YDL100Cp 是在酵母菌Saccharomyces. cerevisiae.中發現的ArsA同源蛋白，在原核生物中，ArsA蛋白是抗砷系統中的催化次單元，但YDL100Cp在酵母菌中的功能卻仍然是未知的。初步研究結果顯示，在酵母菌中YDL100C基因的缺失並不會對細胞產生致命的傷害，而且此缺失對細胞在30℃下對砷的敏感性以及細胞的生長並沒有顯著的影響；但在40℃的高溫下，YDL100C基因的缺失卻會抑制細胞的生長及繁殖。在此研究中，經過了50℃ 15分鐘的致死性熱休克處理後，發現缺乏YDL100C基因的細胞對致死性的高溫有較高的敏感性，其存活率大約為野生菌種的二分之一，而且此差異可經由轉殖攜有YDL100C基因的載體彌補，證實了YDL100C基因在致死性的高溫下對酵母菌細胞具有保護的作用。
雖然在高溫下造成酵母菌細胞死亡的真正因素仍然是未知的，但有兩個因素是被認為與細胞在高溫下的死亡相關的，即高熱的傷害與氧化性的傷害。因此，為了進一步的確認YDL100C基因在細胞中的作用機制，在此研究中便針對一些已知在酵母菌中與這兩種傷害相關的抵禦機制的反應作進一步的探索。而在與高熱傷害有關的防禦機制的研究中，包含了Trehalose的累積、Hsp70的作用、以及Hsp104的作用等，發現YDL100C基因的缺失對這些機制的表現並無顯著的影響，因此初步的判定這些相關的防禦機制並非造成YDL100C基因缺失的菌株在致死性熱休克中存活率下降的主要原因。而在氧化性傷害相關的研究上，初步的發現在YDL100C基因缺失的菌株中，其細胞中所受到自由基的傷害較野生菌株低，這也許反映了YDL100C基因缺失菌株在高溫下較高的死亡率。而在測量其細胞中的還原性物質glutathione的含量比後，發現在YDL100C基因缺失的菌株中其所受到的氧化性傷害較為嚴重，但再進一歩以RT-PCR以及酵素活性測試檢視幾個與熱休克相關的抗氧化基因的表現，如catalase、superoxide dismutase、以及γ-glutamylcysteine synthetase等，卻未發現有顯著的差異，因此推測YDL100C基因與這些防禦機制的啟動應該沒有直接的關係。經由以上的結果，可以得知YDL100Cp可以降低酵母菌細胞在高溫下受到氧化性傷害的程度，進而使細胞渡過高溫的傷害；而YDL100Cp的作用並非經由啟動其他已知的自由基清除系統，此蛋白本身或許具有修復受氧化性傷害之蛋白的能力。

YDL100Cp is the ArsA homologue protein found in S. cerevisiae. In bacteria, ArsA protein is involved in As3+detoxification but the function of YDL100Cp is still unknown. Previous studies show that deletion of YDL100C in S. cerevisiae was not lethal and had no effect on As3+ sensitivity or growth at 30℃. However, when grown at 40℃, growth of YDL100C disrupted strain (JSY1) was inhibited. To study the role of YDL100C in response to lethal heat shock, wild type (W303-1B) and JSY1 cells were exposed to 50℃ for 15 min. The survival rate of JSY1 cells was half of W303-1B cells and the difference in survival rate was complemented by introduction of plasmid carrying YDL100C. It suggests that YDL100Cp plays a role in acquisition of thermotolerance to lethal heat shock. It is believed that there are two factors involved in heat-induced cell death: the heat damage and the oxidative damage. Determinations of heat-damage related defense system in S. cerevisiae, including trehalose (a thermoprotectant) content, Hsp70 expression and Hsp104 expression, demonstrate that heat damage should not be the major cause of JSY1 cell death during heat shock. For the oxidative damage, the measurement of in vivo reactive oxygen species reveal the lower protein damage caused by reactive oxygen species (ROS) in JSY-1 after 50℃ 15 min heat shock, this might reflect the difference in viability of three strains under lethal heat shock. And with the intra cellular content of glutathione, it revels that the YDL100C deficient caused cell got more serious oxidative damage under 50℃ heat shock. But the observation of thermotolerance related ROS scavenger system (including the catalase, and superoxide dismutase) expression with reverse transcription polymerase chain reaction suggested that YDL100C deficient had no effect on triggering these system. As the result, it is suggested that the function of YDL100Cp in S. cerevisiae might be an oxidative damage repair system, such as the glutathione peroxidase. It might react with the oxidative damage substance and function as a deoxidizer.

Abstract in Chinese ------------------------------------ 1
Abstract in English ------------------------------------ 3
Table of Contents -------------------------------------- 4
List of Tables and Figures ----------------------------- 5
Introduction ------------------------------------------- 6
Experimental Procedures ------------------------------- 12
Results ----------------------------------------------- 17
Discussion -------------------------------------------- 20
Tables ------------------------------------------------ 23
Figures ----------------------------------------------- 27
References -------------------------------------------- 35

Bhattacharjee, H., Ghosh, M., Mukhopadhyay, R., Rosen, B. P. (1999). Arsenic transporters from E. coli to humans in Broome-Smith JK, Baumberg, S., Sterling, C. J., and Ward, F.B. eds, Transport of molecules across microbial membranes, Society for General Microbiology. pp. 58–79.
Bhattacharjee, H., Ho, Y. S., Rosen, B. P. (2001). Genomic organization and chromosomal localization of the Asna1 gene, a mouse homologue of a bacterial arsenic-translocating ATPase gene. Gene. 272, 291-299.
Bruhn, D. F., Li, J., Silver, S., Roberto, F., Rosen, B. P., (1996). The arsenical resistance operon of IncN plasmid R46. FEMS Microbiol. Lett. 139, 149-153.
Cabib, E., Leloir, L. F. (1958). The biosynthesis of trehalose phosphate. J. Biol. Chem. 231, 259-275.
Cohen, G., Fessl, F., Traczyk, A., Rytka, J., Ruis, H. (1985). Isolation of the catalase A gene of Saccharomyces cerevisiae by complementation of the cta1 mutation. Mol. Gen. Genet. 200, 74-79.
Coote, P. J., Cole, M. B., Jones, M. V. (1991). Induction of increased thermotolerance in Saccharomyces cerevisiae may be triggered by a mechanism involving intracellular pH. J. Gen. Microbiol. 137, 1701-1708.
Coote, P. J., Jones, M. V., Edgar, K., Cole, M. B. (1992). TPK gene products mediate cAMP-independent thermotolerance in Saccharomyces cerevisiae. J. Gen. Microbiol. 138, 2551-2557.
Coote, P. J., Jones, M. V., Seymour, I. J., Rowe, D. L., Ferdinando, D. P., McArthur, A. J., Cole, M. B. (1994). Activity of the plasma membrane H(+)-ATPase is a key physiological determinant of thermotolerance in Saccharomyces cerevisiae. Microbiology. 140, 1881-1890.
Craig, E. A., Gross, C. A. (1991). Is hsp70 the cellular thermometer? Trends Biochem. Sci. 16, 135-140.
Davidson, J. F., Whyte, B., Bissinger, P. H., Schiestl, R. H. (1996). Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 93, 5116-5121.
De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T., Wiemken, A. (1994). The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant. Eur. J. Biochem. 219, 179-186.
Demple, B., Harrison, L. (1994). Repair of oxidative damage to DNA: enzymology and biology. Annu. Rev. Biochem. 63, 915-948.
Dey, S., Dou, D., Rosen, B. P., (1994). ATP-dependent arsenite transport in everted membrane vesicles of Escherichia coli. J. Biol. Chem. 269, 25442-25446.
Estruch, F. (2000). Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol Rev. 24, 469-486.
Gatti, D., Mitra, B., Rosen, B. P., (2000). Escherichia coli soft metal ion-translocating ATPases. J. Biol. Chem. 275, 34009-34012.
Glover, J. R., Lindquist, S. (1998). Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell. 94, 73-82.
Gottesman, S., Squires, C., Pichersky, E., Carrington, M., Hobbs, M., Mattick, J. S., Dalrymple, B., Kuramitsu, H., Shiroza, T., Foster, T. (1990). Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes. Proc. Natl. Acad. Sci. USA 87, 3513-3517.
Gralla, E. B., Kosman, D. J. (1992) Molecular genetics of superoxide dismutases in yeasts and related fungi. Adv. Genet. 30, 251-319.
Guidot, D. M., McCord, J. M., Wright, R. M., Repine, J. E. (1993) Absence of electron transport (Rho 0 state) restores growth of a manganese-superoxide dismutase-deficient Saccharomyces cerevisiae in hyperoxia. Evidence for electron transport as a major source of superoxide generation in vivo. J. Biol. Chem. 268, 26699-26703.
Holyoak, C. D., Stratford, M., McMullin, Z., Cole, M. B., Crimmins, K., Brown, A. J., Coote, P. J. (1996). Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid. Appl. Environ. Microbiol. 62, 3158-3164.
Hottiger, T., De Virgilio, C., Hall, M. N., Boller, T., Wiemken, A. (1994). The role of trehalose synthesis for the acquisition of thermotolerance in yeast. II. Physiological concentrations of trehalose increase the thermal stability of proteins in vitro. Eur. J. Biochem. 219, 187-193.
Inoue, Y., Kimura, A. (1995). Methylglyoxal and regulation of its metabolism in microorganisms. Adv. Microb. Physiol. 37, 177-227.
Izawa, S., Inoue, Y., Kimura, A. (1995) Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae. FEBS Lett. 368, 73-76.
Jamieson, D. J. (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast. 14, 1511-1527.
Jozwiak, Z. (1994). Involvement of heat shock proteins and cellular membranes in the development of thermotolerance. Arch. Immunol. Ther. Exp. 42, 247-252.
Jozwiak, Z., Leyko, W. (1992). Role of membrane components in thermal injury of cells and development of thermotolerance. Int. J. Radiat. Biol. 62, 743-756.
Kane, S. M., Roth, R. (1974). Carbohydrate metabolism during ascospore development in yeast. J. Bacteriol. 118, 8-14.
Kim, J., Alizadeh, P., Harding, T., Hefner-Gravink, A., Klionsky, D. J. (1996). Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications. Appl Environ. Microbiol. 62, 1563-1569.
Lee, J., Godon, C., Lagniel, G., Spector, D., Garin, J., Labarre, J., Toledano, M. B. (1999). Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J. Biol. Chem. 274, 16040-16046.
Lee, S. M., Park, J. W. (1998). Thermosensitive phenotype of yeast mutant lacking thioredoxin peroxidase. Arch. Biochem. Biophys. 359, 99-106.
Lillie, S. H., Pringle, J. R. (1980). Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J. Bacteriol. 143, 1384-1394.
Lis, J., Wu, C. (1993). Protein traffic on the heat shock promoter: parking, stalling, and trucking along. Cell. 74, 1-4.
Mager, W. H., Ferreira, P. M. (1993). Stress response of yeast. Biochem J. 290, 1-13.
Meister, A. (1985). Methods for the selective modification of glutathione metabolism and study of glutathione transport. Methods Enzymol. 113, 571-585.
Miller, M. J., Xuong, N. H., Geiduschek, E. P. (1982). J. Bacteriol. 151, 311-327.
Morimoto, R. I., Jaravich, D. A., Kroeger, P. E., Mathur, S. K., Murphy, S. P., Nakai, A., Sarge, K., Abravaya, K. & Sistoren, L. T. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones, eds. Morimoto, R. I., Tissieres, A. & Georgopoulos, C. (Cold Spring Harbor Lab. Press, New York), pp. 417–456.
Nwaka, S., Holzer, H. (1998). Molecular biology of trehalose and the trehalases in the yeast Saccharomyces cerevisiae. Prog. Nucleic Acid Res. Mol. Biol. 58, 197-237.
Parsell, D. A., Kowal, A. S., Singer, M. A., Lindquist, S. (1994). Protein disaggregation mediated by heat-shock protein Hsp104. Nature. 372, 475-478.
Parsell, D. A., Lindquist, S. (1993). The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet. 27, 437-496.
Parsell, D. A., Sanchez, Y., Stitzel, J. D., Lindquist, S. (1991). Hsp104 is a highly conserved protein with two essential nucleotide-binding sites. Nature. 353, 270-273.
Rothstein RJ. (1983). One-step gene disruption in yeast. Methods. Enzymol. 101, 202–211.
Royall, J. A., Ischiropoulos, H. (1993). Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch. Biochem. Biophys. 302, 348-355.
Russell, J., Ness, J., Chopra, M., McMurray, J., Smith, W. E. (1994). The assessment of the HO. scavenging action of therapeutic agents. J. Pharm. Biomed. Anal. 12, 863-866.
Sanchez, Y., Lindquist, S. L. (1990). HSP104 required for induced thermotolerance. Science. 248, 1112-1115.
Sanchez, Y., Taulien, J., Borkovich, K. A., Lindquist, S. (1992). Hsp104 is required for tolerance to many forms of stress. EMBO J. 11, 2357-2364.
Schirmer, E. C., Glover, J. R., Singer, M. A., Lindquist, S. (1996). HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21, 289-296.
Seah, T. C., Bhatti, A. R., Kaplan, J. G. (1973). Novel catalatic proteins of bakers' yeast. I. An atypical catalase. Can J. Biochem. 51, 1551-1555.
Seah, T. C., Kaplan, J. G. (1973). Purification and properties of the catalase of bakers' yeast. J. Biol. Chem. 248, 2889-2893.
Shen, J., Hsu, C. M., Kang, B. K., Rosen, B. P., Bhattacharjee, H. (2003). The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance. Biometals 16, 369-378.
Singer, M. A., Lindquist, S. (1998). Multiple effects of trehalose on protein folding in vitro and in vivo. Mol. Cell. 1, 639-648.
Sorger, P. K., Pelham, H. R. (1988). Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell. 54, 855-864.
Spevak, W., Fessl, F., Rytka, J., Traczyk, A., Skoneczny, M., Ruis, H. (1983). Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation. Mol. Cell. Biol. 3, 1545-1551.
Strain, J., Lorenz, C. R., Bode, J., Garland, S., Smolen, G. A., Ta, D. T., Vickery, L. E., Culotta, V. C. (1998). Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae. Identification of proteins predicted to mediate iron-sulfur cluster assembly. J. Biol. Chem. 273, 31138-31144.
Sugiyama, K., Kawamura, A., Izawa, S., Inoue, Y. (2000). Role of glutathione in heat-shock-induced cell death of Saccharomyces cerevisiae. Biochem J. 352, 71-78.
Suzuki, K., Wakao, N., Kimura, T., Sakka, K., Ohmiya, K. (1998). Expression and regulation of the arsenic resistance operon of Acidiphilium multivorum AIU 301 plasmid pKW301 in Escherichia coli. Appl. Environ. Microbiol. 64, 411-418.
Thevelein, J. M. (1984). Regulation of trehalose mobilization in fungi. Microbiol Rev. 48, 42-59.
Tissieres, A., Mitchell, H. K., Tracy, U. M. (1974). Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs. J. Mol. Biol. 84, 389-398.
Van Loon, A. P., Pesold-Hurt, B., Schatz, G. (1986). A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen. Proc. Natl. Acad. Sci. U S A. 83, 3820-3824.
Walker, J.E., Saraste, M., Runswick, M. J., Gay, N. J. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945-951.
Walmsley, A. R., Zhou, T., Borges-Walmsley, M. I., Rosen, B. P. (1999). The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump. J. Biol. Chem. 274, 16153-16161.
Wiederrecht, G., Seto, D., Parker, C. S. (1988). Isolation of the gene encoding the S. cerevisiae heat shock transcription factor. Cell. 54, 841-853.
Wiemken, A. (1990). Trehalose in yeast, stress protectant rather than reserve carbohydrate. Antonie Van Leeuwenhoek. 58, 209-217.
Zhou, T., Radaev, S., Rosen, B. P., Gatti, D. L. (2000). Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump. EMBO J. 19, 4838-4845.
Zhou, T., Rosen, B. P. (1997). Tryptophan fluorescence reports nucleotide-induced conformational changes in a domain of the ArsA ATPase. J. Biol. Chem. 272, 19731-19737.
Ziegelhoffer, T., Lopez-Buesa, P., Craig, E. A. (1995). The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates. J. Biol. Chem. 270, 10412-10419.
Zuniga, S., Boskovic, J., Jimenez, A., Ballesta, J. P., Remacha, M. (1999). Disruption of six Saccharomyces cerevisiae novel genes and phenotypic analysis of the deletants. Yeast 15, 945-953.
葉瓊霞, (2001). ArsA 同源蛋白在酵母菌內可能功能的探討, 國立中山大學生物科學研究所碩士論文.
洪詩雅, (2002). 酵母菌中YDL100c基因之表現及其可能功能探討, 國立中山大學生物科學研究所碩士論文.