FIG. 1.

Disruption of Tri16 in F. sporotrichioides NRRL 3299. (A) Expected result for insertion of the Tri16 gene disruption construct pKO16 into the host genome via a single homologous integration event. B, BglII restriction sites. (B) Southern blot analysis of selected transformant strains. Wild-type (WT) and transformant genomic DNAs were cut with BglII, and blots were probed with a doubly truncated fragment of the Tri16 gene. M, lambda DNA cut with HindIII. All transformants except Tx #22 lack the wild-type Tri16 BglII fragment. Instead, transformants Tx #7 and Tx #20 contain a single fragment consistent with the expected combined size (approximately 13 kb) of the wild-type Tri16 BglII fragment and one homologously integrated copy of pKO16. The remaining transformants contain a larger fragment indicative of the presence of two (expected size, approximately 20 kb) or more tandem, homologously integrated copies of pKO16. Band sizes (bases) of the marker are shown at the right. (C) PCR analysis of wild-type and selected transformant strains. PCRs were performed with primers A-90 and A-91 and primers A-90 and S-12 (positions are shown in panel A). PCR products were resolved on an agarose gel and stained with ethidium bromide. M, lambda DNA cut with BstEII. Band sizes (bases) of the marker are shown at the left, and band sizes of the PCR products are shown at the right.