Table 1.

Summary of methods for disruption of ALS5, ALS6 or ALS7 in C. albicans^a

PCR Deletion Cassetteb				hisG-URA3-hisG Deletion Cassettec				Reintegration of Wild-Type Alleled				Southern Blot Probe
			alsΔ/ALS				alsΔ/alsΔ			alsΔ/alsΔ::□□□
Gene	Allelee	Primer Namesf	Strain Name	Allele	Upstream Primersf	Downstream Primersf	Strain Name	Allele	Upstream and Coding Region Primersf	Downstream Primersf	Strain Name	Primer Namesf
ALS5	N/Ag			ALS5SA	ALS5upF ALS5upR	ALS5dnF ALS5dnR	1447					ALS5gfpF ALS5gfpR
				ALS5LA			2373	ALS5SA	ALS5upF ALS5R	ALS5dnF ALS5dnF	2407
ALS6	ALS6-1	ALS6-5DR ALS6-3DR	1268	ALS6-2	ALS6upF ALS6upR	ALS6dnF ALS6dnR	1420	ALS6	ALS6upF ALS6R	ALS6dnF ALS6dnR	1522	ALS6gfpF ALS6gfpR
ALS7	ALS7-1	ALS7-5DR ALS7-3DR	1275	ALS7-2	ALS7upF ALS7upR	ALS7dnF ALS7dnR	1429	ALS7	ALS7upF ALS7R	ALS7dnF ALS7dnR	1964	ALS7gfpF ALS7gfpR

^aAll deletions were constructed in strain CAI4 (iro1-ura3Δ::λimm⁴³⁴/ iro1-ura3Δ::λimm⁴³⁴) [7], which was a generous gift from William Fonzi (Georgetown University).

^bAmplification of a deletion cassette using plasmid pDDB57 as template was described by Wilson et al. [8]. Plasmid pBBD57, which contains a PCR-amplifiable copy of a deletion cassette that encodes a URA3 selectable marker and direct-repeat flanking sequences to promote excision of the cassette, was provided by Aaron Mitchell (Columbia University).

^cThis method for gene deletion was described by Fonzi and Irwin [6]. Deletion of ALS genes uses plasmid pHUL [3], which is a modified version of pMB7 [7] that contains HindIII-AvrII-XhoI-SpeI restriction sites 5′ of the hisG-URA3-hisG cassette, and KpnI-SstII-NgoMIV-SstI sites downstream of the cassette. To make a disruption cassette, sequences upstream and downstream of the target gene are cloned into pHUL using the AvrII-XhoI and SstII-NgoMIV restriction sites, respectively. The final cassette is excised by AvrII-NgoMIV digestion prior to transformation into a Uri^- strain.

^dReintegration of a wild-type ALS allele was accomplished using plasmid pUL [3]. Plasmid pUL differs from pHUL in that the hisG-URA3-hisG cassette in pHUL is replaced by URA3 in pUL.

^eIn SC5314, the sequence of the ALS5 alleles is nearly identical except that the small allele (ALS5SA) has 4 tandem copies of the repeated sequence within the central domain while the large allele (ALS5LA) has 5 tandem copies [8]. ALS6 alleles each encode 4 tandem repeat copies and are likely to have only minor sequence differences [9]. SC5314 ALS7 alleles were described by Zhang et al. [10] and have the same number of tandem repeat copies and same configuration of repeated sequences within the 3′ domain.

^fPrimer names correspond to sequences shown in Table 2.

^gN/A = not applicable. This method was not used to disrupt this gene.