Histone H4 Dosage Effects Figure 5. Histone deficit causes alterations in colony morphology. Examples of morphologically altered colonies that arise in mutants with only one HHF1 (DAY1072) or one HHF22 (DAY1079) allele after overnight incubation at 30uC on YPD (A). Partial penetrance of the morphological change in the small colonies (B). Stable morphology of the large smooth suppressor colonies after subculturing (C). doi:10.1371/journal.pone.0010629.g005 defect that can be overcome through whole chromosome aneuploidy to increase histone H4 copy number. While we cannot rule out segmental aneuploidy as a mechanism to restore histone H4 dosage, whole chromosome aneuploidy is clearly one mechanism that can restore histone H4 dosage. We noted two additional aneuploidies in our CGH analysis. First, a segmental monosomy of one end of chromosome 5 in both the small and large colony (Figure 7). This is an attribute of the RM1000 background from which these strains are descended, which is known to have a stable deletion in one arm of chromosome 5 [12,48]. Second, a whole chromosome monosomy in chromosome 3 was observed in the hhf22-hht2D/D HHF1/hhf1D small colony, but not the large suppressor colony derivative. Defects on histone dosage has been implicated in the generation of aneuploidies and chromosome instability, and this may reflect evidence of that phenomenon in C. albicans. However, it is also possible that reduced chromosome 3 dosage provides some advantage to cells containing one histone H4 locus. It is noteworthy that not all large colonies gave the expected 1:,2 ratio of mutant to wild-type histone H4 by quantitative Southern blot (Figure 6C). We found a 0:1 ratio (arbitrarily set to 1 as there was no mutant allele to normalize to), which lost the mutant histone H4 allele, a 1:1 ratio, which maintained the ratio of the starting strain, and a ,2:1 ratio, which duplicated the mutant histone H4 allele. The 0:1 ratio found in large colonies indicates that the mutated version of the histone H4 was replaced with the wild-type histone H4 by mitotic recombination or by loss of the chromosome carrying the mutant allele followed by duplication of the remaining chromosome containing the wild type allele. The 1:1 ratio found in large colonies indicates that either the strain became tetrasomic, duplicating both loci, or that there exist alternative suppressor mechanisms. The ,2:1 ratio found in large colonies indicates that the mutated version of the histone H4 was duplicated, perhaps causing a trisomy of part or all of chromosome R. This type of aneuploidy may restore growth, however we cannot rule out the possibility that chromosome R is found in a 4:2 mutant to wild-type ratio in these cells. In fact this latter possibility seems to be supported by the increased hhf22D and HHF22 signals observed in this sample (Figure 6C). We noted that the large colonies carrying a 0:1, 1:1, and 1:2 ratio of mutant to wild-type histone H4 had the typical smooth morphology, but the colonies carrying a 2:1 ratio showed a wrinkly top and were more heterogeneous in size (Figure 6D). These differences in colony morphology are in agreement with the formation of alternative karyotypes (see below). The 0:1, 1:1, and 2:1 ratio for large colonies arose from the HHF22-HHT2/hhf22-hht2D hhf1D/D mutant but not from the hhf22-hht2D/D HHF1/hhf1D mutant, which suggests that Chromosome R is less stable than Chromosome 1. Since maintenance of genomic integrity is critical for survival, cells have different mechanisms to compensate for histone dosage defects, including genomic rearrangements and, more commonly, transcriptional alterations. Genomic rearrangements have been observed in S. cerevisiae, which can increase histone H2A-H2B copy number by forming a small circular chromosome [80]. Dosage compensation through transcriptional up-regulation of histone PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10629