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Nucleic Acids Research, 1987, Vol. 15, No. 22 9461-9469
© 1987

Articles

Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene

Lin Liu, William Whalen1, Asis Das1 and Claire M. Berg*

Department of Molecular and Cell Biology (U-131), University of Connecticut Storrs, CT 06268, USA 1Department of Microbiology, University of Connecticut Health Center Farmington, CT 06032, USA

*To whom reprint requests should be sent

Received September 14, 1987. Accepted October 22, 1987.

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.


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