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Yea890.dvi

Yeast
Yeast 2002; 19: 991–994.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.890
Yeast Sequencing Report
Genomic differences between Candida glabrata and
Saccharomyces cerevisiae
around the MRPL28 and
GCN3
loci
David W. Walsh,1 Kenneth H. Wolfe2 and Geraldine Butler1*1 Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4,Ireland2 Department of Genetics, Smurfit Institute, University of Dublin, Trinity College, Dublin 2, Ireland Abstract
Geraldine Butler, Department ofBiochemistry, University College We report the sequences of two genomic regions from the pathogenic yeast Candida
Dublin, Belfield, Dublin 4, Ireland. glabrata and their comparison to Saccharomyces cerevisiae. A 3 kb region from
C. glabrata was sequenced that contains homologues of the S. cerevisiae genes
TFB3, MRPL28
and STP1. The equivalent region in S. cerevisiae includes a fourth
gene, MFA1
, coding for mating factor a. The absence of MFA1 is consistent with
C. glabrata
’s asexual life cycle, although we cannot exclude the possibility that a-
factor gene(s) are located somewhere else in its genome. We also report the sequence
of a 16 kb region from C. glabrata
that contains a five-gene cluster similar to
S. cerevisiae
chromosome XI (including GCN3) followed by a four-gene cluster similar
to chromosome XV (including HIS3
). A small-scale rearrangement of gene order has
occurred in the chromosome XI-like section. The sequences have been deposited in
the GenBank database with Accession Nos AY083606 and AY083607. Copyright

2002 John Wiley & Sons, Ltd.
Received: 16 March 2002Accepted: 10 May 2002 Keywords:
Candida glabrata; Saccharomyces cerevisiae; gene order; mating
pheromone
Introduction
less susceptible than other Candida species. C.
glabrata
, like all Candida species, is an imper- The yeast Candida glabrata has historically been fect yeast lacking an apparent sexual cycle. How- considered as a commensal organism, and is part of ever, while C. albicans and other related species the normal flora of healthy individuals. However, are always diploid when isolated, C. glabrata is in recent years the incidence of infection caused by haploid (Whelan et al., 1984). C. glabrata is also C. glabrata has greatly increased, particularly in much more closely related to S. cerevisiae and immunocompromised patients. Although candidia- other members of the genus Saccharomyces fam- sis is usually associated with C. albicans, recent ily than it is to other Candida species (Cai et al., reports have shown that C. glabrata is now the 1996). This suggests that C. glabrata may have lost second or third most common cause, accounting the ability to mate relatively recently. To date, the for 12–20% of infections (Pfaller et al., 1999). In available data from C. glabrata suggests that gene some US hospitals C. glabrata is now more fre- order and gene sequence are strongly conserved quently isolated from bloodstream infections than with S. cerevisiae (e.g. Nagahashi et al., 1998).
C. albicans (Berrouane et al., 1999). The increas- Here we report two cases of disruption to con- ing incidence of infection has been associated with served gene order, caused by probable gene loss in widespread use of azole antifungal drugs (specif- C. glabrata (MFA1), and by a local rearrangement ically fluconazole), as C. glabrata is inherently within a five-gene cluster near the GCN3 locus.
Copyright  2002 John Wiley & Sons, Ltd.
D. W. Walsh, K. H. Wolfe and G. Butler
Materials and methods
is encoded by two duplicated genes, MFA1 andMFA2 (Brake et al., 1986). The pheromone genes Plasmids pH1 and pH4, with overlapping inserts have no known role outside of the mating process.
totalling 16.4 kb surrounding the C. glabrata We tried to isolate the C. glabrata MFA1 locus HIS3 locus (Kitada et al., 1995), were gifts from by virtue of sequence conservation in neighbour- Dr K. Kitada. The region between TFB3 and STP1 ing genes. Sequence data from multiple alignments was isolated on a 3.1 kb fragment from C. glabrata with related proteins was used to design oligonu- strain CBS138 by PCR. Degenerate oligonucleotide cleotide primers from conserved parts of the genes primers were designed using CODEHOP (Rose TFB3 and STP1, which flank MFA1 and MRPL28 et al., 1998) from multiple alignments of pro- on S. cerevisiae chromosome IV (Figure 1).
teins from several species. The primers used were A 3.1 kb fragment of genomic DNA from C. glabrata was isolated by PCR as described in GTNGAYRT-3 (for TFB3 ) and 5 -AATAACCT- Materials and methods. Sequence analysis indi- cated that this region encodes two partial and one CA-3 (for STP1). The reaction was performed at complete ORF (Figure 1, Table 1). One end of the an annealing temperature of 45 ◦C using a mix- fragment contains the 3 end (234 residues) of a ture of Taq and Pwo DNA polymerises (Expand, homologue of TFB3 (component of TFIIH). This Roche Diagnostics). The resulting fragment was is followed by a long intergenic region of 1.2 kb ligated into EcoRV-digested pBluescript to gen- with no large ORFs, and then a homologue of the mitochondrial ribosomal protein gene MRPL28 of the pH1/pH4 and pDW1 inserts was deter-mined commercially by Agowa (Berlin, Ger- Table 1. Sequence identity between C. glabrata and many). ORFs were located using the NCBI ORF Finder (www.ncbi.nlm.nih.gov). Sequence align-
ments were performed using ClustalW (Thompson
Identity %
Open reading frame
Nucleic acid
Results and discussion
The biochemical basis of the apparent mating defect in C. glabrata is not known, but if this species has been asexual for a significant evolu- tionary period, it is likely to have lost homologues of S. cerevisiae genes that function exclusively in mating. To investigate this, we searched for a C. glabrata locus homologous to S. cerevisiae MFA1.
In S. cerevisiae, the mating pheromone a-factor
Figure 1. Comparison of the TFB3– STP1 interval in C. glabrata and S. cerevisiae. The scale bar indicated the distance in
base pairs. Only partial sequence is available for the CgTFB3 and CgSTP1 ORFs
Copyright  2002 John Wiley & Sons, Ltd.
Yeast 2002; 19: 991–994.
Gene order in Candida glabrata
(146 residues). The end of the fragment encodes a been proposed for C. albicans (Tzung et al., 2001).
short partial ORF which is homologous to STP1 In Z. rouxii, the a-factor gene identified in Acces-
(pre tRNA splicing). The similarity is clear when sion No. AL394565 is adjacent to a homologue of the sequence corresponding to the oligonucleotide YNL144C, similar to S. cerevisiae MFA2. Z. rouxii used in the PCR reaction is included. The gene TFB3 and MRPL28 genes are linked to each other order in this region is identical with part of chro- (at the two ends of plasmid AR0AA004F02; de mosome IV in S. cerevisiae (Figure 1), except that Montigny et al., 2000) but the region between them there is no equivalent of MFA1 in C. glabrata.
has not been sequenced so we do not know whether The a-factor protein is small (36 residues) but the
a MFA1 homologue is present at the syntenic posi- gene is well-conserved in Saccharomyces castel- lii and Zygosaccharomyces rouxii (71% and 65% Our results show that apart from the loss of identity, respectively; data from GenBank Acces- MFA1 the order of genes in the TFB3–STP1 region sion Nos AZ927101 and AL394565; Cliften et al., is co-linear in C. glabrata and S. cerevisiae. This 2001; de Montigny et al., 2000). As Z. rouxii is is also true for almost all published examples from probably more distantly related to S. cerevisiae C. glabrata where the gene order is known. To test than is C. glabrata (Belloch et al., 2000), we how widespread this conservation is, we analysed should have been able to identify a C. glabrata gene order in a larger (16 kb) region surround- homologue of MFA1 if it were present in this part ing the HIS3 gene in C. glabrata. The fragment of the genome. The 1.2 kb spacer in C. glabrata contains nine partial or complete ORFs (Figure 2, contains several ORFs 30–40 codons in size, but Table 1). The first five are homologous to genes on none has significant sequence similarity to MFA1 S. cerevisiae chromosome XI. The fragment begins and none has strong codon bias like MFA1. Nei- with a partial ORF encoding 51 amino acids from ther is a MFA1 pseudogene present. We cannot, the C-terminal region of a protein with 28% iden- however, exclude the possibility that C. glabrata tity to YKR023Wp (a protein of unknown func- produces a-factor either from an MFA2 locus, or
tion). This is followed by homologues of DBP7 (a from an MFA1 gene that has transposed to some- DEAD box RNA helicase involved in biogenesis of where else in the genome. Further analysis of the the 60S ribosomal subunit; 715 residues), RPC37 C. glabrata genome will be necessary to deter- (C37 subunit of RNA polymerase III; 241 residues) mine whether it has a cryptic sexual cycle, as has GCN3 (α-subunit of translation initiation factor Figure 2. Comparison of a C. glabrata region containing CgGCN3 and CgHIS3 to parts of S. cerevisiae chromosomes XI and
XV. The ORF YOR203W on S. cerevisiae chromosome XV, which overlaps both HIS3 and DED1, is not shown because it is
designated as a ‘spurious ORF’ by Wood et al. (2001) and as a ‘questionable ORF’ in the MIPS database
Copyright  2002 John Wiley & Sons, Ltd.
Yeast 2002; 19: 991–994.
D. W. Walsh, K. H. Wolfe and G. Butler
eIF2B; 305 residues) and YKR021W (unknown Brettanomyces, Debaryomyces, Dekkera and Kluyveromyces function; 694 residues). The first four genes are co- deduced by small-subunit rRNA gene sequences. Int J Syst Bac-
teriol
46: 542–549.
linear in S. cerevisiae and C. glabrata (Figure 2).
Cliften PF, Hillier LW, Fulton L et al. 2001. Surveying Saccha- CgYKR021W, however, is out of position and in romyces genomes to identify functional elements by compara- inverted orientation. This was probably caused by tive DNA sequence analysis. Genome Res 11: 1175–1186.
either a short-range transposition of CgYKR021W or by inversion of a five-gene region (YKR022W to illegitimate recombination in the opportunistic yeast pathogen
Candida glabrata. Genetics 151: 979–987.
GCN3 ) in one of the species. The remaining genes de Montigny J, Straub M, Potier S et al. 2000. Genomic explo- are co-linear with part of chromosome XV of S. ration of the hemiascomycetous yeasts: 8. Zygosaccharomyces cerevisiae. These include the previously reported rouxii. FEBS Lett 487: 52–55.
CgHIS3 and CgDED1 (Kitada, et al., 1995; Cor- Hanic-Joyce PJ, Joyce PBM. 1998. A high-copy-number ADE2- mack and Falkow, 1999). These are followed by bearing plasmid for transformation of Candida glabrata. Gene
211: 395–400.
CgYOR205C, predicted to encode a protein of Kitada K, Yamaguchi E, Arisawa M. 1995. Cloning of the 526 amino acids with 43% identity to S. cere- Candida glabrata TRP1 and HIS3 genes, and construction of visiae YOR205C, a gene of unknown function. The their disruptant strains by sequential integrative transformation.
remainder of the fragment contains an incomplete Gene 165: 203–206.
ORF encoding 633 residues of CgNoc2p, with 69% Nagahashi S, Lussier M, Bussey H. 1998. Isolation of Candida glabrata homologues of the Saccharomyces cerevisiae KRE9 identity to S. cerevisiae Noc2p, another protein and KNH1 genes and their involvement in cell wall β-1,6-glucan involved in biogenesis of the 60S ribosome subunit.
synthesis. J Bacteriol 180: 5020–5029.
surveillance of blood stream infections due to Candida speciesin the European SENTRY Program: species distribution and We thank Dr K. Kitada for plasmids. This study was antifungal susceptibility including the investigational triazole supported by the Health Research Board (to G.B.) and and echinocandin agents. SENTRY Participant Group (Europe).
Science Foundation Ireland (to K.W.).
Diagn Microb Infect Dis 35: 19–25.
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ships among members of the ascomycetous yeast genera Copyright  2002 John Wiley & Sons, Ltd.
Yeast 2002; 19: 991–994.

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