The role of post translational modifications in the regulation of binding of linker histone Hho1 to chromatin in Saccharomyces cerevisiae
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Date
2015-02
Authors
Sharma, Pankaj
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
The eukaryotic genome is functionally organized into a highly ordered nucleoprotein
structure, chromatin. Apart from the four nucleosomal core histones, the linker histone is
pivotal to fluidity and compaction of chromatin. Yeast, a single cell eukaryote, has a short
nucleosomal repeat length, and possesses a structurally unique H1. This thesis is an attempt
to study the influence of post translational modifications in regulating the interactions of
yeast linker histone with chromatin. Chapter 1 gives a broad overview of available literature.
Chapter 2 presents the results from cloning, expression, purification and partial
characterization of Hho1p. The physiochemical characteristics of recombinant protein were
studied with a view to perform subsequent purification of native Hho1p from yeast strains.
The solubility and precipitation of rHho1p were explored under several conditions. Unlike
canonical linker histone, Hho1p was found to be insoluble in Perchloric acid. Chapter 3
comprises generation and characterization of polyclonal antibody against Hho1p. Affinity
purified monospecific antibodies were used to optimize semi-quantitative Western blotting.
Preliminary results from Western blotting analysis suggest that Hho1p is capable of
oligomeric interactions. Chapter 4 presents results for optimization of conditions for Hho1p
extraction from yeast cells or nuclei. For nuclear extract preparation, a standard method
and its rapid variation, developed to limit Hho1p proteolysis, were used. The acid extraction
of Hho1p from nuclei followed by its purification by reverse phase chromatography
provided low yield for downstream analysis. However, sufficient quantities of native Hho1p
could be extracted from cell lysate using affinity matrices prepared with the monospecific
αHho1p antibodies generated in-house. Several novel post translational modification sites
and binding partners of Hho1p were identified from both logarithmic and stationary phase
of yeast cell growth, providing an insight into the stage specific regulation of Hho1p
chromatin binding.
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Keywords
Thesis (Ph.D. (Biochemistry))--University of the Free State, 2015, Chromatin, Yeast