Figure 1.

I-SceI DSB induction and chromosomal survey device. (A) I-SceI mediated DSB targeting in S. ambofaciens chromosome. The target sequences for I-SceI meganuclease are carried by the IKI cassette designed with neo gene, conferring resistance to kanamycine, flanked by two convergent I-SceI restriction sites (dark triangle). Red and blue boxes represent left and right replichores of the chromosome. Grey arrows represent the terminal inverted repeats at each chromosome extremity. The IKI cassette was integrated either in the core genome (IKI-C, position 4.94 Mb), in the left chromosomal arm (IKI-L, position 0.32 Mb) or in the right chromosomal arm (IKI-R, position 7.91 Mb). The cassette was inserted at IKI-L or IKI-R loci through homologous recombination. The IKI-C locus corresponds to the attachment site of phage φBT1 (attB φBT1) where pMS82 conjugative plasmid carrying IKI cassette (therefore named pMS82-IKI) was integrated. Meganuclease encoding I-SceI gene was carried by pDYN6902 conjugative plasmid and integrated in the chromosome at the attachment site of phage φC31 (attB φC31). Scissors symbolize the DSB (I-SceI restriction sites) triggered by the meganuclease. (B) Distribution of the loci used for chromosome mapping after DSB induction. T stands for loci included in the TIRs. L, C and R for left, central and right loci, respectively. The data analysis is schematically represented on Fig. 4.