Figure 6
Configuration of the excision products at 37°C. (a) Southern blot analysis of excision products obtained by incubating pBC-3Kana3 with MOS1 at 37°C, for 15 (lanes 1 and 5), 30 (lanes 2 and 6), 60 (lanes 3 and 7), and 120 minutes (lanes 4 and 8). After deproteinizing, DNA samples were digested (lanes 5 to 8) by ClaI. Products were finally loaded onto 0.8% agar gel (20 × 24 cm), separated by electrophoresis, and blotted onto Nylon membrane. Hybridization was done with a P32-labelled fragment containing the kanamycin resistance gene. Ip: integration products, OC: open circle, Lin: linear; SC: super-coiled. Molecular weights (MW) are indicated in the left margin. Grey diamond: linear excised 3Kana3. Black diamond: circular excised 3Kana3. (b) Nucleic acid sequence of the kanamycin resistance gene cloned within the pBC-3Kana3. The motifs used to design the KanaSal1 and KanaHind3 primers are boxed at both ends of the sequence (Table 3). Motifs highlighted in black with the sequence typed in white were used to design the primers KanaJunc1 and KanJunc2 (Table 3). The start and stop codons of the kanamycin resistance gene are highlighted in dark gray and typed in white. The Cla I site is typed in black and boxed in gray. (c) Diagrammatic representation of the excision products obtained after incubation of pBC-3Kana3 with MOS1 at 37°C. Three forms of the excised transposon are drawn: linear (1) and circular (2) molecules containing a full-length transposon, and (3) circular molecules containing a truncated transposon, due to intra-molecular auto-integration after excision. ITR are represented by light gray arrows and promoters by 90°-angled arrows. KanaR: Kanamycin resistance gene. ChlR: CAT gene. (d) Sequences of the twelve excision products analyzed. The 3'ITR is in bold uppercase letters. On the right, the pBC MCS is in lowercase letters. On the left, the dinucleotide of the auto-integration site is in gray-boxed uppercase letters. Numbers flanking the insertion sites correspond to the position of insertion in the 3Kana3 pseudo-Mos1. They are the same as in (b). The sequence in 5' of the insertion site corresponds to the inner sequence for 3Kana3.