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New Methods for the isolation of disomic strains of Saccharomyces cerevisiae and quantitation of chromosome segregation fidelity

by
David C. Zebrowski
Microbiology and Molecular Genetics

B.S. 1996, Carnegie Mellon University


Thesis Advisor: David Kaback, Ph.D.

Professor

Department: Microbiology and Molecular Genetics

Tuesday, August 18, 2009
10:30 pm. ICPH TB Conference Room


Abstract

During meiosis a round of DNA replication is followed by two nuclear divisions, meiosis I (MI) and meiosis II (MII) where homologues and sister chromatids segregate, respectively, resulting in daughter nuclei that have one half the number of chromosomes as the original parent nucleus. Errors in chromosome segregation, or nondisjunction, can occur during either meiotic division resulting in daughter nuclei with abnormal chromosome content. To investigate chromosome segregation during meiosis, a new method was developed for detecting and selecting cells of Saccharymyces cerevisiae that have undergone meiotic nondisjunction. The method can also be used to isolate strains that are disomic for almost any chromosome. A diploid heterozygous for a KanMX knock-out mutation in virtually any essential gene is sporulated and viable G418-resistant colonies selected. Disomic products of a missegregation or non-disjunction event containing a copy of both the wild-type essential gene and its complementary KanMX knock-out allele were found to make up most of the colonies that were selected. As disome production using this method involves chromosome non-disjunction or missegregation, the method has been aptly termed missegregation associated restoration of viability (MARV). 100% of colonies isolated for smaller chromosomes (I and IX) were singly disomic. In contrast, single disomes for the two larger chromosomes tested (X and XIV) were not found 100% of the time and required screening of multiple colonies. Based on high reliability of chromosome I disome isolation, meiotic and mitotic segregation fidelity for a number of mutations and conditions were quantitated. Using MARV I found that chromosome I segregation errors appeared to occur much more frequently during MI than during MII. In addition, deletion of NDJ1 increased the disome isolation frequency, while deletion of subtelomeric DNA did not appear to produce a measurable effect in chromosome segregation fidelity. Furthermore, I found treatment of cells with the mutagen methyl methanesulfonate or the actin antagonist Latrunculin B resulted in an increase in the number of disomes selected suggesting that MARV is suitable for screening potential aneugens. Thus, MARV should be applicable to a wide variety of studies where segregation fidelity measured or disomic strains for almost any particular chromosome are required.


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