以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：28

、訪客IP：3.144.46.90

姓名	葉蟬嫻(Chan-Hsien Yeh)  查詢紙本館藏	畢業系所	生命科學系
論文名稱	探討酵母菌ALA1基因的non-AUG轉譯機制 (Mechamism of non-AUG initiation in yeast ALA1)
相關論文	★ Kineosphaera limosa 菌株中 phaC 基因之序列分析 ★ 剪力和組織蛋白去乙醯酶在動靜脈廔管失效扮演的角色 ★ Classification of powdery mildews on ornamental plants in northern Taiwan ★ 秀麗隱桿線蟲線粒體AlaRS通過非傳統模式識別T型無臂tRNAAla ★ Bacillus thuringiensis contains two prolyl-tRNA synthetases of different origins ★ Recognition of tRNA His isoacceptors by human HisRS isoforms ★ Functional replacement of yeast nuclear and mitochondrial RNase P by a protein-only RNase P ★ Functional characterization of a noncanonical ProRS in Toxoplasma gondii ★ tRNA aminoacylation by a naturally occurring mini-AlaRS ★ Functional Repurposing of C-Ala Domains ★ Recognition of a non-canonical tRNAAla by a non-canonical alanyl-tRNA synthetase ★ 探討Alanyl-tRNA synthetase的演化及專一性 ★ 酵母菌valyl-tRNA synthetase附加區段的 生物功能之探討 ★ 探討酵母菌glycyl-tRNA合成酵素的非傳統生物功能 ★ 探討酵母菌Valyl-tRNA synthetase的生化活性 ★ 酵母菌轉譯起始機制的研究
檔案	[Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   [檢視]  [下載] 本電子論文使用權限為同意立即開放。 已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。 請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。
摘要(中)	ALA1是酵母菌唯一的alanyl-tRNA synthetase (AlaRS) 基因，其5’端只有一個ATG起始密碼 (即ATG1)，卻可以轉譯出兩種型的AlaRS，分別作用在細胞質及粒腺體內。ALA1利用ATG1轉譯出細胞質的AlaRS，利用ATG1上游的二個重複non-ATG (即ACG-25和ACG-24) 作出序列較長的粒腺體AlaRS。本實驗著重在進一步研究酵母菌的轉譯機制，已知ALA1可以轉錄出三條長短不同的messenger RNA (簡稱mRNA)，其5’端分別座落在核苷酸-143，-105和-54上；利用功能性互補試驗 (complementaion) 及西方點漬法 (western blotting)，我發現ALA1會利用選擇性轉錄及轉譯的方式，由長度不同的mRNA做出大小不同的兩種AlaRS，也可用leaky scanning的方式由同一條mRNA合成細胞質及粒腺體AlaRS，一般而言這兩種機制甚少同時出現，因此ALA1是相當獨特的例子。除此之外，我也證實non-ATG下游的二級結構，對於non-ATG轉譯機制並非絕對需要，當此結構被破壞時，non-ATG依舊可以有效的產生粒腺體AlaRS，維持酵母菌的生
摘要(英)	It was recently shown that ALA1, the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase (AlaRS), encodes both cytoplasmic and mitochondrial forms. The former is translationally initiated at the ATG codon (designated ATG1) at the 5’-end of its open reading frame, while the latter is initiated from upstream in-frame redundant non-ATG codons (i.e., ACG-25 and ACG-24). In this thesis, I investigated the translational mechanism of ALA1 by which long and short protein isoforms were produced from the single gene. Like many known non-ATG initiators, a secondary structure is identified downstream of ACG-25. However, mutations that destroy the secondary structure do not impair its initiating activity. Functional tests, in combination with Western blot analysis, suggest that the isoforms of AlaRS can be translated from long and short transcripts by alternative transcription/translation, or from a single transcript by leaky scanning. To our knowledge, this appears to be a novel case where both leaky scanning and alternative transcription/translation are involved in the production of protein isoforms.
關鍵字(中)	★ 酵母菌 ★ 轉譯機制	關鍵字(英)	★ ALA1 ★ AlaRS ★ non-AUG translation
論文目次	目錄……………………………………………………………..….....I 縮寫檢索表…………………………………………………….…....Ⅲ 中文摘要……………………………………………………….…......1 英文摘要……………………………………………………………...2 第一章 緒論………………………………………………………...3 第二章 材料與方法 l 使用之菌株、載體....………………………………………..…..8 l 建構質體……………………………………………………..…..8 l 用PCR方式破壞pseudoknot 二級結構……………..……….9 l 功能性互補試驗 (Complementation ) ― 細胞質AlaRS的功能測試……………………………..……..10 l 功能性互補試驗 ― 粒腺體AlaRS的功能測試………….…12 l 西方點漬法 (Western Blotting) A. 蛋白質製備 (Protein Preparation) …………….…..….....13 B. 西方點漬法……………………………………………..….15 l 5’ RACE (Rapid Amplification of cDNA Ends) ……….….…17 第三章 結果 I. ALA1基因可以選擇性轉錄及轉譯方式來產生 粒腺體及細胞質兩種型的AlaRS…………….……..……...20 II. ALA1基因可以leaky scanning的方式由同一條 mRNA同時轉譯粒腺體及細胞質AlaRS…………….......21 III. 破壞mRNA上的pseudoknot二級結構，對於 粒腺體蛋白質的轉譯並無明顯的影響……………............22 Ⅳ. 以西方點漬法證實non-AUG可以做為轉譯起始點.…....23 第四章 討論………………………………………………………26 第五章 參考文獻…………………………………………………29 圖表…………………………………………………………………35 附錄一………………………………………………………………41
參考文獻	Acland, P., Dixon, M., Peters, G., and Dickson, C. (1990) Subcellular fate of the int-2 oncoprotein is determined by choice of initiation codon. Nature 343, 662-665 Arnaud, E., Touriol, C., Boutonnet, C., Gensac, M. C., Vagner, S., Prats, H., and Prats, A. C. (1999) A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol. Cell. Biol. 19, 505–514. Birnboim, H. C. and Doly, J. (1980) A rapid alkaline extraction procedure for screening recombinant plamid DNA. Nucleic acid Res. 7: 1513-1523 Bradford, M. M. (1976) A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Carroll, R. and Derse, D. (1993) Translation of equine infectious anemia virus bicistronic tat-rev mRNA requires leaky ribosome scanning of the tat CTG initiation codon. J. Virol. 67, 1433–1440. Carter, C. W. Jr. (1993) Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu. Rev. Biochem. 62: 715-748 Chatton, B., Walter, P., Ebel, J. P., Lacroute, F., and Fasiolo, F. (1988) The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. J. Biol. Chem. 263: 52-57. Cigan, A. M., Pabich, E. K., and Donahue, T. F. (1988) Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae. Mol. Cell. Biol. 8: 2964-2975. Clements, J. M., Laz, T. M., and Sherman, F. (1988) Efficiency of translation initiation by non-AUG codons in Saccharomyces cerevisiae. Mol. Cell. Biol. 8: 4533-4536. Donahue, T. F. and Cigan, A. M. (1988) Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region. Mol. Cell. Biol. 8: 2955-2963 Fajardo, J. E., Birge, R. B., and Hanafusa, H. (1993) A 31-amino acid N-terminal extension regulates c-Crk binding to tyrosine- phosphorylated proteins. Mol. Cell. Biol. 13, 7295–7302. Felter, S., Diatewa, M., Schneider, C., and Stahl, A. J. (1981) Yeast mitochondrial and cytoplasmic valyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 98: 727-734. Florkiewicz, R. Z. and Sommer, A. (1989) Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non- AUG codons. Proc. Natl. Acad. Sci. USA 86, 3978–398 Fu¨tterer, J., Potrykus, I., Bao, Y., Li, L., Burns, T. M., Hull, R., and Hohn (1996) Position-dependent ATT initiation during plant pararetrovirus rice tungro bacilliform virus translation. J. Virol. 70, 2999–3010. Fuxe, J., Raschperger, E., and Pettersson, R. F. (2000) Translation of p15.5INK4B, an N-terminally extended and fully active form of p15INK4B, is initiated from an upstream GUG codon. Oncogene 19, 1724–1728. Giegé, R., Sissler, M., and Florentz, C. (1998) Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 26: 5017-5035 Hackett, P. B., Petersen, R. B., Hensel, C. H., Albericio, F., Gunderson, S. I., Palmenberg, A. C., and Barany, G. (1986) Synthesis in vitro of a seven amino acid peptide encoded in the leader RNA of Rous sarcoma virus. J. Mol. Biol. 190, 45–57. Hann, S. R., Aloan-Brown, K., and Spotts, G. D. (1992) Translational activation of the non-AUG-initiated c-myc 1 protein at high cell densities due to methionine deprivation. Genes Dev. 6, 1229-1240 Kozak, M. (1989) Context effects and inefficient initiation at non-AUG codons in eukaryotic cell-free translation systems. Mol. Cell. Biol. 9: 5073-5080. Kozak, M. (1990) Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc. Natl. Acad. Sci. USA 87: 8301-8305. Kozak, M. (1991) An analysis of vertebrate mRNA sequences: intimationsof translational control. J. Cell Biol. 115, 887–903. Kozak, M. (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem. 266: 19867-19870. Kozak, M. (1997) Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO J 16(9):2482-92 Kozak, M. (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234: 187-208 Lock, P., Ralph, S., Stanley, E., Boulet, I., Ramsay, R., and Dunn, A. R. (1991) Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization. Mol. Cell. Biol. 11, 4363–4370. Manistic, T., et al. (1989) Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory. Martinis, S. A. and Schimmel, P. (1996) In Escherichia coli and Salmonella Cellular and Molecular Biology, ed. Neidhardt, F. C. (Am. Soc. Microbiol., Washington, DC), 2nd Ed., pp. 887-901 Maréchal-Drouard, L., Weil, J. H., and Dietrich, A. (1993) Transfer RNAs and transfer RNA genes in plants. Annu. Rev. Cell. Biol. 8: 115-131. Mireau, H., Lancelin, D., and Small, I. D. (1996) The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases The Plant Cell 8: 1027-1039 Muralidhar, S., Becerra, S. P., and Rose, J. A. (1994) Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity. J. Virol. 68, 170–176. Nakai, K. and Horton, P. (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization Trends Biochem. Sci. 24: 34-36 Natsoulis, G., Hilger, F., and Fink, G. R. (1986) The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae. Cell 46: 235-243. Nett, J. H., Kessl, J., Wenz, T., and Trumpower, B. L. (2001) The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons. Eur. J. Biochem. 268: 5209-5214 Packham, G., Brimmell, M., and Cleveland, J. L. (1997) Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation. Biochem. J. 328, 807-813 Pelchat, M. and Lapointe, J. (1999) Aminoacyl-tRNA synthetase genes of Bacillus subtilis: organization and regulation. Biochem. Cell Biol. 77: 343-347 Portis, J. L., Spangrude, G. J., and McAtee, F. J. (1994) Identification of a sequence in the unique 5’ open reading frame of the gene encoding glycosylated gag which influences the incubation period of neurodegenerative disease induced by a murine retrovirus. J. Virol. 68, 3879–3887. Raney, A., Baron, A. C., Mize, G. J., Law, G. L., and Morris, D. R. (2000) In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase. J. Biol. Chem. 275, 24444–24450. Ripmaster, T. L., Shiba, K., and Schimmel, P. (1995) Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen. Proc. Natl. Acad. Sci. USA 92: 4932-4936 Saris, C. J., Domen, J., and Berns, A. (1991) The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG. EMBO J. 10, 655-664 Sherman, F., Stewart, J. W., and Schweingruber, A. M. (1980) Mutants of yeast initiating translation of iso-1-cytochrome c within a region spanning 37 nucleotides. Cell 20: 215-222. Sikorski, R. S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27 Souciet, G., Menand, B., Ovesna, J., Cosset, A., Dietrich, A., and Wintz, H. (1999) Characterization of two bifunctional Arabdopsis thaliana genes coding for mitochondrial and cytosolic forms of valyl-tRNA synthetase and threonyl-tRNA synthetase by alternative use of two in-frame AUGs. Eur. J. Biochem. 266: 848-854. Spotts, G. D., Patel, S. V., Xiao, Q., and Hann, S. R. (1997) Identification downstream-initiated c-myc proteins which are dominant-negative inhibitors of transactivation by full-length c-myc proteins. Mol. Cell. Biol. 17, 1459–1468. Wang, L. and Wessler, S. R. (2001) Role of mRNA secondary structure in translational repression of the maize transcriptional activator Lc. Plant Physiol. 125, 1380–1387. Yoon, H. and Donahue, T. F. (1992) Control of translation initiation in Saccharomyces cerevisiae. Mol. Biol. 6: 1413-1419 唐蕙苓 (2002) 酵母菌轉譯起始機制的研究。中央大學碩士論文。
指導教授	王健家(Chien-Chia Wang)	審核日期	2003-7-21
推文	facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu
網路書籤	Google bookmarks   del.icio.us   hemidemi   myshare