Measurement of Spatial Proximity and Accessibility of Chromosomal Loci in Saccharomyces cerevisiae Using Cre /loxP Site-Specific Recombination
Several methods have been developed to measure interactions between homologous chromosomes during meiosis in budding yeast. These include cytological analysis of fixed, spread nuclei using fluorescence in situ hybridization (FISH) (1, 2), visualization of GFP-labeled chromosomal loci in living cells (3), and Chromosome-Conformation Capture (3C) (4). Here we describe a quantitative genetic assay that uses exogenous site-specific recombination to monitor the level of homolog associations between two defined loci in living cells of budding yeast (5). We have used the Cre/loxP assay to genetically dissect nuclear architecture and meiotic homolog pairing in budding yeast. Data obtained from this assay report on the relative spatial proximity or accessibility of two chromosomal loci located within the same strain and can be compared to measurements from different mutated strains.
Key wordsbudding yeast meiosis homolog pairing chromosome collision assay site-specific recombination Cre/loxP
The authors would like to give a special thanks to Tamara Peoples-Holst, Eric Dean, and Joshua Chang Mell for their contribution to the optimization of this protocol over the years. This work has been supported by the National Institutes of Health (NIH) grant NIH R01 GM075119 (S.M.B), the American Cancer Society RSG-01-053-01-CCG (S.M.B), and the NIH-Environmental Health Sciences training grant NIH T 32 ES07059 (D.Y.L).
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