Figure 7

Determination of minimal attB and attP lengths required for efficient recombination. (A) Sequence of the resected attP substrates and their abilities to support in vitro intermolecular integration (for example (C)) or in vivo intramolecular deletion (for example (E)). Symmetry-related segments denoted above the attP and attB sequences, and the GG dinucleotide crossover segments (see Figure 8) are boxed. In vitro data: (+), >60% recombinants; (−), little–no detectable products in 40-minute reactions. In vivo data: (+), >90% white colonies (deletion products); (−), <10% white colonies. Sequences in light grey are absent in the in vitro reactions and replaced with different nucleotides in the in vivo reactions. (B) Sequence of the resected attB substrates and their abilities to support in vitro intermolecular integration (for example (D)) or in vivo intramolecular deletion. Activities are designated as in (A); in vitro reaction rates for attB 41 (+/−) were about 15% of those of the longer substrates. NA, not analyzed. (C), (D) Representative gels showing in vitro intermolecular integration reactions (40 minutes) between supercoiled attB (pRJ2215) and resected attP fragments (C) or between supercoiled attP (pRJ2214) and resected attB fragments. (E) Illustration of the in vivo attP×attB deletion reaction with pBCPB-A1+ [31]. Deletion generates two circular products that may be topologically linked, but only the lacZ^–Cam^r product containing the replication origin will be maintained.