Analysis of a clonal selection event during transposon-mediated nested-deletion formation in rare BAC and PAC clones

Nested deletions from one end of the genomic DNA in bacterial artificial ch romosomes (BACs) and P1 artificial chromosomes (PACs) are readily generated by inserting a loxP site-containing Tn10 minitransposon into the recombina nt clone and transducing with P1 phage, Although the size of clones in the deletion series is largely random, in about 5% of BACs and PACs the distrib ution appears skewed to a certain length, and in rare cases (<1%) is defini tely skewed to a particular size. Here we investigate this relatively rare phenomenon and validate that sequence-specific transposon insertions are no t the cause of such skewed nested-deletion libraries. Instead, a detailed a nalysis of our experiments with a BAC clone demonstrating this unusual feat ure indicates that deletions of a certain size arise from clonal expansion of a transposon insertion as a result of transient derepression of the tran sposase gene prior to IPTG induction. Transposition itself shows no bias to any particular region of insert DNA in the clone. We suggest a simple modi fication to the procedure for generating nested-deletions that allows all B ACs and PACs to produce nested-deletions of random size. These findings sho uld provide additional insight into the causes of site selectivity in genom ic clones with other inducible transposon-systems, (C) 2000 Academic Press.