Fig. 1.

Construction and Southern blot verification of C. albicans als4Δ/als4Δ and als4Δ/ALS4SA strains. The locations of the primers from Table 2 are shown by numbers in parentheses. Numbers placed above the line drawings denote forward primers; reverse primers are shown below the line drawings. (a) ALS4 alleles from strain SC5314. The length of the alleles differs due to variation in the number of tandem copies of the 108 bp sequence in the central domain. ALS4 alleles in strain SC5314 are approximately 6300 bp (large allele; LA) and 4569 bp (small allele; SA). (b) The hisG-URA3-hisG cassette constructed by cloning PCR-amplified ALS4 upstream and downstream sequences on either side of the parent cassette. (c) The ALS4 locus with the disruption cassette integrated to replace the ALS4LA coding region as in strain 1517. Growth of this strain on 5-FOA, followed by transformation of the resulting strain with the disruption cassette shown in (b), eliminated the second ALS4 allele to create strain 2034. (d) Construct built to replace a wild-type copy ALS4SA in the mutant strain. (e) ALS4 locus with the replacement wild-type allele integrated. (f) Southern blot of Bg/II-digested genomic DNA from the indicated strains hybridized with the ALS4 upstream probe to demonstrate correct strain construction. Molecular sizes (in kb) are shown on the left. Cross-hybridization between sequences upstream of ALS2 and ALS4 resulted in the appearance of the band above 12 kb, which corresponds to ALS2.