California Sea Grant College Program

Histone H4 is one of the most conserved proteins known. The low rate of non­ synonymous substitutions over long evolutionary periods suggests that histone H4 is under severe selective constraints. Previous mutational analyses of histone H4 suggest that the central core region of the protein is less permissive to modification than the amino or carboxy termini. The focus of this dissertation was to introduce novel mutations to the core of histone H4 and develop an in vivo assay for viability for each of these modifications in the yeast Saccharomyces cerevisiae.

A background on the evolutionary history of histone H4 is included in Chapter 1. In order to test a large number of mutations simultaneously in histone H4, the wild-type yeast histone H4 was replaced by the highly divergent histone H4 from Tetrahymena thermophila. This replacement forces yeast to utilize a histone with 21 amino acid replacements out of 102 amino acids relative to the wild-type protein. Growth characteristics of these yeast were assayed over a range of temperatures suggesting that histone H4 still functions in yeast even after a large number of alterations were scattered throughout the protein.

To test the relative growth rates of yeast utilizing modified histone H4 proteins, a temperature gradient incubator was developed. In combination with computer imaging technology, this incubator can be used to assay 24 yeast strains simultaneously across a user-specified temperature gradient.

By using oligonucleotide cassette mutagenesis, a series of modifications were introduced to a universally conserved core region in histone H4. Using the first cassette, a sequence space of 256 possible histone H4 proteins was assayed in both the yeast histone H4 and the Tetrahymena histone H4 contexts. The analysis suggests that different sets of modifications will work in each of these contexts. Using the second cassette, a sequence space of 13,824 possible histone H4 proteins was assayed in the yeast histone H4 background. 9.2% of these sequences were viable in yeast. This suggests that there is a much larger space of potentially viable histone H4 sequences than has been realized in light of the extreme historical conservation of this protein.