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The centromeric DNA (CEN3) from yeast chromosome III has been isolated on a 1.6 kilobase-pair segment of DNA located near the centromere-linked CDC10 locus of Saccharomyces cerevisiae. When present on a plasmid carrying a yeast chromosomal replicator, CEN3 enables that plasmid to function as a chromosome both mitotically and meiotically. Minichromosomes containing CEN3 are stable in mitosis and segregate as ordinary yeast chromosomes in the first and second meiotic divisions.

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In an approach to clone and characterize centromeric DNA sequences of Candida albicans by chromatin immunoprecipitation, we have used antibodies directed against an evolutionarily conserved histone H3-like protein, CaCse4p (CENP-A homolog). Sequence analysis of clones obtained by this procedure reveals that only eight relatively small regions (Ϸ3 kb each) of the Can. albicans genome are selectively enriched. These CaCse4-bound sequences are located within 4-to 18-kb regions lacking ORFs and occur once in each of the eight chromosomes of Can. albicans. Binding of another evolutionarily conserved kinetochore protein, CaMif2p (CENP-C homolog), colocalizes with CaCse4p. Deletion of the CaCse4p-binding region of chromosome 7 results in a high rate of loss of the altered chromosome, confirming that CaCse4p, a centromeric histone in the CENP-A family, indeed identifies the functional centromeric DNA of Can. albicans. The CaCse4p-rich regions not only lack conserved DNA motifs of point (<400 bp) centromeres and repeated elements of regional (>40 kb) centromeres, but also each chromosome of Can. albicans contains a different and unique CaCse4p-rich centromeric DNA sequence, a centromeric property previously unobserved in other organisms.

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In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (⌿) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known ⌿ synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative ⌿ synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative ⌿ synthase domain of Cbf5p. Yeast strains expressing these mutated cbf5 genes in a cbf5⌬ null background are viable at 25°C but display pronounced cold-and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of ⌿ in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of ⌿ in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks ⌿ in rRNA. A subset of mutations in the ⌿ synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the ⌿ synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.In eukaryotes the biosynthesis of rRNA occurs in a specialized organelle known as the nucleolus (33,41,46,56). rRNA is transcribed by RNA polymerase I (Pol I) as a single large precursor, which undergoes a series of endo-and exonucleolytic cleavages to produce mature rRNA species. In the yeast Saccharomyces cerevisiae, the 35S pre-rRNA precursor is processed to produce mature 18S, 5.8S, and 25S RNAs (54). The 5S rRNA and ribosomal proteins are imported into the nucleolus for assembly into precursors of the 40S and 60S ribosomal subunits before their export to the cytoplasm (16,41,46). An interesting feature of rRNA maturation is the extensive modification the 35S precursor undergoes prior to subsequent cleavage events (29,40,39). One such modification, isomerization of uridine to pseudouridine (⌿), is by far the most abundant posttranscriptional modification of rRNA (29,40,39). Formation of ⌿ is also known to occur in tRNAs (49), small nuclear RNAs (sn...