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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

  • Ji, Sang-Chun (Department of Biological Science, College of Biological Sciences and Biotechnology, Chungnam National University) ;
  • Wang, Xun (Department of Biological Science, College of Biological Sciences and Biotechnology, Chungnam National University) ;
  • Jeon, Heung-Jin (Department of Biological Science, College of Biological Sciences and Biotechnology, Chungnam National University) ;
  • Yun, Sang-Hoon (Department of Biological Science, College of Biological Sciences and Biotechnology, Chungnam National University) ;
  • Lee, Hee-Jung (Department of Biological Science, College of Biological Sciences and Biotechnology, Chungnam National University) ;
  • Lim, Heon-M. (Department of Biological Science, College of Biological Sciences and Biotechnology, Chungnam National University)
  • Received : 2010.05.05
  • Accepted : 2010.06.04
  • Published : 2010.07.31
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Abstract

Three assay methods for quantification of the two galactose-operon mRNAs that only differ by 5 bases in their 5'-end are presented. The 5' ends of each mRNA were extended by ligating the 3'-end of the abundant 5S rRNA. This ligation extends the 5' ends of the two gal mRNAs long enough to be distinguished by the specific PCR primers in the following quantification reactions. Quantification of the corresponding cDNAs was performed either by primer extension assay or real-time qPCR. To circumvent the problem of the RNA ligation reaction (i.e. very low ligation efficiency), we devised a new method that employs real-time qPCR directly for the quantification of the gal transcripts which differ by 5 bases in their 5'-ends.

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References

  1. Adhya, S. and Miller, W. (1979). Modulation of the two promoters of the galactose operon of Escherichia coli. Nature 279, 492-494. https://doi.org/10.1038/279492a0
  2. Musso, R. E., Di Lauro, R., Adhya, S. and de Crombrugghe, B. (1977). Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters. Cell 12, 847-854. https://doi.org/10.1016/0092-8674(77)90283-5
  3. Geanacopoulos, M., Vasmatzis, G., Zhurkin, V. B. and Adhya, S. (2001). Gal repressosome contains an antiparallel DNA loop. Nat. Struct. Biol. 8, 432-436. https://doi.org/10.1038/87595
  4. Lewis, D. E. and Adhya, S. (2002). In vitro repression of the gal promoters by GalR and HU depends on the proper helical phasing of the two operators. J. Biol. Chem. 277, 2498-2504. https://doi.org/10.1074/jbc.M108456200
  5. Aki, T. and Adhya, S. (1997). Repressor induced site-specific binding of HU for transcriptional regulation. EMBO J. 16, 3666-3674. https://doi.org/10.1093/emboj/16.12.3666
  6. Semsey, S., Virnik, K. and Adhya, S. (2006). Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA. J. Mol. Biol. 358, 355-363. https://doi.org/10.1016/j.jmb.2006.02.022
  7. Semsey, S., Krishna, S., Sneppen, K. and Adhya, S. (2007). Signal integration in the galactose network of Escherichia coli. Mol. Microbiol. 65, 465-476. https://doi.org/10.1111/j.1365-2958.2007.05798.x
  8. Heid, C. A., Stevens, J., Livak, K. J. and Williams, P. M. (1996). Real time quantitative PCR. Genome Res. 6, 986-994. https://doi.org/10.1101/gr.6.10.986
  9. Frohman, M. A., Dush, M. K. and Martin, G. R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. U.S.A. 85, 8998-9002. https://doi.org/10.1073/pnas.85.23.8998
  10. Bingham, A. H., Ponnambalam, S., Chan, B. and Busby, S. (1986). Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon. Gene 41, 67-74. https://doi.org/10.1016/0378-1119(86)90268-4
  11. Dreyfus, M., Kotlarz, D. and Busby, S. (1985). Point mutations that affect translation initiation in the Escherichia coli galE gene. J. Mol. Biol. 182, 411-417. https://doi.org/10.1016/0022-2836(85)90200-1
  12. Lee, J. J. and Costlow, N. A. (1987). A molecular titration assay to measure transcript prevalence levels. Methods Enzymol. 152, 633-648. https://doi.org/10.1016/0076-6879(87)52070-5
  13. Schmittgen, T. D., Zakrajsek, B. A., Mills, A. G., Gorn, V., Singer, M. J. and Reed, M. W. (2000). Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal. Biochem. 285, 194-204. https://doi.org/10.1006/abio.2000.4753
  14. Datsenko, K. A. and Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645. https://doi.org/10.1073/pnas.120163297
  15. Lee, H. J., Jeon, H. J., Ji, S. C., Yun, S. H. and Lim, H. M. (2008). Establishment of an mRNA gradient depends on the promoter: an investigation of polarity in gene expression. J. Mol. Biol. 378, 318-327. https://doi.org/10.1016/j.jmb.2008.02.067
  16. Rozen, S. and Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365-386.

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