Expression of lexA targeted ribozyme in Escherichia coli BL-21 (DE3) cells |
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| Authors: | Ramesh Singh Yadava Ravindra Kumar Pramod Kumar Yadava |
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| Institution: | (1) Md-Path Pathology Admin, University of Virginia, USA;(2) Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India;(3) Present address: 403 Wartik Laboratory/104 Chemistry Building, The Pennsylvania State University, University Park, PA, U.S.A. |
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| Abstract: | 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. (Mol Cell Biochem 271: 197–203, 2005) |
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| Keywords: | hammerhead ribozyme lexA SOS response E coli |
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