Department of Microbiology and Immunology, Georgetown University, 3900 Reservoir Road NW, Washington, DC 20007-2197, USA1
Author for correspondence: William A. Fonzi. Tel: +1 202 687 1135. Fax: +1 202 687 1800. e-mail: fonziw{at}gusun.georgetown.edu
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ABSTRACT |
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Keywords: Candida albicans, recombination, gene targeting, heterozygosity
The GenBank accession numbers for the sequences reported in this paper are AF247189 and AF247190 for PHR1-1 and PHR1-2, respectively.
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INTRODUCTION |
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Currently, genetic manipulation of C. albicans relies almost exclusively on reverse genetic methods. The demonstration of transformation (Kelly et al., 1988 , 1987
) coupled with workable methods of gene replacement (Fonzi & Irwin, 1993
; Gorman et al., 1991
) has fostered numerous molecular genetic studies of C. albicans biology. Although improved methods of gene replacement have been developed (Morschhauser et al., 1999
; Wilson et al., 1999
, 2000
), most studies to date have relied on recycling of a URA3 marker using positive and negative selection (Fonzi & Irwin, 1993
). This method, first developed for Saccharomyces cerevisiae, employs a cassette consisting of direct repeats of Salmonella typhimurium hisG flanking a URA3 marker gene (Alani et al., 1987
; Fonzi & Irwin, 1993
). The cassette is inserted within the cloned gene of interest, which provides homologous flanking sequences to direct targeted gene replacement. Integration of transforming DNA in C. albicans occurs largely via homologous recombination (Kelly et al., 1987
). The integrated DNA restores uridine prototrophy to the ura3 recipient cells. Spontaneous recombination between the hisG sequences results in loss of the URA3 marker and retention of a single copy of hisG at the disrupted locus (Fonzi & Irwin, 1993
). The Uri- uridine auxotrophs resulting from this excision event are selected on 5-fluoroorotic-acid-containing medium (Boeke et al., 1984
).
Because C. albicans is diploid, expression of recessive mutations requires disruption of two alleles. The heterozygous Uri- mutants are transformed with the same cassette construction, selecting again for uridine prototrophy. Statistically, the cassette is expected to integrate with equal probability into either the previously disrupted allele or the remaining wild-type allele. In practice this is not the case. The ratio of targeting to the wild-type versus the disrupted allele can vary from 1 in 3 to 1 in 35, depending on the locus under investigation (Fonzi & Irwin, 1993 ; Sentandreu et al., 1998
). The factors responsible for this allele specificity and locus-specific variation are unknown.
In this study we examined allele-specific integration at the PHR1 locus (Saporito-Irwin et al., 1995 ). In a heterozygous mutant with the genotype phr1
::hisG/PHR1, transforming DNA recombined with the phr1
::hisG allele approximately 50-times more frequently than with the wild-type allele. This selectivity was only partially due to the additional homology between the disrupted allele and the transforming DNA provided by the hisG sequences. Restriction site polymorphisms permitted identification and cloning of both wild-type alleles of PHR1, designated PHR1-1 and PHR1-2. Transformation with the cloned alleles demonstrated that PHR1-1 preferentially recombined with the resident copy of allele 1 and that PHR1-2 preferentially recombined with the resident copy of allele 2. The nucleotide sequences of the two alleles were approximately 1% divergent overall and these heterologies were concentrated in the 5' and 3' flanking regions of the transforming DNAs. Transformation with PHR1 sequences that were identical in both alleles eliminated the recombinational bias. The results suggest that in C. albicans, as in other organisms, homeologous recombination is less efficient than homologous recombination and that this imparts allele specificity to targeted integration events.
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METHODS |
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Gene cloning and plasmid constructions.
Both alleles of PHR1 were cloned by hybridization screening of a GEM-12 genomic library derived from strain SC5314 (Gillum et al., 1984
). The library and screening conditions have been described by Birse et al. (1993)
. The 2·1 kb insert from plasmid pSMS-24 (Saporito-Irwin et al., 1995
) was used as the hybridization probe to detect PHR1 sequences. The two alleles were distinguished by a BamHI restriction site polymorphism, which resulted in hybridization bands of 8·4 and 7·4 kb on Southern blots of genomic DNA.
clones containing the corresponding fragments were isolated and the inserts were subcloned into the BamHI site of plasmid pBSK+ (Stratagene). The alleles contained on the 7·4 and 8·4 kb fragments were designated PHR1-1 and PHR1-2, respectively.
Plasmids pPHR1-1::URA3 and p
PHR1-2::URA3 contained analogous deletions in alleles 1 and 2, respectively. These were constructed by first subcloning a 2·5 kb EcoRIEcoRV fragment containing PHR1 into a modified pBSK+ vector (Stratagene). The vector lacked the ClaI recognition site in the polylinker region, which was destroyed by Klenow polymerase fill-in of the restricted site followed by ligation. The subclones were digested with ClaI to remove nt +397 to +1235 of the PHR1 coding region and ligated with a 1·1 kb ClaI fragment containing URA3. The URA3 fragment was generated by PCR amplification using the forward primer USP-1 (5'-CCATCGATTGCTGTAGTGCCATTGAT-3') and the reverse primer RP-2 (5'-CCATCGATAAAGTGAAAATCTCCCCCT-3'). The amplification product encompassed nt 283 to +840, relative to the start codon of URA3 and incorporated flanking ClaI restriction sites. URA3 was cloned in the same transcriptional orientation as PHR1 in both plasmids.
Plasmid pSMS-23 contains a deletion mutation of PHR1 similar to that of pPHR1-1::URA3 and p
PHR1-2::URA3. It differs in that nt +397 to +1235 of the PHR1 coding region were substituted with hisG-URA3-hisG sequences (Fonzi & Irwin, 1993
). Construction of this plasmid was described previously (Saporito-Irwin et al., 1995
).
Plasmid pSMS24-URA contained an insertional disruption of PHR1-2. This plasmid was constructed by blunt-end ligation of a 1·3 kb ScaIXbaI fragment containing URA3 into the BspEI site located at nt +889 of the PHR1 coding region in plasmid pSMS-24 (Saporito-Irwin et al., 1995 ). The BspEI and XbaI ends were made blunt by Klenow polymerase fill-in.
Transformation and screening.
Strains were transformed as described previously (Mühlschlegel & Fonzi, 1997 ) and Uri+ transformants were selected on YNB plates. For phenotypic screening of strains CAS-6, YZP1-3 and YZP1-9, transformants were patched to Medium 199 plates buffered to pH 8. These were incubated at 37 °C for 48 h. The resulting colonies were examined macroscopically for the presence of hyphae and microscopically for cell morphology. Colonies lacking filamentation and containing enlarged, highly vacuolated cells were scored as PHR1 null mutants (Saporito-Irwin et al., 1995
). Between 100 and 500 colonies were scored for each transformation. The results are presented as the fraction of transformants exhibiting the null mutant phenotype.
Genotypic screening of CAF3-1 transformants was performed by Southern blot analysis. The methods for Southern blot preparation and hybridization were described previously (Mühlschlegel & Fonzi, 1997 ). The 2·1 kb EcoRI fragment of PHR1 (Saporito-Irwin et al., 1995
) was used as the hybridization probe. Genomic DNA from cells transformed with p
PHR1-1::URA3 or p
PHR1-2::URA3 was digested sequentially with EcoRI and MscI. The MscI site is located at position +23 of allele 2, but is absent from allele 1. Replacement of PHR1-1 was distinguished by the loss of the native 9 kb EcoRIEcoRI hybridizing band and acquisition of a 1·21 kb EcoRIEcoRI band due to the proximal EcoRI introduced by integration of URA3. The loss of the native 8·7 kb MscIEcoRI band and acquisition of a 0·9 kb MscIEcoRI band distinguished replacement of PHR1-2.
Genomic DNA from cells transformed with pSMS24-URA was digested with MscI and PvuII. Replacement of PHR1-1 was distinguished by loss of a native 3·2 kb MscIPvuII hybridization band and acquisition of a 2·8 kb MscIMscI band due to insertion of a proximal MscI site present in URA3. Loss of a native 1·4 kb MscIPvuII band and acquisition of a 0·99 kb MscIMscI band distinguished replacement of PHR1-2.
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RESULTS |
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Effect of hisG sequences on recombination
Preferential targeting of the disrupted allele might reflect the simultaneous presence of hisG sequences in the disrupted allele and the exogenous transforming DNA. As a consequence, the disrupted allele has homology with the exogenous DNA that the wild-type allele does not. By transforming with DNA lacking these sequences the effect of hisG on recombinational preference was assessed. Plasmid pPHR1-2::URA3 was analogous to pSMS-23 used in the previous experiment in that both had deletions of the same PHR1 sequences and, as discussed subsequently, both were derived from the same allele of PHR1. However, instead of a hisG-URA3-hisG insert, p
PHR1-2::URA3 contained an insert of URA3 alone. Digestion of p
PHR1-2::URA3 with PstI and PvuII released a DNA fragment in which URA3 was flanked by PHR1 sequences essentially identical to those of the HindIIIPvuII digest of pSMS-23. The fragments differed in only a few nucleotides at the 5' end resulting from cleavage in the polylinker of the plasmid. Despite the absence of hisG sequences, preferential recombination with the disrupted allele was still evident (Table 1
). The median frequency of mutant transformants was 15x10-2, a roughly sixfold increase in comparison with the previous transformations using hisG-containing DNA. However, the disrupted allele was still targeted at a sixfold higher frequency than the wild-type allele. This indicates that the simultaneous presence of hisG sequences in the disrupted allele and on the transforming DNA does have a selective influence on recombination. However, it does not fully account for the allelic preference of recombination at the PHR1 locus.
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By transforming with identical DNA fragments derived from either PHR1-1 or PHR1-2 the relationship between the allelic source of the transforming DNA and the allele specificity of recombination was tested. Plasmids pPHR1-1::URA3 and p
PHR1-2::URA3 were identically constructed, except that the PHR1 sequences were derived from the cloned allele 1 or 2, respectively. In the preceding experiment, transformation of the heterozygous disruptants with a PstIPvuII digest of p
PHR1-2::URA3 resulted in preferential recombination with the disrupted allele as indicated by the low frequency of null mutants (Table 1
). The analogous transformation using p
PHR1-1::URA3 DNA yielded the reciprocal results; the mutant frequency increased to 81x10-2, indicating preferential replacement of the functional allele. Since the functional allele was PHR1-1 in these strains, p
PHR1-1::URA3 DNA selectively recombined with the corresponding allele in the genome. The magnitude of this bias was similar to that of p
PHR1-2::URA3 DNA for allele 2. Several other restriction enzyme digests, which altered the length of the flanking sequences, were tested in the transformations and in each case the allele specificity of integration was maintained (Table 1
).
To ensure that these results were not an artefact of the locus asymmetry in the heterozygous mutants, DNA from allele 1 or 2 was transformed into the parental strain CAF3-1. Plasmid pPHR1-1::URA3 was digested with BsaBI and AhdI. The recognition sites for these enzymes are located at positions +313 and +1306, respectively. Plasmid p
PHR1-2::URA3 was digested with BsaBI alone since two recognition sites are present, one at +313 and the other +1300. The 3' ends differ due to a restriction site polymorphism. The transformants were screened by Southern blot hybridization using an MscI restriction site polymorphism to distinguish which allele had recombined. Of 24 isolates recovered from transformation with p
PHR1-1::URA3 DNA, 22 were disrupted in allele 1. Conversely, 15 of 16 transformants recovered from transformation with p
PHR1-2::URA3 DNA were disrupted in allele 2. Therefore, exogenous DNA derived from allele 1 preferentially recombines with allele 1 of the genome and the inverse preference is observed for allele 2, irrespective of the presence or absence of a non-homologous insert within one of the alleles.
Allele-specific recombination due to sequence heterology
One obvious explanation for the self-preference of allele targeting lies in the diverged nucleotide sequence of the two alleles as reflected in their multiple restriction site polymorphisms (Fig. 2). As a consequence, recombination between the exogenous DNA and its genomic counterpart is a homologous event, but recombination with the alternate allele would be a homeologous event. Homeologous recombination is less efficient than homologous recombination (Chen & Jinks-Robertson, 1998
; Elliott et al., 1998
; Nassif & Engels, 1993
; Stambuk & Radman, 1998
) and this could account for the bias in gene replacement.
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DISCUSSION |
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The hisG insert in the disrupted allele could potentially affect recombination by other mechanisms as well. For instance, the insert increased by >500 bp, the distance between PHR1 homologous sequences, and may alter the chromatin structure, nucleosome positioning or rate of transcription initiation, any of which might influence recombination frequency (Grigoriev & Hsieh, 1997 ; Kirkpatrick et al., 1999
; Wu & Lichten, 1994
). However, the significance of these effects appear minimal since they would be predicted to have a similar effect on recombination with transforming DNA from either PHR1-1 or PHR1-2 and this was not the case. Exogenous PHR1-1 DNA replaced the endogenous PHR1-1 at a similar frequency in both parental and heterozygous strains.
The effect of allelic sequence divergence, which was approximately 1·2% overall for PHR1, presumably reflects the greater efficiency of homologous recombination in comparison with homeologous transformation. Recombination between like alleles was a homologous event while recombination between the diverged alleles was a homeologous event. It is well established that sequence heterology interferes with recombination in prokaryotic and eukaryotic species (Chen & Jinks-Robertson, 1998 ; Elliott et al., 1998
; Nassif & Engels, 1993
; Stambuk & Radman, 1998
) and this includes targeted gene replacements (Leung et al., 1997
; Negritto et al., 1996
). In S. cerevisiae a single nucleotide difference within a 350 bp region can reduce recombination fourfold (Datta et al., 1997
). Reduced recombination is due to interference by the mismatch repair system with heteroduplex formation between diverged sequences (Chen & Jinks-Robertson, 1998
; Leung et al., 1997
).
The results of this study imply that the mismatch repair system is similarly effective in Candida albicans. As shown by the data in Table 1, a single nucleotide difference was sufficient to bias recombination frequencies. In all of these transformations, recombination with the homologous allele was favoured four- to fivefold over recombination with the heterologous allele. The only exception occurred in transformations using the BsaBI fragment of p
PHR1-2::URA3, which contained only a single nucleotide difference from PHR1-1. This fragment unexpectedly recombined 30-times more frequently with the homologous allele. Preliminary experiments suggest that this atypical behaviour is a peculiarity of this particular DNA fragment (unpublished results). In these experiments, designed to test reciprocity, a heterozygous mutant was constructed in which PHR1-1 was disrupted instead of PHR1-2 as in the present experiments. Transformations of this heterozygote analogous to those in Table 1
yielded quantitatively inverted mutant frequencies as expected. The only exception was again with the BsaBI fragment of p
PHR1-2::URA3. The frequency of mutants in these experiments was 14% rather than the expected 97%. However, it should be noted that the mutant frequency increased about fivefold.
Allele-specific gene targeting has been reported for other loci in C. albicans (Sentandreu et al., 1998 ) and this is presumably related to the same factors influencing gene replacement of PHR1. Although few loci in C. albicans have been examined for heteroallelic differences at the nucleotide level, heterology has been evident at all loci examined. Both alleles of SAP4, ALS2 and ALS4 have been completely or partially sequenced (Hoyer et al., 1998
; Miyasaki et al., 1994
) and roughly 0·5% divergence was found for each locus. This compares to the 1·2% divergence of PHR1 alleles. Variations in the degree and distribution of these heterologies could account for the observed locus to locus variations in allele-specific gene targeting. Of course this does not rule out additional factors. In C. albicans the frequency of recombination with exogenous DNA correlates with the transcriptional activity of the targeted locus (Srikantha et al., 1995
). Given that C. albicans is naturally heterozygous for many recessive mutations (Whelan & Magee, 1981
; Whelan et al., 1980
; Whelan & Soll, 1982
), it would not be surprising to find allelic variations in transcription or perhaps DNA topology, which could influence recombination (Paques & Haber, 1999
).
There are both practical and theoretical implications to the results reported here. From a pragmatic perspective, the inability to inactivate both alleles of a locus cannot be taken as prima facie evidence that the gene is essential. Since a single cloned allele is typically employed in such experiments, sequence divergence between alleles may greatly reduce recombination and prevent production of the homozygous replacement. Of more biological significance is the implied effect of the mismatch repair system in generating genomic diversity in C. albicans. As discussed by Vulic et al. (1997 ), the mismatch repair system serves dual roles in the evolution of genetic diversity, it prevents the accumulation of mutations and inhibits genetic exchange between diverged sequences. In an asexual diploid like C. albicans this may have the interesting consequence of fostering allelic divergence. Although the recombination and repair system would limit the rate of accumulation of mutations, once a mutation did occur, mismatch repair would limit interchromosomal recombination and act to maintain heterozygosity. This would promote the accumulation of additional heterozygous mutations, which would further inhibit interchromosomal recombination. The net effect would be divergence of alleles. Environmental and physiological influences on expression of the mismatch repair system would periodically relax the barrier on chromosomal exchange and allow improved traits to be brought to homozygosity and deleterious mutations to be lost (Vulic et al., 1997
). This periodic recombination between diverged alleles may serve a genetic role analogous to sexual recombination in other organisms.
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ACKNOWLEDGEMENTS |
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Received 30 March 2000;
revised 30 June 2000;
accepted 4 July 2000.