Date of Graduation

5-2012

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Ines Pinto

Committee Member

Suresh K. Thallapuranam

Second Committee Member

T.K.S. Kumar

Third Committee Member

Dan Lessner

Abstract

Histones are small basic proteins that associate with DNA to form the basic unit of chromatin, the nucleosome. Histones H3 and H4 form a tetramer that is bound by two H2A-H2B dimers to form the histone octamer, to which approximately 146 bp of DNA wrap around to form the nucleosome. High resolution structural information and recent advances in the understanding of histone post-translational modifications have illuminated the many regulatory functions chromatin exerts in the cell, from the transcriptional control of gene expression to chromosome segregation. However, the specific role that histones play in these processes is not well understood. Previous work from this laboratory identified histone H2A mutants that cause chromosome segregation defects associated with altered centromeric chromatin. As a continuation of that work, this project focuses on the identification of histone H2B mutants that cause severe defects in ploidy maintenance and mitotic chromosome transmission. A library of plasmids containing mutagenized copies of the H2B encoding gene, htb1, was built in E.coli by PCR Mutagenesis. The library was introduced into an S.cerevisiae strain that carries deletions of the genomic loci that encode H2B. The yeast transformants were screened for phenotypes that would likely indicate chromosome segregation defects. Fourteen novel H2B mutants were identified that displayed clear defects in chromosome segregation. The location of the amino acid replacements on the nucleosome model suggests domain(s) within the histone octamer are important for chromosome segregation.

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