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 chromosomes (BACs) and P1 artificial chromosomes (PACs) are readily generated by inserting a loxP site-containing Tn10 minitransposon into the recombinant 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 distribution appears skewed to a certain length, and in rare cases (<1%) is definitely skewed to a particular size. Here we investigate this relatively rare phenomenon and validate that sequence-specific transposon insertions are not the cause of such skewed nested-deletion libraries. Instead, a detailed analysis of our experiments with a BAC clone demonstrating this unusual feature indicates that deletions of a certain size arise from clonal expansion of a transposon insertion as a result of transient derepression of the transposase gene prior to IPTG induction. Transposition itself shows no bias to any particular region of insert DNA in the clone. We suggest a simple modification to the procedure for generating nested-deletions that allows all BACs and PACs to produce nested-deletions of random size. These findings should provide additional insight into the causes of site selectivity in genomic clones with other inducible transposon systems.
Chatterjee
P K
PK
Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, North Carolina 27707, USA. pchatterjee@wpo.nccu.edu
Briley
L P
LP
D022201:Chromosomes, Artificial; D022202:Chromosomes, Artificial, Bacterial; D004251:DNA Transposable Elements; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D017384:Sequence Deletion
D004251:DNA Transposable Elements; D007544:Isopropyl Thiogalactoside