Figure 8

Determination of the crossover site. (A) Sequence of the minimal attP and attB sites, with common nucleotides marked with dots. The central three nucleotides are numbered 1 to 3 in this figure. (B) In vitro intramolecular deletion reactions (30 minutes) on supercoiled plasmids with attB sites containing mutations at the central three nucleotides as designated. P×B, wild-type control. The products were digested with Xho I and Bam H1 to reveal deletion products (Figure 1D). (C) Results of in vivo deletion reactions between attP and mutated attB sites. White colonies result from site-specific deletions and blue colonies signify absence of deletions. (D) In vitro intramolecular recombination reactions (30 minutes) with plasmid substrates containing identical changes within attP and attB at the designated positions. Parental and deletion product bands are denoted. (E) Results of in vivo recombination reactions between mutated attP and attB sites. Because G1C and G2C mutations create symmetrical cores, inversions between att sites oriented in an antiparallel configuration (G) form along with deletions. Inversion products retain the Lac⁺ (blue) phenotype. (F) Diagram of synapsis between attP(G1C) and attB(G1C) in the standard parallel orientation generating attL and attR upon DNA exchange. Productive recombination between wild-type attP and attB sites containing the asymmetric GG core nucleotides only occur by this pathway. (G) Diagram of an antiparallel synapsis between attP(G1C) and attB(G1C) generating the hybrid attL* and attR* sites upon DNA exchange. (H) In vitro intramolecular recombination reactions (30 minutes) with plasmid substrates containing identical changes within attP and attB within the core nucleotides. The reaction products were digested with Bam HI, Nde I, and Sca I to reveal both deletions (doublet bands) and inversions as denoted.