FIG. 1.

Construction of the aflR disruption vector pHXSE. The aflR disruption vector pXHSE was constructed by ligating a 2.8-kb SacI-HindIII fragment of pHX1 containing A. parasiticus aflR (3) into the corresponding sites in pUC18 to generate pACH. A 6.7-kb XbaI fragment of pSL82 (10) was subcloned into the XbaI site of a pUC18 derivative from which the HindIII site had been removed. Then, the HindIII site of a 6.7-kb XbaI fragment in the niaD-niiA intergenic region (2) was eliminated by partial digestion with HindIII and self-ligation, yielding pXN1, which contained only one HindIII site. The 6.7-kb niaD-containing HindIII-XbaI fragment of pXN1 was blunt ended with T4 DNA polymerase, ligated to pACH, pretreated with SmaI and EcoRV, blunt ended, and dephosphorylated to yield pXHSE. EV, EcoRV; H, HindIII; S, SalI; Sm, SmaI; Sp, SphI; X, XbaI.