FIG. 4.

Construction of the plasmid for cloning and disruption of epoA. A HindIII-XbaI fragment (2.3 kb) containing epoA was isolated from cosmid 7 and inserted into pUC119 to generate pFOM3. pFOM4 was prepared by inserting the hph cassette [on an XbaI fragment isolated from pDH25Xba after EcoRI/XbaI linkers were added to pDH25 (3)] into the XbaI site of pFOM3 located downstream of epoA. To prepare pFOM13, pFOM3 was digested to eliminate an HpaI-BglII fragment encoding about 400 bp of the epoA gene. The digested plasmid was filled in and ligated to the hph cassette (isolated from pDH25Xba as a filled-in XbaI fragment). pFOM XbaI/HindIII fragments are labelled A, B, and C (observed in Southern blot of Fig. 5). Abbreviations: PtrpC, the A. nidulans trpC promoter region; TtrpC, the A. nidulans trpC terminator region; hph, the E. coli hygromycin B phosphotransferase gene.