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Identification and characterization of a suppressor of the kinetochore mutant ctf13-30 Law, Elaine Wing Ping

Abstract

CTF13 is a component of the inner kinetochore complex CBF3. A screen was carried out to isolate spontaneous mutants that could rescue a ctf13 mutant, ctf13-30. Strains harboring this mutation are temperature sensitive at 37 degrees and have elevated chromosome loss rate. The screen identified two major classes of suppressors, one of which rescued the ctf13-30 defect by increasing mutant mRNA levels. The other compensated for the ctf13-30 mutation by genetic interaction. This extragenic suppressor ick1 was able to restore both the temperature sensitivity and the chromosome loss (as assayed by a colony sectoring assay) of ctf13-30 to a wild type profile. My hypothesis is this suppressor mutant may represent a novel kinetochore protein that is either a structural component or a regulator of events at the kinetochore. Yeast strains carrying the ick1 mutation alone did not have a temperature sensitive phenotype, and were not sensitive to the microtubule disrupting agent benomyl, or the DNA damaging agents bleomycin, hydroxyurea, and methylmethane sulfonate. Cloning of ICK1 by wild type complementation using a yeast genomic library yielded many candidates for this gene. However, when two highly represented candidates, GLC7 and SDS22, were analyzed for linkage to ICK1, both genes demonstrated random segregation. I undertook a large scale positional mapping experiment to clone ICK1 using the yeast non-essential haploid deletion mutant collection. The 4,700 deletion mutants were used as markers along the 16 budding yeast chromosomes and linkage between the markers and ICK1 was determined by assaying for growth versus a synthetic lethal phenotype. The screen produced a candidate region on Chromosome TV where ICK1 might be located, which contains two kinetochore-related genes.

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