目前大部分分子演化的研究主要著重於對DNA序列上蛋白質編碼序列的差異，而較少關注基因調控機制的變異。調控機制的變異可能也是導致演化的主要原因。本論文主要是以酵母菌(Saccharomyces cerevisiae)為模式生物，探討同序列調控因子(cis regulatory element)與外部調控因子(trans regulatory element)變異性對氧化逆境反應基因的影響。酵母菌在過氧化氫所產生的氧化壓力下有428個基因的表現量是正常狀況下的兩倍以上。我從其中373個具有單一核苷酸多型性的基因中隨機挑選了75個基因以焦磷酸定序定量分析技術研究兩酵母菌間(BY和RM品系)基因表現量的差異以探討同序列調控因子及外部調控因子變異性對酵母菌氧化逆境反應基因表現量差異的影響。我的研究結果顯示BY和RM品系酵母菌不管是在一般正常環境下或是在氧化壓力下，BY和RM對偶基因表現量的差異主要還是由外部調控因子變異性(trans major effect [trans variation effect alone + major trans variation effect])所造成的，在一般正常環境下與氧化壓力環境下受外部調控因子變異性(trans major effect)影響的基因比例分別是77% (57/74)和57.3%(43/75)。然而酵母菌細胞在經歷氧化壓力刺激的反應下，BY和RM對偶基因同序列調控因子變異性對BY和RM氧化壓力反應基因表現量的差異有更顯著的影響。經分析同序列調控因子變異性及外部調控因子變異性對這75個氧化壓力誘導基因在經歷氧化壓力後對BY和RM對偶基因表現量差異的影響，我發現有48% (36/75)的基因其影響BY和RM對偶基因表現量差異的因素不因氧化壓力刺激而改變，37.3% (28/75)的基因其影響BY和RM對偶基因表現量差異則因氧化壓力而使外部調控因子變異性的效應減弱(同序列調控因子變異性效應增強)，14.7% (11/75)的基因其影響BY和RM對偶基因表現量差異則因氧化壓力而使外部調控因子變異性的效應增強。分析基因在經歷氧化壓力後影響BY和RM基因表現量差異的因素不因氧化壓力刺激而改變或使外部調控因子變異性的效應增強、減弱可能與哪些因素有關，我發現調控基因之轉錄因子的數量多寡、氧化壓力反應轉錄因子種類的不同、TATA box 調控元件的有無、基因在染色體的位置(染色體左、右臂)，以及基因在DNA雙股上的位置(華生、克利克股) 都與外部調控因子變異性效應的改變與否有關聯性。根據實驗的結果我認為雖然在一般正常環境下或是在氧化壓力環境下，BY和RM基因表現量的差異主要都是受到外部調控因子變異性的影響，然而在氧化壓力反應下外部調控因子變異性效應則有顯著減弱的現象，顯示同序列調控因子變異性對影響酵母菌族群在氧化壓力的適應性上扮演了重要的角色。

Variation(s) in both protein coding sequence and expression contributes to phenotypic evolution. However, most studies of molecular evolution have examined changes in protein coding sequences; little was known on the regulatory roles on species diversity. Recently, several reviews pointed out that mutations in transcriptional regulation may have been a major cause for phenotypic evolution. In this study, I used yeast Saccharomyces cerevisae as a model organism to study the contribution of cis regulatory variation and trans regulatory variation to the expression divergence between two yeast strains, BY and RM, under oxidative stress condition. Previous microarray studies showed that the expression of about 428 genes were induced and about 457 genes whose expression was repressed at least two folds in response to oxidative stress in S. cerevisiae. In this study I mainly focus on the oxidative stress induced genes because these genes could be responsible for the adaption for oxidative stress. I randomly selected 75 genes for pyrosequencing analysis to determine the contribution of cis and trans regulatory variation to the expression divergence of two different yeast strains, BY and RM strains. My results indicated the expression divergence of BY and RM were mainly due to trans regulatory variations in both normal condition and in oxidation stress condition, 77% (57/74) and 57.3%(43/75) of the genes were affected by the trans major regulatory variations, respectively. However, cis regulatory variation seems to play a very important role in oxidation stress. My results indicated that 48% (36/75) of genes showed the same trend of cis or trans variation effect even after oxidation stress, 37.3% (28/75) of genes showed decrease of trans variation effect and 14.7% (11/75) of genes showed increase of trans variation effect after oxidation stress. Although the expression divergence of BY and RM were mainly due to trans regulatory variations in both normal condition and in oxidation stress condition, however trans regulatory variations showed significant decrease under oxidation stress condition. I think cis regulatory variation was important for yeast adaptate to oxidation stress enviroment.

Basehoar, A.D., S.J. Zanton, and B.F. Pugh. Identification and distinct regulation of yeast TATA box-containing genes. Cell 116: 699-709. 2004.
Beck, T. and M.N. Hall. The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402: 689-692. 1999.
Boy-Marcotte, E., G. Lagniel, M. Perrot, F. Bussereau, A. Boudsocq, M. Jacquet, and J. Labarre. The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons. Mol Microbiol 33: 274-283. 1999.
Brem, R.B., G. Yvert, R. Clinton, and L. Kruglyak. Genetic dissection of transcriptional regulation in budding yeast. Science 296: 752-755. 2002.
Brown, J.L., H. Bussey, and R.C. Stewart. Yeast Skn7p functions in a eukaryotic two-component regulatory pathway. EMBO J 13: 5186-5194. 1994.
Bush, R. M., and K. Paigen. Evolution of β-glucuronidase regulation in the genus Mus. Evolution 46:1–15. 1992.
Carroll, S.B. Endless forms: the evolution of gene regulation and morphological diversity. Cell 101: 577-580. 2000.
Causton, H.C., B. Ren, S.S. Koh, C.T. Harbison, E. Kanin, E.G. Jennings, T.I. Lee, H.L. True, E.S. Lander, and R.A. Young. Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell 12: 323-337. 2001.
Cliften, P., P. Sudarsanam, A. Desikan, L. Fulton, B. Fulton, J. Majors, R. Waterston, B.A. Cohen, and M. Johnston. Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science 301: 71-76. 2003.
Coleman, S.T., E.A. Epping, S.M. Steggerda, and W.S. Moye-Rowley. Yap1p activates gene transcription in an oxidant-specific fashion. Mol Cell Biol 19: 8302-8313. 1999.
Cowles, C.R., J.N. Hirschhorn, D. Altshuler, and E.S. Lander. Detection of regulatory variation in mouse genes. Nat Genet 32: 432-437. 2002.
Daborn, P.J., J.L. Yen, M.R. Bogwitz, G. Le Goff, E. Feil, S. Jeffers, N. Tijet, T. Perry, D. Heckel, P. Batterham et al. A single p450 allele associated with insecticide resistance in Drosophila. Science 297: 2253-2256. 2002.
Daryi Wang, Huang-Mo Sung, Tzi-Yuan Wang, Chih-Jen Huang, Peggy Yang, Tiffany Chang, Yang-Chao Wang, Da-Lun Tseng, Jen-Pey Wu, Tso-Ching Lee, Ming-Che Shih, and Wen-Hsiung Li. Expression evolution in yeast genes of single-input modules is mainly due to changes in trans-acting factors. Genome Research 17: 1161-1169. 2007.
Eastmond, D.L. and H.C. Nelson. Genome-wide analysis reveals new roles for the activation domains of the Saccharomyces cerevisiae heat shock transcription factor (Hsf1) during the transient heat shock response. J Biol Chem 281: 32909-32921. 2006.
Estruch, F. Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol Rev 24: 469-486. 2000.
Estruch, F. and M. Carlson. Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae. Mol Cell Biol 13: 3872-3881. 1993.
Gasch, A.P., P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, and P.O. Brown. Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11: 4241-4257. 2000.
Georgiou, G. 2002. How to flip the (redox) switch. Cell 111: 607-610.
Godon, C., G. Lagniel, J. Lee, J.M. Buhler, S. Kieffer, M. Perrot, H. Boucherie, M.B. Toledano, and J. Labarre. The H2O2 stimulon in Saccharomyces cerevisiae. J Biol Chem 273: 22480-22489. 1998.
Görner, W., E. Durchschlag, M.T. Martinez-Pastor, F. Estruch, G. Ammerer, B. Hamilton, H. Ruis, and C. Schüller. Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 12: 586-597. 1998.
Herrero E, Ros J, Bellí G, Cabiscol E. Redox control and oxidative stress in yeast cells. Biochim Biophys Acta. 1780(11):1217-35. 2008.
Hohmann, S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66: 300-372. 2002.
Ikner, A. and K. Shiozaki. Yeast signaling pathways in the oxidative stress response. Mutat Res 569: 13-27. 2005.
Kobayashi, N. and K. McEntee. Evidence for a heat shock transcription factor-independent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 87: 6550-6554. 1990.
Kobayashi, N. and K. McEntee. Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae. Mol Cell Biol 13: 248-256. 1993.
Kuge, S. and N. Jones. YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO J 13: 655-664. 1994.
Landry, C.R., B. Lemos, S.A. Rifkin, W.J. Dickinson, and D.L. Hartl. Genetic properties influencing the evolvability of gene expression. Science 317: 118-121. 2007.
Laurie-Ahlberg, C.C. and G.C. Bewley. Naturally occurring genetic variation affecting the expression of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster. Biochem Genet 21: 943-961. 1983.
Lee, J., C. Godon, G. Lagniel, D. Spector, J. Garin, J. Labarre, and M.B. Toledano. Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J Biol Chem 274: 16040-16046. 1999.
Li, W.H. Molecular evolution. Sinauer Associated, Inc., Sunderland Massachusettes.
Lushchak VI. 2010. Oxidative stress in yeast. Biochemistry (Mosc) 75: 281-96. 1997.
Mager, W.H. and A.J. De Kruijff. Stress-induced transcriptional activation. Microbiol Rev 59: 506-531. 1995.
Martinez-Pastor, M.T., G. Marchler, C. Schüller, A. Marchler-Bauer, H. Ruis, and F. Estruch. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15: 2227-2235. 1996.
Morgan, B.A., G.R. Banks, W.M. Toone, D. Raitt, S. Kuge, and L.H. Johnston. The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. EMBO J 16: 1035-1044. 1997.
Moskvina, E., C. Schüller, C.T. Maurer, W.H. Mager, and H. Ruis. A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements. Yeast 14: 1041-1050. 1998.
Moye-Rowley, W.S. Regulation of the transcriptional response to oxidative stress in fungi: similarities and differences. Eukaryot Cell 2: 381-389. 2003.
Moye-Rowley, W.S., K.D. Harshman, and C.S. Parker. Yeast YAP1 encodes a novel form of the jun family of transcriptional activator proteins. Genes Dev 3: 283-292. 1989.
O'Rourke, S.M., I. Herskowitz, and E.K. O'Shea. Yeast go the whole HOG for the hyperosmotic response. Trends Genet 18: 405-412. 2002.
Osada, N., M.H. Kohn, and C.I. Wu. Genomic inferences of the cis-regulatory nucleotide polymorphisms underlying gene expression differences between Drosophila melanogaster mating races. Mol Biol Evol 23: 1585-1591. 2006.
Purugganan, M.D. The molecular population genetics of regulatory genes. Mol Ecol 9: 1451-1461. 2000.
Rifkin, S.A., J. Kim, and K.P. White. Evolution of gene expression in the Drosophila melanogaster subgroup. Nat Genet 33: 138-144. 2003.
Rockman, M.V. and L. Kruglyak. Genetics of global gene expression. Nat Rev Genet 7: 862-872. 2006.
Rockman, M.V. and G.A. Wray. Abundant raw material for cis-regulatory evolution in humans. Mol Biol Evol 19: 1991-2004. 2002.
Ronald, J., R.B. Brem, J. Whittle, and L. Kruglyak. Local regulatory variation in Saccharomyces cerevisiae. PLoS Genet 1: e25. 2005.
Ruis, H. and C. Schüller. Stress signaling in yeast. Bioessays 17: 959-965. 1995.
Schadt, E.E., S.A. Monks, T.A. Drake, A.J. Lusis, N. Che, V. Colinayo, T.G. Ruff, S.B. Milligan, J.R. Lamb, G. Cavet et al. Genetics of gene expression surveyed in maize, mouse and man. Nature 422: 297-302. 2003.
Schmitt, A.P. and K. McEntee. Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 93: 5777-5782. 1996.
Stern, D.L. Evolutionary developmental biology and the problem of variation. Evolution 54: 1079-1091. 2000.
Streelman, J.T. and T.D. Kocher. 2002. Microsatellite variation associated with prolactin expression and growth of salt-challenged tilapia. Physiol Genomics 9: 1-4.
Sung HM, Wang TY, Wang D, Huang YS, Wu JP, Tsai HK, Tzeng J, Huang CJ, Lee YC, Yang P, Hsu J, Chang T, Cho CY, Weng LC, Lee TC, Chang TH, Li WH, Shih MC. Roles of trans and cis variation in yeast intraspecies evolution of gene expression. Mol Biol Evol. 26:2533-8. 2009.
Tautz, D. Evolution of transcriptional regulation. Curr Opin Genet Dev 10: 575-579. 2000.
Temple, M.D., G.G. Perrone, and I.W. Dawes. Complex cellular responses to reactive oxygen species. Trends Cell Biol 15: 319-326. 2005.
Tirosh, I., A. Weinberger, M. Carmi, and N. Barkai. A genetic signature of interspecies variations in gene expression. Nat Genet 38: 830-834. 2006.
Tirosh, I., S. Reikhav, A. A. Levy, and N. Barkai. A yeast hybrid provides insight into the evolution of gene expression regulation. Science 324:659-662. 2009.
Treger, J.M., T.R. Magee, and K. McEntee. Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae. Biochem Biophys Res Commun 243: 13-19. 1998.
Wang D, Sung HM, Wang TY, et al. (12 co-authors). Expression evolution in yeast genes of single-input modules is mainly due to changes in trans-acting factors. Genome Res. 17:1161–1169. 2007.
Wieser, R., G. Adam, A. Wagner, C. Schüller, G. Marchler, H. Ruis, Z. Krawiec, and T. Bilinski. Heat shock factor-independent heat control of transcription of the CTT1 gene encoding the cytosolic catalase T of Saccharomyces cerevisiae. J Biol Chem 266: 12406-12411. 1991.
Wittkopp, P.J., B.K. Haerum, and A.G. Clark. Evolutionary changes in cis and trans gene regulation. Nature 430: 85-88. 2004.
Wray, G.A., M.W. Hahn, E. Abouheif, J.P. Balhoff, M. Pizer, M.V. Rockman, and L.A. Romano. The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol 20: 1377-1419. 2003.
Wu, A.L. and W.S. Moye-Rowley. GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation. Mol Cell Biol 14: 5832-5839. 1994.
Yvert, G., R.B. Brem, J. Whittle, J.M. Akey, E. Foss, E.N. Smith, R. Mackelprang, and L. Kruglyak. Trans-acting regulatory variation in Saccharomyces cerevisiae and the role of transcription factors. Nat Genet 35: 57-64. 2003.