Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR.
Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins.
Rao
Shilpakala Sainath
SS
Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, 600 113, India.
Savithri
H S
HS
Raghunathan
Malathi
M
D027081:DNA Gyrase / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D016372:RNA, Antisense / Q000737:chemistry*; D012329:RNA, Bacterial / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043262:Ribonuclease P / Q000378:metabolism
D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D043262:Ribonuclease P; D027081:DNA Gyrase
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