Current Genetics
Curr Genet (1992)22: 501-503
9 Springer-Verlag 1992
Short communications
The Candida albicans P M M 1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation David J. Smith, Michelle Cooper, Mariastella DeTiani, Christophe Losberger, and Mark A. Payton Glaxo Institute for Molecular Biology, Chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland Received March 24, 1992/Accepted May 29, 1992
Abstract. We have constructed an ordered-array genomic D N A library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, P M M 1 , and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans P M M 1 gene, in single copy, to transform temperaturesensitive S. cerevisiae sec53-6 mutant cells, which are defective in P M M activity at 37 ~ to growth at 37 ~ The C. albicans P M M 1 gene is thus the structural and functional equivalent of the SEC53 gene. Key words: Candida albicans - Phosphomannomutase Mannosylation - Gene library
Introduction An early step in the pathway of yeast O- and N-linked mannosylation (Tanner and Lehle 1987) is that performed by the enzyme phosphomannomutase (PMM) which catalyses the reversible interconversion of mannose-6-phosphate and mannose-l-phosphate. Saccharomyces eerevisiae strains containing the sec53-6 temperature-sensitive mutation lack P M M activity at 37 ~ and are defective in secretion (Kepes and Schekman 1988). In order to extend studies on yeast mannosylation to the medically important pathogenic fungus Candida albicans we have constructed an ordered-array genomic library of this organism which permits the rapid cloning of C. albicans genes by hybridisation with heterologous probes. We have screened this library using the S. eerevisiae SEC53 gene encoding P M M (Bernstein et al. 1985) as a probe to isolate the C. albicans homologue. The Correspondence to: D. Smith
sequence of the gene has been determined and we show that it is capable of complementing the temperature-sensitive growth defect of the S. cerevisiae sec53-6 mutant.
Materials and methods Strains and culture conditions. C. albicans ATCC 10261 (His-) was used as a reference strain. The S. cerevisiae strain RSY12 (sec53-6, ura3-52, leu2-3,112) was a kind gift from R. Schekman. Escherichia coli DH1 and yeasts were cultured as previously described (Smith et al. 1992). Molecular biology techniques. Small and large scale plasmid isolation from E. coli, restriction enzyme digestion, gene bank construction, E. coli transformation, Southern blotting onto HybondTM N membrane (Amersham International, Ztirich, Switzerland), hybridisation, E. coli colony lysis, agarose-gel electrophoresis and DNA isolation from gels were all performed according to standard techniques (Sambrook et al. 1989). DNA probes were prepared using an Amersham Multiprime DNA labelling kit and [~-32p]dCTP at approximately 3000 Ci/mMol (Amersham). Isolation of total DNA from C. albicans was by procedures established for S. cerevisiae (Sherman et al. 1982). S. cerevisiae transformation was by the lithium acetate technique (Ito et al. 1983). DNA sequencing was performed as previously described (Smith et al. 1992).
Results and discussion Construction o f a C. albieans A T C C 10261 ordered-array genomic library
A bank o f partial Sau3AI fragments (average size 9 12 kb) from total D N A of C. albicans ATCC 10261 was constructed in the S. cerevisiae centromeric shuttle vector YCp50 (Rose et al./987). The 6,000 primary E. coli DH1 transformants were picked into individual wells on 96well microtitre plates containing 100 gl of LB-broth plus 100 gg/ml of ampicillin. The microtitre plates were incubated at 37 ~ overnight with gentle shaking and a 48point inoculator was used to transfer some of the contents of the wells to duplicate H y b o n d N membrane
502 (Amersham) placed on LB-agar plus ampicillin contained in Nunc 243 mm x 243 mm bioassay plates. Following incubation for 10 h the bacterial colonies were lysed and the D N A fixed to the filter. All 6,000 clones were in this way allocated to identifiable positions on specific membranes. Glycerol (to 25% v/v) was added to the microcultures which remained in the microtitre wells and the plates were stored at - 8 0 ~ Insert frequency in the library was estimated at approximately 80% with an insert size between 8 and 12 kb. Using the formula of Clarke and Carbon (1976) this indicates there is a 95% probability of finding a given D N A sequence in the ordered-array gene library. The advantages of such a library include the rapid isolation of plasraids from positively hybridising colonies, the ability to reuse the filters after removing the probe by very high stringency washing, and increased sensitivity. Very weak hybridisation signals, as a result of low homology, can be readily detected over background due to the large quantities of cloned D N A fixed onto the hybridisation membranes.
Fig. 1. Autoradiograph of a representative hybridisation membrane with DNA from 576 independent E. coli colonies containing C. albicans ATCC 10261 gene library plasmids. The filter was probed with a 1.6 kb BglII fragment, containing all of the S. cerevisiae SEC53 gene from plasmid pSEC5310, at a hybridisation wash stringency of 1 x SSC, 0.1% (w/v) sodium dodecyl sulphate (SDS) at 60 ~ Two strongly hybridising colonies were identified on this membrane against a background of faint non-specific hybridisation
Isolation o f the C. albicans SEC53 homologue
A 1.6 kb BglII fragment containing the S. cerevisiae SEC53 gene was isolated from plasmid pSEC5310 (Bernstein et al. 1985) and used as a hybridisation probe for screening duplicate filters containing the C. albieans gene library. A total of 14 positive signals were identified and an autoradiograph of one such filter is shown in Fig. 1. The E. coli cells from the corresponding position on the designated microtitre plate were collected and used to prepare plasmid DNA. The region of C. albicans D N A in one of these plasmids (p209/4B) to which the SEC53 gene hybridised was located by Southern-blot analysis and the complete nucleotide sequence of the hybridising region and flanking sequences was determined on both strands. The nucleotide sequence data will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number M96770.
Fig. 2. S. cerevisiae RSY12 (sec53-6) transformed with (A) control YCp50 plasmid and (B) p209/4B containing the C. albicans PMM! gene plated on YPD medium and incubated overnight at 25 ~ (plate 1) and 37 ~ (plate 2). The growth of RSY12 at the restrictive temperature of 37 ~ when transformed with the plasmid containing the C. albicans PMM1 gene can be seen on plate 2
The C. albicans P M M I gene
The D N A sequence containing the putative C. albicans SEC53 homologue contains an open reading frame (ORF) of 755 bp which is 76.2% identical to the SEC53 gene from S. cerevisiae and encodes a protein of 252 amino acids with a molecular mass of 29,018 Da which is 77.7% identical to the SEC53 protein. These data suggested that we had cloned the structural homologue of SEC53, which we termed P M M 1 .
The C. albicans P M M 1 gene complements the S. cerevisiae sec53-6 mutation
The high sequence similarity between the S. cerevisiae SEC53 and C. albicans PMM1 genes indicated that they are probably not only structural, but also functional ho-
mologues. To determine whether this was the case we used plasmid p209/4B, containing the P M M 1 gene on the centromere-based vector YCp50 (one copy per cell), to transform S. cerevisiae RSY12 carrying the sec53-6 mutation. Ura § transformants were selected for growth on uracil-free medium at 25 ~ and a number of these, with controls transformed with YCp50 only, were streaked onto Y P D medium at 25 ~ and at 37 ~ a temperature at which the S. cerevisiae sec53-6 mutant will not grow (Kepes and Schekman 1988; Fig. 2). The results of such an experiment show that the C. albicans P M M 1 gene is able to completely restore wild-type growth to the S. cerevisiae sec53-6 mutant at 37 ~ even when present as a single copy. P M M 1 is thus the functional homologue of the SEC53 gene. This approach to gene isolation, and the development of gene disruption techniques applicable to C. albicans
503 ( K e l l y et al. 1987; G o r m a n et al. 1991), s h o u l d facilitate m o l e c u l a r studies o f this p a t h o g e n i c yeast. Acknowledgements'. W'e thank Randy Schekman for providing the S. cerevisiae RSY12 strain and plasmid pSEC5310 containing the SEC53 gene. We also thank Gerhard Paravicini for comments on
the manuscript.
References Bernstein M, Hoffmann W, Ammerer G, Schekman R (1985) J Cell Biol 101:2374-2382 Clarke L, Carbon J (1976) Cell 9:91-99 Gorman JA, Chan W, Gorman JW (1991) Genetics 129:19-24 Ito H, Fukada Y, Murata K, Kimura A (1983) J Bacteriol 153:163 168
Kelly R, Miller SM, Kurtz MB, Kirsch DR (1987) Mol Cell Biol 7:199-207 Kepes F, Schekman R (1988) J Biol Chem 263:9155-9161 Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) Gene 60:237 -243 Sambrook JF, Maniatis T, Fritsch EF (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Sherman F, Fink GR, Hicks JB (1982) Methods in yeast genetics: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Smith D J, Proudfoot A, Friedli L, Klig LS, Paravicini G, Payton MA (1992) Mol Cell Biol 12:2924 2930 Tanner W, Lehle L (1987) Biochem Biophys Acta 906:81-99
C o m m u n i c a t e d b y C. P. H o l l e n b e r g
Curr Genet (1992)22: 501-503
9 Springer-Verlag 1992
Short communications
The Candida albicans P M M 1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation David J. Smith, Michelle Cooper, Mariastella DeTiani, Christophe Losberger, and Mark A. Payton Glaxo Institute for Molecular Biology, Chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland Received March 24, 1992/Accepted May 29, 1992
Abstract. We have constructed an ordered-array genomic D N A library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, P M M 1 , and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans P M M 1 gene, in single copy, to transform temperaturesensitive S. cerevisiae sec53-6 mutant cells, which are defective in P M M activity at 37 ~ to growth at 37 ~ The C. albicans P M M 1 gene is thus the structural and functional equivalent of the SEC53 gene. Key words: Candida albicans - Phosphomannomutase Mannosylation - Gene library
Introduction An early step in the pathway of yeast O- and N-linked mannosylation (Tanner and Lehle 1987) is that performed by the enzyme phosphomannomutase (PMM) which catalyses the reversible interconversion of mannose-6-phosphate and mannose-l-phosphate. Saccharomyces eerevisiae strains containing the sec53-6 temperature-sensitive mutation lack P M M activity at 37 ~ and are defective in secretion (Kepes and Schekman 1988). In order to extend studies on yeast mannosylation to the medically important pathogenic fungus Candida albicans we have constructed an ordered-array genomic library of this organism which permits the rapid cloning of C. albicans genes by hybridisation with heterologous probes. We have screened this library using the S. eerevisiae SEC53 gene encoding P M M (Bernstein et al. 1985) as a probe to isolate the C. albicans homologue. The Correspondence to: D. Smith
sequence of the gene has been determined and we show that it is capable of complementing the temperature-sensitive growth defect of the S. cerevisiae sec53-6 mutant.
Materials and methods Strains and culture conditions. C. albicans ATCC 10261 (His-) was used as a reference strain. The S. cerevisiae strain RSY12 (sec53-6, ura3-52, leu2-3,112) was a kind gift from R. Schekman. Escherichia coli DH1 and yeasts were cultured as previously described (Smith et al. 1992). Molecular biology techniques. Small and large scale plasmid isolation from E. coli, restriction enzyme digestion, gene bank construction, E. coli transformation, Southern blotting onto HybondTM N membrane (Amersham International, Ztirich, Switzerland), hybridisation, E. coli colony lysis, agarose-gel electrophoresis and DNA isolation from gels were all performed according to standard techniques (Sambrook et al. 1989). DNA probes were prepared using an Amersham Multiprime DNA labelling kit and [~-32p]dCTP at approximately 3000 Ci/mMol (Amersham). Isolation of total DNA from C. albicans was by procedures established for S. cerevisiae (Sherman et al. 1982). S. cerevisiae transformation was by the lithium acetate technique (Ito et al. 1983). DNA sequencing was performed as previously described (Smith et al. 1992).
Results and discussion Construction o f a C. albieans A T C C 10261 ordered-array genomic library
A bank o f partial Sau3AI fragments (average size 9 12 kb) from total D N A of C. albicans ATCC 10261 was constructed in the S. cerevisiae centromeric shuttle vector YCp50 (Rose et al./987). The 6,000 primary E. coli DH1 transformants were picked into individual wells on 96well microtitre plates containing 100 gl of LB-broth plus 100 gg/ml of ampicillin. The microtitre plates were incubated at 37 ~ overnight with gentle shaking and a 48point inoculator was used to transfer some of the contents of the wells to duplicate H y b o n d N membrane
502 (Amersham) placed on LB-agar plus ampicillin contained in Nunc 243 mm x 243 mm bioassay plates. Following incubation for 10 h the bacterial colonies were lysed and the D N A fixed to the filter. All 6,000 clones were in this way allocated to identifiable positions on specific membranes. Glycerol (to 25% v/v) was added to the microcultures which remained in the microtitre wells and the plates were stored at - 8 0 ~ Insert frequency in the library was estimated at approximately 80% with an insert size between 8 and 12 kb. Using the formula of Clarke and Carbon (1976) this indicates there is a 95% probability of finding a given D N A sequence in the ordered-array gene library. The advantages of such a library include the rapid isolation of plasraids from positively hybridising colonies, the ability to reuse the filters after removing the probe by very high stringency washing, and increased sensitivity. Very weak hybridisation signals, as a result of low homology, can be readily detected over background due to the large quantities of cloned D N A fixed onto the hybridisation membranes.
Fig. 1. Autoradiograph of a representative hybridisation membrane with DNA from 576 independent E. coli colonies containing C. albicans ATCC 10261 gene library plasmids. The filter was probed with a 1.6 kb BglII fragment, containing all of the S. cerevisiae SEC53 gene from plasmid pSEC5310, at a hybridisation wash stringency of 1 x SSC, 0.1% (w/v) sodium dodecyl sulphate (SDS) at 60 ~ Two strongly hybridising colonies were identified on this membrane against a background of faint non-specific hybridisation
Isolation o f the C. albicans SEC53 homologue
A 1.6 kb BglII fragment containing the S. cerevisiae SEC53 gene was isolated from plasmid pSEC5310 (Bernstein et al. 1985) and used as a hybridisation probe for screening duplicate filters containing the C. albieans gene library. A total of 14 positive signals were identified and an autoradiograph of one such filter is shown in Fig. 1. The E. coli cells from the corresponding position on the designated microtitre plate were collected and used to prepare plasmid DNA. The region of C. albicans D N A in one of these plasmids (p209/4B) to which the SEC53 gene hybridised was located by Southern-blot analysis and the complete nucleotide sequence of the hybridising region and flanking sequences was determined on both strands. The nucleotide sequence data will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number M96770.
Fig. 2. S. cerevisiae RSY12 (sec53-6) transformed with (A) control YCp50 plasmid and (B) p209/4B containing the C. albicans PMM! gene plated on YPD medium and incubated overnight at 25 ~ (plate 1) and 37 ~ (plate 2). The growth of RSY12 at the restrictive temperature of 37 ~ when transformed with the plasmid containing the C. albicans PMM1 gene can be seen on plate 2
The C. albicans P M M I gene
The D N A sequence containing the putative C. albicans SEC53 homologue contains an open reading frame (ORF) of 755 bp which is 76.2% identical to the SEC53 gene from S. cerevisiae and encodes a protein of 252 amino acids with a molecular mass of 29,018 Da which is 77.7% identical to the SEC53 protein. These data suggested that we had cloned the structural homologue of SEC53, which we termed P M M 1 .
The C. albicans P M M 1 gene complements the S. cerevisiae sec53-6 mutation
The high sequence similarity between the S. cerevisiae SEC53 and C. albicans PMM1 genes indicated that they are probably not only structural, but also functional ho-
mologues. To determine whether this was the case we used plasmid p209/4B, containing the P M M 1 gene on the centromere-based vector YCp50 (one copy per cell), to transform S. cerevisiae RSY12 carrying the sec53-6 mutation. Ura § transformants were selected for growth on uracil-free medium at 25 ~ and a number of these, with controls transformed with YCp50 only, were streaked onto Y P D medium at 25 ~ and at 37 ~ a temperature at which the S. cerevisiae sec53-6 mutant will not grow (Kepes and Schekman 1988; Fig. 2). The results of such an experiment show that the C. albicans P M M 1 gene is able to completely restore wild-type growth to the S. cerevisiae sec53-6 mutant at 37 ~ even when present as a single copy. P M M 1 is thus the functional homologue of the SEC53 gene. This approach to gene isolation, and the development of gene disruption techniques applicable to C. albicans
503 ( K e l l y et al. 1987; G o r m a n et al. 1991), s h o u l d facilitate m o l e c u l a r studies o f this p a t h o g e n i c yeast. Acknowledgements'. W'e thank Randy Schekman for providing the S. cerevisiae RSY12 strain and plasmid pSEC5310 containing the SEC53 gene. We also thank Gerhard Paravicini for comments on
the manuscript.
References Bernstein M, Hoffmann W, Ammerer G, Schekman R (1985) J Cell Biol 101:2374-2382 Clarke L, Carbon J (1976) Cell 9:91-99 Gorman JA, Chan W, Gorman JW (1991) Genetics 129:19-24 Ito H, Fukada Y, Murata K, Kimura A (1983) J Bacteriol 153:163 168
Kelly R, Miller SM, Kurtz MB, Kirsch DR (1987) Mol Cell Biol 7:199-207 Kepes F, Schekman R (1988) J Biol Chem 263:9155-9161 Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) Gene 60:237 -243 Sambrook JF, Maniatis T, Fritsch EF (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Sherman F, Fink GR, Hicks JB (1982) Methods in yeast genetics: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Smith D J, Proudfoot A, Friedli L, Klig LS, Paravicini G, Payton MA (1992) Mol Cell Biol 12:2924 2930 Tanner W, Lehle L (1987) Biochem Biophys Acta 906:81-99
C o m m u n i c a t e d b y C. P. H o l l e n b e r g
