Detection of the aphVIII gene and AphVIII protein in transformants. (A-C) Paromomycin-resistant transformants and the parent wild type strain were analyzed by genomic PCR. Only transformants that were produced using plasmid paphG (A) or pPmr3 (B) were expected to yield a 422 bp fragment of the aphVIII gene. Lane M, molecular weight marker. (C) Sequence obtained from cloned aphVIII fragments. The positions of the primers and the stop codon (boxed) are indicated. (D) Southern blot analysis of genomic DNA from transformant T-J37, which was produced using plasmid pPmr3, and from the parent wild type strain. The blot was probed using an aphVIII fragment. H, HindIII; P, PstI; S, SalI. (E) Western blot analysis of cell lysates of T-J37 and the parent wild type strain (middle), and purified AphVIII protein as a reference (right). The anti-AphVIII antibody was used for detection. As a control, the cell lysates were also stained with coomassie blue (left).