FIG. 5.

Southern blot and PCR analysis of traM homologues in various A. tumefaciens strains. (A) Southern blot hybridization of nopaline-type strains (C58, C58C1RS, K749, and K588) and octopine-type strains (Ach5C3 and A6) using traM probe under low-stringency conditions (Materials and Methods). (B) Designing PCR primers to amplify the traM homologue from chromosomal DNA based on the traM flanking sequences in pTiA6. The relative locations of the final PCR primer pair used for amplification in panel C are indicated by arrows. (C) PCR fragments amplified from different strains as indicated in panel A. The 0.9-kb band was the expected DNA fragment amplified from individual octopine-type Ti plasmid, which was absent from three Ti plasmid-free strains, i.e., C58C1RS, K749, and Ach5C3. The band could not be amplified from Ti plasmid-containing nopaline strain C58 because of low homology in the region. The about 0.4-kb bands from strain A6 and Ach5C3 were shown to contain traM homologues. The similarly sized nonspecific bands were also amplified from the 4 nopaline-type derivatives (see text). (D) High-stringency Southern blot analysis using traM2 as probe. In panels C and D, the samples used were the same as in panel A.