H4 that overcome a sir3 silencing defect and likely represent amino acid residues involved in Sir-histone interactions. These mutations have been successfully integrated into the yeast genome and display viability in the absence of the wild type copy of the gene. We will determine whether the mutations affect growth and mitotic division and then test the ability of the mutants to suppress sir3 silencing defects. Finally we will assay for genetic interaction with other histone mutants.
ROY3508 (where one copy of the H3:H4 gene is replaced with URA3) was transformed with DNA fragments containing various mutant H3:H4 gene fragments. Transformants were selected by growth of cells on 5-FOA plates and confirmed using genomic PCR and sequencing. Stable integrants have been generated and these integrations have proven stable. Next we will determine whether the mutants have growth defects and whether they can restore silencing in strains lacking the sir3 protein. The ultimate goal of these experiments is to understand how the Sir proteins interact with the histones to silence the transcription of genes.