跳到主要內容

臺灣博碩士論文加值系統

(3.236.23.193) 您好!臺灣時間:2021/07/24 13:06
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:程正儀
研究生(外文):Cheng-Yi Cheng
論文名稱:人類苯丙胺酸羥化酶基因剪接調控之分析
論文名稱(外文):Analysis of the Splicing Regulation of the Human Phenylalanine Hydroxylase Gene
指導教授:蘇宗笙
指導教授(外文):Tsung-Sheng Su
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:苯丙胺酸羥化酶
外文關鍵詞:phenylalanine hydroxylaseexon skipping
相關次數:
  • 被引用被引用:0
  • 點閱點閱:136
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
人類苯丙胺酸羥化酶(phenylalanine hydroxylase, PAH)具有肝臟表現的專一性,是負責將體內的苯丙胺酸(phenylalanine)代謝轉化為酪胺酸(tyrosine)。一旦發生缺失即會導致苯酮尿症的產生。
研究指出原生型PAH mRNA的外顯子11在進行剪接時會發生被略過的狀況,而當外顯子11上具有c.1197A>T (V399V) silent mutation時,會導致mRNA剪接異常,使得外顯子11完全的被略過。為了探究其原因,本研究利用S1 nuclease mapping的方法確定肝臟細胞中內生性原生型PAH mRNA有相同的現象後,我們針對外顯子11剪接位的序列進行分析,然後以定點突變的方式,改善位在內含子10的剪接接受者序列。之後將這樣的minigene質體送入人類肝癌細胞株,HuH-7,取得RNA,以RT-PCR的方式分析其RNA剪接的狀況。結果發現,PAH外顯子11的剪接錯誤和剪接位的不理想有關。而且改善之剪接位足以彌補c.1197A>T突變所造成之剪接錯誤。
由於剪接位的不理想,我們預期在外顯子11或內含子10區域,可能有輔助剪接位使用的剪接加強子存在,因此利用bioinformatics的方式,進行剪接加強子SR protein結合序列比對,企圖找出其位置。我們選擇了四個預測的位置進行分析。以定點突變的方式,破壞其與SR蛋白質的結合位,發現經由這樣的改變後,有部分確會造成外顯子11被略過的情況加劇,但並非所有預測的位置皆是如此,顯示bioinformatics的預測並非全然可靠。
爲了更廣泛尋找剪接加強子的位置,我們利用被登錄在外顯子11上的各種突變,觀察他們對剪接所造成的影響,以期歸納出剪接加強子的位置。在這研究中,我們發現了幾個誤義突變及中止譯碼突變會對剪接造成正面或負面的影響,顯示許多突變表面上改變了蛋白質的組成,但是其造成的影響早在RNA階段就開始了。而這些突變所在的序列可能與調控外顯子11的被確認有關,似乎也意味著,PAH外顯子11的剪接是由許多不同的調控模組所負責的。
Human phenylalanine hydroxylase (PAH) is a liver-specific enzyme, which involved in the rate-limiting step of phenylalanine catabolism. Deficiency of PAH activity results in phenylketonuria, an autosomal recessive disorder.
Study has shown that PAH exon 11 skipping occurs in a fraction of PAH mRNA when illegitimate PAH RNA from lymphoblasts was analyzed by RT-PCR. And, exon 11 is totally skipped when the exon 11 mutation, c.1197A>T, occurs. By S1 nuclease mapping, we confirmed that exon 11 skipping indeed occurs in RNA isolated from liver cells. To study the mechanism of exon 11 skipping, we compared sequence of splice site to that of consensus, and found that the splicing acceptor site of intron 10 has poor pyrimidine content. When we modified such site in the minigene to become optimal by site-directed mutagenesis, we found that exon 11 recognition improved greatly. Thus, the poor exon 11 recognition in the human PAH gene may be mainly due to the sub-optimal splice acceptor site in intron 10.
Because of weak splice site, one would predict the existence of splicing enhancer on exon 11 and/or intron 10 to facilitate exon 11 recognition. Two strategies were used to search for such sequences: (1) to correlate between SR protein score matrices and PAH exon 11 splicing; (2) to evaluate naturally occurring missense, nonsense, frameshift and silent mutations on PAH exon 11 recognition. The nucleotide substitutions of predicted SR protein binding site or sequences correspond to the nature mutations were introduced into minigene, RNA splicing pattern was then analyzed by RT-PCR. Our results show the lack of the predictive capacity of SR protein score matrices on the PAH exon 11 recognition. On the other hand, several nature mutations induce changes in the splicing pattern with either positive or negative effect on exon 11 recognition. The results suggest that human PAH exon 11 recognition may act through multiple sequence motifs.
Ars, E., Serra, E., Garcia, J., Kruyer, H., Gaona, A., Lazaro, C., and Estivill, X. (2000). Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum Mol Genet 9, 237-247.
Berget, S. M. (1995). Exon recognition in vertebrate splicing. J Biol Chem 270, 2411-2414.
Blau, N., Thony, B., Spada, M., and Ponzone, A. (1996). Tetrahydrobiopterin and inherited hyperphenylalaninemias. Turk J Pediatr 38, 19-35.
Blencowe, B. J. (2000). Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci 25, 106-110.
Caceres, J. F., and Kornblihtt, A. R. (2002). Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Genet 18, 186-193.
Cartegni, L., Chew, S. L., and Krainer, A. R. (2002). Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3, 285-298.
Cartegni, L., and Krainer, A. R. (2002). Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 30, 377-384.
Chao, H. K., Hsiao, K. J., and Su, T. S. (2001). A silent mutation induces exon skipping in the phenylalanine hydroxylase gene in phenylketonuria. Hum Genet 108, 14-19.
DiLella, A. G., Kwok, S. C., Ledley, F. D., Marvit, J., and Woo, S. L. (1986). Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene. Biochemistry 25, 743-749.
Ellingsen, S., Knappskog, P. M., and Eiken, H. G. (1997). Phenylketonuria splice mutation (EXON6nt-96A-->g) masquerading as missense mutation (Y204C). Hum Mutat 9, 88-90.
Erlandsen, H., Fusetti, F., Martinez, A., Hough, E., Flatmark, T., and Stevens, R. C. (1997). Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria. Nat Struct Biol 4, 995-1000.
Erlandsen, H., and Stevens, R. C. (1999). The structural basis of phenylketonuria. Mol Genet Metab 68, 103-125.
Fackenthal, J. D., Cartegni, L., Krainer, A. R., and Olopade, O. I. (2002). BRCA2 T2722R is a deleterious allele that causes exon skipping. Am J Hum Genet 71, 625-631.
Fairbrother, W. G., Yeh, R. F., Sharp, P. A., and Burge, C. B. (2002). Predictive identification of exonic splicing enhancers in human genes. Science 297, 1007-1013.
Fairbrother, W. G., Yeo, G. W., Yeh, R., Goldstein, P., Mawson, M., Sharp, P. A., and Burge, C. B. (2004). RESCUE-ESE identifies candidate exonic splicing enhancers in vertebrate exons. Nucleic Acids Res 32, W187-190.
Goltsov, A. A., Eisensmith, R. C., Konecki, D. S., Lichter-Konecki, U., and Woo, S. L. (1992). Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene. Am J Hum Genet 51, 627-636.
Graveley, B. R. (2000). Sorting out the complexity of SR protein functions. RNA 6, 1197-1211.
Graveley, B. R., and Maniatis, T. (1998). Arginine/serine-rich domains of SR proteins can function as activators of pre-mRNA splicing. Mol Cell 1, 765-771.
Guthrie, R., and Susi, A. (1963). A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 32, 338-343.
Hsiao, K. J. (1992). Genetic disorders and neonatal screening. In Miyai K, Kanno T, Ishikawa E (eds): Progress in clinical biochemstry. Amesterdam: Elsevier, pp. 289-292.
Jervis, G. A. (1953). Phenylpyruvic oligophrenia deficiency of phenylalanine-oxidizing system. Proc Soc Exp Biol Med 82, 514-515.
Kashima, T., and Manley, J. L. (2003). A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nat Genet 34, 460-463.
Krecic, A. M., and Swanson, M. S. (1999). hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol 11, 363-371.
Kwok, S. C., Ledley, F. D., DiLella, A. G., Robson, K. J., and Woo, S. L. (1985). Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase. Biochemistry 24, 556-561.
Ledley, F. D., Grenett, H. E., DiLella, A. G., Kwok, S. C., and Woo, S. L. (1985). Gene transfer and expression of human phenylalanine hydroxylase. Science 228, 77-79.
Lidsky, A. S., Law, M. L., Morse, H. G., Kao, F. T., Rabin, M., Ruddle, F. H., and Woo, S. L. (1985). Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome. Proc Natl Acad Sci U S A 82, 6221-6225.
Liu, H. X., Cartegni, L., Zhang, M. Q., and Krainer, A. R. (2001). A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat Genet 27, 55-58.
Liu, H. X., Chew, S. L., Cartegni, L., Zhang, M. Q., and Krainer, A. R. (2000). Exonic splicing enhancer motif recognized by human SC35 under splicing conditions. Mol Cell Biol 20, 1063-1071.
Liu, H. X., Zhang, M., and Krainer, A. R. (1998). Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev 12, 1998-2012.
Maniatis, T., and Tasic, B. (2002). Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418, 236-243.
Maquat, L. E. (2001). The power of point mutations. Nat Genet 27, 5-6.
Mayeda, A., and Krainer, A. R. (1992). Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell 68, 365-375.
Pagani, F., Buratti, E., Stuani, C., and Baralle, F. E. (2003). Missense, nonsense, and neutral mutations define juxtaposed regulatory elements of splicing in cystic fibrosis transmembrane regulator exon 9. J Biol Chem 278, 26580-26588.
Sambrook, J. and Russell, D. W. (2001) Molecular cloning: a laboratory manual, 3rd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Singh, N. N., Androphy, E. J., and Singh, R. N. (2004). An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy. Biochem Biophys Res Commun 315, 381-388.
Smith, C. W., and Valcarcel, J. (2000). Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci 25, 381-388.
Snow, B. E., Heng, H. H., Shi, X. M., Zhou, Y., Du, K., Taub, R., Tsui, L. C., and McInnes, R. R. (1997). Expression analysis and chromosomal assignment of the human SFRS5/SRp40 gene. Genomics 43, 165-170.
Staffa, A., Acheson, N. H., and Cochrane, A. (1997). Novel exonic elements that modulate splicing of the human fibronectin EDA exon. J Biol Chem 272, 33394-33401.
Svensson, E., Eisensmith, R. C., Dworniczak, B., von Dobeln, U., Hagenfeldt, L., Horst, J., and Woo, S. L. (1992). Two missense mutations causing mild hyperphenylalaninemia associated with DNA haplotype 12. Hum Mutat 1, 129-137.
Teraoka, S. N., Telatar, M., Becker-Catania, S., Liang, T., Onengut, S., Tolun, A., Chessa, L., Sanal, O., Bernatowska, E., Gatti, R. A., and Concannon, P. (1999). Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences. Am J Hum Genet 64, 1617-1631.
Thisted, T., Lyakhov, D. L., and Liebhaber, S. A. (2001). Optimized RNA targets of two closely related triple KH domain proteins, heterogeneous nuclear ribonucleoprotein K and alphaCP-2KL, suggest distinct modes of RNA recognition. J Biol Chem 276, 17484-17496.
Williamson, M. L., Koch, R., Azen, C., and Chang, C. (1981). Correlates of intelligence test results in treated phenylketonuric children. Pediatrics 68, 161-167.
Zschocke, J., and Hoffmann, G. F. (1999). Phenylketonuria mutations in Germany. Hum Genet 104, 390-398.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top