Investigating the viability of yeast expressing histone from extrachromosomal DNA : progress in construction of a plasmid bearing one copy of each yeast histone gene

Much of the current information on histones has been based on in vitro studies. The original goal of this research was to construct a mutant strain of Saccharomyces cerevisiae containing all chromosomal copies of the yeast histone genes made nonfunctional. This project would demonstrate whether such a strain could be rescued by a plasmid carrying the wild-type copies of the four core histone genes. Furthermore, this yeast strain construct would allow future investigations to take advantage of histone mutant analysis in vivo. The critical step in beginning this work was the construction of a plasmid which contained the genes coding for histones H2A, H2B, H3, and H4. Construction of this plasmid proved difficult to complete due to such problems as using a nontransformable bacterial single colony, utilizing a plasmid which was subsequently found to be in fact another plasmid. This thesis describes specific methods used for transformation, partial digestion and DNA recovery techniques which were attempted and the results of these attempts. The approaches developed should simplify future construction of the plasmid and contribute to further studies investigating the viability of yeast using histone expressed from extrachromosomal DNA