Expression of lexA targeted ribozyme in Escherichia coli BL-21 (DE3) cells.
Coding sequences for a hammerhead ribozyme designed to cleave lexA mRNA in a targeted manner was cloned under phage T7 promoter and expressed in E. coli strain BL-21 (DE3) expressing T7 RNA polymerase under the control of IPTG-inducible lac UV-5 promoter. Ribozyme expression in vivo was demonstrated by RNase protection assay. Also, total RNA extracted from these transformed cells following induction by IPTG, displays site-specific cleavage of labeled lexA RNA in an in vitro reaction. The result demonstrates the active ribozyme in extracts of cell transformed with a recombinant cassette and goes beyond the earlier demonstration of the stability of in vitro synthesized ribozyme in cell extracts. The observed rise in lexA mRNA rules out any role for protease activity or resulting fragments of lexA protein in de-repression of RNA.
Yadava
Ramesh Singh
RS
Md-Path Pathology Admin, University of Virginia, USA.
Kumar
Ravindra
R
Yadava
Pramod Kumar
PK
D001426:Bacterial Proteins / Q000235:genetics* / Q000528:radiation effects; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000528:radiation effects; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D012321:DNA-Directed RNA Polymerases; D007544:Isopropyl Thiogalactoside; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012697:Serine Endopeptidases; D014764:Viral Proteins; D000099160:LexA Repressor Protein; C427204:hammerhead ribozyme; C062210:bacteriophage T7 RNA polymerase