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，並已建立吳郭魚腦表現序列標記( expressed sequence tag , EST)資料庫，故針對低溫誘發性蛋白進行研究，其為一個富含甘氨酸的蛋白質( glycine-rich protein )。因此本實驗針對低溫誘發性蛋白基因啟動子( promoter )進行瞭解。為
瞭解低溫誘發性蛋白的基因調控，我們經由5’ rapid amplification of cD
NA end ( 5’ -RACE )與Genome Walker定出轉錄起始點( transcription start site, TSS )所在位置，並往前定出低溫誘發性蛋白的promoter序列，透過生物資訊分析預測可能的轉錄因子結合位置。在電泳移動遲滯分析實驗( Electrophoresis mobility shift assay )進行分析，先利用限制酵素切割可能的結合位，找到在-157 bp至-149 bp可能有POU3F2的結合位置，實驗結果得知在-157bp至-149 bp可能有蛋白質之鍵結。然而，針對此結合位進行片段構築在含有螢火蟲冷光( firefly luciferase )報導基因的載體，並進行轉染( transfection )至莫三比克吳郭魚星狀神經膠細胞中表現。藉由冷光( luciferase )的活性分析，構築片段野生型有顯著的提升啟動子活性，但其定位點突變( site-direct mutagenesis )沒有降低啟動子的活性。

Temperature affects the sexual differentiation and the development of brain of tilapia, Oreochromis mossambicus. Expressed sequence tags ( EST ) derived from the developing tilapia brain had been cloned in our laboratory. In the present study, we focus on the promoter of cold-induced protein. The promoter sequence of cold-induced protein from the transcription start site ( TSS ) were cloned by 5’ rapid amplification of cDNA end ( 5’-RACE ) and Genome Walker DNA Walking. The bioinformatic analysis was performed on the fragment for predicting the transcription factor binding site. We used the digestion method of restriction enzymes and an electrophoresis mobility shift assay to find transcription factor binding site. The results indicated that there is a putative POU3F2 binding site in the fragment form -157 bp to -149 bp. The luciferase activity assay was performed on this site and results indicated that wild type showed the enhanced promoter activity. However, site-direct mutagenesis of this site did not result in the reduction of the promoter activity.

中文摘要---------------------------------------------------------------------- 1
英文摘要---------------------------------------------------------------------- 2
目錄---------------------------------------------------------------------------- 3
附圖目錄---------------------------------------------------------------------- 4
前言---------------------------------------------------------------------------- 6
實驗材料與方法------------------------------------------------------------- 8
結果---------------------------------------------------------------------------- 27
討論---------------------------------------------------------------------------- 30
參考文獻---------------------------------------------------------------------- 33
附圖---------------------------------------------------------------------------- 37

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.
Fiona Smart, Armaz Aschrafi, Annette Atkins, Geoffrey C. Owens, Julie Pilotte, Bruce A. Cunningham and Peter W. Vanderklish, (2007). "Two isoforms of the cold-inducible mRNA-binding protein RBM3 localize to dendrites and promote translation." J. Neurochem. 101, 1367–1379.
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.