Promoter-detection vectors for Escherichia coli with multiple useful features.
Two promoter-detection vectors have been constructed which enable the cloning and characterization of promoters recognized by the RNA polymerase of Escherichia coli K-12. The intergenic region of phage M13 DNA, present in opposite orientations in the two vectors, permits the preparation of single-stranded DNA of either strand of the insert thus facilitating oligodeoxyribonucleotide heteroduplex mutagenesis and sequencing of both strands by the dideoxy method of chain termination. After mutagenesis, isolates can be screened for changed function by replica-plating colonies to plates containing XGal. Selected isolates can be characterized by nucleotide sequence analysis, to determine the change in structure, and by beta-galactosidase assays, thus measuring the effect of mutagenesis on promoter function. The vectors could also be used like other protein-fusion vectors.
Malo
M S
MS
Department of Biochemistry, University of Sydney, N.S.W., Australia.
Loughlin
R E
RE
D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D004262:DNA Restriction Enzymes; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic
D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes