族群中的個體跟個體間都存在的差異，且絕大部分是先天個體之間遺傳物質(DNA)的變異所造成，這其中包含了基因去氧核醣核苷酸(DNA)序列中蛋白質編譯區域(coding region)存在的變異(nucleotide polymorphism)，或是在DNA序列中控制基因表現調控(regulatory)區段的不同所造成的。目前分子演化的研究較少著墨於基因調控機制的變異，所以轉錄調控機制的改變在演化上還有很多是目前尚未明瞭的。本論文將探討酵母菌族群間在滲透壓(osmotic stress)逆境壓力下，基因調控機制的演化，尤其是同序列調控因子變異性(cis regulatory variations)及外部調控因子變異性(trans regulatory variations)對酵母菌在高滲透逆境壓力下對基因調控的影響。酵母菌在高滲透壓逆境下大約有378個基因會被誘發表現，以及約有365個基因的表現被抑制。本論文從378個被誘發表現基因中隨機挑選75個具BY-RM基因多型性(polymorphism)的基因，利用焦磷酸定序定量分析技術檢測BY-RM對偶基因的相對表現量以推論同序列調控因子變異性及外部調控因子變異性對BY-RM基因表現量差異的相對貢獻程度。本論文的研究結果顯示BY和RM品系酵母菌不管是在一般正常環境下還是在高滲透壓力環境下，導致BY和RM對偶基因表現量差異的主要還是由外部調控因子變異性(trans major variation effect [trans variation effect alone + major trans variation effect])所造成的，其受外部調控因子變異性影響的基因分別佔76.0%以及62.2%。進一步分析這75個高滲透壓反應基因在經歷高滲透壓力後對BY和RM對偶基因表現量差異的影響，有41.3% (31/75)的基因其影響BY和RM對偶基因表現量差異的因素(同序列調控因子變異性或外部調控因子變異性)並不因高滲透壓刺激而改變、33.3% (25/75)的基因其影響BY和RM對偶基因表現量差異的因素則因高滲透壓刺激而使外部調控因子變異性的效應減弱以及25.3% (19/75)的基因其影響BY和RM對偶基因表現量差異的因素則因高滲透壓刺激而使外部調控因子變異性的效應增強。經分析顯示，外部調控因子變異性效應改變，可能跟基因本身上游轉錄因子個數多寡，滲透壓反應轉錄因子(Sko1、Cin5、Msn2、Msn4和Skn7)，基因所在之染色體位置(左、右臂位置)，以及基因所在之DNA雙股位置(華生股、克利克股)有關聯性。根據分析結果在一般正常環境下或是在高滲透壓環境下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，然而在高滲透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適應高滲透壓力上扮演了重要的角色。

Nucleotide differences in coding regions or in regulatory elements could cause variation among individuals in a species. However, evolutionary changes in transcription regulation are still poorly understood on species divergence. In this thesis, I studied the relative evolutionary roles of cis and/or trans regulatory variation for yeast cells to cope with environmental changes, specifically focusing on the osmotic stress response. I used yeast Saccharomyces cerevisiae BY and RM strains to study the expression evolution when cells are under osmotic stress condition. It has been shown that the expression of about 378 genes were induced and about 365 genes whose expression was repressed at least two folds in response to osmotic stress in S. cerevisiae from microarray studies. I randomly selected 75 genes from 378 genes which induced overexpression under osmotic stress for further pyrosequencing analysis to determine the relative contributions of cis and trans regulatory variations to the expression divergences between BY and RM. My results indicated the expression divergence of BY and RM were mainly due to trans regulatory variations under both standard growth condition and osmotic stress condition, 76.0% and 62.2% of the genes were affected by the trans major regulatory variations, respectively. My results showed that 41.3% (31/75) of genes showed the same trend of cis or trans variation effect even after osmotic stress, 33.3% (25/75) of genes showed decrease of trans variation effect and 25.3% (19/75) of genes showed increase of trans variation effect after osmotic stress. Further analysis showed a positive correlation between the upstream TF numbers and the changes in trans variation effect, the more TF the easier to show the changes in trans variation effect. My results also indicated that the Sko1 and Msn4 may exhitbit greater expression divergence between BY and RM strains under osmotic stress condition compared to the normal unstress condition. More detail analysis is presented in the main text. According to my analysis results, most of genes showed decrease of trans variation effect in osmotic stress condition. Thus my conclusion for this thesis is cis regulatory variations played an important role when yeast adapt to environment of osmotic stress.

Alepuz, P.M., E. de Nadal, M. Zapater, G. Ammerer, and F. Posas. Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II. EMBO J 22: 2433-2442. 2003.
Alepuz, P.M., A. Jovanovic, V. Reiser, and G. Ammerer. Stress-induced map kinase Hog1 is part of transcription activation complexes. Mol Cell 7: 767-777. 2001.
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.
Blomberg, A., and L. Adler. Physiology of osmotolerance in fungi. Adv Microb Physiol 33:145-212. 1992.
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.
Carroll, S.B. Endless forms: the evolution of gene regulation and morphological diversity. Cell 101: 577-580. 2000.
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.
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.
Csonka, L. N., and A. D. Hanson. Prokaryotic osmoregulation: genetics and physiology. Annu Rev Microbiol 45:569-606. 1991.
de Nadal, E., L. Casadome, and F. Posas. Targeting the MEF2-like transcription factor Smp1 by the stress-activated Hog1 mitogen-activated protein kinase. Mol Cell Biol 23: 229-237. 2003.
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.
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.
Hohmann, S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66: 300-372. 2002.
Hohmann, S., M. Krantz, and B. Nordlander. Yeast osmoregulation. Methods Enzymol 428: 29-45. 2007.
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.
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.
Lewis, J. G., R. P. Learmonth, and K. Watson. Induction of heat, freezing and salt tolerance by heat and salt shock in Saccharomyces cerevisiae. Microbiology 141: 687–694. 1995.
Li, W.H. 1997. Molecular evolution. Sinauer Associated, Inc., Sunderland Massachusettes.
Mager, W.H. and A.J. De Kruijff. Stress-induced transcriptional activation. Microbiol Rev 59: 506-531. 1995.
Mager, W.H. and A.J. De Kruijff. Stress-induced transcriptional activation. Microbiol Rev 59: 506-531. 1995.
Marchler, G., Schu¨ ller, C., Adam, G. and Ruis, H. A Saccharomyces cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions. EMBO J. 12: 1997–2003. 1993.
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.
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.
Park, J. I., C. M. Grant, P. V. Attfield, and I. W. Dawes. The freeze-thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway. Appl Environ Microbiol 63: 3818–3824. 1997.
Posas, F., S.M. Wurgler-Murphy, T. Maeda, E.A. Witten, T.C. Thai, and H. Saito. Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor. Cell 86: 865-875. 1996.
Proft, M., F.D. Gibbons, M. Copeland, F.P. Roth, and K. Struhl. Genomewide identification of Sko1 target promoters reveals a regulatory network that operates in response to osmotic stress in Saccharomyces cerevisiae. Eukaryot Cell 4: 1343-1352. 2005.
Proft, M., A. Pascual-Ahuir, E. de Nadal, J. Arino, R. Serrano, and F. Posas. Regulation of the Sko1 transcriptional repressor by the Hog1 MAP kinase in response to osmotic stress. EMBO J 20: 1123-1133. 2001.
Proft, M. and K. Struhl. Hog1 kinase converts the Sko1-Cyc8-Tup1 repressor complex into an activator that recruits SAGA and SWI/SNF in response to osmotic stress. Mol Cell 9: 1307-1317. 2002.
Purugganan, M.D. The molecular population genetics of regulatory genes. Mol Ecol 9: 1451-1461. 2000.
Raitt, D.C., F. Posas, and H. Saito. Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway. EMBO J 19: 4623-4631. 2000.
Rep, M., M. Proft, F. Remize, M. Tamas, R. Serrano, J.M. Thevelein, and S. Hohmann. The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage. Mol Microbiol 40: 1067-1083. 2001.
Rep, M., V. Reiser, U. Gartner, J.M. Thevelein, S. Hohmann, G. Ammerer, and H. Ruis. Osmotic stress-induced gene expression in Saccharomyces cerevisiae requires Msn1p and the novel nuclear factor Hot1p. Mol Cell Biol 19: 5474-5485. 1999.
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.
Saito, H. and K. Tatebayashi. Regulation of the osmoregulatory HOG MAPK cascade in yeast. J Biochem 136: 267-272. 2004.
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.
Struhl, K. Molecular mechanisms of transcriptional regulation in yeast. Annu Rev Biochem 58:1051-1077. 1989.
Sung, H. M., T. Y. Wang, D. Wang, Y. S. Huang, J. P. Wu, H. K. Tsai, J. Tzeng, C. J. Huang, Y. C. Lee, P. Yang, J. Hsu, T. Chang, C. Y. Cho, L. C. Weng, T. C. Lee, T. H. Chang, W. H. Li, and M. C. Shih. Roles of trans and cis variation in yeast intraspecies evolution of gene expression. Mol Biol Evol 26:2533-2538. 2009.
Tatebayashi, K., K. Yamamoto, K. Tanaka, T. Tomida, T. Maruoka, E. Kasukawa, and H. Saito. Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway. EMBO J 25: 3033-3044. 2006.
Tautz, D. Evolution of transcriptional regulation. Curr Opin Genet Dev 10: 575-579. 2000.
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.
Trollmo, C., L. André, A. Blomberg, and L. Adler. Physiological overlap between osmotolerance and thermotolerance in Saccharomyces cerevisiae. FEMS Microbiol. Lett 56: 321–326. 1988.
Tsai, H.K., M.Y. Chou, C.H. Shih, G.T. Huang, T.H. Chang, and W.H. Li. MYBS: a comprehensive web server for mining transcription factor binding sites in yeast. Nucleic Acids Res 35: W221-226. 2007.
Wang, D., H. M. Sung, T. Y. Wang, C. J. Huang, P. Yang, T. Chang, Y. C. Wang, D. L. Tseng, J. P. Wu, T. C. Lee, M. C. Shih, and W. H. Li. 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. Schuller, 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.
Wood, J. M. Osmosensing by bacteria: signals and membrane-based sensors. Microbiol Mol Biol Rev 63:230-262. 1999.
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 WS, Li WH. Systematic identification of yeast cell cycle transcription factors using multiple data sources. BMC Bioinformatics 9: 522. 2008.
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.