Histone H4 Dosage Effects Figure 2. Amino acid sequence alignment of histone H3 and H4 genes. Comparison of the amino acid sequences of histone H3 (A) and histone H4 (B) alleles from C. albicans and S. cerevisiae using ClustalW. doi:10.1371/journal.pone.0010629.g002 (Figure 3A). This suggests that Hhf22 and Hht2 are not essential for growth. S. cerevisiae diploids containing a single H3-H4 locus have a longer generation time and a more prolonged G1 phase than wild- type cells [70]. Further, deletion of HHT2-HHF2 in S. cerevisiae, but not HHT1-HHF1, causes greater minichromosome loss compared to wild-type [70], indicating that, although both H3-H4 loci are generally functionally redundant, there are differences between both loci. To determine if C. albicans has a different requirement for HHF1 or HHF22 for growth, we constructed a strain in which both copies of HHF1 were deleted. We were readily able to generate HHF1/hhf1D and hhf1D/D strains, and these strains did not show growth or morphological defects (Figure 3B and data not shown), indicating that Hhf1 is also not essential for growth in C. albicans. Therefore, unlike in S. cerevisiae, in C. albicans the presence of either one of the histone H4 gene is sufficient to ensure normal growth and colony morphology. When we attempted to mutate one copy of HHF1 in the hhf22- hht2D/D background, we only recovered a single homologous recombinant (1 homologous recombinant/395 transformants screened). Similarly, when we attempted to mutate the remaining copy of HHF22-HHT2 from a HHF22-HHT2/hhf22-hht2D HHF1/hhf1D double heterozygote, we only recovered homologous recombinants 7% of the time (32 homologous recombinants/437 transformants screened). However, all of these recombinants retained a wild-type HHF22-HHT2 copy and arose by a marker exchange (29/32) or a by a increased HHF22-HHT2 copy number (3/32). To increase the rate of homologous recombination, we generated an hhf1::URA3 disruption cassette containing regions of homology ,9x larger than the original cassettes (Table 2). Using this extended disruption cassette we increased homologous recombination in the hhf22-hht2D/D strain to 50% (11/22). All hhf22-hht2D/D HHF1/hhf1D transformants had a pronounced growth defect (Figure 3A) and gave rise to both smooth and wrinkly colonies of heterogeneous size (Figure 4A and B). The difficulty in eliminating a third histone H4 gene and the severe growth defects observed in the mutant with only one HHF1 copy suggested that C. albicans might require at least two copies of histone H4 for normal growth. Alternatively, HHF22 might be the primary histone H4 gene in C. albicans. If HHF22 is indeed the primary histone H4 gene in C. albicans, we predicted that we could construct an HHF22/hhf22D hhf1D/D mutant without impacting growth. To construct this mutant, we eliminated the last HHF1 copy from a HHF22-HHT2/hhf22-hht2D HHF1/hhf1D double heterozygous strain. As before, the use of longer regions of homology increased the rate of homologous recombinants (44/45). However, in most cases (40/44) the wild- type HHF1 copy was retained. While the HHF22-HHT2/hhf22- hht2D HHF1/hhf1D strain had no overt growth defects compared to wild-type colonies, the four HHF22-HHT2/hhf22-hht2D hhf1 D/D mutants obtained had severe growth defects (Figure 3B). HHF22-HHT2/hhf22-hht2D hhf1D/D strains also gave rise to PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10629