Histone H4 Dosage Effects Figure 3. Growth of (A) wild-type (DAY286), HHF22-HHT2/hhf22-hht2D (DAY1067), hhf22-hht2D/hhf22-hht2D (DAY1069), and hhf22- hht2D/hhf22-hht2D HHF1/hhf1D (DAY1072) or (B) wild-type (DAY286), HHF1/hhf1D (DAY1066), HHF1/hhf1D HHF22-HHT2/hhf22-hht2D (DAY1068), and hhf1D/hhf1D HHF22-HHT2/hhf22-hht2D (DAY1074) histone H4 mutants in rich medium. All strains were grown overnight at 30uC in liquid YPD, streaked on YPD, and incubated at 30uC for 48 hrs. doi:10.1371/journal.pone.0010629.g003 heterogeneous colony sizes with both smooth and wrinkly morphologies (Figure 4 and 5). The similar growth defects in the HHF22-HHT2/hhf22-hht2D hhf1D/D and hhf22-hht2D/D HHF1/ hhf1D strains suggest that HHF22 is not the primary histone H4 gene. Rather, C. albicans growth is dependent on the presence of at least two copies of histone H4. We noticed that both hhf22-hht2D/D HHF1/hhf1D and HHF22- HHT2/hhf22-hht2D hhf1D/D strains gave rise to larger colonies at a low frequency (Figure 3 and 4). We considered the possibility that the larger colonies contained stable suppressor mutations. To address this, we re-isolated small and large colonies on fresh medium. When re-isolated, hhf22-hht2D/D HHF1/hhf1D small colonies gave rise to primarily small colonies with distinct morphologies (Figure 5), and a few large and always smooth colonies (Figure 4C). However, hhf22-hht2D/D HHF1/hhf1D large colonies gave rise to uniformly large and smooth colonies (Figure 4C). Re-isolation of small colonies from the HHF22- HHT2/hf22-hht2D hhf1/hhf1D strain also gave rise to small colonies with distinct morphologies, and to large colonies (Figure 4D and 5). In contrast to the hhf22-hht2D/D HHF1/hhf1D strain (Figure 4E), re-isolation of large colonies from the HHF22- HHT2/hhf22-hht2D hhf1D/D strain gave rise to primarily large colonies, but also occasionally to small colonies (Figure 4F). When re-isolated, these small colonies behaved like the parental HHF22- PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10629