies on which numerous papillae appear. Strain shown is KAY614. Bar is 5 mm.

Figure 5

Karyotypes of Ura⁺His⁺ candidate disomes. Haploid strains carrying a modified chromosome were treated with galactose and plated to select Ura⁺His⁺ papillae as described in the text. To assess chromosome copy number, DNA from Ura⁺His⁺ isolates (red) was combined with DNA from a haploid parent strain (green) and hybridized to microarrays containing the genomic collection of yeast open reading frames. Log-transformed red:green ratios are displayed in histogram format for each gene along each chromosome, using the Karyoscope viewer of Java Treeview [44]. Results from representative arrays are shown (disomic III: KAY495; disomic IV: KAY638; disomic VI: KAY605; disomic VI, XII: KAY679; disomic II, VI, XII: KAY681). Complete data from all arrays are deposited at GEO [45].