We describe here a rapid method for disruption of C. albicans
genes. The method is based upon PCR primer-directed gene disruption in S. cerevisiae
) and upon the idea of using two markers to create homozygous disruptions in C. albicans
). We were able to disrupt four different genes with PCR products, so it is likely that the method will be applicable to many other loci.
We had expected that the ARG4
cassettes would integrate more efficiently than HIS1
at targeted loci. The URA3
cassette in plasmid pGEM-URA3 lacks sequences to direct integration at the URA3
locus, because the entire URA3
locus has been deleted in strain CAI4 (2
). The ARG4
cassette in plasmid pRS-ARG4ΔSpeI also lacks sequences to direct integration at the ARG4
locus, because it does not extend beyond one end of the arg4
insertion/deletion allele in strain BWP17. On the other hand, the HIS1
cassette in plasmid pGEM-HIS1 extends far beyond the HIS1
coding region, so it should be capable of integration into the his1
allele. However, targeted integration of hrm101
in strain BWP17 occurred with efficiency comparable to that of arg5
in strain RM1000. These results are not directly comparable, because they involve different targeted loci and different transformation recipients. However, they suggest that all three markers may be equally useful for future gene disruption experiments.
cassettes lack known sequences for homologous integration at each respective locus in strain BWP17, yet we recovered transformants that did not carry the markers at targeted loci. We are uncertain whether the PCR products integrate into the genome or are maintained in an extrachromosomal state. However, the markers are much more stable than the ARS plasmid pRC2312 (4
) in our hands (unpublished results), so we believe that they are integrated. These observations underscore the importance of verifying targeted integration through either Southern analysis or PCRs with outside primers.
We observed a significant allelic integration bias in the second of successive transformations. For example, transformation of the arg5::HIS1/ARG5 strain with the arg5::URA3 PCR product yielded more frequent integration into the arg5::HIS1 allele than into the ARG5 allele. A similar bias, though less severe, was observed during disruption of ADE2. The bias may reflect a greater recombination efficiency between molecules that have more extensive homology: the ends of the arg5::URA3 PCR product have 160 and 210 bp of homology to the arg5::HIS1 allele but 60 bp (at each end) to the ARG5 allele. Also, our use of unpurified PCR primers for creation of disruption constructs may contribute to allelic integration bias: contaminating primers with 5′ truncations will yield PCR products with little or no homology to a wild-type allele. In practice, this problem is not a significant impediment because undesired integrants may be eliminated by selection. Where cost is not a factor, the problem might be eliminated entirely through use of nonhomologous primers for amplification of each disruption cassette.
The main value of a rapid gene disruption method in C. albicans
is to provide functional information about a sequence before investing significant effort in its characterization. In this study, we examined the function of two C. albicans
genomic segments that were identified through Blast searches of the genomic sequence database. They were relatively short for traditional gene disruption strategies (HRM101
, 627 bp; ENX3
, 753 bp); in addition, both sequence records include uncertain nucleotides, so their isolation by PCR amplification might be difficult. Both sequences are also too short to specify an entire protein. By primer-directed disruption, we created homozygous mutant strains within 4 weeks of sequence identification. Our hypothesis predicted that hrm101
homozygotes would have similar phenotypes, and this prediction was verified. However, we might have found that enx3
homozygotes have different phenotypes, thus suggesting that the respective gene products have distinct physiological functions. We might have found that one or both homozygotes have no detectable phenotype, thus suggesting that the sequences may be pseudogenes, that they may have functional homologs elsewhere in the C. albicans
genome, or that our phenotypic assays are too crude to detect their function. We might have been unable to isolate homozygous mutants, thus suggesting that the respective gene products may be essential for growth, essential for recovery from transformation, or perhaps essential for completion of recombination. Each of these possible outcomes would affect our priorities for further characterization of a possible RIM101
pathway in C. albicans
. Also, the outcomes might change our view of the RIM101
pathway in S. cerevisiae
). Thus it is extremely valuable to be able to characterize a homozygous mutant at the start of a research effort.
Our information about HRM101 and ENX3 function is preliminary, because we have not complemented or reverted the defects. It is formally possible that the phenotypes of mutant strains arise from coincident mutations that were inadvertently isolated during transformation. However, the finding that three independent hrm101/hrm101 homozygotes have a filamentation defect supports the idea that the phenotype arises from the hrm101 mutation, not from a coincidental secondary mutation. A similar argument applies to the two enx3/enx3 homozygotes. These observations provide preliminary support for the idea that HRM101 and ENX3 sequence fragments are parts of genes, that they specify products, and that Hrm101p and Enx3p may have related functions.