Chatterjee P K, Briley L P
Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, North Carolina 27707, USA.
Anal Biochem. 2000 Oct 1;285(1):121-6. doi: 10.1006/abio.2000.4750.
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.
通过将含loxP位点的Tn10微型转座子插入重组克隆并用P1噬菌体转导，可轻松从细菌人工染色体（BAC）和P1人工染色体（PAC）的基因组DNA一端产生嵌套缺失。尽管缺失系列中克隆的大小在很大程度上是随机的，但在约5%的BAC和PAC中，分布似乎偏向于某个长度，在极少数情况下（<1%）则明确偏向于特定大小。在此，我们研究了这种相对罕见的现象，并证实序列特异性转座子插入并非此类偏向性嵌套缺失文库的原因。相反，对一个显示出这种异常特征的BAC克隆实验的详细分析表明，特定大小的缺失源于转座子插入的克隆扩增，这是由于在IPTG诱导之前转座酶基因的瞬时去抑制所致。转座本身对克隆中插入DNA的任何特定区域均无偏向性。我们建议对产生嵌套缺失的程序进行一项简单修改，以使所有BAC和PAC都能产生随机大小的嵌套缺失。这些发现应能为其他可诱导转座子系统的基因组克隆中的位点选择性原因提供更多见解。
