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. 2002 Mar;68(3):1232–1239. doi: 10.1128/AEM.68.3.1232-1239.2002

FIG. 3.

FIG. 3.

Construction of XYL3 gene disruption cassette and confirmation of XYL3 disruption by PCR and Southern blotting. (A) Systematic representation of the disruption cassette (pYS03). The linear disruption cassette was amplified by PCR from pYS03. It was then introduced into the chromosome by gene replacement. The pYS03 contains 1.2 kbp of the P. stipitis URA3 gene in the middle of the XYL3 coding region. (B) PCR with specific primers for amplification of the 1.7-kbp XYL3. The templates were used as follows: lane 1, markers; lane 2, P. stipitis FPL-UC7 genomic DNA; lane 3, P. stipitis FPL-YS30 genomic DNA. (C) Southern blot analysis of XYL3 disruption with the probe made from the XYL3 coding region. The genomic DNAs from P. stipitis FPL-UC7 (lane 1) and P. stipitis YS30 (lane 2) were digested with XmnI. The parental strain, P. stipitis FPL-UC7, showed a 2.2-kbp band and the XYL3 disruptant, P. stipitis FPL-YS30 (xyl3::URA3), showed a 3.4-kbp band. Positions of size markers are shown to the left.