Analysis of Gross-Chromosomal Rearrangements in Saccharomyces cerevisiae

Ludwig Institute for Cancer Research, University of North Texas, Health Science Center, Fort Worth, USA.
Methods in Enzymology (Impact Factor: 2.09). 02/2006; 409:462-76. DOI: 10.1016/S0076-6879(05)09027-0
Source: PubMed


Cells utilize numerous DNA metabolic pathways and cell-cycle checkpoints to maintain the integrity of their genome. Failure of these mechanisms can lead to genome instability, abnormal cell proliferation, and cell death. This chapter describes a method for the measurement of the rate of accumulating gross-chromosomal rearrangements (GCRs) in haploid cells of the yeast Saccharomyces cerevisiae. The isolation of cells with GCRs relies on the simultaneous loss of two counterselectable markers, CAN1 and URA3, within a nonessential region on the left arm of chromosome V. Healing of DNA breaks by de novo telomere addition, translocations, large interstitial deletions, and chromosome fusion has been detected using a PCR-based procedure for the mapping and amplification of breakpoint junctions, which is also described in detail here. This GCR analysis provides an effective tool for the assessment of the contribution by multiple cellular mechanisms to the maintenance of genome integrity.

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    • "To better understand the types of GCRs in the rfa1-t33 and sae2D derivatives, we utilized a previously described method to identify the location of the breakpoint by overlap PCR using Ch V primers and then attempted to sequence across the junction using an arbitrary PCR strategy (Figure S3) (Schmidt et al., 2006). We characterized 12 GCR isolates from WT cells and found that repair occurred primarily by telomere addition (Table 1). "
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    • "Gross-chromosomal rearrangement (GCR) assay GCR rates were determined by fluctuation analysis by taking the median rate of at least 15 cultures from at least two isolates [52] [53] and are shown with 95% confidence intervals [54]. Cells with GCRs were identified by their resistance to canavanine and 5-fluoro-orotic acid (Can r 5-FOA r ), which is indicative of simultaneous inactivation of CAN1 and URA3 on chromosome V. Selective media for the GCR assay was prepared as previously described [55] "
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    Full-text · Article · Oct 2014 · DNA Repair
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    • "To test nuclear genome instability in a large number of samples, we designed a quantitative and highly sensitive assay called CINA (Figure 1). It is based on the gross chromosomal rearrangement (GCR) assay (Schmidt et al. 2006) but is 10,000-times more sensitive. To improve the sensitivity, we designed the assay based on diploid cells (Figure 1A) with the following features: as in the original GCR assay, chromosome instability is measured by positive selection of two marker losses (URA3 and CAN1, linked in the distal part of chromosome V) (Chen and Kolodner 1999); it uses a diploid, allowing either breakage or full chromosome loss to occur without loss of viability, in contrast to the original GCR assay in which haploid cells can only break within a limited area around the URA3-CAN1 markers and survive; the addition of a LEU2 marker close to CEN V allows the ability to distinguish between chromosome breakage and chromosome loss (Figure 1B) and CINA is performed starting from single cells grown to colonies on the plate of choice (clonal assay). "
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