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. 2006 Jul;188(13):4871–4878. doi: 10.1128/JB.00210-06

FIG. 2.

FIG. 2.

Analysis of the target site choices of Tn5397 in B. subtilis. (A to D) Schematic showing the insertion of Tn5397 into different target sites used here. The annotations are as follows: the light-blue shaded boxes represent the target inserted into the B. subtilis chromosome and the 5′GA dinucleotide that is thought to be the crossover region. The thin bands represent the flanking vector sequence. The expected product(s) after the conjugation and insertion of Tn5397 (dark blue), flanked by attL and attR, is shown underneath. The tndX gene was used as a probe in Southern blotting experiments with HindIII-digested chromosomal DNA. HindIII has one recognition site in tndX. If Tn5397 had inserted into any of these targets, two bands would hybridize to the probe (as shown). The different target sites used were attBCd (A), attBCd2 (B), attBCd(TC) (C), and the 50-bp attB target, attBCd50 (D). Panels E and F show results from Southern blotting of transconjugants containing various insertions of Tn5397 at different target sites. The DNA was digested with HindIII and probed with tndX. For clarity, only one clone from each cross is shown here. (E) Lane 1 shows DNA from an insertion into attBCd (see panel A); lanes 2 and 3 show DNA from an insertion into attBCd (see panel B); lane 4 shows DNA from an insertion into attBCd(CT) (lane 5) DNA in which the target site had been reduced to 50 bp (see panel D). (F) Southern blot analysis of B. subtilis DNA (containing unmodified pSWEET integrated into the chromosome) from six transconjugants containing insertions of Tn5397 isolated from independent mating experiments. If the element were integrating into the genome at random, one would expect to see two hybridizing fragments, one of 5,425 bp internal to Tn5397 and one of variable size representing the junction region.