Construction of a vector for the study of regulatory mechanism of gene expression and its utilization in the melibiose operon of Escherichia coli.
We constructed a vector to evaluate terminator or attenuator of transcription quantitatively. This vector is a plasmid possessing lac promoter-polylinker-CAT(chloramphenicol acetyltransferase) gene. DNA fragment of interest can be inserted into the polylinker site, and the effect of the DNA fragment on the expression of the downstream gene is evaluated from the CAT activity. We analyzed gene expression of the melibiose operon of Escherichia coli using this plasmid, and found that a DNA fragment containing a large stem-loop structure with boxA sequence in it, which was present between melA and melB, caused strong reduction of gene expression. This DNA portion seems to be responsible for reduced expression of melB, the second gene of the melibiose operon.
Shimamoto
T
T
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Noguchi
K
K
Kuroda
M
M
Tsuda
M
M
Tsuchiya
T
T
D003001:Cloning, Molecular / Q000379:methods; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005822:Genetic Vectors; D008553:Melibiose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics; D009876:Operon; D027981:Symporters; D014158:Transcription, Genetic; D000519:alpha-Galactosidase / Q000235:genetics
D004187:Disaccharides; D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D000519:alpha-Galactosidase