Four new Candida species from geographically diverse locations

Four new species of Candida are described based on their unique nucleotide sequences in the D1/D2 domain of large subunit (26S) ribosomal DNA. Candida peoriaensis (type strain NRRL YB-1497, CBS 8800) and C. ponderosae (type strain NRRL YB-2307, CBS 8801) are members of the Pichia anomala clade and were isolated in the U.S. from, respectively, the stump of an elm tree (Ulmus sp.) and from insect frass of a Ponderosa pine (Pinus ponderosa). Candida ghanaensis (type strain NRRL YB-1486, CBS 8798) is a phylogenetically divergent species from soil in Ghana and appears related to the Dipodascus/Geotrichum clade. Candida litsaeae (type strain NRRL YB-3246, CBS 8799) was isolated from the frass of an insect-infested Litsaea polyantha tree from India, and is a divergent species that is most closely related to Candida ontarioensis.     

Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.     

Phylogenetic relationships among yeasts of the 'Saccharomyces complex' determined from multigene sequence analyses

Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C. castellii, C. glabrata, C. humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1alpha, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II). Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved. Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.     

Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences

Approximately 500 species of ascomycetous yeasts, including members of Candida and other anamorphic genera, were analyzed for extent of divergence in the variable D1/D2 domain of large subunit (26S) ribosomal DNA. Divergence in this domain is generally sufficient to resolve individual species, resulting in the prediction that 55 currently recognized taxa are synonyms of earlier described species. Phylogenetic relationships among the ascomycetous yeasts were analyzed from D1/D2 sequence divergence. For comparison, the phylogeny of selected members of the Saccharomyces clade was determined from 18S rDNA sequences. Species relationships were highly concordant between the D1/D2 and 18S trees when branches were statistically well supported.     

Candida arabinofermentans, a new L-arabinose fermenting yeast

Candida arabinofermentans (type strain NRRL YB-2248, CBS 8468), a new yeast that ferments the pentose L-arabinose, is described. The three known strains of this new species were isolated from insect frass of pine and larch trees in the U.S. Phylogenetic analysis of nucleotide sequences from the D1/D2 domain of large subunit (26S) ribosomal DNA places C. arabinofermentans among the methanol-assimilating yeasts and most closely related to Candida ovalis. Strains of the new species produce 0.7-1.9 g/l ethanol from L-arabinose.     

Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106^T, NRRL Y-27775, DQ839395; C. cretensis CBS 9453^T, NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107^T, NRRL Y-27665, DQ839394; C. vadensis CBS 9454^T, NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.     

Yeast species recognition from gene sequence analyses and other molecular methods

This review discusses DNA-based methods used for identification of yeasts. Nuclear DNA reassociation was the first quantitative molecular method employed for recognition of yeast species and has provided a baseline for interpretation of other molecular comparisons. Among these, gene sequencing is the most definitive method, with ribosomal RNA gene sequences providing the preponderance of available data. Multigene analyses that include the sequences of protein encoding genes are being increasingly developed to provide a more definitive resolution of species. A number of rapid identification methods, such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and flow cytometry, which are based on species-specific gene sequences, are available for use in diagnostic laboratories.     

Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.