Sequence (59to 39) 59-taaacgtcacagaaagattttaagagataacattcaaggtattacaaaaccagctatcagtttcccagtcacgacgtt 59-ttaatactatacaataaagaaaacgaactaaaaagacaattagaaatacaacccagtttagtggaattgtgagcggata 59-cttctagctaattgcatatctttcttttgaatggtaactctcttagcatggatagcacactttcccagtcacgacgtt Table 2. Primers used in this study. Name HHF1 5 DR 100 in HHF1 3 DR new HHT2-HHF22 5 DR HHF1 5 detect HHF1 3 detect HHT2-HHF22 5 detect HHF1 59 fragm DDB78 HHF1 59fragm 39 HHF1 39fragm 59 HHF1 39 fragm DDB78 HHF1 59fragm 5 HHF1 39 for DDB78 HHF22 5 SB HHF22 3 detect 59-tcttagtgtaaggaacctcc 59-acgattataaaggagaaggtg 59-aaatgtccaataccagcacc 59-ctgatagctggttttgtaatacc 59-actgggttgtatttctaattgtc 59-accttgtatggtttcggtgg 59-gttattcggttagaaagcgg HHT2-HHF22 3 DR new 59-taatctaaaaatacagttatcatgaatcgaaaaacataaagaaaagaagatatttctttagtggaattgtgagcggata HHT2-HHF22 39detect-new 59-ccgaaaataatttgcttgccttgcc 59-acgacggccagtgaattgtaatacgactcactatagggcggctcactcttagtgtaaggaacctcc HHF1 59 100 in for DDB78 59-taaacgtcacagaaagattttaagagataacattcaaggtattacaaaaccagctatcaggggcgaattggggagctccc 59-aagctcggaattaaccctcactaaagggaacaaaagctggtctcagtgagctgttacgaggc 59-ctcactcttagtgtaaggaacctcc 59 -ttaatactatacaataaagaaaacgaactaaaaagacaattagaaatacaacccagtttacgataagcttcatctagaagg Histone H4 Dosage Effects Reference This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study doi:10.1371/journal.pone.0010629.t002 Plasmid DDB383 was generated by in vivo recombination as follows. An hhf1::URA3 disruption cassette was amplified in a PCR from DDB245 [45] using primers HHF1 59 100 in for DDB78 and HHF1 39 for DDB78 (Table 2). The two flanking HHF1 regions of 570 bp and 523 bp with homology upstream (including the first 113 nucleotides of HHF1) and downstream of HHF1, respectively, were amplified in two high fidelity PCRs (Pfu Turbo DNA polymerase, Stratagene) from BWP17 genomic DNA using the primer pairs HHF1 59 fragm DDB78 and HHF1 59 fragm 39, and HHF1 39 fragm DDB78 and HHF1 39 fragm 59, respectively. The three PCR products were co-transformed with a NotI/EcoRI double digestion of DDB78 into the Trp- Saccharomyces cerevisiae L40 strain to generate DDB383. Media and growth conditions C. albicans was routinely grown at 30uC in YPD supple- mented with uridine (2% bacto-peptone, 1% yeast extract, 2% dextrose, and 80 mg ml21 of uridine). Mutants were selected on synthetic medium (0.17% yeast nitrogen base without ammo- nium sulfate (Q-BioGene), 0.5% ammonium sulfate, 2% dextrose, and supplemented with a dropout mix containing amino and nucleic acids except those necessary for the Figure 1. Genomic organization of the histone H3 and H4 loci in Candida albicans. The black boxes indicate the extent of the deleted regions in the mutants, which are replaced by the auxotrophic markers ARG4, URA3, URA3-dpl200 or the dpl200 loop-out. HHF1 is deleted from nucleotide +114 to the STOP codon (65% of the gene) and the HHF22-HHT2 cluster is deleted from the STOP codon of HHF22 to nucleotide +329 of HHT2 (80% of the HHT2 gene). orf19.1060 is a possible spurious ORF. In red, the regions recognized by the probes D and G used for determining HHF1 or HHF22 copy dosage by Southern Blot are shown. H: HindIII; N: NcoI. doi:10.1371/journal.pone.0010629.g001 PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10629