Rapid discrimination between Candida glabrata, Candida nivariensis, and Candida bracarensis by use of a singleplex PCR.

We report here a PCR-based assay using a single primer pair targeting the RPL31 gene that allows discrimination between Candida glabrata, Candida bracarensis, and Candida nivariensis according to the size of the generated amplicon.

Multilocus microsatellite markers for molecular typing of Candida glabrata: application to analysis of genetic relationships between bloodstream and digestive system isolates.

Candida glabrata has emerged as the second most common etiologic agent, after Candida albicans, of superficial and invasive candidiasis in adults. Strain typing is essential for epidemiological investigation, but easy-to-use and reliable typing methods are still lacking. We report the use of a multilocus microsatellite typing method with a set of eight markers on a panel of 180 strains, including 136 blood isolates from hospitalized patients and 34 digestive tract isolates from nonhospitalized patients. A total of 44 different alleles were observed, generating 87 distinct genotypes. In addition to perfect reproducibility, typing ability, and stability, the method had a discriminatory power calculated at 0.97 when all 8 markers were associated, making it suitable for tracing strains. In addition, it is shown that digestive tract isolates differed from blood culture isolates by exhibiting a higher genotypic diversity associated with different allelic frequencies and preferentially did not group in clonal complexes (CCs). The demonstration of the occurrence of microevolution in digestive strains supports the idea that C. glabrata can be a persistent commensal of the human gut.

Large telomerase RNA, telomere length heterogeneity and escape from senescence in Candida glabrata.

Telomerase, the key enzyme essential for the maintenance of eukaryotic chromosome ends, contains a reverse transcriptase and an RNA that provides the template for the synthesis of telomeric repeats. Here, we characterize the telomerase subunits in the hemiascomycete yeast Candida glabrata. We propose a secondary structure model for the telomerase RNA that is the largest described to date. Telomerase deletion mutants show a progressive shortening of telomeres and a modest loss of viability. Frequent post-senescence survivors emerge that possess long telomeric repeat tracts. We suggest that the high telomere length heterogeneity accounts for this distinct senescence phenotype.

Mass-murder deletion of 19 ORFs from Saccharomyces cerevisiae chromosome XI.

Nineteen open reading frames (ORFs) in the left arm of chromosome XI of the yeast Saccharomyces cerevisiae were inactivated. This was done by producing single-gene or contiguous-gene deletions in haploid and diploid strains. Four deletions are lethal to the corresponding haploid strains, and two result in a failure to grow on a rich glycerol medium. Complementation experiments showed that five of the six identified phenotypes were due to deletion of a single gene (ORFs YKL173w, YKL172w, YKL165c, YKL154w are essential, and YKL160w is required for growth on glycerol medium). One of the phenotypes observed on glycerol medium was not suppressed by the corresponding deleted genes. None of the other deletions, covering 13 ORFs in all, gave rise to any obvious phenotype when the cells were grown at three different temperatures on rich glycerol or glucose medium or on minimal synthetic medium.

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes.

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.

Genetic redundancy and gene fusion in the genome of the Baker's yeast Saccharomyces cerevisiae: functional characterization of a three-member gene family involved in the thiamine biosynthetic pathway.

Redundancy is a salient feature of all living organisms' genome. The yeast genome contains a large number of gene families of previously uncharacterized functions that can be used to explore the functional significance of structural redundancy in a systematic manner. In this work, we describe results on a three-member gene family with moderately divergent sequences (YOL055c, YPL258c and YPR121w ). We demonstrate that two members are isofunctional and encode a hydroxymethylpyrimidine phosphate (HMP-P) kinase (EC 2.7.4.7), an activity required for the final steps of thiamine biosynthesis, whose genes were not previously known in yeast. In addition, we show that the three genes are each composed of two distinct domains, each corresponding to individual genes in prokaryotes, suggesting gene fusion during evolution. The function of the carboxy-terminal part of the proteins is not yet understood, but it is not required for HMP-P kinase activity. Expression of all three genes is regulated in the same way. Several other examples of gene fusions exist in the same biosynthetic pathway when eukaryotic genes are compared with prokaryotic ones.

Mass-murdering: deletion of twenty-three ORFs from Saccharomyces cerevisiae chromosome XI reveals five genes essential for growth and three genes conferring detectable mutant phenotype.

In the frame of the European Network for Functional Analysis (EUROFAN), two regions from chromosome XI covering 54kb have been subjected to 'mass-murder'. Ten deletions covering 23 novel open reading frames (ORFs) were constructed in haploid and diploid strains. Six deletions were lethal in haploid strains. One deletion caused slow germination of spores and slow cellular growth, and another one was associated with both cellular growth thermosensitivity and poor growth on glycerol. These two defects were assigned to two different genes. All mutant phenotypes were complemented by a single gene, enabling us to identify five genes essential for vegetative growth, three genes with detectable phenotype and 15 dispensable genes under standard physiological conditions.

'Mass-murder' of ORFs from three regions of chromosome XI from Saccharomyces cerevisiae.

The complete sequence of the yeast Saccharomyces cerevisiae reveals the presence of many new genes, many of which are without homologs in databases. Characterisation of these genes by novel methods includes systematic deletion followed by phenotypic analysis of mutant strains. We have developed a hierarchical strategy for such a functional analysis of genes, in which the primary phenotypic screening is performed on groups of contiguous genes which are then reinvestigated down to the single gene level. This strategy is applied to the whole chromosome XI as part of EUROFAN (the EUROpean Functional ANalysis) program, and we present here our results on a group of 22 genes from this chromosome. This sample is representative of the results that are emerging for the whole chromosome. Out of the 22 genes deleted, three were shown to be essential, and another three genes confer a mutant growth phenotype to cells when deleted. All phenotypes have been complemented. These figures are in accordance with the previously published fraction of lethal and growth-defective deletions of single genes. We have found no synthetic phenotypes resulting from a combination of deleted genes and have always been able to attribute a mutant phenotype to a single gene.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XV.

Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.

Complete transcriptional map of yeast chromosome XI in different life conditions.

Systematic sequencing of the genome of Saccharomyces cerevisiae has demonstrated the existence of many novel genes, whose functions need to be studied. Entire chromosome sequences also offer the possibility to examine functional properties of the genome at a higher hierarchical level than the genes themselves. We used ordered DNA fragments of chromosome XI to systematically probe yeast DNA and total RNA extracted from MAT a, MAT alpha and diploid cells grown under three different conditions. Taking into account transcript sizes and uniqueness of probes, we attributed 94 transcripts to sequence-predicted open reading frames (ORFs) or tRNA genes; another 83 being tentatively assigned. The remaining 187 ORFs on chromosome XI do not correspond to transcripts detected under our conditions. More than 80% of transcripts are constitutively expressed, others are regulated by medium composition or cell type, the most frequent regulations being determined by carbon source (glycerol/glucose) or rich versus synthetic medium. Moreover, we show that transcript levels and regulation patterns are not statistically different between ORFs of unknown function, which constitute ca. 40% of the total, and previously identified genes (ca. 30%) or their structural homologues.

Sequence and analysis of a 26.9 kb fragment from chromosome XV of the yeast Saccharomyces cerevisiae.

We have determined the nucleotide sequence of a fragment of chromosome XV of Saccharomyces cerevisiae cloned into cosmid pEOA048. The analysis of the 26,857 bp sequence reveals the presence of 19 open reading frames (ORFs), and of one RNA-coding gene (SNR17A). Six ORFs correspond to previously known genes (MKK1/SSP32, YGE1/GRPE/MGE1, KIN4/KIN31/KIN3, RPL37B, DFR1 and HES1, respectively), all others were discovered in this work. Only five of the new ORFs have significant homologs in public databases, the remaining eight correspond to orphans (two of them are questionable). O5248 is a probable folypolyglutamate synthetase, having two structural homologs already sequenced in the yeast genome. O5273 shows homology with a yeast protein required for vanadate resistance. O5268 shows homology with putative oxidoreductases of different organisms. O5257 shows homology with the SAS2 protein and another hypothetical protein from yeast. The last one, O5245, shows homology with a putative protein of Caenorhabditis elegans of unknown function. The present sequence corresponds to coordinates 772,331 to 799,187 of the entire chromosome XV sequence which can be retrieved by anonymous ftp (ftp. mips. embnet. org).

New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using 'split-marker' recombination.

New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome. We have developed a novel transformation strategy, based on 'split-marker' recombination, which allows generation of chromosomal deletions and direct gene cloning. For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large-scale applications such as one-step cloning of target sequence homologs and combinatorial use. Gene deletions or gap-repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids. Gap-repair vectors are based on the URA3 marker. Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I-Scel sites, which allow their subsequent elimination from the transformant without the need for counter-selection. The application of the "split-marker' vectors to the analysis of a few open reading frames of chromosome XI is described.

Transcript map of two regions from chromosome XI of Saccharomyces cerevisiae for interpretation of systematic sequencing results.

A detailed and systematic transcript map is a first and necessary step to characterize new genes revealed by systematic sequencing. Chromosome XI of Saccharomyces cerevisiae contains 331 open reading frames (ORFs) of which 44% are of unknown function (Dujon et al., 1994). As a first study towards complete transcript analysis of chromosome XI, we have extracted RNA from three isogenic strains (a, alpha and 2n) grown in three standard laboratory media, and have analysed them using contiguous probes covering two regions of 17 and 19 kilobases, respectively. All 20 predicted ORFs in the sequences correspond to expressed genes, six of which have no predicted function. Four short ORFs which were suspected as not being real genes on the basis of their sequence are not expressed in our growth conditions. An additional transcript which does not correspond to a large ORF was found. Steady-state RNA level of most ORFs is 10 to 100 times than that of the actin gene, only three are transcribed in comparable amounts. Three ORFs show variable levels of transcripts in the different growth conditions, all patterns being different from one another. Extrapolation of these results to systematic transcript analysis of chromosome XI and other yeast chromosomes is presented.

Consequences of unique double-stranded breaks in yeast chromosomes: death or homozygosis.

We have developed a system in which a unique double-stranded break (DSB) can be introduced into a yeast chromosome during mitotic growth. The recognition site for the endonuclease I-SceI was inserted at different places in the yeast genome in haploid and diploid cells expressing this endonuclease. Induction of the break in haploids results in cell death if no intact copy of the cleaved region is present in the cell. If such a copy is provided on a plasmid, as an ectopic gene duplication, or on a homologous chromosome, the break can be repaired. Repair results in two identical copies in the genome of the locus which has been cut. We call this phenomenon homozygotization by reference to diploids heterozygous for the cut site in which repair leads to homozygosis at this site. We have compared the efficiencies of repair in the various topological situations examined, and conclude that some mechanism must search for regions of homology to both sides of the DSB and that repair is successful only if the homologies are provided by the same template molecule.

The complete sequence of the 8.2 kb segment left of MAT on chromosome III reveals five ORFs, including a gene for a yeast ribokinase.

We report here the DNA sequence of a segment of chromosome III extending over 8.2 kb. The sequence was determined using the random clone strategy followed by oligonucleotide-directed sequencing. The segment contains five long open reading frames, YCR521, 522, 523, 524 and 526, with only short distances between them. YCR523 (333 codons) encodes a ribokinase, a new function for yeast. YCR526 originates inside the MAT cassette, which is in continuity with the present segment, and extends over 358 codons outside of MAT. YCR524 (923 codons) codes for a putative membrane protein. YCR521, 522 and 524, have each been disrupted by insertion of a URA3 cassette and are non-essential genes. An active ARS element is located within YCR523 or its vicinity.