Fig. 1. Construction of integration vector and isolation of cadA-null strains. (A) The 3.8 kb genomic DNA containing the cadA gene. The three black rectangles represent the three exons encoding DdCAD-1. Several unique restriction enzyme cut sites are also indicated. (B) The sequential steps involved in the construction of the cadA-null mutant strains. First, the genomic fragment containing the cadA gene was circularized and then cut at the HincII site. The linearized fragment was ligated to the SmaI site of the integration vector pbsrEco to generate pbsr/cadA. Next, the plasmid DNA was cut with EcoRI and used to transfect KAX3 cells by electroporation in the presence of excess EcoRI. Gene disruption was achieved by homologous recombination between the endogenous DNA and the plasmid DNA. (C) Protein blot analysis of blasticidin-resistant mutant clones. Total cell protein from 2x10⁵ cells collected at 3 hours of development was separated by SDS-PAGE and the protein blots were probed with rabbit antibodies raised against DdCAD-1. (D) DNA blot analysis of the two cadA^– clones. Genomic DNA isolated from wild-type or cadA^– cells was digested with EcoRI and then separated by electrophoresis in a 1% agarose gel and the DNA blot was hybridized with either ³²P-labeled cadA cDNA or pbsrEco DNA.