利用甲基化核糖核酸多型分析法找尋印痕基因鄭又瑋、李泰林

(1)

利用甲基化核糖核酸多型分析法找尋印痕基因鄭又瑋、李泰林

摘要

DNA甲基化是一種epigenetic的機制，在哺乳類動物中它主要是利用甲基化轉換? ( Methyltransferase )，將甲基共價鍵結在5

，-CG-3，序列上胞嘧啶環上的第5個碳的位置。DNA甲基化這種特殊的DNA印痕和調控基因的表現與組織專一性基因之表達有密切的關係，並且也調節一些細胞功能的表現，如染色體印痕基因的表現、DNA的突變及癌症的形成。儘管DNA 甲基化是那麼重要，但是即將定序完成的基因序列卻不能代表任何DNA甲基化的訊息，又加上過去的甲基化研究都只限於已知的基因。有鑑於此，利用10組AP引子進行甲基化敏感性PCR ( AP-PCR ) 的方法，能夠廣泛地在基因組中來尋找組織專一性的甲基化片段和胚胎組織中的甲基化片段。在此，我們將基因組DNA以Hind III限制酵素作用，再分別以甲基化敏感性限制酵素Hpa II及甲基化非敏感性限制酵素Msp I處理，以AP引子做PCR反應，利用這種技術可於組織中尋找甲基化差異片段，以AP-1引子作用所得到差異片段，分別為AP101、 AP102、AP103、AP104、AP105，而AP104片段順利的選殖並定序，但經比對並無相似片段；以AP-2引子作用得到AP201片段，經選殖後定序，比對後於DNA序列中未有DNA甲基化Hpa II ( CCGG ) 酵素之切位，因此沒有進一步分析；而以AP-3及AP-8引子PCR得到的差異片段AP301及AP803，選殖後定序之結果，DNA序列與NCBI基因庫小鼠基因組序列比對相似度高達98 % 及99 %，並且序列中具有Hpa II酵素切位，表示此二片段DNA序列中確實為甲基化差異性片段，因此，將此二片段由放射線標定後進一步以南方點墨雜合分析法分析甲基化差異片段之相似度。

關鍵詞 : 甲基化 ; 印痕基因 ; 基因組DNA ; Arbitrary Primed PCR 目錄

中文摘要……….. iv 英文摘要……….

…. vi 目錄……….………..… viii 圖目錄……….……

…...…………. xi 第一章前言……….……. 1 1.1 基因印痕……….………

……..…...…4 1.2 DNA甲基化酵素系統………..…………....…..……. 8 1.3 DNA甲基化與基因調控……..……….……..………

8 1.4 DNA甲基化與癌症關係…….…...……..……..…... 10 1.4.1 DNA甲基化與基因突變…….….…………..… 14 1.4.2 DNA甲基化不平衡與癌症之形成…..……….. 16 1.4.2.1 DNA過度甲基化….………..….……...….. 16 1.4.2.2 DNA低度甲基化….……

……..……...….. 19 1.5 DNA甲基化研究方法…..….………...….… 20 1.6 研究動機與目的……….……..…....

…..… 24 1.6.1 實驗老鼠品系及組織器官之選擇…...…..……. 24 第二章研究方法……….……...

…. 27 2.1 材料……….…..……...…... 27 2.2 方法……….…….………...….. 30 2.2.1 組織中DNA甲基化差異片段之篩選….…..…. 30 2.2.1.1 實驗流程……….……….. 30 2.2.1.2 小鼠養殖及犧牲

….……….…. 32 2.2.1.3 組織中基因組DNA萃取及純化………... 32 2.2.1.4 甲基化敏感性限制?反應…………...

…… 33 2.2.1.5 甲基化敏感性AP-PCR………...…. 33 2.2.2 DNA甲基化差異片段之選殖………..……….. 34 2.2.2.1 勝任細胞之製備……….…...…. 34 2.2.2.2 DNA片段分離與接合……...…..……...…. 35 2.2.2.3 轉形作用及質體選殖…

…..……….… 36 2.2.2.4 質體DNA小量製備……….… 36 2.2.3 DNA甲基化差異片段之分析….…….……..…

37 2.2.3.1 限制?消化反應及瓊脂凝膠電泳…...…… 37 2.2.3.2 南方點墨雜合反應…………...……… 38 2.2.3.2.1 前雜合反應……….……….…… 39 2.2.3.2.2 探針製作……….…………... 39 2.2.3.2.3 放射線顯影……….………….…

40 第三章結果……….………..……….…….……... 41 3.1 組織中基因組DNA抽取及限制酵素反應結果.…. 41 3.2 組織間DNA甲基化差異表現………..…. 44 3.2.1 組織間DNA甲基化差異表現 ( AP-1引子 PCR )……….……

……….. 44 3.2.2 組織間DNA甲基化差異表現 ( AP-2引子 PCR )………...…… 49 3.2.3 組織間DNA甲基化差異表現 ( AP-3引子 PCR )……….………..… 54 3.2.4 組織間DNA甲基化差異表現 ( AP-5引子 PCR )……….……..…… 59 3.2.5 組織間DNA甲基化差異表現 ( AP-8引子 PCR )………

………...… 61 3.2.6 組織間DNA甲基化差異表現………….…...… 67 3.3 利用甲基化敏感性AP-PCR證明DNA 甲基化之遺傳性………….……….……….….. 68 3.4 組織專一性DNA甲基化表現……...………..…….. 68 3.5 DNA甲基化片段之blast分析……….….….. 69 第四章討論……….………..……….…….……... 74 4.1 差異性甲基化基因……….……… 74 4.2 差異性甲基化之遺傳……….…...…….…….……... 75 4.3 南方點墨雜合反應……..…….…….………….…... 76 第五章結論……….………..……….…….……... 77 參考文獻………

….………...… 78 附錄………...………. 91

(2)

參考文獻

吳岱珍。2002。肝細胞癌內FAP-1及其染色體鄰近區域之分子遺傳學研究。國立陽明大學遺傳學研究所。碩士論文。何康潔。1999。基因印刻。當代醫學。26(11):924-927。李中芬。1993。DNA cytosine甲基化和基因表現的關係。中國文化大學生命科技研究所。碩士論文

。林秋宏。2000。DNA甲基化異常與肝細胞腫瘤的關係。私立長庚大學基礎醫學研究所。碩士論文。柯順耀。1999。口腔癌基因甲基化異常之鑑定。國立陽明大學口腔生物研究所。碩士論文。蘇家興。2001。利用Alu-PCR指紋分析及Bisulfite定序尋找及選殖小鼠組織基因體DNA中的甲基化的片段:p53啟動子到s27基因之間組織特異性的CpG甲基化的分析。國立陽明大學生物化學研究所。碩士論文。

Antequera, F., and Bird, A. 1993. Number of CpG islands and genes in human and mouse. Proc. Natl. Acad. Sci. USA. 90: 11995-11999. Bagwe, A.N., Kay, P.H., Spagnolo, and D.V. 1997. Evidence that DNA methylation imbalance is not involved in the development of malignant

mesothelioma. Anticancer Res. 17: 3341-3344. Bartolomei, M.S., Webber A.C., Brunkow M.F., and Tilghman S. 1994. The search for imprinted genes. Nature Genet 6:220-221. Baylin, S.B., Herman, J.G., Graff, J.R., Vertino, P.M., and Issa, J.P. 1998. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 72:141-196. Baylin, S.B., Makos, M., and Wu, J. 1991. Abnormal patterns of DNA methylation in human neoplasia: Potential consequence for tumor progression. Cancer Cell 3:383-390. Bhattacharya, S.K., Ramchandani, S., Cervoni, N., and Szyf, M. 1999. A mammalian protein with specific demethylase activity for mCpG DNA. Nature 397:579-583. Bird, A.P. 1995. Gene number, noise reduction and biological complexity. Trends Genet. 11:94-100. Bird, A. 1992. The essentials of DNA methylation. Cell 70:5-8. Bird, A.P., and Southern, E.M. 1978. Use of restriction enzymes to study eukaryotic DNA methylation: I. The methylation pattern in ribosomal DNA from Xenopus laevis. J. Mol. Biol. 118:27-47. Cervoni, N., Bhattacharya, S., and Szyf, M. 1999. DNA demethylase is a processive enzyme. J. Biol.

Chem. 274:8363-8366. Christman, J.K., Sheikhnejad, G., Marasco, C.J., and Sufrin, J.R. 1995. 5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation. Proc. Natl. Acad. Sci. USA. 92:7347-7351. Clark, S.J., Harrison, J., and Frommer, M. 1995.

CpNpG methylation in mammalian cells. Nat. Genet. 10:20-27. Counts, J.L., and Goodman, J.I. 1995. Alteration in DNA methylation may play a variety of roles in carcinogenesis. Cell 83:13-15. Cravo, M., Pinto, R., Fidalgo, P., Chaves, P., Gloria, L., Nobre-Leitao, C., and Mira, F. 1996.

Global DNA hypomethylation occurs in the early stages of intestinal type gastric carcinoma. Gut 39:434-438. Denissenko, M.F., Chen, J.X., Tang, M.S., and Pfeifer, G.P. 1997. Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc. Natl. Acad. Sci. USA.

94:3893-9838. Dieguez, M.J., M, Bellotto, K., Afsar, O., Mittelsten, Scheid., and J, Paszkowski. 1997. Methylation of cytosine in nonconventional methylation acceptor sites can contribution to reduced gene expression. Mol. Gen. Genet. 253:581-588. Ferguson, A.T., Vertino, P.M., Spitzner, J.R., Baylin, S.B., Muller, M.T., and Davidson, N.E., 1997. Role of estrogen receptor gene demethylation and DNA methyltransferase DNA adducts formation in 5-aza-2'-deoxycytidine-induced cytotoxicity in human breast cancer cells. Biol. Chem. 272:32260-32266. Ferguson, A.T., Lapidus, R.G., Baylin, S.B., and Davidson, N.E. 1995. Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res. 55:2279-2283. Fremont, M., Siegmann, M., Gaulis, S., Matthies, R., Hess, D., and Jost, J.P. 1997. Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA. Nucleic Acids Res. 25:2375-2380. Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., and Paul, C.L. 1992. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues inindividual DNA strands. Proc. Natl. Acad. Sci. USA.

89:1827-1831. Gonzalez-Zulueta, M., Bender, C.M., Yang, A.S., Nguyen, T., Beart, R.W., Van Tornout, J.M., and Jones, P.A. 1995. Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissue correlates with gene silencing. Cancer Res. 55:4531-4535. Gonzalgo, M.L., Bender, C.M., You, E.H., Glendening, J.M., Flores, J.F., Walker, G.J., Hayward, N.K., Jones, P.A., and Fountain, J.W. 1997. Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors. Cancer Res. 57:5336-5347. Gonzalgo, M.L., Liang, G., Spruck III, C.H., Zingg, J.M., Rideout, III, W.M., and Jones, P.A. 1997.

Identification and characterization of differentially methylated regions of genomic DNA by methylation-sensitive arbitrary primed PCR. Cancer Res. 57:594-599. Greenblatt, M.S., Bennett, W.P., Hollstein, M., and Harris, C.C. 1994. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54:4855-4878. Herman, J.G., Merlo, A., Mao, L., Lapidus, R.G., Issa, J-P., Davidson, N.E., Sidransky, D., and Baylin, S.B. 1995. Inactivation of the CDKN2/p16/MTS 1 gene is frequently associated with aberrant DNA methylation in all common human cancer. Cancer Res. 55:4525-4530. Hess, J.F., Fox, M., Schmid, C., and Shen, C.K. 1983. Molecular evolution of the human adult alpha-globin-like gene region: insertion and deletion of Alu family repeats and non-Alu DNA sequences. Proc. Natl. Acad. Sci. USA.

80:5970-5974. Hiltunen, M.O., Alhonen, L., Koistinaho, J., Myohanen, S., Paakkonen, M., Marin, S., Kosma, V.M., and Janne, J. 1997.

Hypermethylation of the APC ( Adenomatous polyposis coli ) gene promoter region in human colorectal carcinoma. Int. J. Cancer 70:644-648.

Holliday, R., and Grigg, G.W. 1993. DNA methylation and mutation. Mutat. Res. 285:61-67. Hollstein, M., Shomer, B., Greenblatt, M., Soussi, T., Hovig, E., Montesano, R., and Harris, C.C. 1996. Somatic point mutations in the p53 gene of human tumors and cell lines: updated

compilation. Nucleic Acids Res. 24:141-146. Hollstein, M., Rice, K., Greenblatt, M.S., Soussi, T., Fuchs, R., Sorlie, T., Hovig, E., Smith-Sorensen, B., Montesano, R., and Harris, C.C. 1994. Database of p53 gene somatic mutations in human tumors and cell lines. Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res. 22:3551-3555. Huang, T. H-M., Laux, D.E., Hamlin, B.C., Tran, P., Tran, H., and Lubahn, D.B. 1997. Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction

fingerprinting technique. Cancer Res. 57: 1030-1034. Huynh, L., Alpert, L., and Pollak, M. 1996. Silencing of the mammary-derived growth inhibitor ( MDGI ) gene in breast neoplasms is associated with epigenetic changes. Cancer Res. 56: 4865-4870. Issa, J.P., Zehnauer, B.A., Civin,

(3)

C.I., Collector, M.I., Sharkis, S.J. Davidson, N.E., Kaufmann, S.H., and Baylin, S.B. 1996. The estrogen receptor CpG island is methylated in most hematopoietic neoplasms. Cancer Res. 56:973-977. Issa, J.P., Vertino, P.M., Wu, J., Sazawal, S., Celano, P., Nelkin, B.D., Hamilton, S.R., and Baylin, S.B. 1993. Increased cytosine DNA-methyltransferase activity during colon cancer progression. J. Natl Cancer Inst. 85:1235-1240.

Jones, P.A., Wolkowiz, M.J., Rideut III, W.M., Gonzales, F.A., Marziasz, C.M., Coetzee, G.A., and Tapscott, S.J., 1990. De novo methylation of the Moy D, CpG island during the establishment of immortal cell lines. Proc. Natl. Acad. Sci. USA. 87:6117-6121. Kautiainen, T.L., and Jones, P.A., 1986. DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture. J. Biol. Chem. 261:1594-1598. Keshet, I., Lieman-Hurwitz, J., and Cedar, H. 1986. DNA methylation affects the formation of active chromatin. Cell 44:535-543. Keverne, E.B. 1997.

Genomic imprinting in the brain. Curr. Opion. In Neurobiology 7:463-468. Keverne, E.B., Fundele, R., Narasimha, M., Barton, S.C., and Surani, M.A. 1996. Genomic imprinting and the differential roles of parental genomes in brain development. Devel. Brain Res. 92:91-100. Klein, C.B., and M, Costa. 1997. DNA methylation and gene expression: Introduction and overiew. Mut. Res. 286:103-105. Kondo, M., Suzuki, H., Ueda, R., Osada, H., Takagi, K., Takahashi, T., and Takahahi, T. 1995. Frequent loss of imprinting of the H19 gene is often associated with its

overexpression in human lung cancer. Oncogene 10:1193-1198. Kono, T., Obata, Y., Wu, Q., Nlwa, K., Ono, Y., Yamamoto, Y., Park, E.S., S, J-S., and Oagawa, H. 2004. Birth of parthenogenetic mice that can develop to adulthood. Nature Publlshing Group 428:860-864. Laird, P.W., and Jaenisch, R. 1996. The role of DNA methylation in cancer genetics and epigenetics. Ann. Rev. Genet 30:441-464. Laird, P.W., Jackson-Grusby, L., Fazeli, A., Dickinson, S.L., Jung, W.E., Li, E., Weinberg, R.A., and Jaenisch, R. 1995. Suppression of intestinal neoplasia by DNA

hypomethylation. Cell 81:197-205. Laird, P.W., and Jaenisch, R. 1994. DNA methylation and cancer. Hum. Mol. Genet 3:1487-1495. Larsen, F., Gundersen, G., Lopez, R., and Prydz, H., 1992. CpG island as gene markers in the human genome. Genomics 13:1095-1107. Lee, W-H., Morton, R.A., Epstein, J.I., Brook, J.D., Campbell, P.A., Boca, G.S., Hsieh, W.S., Issacs, W.B., and Nelson, W.G. 1994 Cytidine methylation of regulatory sequences near theπ-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc. Natl. Acad. Sci. USA.

91:11733-11777. Liu, C., Xu, D., Sjoberg, J., Forsell, P., Bjorkholm, M., and Claesson, H.E. 2004 Transcriptional regulation of 15-lipoxygenase expression by promoter methylation. Cell Res. 297:61-67. Mancini, M.A., Shan, B., Nickerson, J.A., Penman, S., and Lee, W.H. 1994. The retinoblastoma gene product is cell cycle-dependent nuclear matrix-associated protein. Proc. Natl. Acad. Sci. USA. 91:418-422. Mannens, M., Hoovers, J.M., Redeker, E., Verjaal, M., and Feinberg, A.P. 1994. Parental imprinting of human chromosome region 11p15.3 involved in the Beckwith-Wiedmann syndrome and various human neoplasia. Eur. J. Hum. Genet 2:3-23. McGhee, J.D., and Ginder, G.D. 1979. Specific DNA methylation sites in the vicinity of the chicken beta-globin genes. Nature 280:419-420. Meehan, R.R., Lewis, J.D., and Bird, A.P. 1992.

Characterization of MeCP 2, a vertebrate DNA binding protein with affinity for methylated DNA. Nucleic Acids Res. 20:5085-5092. Meehan, R.R., Lewis, J.D., McKay, S., Kleiner, E.L., and Bird, A.P. 1989. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 58:499-507. Merlo, A., Herman, J.G., Mao, L., Lee, D.J., Gabrielson, E., Burger, P.C., Baylin, S.B., and Sidransky, D.

1995. 5'CpG island methylation is associated with transcriptional silencing of the tumor suppressor p16/CDK/MTS1 in human cancers. Nat.

Med. 1:686-692. Nan, X., Campoy, F.J., and Bird, A. 1997. MeCP 2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 88:471-481. Nan, X., Tate, P., Li, E., and Bird, A. 1996. DNA methylation specifies chromosomal localization of MeCP 2. Mol.

Cell. Biol. 16:414-421. Ohtani-Fujita, N., Dryja, T.P., Rapaport, J.M., Fujita, T., Matsumura, S., Ozasa, K., Watanabe, Y., Hayashi, K., Maeda, K., Kinoshita, S., Matsumura, T., Ohnishi, Y., Hotta, Y., Takahashi, R., Kato, M.V., shizaki, K., Sasaki, M.S., Horsthemke, B., Minoda, K., and Sakai, T. 1997. Hypermethylation in the retinoblastoma gene is associated with unilateral, sporadic retinoblastoma. Cancer Genet. Cytogenet.

98:43-49. Panning, B., and Jaeniscch, R. 1996. DNA hypomethylation can activate Xist expression and silence X-linked genes. Genes Dev.

10:1991-2002. Poirier, F., Chen, C.T.J., Timmons, D.M., Robertson, E.J., Evans, M.J., and Rigby, P.W.J. 1991. The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo. Dev. 113:1105-1114. Prowse, A.H., Webster, A.R., Richards, F.M., Richard, S., Olschwang, S., Resche, F., Affara, N.A., and Maher, E.R. 1997. Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors. Am. J. Hum. Genet. 60:765-771. Pulford, D.J., Falls, J.G., Killian, J.K., and Jirtle, R.L. 1999.

Polymorphism, genomic imprinting and cancer susceptibility. Mut. Res. 436:59-67. Razin, A., and Cedar, H. 1991. DNA methylation and gene expression. Microbiol. Rev. 55:451-458. Razin, A. 1988. Assay for studying DNA methylation. Plant Mol Biol. Manual. B3:1-28. Razin, A., and Riggs, A.D. 1980. DNA methylation and gene function. Science 210:604-610. Recillas-Targa, F., 2002. DNA methylation, chromatin boundaries, and mechanisms of genomic imprinting. Archi. Med. Res. 33:428-438. Rice, J.C., Massey-Brown, K.S., and Futscher, B.W. 1998. Aberrant methylation of the BRCA 1 CpG island promoter is associated with decreased BRCA 1 mRNA in sporadic breast cancer cells. Oncogene 17:807-812. Robertson, K.D., and Jones, P.A. 2000. DNA methylation: past, present and future directions. Carcinogenesis 21:461-467. Sakai, T., Toguchida, J., Ohtani, N., Yandell, D.W., Rapaport, J.M., and Dryja, T.P. 1991. Allele-specific hypermethylation of the retinoblastoma

tumor-suppressor gene. Am. J. Hum. Genet. 48:880-888. Shen, J.C., Rideout, W.M., and Jones, P.A. 1994. The role of hydrolytic deamination of 5-methylcytosine in double-stranded DNA. Nucleic Acids Res. 22:972-976. Singer-Sam, J., Grant, M., LeBon, J.M., Okuyama, K., Chapman, V., Monk, M., and Riggs, A.D. 1990. Use of a Hpa II-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation. Mol. Cell. Biol. 10:4987-4989. Stopper, H., Pechan, R., and Schiffman, D. 1992.

5-Azacytidine induces micronuclei and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis. Mut. Res. 283:21-28. Tate, P., Skarnes, W. and Bird, A. 1996. The methyl-CpG binding protein MeCP2 is essential for embryonic development in the mouse. Nat. Genet 12:205-208. Tate, P.H. and Bird, A.P. 1993. Effects of DNA methylation on DNA-binding protein and gene

(4)

expression. Curr Opin Genet Dev 3:226-231. Tollefsbol, T.O., and Hutchison, C.A. 1997. Control of methylation spreading in synthetic DNA sequences by the murine DNA methyltransferase. J. Mol. Biol. 269:494-504. Tornaletti, S., and Pfeifer, G.P. 1995. Complete and tissue-

independent methylation of CpG sites in the p53 gene: implications for mutations in human cancers. Oncogene 10:1493-1499. Toyota, M., Ahuja, N., Ohe-Toyota, M., Herman, J.G., Baylin, S.B., and Issa, J-P.J. 1999. CpG island methylation phenotype in colorectal cancer. Proc. Natl. Acad.

Sci. USA. 96:8681-8686. Trinh, B.N., Long, T.I., and Laird, P.W. 2001. DNA methylation analysis by methyligh technology. Methods 25:456-462. Vachtenheim, J., Horakova, I., and Novotna, H. 1994. Hypomethylation of CCGG sites in the 3' region of H-ras protoonco gene is frequent and is associated with H-ras allele loss in non-small cell lung cancer. Cancer Res. 54:1145-1148. Van der Ploeg, L.H., and Flavell, R.A.

1980. DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell 19:947-958. Waalwijk, C., and Flavell, R.A. 1978. MspI, an isoschizomer of Hpa II which cleaves both unmethylated and methylated Hpa II sites. Nucleic Acids Res.

5:3231-3236. Yoder, J.A., Walsh, C.P., and Bestor, T.H. 1997. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet.

13:335-340. Yoder, J.A., Walsh, C.P., and Bestor, T.H. 1997. Cytosine methylation and the ecology of intragenomic parasites. Trends in Genet.

13:335-340. Yoshiura, K., Kanai, Y., Ochiai, A., Shimcyama, Y., Sugimura, T., and Hirohashi, S. 1995. Silencing of the E-cadherin invasion- suppressor gene by CpG methylation in human carcinomas. Proc. Natl. Acad. Sci. USA. 92:7416-9419. Zemel, S., Bartolomei, M.S., and Tilghman, S.M. 1992. Physical linkage of two mammalian imprinting genes, H19 and insulin-like growth factor 2. Nature Genet. 2:61-65. Zingg, J-M., and Jones, P.A. 1997. Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis.

Carcinogenesis 18:869-882. Zrihan-Licht, S., Weiss, M., Keydar, I., and Wreschner, D.H. 1995. DNA methylation status of the MUC 1 gene coding for a breast- cancer-associated protein. Int. J. Cancer 62:245-251.

利用甲基化核糖核酸多型分析法找尋印痕基因 鄭又瑋、李泰林