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Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR

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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.

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References

  1. Rossi JJ (1999) Ribozymes, genomics and therapeutics. Chem Biol 6:R33–R37

    Article  PubMed  CAS  Google Scholar 

  2. Stein CA, Cheng YC (1993) Antisense oligonucleotides as therapeutic agents–is the bullet really magical? Science 261:1004–1012

    Article  PubMed  CAS  Google Scholar 

  3. Altman S, Kirsebom LA (1999) In: Gesteland RF, Cech TR, Atkins JF (eds) The RNA world. Cold Spring Harbor Lab. Press, Plainview, NY, pp 351–380

  4. Altman S (1993) RNA enzyme-directed gene therapy. Proc Natl Acad Sci USA 90:10898–10900

    Article  PubMed  CAS  Google Scholar 

  5. Ma M, Benimetskaya L, Lebedeva I, Dignam J, Takle G, Stein CA (2000) Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences. Nat Biotechnol 18:58–61

    Article  PubMed  CAS  Google Scholar 

  6. Yuan Y, Hwang ES, Altman S (1992) Targeted cleavage of mRNA by human RNase P. Proc Natl Acad Sci USA 89:8006–8010

    Article  PubMed  CAS  Google Scholar 

  7. Altman S, Kirsebom L, Talbot S (1993) Recent studies of ribonuclease P. FASEB J 7:7–14

    PubMed  CAS  Google Scholar 

  8. Altman S, Wesolowski D, Puranam RS (1993) Nucleotide sequences of the RNA subunit of RNase P from several mammals. Genomics 18:418–422

    Article  PubMed  CAS  Google Scholar 

  9. Guerrier-Takada C, Altman S (2000) Inactivation of gene expression using ribonuclease P and external guide sequences. Meth Enzymol 313:442–456

    Article  PubMed  CAS  Google Scholar 

  10. Levine C, Hiasa H, Marians KJ (1998) DNA gyrase and topoisomerase IV: biochemical activities, physiological role. Biochim Biophys Acta 1400:29–43

    PubMed  CAS  Google Scholar 

  11. Hooper DC, Wolfson JS (1993) Mechanism of quinolone action and bacterial killing. In: Hooper DC, Wolfson JS (eds) Quinolone antimicrobial agents. Am Soc Microbiol, Washington, DC, pp 53–75

    Google Scholar 

  12. Cullen ME, Wyke AW, Kuroda R, Fisher LM (1989) Cloning and characterization of a DNA gyrase A gene from E. coli that confers clinical resistance to 4-quinolones. Antimicrob Agents Chemother 33:886–894

    PubMed  CAS  Google Scholar 

  13. McKinney J, Guerrier-Takada C, Wesolowski D, Altman S (2001) Inhibition of E. coli viability by external guide sequences complementary to two essential genes. Proc Natl Acad Sci USA 98:6605–6610

    Article  PubMed  CAS  Google Scholar 

  14. Gilbert DN, Moellering RC, Sande MA (1998) The sanford guide to antimicrobial therapy. Antimicroial Therapy, Vienna, VA, pp 52–54

    Google Scholar 

  15. Gowrishankar J (1985) Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation. J Bacterial 164:434

    CAS  Google Scholar 

  16. Studier WF, Rosenberg AH, Dunn JJ, Dudendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Meth Enzymol 185:60

    Article  PubMed  CAS  Google Scholar 

  17. Yuan Y, Altman S (1994) Selection of guide sequences that direct efficient cleavage of mRNA by human ribonuclease P. Science 263:1269–1273

    Article  PubMed  CAS  Google Scholar 

  18. Ma MY, Jacob-Samuel B, Dignam JC, Pace U, Goldberg AR, George ST (1998) Nuclease-resistant external guide sequence-induced cleavage of target RNA by human ribonuclease P. Antisense Nucleic Acid Drug Dev 8:415–426

    PubMed  CAS  Google Scholar 

  19. Guerrier-Takada C, Li Y, Altman S (1995) Artificial Regulation of Gene expression in Escherichia coli by RNase P. Proc Natl Acad Sci USA 92:1115–1119

    Article  Google Scholar 

  20. Kawa D, Wang J, Yuan Y, Liu F (1998) Inhibition of viral gene expression by human ribonuclease P. RNA 4:1397–1406

    Article  PubMed  CAS  Google Scholar 

  21. Guerrier-Takada C, Salavati R, Altman S (1997) Phenotypic conversion of drug resistant bacteria to drug sensitivity. Proc Natl Acad Sci USA 94:8468–8472

    Article  PubMed  CAS  Google Scholar 

  22. Wyekoff E, Natahe D, Nolan JM, Lee M, Hsteh T (1989) Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II. J Mol Biol 205:6–13

    Google Scholar 

  23. Hannon GJ (2004) RNAi: a guide to gene silencing, 1st ed. Photolithograph book. Chemistry industry press, Beijing, pp 23–24

  24. Hannon GJ (2004) RNAi: a guide to gene silencing, 1st ed. Photolithograph book. Chemistry industry press, Beijing, pp 243–246

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Acknowledgement

We are greatly indebted to Dr. McKinney for the plasmids and suggestions.

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Authors and Affiliations

  1. Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, 600 113, India

    Shilpakala Sainath Rao & Malathi Raghunathan

  2. Department of Biochemistry, Indian Institute of Science, Bangalore, India

    H. S. Savithri

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  1. Shilpakala Sainath Rao
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  2. H. S. Savithri
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  3. Malathi Raghunathan
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Correspondence to Malathi Raghunathan.

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Rao, S.S., Savithri, H.S. & Raghunathan, M. Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR. Mol Biol Rep 35, 575–578 (2008). https://doi.org/10.1007/s11033-007-9126-y

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  • DOI: https://doi.org/10.1007/s11033-007-9126-y

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