Bart Nelissen

Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae

We present the complete inventory of currently recognized and putative transporters encoded within the genome of Saccharomyces cerevisiae. These 258 transporters are classified into 42 families according to phylogenetic and substrate specificity criteria. Twelve of these yeast families are found only in eukaryotic organisms, and four are so far unique to yeast. Putative yeast‐specific families transport heavy metals, arsenite and calcium. The phylogenetic analyses reported allow classification of 139 functionally uncharacterized yeast transporters into families of known functions. The relative proportions of yeast transporters specific for different classes of substrates differ only slightly from those reported for Escherichia coli. However, the ratio of secondary transporters (uniporters, cation symporters and antiporters) to primary ATP‐driven transporters is much higher for yeast than for bacteria.

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Wenonah Vercoutere, Stephen Winters-Hilt, Hugh Olsen, David Deamer, David Haussler, and Mark Akeson Nat. Biotechnol. 19, 248–252 (2001). The URL given for the DNA mfold server in Table 1 (p. 249 ) and in the text (p. 251) is incorrect. The correct URL is http://bioinfo.math.rpi.edu/~mfold/dna/form1.cgiConverting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis.

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish polymerase chain reaction (PCR) products of the cyanobacterial rRNA operons from those resulting from amplification of contaminating bacteria. Using this screening method the 16S rRNA genes of four nonaxenic filamentous cyanobacterial strains belonging to the generaLeptolyngbya andOscillatoria were cloned and sequenced. For the genusLeptolyngbya, the 16S rRNA sequence of the axenic strain PCC 73110 was also determined. Phylogenetic trees were constructed based on complete and partial sequences. The results show that the strainsLeptolyngbya foveolarum Komárek 1964/112,Leptolyngbya sp. VRUC 135 Albertano 1985/1, andLeptolyngbya boryanum PCC 73110 belong to the same cluster. StrainOscillatoria cf.corallinae SAG 8.92, which contains the rare photosynthetic pigment CU-phycoerythrin, is not closely related to other CU-phycoerythrin-containing cyanobacteria.Oscillatoria agardhii CYA 18, which is a representative of planktonicOscillatoria species that form toxic blooms in Norwegian inland waters, has no close relatives in the tree.

Evolutionary Relationships Among Higher Fungi Inferred from Small Ribosomal Subunit RNA Sequence Analysis

The primary structure of the small ribosomal subunit RNA. (SSU rRNA) was determined for 13 species belonging to 10 ascomycete families and for the basidiomycetous anamorphic yeast Rhodotorula glutinis. The sequences were fitted into an alignment of all hitherto published complete or nearly complete eukaryotic small subunit rRNA sequences. The evolutionary relationships within the fungi were examined by construction of a tree from 87 SSU rRNA sequences, corresponding to 71 different species, by means of a distance matrix method and bootstrap analysis. It confirms the early divergence of the zygomycetes and the classical division of the higher fungi into basidiomycetes and ascomycetes. The basidiomycetes are divided into true basidiomycetes and ustomycetes. Within the ascomycetes, the major subdivisions hemiascomycetes and euascomycetes can be recognized. However, Schizosaccharomyces pombe does not belong to the cluster of the hemiascomycetes, to which it is assigned in classical taxonomic schemes, but forms a distinct lineage. Among the euascomycetes, the plectomycetes and the pyrenomycetes can be distinguished. Within the hemiascomycetes, the polyphyly of genera like Pichia or Candida and of families like the Dipodascaceae and the Saccharomycetaceae can be observed.

Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing

From the approximately 5000 open reading frames presently identified by systematic sequencing of the yeast genome, 100 Saccharomyces cerevisiae transport proteins belonging to the major facilitator superfamily (MFS), were assigned to 17 families on the basis of extensive database searches and binary comparisons. These families include multidrug resistance proteins and transport proteins for sugars, amino acids, uracil/allantoin, allantoate, phosphate, purine/cytosine, proteins, peptides, potassium, sulfate, and urea. Four new families of unknown function have been identified. For the sugar and amino acid transport proteins, alignments were made and phylogenetic trees were constructed allowing the identification of several clusters of proteins presumably exhibiting similar transport functions.

Phylogenetic Relationships Among Filamentous Helical Cyanobacteria Investigated on the Basis of 16S Ribosomal RNA Gene Sequence Analysis

Summary The cyanobacterial genera Spirulina and Arthrospira are both characterized by helical trichomes but their taxonomy is controversial and a phylogenetic study is necessary. The sequence of the 16S rRNA gene and the spacer between the 16S and 23S rRNA genes (ITS) was determined for three filamentous coiled cyanobacteria: Spirulina PCC 6313, Arthrospira PCC 7345 and Arthrospira PCC 8005. A distance tree based on the 16S rRNA sequences was constructed using the neighbor-joining method. This tree shows that the two Arthrospira strains are not closely related to the Spirulina strain but belong to a cluster of strains assigned to the genera Oscillatoria, Lyngbya, and Microcoleus. The strain Spirulina PCC 6313 belongs to a branching containing unicellular cyanobacteria. For the two Arthrospira strains, the sequenced ITS region contains the tRNAIle and tRNAAla genes, whereas the spacer region of strain Spirulina PCC 6313 contains only the tRNAIle gene.