Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Genome-wide screen to identify genes involved in inverted repeat and GAA/TTC-mediated fragility
Previously we have found that inverted Alu repeats and long GAA/TTC tracts trigger gross chromosomal rearrangements (GCRs) in yeast, Saccharomyces cerevisiae. Chromosomal aberrations result from double strand break (DSB) formation at the site of unstable sequences. However, mode of breakage and consequences for the genome integrity are different for these two types of repeats.
We have developed experimental system that allows to carry out systematic analysis of the complete set of deletion mutations and 800 essential genes for which expression is regulated by doxycycline or compromised due to mRNA perturbation, to get better insights into the mechanism of palindrome and/or GAA/TTC-mediated breakage. This experimental approach is based on the method developed by C.Boone?s laboratory with further modification by K.Myung?s laboratory. The query strains contain modified chromosome V which carries LYS2 cassette with 100% and 94% homologous Alu-IRs or 230 copies of GAA/TTC repeats inserted centromere-proximal to CAN1 gene. The hygromycin resistance cassette (hphMX) was placed centromere proximal to LYS2. The cassette allows for the selection of diploids as well haploids that have both the GCR construct and tester ORF marked with G418-resistant cassette (kanMX) from the yeast collection. In addition, query strains carry a reporter mfa1::MFA1pr-HIS3 and the recessive cyh2-1 mutation. Following sporulation, the reporters allow for the growth of only Mata cells that have the GCR construct on media lacking histidine and containing cyclohexamide. GCR level was tested by plating isolates on media containing canavanine. In the preliminary screen we have identified 3 groups of mutants exhibiting increased level of GCRs: i) affecting only IR-mediated fragility; ii) affecting only GAA/TTC ?mediated breakage and iii) affecting fragility induced by both sequence motifs.
School of Biology and Institute for Bioengineering and Biosciences