Histone H4 Dosage Effects Figure 6. Quantitative Southern blot of histone H4 alleles. DNA samples were obtained from 30uC overnight cultures of small and large colonies isolated from (A) hhf22-hht2D/D HHF1/hhf1D (DAY1072) and (B and C) HHF22-HHT2/hhf22-hht2D hhf1D/D (DAY1074, DAY1075, DAY1076, DAY1078 and DAY1079). Due to the formation of suppressors, the proportion of small to large colonies was verified for each overnight culture by colony count on YPD to ensure that the DNA extraction was representative of a small or a large colony population. Overnight cultures from small colonies with a minimum of 80% of small colonies were used to prepare DNA for the Southern blot. Representative colonies from these plates corresponding to different ratios of mutated to wild-type histone H4 ratio are shown (D). Probes D and G were used to detect the alleles: hhf1::URA3 and HHF1, and hhf22::ARG4 and HHF22, respectively. HHF22-HHT2/hhf22-hht2D HHF1/hhf1D (DAY1070) was used in (B) as a control for a 1:1 mutated to wild-type HHF22 ratio. doi:10.1371/journal.pone.0010629.g006 gene expression has been observed both in S. cerevisiae, where the expression of one of the H2A-H2B loci (HTA1-HTB1) is regulated by the availability of histones H2A-H2B in the cell [73], as well as in S. pombe [81]. Thus, while we observed an increase in gene copy number in the large colonies screened, we cannot rule out the possibility that C. albicans can increase histone H4 availability by transcriptional mechanisms. We observed that while the large colonies of the hhf22-hht2D/D HHF1/hhf1D strain were phenotypically stable, the large colonies isolated from the HHF22-HHT2/hhf22-hht2D hhf1D/D strain often gave rise to small colonies. This observation suggests that a Chromosome R trisomy (HHF22) is less stable than a Chromo- some 1 trisomy (HHF1). Chromosome R has a variable electrophoretic mobility. These variations are found in natural isolates, in spontaneous morphological mutants, and in randomly selected colonies from a clonal population of a reference strain [82–84]. However, chromosome R variations are attributed to changes in the number of rDNA repeats, and not to ploidy changes [84,85]. A random gain and subsequent loss of an extra copy of Chromosome R might explain the slightly unstable phenotype of the large colonies with only HHF22. Acquiring a third Chromosome R copy would provide a selective growth advantage because it increases histone H4 copy number. However, excess of rDNA synthesis, or other chromosome R associated loci, could be deleterious for cells [84,86,87]. Thus, some suppressor cells might randomly lose the extra Chromosome R copy and revert to a slow growth small colony phenotype. Trisomy in Chromosome 1 might not have as negative an effect on growth, explaining the more stable colony morphology of the suppressors from strains with only a single allele of HHF1. In fact, some stocks of the routinely used laboratory strain CAI-4 are stably trisomic for Chromosome 1 under non-stress conditions PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10629