ABSTRACT
Previous work in Saccharomyces cerevisiae identified three residues located in close proximity to each other on the side of the nucleosome whose integrity is required for proper association of the Spt16 component of the FACT complex across transcribed genes. In an effort to gain further insights into the parameters that control Spt16 interactions with genes in vivo, we tested the effects of additional histone mutants on Spt16 occupancy across two constitutively transcribed genes. These studies revealed that mutations in several charged residues in the vicinity of the three residues originally identified as important for Spt16-gene interactions also significantly perturb normal association of Spt16 across genes. Based on these and our previous findings, we propose that the charge landscape across the region encompassed by these residues, which we refer to as the Influences Spt16-Gene Interactions or ISGI region, is an important contributor to proper Spt16-gene interactions in vivo.
Subject(s)
Histones/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/metabolism , Alanine/genetics , Amino Acid Substitution , Chromatin Immunoprecipitation , Gene Expression Regulation, Fungal , Histones/chemistry , Histones/genetics , Mutation , Nucleosomes/chemistry , Nucleosomes/genetics , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Transcriptional Elongation Factors/chemistryABSTRACT
We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.
