Alterations in histone availability have profound effects on the cell. Unbalanced histone dimer stoichiometry causes defects in the segregation of mitotic chromosomes, increases recombination and genetic instability, and leads to sporulation defects in Saccharomyces cerevisiae [28–37]. Furthermore, incomplete nucleosomal occupan- cy due to histone dosage defects directly impacts transcriptional regulation [38–44]. Therefore, alterations in histone stoichiometry have pleiotropic effects in cells. In this study we performed serial deletions of C. albicans histone H4 genes and determined the effect of a deficit in histone H4 on growth. We found that reduced histone H4 dosage caused a severe growth defect and the formation of colony morphology variants. C. albicans primarily counterbalanced the low dosage of histone H4 by increasing histone H4 gene copy number through the formation of aneuploidies. Suppression of the growth defect associated with low histone H4 dosage also restored colony morphology to the wild- type morphology. This is the first study on core histones in C. albicans, which provides background genetic information for future experiments that address the role of chromatin structure and function in C. albicans biology and pathogenesis. Materials and Methods Strains and plasmids All strains used in this study are listed in Table 1. Strain DAY1069 was generated as follows. BWP17 was transformed with a hht2-hhf22::URA3-dpl200 disruption cassette amplified in a PCR using primers HHT2-HHF22 5DR and HHT2-HHF22 3DR new (Table 2) to give strain DAY1067. DAY1067 was then Table 1. Strains used in this study. Parent/Background Genotype Histone H4 Dosage Effects transformed with a hht2-hhf22::ARG4 disruption cassette amplified as above. The hht2-hhf22 disruption cassettes delete the region from nucleotide +329 of HHT2 to the stop codon of HHF22 (Figure 1). The URA3-dpl200 marker from strain DAY1069 was recycled by plating the cells in synthetic medium supplemented with 5-fluoroorotic acid (5-FOA) to obtain strain DAY1071. Strain DAY1072 was generated by replacing nucleotide +114 to the stop codon of one HHF1 alleles in DAY1071 using the hhf1::URA3 disruption cassette, which was amplified in a PCR using primers HHF1 5DR 100 in and HHF1 3DR new (Figure 1 and Table 2). DAY1068 was generated by transforming strain BWP17 with the hhf1::URA3-dpl200 disruption cassette (Figure 1) to generate strain DAY1066, followed by transformation with the hht2- hhf22::ARG4 disruption cassette (Table 2). Strain DAY1070 was obtained by plating DAY1068 in synthetic medium supplemented with 5-FOA to recycle the URA3-dpl200 marker. Strains DAY1074, DAY1076 and DAY1078 were generated by partially deleting the last HHF1 copy from DAY1070 using an hhf1::URA3 disruption cassette amplified from plasmid DDB383 with primers HHF1 59 fragm DDB78 and HHF1 39 fragm DDB78. Plasmid DDB383 contains the disruption cassette bordered by additional HHF1 flanking regions in order to increase the efficiency of integration into the HHF1 locus. Strains DAY1075 and DAY1079 are large colony revertants of strains DAY1074 and 1078, respectively. The genotypes of the strains and the correct integration of the disruption cassettes were verified by the PCR using primers HHF1 5 detect, HHF1 3 detect, HHT2-HHF22 5 detect and HHT2-HHF22 39detect-new that flank the integration site (Table 2), and by Southern blot. Strain DAY1 DAY286 DAY963 DAY1066 DAY1067 DAY1068 DAY1069 DAY1070 DAY1071 DAY1072 DAY1074 DAY1075 DAY1076 DAY1078 DAY1079 BWP17 DAY1 SC5314 DAY1 DAY1 DAY1066 DAY1067 DAY1068 DAY1069 DAY1071 DAY1070 DAY1074 DAY1070 DAY1070 DAY1078 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG ura3::limm434/ura3::limm434 his1::hisG/his1::hisG ARG4::URA3::arg4::hisG/arg4::hisG Prototrophic clinical isolate ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG HHF1/hhf1::URA3-dpl200 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG HHT2-HHF22/hht2-hhf22::URA3-dpl200 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG HHF1/hhf1::URA3-dpl200 HHT2-HHF22/hht2-hhf22::ARG4 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG hht2-hhf22::ARG4/hht2- hhf22::URA3-dpl200 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG HHF1/hhf1::dpl200 HHT2-HHF22/hht2-hhf22::ARG4 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG hht2-hhf22::ARG4/hht2- hhf22::dpl200 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG HHF1/hhf1::URA3 hht2-hhf22::ARG4/hht2-hhf22::dpl200 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG hhf1::URA3/hhf1::dpl200 HHT2-HHF22/hht2-hhf22::ARG4 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG hhf1::URA3/hhf1::dpl200 HHT2-HHF22/hht2-hhf22::ARG4 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG hhf1::URA3/hhf1::dpl200 HHT2-HHF22/hht2-hhf22::ARG4 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG hhf1::URA3/hhf1::dpl200 HHT2-HHF22/hht2-hhf22::ARG4 ura3::limm434/ura3::limm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG hhf1::URA3/hhf1::dpl200 HHT2-HHF22/hht2-hhf22::ARG4 DAY414 (L40) S. cerevisiae MATa his3D200 trp1-901 leu2-3, 112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ GAL4 doi:10.1371/journal.pone.0010629.t001 PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10629 Reference [97] [98] [99] This study This study This study This study This study This study This study This study This study This study This study This study [100]