FIG. 5.

Construction and Southern blot analysis confirming replacement of PsADH genes. Linear fragments carrying disrupted copies of the PsADH1 (pJY102) (A) and PsADH2 (pJY202) (B) genes were constructed and introduced into the genome as described in the text. These disruption constructs contain approximately 1.0 kbp of 5′- and 3′-flanking PsADH1 (A) and PsADH2 (B) homology. Bold lines represent fragments containing 5′- and 3′-flanking regions used for gene replacement. White boxes indicate PsADH1 (A) and PsADH2 (B) coding regions, and striped boxes represent functional LEU2 (A) and URA3 (B) genes from P. stipitis. Southern hybridization results are shown at the bottom. Strain CBS 6054 (lane 1) is the P. stipitis wild type. P. stipitis PSU1 (lane 2) and PLU20 (lane 3) are parental strains used for PsADH1 and PsADH2 gene replacements, respectively. PSU218 (lane 4) and PLU123 (lane 5) are strains disrupted in PsADH1 and PsADH2, respectively. PLU1209 (lane 6) is disrupted in both PsADH1 and PsADH2.