Gene Copy-Number Variation in Haploid and Diploid Strains of the Yeast Saccharomyces cerevisiae

Abstract

The increasing ability to sequence and compare multiple individual genomes within a species has highlighted the fact that copy-number variation (CNV) is a substantial and underappreciated source of genetic diversity. Chromosome-scale mutations occur at rates orders of magnitude higher than base substitutions, yet our understanding of the mechanisms leading to CNVs has been lagging. We examined CNV in a region of chromosome 5 (chr5) in haploid and diploid strains of Saccharomyces cerevisiae. We optimized a CNV detection assay based on a reporter cassette containing the SFA1 and CUP1 genes that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. This optimized reporter allowed the selection of low-order gene amplification events, going from one copy to two copies in haploids and from two to three copies in diploids. In haploid strains, most events involved tandem segmental duplications mediated by nonallelic homologous recombination between flanking direct repeats, primarily Ty1 elements. In diploids, most events involved the formation of a recurrent nonreciprocal translocation between a chr5 Ty1 element and another Ty1 repeat on chr13. In addition to amplification events, a subset of clones displaying elevated resistance to formaldehyde had point mutations within the SFA1 coding sequence. These mutations were all dominant and are proposed to result in hyperactive forms of the formaldehyde dehydrogenase enzyme.A S a consequence of studies that utilize genomic microarrays and next-generation DNA sequencing, it has become clear that much of the natural genetic variation that exists between individuals is due to alterations in the number of copies of genes rather than differences in the nucleotide sequence (Girirajan et al. 2011;Veltman and Brunner 2012). It has been calculated that 8-25 kb of DNA are deleted or duplicated per generation in humans compared to about 100 bp of point mutations (Itsara et al. 2010). Although deletions and duplications vary in size from a few base pairs (for example, changes in the lengths of microsatellites) to many megabases (for example, changes in ploidy), Girirajan et al. (2011) define copy-number variants (CNVs) in humans as changes that vary in size between 50 bp and 1 Mb. While most CNV events have no obvious effect, a significant number are associated with human diseases including Charcot-Marie-Tooth syndrome, autosomal dominant leukodystrophy, Williams syndrome, several cancer predispositions, and autism-related and other neurodevelopmental disorders (Abrahams and Geschwind 2008;Girirajan et al. 2011;Krepischi et al. 2012;Malhotra and Sebat 2012;Sullivan et al. 2012). There is also strong evidence suggesting that CNVs played a significant role in human evolution (Iskow et al. 2012). Finally, multiple somatic CNV events are frequently observed in the altered karyotypes of cancer cells (Stratton et al. 2009).CNVs have also been widely observed in natural populations and have been studied in detail in model organisms. In the discussion below, w...

Too Much of a Good Thing: The Unique and Repeated Paths Toward Copper Adaptation

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Stimulation of Chromosomal Rearrangements by Ribonucleotides

“…The first assay was designed to characterize the LOH effect observed in the mutation accumulation experiment described above. We used isogenic diploids ( Figure 1; Table S1) that were homozygous at all positions on Chr7, except that they contained a single hemizygous copy of the CORE2 counter-selectable cassette with two diverged orthologous copies of the URA3 gene and a marker for geneticin resistance (Zhang et al 2013) inserted near the right end of Chr7. An allelic interhomolog recombination event occurring anywhere within the 575-kb region between CEN7 and the CORE2 insertion can result in LOH that may cause a diploid to become homozygous for the distal regions of the right arm of Chr7.…”

“…There are 60,000 single nucleotide polymorphisms (SNPs) between two strains' genomes, some of which are associated with restriction fragment length polymorphisms (RFLPs) that can be used to monitor recombination between homologous chromosomes. For the isogenic and hybrid LOH assays, the CORE2 cassette containing the Kluyveromyces lactis URA3 gene, the S. cerevisiae URA3 gene, and the kanMX geneticin resistance gene was amplified from plasmid pJA40 (Zhang et al 2013) and integrated at chromosome 7 (Chr7) downstream of the MAL13 gene (distal side), 20 kb from the right telomere. For the NAHR assay, we used a PCR-based approach to delete a 180-bp segment spanning the 39 end of the open reading frame (ORF) and the immediate downstream sequence of the URA3 gene at its native position on Chr5.…”

“…Independent Ura + clones were picked from uracil drop-out plates, purified to single colonies in YPD plates, and then patched on to diagnostic plates containing uracil drop-out, 5-FOA, YPD geneticin, and YPD media. Full-length chromosomal DNA embedded in agarose was prepared and fractionated by pulse field gel electrophoresis (PFGE), and in some cases the DNA was used for comparative genome hybridization microarrays (array-CGH) using the methods and microarray design described previously (Zhang et al 2013). …”

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“…Genomic DNA preparation, labeling, hybridization, and data analysis procedures were as described earlier (Zhang et al 2013). …”