FIG. 6.

Southern blot evidence for pMAD10-mediated URA5_Hc gene disruption in strain G184AS. Genomic DNAs from transformation recipient strain G184AS (lanes 1) and the four hygromycin B-resistant, uracil-auxotrophic transformants (lanes 2 to 5) were digested with ClaI, electrophoresed, Southern blotted, and probed with radiolabeled fragments of the URA5_Hc gene, the 0.4-kb internal URA5_Hc fragment deleted from pMAD10, and the inserted selectable marker hph. The four uracil auxotrophs (lanes 2 to 5) have lost the native genomic band that hybridizes with the URA5_Hc probe and gained a new, larger band due to hph insertion that hybridizes with both the URA5_Hc and hph probes, as well as failing to show any hybridization with the 0.4-kb fragment deleted from the disruption construct pMAD10. Fragment sizes were estimated by comparison to molecular size standards in the ethidium bromide-stained gel.