Figure 1.

Disruption of stuA. (A) Split marker strategy for the deletion of stuA. Two deletion fragments were generated by successive rounds of PCR. First flanking regions were amplified containing M13 sequences at the stuA flanking ends. Next, a fusion PCR was performed using each fragment and the HYG resistance cassette, both containing homologous M13 sequences, as template to generate the final deletion fragments. Finally, Af293 was cotransformed with the two deletion fragments. A triple crossover event resulted in replacement of stuA by the HYG resistance cassette. F1, F2, F3, F4, HY, and YG, primers used for amplification. HYG, hygromycin resistance cassette amplified from pAN7-1. (B) Northern blot of Af293 and the stuA null mutant strain. Total RNA from hyphae grown in YPD at 37°C was probed with the entire open reading frame of stuA, confirming an absence of stuA mRNA in the stuA null mutant strain.