| Title | The Influence of the Nucleotide Sequences of Random Shine-Dalgarno and Spacer Region on Bovine Growth Hormone Gene Expression |
|---|---|
| Author | Soon-Young Paik1, Kyung Soo Ra2, Hoon Sik Cho3, Kwang Bon Koo3, Hyung Suk Baik4, Myung Chul Lee5, Jong Won Yun6, and Jang Won Choi3,* |
| Address | 1Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea, 2Department of Food and Nutrition, Daegu Technical College, Daegu 704-721, Republic of Korea, 3Department of Bioindustry, College of Life & Environment, Daegu University, Kyungbuk 712-714, Republic of Korea, 4Department of Microbiology, Busan National University, Busan 609-735, Republic of Korea, 5Rice Functional Genomics Team, National Institute of Agricultural Biotechnology, Rural Development Administration,Suwon 441-707, Republic of Korea, 6Department of Biotechnology, College of Engineering, Daegu University, Kyungbuk 712-714, Republic of Korea |
| Bibliography | Journal of Microbiology, 44(1),64-71, 2006, |
| DOI | |
| Key Words | SD sequence, spacer region, bGH expression, mRNA secondary structure |
| Abstract | 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. |
| Download PDF | 10 JM2005-167.pdf |