Figure 4.

Genomic PCR analyses of transformants obtained from conjugating ATU knockout heterokaryons transformed with a wild-type ATU gene. Conjugating knockout heterokaryons were transformed with linearized pTUB100E3-PvuII (see Fig. 2A). Genomic PCR was performed with primers 5′TUBx and 3′TUBz (shown in Fig. 2A) for two transformants (1 and 2) which survived in pm. The 1.8-kb band of the undigested PCR product could be either the endogenous or the transforming ATU. The 1.2-kb band corresponds to the knockout fragment containing the neo gene. The 1.8-kb band in both transformants completely shifts to small sizes after digestion by either enzyme, indicating that the cells contain only the transforming ATU gene. The 1.2-kb band was also cut by the two enzymes into small fragments as expected. Lane M, PUC19 marker (Biosynthesis, Lewisville, TX).