Abstract
BackgroundMost 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.ResultsThe 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.ConclusionThis 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.
Highlights
Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity
The methods described above are clearly effective at isolating disomic strains of yeast, each of them requires a spontaneous failure of chromosome segregation during cell division
The polymerase chain reaction (PCR) fragment is transformed into yeast and integrated into the target site by homologous recombination, replacing the endogenous centromere [15] (Figure 1A)
Summary
Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. Specific disomes (haploids carrying an extra chromosome, karyotype N+1) have been isolated by differentially marking two homologs in a diploid, selecting for meiotic segregants that contain both homologs (for example, [7]). By differentially marking homologs in the parents and selecting for progeny cells that retain both homologs, haploid progeny carrying a disomic chromosome have been isolated [9,10] This method, termed chromoduction, was used to select for 14 of the possible 16 disomes of yeast in a recent systematic study of aneuploidy [3]. The methods described above are clearly effective at isolating disomic strains of yeast, each of them requires a spontaneous failure of chromosome segregation during cell division. The mechanisms that underlie these failures (the breakdown of nuclear integrity in a cell containing multiple nuclei or the bypass of the spindle assembly checkpoint to allow nondisjunction [11,12]) are not well understood, and may lead to additional, unplanned genetic changes
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