selection [46]. Solid media were prepared by addition of 2% Bacto-agar. Southern blot and comparative genomic hybridizations For the HHF1 and HHF22 dosage experiments, genomic DNA was digested with HindIII or NcoI, respectively (Figure 1), separated in a 1.2% agarose gel by electrophoresis, and transferred by capillary action to a nylon membrane. Probes D (for HHF1) and G (for HHF22) were PCR amplified from C. albicans BWP17 genomic DNA using primer pairs HHF1 59 fragm 59 and HHF1 59 fragm 39, and HHF22 59 SB and HHF22 39 detect, respectively (Figure 1 and Table 2). The probes were radiolabelled with [a-32P]-dCTP using the Prime-a-Gene labeling system (Promega). Blots were developed with a phosphoimager (STORM system). Densitometry analysis of the images was performed using ImageJ 1.30v (Wayne Rasband, NIH, USA). For comparative genomic hybridizations (CGH), genomic DNA was prepared from C. albicans strains grown overnight to saturation in 5 ml YPAD medium using phenol/chloroform as described [47]. 3 mg of DNA was digested with HaeIII (Invitrogen), labeled with Cy3 (experimental strains) or Cy5 (reference strain, SC5314), and hybridized to microarrays as described previously [48]. The microarrays were printed in-house and contain 14,688 total spots, representing 6175 ORFs, designed using Assembly six C. albicans ORFs [49] and updated with Assembly 19 ORFs. Arrays were scanned (ScanArray 5000) using QuantArray v.2.01 software (GSI Lumonics, Watertown, MA). Data were analyzed using GenePixPro 5.1 and GeneTraffic 3.1. The average mean log2 ratio average of two duplicate spots per microarray slide was calculated and plotted as a function of chromosome position using Chromosome_Map [48]. Results and Discussion Histone H3 and H4 genes in Candida albicans Several characteristics of histones H3 and H4 prompted us to focus on them for the purpose of this study. First, histones H3 and H4 show greater conservation than histones H2A and H2B [50]. Second, the H3/H4 tetramer has a more critical role in nucleosome assembly and chromatin condensation, as the H3/ H4 tetramer interacts more strongly with the central portion of the nucleosomal DNA than the H2A/H2B dimers [51,52], and it has the ability to block transcriptional elongation in vitro [53]. Third, the effect of histone H3 and H4 posttranslational modification is better understood and includes modifications in both the amino terminal tail and globular core domains [54,55]. Based on the higher conservation, the prominent role in chromatin structure, and the extensive knowledge on histone H3 and H4 posttransla- tional modifications, we focused on the histone H3-H4 cluster and, in particular, on histone H4, for reasons discussed below. Most eukaryotes, including C. albicans, contain multiple copies of histone H3 and H4 genes, which are generally organized in clusters [56]. The S. cerevisiae genome contains two genes, HHT1 and HHT2, which encode identical proteins for the canonical histone H3 isoform. The C. albicans genome contains three genes that encode histone H3: HHT1 (orf19.6791), HHT2 (orf19.1853), and HHT21 (orf19.1061). HHT2 and HHT21 encode identical histone H3 isoforms, whereas HHT1 encodes a histone H3 with three differences when compared to HHT2/21: a non-conserved S32V change, and two conserved S33T and S81T changes (Figure 2A). When compared to S. cerevisiae HHT1/2, there are five amino acid differences, which are found in all three C. albicans histone H3 genes and are located in the C terminal half of the protein (Figure 2A). Histone H4 Dosage Effects Similar to S. cerevisiae and Schizosaccharomyces pombe [57,58], C. albicans HHT2 and HHT21 are divergently transcribed from the histone H4 genes HHF22 (orf19.1059) and HHF1 (orf19.1854), respectively. HHT2-HHF22 is on chromosome R; HHT21-HHF1 is on chromosome 1 (Figure 1). HHT1, on chromosome 3, is unique in that it is not paired with a histone H4 gene. Unpaired histone genes are also observed in other fungi, including S. pombe, which has an unpaired histone H2A [57,59]. HHT2 and HHT21 are located 900–1000 bp from their cognate histone H4 gene and likely share the same promoter. However, we noted a potential open reading frame (ORF), orf19.1060, within the HHT21-HHF1 intergenic region (Figure 1). This ORF can encode a protein of 169 residues. However, this predicted protein has no significant homology to other proteins or identifiable motifs, suggesting that orf19.1060 is a spurious ORF. Therefore, the genomic arrange- ment of the H3/H4 cluster in C. albicans is similar to S. cerevisiae, except for the presence in C. albicans of a single unpaired, divergent third histone H3 gene. Since Hht1 has a divergent amino acid sequence compared to Hht2/21 and is expressed independent of a histone H4, we propose that Hht1 is a histone H3 variant. Histone variants are usually replication-independent, diverge from the canonical histone sequences, and have a different function [60,61]. For example, Drosophila melanogaster and vertebrates express histone H3.3, a canonical histone H3 variant that differs in four amino acids, three of which are clustered in the histone fold domain [62–65]. Although all three C. albicans histone H3s are of the H3.3 class and the amino acid differences in Hht1 are not within the histone fold domain, Hht1 might still constitute a histone H3 variant. In particular, the S32V change in Hht1 could affect epigenetic regulatory events as it eliminates a potential phosphor- ylation site in the N-terminus tail of Hht1 [66,67]. Thus, HHT1 may encode a histone H3 homomorphic variant. The C. albicans genome contains two genes, HHF1 and HHF22, that encode identical histone H4 proteins. The S. cerevisiae genome also encodes two identical histone H4 proteins. Comparison of C. albicans Hhf1/22 to S. cerevisiae Hhf1/2, revealed seven amino acids differences (Figure 2B). Therefore, the genomic arrangement of histone H4 genes in C. albicans is similar to S. cerevisiae. Since C. albicans contains three non-allelic histone H3 genes as well as a heteromorphic histone H3 variant Cse4, which replaces histone H3 in centromeric chromatin [68,69], we focused our studies on histone H4, which is encoded by two genes and is present in all nucleosomes. Reduced histone H4 dosage impairs C. albicans growth Previous work in S. cerevisiae, S. pombe, and D. melanogaster showed that alterations in the dosage of the core nucleosomal histones can lead to pleiotropic phenotypes, including growth and cell cycle defects, chromosomal and telomere instability and gene expression deregulation [28,29,34,36,38,39,41,70–72]. In order to under- stand the effects of altering histone H4 dosage in C. albicans, sequential deletions of three of the four histone H4 alleles were performed. First, we generated a deletion of the HHF22-HHT2 region, which lacks additional ORFs (Figure 1). HHF22 and HHT2 were simultaneously deleted in order to reduce any potentially harmful effects of changing the histone H3/histone H4 ratio [36,41,71,73]. The entire HHF22 ORF, 80% of the HHT2 ORF, and the intergenic region were replaced by an auxotrophic marker cassette (Figure 1). We were able to generate both HHF22-HHT2/hhf22- hht2D heterozygous and hhf22-hht2D/D homozygous strains, and these strains had no overt growth or morphological phenotypes PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10629