Journal of Microbiology

  • 제44권1호
  • /
  • Pages.64-71
  • /
  • 2006
  • /
  • 1225-8873(pISSN)
  • /
  • 1976-3794(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

The Influence of the Nucleotide Sequences of Random Shine-Dalgarno and Spacer Region on Bovine Growth Hormone Gene Expression

  • Paik Soon-Young (Department of Microbiology, College of Medicine, The Catholic University of Korea) ;
  • Ra Kyung Soo (Department of Food and Nutrition, Daegu Technical College) ;
  • Cho Hoon Sik (Department of Bioindustry, College of Liefe & Environment, Daegu University) ;
  • Koo Kwang Bon (Department of Bioindustry, College of Liefe & Environment, Daegu University) ;
  • Baik Hyung Suk (Department of Microbiology, Busan National University) ;
  • Lee Myung Chul (Rice Functional Genomics Team, National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Yun Jong Won (Department of Biotechnology, College of Engineering, Daegu University) ;
  • Choi Jang Won (Department of Bioindustry, College of Liefe & Environment, Daegu University)
  • 발행 : 2006.02.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

To investigate the effects of the nucleotide sequences in Shine-Dalgarno (SD) and the spacer region (SD-ATG) on bovine growth hormone (bGH) gene expression, the expression vectors under the control of the T7 promoter (pT7-7 vector) were constructed using bGH derivatives (bGH1 & bGH14) which have different 5'-coding regions and were induced in E. coli BL21 (DE3). Oligonucleotides containing random SD sequences and a spacer region were chemically synthesized and the distance between the SD region and the initiation codon were fixed to nine bases in length. The oligonucleotides were annealed and fused to the bGH1 and bGH14 cDNA, respectively. When the bGH gene was induced with IPTG in E. coli BL21(DE3), some clones containing only bGH14 cDNA produced considerable levels of bGH in the range of $6.9\%\;to\;8.5\%$ of total cell proteins by SDS-PAGE and Western blot. Otherwise, the bGH was not detected in any clones with bGH1 cDNA. Accordingly, the nucleotide sequences of SD and the spacer region affect on bGH expression indicates that the sequences sufficiently destabilize the mRNA secondary structure of the bGH14 gene. When the free energy was calculated from the transcription initiation site to the +51 nucleotide of bGH cDNA using a program of nucleic acid folding and hybridization prediction, the constructs with values below -26.3 kcal/mole (toward minus direction) were not expressed. The constructs with the original sequence of bGH cDNA also did not show any expression, regardless of the free energy values. Thus, the disruption of the mRNA secondary structure may be a major factor regulating bGH expression in the translation initiation process. Accordingly, the first stem-loop among two secondary structures present in the 5'-end region of the bGH gene should be disrupted for the effective expression of bGH.

키워드

참고문헌

  1. Baumann, D.E., C.J. Peel, W.D. Steinhour, P.J. Reynolds, H.F. Tyrrell, A.C.G. Brown, and G.L. Haaland. 1988. Effect of bovine somatotropin on metabolism of lactating dairy cow. J. Nutr. 118, 1031-1040 https://doi.org/10.1093/jn/118.8.1031
  2. Chen, H., M. Bjerknes, R. Kumar, and E. Jay. 1994. Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res. 22, 4953-4957 https://doi.org/10.1093/nar/22.23.4953
  3. Choi, J.W. and S.Y. Lee. 1996. Optimization of bovine growth hormone gene expression in E. coli. Mol. Cells. 6, 712-718
  4. Choi, J.W., K.S. Ra, and Y.S. Lee. 1999a. Enhancement of bovine growth hormone gene expression by increasing the plasmid copy number. Biotechnology Letters. 21, 1-5 https://doi.org/10.1023/A:1005479907709
  5. Choi, J.W., H.S. Park, J.W. Yun, and Y.S. Lee. 1999b. The artificial AT-rich block enhanced the production of bovine growth hormone in Escherichia coli. Bioprocess Engineering. 21, 293-298
  6. de Smit, M.H. and J. van Duin. 1990. Control of prokaryotic translational initiation by mRNA secondary structure. Prog. Nucleic Acids Res. Mol. Biol. 38, 1-35 https://doi.org/10.1016/S0079-6603(08)60707-2
  7. de Smit, M.H. and J. van Duin. 1994. Translational initiation on structured messengers. Another role for the Shine-Dalgarno interaction. J. Mol. Biol. 235, 173-184 https://doi.org/10.1016/S0022-2836(05)80024-5
  8. Draper, D.E. 1996. Translational initiation, p. 902-908. In Neidhardt, F.C., R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger. (ed), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C
  9. George, H.J., J.J. L'Italien, W.P. Pilacinski, D.L. Glassman, and R.A. Krzyzek. 1985. High level expression in E. coli of biologically active bovine growth hormone. DNA. 4, 273-281
  10. Gold, L., D. Pribnow, T. Schneider, S. Shinedling, B.S. Singer, and G. Stormo. 1981. Translational initiation in prokaryotes. Annu. Rev. Microbiol. 35, 365-403 https://doi.org/10.1146/annurev.mi.35.100181.002053
  11. Gold, L. 1988. Posttranslational regulatory mechanisms in Escherichia coli. Annu. Rev. Biochem. 57, 199-233 https://doi.org/10.1146/annurev.bi.57.070188.001215
  12. Green, P.J. and M. Inouye. 1984. Roles of the 5' leader region of the ompA mRNA. J. Mol. Biol. 176, 431-442 https://doi.org/10.1016/0022-2836(84)90499-6
  13. Hsiung, H.M. and W.C. Mackellar. 1987. Expression of bovine growth hormone derivatives in E. coli and the use of the derivatives to produce natural sequence growth hormone by cathepsin C cleavage. Methods Enzymol. 153, 390-401 https://doi.org/10.1016/0076-6879(87)53067-1
  14. Hui, A., J. Hayflick, K. Dinkelspiel, and H.A. de Boer. 1984. Mutagenesis of the three base pair preceding the start codon of the $\beta$-galactosidase mRNA and its effect on translation in E. coli. EMBO J. 3, 623-629
  15. Ikemura, T. 1985. Codon usage and tRNA content in unicellular and multicellular organisms. Mol. Biol. Evol. 2(1), 13-34
  16. Kanaya, S., Y. Yamada, Y. Kudo, and T. Ikemura. 1999. Studies of codon usage and tRNA genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: Gene expression level and species-specific diversity of codon usage based on multivariate analysis. Gene 238, 143-155 https://doi.org/10.1016/S0378-1119(99)00225-5
  17. Karlin, S. and J. Mrazek. 2000. Predicted highly expressed genes of diverse prokaryotic genomes. J. Bacteriol. 182(18), 5238-5250 https://doi.org/10.1128/JB.182.18.5238-5250.2000
  18. Karlin, S., J. Mrazek, A. Campbell, and D. Kaiser. 2001. Characterization of highly expressed genes of four fast-growing bacteria. J. Bacteriol. 183, 5025-5040 https://doi.org/10.1128/JB.183.17.5025-5040.2001
  19. Kim, E.Y., M.S. Shin, J.H. Rhee, and H.E. Choy. 2004a. Factors influencing preferential utilization of RNA polymerase containing sigma-38 in stationary-phase gene expression in Escherichia coli. J. Microbiol. 42, 103-110
  20. Kim, H.G., T.N. Phan, T.S. Jang, M.J. Koh, and S.W. Kim. 2005. Characterization of Methylophaga sp. Strain SK1 cytochrome cL expressed in Escherichia coli. J. Microbiol. 43, 499-502
  21. Kim, Y.M., K. Park, S.H. Jung, J.H. Choi, W.C. Kim, G.J. Joo, and I.G. Rhee. 2004b. Chlorothalonil-biotransformation by glutathione s-transferase of Escherichia coli. J. Microbiol. 42, 42-46
  22. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  23. Ma, J., A. Campbell, and S. Karlin. 2002. Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures. J. Bacteriol. 184(20), 5733-5745 https://doi.org/10.1128/JB.184.20.5733-5745.2002
  24. Morikawa, M., T. Nixon, and H. Green. 1982. Growth hormone and adipose conversion of 3T3 cells. Cell 29, 783-789 https://doi.org/10.1016/0092-8674(82)90440-8
  25. Osada, Y., R. Saito, and M. Tomita. 1999. Analysis of base-pairing potentials between 16S rRNA and 5'UTR for translation initiation in various prokaryotes. Bioinformatics 15, 578-581 https://doi.org/10.1093/bioinformatics/15.7.578
  26. Paladini, A.C., C. Pena, and E. Poskus. 1983. Molecular biology of growth hormone. CRC Crit. Rev. Biochem. 15, 25-56 https://doi.org/10.3109/10409238309102800
  27. Park, S.H. and J.I. Kim. 2004. Characterization of recombinant Drosophila melanogaster Myo-inositol-1-phosphate synthase expressed in Escherichia coli. J. Microbiol. 42, 20-24
  28. Poole, E.S., C.M. Brown, and W.P Tate. 1995. The identity of the base following the stop codon determines the efficiency of in vivo translational termination in E. coli. EMBO J. 14, 151-158
  29. Ringquist, S., S. Shinedling, D. Barrick, L. Green, J. Binkley, G.D. Stormo, and L. Gold. 1992. Translation initiation in Escherichia coli: sequences within the ribosome binding site. Mol. Microbiol. 6, 1219-1229 https://doi.org/10.1111/j.1365-2958.1992.tb01561.x
  30. Saito, Y., Y. Ishii, S. Koyama, K. Tsuji, H. Yamada, T. Terai, M. Kobayashi, T. Ono, M. Niwa, I. Ueda, and H. Kikuchi. 1987. Bacterial synthesis of recombinant alpha-human atrial natriuretic polypeptide. J. Biochem. 102, 11-122
  31. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning : A laboratory Manual, Cold Spring Harbor Lab. New York
  32. Sanger, F., S. Niklen, and A.R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74, 5463-5467
  33. Scherer, G.E.F., M.D. Walkinshaw, S. Arnott, and D.J. Morre. 1980. The ribosome binding sites recognized by E. coli ribosomes have regions with signal character in both the leader and protein coding segments. Nucleic Acids Res. 8, 3895-3906 https://doi.org/10.1093/nar/8.17.3895
  34. Schoner, B.E., H.M. Hsiung, R.M. Belagaje, N.G. Mayne, and R.G. Schoner. 1984. Role of mRNA translational efficiency in bovine growth hormone expression in E. coli. Proc. Natl. Acad. Sci. USA. 81, 5403-5407
  35. Secchi, C. and V. Borromeo. 1997. Bovine growth hormone as an experimental model for studies of protein-protein interactions. J. Chromatogr. Biomed. Sci. 688(2), 161-177 https://doi.org/10.1016/S0378-4347(96)00296-4
  36. Seeburg, P.H., S. Sias, J. Adelman, H.A. de Boer, J. Hayflick, P. Jhurani, D.V. Goeddel, and H.L. Heyneker. 1983. Efficient bacterial expression of bovine and porcine growth hormones. DNA 2, 37-45 https://doi.org/10.1089/dna.1.1983.2.37
  37. Selinger, D.W., K.J. Cheung, R. Mei, E.M. Johansson, C.S. Richmond, F.R. Blattner, D.J. Lockhart, and G.M. Church. 2000. RNA expression analysis using a 30 base pair resolution Escherichia coli genome array. Nat. Biotechnol. 18, 1262-1268 https://doi.org/10.1038/82367
  38. Shine, J. and L. Dalgarno. 1974. The 3'-terminal sequence logos: prediction of major groove binding by information theory. Methods Enzymol. 274, 445-455
  39. Studier, F.W., A.H. Rosenberg, J.J. Dunn, and J.W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89 https://doi.org/10.1016/0076-6879(90)85008-C
  40. Tabor, S. and C.C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA. 82, 1074-1078
  41. Tomich, C.S.C., E.R. Olsen, M.K. Olsen, P.S. Kaytes, S.K. Rockenbach, and N.T. Hazenbuhler. 1989. Effect of the nucleotide sequence directly downstream from the AUG on the expression of bovine somatotropin in E. coli. Nucleic Acids Res. 17, 3179-3197 https://doi.org/10.1093/nar/17.8.3179
  42. Uhlin, B.E., S. Molin, P. Gustafsson, and K. Nordstrom. 1979. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene 6, 91-106 https://doi.org/10.1016/0378-1119(79)90065-9
  43. Warburton, N., P.G. Bosely, and A.G. Porter. 1983. Increased expression of a cloned gene by local mutagenesis of its promoter and ribosome binding site. Nucleic Acids Res. 11, 5837-5854 https://doi.org/10.1093/nar/11.17.5837
  44. Wingfield, P.T., P. Graber, G. Buell, K. Rose, M. Simona, and B.D. Burleigh. 1987. Preparation and characterization of bovine growth hormones produced in recombinant E. coli. Biochem. J. 243, 829-839 https://doi.org/10.1042/bj2430829
  45. Wood, C.R., M.A. Boss, T.P. Patel, and J.S. Emtage. 1984. The influence of messenger RNA secondary structure on expression of an immunoglobulin heavy chain in Escherichia coli. Nucleic Acids Res. 12, 3937-3950 https://doi.org/10.1093/nar/12.9.3937
  46. Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-3415 https://doi.org/10.1093/nar/gkg595