Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96398
標題: 酵母菌耐高溫之實驗演化
Experimental Evolution of Yeast for High Temperature Tolerance
作者: 黃志仁
Chih-Jen Huang
關鍵字: 耐熱
逆境適應
實驗演化學
集群分離分析法
數量性狀基因座
thermotolerance
stress adaptation
experimental evolution
high temperature growth
protein complexes
pooled segregant analysis
引用: Adams J, Puskas-Rozsa S, Simlar J, Wilke CM. 1992. Adaptation and major chromosomal changes in populations of Saccharomyces cerevisiae. Curr Genet 22:13-19. Alcazar-Roman AR, Tran EJ, Guo S, Wente SR. 2006. Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export. Nat Cell Biol 8:711-716. Altmann A, Weber P, Bader D, Preuss M, Binder EB, Muller-Myhsok B. 2012. A beginners guide to SNP calling from high-throughput DNA-sequencing data. Hum Genet 131:1541-1554. Benedetti H, Raths S, Crausaz F, Riezman H. 1994. The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast. Mol Biol Cell 5:1023-1037. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120. Bolotinfukuhara M, Faye G, Fukuhara H. 1977. Temperature-Sensitive Respiratory-Deficient Mitochondrial Mutations - Isolation and Genetic-Mapping. Molecular & General Genetics 152:295-305. Bozza WP, Zhuang ZH. 2011. Biochemical Characterization of a Multidomain Deubiquitinating Enzyme Ubp15 and the Regulatory Role of Its Terminal Domains. Biochemistry 50:6423-6432. Bricmont PA, Daugherty JR, Cooper TG. 1991. The Dal81 Gene-Product Is Required for Induced Expression of 2 Differently Regulated Nitrogen Catabolic Genes in Saccharomyces-Cerevisiae. Mol Cell Biol 11:1161-1166. Buchan JR, Muhlrad D, Parker R. 2008. P bodies promote stress granule assembly in Saccharomyces cerevisiae. J Cell Biol 183:441-455. Carmona-Gutierrez D, Alavian-Ghavanini A, Habernig L, Bauer MA, Hammer A, Rossmann C, Zimmermann AS, Ruckenstuhl C, Buttner S, Eisenberg T, et al. 2013. The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast. Cell Cycle 12:1704-1712. Caspeta L, Chen Y, Ghiaci P, Feizi A, Buskov S, Hallstrom BM, Petranovic D, Nielsen J. 2014. Biofuels. Altered sterol composition renders yeast thermotolerant. Science 346:75-78. Celenza JL, Carlson M. 1984. Cloning and Genetic-Mapping of Snf1, a Gene Required for Expression of Glucose-Repressible Genes in Saccharomyces-Cerevisiae. Mol Cell Biol 4:49-53. Chaleff DT, Tatchell K. 1985. Molecular-Cloning and Characterization of the Ste7 and Ste11 Genes of Saccharomyces-Cerevisiae. Mol Cell Biol 5:1878-1886. Chen YL, Konieczka JH, Springer DJ, Bowen SE, Zhang J, Silao FG, Bungay AA, Bigol UG, Nicolas MG, Abraham SN, et al. 2012. Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata. G3 (Bethesda) 2:675-691. Clemons KV, McCusker JH, Davis RW, Stevens DA. 1994. Comparative pathogenesis of clinical and nonclinical isolates of Saccharomyces cerevisiae. J Infect Dis 169:859-867. Cubillos FA, Parts L, Salinas F, Bergstrom A, Scovacricchi E, Zia A, Illingworth CJR, Mustonen V, Ibstedt S, Warringer J, et al. 2013. High-Resolution Mapping of Complex Traits with a Four-Parent Advanced Intercross Yeast Population. Genetics 195:1141-1141. Dawes IW, Hardie ID. 1974. Selective Killing of Vegetative Cells in Sporulated Yeast Cultures by Exposure to Diethyl-Ether. Molecular & General Genetics 131:281-289. de Jesus Ferreira MC, Bao X, Laize V, Hohmann S. 2001. Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperature. Curr Genet 40:27-39. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, et al. 2011. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491-498. Devenish RJ, Prescott M, Roucou X, Nagley P. 2000. Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex. Biochimica Et Biophysica Acta-Bioenergetics 1458:428-442. DeZwaan DC, Freeman BC. 2010. HSP90 manages the ends. Trends Biochem Sci 35:384-391. Drake JW. 1991. A constant rate of spontaneous mutation in DNA-based microbes. Proc Natl Acad Sci U S A 88:7160-7164. Duc C, Pradal M, Sanchez I, Noble J, Tesniere C, Blondin B. 2017. A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation. PLoS One 12(9):e0184838. Fitch PG, Gammie AE, Lee DJ, de Candal VB, Rose MD. 2004. Lrg1p is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast. Genetics 168:733-746. Friedman KL, Heit JJ, Long DM, Cech TR. 2003. N-terminal domain of yeast telomerase reverse transcriptase: Recruitment of Est3p to the telomerase complex. Mol Biol Cell 14:1-13. Hahn JS, Thiele DJ. 2004. Activation of the Saccharomyces cerevisiae heat shock transcription factor under glucose starvation conditions by Snf1 protein kinase. Journal of Biological Chemistry 279:5169-5176. Harrison JC, Bardes EG, Zyla TR, Lew DJ. 2002. Stress-specific activation mechanisms for the 'cell integrity' MAPK pathway in Saccharomyces cerevisiae. Mol Biol Cell 13:294a-294a. Harrison JC, Zyla TR, Bardes ESG, Lew DJ. 2004. Stress-specific activation mechanisms for the 'cell integrity' MAPK pathway. Journal of Biological Chemistry 279:2616-2622. Herskowitz I, Jensen RE. 1991. Putting the Ho Gene to Work - Practical Uses for Mating-Type Switching. Methods in Enzymology 194:132-146. Heude M, Fukuhara H, Moustacchi E. 1979. Spontaneous and induced rho mutants of Saccharomyces cerevisiae: patterns of loss of mitochondrial genetic markers. J Bacteriol 139:460-467. Huang da W, Sherman BT, Lempicki RA. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44-57. Huang DW, Sherman BT, Tan Q, Kir J, Liu D, Bryant D, Guo Y, Stephens R, Baseler MW, Lane HC, et al. 2007. DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Res 35:W169-175. Huxley C, Green ED, Dunham I. 1990. Rapid Assessment of Saccharomyces-Cerevisiae Mating Type by Pcr. Trends in Genetics 6:236-236. Illingworth CJR, Parts L, Bergstrom A, Liti G, Mustonen V. 2013. Inferring Genome-Wide Recombination Landscapes from Advanced Intercross Lines: Application to Yeast Crosses. PLoS One 8(5):e62266. Jimenez J, Benitez T. 1988. Yeast cell viability under conditions of high temperature and ethanol concentrations depends on the mitochondrial genome. Curr Genet 13:461-469. Kamada Y, Jung US, Piotrowski R, Levin DE. 1995. The Protein-Kinase C-Activated Map Kinase Pathway of Saccharomyces-Cerevisiae Mediates a Novel Aspect of the Heat-Shock Response. Genes Dev 9:1559-1571. Koszul R, Caburet S, Dujon B, Fischer G. 2004. Eucaryotic genome evolution through the spontaneous duplication of large chromosomal segments. EMBO J 23:234-243. Krysan DJ, Ting EL, Abeijon C, Kroos L, Fuller RS. 2005. Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae. Eukaryot Cell 4:1364-1374. Lang GI, Murray AW. 2008. Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae. Genetics 178:67-82. Langle-Rouault F, Jacobs E. 1995. A method for performing precise alterations in the yeast genome using a recycable selectable marker. Nucleic Acids Res 23:3079-3081. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357-359. Layfield R, Franklin K, Landon M, Walker G, Wang P, Ramage R, Brown A, Love S, Urquhart K, Muir T, et al. 1999. Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes. Analytical Biochemistry 274:40-49. Levin DE. 2005. Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 69:262-262. Li H. 2011. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27:2987-2993. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754-1760. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078-2079. Li WH. 1993. Unbiased Estimation of the Rates of Synonymous and Nonsynonymous Substitution. Journal of Molecular Evolution 36:96-99. Lingner J, Cech TR, Hughes TR, Lundblad V. 1997. Three ever shorter telomere (EST) genes are dispensable for in vitro yeast telomerase activity. Proc Natl Acad Sci U S A 94:11190-11195. Long A, Liti G, Luptak A, Tenaillon O. 2015. Elucidating the molecular architecture of adaptation via evolve and resequence experiments. Nat Rev Genet 16:567-582. Lorberg A, Schmitz HP, Jacoby JJ, Heinisch JJ. 2001. Lrg1p functions as a putative GTPase-activating protein in the Pkc1p-mediated cell integrity pathway in Saccharomyces cerevisiae. Molecular Genetics and Genomics 266:514-526. Lussier M, White AM, Sheraton J, di Paolo T, Treadwell J, Southard SB, Horenstein CI, Chen-Weiner J, Ram AF, Kapteyn JC, et al. 1997. Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147:435-450. Lynch M, Sung W, Morris K, Coffey N, Landry CR, Dopman EB, Dickinson WJ, Okamoto K, Kulkarni S, Hartl DL, et al. 2008. A genome-wide view of the spectrum of spontaneous mutations in yeast. Proc Natl Acad Sci U S A 105:9272-9277. Maclean CJ, Metzger BPH, Yang JR, Ho WC, Moyers B, Zhang J. 2017. Deciphering the Genic Basis of Yeast Fitness Variation by Simultaneous Forward and Reverse Genetics. Mol Biol Evol 34:2486-2502. Madden K, Snyder M. 1998. Cell polarity and morphogenesis in budding yeast. Annu Rev Microbiol 52:687-744. Maeta K, Izawa S, Inoue Y. 2005. Methylglyoxal, a metabolite derived from glycolysis, functions as a signal initiator of the high osmolarity glycerol-mitogen-activated protein kinase cascade and Calcineurin/Crz1-mediated pathway in Saccharomyces cerevisiae. Journal of Biological Chemistry 280:253-260. McCusker JH, Clemons KV, Stevens DA, Davis RW. 1994a. Genetic characterization of pathogenic Saccharomyces cerevisiae isolates. Genetics 136:1261-1269. McCusker JH, Clemons KV, Stevens DA, Davis RW. 1994b. Saccharomyces cerevisiae virulence phenotype as determined with CD-1 mice is associated with the ability to grow at 42 degrees C and form pseudohyphae. Infect Immun 62:5447-5455. Murthi A, Shaheen HH, Huang HY, Preston MA, Lai TP, Phizicky EM, Hopper AK. 2010. Regulation of tRNA bidirectional nuclear-cytoplasmic trafficking in Saccharomyces cerevisiae. Mol Biol Cell 21:639-649. Nandakumar J, Cech TR. 2013. Finding the end: recruitment of telomerase to telomeres. Nature Reviews Molecular Cell Biology 14:71-71. Nei M, Gojobori T. 1986. Simple Methods for Estimating the Numbers of Synonymous and Nonsynonymous Nucleotide Substitutions. Mol Biol Evol 3:418-426. Pamilo P, Bianchi NO. 1993. Evolution of the Zfx and Zfy Genes - Rates and Interdependence between the Genes. Mol Biol Evol 10:271-281. Parts L, Cubillos FA, Warringer J, Jain K, Salinas F, Bumpstead SJ, Molin M, Zia A, Simpson JT, Quail MA, et al. 2011. Revealing the genetic structure of a trait by sequencing a population under selection. Genome Res 21:1131-1138. Piper PW, Ortiz-Calderon C, Holyoak C, Coote P, Cole M. 1997. Hsp30, the integral plasma membrane heat shock protein of Saccharomyces cerevisiae, is a stress-inducible regulator of plasma membrane H(+)-ATPase. Cell Stress Chaperones 2:12-24. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. 2011. Integrative genomics viewer. Nat Biotechnol 29:24-26. Roelants FM, Torrance PD, Bezman N, Thorner J. 2002. Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity. Mol Biol Cell 13:3005-3028. Schwarz SE, Matuschewski K, Liakopoulos D, Scheffner M, Jentsch S. 1998. The ubiquitin-like proteins SMT3 and SUMO-1 are conjugated by the UBC9 E2 enzyme. Proc Natl Acad Sci U S A 95:560-564. Sherman F. 1959. The effects of elevated temperatures on yeast. II. Induction of respiratory-deficient mutants. J Cell Comp Physiol 54:37-52. Shitamukai A, Hirata D, Sonobe S, Miyakawa T. 2004. Evidence for antagonistic regulation of cell growth by the calcineurin and high osmolarity glycerol pathways in Saccharomyces cerevisiae. Journal of Biological Chemistry 279:3651-3661. Shivaswamy S, Iyer VR. 2008. Stress-dependent dynamics of global chromatin remodeling in yeast: Dual role for SWI/SNF in the heat shock stress response. Mol Cell Biol 28:2221-2234. Singh RK, Gonzalez M, Kabbaj MHM, Gunjan A. 2012. Novel E3 Ubiquitin Ligases That Regulate Histone Protein Levels in the Budding Yeast Saccharomyces cerevisiae. PLoS One 7(5):e36295. Singh RK, Kabbaj MHM, Paik J, Gunjan A. 2009. Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis. Nature Cell Biology 11:925-U940. Sinha H, David L, Pascon RC, Clauder-Munster S, Krishnakumar S, Nguyen M, Shi G, Dean J, Davis RW, Oefner PJ, et al. 2008. Sequential elimination of major-effect contributors identifies additional quantitative trait loci conditioning high-temperature growth in yeast. Genetics 180:1661-1670. Sinha H, Nicholson BP, Steinmetz LM, McCusker JH. 2006. Complex genetic interactions in a quantitative trait locus. PLoS Genet 2:e0020013. Smith A, Ward MP, Garrett S. 1998. Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation. Embo Journal 17:3556-3564. Spor A, Kvitek DJ, Nidelet T, Martin J, Legrand J, Dillmann C, Bourgais A, de Vienne D, Sherlock G, Sicard D. 2014. Phenotypic and Genotypic Convergences Are Influenced by Historical Contingency and Environment in Yeast. Evolution 68:772-790. Steels EL, Learmonth RP, Watson K. 1994. Stress Tolerance and Membrane Lipid Unsaturation in Saccharomyces-Cerevisiae Grown Aerobically or Anaerobically. Microbiology-Uk 140:569-576. Steinmetz LM, Sinha H, Richards DR, Spiegelman JI, Oefner PJ, McCusker JH, Davis RW. 2002. Dissecting the architecture of a quantitative trait locus in yeast. Nature 416:326-330. Swiecilo A. 2016. Cross-stress resistance in Saccharomyces cerevisiae yeast-new insight into an old phenomenon. Cell Stress Chaperones 21:187-200. Tang HY, Munn A, Cai M. 1997. EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae. Mol Cell Biol 17:4294-4304. Tang HY, Xu J, Cai M. 2000. Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis. Mol Cell Biol 20:12-25. Tenaillon O, Rodriguez-Verdugo A, Gaut RL, McDonald P, Bennett AF, Long AD, Gaut BS. 2012. The Molecular Diversity of Adaptive Convergence. Science 335:457-461. Tesniere C, Delobel P, Pradal M, Blondin B. 2013. Impact of Nutrient Imbalance on Wine Alcoholic Fermentations: Nitrogen Excess Enhances Yeast Cell Death in Lipid-Limited Must. PLoS One 8(4):e61645. Thewes S. 2014. Calcineurin-Crz1 Signaling in Lower Eukaryotes. Eukaryot Cell 13:694-705. Traven A, Wong JM, Xu D, Sopta M, Ingles CJ. 2001. Interorganellar communication. Altered nuclear gene expression profiles in a yeast mitochondrial dna mutant. J Biol Chem 276:4020-4027. Verna J, Lodder A, Lee K, Vagts A, Ballester R. 1997. A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 94:13804-13809. Walden H, Podgorski MS, Huang DT, Miller DW, Howard RJ, Minor DL, Holton JM, Schulman BA. 2003. The structure of the APPBP1-UBA3-NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1. Mol Cell 12:1427-1437. Wang D, Zhang Y, Zhang Z, Zhu J, Yu J. 2010. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genomics Proteomics Bioinformatics 8:77-80. Wang K, Li M, Hakonarson H. 2010. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38(16):e164. Watanabe D, Abe M, Ohya Y. 2001. Yeast Lrg1p acts as a specialized RhoGAP regulating 1,3-beta-glucan synthesis. Yeast 18:943-951. Winkler A, Arkind C, Mattison CP, Burkholder A, Knoche K, Ota I. 2002. Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway, and protein tyrosine phosphatases are essential under heat stress. Eukaryot Cell 1:163-173. Xie C, Tammi MT. 2009. CNV-seq, a new method to detect copy number variation using high-throughput sequencing. BMC Bioinformatics 10:80. Yang Y, Foulquie-Moreno MR, Clement L, Erdei E, Tanghe A, Schaerlaekens K, Dumortier F, Thevelein JM. 2013. QTL analysis of high thermotolerance with superior and downgraded parental yeast strains reveals new minor QTLs and converges on novel causative alleles involved in RNA processing. PLoS Genet 9:e1003693. Yona AH, Manor YS, Herbst RH, Romano GH, Mitchell A, Kupiec M, Pilpel Y, Dahan O. 2012. Chromosomal duplication is a transient evolutionary solution to stress. Proc Natl Acad Sci U S A 109:21010-21015. Zahringer H, Burgert M, Holzer H, Nwaka S. 1997. Neutral trehalase Nth1p of Saccharomyces cerevisiae encoded by the NTH1 gene is a multiple stress responsive protein. FEBS Lett 412:615-620. Zhao C, Jung US, Garrett-Engele P, Roe T, Cyert MS, Levin DE. 1998. Temperature-induced expression of yeast FKS2 is under the dual control of protein kinase C and calcineurin. Mol Cell Biol 18:1013-1022. Zubko EI, Zubko MK. 2014. Deficiencies in mitochondrial DNA compromise the survival of yeast cells at critically high temperatures. Microbiol Res 169:185-195.
摘要: '耐熱'是一種由多基因決定的性狀,用以描述在高溫的環境下,生物體的存活的能力與生長的情形。即便有零星的耐熱相關基因被發現,生物如何逐漸累積自身的突變以適應高溫的環境卻還不是那麼的清楚。在本研究中,我利用實驗演化學的方法以階梯式的漸進升溫,成功地將酵母菌的耐熱生長溫度提高到了42度。利用全基因體定序技術,我解序了14株親本及序列演化品系,並從其中發掘出153個不同的突變,囊括了各種點突變、小插入缺失以及長片段的染色體區段性複製/缺失變異。這些突變發生之後,有些突變隨溫度上升至42度的演化過程中被保留下來,某種程度上代表了這些突變與耐熱生長的性狀有所關聯。利用功能性分類工具,我發現到一些突變傾向於發生在某些蛋白質複合體上,例如SWI/SNF 複合體以及F型三磷酸腺苷合成酶。同時,也看到一些突變發生在逆境調控相關的訊息傳遞路徑上,例如Hog1, RAS-cAMP, 以及 Rho1-Pkc 等。這些研究結果顯示,要達到耐熱生長,部分可藉由修改現有的逆境調控機制來達成。利用集群分離分析法,我進一步分析了五組不同耐熱程度的分離子樣本群,找到了42度耐熱的關鍵基因突變組,並進一步定義了其中較具高顯性的突變。利用分子遺傳學方法,也成功的在親本的遺傳背景下,以功能性實驗驗證了這些突變所帶來的耐熱效果。本研究顯著地拓展了我們對酵母菌耐熱遺傳基礎的了解。
Thermotolerance is a polygenic trait that contributes to cell survival and growth under unusually high temperatures. Although some genes associated with high temperature growth (Htg+) have been identified, how cells accumulate mutations to achieve prolonged thermotolerance is still mysterious. Here I conducted experimental evolution of a Saccharomyces cerevisiae laboratory strain with stepwise temperature increases for it to grow at 42 °C. Whole genome resequencing of 14 evolved strains and the parental strain revealed a total of 153 mutations in the evolved strains, including single nucleotide variants, small INDELs, and segmental duplication/deletion events. Some mutations persisted from an intermediate temperature to 42 °C, so they might be Htg+ mutations. Functional categorization of mutations revealed enrichment of exonic mutations in the SWI/SNF complex and F-type ATPase, pointing to their involvement in high temperature tolerance. In addition, multiple mutations were found in a general stress associated signal transduction network consisting of Hog1 mediated pathway, RAS-cAMP pathway, and Rho1-Pkc1 mediated cell wall integrity pathway, implying that cells can achieve Htg+ partly through modifying existing stress regulatory mechanisms. Using pooled segregant analysis of 5 Htg+ phenotype-orientated pools, I inferred causative mutations for growth at 42 °C and identified those mutations with stronger impacts on the phenotype. Finally, I experimentally validated a number of the candidate Htg+ mutations. This study increased our understanding of the genetic basis of yeast tolerance to high temperature.
URI: http://hdl.handle.net/11455/96398
文章公開時間: 2019-07-09
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