Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Biological Studies on the Stress Protein HSP90$\beta$ Gene from Flounder (Paralichthys olivaceus)

넙치(Paralichthys olivaceus) HSP90$\beta$ 유전자의 분자생물학적 연구

  • Lee, Jae-Hyeong (Department of Microbiology, Pukyong National University) ;
  • Kim, Yeong-Tae (Department of Microbiology, Pukyong National University)
  • Published : 2004.12.01
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Abstract

Heat shock proteins (HSPs) were induced in cells in the thermal stress, and the HSP90 family is one of the major classes of HSPs. Gene encoding HSPs have been characterized from various mammals and piscine. We have cloned and sequenced the HSP90 cDNA from a brain cDNA library constructed from flounder (Paralichthys oliThe result of sequence analysis shows it to be the HSP90~. The nucleotide sequence of the HSP90$\beta$ was composed of 2791 long, encoding 726 amino acid residues. The flounder hsp90$\beta$ gene showed very high sequence homology with hsp90f3 of European sea bass (96.6%), zebrafish (92.9%), Atlantic salmon (92.0%) and human (89.5%). We also constructed a phylogenetic tree based on HSP90 amino acid sequences from vertebrate species. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal hsp90$\beta$ mRNA. The hsp90f3 gene is constitutively expressed at a fairly high level in all examined tissues (brain, liver, kidney, muscle, and spleen). In order to express protein of flounder hsp90$\beta$ in E. coli, we used the His-tagged pETvector. Then, the expression of flounder HSP90$\beta$ was confirmed by Western blot analysis.

열 충격단백질(Heat shock protein : HSP)은 온도 스트레스에 대하여 세포 내에서 발현되는 단백질이다. HSP의 중요 분류군의 하나가 HSP90 family 이다. 여러 종류의 포유동물과 조류에서 HSP 유전자 특성에 대한 연구가 많이 진행되었다. 본 연구에서는 넙치(Paralichthys olivaceus)로부터 제조한 넙치 뇌 cDNA 유전자 은행을 이용하여 넙치 HSP90 cDNA 유전자를 분리하여 구성 염기서열의 특성을 밝혀 내었다. 염기서열의 분석결과 넙치의 hsp90$\beta$ 유전자는 2,791 개의 뉴클레오타이드로 구성되어 있고, 726개의 아미노산 잔기가 암호화되어 있었다. 넙치 hsp90$\beta$ 유전자는 European sea bass와 96.6% zebrafish와 92.9%, Atlantic salmon와 92.0%, 그리고 사람과는 89.5%의 염기서열 상동성을 지니고 있었다. 또한 HSP90 아미노산 서열을 바탕으로 척추동물 종들과의 진화계통수를 구축하였다. 넙치 hsp90$\beta$ 유전자의 mRNA의 분포 정도를 RT-PCR를 이용하여 조사하였다. hsp90$\beta$ 유전자는 조사한 모든 조직(뇌, 간, 신장, 근육, 비장)에서 높은 수준으로 발현이 되고 있었다. 또한, 넙치 hsp90$\beta$ 단백질을 대량발현하기 위하여 대장균에서 발현을 유도하였다.

Keywords

References

  1. Ashburner, M. and J. J. Bonner. 1979. The induction of gene activity in drosophilia by heat shock. Cell 17: 241-254
  2. Binart, N., B. Chambraud, B. Dumas, D. A. Rowlands, C. Bigogne, J. M. Levin, J. Garnier, E. E. Baulieu, and M. G. Catelli. 1989. The cDNA-derived amino acid sequence of chick heat shock protein Mr 90,000 (HSP 90) reveals a 'DNA like' structure: potential site of interaction with steroid receptors. Biochem. Biophys. Res. Commun. 159: 140-147
  3. Buchner, J. 1996. Supervising the fold: functional principles of molecular chaperones. FASEB J. 10: 10-19
  4. Carrello, A., E. lngley, R. F. Minchin, S. Tsai, and T. Ratajczak. 1999. The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and hop is located in the dimerization domain of Hsp90. J. Biol. Chem. 274: 2682-2689
  5. Cho, J. J., J. H. Lee, S. K. Kim, T.-J. Choi, and Y. T. Kim. 1999. cDNA encoding nm23/NDP kinase gene from Korean tiger shark, Scyliorhinus torazame. Mar Biotechnol 1: 131- 136
  6. Cho, J. J., B. K. Sung, J. H. Lee, J. K. Chung, T.-J. Choi, and Y. T. Kim. 2001. cDNA for an immune response gene encoding low molecular weight polypeptide from flounder, Paralichthys olivaceus. Mol. Cells 11: 226-230
  7. Craig, E. A., J. S. Weissman, and A. L. Horwich. 1994. Heat shock proteins and molecular chaperones: mediators of protein conformation and turnover in the cell. Cell 78: 365-372
  8. Csermely, P. and C. R. Kahn. 1991. The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity. J. Biol. Chem. 266: 4943-4950
  9. Csermely, P. T. Schnaider, C. Sõti, Z. Prohászka, and G. Nardai. 1998. The 90-kDa molecular chaperone family: Structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther. 79: 129-168
  10. Grenert, J. P., W. P. Sullivan, P. Fadden, T. A. Haystead, J. Clark, E. Mimnaugh, H. Krutzsch, H. Ochel, T. W. Schulte, E. Sausville, L. M. Neckers, and D. O. Toft. 1997. The amino-terminal domain of heat shock protein 90 (Hsp90) that binds Geldanamycin is an ATP/ADP switch domain that regulates Hsp90 conformation. J. Biol. Chem. 272: 23843- 23850
  11. Gupta, R. S. 1995. Phylogenetic analysis of the 90 kD heat shock family of protein sequences and an examination of the relationship among animals, plants, and fungi species. Mol. Biol. Evol. 12:1063-73
  12. Hermesz, E., M. Árahám, and J. Nemcsók. 2001. Identification of two hsp90 genes in carp. Comp. Biochem. Physiol. (C) 129: 397-407
  13. Hickey, E., S. E. Brandon, G. Smale, D. Lloyd, and L. A. Weber. 1989. Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein. Mol. Cell. Biol. 9: 2615-2626
  14. Hoffmann, T. and B. Hovemann. 1988. Heat-shock proteins, Hsp84 and Hsp86, of mice and men: two related genes encode formerly identified tumor-specific transplantation antigens. Gene 74: 491-501
  15. Kim, D. S. and Y. T. Kim. 1999. Identification of an embryonic growth factor IGF-II from the central nervous system of the teleost, flounder, and its expressions in adult tissues. J. Microbiol. Biotechnol. 9: 113-118
  16. Kim, Y. T. and C. C. Richardson. 1993. Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication. Proc. Natl. Acad. Sci. USA 90: 10173-10177
  17. Kim, Y. T. and C. C. Richardson. 1994. Acidic carboxyl-terminal domain of gene 2.5 protein of bacteriophage T7 is essential for protein-protein interactions. J. Biol. Chem. 269: 5270-5278
  18. Kim, Y. T., Y. H. Song, and J. E. Churchich. 1997. Recombinant brain 4-aminobutyrate aminotransferases: Overexpression, purification, and identification of Lys-330 at the active site. Biochim. Biophys. Acta. 1337: 248-256
  19. Krone, P. H. and J. B. Sass. 1994. HSP 90 alpha and HSP 90 beta genes are present in the zebrafish and are differentially regulated in developing embryos. Biochem. Biophys. Res. Commun. 204: 746-752
  20. Landschulz, W. H., P. F. Johnson, and S. L. McNight. 1988. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240: 1759-1764
  21. Lee, J. H., S. W. Nam, and Y. T. Kim. 2003. Isolation of cDNAs for gondotropin-II of flounder (Paralichthys olivaceus) and its expressions in adult tissues. J. Microbiol. Biotechnol. 13: 710-716
  22. Lindquist, S. and E. A. Craig. 1988. The heat shock proteins. Annu. Rev. Genet. 22: 631-677
  23. Meng, X., V. Jerome, J. Devin, E.E. Bauliea, and M.G. Catelli. 1993. Cloning of chicken hsp90 beta: the only vertebrate hsp90 insensitive to heat shock. Biochem. Biol. Chem. 264: 15006-15011
  24. Moore, S. K. C. Kozak, E. A. Robinson, S. J. Ullrich, and E Appella. 1987. Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences. Gene 56: 29-40
  25. Nathan, D. F., M. H. Vos, and S. Lindquist. 1997. In vivo functions of the Saccharomyces cervisiae HSP90 chaperons. Proc. Natl. Acad. Sci. USA 94: 12949-12956
  26. Ozawa, K., Y. Murakami, T. Eki, E. Soeda, and K. Yokoyama. 1992. Mapping of the gene family for human heat-shock protein 90 alpha to chromosomes 1, 4, 11, and 14. Genomics 12: 214-220
  27. Palmisano, A. N., J. R. Winton, and W. W. Dickhoff. 1999. Sequences features and phylogenetic analysis of the stress protein Hsp90$\alpha$ in chinook salmon (Oncorhynchus tshawytscha), a poikilothermic vertebrate. Biochem Biophys. Res. Commun. 258: 784-791
  28. Parsell, D. A. and S. Lindquist. 1993. The function of heatshock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27: 437-496
  29. Pepin K., F. Momose, N. Ishida, and K. Nagata. 2001. Molecular cloning of horse Hsp90 cDNA and its comparative analysis with other vertebrate Hsp90 sequences. J. Vet. Med. Sci. 63: 115-124
  30. Rebbe, N. F., W. S. Hickman, T. J. Ley, D. W. Stafford, and S. Hickman. 1989. Nuculeotide sequence and regulation of a human 90-kDa heat shock protein gene. J. Biol. Chem. 264: 15006-15011
  31. Stephanou, A. and D. S. Latchman. 1999. Transcriptional regulation of the heat shock protein genes by STAT family transcription factors. Gene Expr. 7: 311-319
  32. Welch, W. J. and J. R. Feramisco. 1982. Purification of the major mammalian heat shock proteins. J. Biol. Chem. 257: 14949-14959