利用转座子进行双向引物介导的克隆DNA快速测序：大肠杆菌K-12 avtA基因的序列
Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene.
作者信息
Liu L, Whalen W, Das A, Berg C M
机构信息
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
出版信息
Nucleic Acids Res. 1987 Nov 25;15(22):9461-9. doi: 10.1093/nar/15.22.9461.
Abstract
A new approach to determining the sequence of cloned DNA is described. Unique regions near each end of the transposable element gamma-delta provide a pair of "portable" primer-specific sites for bidirectional sequencing by the dideoxy chain termination method. A set of gamma-delta insertions positioned about 200 bp apart over the entire cloned DNA allowed us to determine the sequence of both strands in a single parental plasmid without subcloning. The avtA (alanine-valine transaminase) gene of E. coli K-12 was sequenced by this approach. Surprisingly, gamma-delta insertions downstream of the coding region were found to significantly reduce avtA expression. We suggest that these nondisruptive insertions probably change the DNA topology and thereby alter gene expression.
摘要
本文描述了一种确定克隆DNA序列的新方法。转座因子γ-δ两端附近的独特区域为双脱氧链终止法双向测序提供了一对“便携式”引物特异性位点。在整个克隆DNA上相隔约200 bp定位的一组γ-δ插入片段使我们能够在不进行亚克隆的情况下确定单个亲本质粒中两条链的序列。通过这种方法对大肠杆菌K-12的avtA（丙氨酸-缬氨酸转氨酶）基因进行了测序。令人惊讶的是，发现在编码区下游的γ-δ插入片段会显著降低avtA的表达。我们认为这些非破坏性插入可能改变了DNA拓扑结构，从而改变了基因表达。
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