摘 要
ArsA蛋白是大腸桿菌經由質體R773所產生抗砷系統中的催化次單元位，此抗砷系統利用ArsA蛋白水解ATP的能量將進入細胞的砷化物或銻化物排除至細胞外而造成大腸桿菌對砷化物產生抗性。目前，在已知的真核生物基因庫中，當以大腸桿菌的arsA基因進行搜尋與比對時均可在人類，小白鼠，原蟲與酵母菌等細胞中找到arsA基因的同源基因。
但目前除了可確定ArsA蛋白在大腸桿菌的抗砷功能外，其他生物中ArsA同源蛋白的真正功能仍不清楚。初步研究結果顯示，酵母菌的ArsA同源蛋白(yArsA蛋白)缺失並不影響arsA同源基因被破壞的knock-out(KO)菌株對葡萄糖，乙醇或甘油的利用，即yArsA蛋白並不直接參與酵母菌在有氧或厭氧條件下對不同碳源的代謝。而yArsA蛋白缺失也不影響KO菌株在30℃時對砷化物或銻化物的抗性，因此yArsA與抗砷機制無直接的相關性，但KO菌株在30℃與37℃時，對金屬離子稍具敏感性，因此yArsA蛋白可能參與一般排除有毒金屬的機制。
另yArsA蛋白缺失使KO菌株在40℃的生長受到抑制，證明了yArsA蛋白在高溫時扮演保護的角色。而經流式細胞儀分析結果顯示，在40℃時KO菌株細胞大多呈現3N或4N多倍體的情形，因此yArsA蛋白的缺失並不影響酵母細胞DNA的複製，但可能會使細胞無法正常分裂。當利用共焦雷射顯微鏡進行in vivo觀察研究yArsA蛋白在細胞內分佈情形時，發現yArsA與GFP融合蛋白的螢光會形成特殊亮點〝punctate〞構造，但此構造的確切位置仍待colocalization的研究。

Abstract
ArsA protein is the catalytic component of an Escherichia coli plasmid R773-encoded ArsAB pump. In E.coli, the ArsAB pump provides resistance to arsenite and antimonite. At present, comparison with the data banks shows that there are sequences homologous to the bacterial arsA gene in Caenorhabditis elegans, Homo sapiens, Mus Musculus and Saccharomyces cerevisiae genome. The ArsA is proved to be associated with arsenical resistance in E.coli, but the function of ArsA homologous protein in other organisms is still unclear.
Initial studies show that deletion of ArsA homology protein in S.cerevisiae (yArsA protein) has no effect on utilization of different carbon sources, it means that yArsA protein is not directly related to carbon metabolism in aerobic or anaerobic condition. Also, it is probably not directly related to the arsenical resistance mechanism either. Knock-out strain has shown sensitivity to 5 mM Zn+2 when grown at 30℃ and 37℃. Therefore, yArsA protein may be involved in the general detoxification mechanism. Interestingly, the knock-out strain shows thermosensitivity at 40℃, it suggests that the yArsA protein may play a protective role at heat-stress condition. DNA multiploidy was detected in knock-out strain when grown at 40℃, this result suggested that yArsA protein defect may inhibit cytokinesis but not DNA replication when under heat-stress. The subcellular localization of yArsA and GFP fusion protein was determined by confocal microscope of intact cells. In cells expressing the recombinant protein, the fusion protein was found to display specific “punctate” structure, suggesting that the yArsA protein in S.cerevisiae is probably related to function in transport between the prevacuolar compartment and the vacuole or in the signal transduction.

前言-1
材料與方法-6
結果-12
討論-18
參考文獻-24
圖表-28

參考文獻
1.Silver, S. and M. Walderhaug, Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria. Microbiol Rev, 1992 56(1): 195-228.

2.Rosen, B.P., Families of arsenic transporters. Trends Microbiol, 1999 7(5): 207-12.

3.Zhou, T., Radaev, S., Rosen, B. P., Gatti, D. L., Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump. Embo J, 2000 19(17): 4838-45.

4.Rosen, B.P, Bhattacharjee, H., Zhou, T., Walmsley, A. R., Mechanism of the ArsA ATPase. Biochim Biophys Acta, 1999 1461(2): 207-15.

5.Kurdi-Haidar, B., Heath, D., Aebi, S., Howell, S. B., Biochemical characterization of the human arsenite-stimulated ATPase (hASNA-I). J Biol Chem, 1998 273(35): 22173-6.

6.Kurdi-Haidar, B., Aebi, S., Heath, D., Enns, R. E., Naredi, P., Hom, D. K., Howell, S. B., Isolation of the ATP-binding human homolog of the arsA component of the bacterial arsenite transporter. Genomics, 1996 36(3): 486-91.

7.Kurdi-Haidar, B., Heath, D., Naredi, P., Varki, N., Howell, S. B., Immunohistochemical analysis of the distribution of the human ATPase (hASNA-I) in normal tissues and its overexpression in breast adenomas and carcinomas. J Histochem Cytochem, 1998 46(11): 1243-8.



8.Boskovic, J., Soler-Mira, A., Garcia-Cantalejo, J. M., Ballesta, J. P., Jimenez, A., Remacha, M., The sequence of a 16,691 bp segment of Saccharomyces cerevisiae chromosome IV identifies the DUN1, PMT1, PMT5, SRP14 and DPR1 genes, and five new open reading frames. Yeast, 1996 12(13): 1377-84.

9.Zuniga, S., Boskovic, J., Jimenez, A., Ballesta, J. P., Remacha, M., Disruption of six Saccharomyces cerevisiae novel genes and phenotypic analysis of the deletants. Yeast, 1999 15(10B): 945-53.

10.Lowder, M., Unge, A., Maraha, N., Jansson, J. K., Swiggett, J., Oliver, J. D., Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Appl Environ Microbiol, 2000 66(8): 3160-5.

11.Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C., Green fluorescent protein as a marker for gene expression. Science, 1994 263(5148): 802-5.

12.Li, J., Wang, S., VanDusen, W. J., Schultz, L. D., George, H. A., Herber, W. K., Chae, H. J., Bentley, W. E., Rao, G., Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter. Biotechnol Bioeng, 2000 70(2): 187-96.

13.Ye, K., Shibasaki, S., Ueda, M., Murai, T., Kamasawa, N., Osumi, M., Shimizu, K., Tanaka, A., Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface. Appl Microbiol Biotechnol, 2000 54(1): 90-6.

14.Lee, S.M. and J.W. Park, Thermosensitive phenotype of yeast mutant lacking thioredoxin peroxidase. Arch Biochem Biophys, 1998 359(1): 99-106.

15.Grant, C.M., F.H. MacIver, and I.W. Dawes, Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. FEBS Lett, 1997 410(2-3): 219-22.

16.Davidson, J.F., Whyte, B., Bissinger, P. H., Schiestl, R. H., Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A, 1996 93(10): 5116-21.

17.Forgac, M., Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases. J Exp Biol, 2000 203(1): 71-80.

18.Nass, R. and R. Rao, Novel localization of a Na+/H+ exchanger in a late endosomal compartment of yeast. Implications for vacuole biogenesis. J Biol Chem, 1998 273(33): 21054-60.

19.Ramirez, J., A. Pena, and M. Montero-Lomeli, H+/K+ exchange in reconstituted yeast plasma membrane vesicles. Biochim Biophys Acta, 1996 1285(2): 175-82.

20.Altheim, B.A. and M.C. Schultz, Histone modification governs the cell cycle regulation of a replication- independent chromatin assembly pathway in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A, 1999 96(4): 1345-50.

21.Sun, Y., Taniguchi, R., Tanoue, D., Yamaji, T., Takematsu, H., Mori, K., Fujita, T., Kawasaki, T., Kozutsumi, Y., Sli2 (Ypk1), a homologue of mammalian protein kinase SGK, is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. Mol Cell Biol, 2000 20(12): 4411-9.

22.Ahn, S.H., A. Acurio, and S.J. Kron, Regulation of G2/M progression by the STE mitogen-activated protein kinase pathway in budding yeast filamentous growth. Mol Biol Cell, 1999 10(10): 3301-16.

23.Richman, T.J., M.M. Sawyer, and D.I. Johnson, The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast. J Biol Chem, 1999 274(24): 16861-70.

24.Griffioen, G., Anghileri, P., Imre, E., Baroni, M. D., Ruis, H., Nutritional control of nucleocytoplasmic localization of cAMP-dependent protein kinase catalytic and regulatory subunits in Saccharomyces cerevisiae. J Biol Chem, 2000 275(2): 1449-56.

25.Gerhardt, B., Kordas, T. J., Thompson, C. M., Patel, P., Vida, T., The vesicle transport protein Vps33p is an ATP-binding protein that localizes to the cytosol in an energy-dependent manner. J Biol Chem, 1998 273(25): 15818-29.