臺灣博碩士論文加值系統

HTL1為一酵母菌高溫致死基因。利用酵母菌雙雜交篩選、合成致死策略以及多拷貝抑制基因策略找尋其功能相關基因，分別得到RSC8、STH1、RSC3、RSC30、RSC58。這些皆屬於RSC複合物（Remodeling the Structure of Chromatin complex）之成員，目前已知RSC複合物之功能與染色質重組有關。由於已知特定的sth1及rsc3高溫敏感突變株在非容許（non-permissive）之環境下有G2/M階段之細胞生長休止情形，且sth1高溫敏感突變株在非容許溫度下生長以及rsc8刪除株細胞有異常的型態。此外，將htl1刪除株轉移至非容許溫度下生長一段時間後，發現其有G2/M階段之細胞生長休止現象；並在顯微鏡下觀察htl1刪除株和HTL1野生型菌株的細胞型態，並由統計分析方法來證實其二者是有統計上的差異，且將含有HTL1的片段轉殖到htl1刪除株中，發現可以拯救htl1刪除株的細胞之型態異常。此外，因htl1突變株在非容許溫度下生長有G2/M細胞週期發生休止現象，故推測Htl1p也許和細胞週期的調控有關，故將細胞同步化後，觀察在不同的時間點中，發現Htl1p的表現情形似乎對於細胞週期沒有特定的專一性。另一方面，為了了解Htl1p與RSC複合物之關係，除了藉由活體外結合實驗，找出Htl1p和Rsc8p的最小交互作用區域為Rsc8p的胺基酸序列之206-356的區域，且利用共同免疫沉澱法實驗觀察到在活體內：Htl1p和Rsc8p彼此之間有交互作用。接著，更進一步地利用蔗糖梯度分離實驗，觀察到：在HTL1野生型菌株中，Sth1p和Rsc8p在蔗糖梯度溶液中之中層的部分有同時分餾出來的現象，但同時也觀察到Rsc8p也有出現在蔗糖梯度溶液中之上層的部分。就Htl1p在蔗糖梯度溶液中的分布情形，觀察到有三個波峰存在：分別位於蔗糖梯度溶液中的上、中、下層，分布於中、上層的Htl1p似乎和Rsc8p在蔗糖梯度溶液中的分布情形類似；對於Htl1p出現在蔗糖梯度溶液中下層的部分，或許表示Htl1p似乎還有其他未知的功能存在。在htl1刪除株中，觀察到Rsc8p在蔗糖梯度溶液中的分布情形發生改變，即觀察到Rsc8p只分布在蔗糖梯度溶液中的上層部分。由此，得知在htl1刪除株之RSC複合物的結構性質和HTL1野生型菌株的RSC複合物之結構性質有差異，推測Htl1p可能藉由和Rsc8p的交互作用而使RSC複合物更趨於穩定。

HTL1 is essential for high temperature growth in yeast. By yeast two hybrid screening, Rsc8p was identified to have physical interaction with Htl1p. By the synthetic lethal scheme, STH1、RSC30 and RSC58 were found. By multi-copy suppression, RSC3 was able to rescue the temperature-sensitive lethalits of htl1 deletion mutant. Rsc8p、Sth1p、Rsc58p、Rsc30p and Rsc3p are members of RSC complex, mediating chromatin remodeling. Reports have demonstrated that specific sth1-ts and rsc8 strains exhibit G2/M cell cycle arrest and diversed cellular morphologies at non-permissive temperature. Similar phenotype was also observed in the htl1 deletion strain. To study the relationship between Htl1p and RSC complex , a series of analysis, including in vitro binding assay, coimmunoprecipitation analysis, sucrose gradient fractionation analysis and the statistical analysis of cell morphology were performed. Statistical analysis of cell morphology demonstrated that the size of htl1 deletion strain is significantly larger than that of the wildtype strain. By in vitro binding assay to further dissect the interaction region of Rsc8p, the region between a.a. 206 to a.a.356 of Rsc8p was defined to interact with Htl1p. This region contains a ZZ-zinc finger domain and a Myb-DNA binding domain. Furthermore, coimmunoprecipitation analysis showed that Htl1p was co-precipitated with Rsc8p. To examine the intactness of RSC complex in htl1 deletion mutant, sucrose gradient analysis was performed using Sth1p and Rsc8p as indicators. In wild-type yeast cells, Rsc8p and Sth1p were cofractionated in the high molecular weight fractions. However, Rsc8p also appeared in the low molecular weight fractions in which Sth1p was absent. The distribution of Htl1p in sucrose gradient was also examined. Three peaks were observed, one in the low molecular weight fractions and the second in the high molecular weight fractions similar to that of Rsc8p. However, the third peak appeared in fractions with ever higher molecular weight, indicating that Htl1p might possesses different cellular functions. In htl1 deletion strains, unlike Sth1p, the distribution of rsc8p was altered. The high molecular weight peak diminished. Our results suggest that by interacting with Rsc8p, Htl1p may play a role in the formation of stable RSC complex.

目錄
中文摘要…………………………………………………………3
英文摘要…………………………………………………………5
緒論………………………………………………………………7
材料……………………………………………………………..13
方法……………………………………………………………..16
結果……………………………………………………………..26
討掄……………………………………………………………..36
參考文獻…………………………………………………….….45
圖表………………………………………………………..……51

1.林育如。（1997）。利用合成性致死方法找出cas2相關基因。陽明大學，遺傳學研究所碩士班論文。
2.陸耀明。（1996）。酵母菌突變株cas2之分子遺傳特性研究。陽明大學，遺傳學研究所碩士班論文。
3.李朝欽。（2000）。探討酵母菌Rsc8p與Htllp之關係。陽明大學，遺傳學研究所碩士班論文。
4.黃姿雯。（2000）。功能性分析酵母菌SHL1基因。陽明大學，遺傳學研究所碩士班論文。
5.楊孟玲。（2001）。功能性分析酵母菌SHL1基因。陽明大學，生命科學系學士論文。
6.張禕倫。（2001）。探討酵母菌Htllp與RSC複合物之關係。陽明大學，生命科學系學士論文。
7.http://genome-www.stanford.edu/Saccharomtces/
8.http://expasy.ch/
9.http://www.ncbi.nlm.nih.gov/
10.Altschul SF,Gish W,Miller W,Myers EW,Lipman DJ.(1990).Basic local alignment search tool.J Mol Biol.215(3):403-10.
11.Andrew Travers.(1999).An engine for nucleosome remodeling.Cell.96,311-314.
12.Bender A,Pringle JR.(1991).Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae.Mol Cell Biol.11(3):1295-305.
13.Bjorklund S,Almouzni G,Davidson I,Nightingale Kp,Weiss K.(1999).Global transcription regulators of eukaryotes.Cell.98,759-767.
14.Burns N,Grimwade B,Ross-Macdonald PB,Choi EY,Finberg K,Roeder GS,Synder M.Large-scale analysis of gene expression,protein localization,and gene disruption in Saccharomyces cerevisiae.Genes Dev.1994,8(9):1087-105.
15.Cairns BR,Erdjument-Bromage H,Tempst P,Winston F,Kornberg RD,(1998).Two actin-related proteins are shared functional components of the components of the chromatin-remodeling complex RSC and SWI/SNF.Mol.Cell 2:639-651.
16.Cairns BR,Lorch Y,Li Y,Zhang M,Lacomis L,Erdjument-Bromage H,Tempst P,Du J,Laurent B,Kornberg R.(1996).RSC,an essential,abundant chromatin-remodeling complex.Cell.87(7):1249-60.
17.Cairns BR,Schlichter A,Erdjument-Bromage H,Tempst P,Kornberg RD,Winston F.(1999).Two tunctionally distinct forms of the RSC nucleosome remodeling complex,containing essential AT hook,BAH,and bromodomains.Mol cell.4(5):715-23.
18.Cao Y,Cairns BR,Kornberg RD,Laurent BC.(1997).Shlp,a component of a novel chromatin remodeling complex,is required for cell cycle progression.Mol Cell Biol.17(6):3323-34.
19.Chai B, Hsu JM, Du J and Laurent BC.（2002）.Yeast RSC function is required for organizatiion of the cellular cytoskeleton via an alternative PKC1 pathway. Genetics. 161(2):575-84
20.Chervitz SA,Aravind L,Sherlock G,Ball CA,Koonin EV,Dwight SS.Harris MA,Dolinski K,Mohr S,Smith T,Weng S,Cherry JM,Botstein D.(1998).Comparison of the complete protein sets of worm and yeast:orthology and divergence.Science.282(5396):2022-8.
21.Chien CT,Bartel PL,Sternglanz R,Fields S.(1991).The two-hybrid system:a method to identify and clone genes for proteins that interact with a protein of interest.Proc Natl Acad Sci USA.88(21):9578-82.
22.Du J,Nasir I,Benton BK,Kladde MP,Laurent BC.(1998).Sthlp,a Saccharomyces cerevisiae Snf2p/Swi2p homolog,is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins.Genetics.150(3):987-1005.
23.Fields,S. and Sternglanz,R.(1994).The two-hybrid system:an assay for protein-protein interactions.TIG 10,2860291.
24.Hosotani,T.,H.Koyama,M.Uchino,T.Miyakawa,and E.Tsuchiya.(2001).PKC1,a protein kinase C homologue of Saccharomyces cerevisiae,participates in microtubule function through the yeast EB1 homologue,BIM1?Genes Cell 6:775-88.
25.Kornberg RD,Lorch Y.(1991).Chromatin-modifying and remodeling complexes.Current Opinion in Genetics and Development.(9):148-151.
26.Kornberg RD,Lorch Y.(1991).Twenty-five years of the nucleosome,fundamental particle of the eukaryotic chromosome.Cell.98:285-294.
27.Kranz,J.E. and Holm,C.(1990).Cloning by function:an alternative approach for identifying yeast homologs of genes from other organisms.Proc.Natl.Acad.Sci.USA.87,6629-6633.
28.Lanzuolo,C.,S.Ederle,A.Pollice,F.Russo,A.Storlazzi,and J.F.Pulitzer.(2001).The HTL1 gene(YCR020W-b) of Saccharomyces cerevisiae is necessary for growth at 37 degrees C,and for the conservation of chromosome stability and fertility?Yeast 18:1317-30.
29.Lorch Y,Cairns BR,Zhang M,Kornberg RD.(1998).Activated RSC-nucleosome complex and persistently altered form of the nucleosome.Cell.94(1):29-34.
30.Lorch Y,Zhang M,Kornberg RD.(1999).Histone octamer transfer by a chromatin-remodeling complex.Cell.96(3):389-92.
31.Ma,X.J.,Lu,Q. and Grunstein,M.(1996).A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in Saccharomyces cerevisiae.Genes and Development.10,1327-1340.
32.Mackay,J.P. and Crossley,M.(1998).Zinc fingers are sticking together.Trends Biochem.Sci.23,1-4.
33.Melinda L.A.,Alisha S.,Douglas R.,Hediye E.B.,Paul T. and Bradley R.C.(2001).A RSC3/RSC30 Zinc cluster dimer reveals novel roles for the chromatin remodeler RSC in gene expression and cell cycle control.Mol.Cell.7,741-751.
34.Moreira JM,Holmberg S.(1998).Nucleosome structure of the yeast CHA1 promoter:analysis of activation-dependent chromatin remodeling of an RNA-polymerase-Ⅱ-transcribed gene in TBP and RNA polⅡ mutants defective in vivo in response to acidic activators.EMBO J.17(20):6028-38.
35.Moreira JM,Holmberg S.(1999).Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC.EMBO J.18(10):2836-44.
36.Ozier-Kalogeropoulos,O.,Adeline,M.T.,Yang,W.L.,Carman,G.M.and Lacroute,F.(1994).Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae.Mol.Gen.Genet.242,431-439.
37.Pazin MJ,Kadonaga JT.(1997).SWI/SNF and related proteins:ATP-driven motors that disrupt protein-DNA interactions?Cell.88:737-740.
38.Roberts SM,Winston F.(1997).Essential functional interactions of SAGA,a Saccharomyces cerevisiae complex of Spt,Ada,and Gcn5 proteins,with the Snf/Swi and Srb/ mediator complexes.Genetics.147(2):451-65.
39.Schagger H, and von Jagow.（1987）. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 166(2):368-79.
40.Schwartz,K.,K.Richards,and D.Botstein.(1997).BIM1 encodes a microtubule-binding protein in yeast?Mol Biol Cell 8:2677-91.
41.Treich I,Carlson M.(1997).Interaction of a Swi3 homolog with Sth1 provides evidence for a Swi/Snf-related complex with an essential function in Saccharomyes cerevisiae.Mol.Cell Biol.17(4):1768-75.
42.Treich I,Ho L,Carlson M.(1998).Direct interaction between Rsc6 and Rsc8/Swh3,two proteins that are conserved in SWI/SNF-related complexes.Nucleic Acids Res.26(16):3739-45.
43.Tsuchiya E,Hosotani T,Miyakawa T.(1998).A mutation in NPS1/STH1,an essential gene encoding a component of a novel chromatin-remodeling complex RSC,alters the chromatin structure of Saccharomyces cerevisiae centromeres.Nucleic Acids Res.26(13):3286-92.
44.Tsuchiya E,Uno M,Kiguchi A,Masuoka K,Kanemovi Y,Okabe S,Miyakawa T.(1998).The Saccharomyces cerevisiae NPS1 gene,a novel CDC gene which encodes a 150KDa nuclear protein involved in G2 phase control.EMBO J.11,4017-4026.
45.Venables W. N. and B. D. Ripley.（1999）Modern applied statistics with S-PLUS.thired edition
46.Velculescu VE,Zhang L,Zhou W,Vogelstein J,Basrai MA,Bassett DE Jr,Hieter P,Vogelstein B,Kinzler KW.(1997).Characterization of the yeast transcription.Cell.88(2):243-51.
47.Yukawa M,Katoh S,Miyakawa T,Tsuchiya E.(1999).Nps1/Sthlp,a component of an essential chromatin-remodeling complex of Saccharomyces cerevisiae,is required for the maximal expression of early meiotic genes.Genes Cells.4(2):99-110.