(CTG/CAG)n三聯核酸重複序列之擴增突變與多種人類疾病之發生有密切關係。其中，位於染色體19q13.3區域內DMPK基因3’端不轉譯區的此一序列之擴增會造成強直型肌肉萎縮症(myotonic dystrophy)。在此研究中，我們嘗試以線蟲(C. elegans)作為疾病模式動物來研究強直肌肉萎縮症之可能致病機制。首先，我們以PCR-cloning方法將不同長度之CTG三聯核酸重複序列接入pGEM-T質體，再轉至含線蟲肌肉專一性驅動之綠螢光蛋白基因的載體。為模仿人體內真實情形，CTG三聯核酸重複序列係插入綠螢光蛋白基因的3’端不轉譯區。經顯微注射產生之基因轉殖線蟲共有myo3::gfp，myo-3::gfp(CTG)5，及myo-3::gfp(CTG)120三種。其中，myo-3::gfp(CTG)120 line之螢光表現量約只有myo-3::gfp(CTG)5 line之20%以及GFP line之7%，顯示擴增之CTG三聯核酸重複序列會降低其所在基因之表現。 myo-3::gfp(CTG)5 line之C. elegans身體移動速度正常且移動軌跡呈正常規則S型，而myo-3::gfp(CTG)120 line 線蟲之移動則明顯緩慢且不甚規則。此外，我們以線蟲的咽喉肌電圖(EPG)來觀察其肌肉生理發現myo-3::gfp(CTG)120 line之肌肉收縮及舒張明顯異常。這些結果證實擴增之CTG重複序列會造成線蟲肌肉功喪失。

Expansion mutation of CTG repeat length in the 3’-untranslated region of human DMPK gene causes a dominantly inherited muscular disease, called myotonic dystrophy type1 (DM1). In this study we attempt to establish a DM1-disease animal model using transgenic C. elegans. We made transgenic constructs in which (CTG)n was inserted into the 3’-untranslated region of GFP gene driven by muscle-specific promoter of C. elegans. Three lines, including myo-3::gfp, myo-3::gfp(CTG)5, and myo-3::gfp(CTG)120, were generated. As judged from western blotting, the GFP protein level in myo-3::gfp(CTG)120 line is only about 20% of that in myo-3::gfp(CTG)5 line, and is about 7% of that in myo-3::gfp . This result indicates that expanded CTG repeats will greatly decrease the GFP gene expression. In addition, the rate and patterns of body movement as well as electropharyngealgram (EPG) of myo-3::gfp(CTG)120 line is significantly deviated from those observed in myo-3::gfp. myo-3::gfp(CTG)5 line presents normal muscle activity and coordination. These data demonstrate that expanded CTG repeats have pathogenic effects on C. elegans

目次
頁次
中文摘要………………………………………………… 2
英文摘要………………………………………………… 3
序 論………………………………………………… 4
材料與方法……………………………………………… 11
結 果………………………………………………… 23
討 論………………………………………………… 27
圖 表………………………………………………… 31
參考文獻………………………………………………… 39

參考文獻
Berul, C. I., Maguire, C. T., Aronovitz, M. J., Greenwood, J., Miller, C., Gehrmann, J., Housman, D., Mendelsohn, M. E., and Reddy, S. (1999) DMPK dosage alternations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model. J. Clin. Invest. 103, R1-7
Brook, J. D., McCurrach, M. E., Harley, H. G., Buckler, A. J., Church, D., Aburatani, H., Hunter, K., Stanton, V. P., Thirion, J.-P., Hudson, T., Sohn, R., Zemelman, B., Snell, R. G., Rundle, S. A., Crow, S., Davies, J., Shelbourne, P., Buxton, J., Johns, C., Juvonen, V., Johnson, K., Harper, P. S., Shaw, D. J., and Housman D. E. (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3’ end of a transcript encoding a protein kinase family member. Cell 68, 799-808.
Cassata, G., Dozier, C., Burglin, T. R., Kagoshima, H., and Niklaus, G (1999) Function and expression of ceh-32, a sine oculis homologue. International C. elegans Meeting 279.
Cummings, C. J., and Zoghbi, H.Y. (2000) Fourteen and counting: unraveling trinucleotide repeat disease. Hum. Mol Genet. 9, 909-916.
Davis, B. M., McCurrach, M. E., Taneja, K. L., Singer, R. H., and Housman, D. E. (1997) Expansion of a CUG trinucleotide repeat in the 3’ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc. Natl. Acad. Sci., USA 94 , 7388-7393.
Fu, Y.-H., Pizzuti, A., Fenwick, R. G., King, J., Jr., Pajnarayan, S., Dunne, P. W., Dubel, J., Nasser, G. A., Ashizawa, T., de Jong, P., Wieringa, B., Korneluk, R., Perryman, M. B., Epstein, H. F., and Caskey, C. T. (1992) An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255, 1256-1258.
Hoffman-Radvanyi, H., Lavedan, H., Rabes, J. P., Savoy, D., Duros, C., Johnson, K., and Junien, K. (1993) Myotonic dystrophy: absence of CTG enlarged transcript in congenital forms, and low expression of the normal allele. Hum. Mol. Genet. 2, 1263-1266.
Heath, S. K., Carne, S., Hoyle, C., Johnson, K. L., and Wells, D. J. (1997) Characterization of expression of mDMAHP, a homeodomain-encoding gene at the murine DM locus. Hum. Mol. Genet. 6, 651-657.
Hsiao, K.M., Lin, H. M., Pan, H. C., Li, T. C., Chen, S. S., Jou, S. B., Chiu, Y. L., Wu, M. F., Lin, C. C., and Li, S. Y.（1999）Application of FTA® Sample Collection and DNA Purification System on the Determination of CTG Trinucleotide Repeat Size by PCR-Based Sourthern Blotting. Journal of Clinical Laboratory Analysis 13, 188-193.
Jansen, G., Groenen, P. J., T. A., Bachner, D., Jap, P. H. K., Coerwinkel, M., Oerlemans, F., van den Broek, W., Gohlsch, B., Pette, D., Plomp, J. J., Molenaar, P. C., Nederhoff, M. G. J., van Echteld, C. J. A., Dekker, M., Berns, A., Hameister, H., and Wieronga, B. (1996) Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nature Genet.13, 316-324.
Klesert, T. R., Cho, D. H., Clark, J. I., Maylie, J., Adelman, J., Snider, L., Yuen, E. C., Soriano, P., and Tapscott, S. J. (2000) Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nature Genet. 25, 105-109.
Korade-Mirnics, Z., Babtizke, P., and Hoffman, E. (1998) Myotonic dystrophy: molecular windows on a complex etiology. Nucleic Acids Res. 26, 1363-1368.
Liquori CL, Ricker K, Moseley ML, Jacobsen JF, Kress W, Naylor SL, Day JW, Ranum LP. (2001)Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293,864-7.
Mahadevan, M., Tsilfidis, C., sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcero, J., O’Hoy, K., Leblond, S., Earle-Macdonald, J., de Jong, P. J., Wieringa, B., and Korneluk, R. G. (1992) Myotonic dystrophy mutation: an unstable CTG repeat in the 3’ untranslated region of the gene. Science 255, 1253-1255.
Mankodi, A., Logigian, E., Callahan, L., McClain, C., White, R., Henderson, D., Krym, M., and Thornton, C., A. (2000) Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289, 1769-1772.
Miller, J.W., Urbinati, C.R., Teng-Umnuay P, Stenberg, M.G., Byrne, B.J., Thornton. (2000)Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy. EMBO J 19, 4439-48.
Milne, C. A., and Hodgkin, J. (1999) ETR-1, a homologue of a protein linked to myotonic dystrophy, is essential for muscle development in Caenorhabditid elegans. Current Biology 9, 1243-1246.
Morrone, A., Pegoraro, E., Angelini, C., Zammarchi, E., Marconi, G., and Hoffman, E. (1997) RNA metabolism in myotonic dystrophy: patient muscle shows decreased insulin receptor RNA and protein consistent with abnormal insulin resistance. J. Clin. Invest. 99, 1691-1698.
Otten, A.D. and Tapscott, S. J. (1995) Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure. Proc. Natl. Acad. Sci. USA 92, 5465-5469.
Philips, A.V., Timchenko, L.T., and Cooper, T.A. (1998) Disruption of splicing regulated by a CUG-binding protein in myotonic dystophy. Science 280, 737-741.
Reddy, S., Smith, D. B. J., Rich, M. M., Leferovich, J. M., Reilly, P., Davis, B. M., Tran, K., rayburn, H., Bronson, R., Cros D., Balice-Gordon, R. J., and Housman, D. (1996) Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nature Genet. 13, 325-335.
Sarker, P. S., Appukuttan, B., Han, J., Ito, Y., Ai, C., Tsai, W., Chai, Y., Stout, J. T., and Reddy, S. (2000) Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nature Genet. 25, 110-114.
Sergeant, N., Sablonniere, B., Schraen-Maschke, S., Ghestem, A., Claude-alain, M. Wattez, A., Vermersch, P., Delacourte, A.(2001) Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1.Hum.Mol.Genet.10, 2143-55.
Seznec H., Agbulut O, Sergeant N, Savouret C, Ghestem A, Tabti N, Willer JC, Ourth L, Duros C, Brisson E, Fouquet C, Butler-Browne G, Delacourte A, Junien C, Gourdon G. (2001)Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities. Hum. Mo.l Genet. 10, 2717-26.
Thornton C. A., Wymer, J. P., Simmons, Z., McClain, C., and Moxley, R. T. (1997) Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nature Genet. 16, 407-409.
Timchenko, L.T. and Caskey, C.T. (1996) Trinucleotide repeat disorders in humans : discussions of mechanisms and medical issues. FASEB J. 10, 1589-1597.
Tiscornia G, Mahadevan MS. Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios. Mol. Cell. 5, 959-967.
Wang, Y.-H., Amirhaeri, S., Kang, S., Wells, R. D., and Griffith, J. D. (1994) Preferential nucleosome assembly at DNA triplet repeats from the myotonic dystrophy gene. Science 265, 669-671.
Wang, Y.-H. and Griffith, J. (1995) Expanded CTG triplet blocks from the myotonic dystrophy gene create the strongest known natural nucleosome positioningelements. Genomics 25, 570-573.
Wissmann, A., Ingles, J., McGhee J. D., and Mains, P. E. (1997) Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunit of smooth muscle myosin phosphatase to affect cell shape. Genes & Dev. 15, 409-422