本實驗室之前研究指出，溫度影響吳郭魚之性別分化及腦神經的
發育，而本實驗室發展孵化後10天吳郭魚腦基因的表現，並建立吳郭魚腦表現序列標記( expressed sequence tag , EST)資料庫。而編號F10A83為一個低溫誘發表現之基因，F10A83的cDNA全長為1526 bp，轉譯之蛋白質由176個氨基酸所組成，為一個富含甘氨酸的蛋白質( glycine-rich protein )。本研究藉由6 個His作為標記，將F10A83開放閱讀區( open reading frame, ORF )建構於原核表現系統中，再利用親和性管柱將F10A83成功分離純化，所分離之F10A83蛋白質針對吳郭魚腦細胞，探討其生長趨勢分析。在真核生物方面，將F10A83之ORF構築在綠螢光蛋白( green fluorescent protein, GFP )表現載體上，轉染( transfection )至老鼠神經瘤細胞( Neuro-2a )中，使用螢光顯微鏡觀察發現，F10A83大部分都表現於細胞核中，為細胞核蛋白的一種。我們也篩選穩定表現F10A83之Neuro-2a細胞，探討分析F10A83基因對細胞生長的影響。

In tilapia , sex determination is controled by genetic and triggered by the environmental factors. Expressed sequence tags ( EST ) derived from the developing tilapia brain is cloned in our lab. The cDNA full length of the gene, F10A83 was cloned. In the present study, F10A83 is a gene with 1526 bp of cDNA sequence, and deduced 176 amino acids of protein sequence. F10A83 was overexpressed as a GFP fusion protein in mouse neuroblastoma Neuro-2a cells. F10A83 is abundantly distributed in the nucleus of Neuro-2a cell. The protein of F10A83 was expressed in the prokaryotic cell (BL21) and eukaryotic cell (neuro-2a), and purified using a simple purification process, inducing, isolation, and Ni-NTA affinity chromatography. The protein of F10A83 in both E. coli system and neuro-2a cell line expression has been established.

頁次
致謝---------------------------------------------------- 1
中文摘要-------------------------------------------------2
英文摘要-------------------------------------------------3
目錄-----------------------------------------------------4
附圖、表目錄---------------------------------------------5
前言-----------------------------------------------------6
實驗材料與方法-------------------------------------------9
結果----------------------------------------------------41
討論----------------------------------------------------45
參考文獻------------------------------------------------49
附圖、表------------------------------------------------53

Aoki, K., Y. Ishii, et al. (2002). "Methylation of Xenopus CIRP2 regulates its arginine- and glycine-rich region-mediated nucleocytoplasmic distribution." Nucleic Acids Res 30(23): 5182-92.
Aoki, K., K. Matsumoto, et al. (2003). "Xenopus cold-inducible RNA-binding protein 2 interacts with ElrA, the Xenopus homolog of HuR, and inhibits deadenylation of specific mRNAs." J Biol Chem 278(48): 48491-7.
Banks, S., S. A. King, et al. (2005). "Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa." Reproduction 129(4): 505-14.
Baroiller, J. F. and H. D'Cotta (2001). "Environment and sex determination in farmed fish." Comp Biochem Physiol C Toxicol Pharmacol 130(4): 399-409.
Bhatia, R., D. K. Dube, et al. (1999). "Expression of axolotl RNA-binding protein during development of the Mexican axolotl." Cell Tissue Res 297(2): 283-90.
Danno, S., K. Itoh, et al. (2000). "Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of cryptorchid testis." Am J Pathol 156(5): 1685-92.
D'Cotta, H., A. Fostier, et al. (2001). "Search for genes involved in the temperature-induced gonadal sex differentiation in the tilapia, Oreochromis niloticus." J Exp Zool 290(6): 574-85.
D'Cotta, H., A. Fostier, et al. (2001). "Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus." Mol Reprod Dev 59(3): 265-76.
Etchegaray, J. P., P. G. Jones, et al. (1996). "Differential thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of Escherichia coli." Genes Cells 1(2): 171-8.
Felix, G. and T. Boller (2003). "Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco." J Biol Chem 278(8): 6201-8.
Fujita, J. (1999). "Cold shock response in mammalian cells." J Mol Microbiol Biotechnol 1(2): 243-55.
Guiguen, Y., J. F. Baroiller, et al. (1999). "Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus)." Mol Reprod Dev 54(2): 154-62.
Hamid, A. A., M. Mandai, et al. (2003). "Expression of cold-inducible RNA-binding protein in the normal endometrium, endometrial hyperplasia, and endometrial carcinoma." Int J Gynecol Pathol 22(3): 240-7.
Inaba, M., I. Suzuki, et al. (2003). "Gene-engineered rigidification of membrane lipids enhances the cold inducibility of gene expression in synechocystis." J Biol Chem 278(14): 12191-8.
Kim, Y. O., J. S. Kim, et al. (2005). "Cold-inducible zinc finger-containing glycine-rich RNA-binding protein contributes to the enhancement of freezing tolerance in Arabidopsis thaliana." Plant J 42(6): 890-900.
Koizumi, M., K. Yamaguchi-Shinozaki, et al. (1993). "Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana." Gene 129(2): 175-82.
Kondo, K. and M. Inouye (1992). "Yeast NSR1 protein that has structural similarity to mammalian nucleolin is involved in pre-rRNA processing." J Biol Chem 267(23): 16252-8.
Lu, D. and A. J. Hickey (2005). "Liposomal dry powders as aerosols for pulmonary delivery of proteins." AAPS PharmSciTech 6(4): E641-8.
Matsumoto, K., K. Aoki, et al. (2000). "CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes." Nucleic Acids Res 28(23): 4689-97.
Mayr, B., T. Kaplan, et al. (1996). "Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201." J Bacteriol 178(10): 2916-25.
Nishiyama, H., S. Danno, et al. (1998). "Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature." Am J Pathol 152(1): 289-96.
Nishiyama, H., H. Higashitsuji, et al. (1997). "Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene." Gene 204(1-2): 115-20.
Nishiyama, H., K. Itoh, et al. (1997). "A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth." J Cell Biol 137(4): 899-908.
Nishiyama, H., J. H. Xue, et al. (1998). "Diurnal change of the cold-inducible RNA-binding protein (Cirp) expression in mouse brain." Biochem Biophys Res Commun 245(2): 534-8.
Pan, F., J. Zarate, et al. (2004). "Osmotic stress of salmon stimulates upregulation of a cold inducible RNA binding protein (CIRP) similar to that of mammals and amphibians." Biochimie 86(7): 451-61.
Peng, Y., K. H. Kok, et al. (2000). "Maternal cold inducible RNA binding protein is required for embryonic kidney formation in Xenopus laevis." FEBS Lett 482(1-2): 37-43.
Peng, Y., P. H. Yang, et al. (2006). "Cold-inducible RNA binding protein is required for the expression of adhesion molecules and embryonic cell movement in Xenopus laevis." Biochem Biophys Res Commun 344(1): 416-424.
Pirok, E. W., 3rd, M. S. Domowicz, et al. (2005). "APBP-1, a DNA/RNA-binding protein, interacts with the chick aggrecan regulatory region." J Biol Chem 280(42): 35606-16.
Saito, T., K. Sugimoto, et al. (2000). "Cloning and characterization of amphibian cold inducible RNA-binding protein." Comp Biochem Physiol B Biochem Mol Biol 125(2): 237-45.
Sakurai, T., K. Itoh, et al. (2006). "Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signal-regulated kinase." Biochim Biophys Acta 1763(3): 290-295.
Shiue, Y. L., L. H. Wang, et al. (2004). "EST-based identification of genes expressed in the hypothalamus of adult tilapia, Oreochromis mossambicus." Biochem Biophys Res Commun 316(2): 523-7.
Spurrell, J. C., S. Wiehler, et al. (2005). "Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivo upon rhinovirus infection." Am J Physiol Lung Cell Mol Physiol 289(1): L85-95.
Tanaka, K. J., H. Kawamura, et al. (2000). "An ascidian glycine-rich RNA binding protein is not induced by temperature stress but is expressed under a genetic program during embryogenesis." Gene 243(1-2): 207-14.
Tsai, C. L., S. L. Chang, et al. (2003). "Temperature influences the ontogenetic expression of aromatase and oestrogen receptor mRNA in the developing tilapia (Oreochromis mossambicus) brain." J Neuroendocrinol 15(1): 97-102.
Tsai, C. L., L. H. Wang, et al. (2000). "Effects of gonadal steroids on brain serotonergic and aromatase activity during the critical period of sexual differentiation in tilapia, Oreochromis mossambicus." J Neuroendocrinol 12(9): 894-8.
Uochi, T. and M. Asashima (1998). "XCIRP (Xenopus homolog of cold-inducible RNA-binding protein) is expressed transiently in developing pronephros and neural tissue." Gene 211(2): 245-50.
Vasina, J. A., M. S. Peterson, et al. (1998). "Scale-up and optimization of the low-temperature inducible cspA promoter system." Biotechnol Prog 14(5): 714-21.
Wang, L. H. and C. L. Tsai (2000). "Temperature affects the development of central neurotransmitter systems of tilapia, Oreochromis mossambicus." Neurosci Lett 285(2): 95-8.
Wang, L. H. and C. L. Tsai (2000). "Effects of temperature on the deformity and sex differentiation of tilapia, Oreochromis mossambicus." J Exp Zool 286(5): 534-7.
Wellmann, S., C. Buhrer, et al. (2004). "Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism." J Cell Sci 117(Pt 9): 1785-94.
Wu, Q., K. Sugimoto, et al. (2002). "Cloning of hibernation-related genes of bullfrog (Rana catesbeiana) by cDNA subtraction." Comp Biochem Physiol B Biochem Mol Biol 133(1): 85-94.
Xue, J. H., K. Nonoguchi, et al. (1999). "Effects of ischemia and H2O2 on the cold stress protein CIRP expression in rat neuronal cells." Free Radic Biol Med 27(11-12): 1238-44.
Yoon, S. K., J. K. Hong, et al. (2006). "Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production." J Biotechnol 122(4): 463-72.