Figure 4.

φC31 integrase only catalyzes attP/B recombination. Plasmids encoding attP (pHS20 or pHS22), attB (pHS21 or pHS23), attL (pHS50), or attR (pHS52) or attL and attR together (pHS55) were used as substrates for recombination with φC31 integrase in vitro. Recombinant products were obtained only in lane 2 that contained attP and attB as substrates, and the reaction is the same as that shown in Fig. 3. If recombination had occurred, the predicted recombinant products between attP/attP (lane 3) and attB/attB (lane 4) cut with NsiI and PstI and between attB/attL (lane 7) and attB/attR (lane 8) cut with BamHI would have been 6 kbp and 0.4 kbp, respectively. For attP/attL (lane 5) and for attP/attR (lane 6) recombination, the predicted products would have been 6.4 kbp and 0.4 kbp when cut with XhoI and ApaI, respectively. For attL/attR (lane 9) on different plasmids, the predicted product would have been 3.6 kbp when cut with SphI and SmaI, and when attL–attR (lane 10) were on the same plasmid the predicted recombinant product would have been 0.4 kbp detected by restriction with KpnI. Note that pHS52 cut with SphI and SmaI (lane 9) yielded a parental band of a size (3.4 kbp) similar to the predicted recombinant product in the pHS50/pHS52 reaction, but still no recombinant could be detected in gels that had undergone electrophoresis for a longer period. Note also that the 0.4-kbp SphI-SmaI fragment in pHS50 is a parentally derived fragment. Molecular weight markers (M) are as in Fig. 2.