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?????
???. 13: 1375?1381 (1997)
Isolation of a Putative Prolyl-tRNA Synthetase
(CaPRS) Gene from Candida albicans
MARIA SENTANDREU, M. VICTORIA ELORZA AND RAFAEL SENTANDREU*
Departament de Microbiolog??a i Ecolog??a, Facultat de Farmacia, Avgda. Vicent Andre?s Estelle?s s/n,
46100 Burjassot, Vale?ncia, Spain
Received 31 January 1997; accepted 16 April 1997
We have isolated a 4�kb fragment from a genomic library of Candida albicans which contained two open reading
frames (ORFs). One of them is homologous to a prolyl-tRNA synthetase that catalyses the charging of a specific
tRNA by proline (CaPRS). A deduced sequence of 575 amino acids representing a polypeptide of 66�kDa was
determined. A FASTA search indicated that the CaPRSp had an overall similarity of 54� with the product of a
Saccharomyces cerevisiae ORF (YER087) and 43� with the prolyl-tRNA synthetase of Escherichia coli
(COLIPRO). Consensus Class II aminoacyl-tRNA synthetase sequences were identified by the PROSITE program.
CaPRS was localized to chromosome R of the C. albicans genome and CaPRS DNA hybridized to a major RNA
transcript of 1�kb under all conditions tested. The CaPRS sequence submitted to the EMBL data library is
available under Accession Number U86341. ? 1997 John Wiley & Sons, Ltd.
Yeast 13: 1375?1381, 1997.
No. of Figures: 6. No. of Tables: 0.
No. of References: 27.
??? ????? ? Candida albicans; prolyl tRNA synthetase; CaPRS
INTRODUCTION
Candida albicans is an opportunistic pathogen with
increasing medical significance. One of the most
important virulence factors of this microorganism
is its ability to switch between a yeast and a
mycelial phase that may be associated with the
infection of tissues and organs. Changes in cellular
morphology run in parallel with the presence of
morphology-specific cell wall proteins. This phe*Correspondence to: Rafael Sentandreu, Departament de
Microbiolog??a i Ecolog??a, Facultat de Farmacia, Avgda.
Vicent Andre?s Estelle?s s/n, 46100 Burjassot, Vale?ncia,
Spain.
Tel.: +34 (6) 386 4299;
Fax: +34 (6) 386 4686;
e-mail: rafael.sentandreu@uv.es.
Contract grant sponsor: Fondo de Investigaciones Sanitarias de
la Seguridad social del Ministerio de Sanidad y Consumo.
Contract grant sponsor: New Targets for Antifungal
Therapy?Molecular Biology of Dimorphism in the Human
Pathogen Candida albicans.
CCC 0749?503X/97/141375?07 $17.50
? 1997 John Wiley & Sons, Ltd.
nomenon must involve the differential expression
of genes that in turn depend upon the reception
and transduction of environmental signals.
Aminoacyl-tRNA synthetases are ubiquitous
enzymes that catalyse the same overall reaction,
the charging of a specific tRNA by the cognate
amino acid. Despite their common enzymatic function, aminoacyl tRNA synthetases are quite different with respect to their primary structure, size and
subunit composition. Though the synthetases from
eukaryotic cells are larger than their equivalent
prokaryotic enzymes (Schimmel, 1987), those for a
specific amino acid appear to have been highly
conserved during evolution and contain regions
displaying strong similarity.
An unexpected aspect of mammalian synthetases is that some of them are organized in large
multienzyme complexes. One complex includes
?. ?????????? ?? ??.
1376
the synthetases that catalyse the transferences of
arginine, glutamine, glutamic acid, isoleucine,
leucine and methionine (Mirande et al., 1982;
Dang, 1986; Godar et al., 1988; Norcum, 1989).
Other synthetases such as valyl-tRNA are found in
a complex with the translation elongation factor
EF-1 (Bec and Waller, 1989).
Although 34 genes have been detected in
the Saccharomyces cerevisiae genome that code
putative aminoacyl-tRNA synthetases, limited
information has been obtained. Partial physical
mapping of the C. albicans genome has detected
two putative aminoacyl-tRNA synthetase genes
(seryl- and arginyl-tRNA synthetases) but nothing
is known about them. Here we present evidence of
a prolyl-tRNA synthetase of C. albicans.
MATERIALS AND METHODS
Strains and growth conditions
The strains of C. albicans used in this study were
ATCC26555, FC18 (from Chu et al., 1993) and
996 (Institute Pasteur, Paris). Yarrowia lipolytica
(CX39-74 was provided by Dr J. Bassel (Donner
Lab., Berkeley, CA) and S. cerevisiae X2180-1A
from the Yeast Genetic Stock Center (Berkeley,
CA).
Escherichia coli DN5� was used for most E. coli
transformations and was grown at 37)C in LuriaBertani (LB) medium (Sambrook et al., 1989).
Yeasts were routinely grown at 28)C in YPD
(2% peptone, 2% glucose, 1% yeast extract).
C. albicans blastoconidia were obtained from
cultures grown at 30)C on synthetic medium (Lee
et al., 1975). Protoplasts from C. albicans were
obtained as previously described (Elorza et al.,
1988) and cultured for 3 h in Lee medium
stabilized with 0�?-KCl either at 28)C or 37)C.
Cloning procedures and molecular techniques
Screening of a cDNA mycelial library with
rabbit antiserum raised against cell wall material
from mycelial cells was carried out as previously
described (Sentandreu et al., 1995). cDNA fragments from positive clones were isolated by EcoRI
digestion of the recombinant lambda DNA and
employed as probes for the screening of a genomic
library of C. albicans ATCC26555 constructed in
the multi-copy plasmid YRp7 (Nieto et al., 1993).
Portions of a genomic insert that hybridized a
cDNA clone were subcloned into the M13 cloning
vectors (mp18 and mp19) and ssDNA was
?????
???. 13: 1375?1381 (1997)
sequenced by the dideoxy chain-termination
method using T7 DNA polymerase and by the
automated DNA sequencer model 370 A (Applied
Biosystem).
Nucleotide sequence analyses were performed
with the PROSITE program (Bairoch, 1993).
Homology searches of the GenBank database were
conducted with the FASTA program of Pearson
and Lipman (1988).
For Southern analysis, yeast strains were grown
on YPD and yeast genomic DNA was isolated
following the method of Fujimura and Sakuma
(1993). Genomic DNA was digested with either
EcoRI or XbaI and transferred onto a nylon
membrane. The probe used in the hybridization
experiment was the BglII-EcoRI (0�kb) genomic
fragment encoding the 5* end of the gene, labelled
by the digoxigenin method of BoehringerMannheim.
Preparation of chromosomes and pulse-field
electrophoresis were carried out as described
earlier (Nieto et al., 1993). Southern analysis and
hybridization of chromosomes was done after
blotting onto a nylon membrane. The BglII-EcoRI
fragment of 0�kb was used again as hybridization
probe.
For Northern analysis, total RNA from blastoconidia and protoplasts was isolated following the
method of Langford and Gallwitz (1983). RNA
(10 靏/lane) was fractionated on formaldehydeagarose gel and transferred onto a nylon membrane. RNA was fixed at 100)C for 1�h and
hybridizations in Na2HPO4 0� ?, pH 7� EDTA
1 m? pH 8� SDS 20%, blocking reagent 0�
(Boehringer-Mannhein) were performed for 16 h
at 42)C, using as probe the 0�kb fragment that
included the 5* end of the gene. The C. albicans
actin gene used in control hybridizations was
kindly provided by W. A. Fonzi (Georgetown
University Medical Center, Washington DC).
RESULTS AND DISCUSSION
Cloning of prolyl tRNA synthetase
Polyclonal antibodies against blastoconidia and
hyphal cell walls were used to screen cDNA
libraries (Sentandreu et al., 1995). A cDNA insert
from positive clone 24M from the mycelial library
was used in the hybridization of a genomic library
constructed in YRp7 (Nieto et al., 1993) and a
genomic clone pGEV2.1 containing a 9-kb insert
was isolated. Various overlapping restriction
? 1997 John Wiley & Sons, Ltd.
????? ???? ???? ?. ????????
1377
Figure 1. Restriction map of the XhoI-BglII C. albicans genomic fragment that
contains the two identified ORFs. The top shaded line is the scale in bp with
relevant restriction endonuclease sites used to subclone fragments for sequencing. The location, orientation, and lengths of revealed ORFs are indicated by
black arrows.
fragments generated from pGEV2.1 were subcloned into pUC and M13, and the complete
nucleotide sequence of a XhoI-BglII DNA fragment was determined (Figure 1).
The nucleotide sequence (4033 bp) contained
two open reading frames (ORFs; Figure 1). The
first extended 418 bp downstream of a Dra1 site
and ended at nucleotide 1381. The second ORF
was separated from the first one by 528 bp and
extended from nucleotide 1909 to 3633.
Characteristics of the CaPRS gene
In the present paper, characteristics of the
second ORF are reported (Figure 2). In common
with other C. albicans genes, the promoter of the
second ORF was highly AT rich with two potential
TATA boxes, as shown in Figure 2: the first one,
from nucleotide 58 to 72 (CAATAAAGATCATAC) and the second from nucleotide 125 to
139 (CAATATATCCACTTG). The second
TATA box correlated with the same CCAAT box.
No consensus splice signal characteristic of introns
was found within the coding region. The ORF of
the CaPRS (Candida albicans-prolyl-tRNAsynthetase) gene encodes a hypothetical polypeptide of 575 amino acids, with a molecular size of
66 kDA.
Both the nucleotide and the corresponding predicted amino acid sequences (Figure 2) were compared with the EMBL and GenBank databases by
the FASTA and BLAST programs. The polypeptide showed the highest degree of homology to a
protein of 576 amino acids (Yer 087p; accession
number U18839) deduced from sequencing chromosome V of S. cerevisiae (Diethrich et al., unpublished observations). The nucleotide sequence
similarity between the C. albicans gene and that of
? 1997 John Wiley & Sons, Ltd.
chromosome V of S. cerevisiae is 58�. The
identity between both proteins was 36� along
the entire sequence, and the homology 54�
(Figure 3). Yer087p is similar to prolyl-tRNA
synthetase from E. coli that belongs to class II
amino-acyl tRNA synthetases. The E. coli protein
has 572 amino acids and shows 29�% identity
with Yer87p and 27�% with the C. albicans gene.
Aminoacyl tRNA synthetases have been divided
into two classes based on the existence of two
structurally distinct active sites. They differ widely
in size and oligomeric state and have only a limited
sequence homology (Eriani et al., 1990). The Class
I synthetases (ERS, YRS, MRS, QRS, RRS, VRS,
IRS, LRS and WRS) display two short common
consensus sequences (HIGH and KMSKS), which
correspond to the structural domain that binds
ATP.
Class II synthetases (PRS, ARS, NRS, DRS,
GRS, HRS, KRS, SRS and TRS) probably have in
common the folding pattern in their catalytic domain for binding of ATP and the corresponding
amino acid (Cusack et al., 1990). Class II synthetases are also characterized by the fact that they
do not share a high degree of similarity, but at
least some conserved regions (motifs 1, 2, 3) are
present (Eriani et al., 1990; Cusack et al., 1991;
Leveque et al., 1990). The CaPRSp analysed with
the PROSITE program was shown to have the
typical three aminoacyl-tRNA synthetase class II
signatures (Figure 3): from amino acid 67 to 87
(motif 1), from 136 to 169 (motif 2), and from 395
to 432 (motif 3).
The molecular size of the C. albicans gene as
well as those from S. cerevisiae and E. coli prolyltRNA synthetases are small compared with the
equivalent enzymes in human cells (1440 amino
acids, molecular weight 163,026), Drosophila
?????
???. 13: 1375?1381 (1997)
?. ?????????? ?? ??.
1378
Figure 2. Sequence of CaPRS gene and flanking regions. The amino acid sequence
is given below the nucleotide sequence. Putative TATA-box sequences are indicated
by shaded boxes.
melanogaster (1704 amino acids, molecular weight
189, 197) and probably in other higher eukaryotes.
In these species PRSp are proteins with more than
one enzymatic activity. They are multifunctional
aminoacyl-tRNA synthetases presenting three
domains: the amino-terminal catalyses aminoacylation of glutamic acid, the carboxy-terminal
the aminoacylation of proline (Cerini et al., 1991)
and thirdly the human enzyme also possesses in
its central part some regions of similarity with
eukaryotic translation factor EF-1 (Fett and
Knippers, 1991). In the case of D. melanogaster,
the central domain may be responsible for the
formation of the multienzyme complex that is
found in higher eukaryotes but not in yeast (Dang,
1986). This type of molecular organization is
interesting, as the activities they have (glutamyl
and prolyl-RS) belong to different classes of
?????
???. 13: 1375?1381 (1997)
aminoacyl-RS but are expressed in the same
polypeptide, whereas in C. albicans, S. cerevisiae
and E. coli these enzymes are expressed separately,
suggesting that they have evolved along independent evolutionary pathways (Kaiser et al., 1994).
Genomic and chromosomal localization of CaPRS
To obtain information about the genomic organization and the presence of potential homologous genomic sequences to CaPRS, Southern blot
analysis was carried out using genomic DNA from
C. albicans ATCC26555 (Figure 4). Hybridization
with a BglII-EcoRI fragment of 0�kb from the
coding region showed that CaPRSp is probably
encoded by a unique nuclear gene (Figure 4). Only
a single band was detected with the DNA digested
either with EcoRI (1�kb) or XbaI (19 kb). On the
? 1997 John Wiley & Sons, Ltd.
????? ???? ???? ?. ????????
1379
Figure 3. Comparison of prolyl-tRNA synthetase sequences. The amino acid
sequence of C. albicans (CaPRSP) is aligned with the corresponding regions
from S. cerevisiae (YER087P) and E. coli (COLIPRO). The annotations below
the sequences indicate the positions in the alignment which are perfectly matched
(*) or well-conserved (.). Consensus motifs characteristic of Class II aminoacyltRNA synthetases (Eriani et al., 1990) are boxed.
other hand, the probe did not react with the DNA
from S. cerevisiae or Y. lipolytica digested with the
same enzymes (Figure 4). Though the homology
with the PRS of S. cerevisiae is high, the different
codon usage in both species may be the reason for
the negative result. C. albicans presents a strong
preference for an A or T in the third, or ?wobble?
position (Brown et al., 1991) compared to S.
cerevisiae. In the case of Y. lipolytica, the homologous gene has not been described, but again, codon
usage is very different compared to C. albicans,
as the G/C content is approximately 70% in
Y. lipolytica (Kurtzman et al., 1983).
The above result implies that the gene could
be present in a single copy in the genome of
C. albicans, so chromosomal hybridization was
? 1997 John Wiley & Sons, Ltd.
performed to determine chromosomal localization
using C. albicans FC18 and 996 strains (Figure 5).
Hybridization of a chromo-blot of electrophoretically separated chromosomes with the 0�kb
BglII-EcoRI fragment showed that the CAPRS
gene is localized on chromosome R1 and R2+1
(Chu et al., 1993). This result is similar to those
reported for MGLI (Iwaguchi et al., 1990) and
ADEI (Wickes et al., 1991) that migrate with
chromosome R. Strain 996 showed a very peculiar
pattern with an extra band migrating slower than
chromosome 3. Although this could indicate the
presence of several copies of the gene in this strain,
abnormalities that have been described regarding
the behaviour of chromosome R could explain this
result (Wickes et al., 1991).
?????
???. 13: 1375?1381 (1997)
?. ?????????? ?? ??.
1380
Figure 4. Southern-blot analysis. Genomic DNA from C.
albicans ATCC26555 (lanes 1, 2), S. cerevisiae (lanes 3, 4) and
Y. lipolytica (lanes 5, 6) was digested with EcoRI (lanes 1, 3, 5)
or XbaI (lanes 2, 4, 6) and subjected to electrophoresis through
0� agarose. After transfer, the blot was hybridized with the
EcoRI-BglII fragment containing the 5* end of CaPRS labelled
with digoxigenin. The electrophoretic positions of DNA size
markers are indicated on the left in kb.
Figure 6. Northern-blot analysis of the CAPRS transcript.
Total RNA from blastoconidia (lane 1) and from protoplasts,
grown either at 28)C (lane 2) or 37)C (lane 3). The upper panel
displays the results of hybridization with the CaPRS probe and
the lower panel displays the results of hybridization with actin
DNA. Electrophoretic positions of the rRNAs are indicated on
the right.
In conclusion, we have isolated a C. albicans
gene which shows a high degree of homology
with known prolyl-tRNA synthetase genes. The
molecular size of the protein product is consonant
with the S. cerevisiae gene and others from
prokaryotes, indicating evolutive relatedness. This
gene has been localized in chromosome R and
hybridization experiments suggest that it may be in
a single copy in the C. albicans genome.
ACKNOWLEDGEMENTS
Figure 5. Separation of chromosomes of C. albicans 996
(lanes 1, 3) and FC18 (2, 4) by pulse-field gel electrophoresis.
Et-Br stained chromosomes (lanes 1, 2). Chromo-blot probed
with the 0�kb EcoRI-BglII fragment of the CaPRS (lanes 3,
4). C. albicans chromosomal designations (left margin) are
those proposed by Wickes et al. (1991).
Expression of CaPRS
To explore transcription of the CaPRS gene,
total RNA was isolated from exponential cultures
and analysed by Northern-blot hybridization
(Figure 6). Level of CaPRS expression seemed to
be constitutive since the amount of mRNA was the
same in regenerating protoplasts as in blastoconidia. The transcription levels appeared largely
unaffected by the temperature of regeneration
(28)C or 37)C).
?????
???. 13: 1375?1381 (1997)
This work was partially supported by grants
from the Fondo de Investigaciones Sanitarias de
la Seguridad Social del Ministerio de Sanidad y
Consumo (95/1602), Madrid, Spain and New
Targets for Antifungal Therapy-Molecular
Biology of Dimorphism in the Human Pathogen
Candida albicans (BIOMED2 BMH4-CT96-0310,
Brussels). M.V.E. is on leave from the Consejo
Superior de Investigaciones Cient??ficas. M.S. was a
recipient of predoctoral grants from the Direccio?
General de Universitat e Investigacio? de la
Generalitat Valenciana, Vale?ncia, Spain.
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???. 13: 1375?1381 (1997)
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