FIG. 3.

tet(M) is associated with Tn916 and integrates as a single copy through a circular intermediate form. (A) PCR amplification of Tn916 regions. Lane 1, 1-kb DNA ladder ranging between 250 bp and 10 kb; lane 2, tet(M) (primer pair I); lane 3, int (primer pair II); lane 4, tet(M)-int (primer pair III). The numbering of the primer pairs and their positions along Tn916 are given in Table 1 and Fig. 1. (B) Southern blot analysis of EcoRI-digested genomic DNA, probed with 16S (left panel) and tet(M) (right panel) gene fragments. (C) Lane 1, HindIII-digested lambda phage DNA size marker; lane 2, 1-kb DNA ladder plus DNA marker (100 bp to 12 kb); lane 3, 1-kb DNA ladder (250 bp to 10 kb); lane 4, PCR amplification of total DNA with primer pair IV (Table 1). (D) DNA sequence of the joined termini of the L. paracasei Tn916 circular intermediate, obtained using the amplicon in panel C, lane 4, as a template. The coupling sequence (GGCAAA) and the interrupted DraI restriction site (TTTAAA) are indicated with larger lettering. Arrows indicate the initial and terminal nucleotides of the linear Tn916 sequence (GenBank accession no. U09422).