Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene.
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.
Liu
L
L
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
Whalen
W
W
Das
A
A
Berg
C M
CM
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D000637:Transaminases / Q000235:genetics*
D004251:DNA Transposable Elements; D004247:DNA; D000637:Transaminases; C034522:valine-pyruvate transaminase