FIG. 2.

Identification of an A. fumigatus transformant carrying the PpksP-egfp fusion integrated in single copy at the pksP gene locus. (A) Partial restriction map of the genomic region containing pksP and schematic representation of the integration of a single copy of PpksP-egfp. The arrows indicate the orientations of the genes. B and S, BamHI and SacI restriction sites, respectively. Sizes of DNA fragments to which the probe hybridizes and which are important for the molecular characterization of transformants are indicated. arp1 and arp2 are located in the vicinity of pksP and encode scytalone dehydratase and 1,3,6,8-tetrahydroxynaphthalene reductase, respectively. The products of both genes are also involved in 1,8-tetrahydroxynaphthalene–melanin biosynthesis (27, 28). (B) Southern blot analyses. Chromosomal DNAs of the wild-type strain and transformant strains were cut by either BamHI or SacI, as indicated. The DNA was hybridized with a DNA probe of 2.0 kb generated by PCR amplification using oligonucleotides KLNBaB and KLupsNF and chromosomal DNA of the wild-type strain ATCC 46645 as the template. Transformant AfPKSPEGFP4 (lanes 4) showed the expected hybridization pattern. The band characteristic of the wild type (lanes 3) had disappeared and two new bands had appeared, indicating that the gene fusion was integrated in single copy at the chromosomal pksP gene locus. The transformants in lanes 1 and 5 carry the gene fusion integrated in double copy at the chromosomal pksP gene locus. Numbers on the right and left indicate approximate sizes of hybridizing bands in kilobase pairs. Lanes: 1, transformant AfPKSPEGFP2; 2, transformant AfPKSPEGFP3; 3, wild-type strain ATCC 46645; 4, transformant AfPKSPEGFP4; 5, transformant AfPKSPEGFP5.