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,
Ireland
2
Department of Genetics, Smurfit Institute, University of Dublin, Trinity College, Dublin 2, Ireland
*Correspondence to: Abstract
Geraldine Butler, Department of
We report the sequences of two genomic regions from the pathogenic yeast Candida
Biochemistry, University College
glabrata and their comparison to Saccharomyces cerevisiae. A 3 kb region from
Dublin, Belfield, Dublin 4, Ireland.
C. glabrata was sequenced that contains homologues of the S. cerevisiae genes
E-mail: gbutler@ucd.ie
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 2002
Keywords: Candida glabrata; Saccharomyces cerevisiae; gene order; mating
Accepted: 10 May 2002
pheromone
less susceptible than other Candida species. C.
Introduction
glabrata, like all Candida species, is an imper-
fect yeast lacking an apparent sexual cycle. How-
The yeast Candida glabrata has historically been
ever, while C. albicans and other related species
considered as a commensal organism, and is part of
are always diploid when isolated, C. glabrata is
the normal flora of healthy individuals. However,
haploid (Whelan et al., 1984). C. glabrata is also
in recent years the incidence of infection caused by
much more closely related to S. cerevisiae and
C. glabrata has greatly increased, particularly in
other members of the genus Saccharomyces fam-
immunocompromised patients. Although candidia-
ily than it is to other Candida species (Cai et al.,
sis is usually associated with C. albicans, recent
1996). This suggests that C. glabrata may have lost
reports have shown that C. glabrata is now the
the ability to mate relatively recently. To date, the
second or third most common cause, accounting
available data from C. glabrata suggests that gene
for 12 20% of infections (Pfaller et al., 1999). In
some US hospitals C. glabrata is now more fre- order and gene sequence are strongly conserved
with S. cerevisiae (e.g. Nagahashi et al., 1998).
quently isolated from bloodstream infections than
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.
992 D. W. Walsh, K. H. Wolfe and G. Butler
is encoded by two duplicated genes, MFA1 and
Materials and methods
MFA2 (Brake et al., 1986). The pheromone genes
have no known role outside of the mating process.
Plasmids pH1 and pH4, with overlapping inserts
We tried to isolate the C. glabrata MFA1 locus
totalling 16.4 kb surrounding the C. glabrata
by virtue of sequence conservation in neighbour-
HIS3 locus (Kitada et al., 1995), were gifts from
ing genes. Sequence data from multiple alignments
Dr K. Kitada. The region between TFB3 and STP1
with related proteins was used to design oligonu-
was isolated on a 3.1 kb fragment from C. glabrata
cleotide primers from conserved parts of the genes
strain CBS138 by PCR. Degenerate oligonucleotide
TFB3 and STP1, which flank MFA1 and MRPL28
primers were designed using CODEHOP (Rose
on S. cerevisiae chromosome IV (Figure 1).
et al., 1998) from multiple alignments of pro-
A 3.1 kb fragment of genomic DNA from C.
teins from several species. The primers used were
glabrata was isolated by PCR as described in
5 -ATTTGAAGATGCTTAAGTTGAAAAAGAR-
Materials and methods. Sequence analysis indi-
GTNGAYRT-3 (for TFB3 ) and 5 -AATAACCT-
cated that this region encodes two partial and one
CTAATTCTAAATCTAGCATCACARTARTGR-
complete ORF (Figure 1, Table 1). One end of the
CA-3 (for STP1). The reaction was performed at
ć%
fragment contains the 3 end (234 residues) of a
an annealing temperature of 45 C using a mix-
homologue of TFB3 (component of TFIIH). This
ture of Taq and Pwo DNA polymerises (Expand,
is followed by a long intergenic region of 1.2 kb
Roche Diagnostics). The resulting fragment was
with no large ORFs, and then a homologue of
ligated into EcoRV-digested pBluescript to gen-
the mitochondrial ribosomal protein gene MRPL28
erate the plasmid pDW1. The DNA sequence
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
S. cerevisiae open reading frames
Finder (www.ncbi.nlm.nih.gov). Sequence align-
Identity %
ments were performed using ClustalW (Thompson
et al., 1996).
Open reading frame Protein Nucleic acid
CgTFB3" 68 65
CgMRPL28 52 53
Results and discussion
CgYKR023W" 28 50
CgDBP7 63 67
The biochemical basis of the apparent mating
CgRPC37 51 60
defect in C. glabrata is not known, but if this
CgGCN3 81 73
species has been asexual for a significant evolu- CgYKR021W 30 29
CgHIS3 74 54
tionary period, it is likely to have lost homologues
CgDED1 72 70
of S. cerevisiae genes that function exclusively in
CgYOR205C 43 50
mating. To investigate this, we searched for a C.
CgNOC2" 69 70
glabrata locus homologous to S. cerevisiae MFA1.
"
In S. cerevisiae, the mating pheromone a-factor Incomplete open reading frames.
1000 2000
0
CgTFB3 CgMRPL28 CgSTP1
TFB3 MFA1 MRPL28 STP1
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 993
(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- tion in that species.
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 (Ä…-subunit of translation initiation factor
0 2500 5000
S. cerevisiae
chr XI
S. cerevisiae chr XV
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.
994 D. W. Walsh, K. H. Wolfe and G. Butler
Brettanomyces, Debaryomyces, Dekkera and Kluyveromyces
eIF2B; 305 residues) and YKR021W (unknown
deduced by small-subunit rRNA gene sequences. Int J Syst Bac-
function; 694 residues). The first four genes are co-
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.
Cormack BP, Falkow S. 1999. Efficient homologous and
either a short-range transposition of CgYKR021W
illegitimate recombination in the opportunistic yeast pathogen
or by inversion of a five-gene region (YKR022W to
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.
Hanic-Joyce PJ, Joyce PBM. 1998. A high-copy-number ADE2-
CgHIS3 and CgDED1 (Kitada, et al., 1995; Cor-
bearing plasmid for transformation of Candida glabrata. Gene
mack and Falkow, 1999). These are followed by
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.
Gene 165: 203 206.
remainder of the fragment contains an incomplete
Nagahashi S, Lussier M, Bussey H. 1998. Isolation of Candida
ORF encoding 633 residues of CgNoc2p, with 69%
glabrata homologues of the Saccharomyces cerevisiae KRE9
identity to S. cerevisiae Noc2p, another protein
and KNH1 genes and their involvement in cell wall ²-1,6-glucan
involved in biogenesis of the 60S ribosome subunit.
synthesis. J Bacteriol 180: 5020 5029.
Pfaller MA, Jones RN, Doern GV et al. 1999. International
surveillance of blood stream infections due to Candida species
Acknowledgements
in the European SENTRY Program: species distribution and
antifungal susceptibility including the investigational triazole
We thank Dr K. Kitada for plasmids. This study was
and echinocandin agents. SENTRY Participant Group (Europe).
supported by the Health Research Board (to G.B.) and
Diagn Microb Infect Dis 35: 19 25.
Science Foundation Ireland (to K.W.).
Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCal-
lum CM, Henikoff S. 1998. Consensus-degenerate hybrid
oligonucleotide primers for amplification of distantly related
References
sequences. Nucleic Acids Res 26: 1628 1635.
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W:
Belloch C, Querol A, Garcia MD, Barrio E. 2000. Phylogeny improving the sensitivity of progressive multiple sequence
of the genus Kluyveromyces inferred from the mitochondrial alignment through sequence weighting, position-specific gap
cytochrome c oxidase II gene. Int J Syst Evol Microbiol 50: penalties and weight matrix choice. Nucleic Acids Res 11:
405 416. 4673 4680.
Berrouane YF, Herwaldt LA, Pfaller MA. 1999. Trends in Tzung KW, Williams RM, Scherer S et al. 2001. Genomic
antifungal use and epidemiology of nosocomial yeast infections evidence for a complete sexual cycle in Candida albicans. Proc
in a university hospital. J Clin Microbiol 37: 531 537. Natl Acad Sci USA 98: 3249 3253.
Brake A, Brenner C, Najarian R, Laybourn P, Merryweather J. Whelan WL, Simon S, Beneke ES, Rogers AL. 1984. Aux-
1986. Structure of genes encoding precursors of the yeast otrophic variants of Torulopsis glabrata. FEMS Microbiol Lett
peptide mating pheromone a-Factor. In Protein Transport and 24: 1 4.
Secretion, Gething MJ (ed.). Cold Spring Harbor Laboratory Wood V, Rutherford KM, Ivens A, Rajandream M-A, Barrell B.
Press: New York; 103 108. 2001. A re-annotation of the Saccharomyces cerevisiae genome.
Cai J, Roberts IN, Collins MD. 1996. Phylogenetic relation- Comp Funct Genom 2: 143 154.
ships among members of the ascomycetous yeast genera
Copyright © 2002 John Wiley & Sons, Ltd. Yeast 2002; 19: 991 994.


Wyszukiwarka