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Candida dubliniensis is the closest known relative of Candida albicans, the most pathogenic yeast species in humans. However, despite both species sharing many phenotypic characteristics, including the ability to form true hyphae, C. dubliniensis is a significantly less virulent and less versatile pathogen. Therefore, to identify C. albicans-specific genes that may be responsible for an increased capacity to cause disease, we have sequenced the C. dubliniensis genome and compared it with the known C. albicans genome sequence. Although the two genome sequences are highly similar and synteny is conserved throughout, 168 species-specific genes are identified, including some encoding known hyphal-specific virulence factors, such as the aspartyl proteinases Sap4 and Sap5 and the proposed invasin Als3. Among the 115 pseudogenes confirmed in C. dubliniensis are orthologs of several filamentous growth regulator (FGR) genes that also have suspected roles in pathogenesis. However, the principal differences in genomic repertoire concern expansion of the TLO gene family of putative transcription factors and the IFA family of putative transmembrane proteins in C. albicans, which represent novel candidate virulence-associated factors. The results suggest that the recent evolutionary histories of C. albicans and C. dubliniensis are quite different. While gene families instrumental in pathogenesis have been elaborated in C. albicans, C. dubliniensis has lost genomic capacity and key pathogenic functions. This could explain why C. albicans is a more potent pathogen in humans than C. dubliniensis.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.

African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that-along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions-have shaped the genomes of each parasite. Contrary to recent reports, our analyses reveal no evidence that these species are descended from an ancestor that contained a photosynthetic endosymbiont.

Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.

The Trypanosoma brucei reference strain TREU927/4 exhibits some resistance to lysis by normal human serum (NHS), but this resistance is never complete even after selection. The genome of this strain contains a minimum of eight sequences related to the T. brucei rhodesiense-specific serum resistance-associated gene (SRA), which encodes a truncated variant surface glycoprotein (VSG) conferring full resistance to lysis by NHS. We selected two sequences showing the highest similarity to SRA and also preceded by a region ("cotransposed region") present immediately upstream from SRA in the VSG expression site termed R-ES, where SRA is expressed in T. brucei rhodesiense. Whereas one of these sequences appears to be a pseudogene, the other, which is contained within a cluster of VSG basic copies (BCs), encodes a VSG truncated in the C-terminal domain. In the latter gene, an inserted region encoding surface-exposed loops similar to those of the BoTat 1.20 VSG interrupts the full SRA sequence. Therefore, this gene was termed SRA-BC, for the putative VSG basic copy from which SRA was derived. Neither this gene nor other SRA-like sequences appeared to be responsible for the relative resistance of TREU927/4 to NHS, since (i) transfection of SRA-BC in T. brucei brucei did not confer increased resistance; (ii) SRA transcripts could not be detected in this strain, even when focusing the search on the limited SRA sequence necessary to confer resistance and when using strain-specific SRA probes on RNA from cells selected in the presence of NHS.

In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing. All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain. Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear. Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA. In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels. This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression.

Environmental change, such as nutritional starvation, induces physiological and morphological alterations that enable fission yeast cells to survive. We isolated a novel gene, taf1+, required for the response to nitrogen starvation in the fission yeast Schizosaccharomyces pombe. taf1 disruptants could not mate upon nitrogen starvation, but could upon carbon starvation. taf1 disruptants had a defect in inducing stell+ expression under nitrogen starvation conditions. Furthermore, they lost viability quickly in nitrogen-depleted medium. Unlike wild-type cells, starved taf1-cells had nuclear chromatin that were flat and adhered to the cell periphery. These results indicate that tqf1+ is required for nitrogen starvation-induced sexual development and entering the dormant G0 state.

Several regions of the Saccharomyces cerevisiae genome are subject to position-dependent transcriptional repression mediated by a multi-component nucleosome-binding complex of silent information regulator proteins (Sir2p, Sir3p and Sir4p). These proteins are present in limiting amounts in the nucleus and are targeted to specific chromosomal regions by interaction with sequence-specific DNA-binding factors. Different sites of repression compete for Sir complexes, although it is not known how Sir distribution is regulated. In a screen for factors that interact with Sir4p amino terminus, we have cloned SIF2, which encodes a WD40-repeat-containing factor that disrupts telomeric silencing when overexpressed. In contrast to deletion of SIR4, SIF2 deletion improved telomeric repression, suggesting that under normal conditions Sif2p antagonizes Sir4p function at telomeres. Sif2p overexpression altered the subnuclear localization of Sir4p, but not its protein expression level, suggesting that Sif2p may recruit Sir4p to nontelomeric sites or repression. The sif2 mutant strains were hypersensitive to a range of stress conditions, but did not have decreased viability and did not alter repression in the rDNA. In conclusion, Sif2p resembles the Sir4p regulatory proteins Sir1p and Uth4p in that it competes for the functional assembly of Sir4p at telomeres, yet unlike Sir1p or Uth4p, it does not target Sir4p to either mating-type or rDNA loci.

In budding yeast genes integrated near telomeres succumb to a variegated pattern of gene repression that requires the silent information regulatory proteins Sir2p, Sir3p and Sir4p, which form a nucleosome-binding complex. Immunolocalization shows that the Sir proteins co-localize with the telomeric repeat binding protein Rap1p and with telomeric DNA in a limited number of foci near the periphery of interphase nuclei. All conditions tested so far that disrupt telomere proximal repression result in a dispersed staining pattern for Sir2p, Sir3p and Sir4p. Although the focal organization is clearly not sufficient for establishing repression, genetic studies suggest that the high local concentration of Sir proteins at telomeric foci facilitates the formation of repressed chromatin. In addition to its telomeric localization, Sir2p is shown by immunostaining and cross-linking to bind a subdomain of the nucleolus. In strains lacking an intact Sir4p, Sir3p also becomes concentrated in the nucleolus by a pathway requiring SIR2 and UTH4. This unexpected localization correlates with observed effects of sir mutations on rDNA stability and longevity, defining a new site of action for silent information regulatory factors. We report a novel WD40 repeat-containing factor, Sif2p, that binds specifically to the Sir4p N-terminus. Like Sir1p and Uth4p, Sif2p antagonizes telomeric silencing by regulating an equilibrium between alternative assembly pathways at different subnuclear loci.

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.

During meiosis, the pairing of chromosomes is crucial for a successful partitioning of the genetic material and its transmission into the developing gamete. The classical view of meiotic pairing involves recombination between homologues as an integral part of the pairing mechanism. But, in cases where no recombination occurs, how do chromosome partners find one another? And how do they pair up and then segregate appropriately? Recently, a combination of molecular genetics and fluorescence in situ hybridization appears to have provided an answer to these questions by demonstrating a crucial role for heterochromatin in chromosome pairing.