title: Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR.
abstract: 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.
authors: Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, 600 113, India.
authors: Shilpakala Sainath
authors: SS
authors: Rao
authors: H S
authors: HS
authors: Savithri
authors: Malathi
authors: M
authors: Raghunathan
mesh_terms: 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
chemical_list: D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D043262:Ribonuclease P; D027081:DNA Gyrase
references: Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8468-72
references: 9238000
references: Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11115-9
references: 7479948
references: Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10898-900
references: 8248189
references: RNA. 1998 Nov;4(11):1397-406
references: 9814760
references: Antimicrob Agents Chemother. 1989 Jun;33(6):886-94
references: 2548439
references: Methods Enzymol. 2000;313:442-56
references: 10595372
references: Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6605-10
references: 11381134
references: Science. 1993 Aug 20;261(5124):1004-12
references: 8351515
references: Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8006-10
references: 1381505
references: J Mol Biol. 1989 Jan 5;205(1):1-13
references: 2538621
references: Methods Enzymol. 1990;185:60-89
references: 2199796
references: Antisense Nucleic Acid Drug Dev. 1998 Oct;8(5):415-26
references: 9826268
references: Biochim Biophys Acta. 1998 Oct 1;1400(1-3):29-43
references: 9748489
references: Science. 1994 Mar 4;263(5151):1269-73
references: 8122108
references: J Bacteriol. 1985 Oct;164(1):434-45
references: 2995318
references: FASEB J. 1993 Jan;7(1):7-14
references: 7916700
references: Genomics. 1993 Nov;18(2):418-22
references: 7507079
references: Nat Biotechnol. 2000 Jan;18(1):58-61
references: 10625392
references: Chem Biol. 1999 Feb;6(2):R33-7
references: 10021422