臺灣博碩士論文加值系統

生物體內生理代謝的副產物以及一些外在的化合物，均很容易造成DNA的氧化傷害，其中以7,8-dihydro-8-oxoguanine（簡稱GO）是形成的DNA氧化產物中較大量且最穩定的，在大腸桿菌（E. coli）中負責修復此類因DNA的氧化傷害而造成的錯誤配對修補系統，由mutT、fpg、mutY三個基因組成；在人類基因中已找到同源基因，分別是hMTH、hOGG1、hMYH；已知大腸桿菌中，若此系統的基因發生缺失，會造成細胞體內DNA的A/T→C/G或G/C→T/A transversion的突變率增高，在人體內，這些同源基因的缺失，對人體造成的影響仍不清楚。
本論文欲透過探討小鼠的mutY同源基因（即mMYH基因）之結構，以利進一步了解MYH之功能及調控。從含有129SV老鼠基因體庫之噬菌體庫，以含有放射線之mMYH小片段序列為探針，進行篩選，萃取所得到之噬菌體的DNA，利用限制酶作用，得到mMYH genomic DNA的圖譜；利用聚合酵素連鎖反應，複製並選殖出mMYH genomic DNA的不同片段，並加以定序分析；得知mMYH genomic DNA是由15個Exons及14個 Introns組成，全長約5.5 kb。並由第3號噬菌體得到mMYH基因5’端約2.8 kb及3’端約5 kb的DNA片段，以利於日後做knock-out老鼠之DNA材料，藉以瞭解mMYH基因若缺陷對生物體的影響，進而推測在人類基因中hMYH的缺失與癌化或其他疾病的關係。

Metabolic byproducts and exogenous compounds can produce oxidative damages to the genomic DNA in organisms. Among these DNA damages, 7,8-dihydro-8-oxoguanine（GO）is one of the most abundant and stable products. In Escherichia coli, a system composed of mutT、fpg and mutY is responsible for repairing these DNA damages. Mutations in one of these genes cause higher frequency of A/T→C/G or G/C→T/A transversions. The functional homologues of these genes were identified in human genome, namely hMTH、hOGG1 and hMYH. However, the effect of gene dificiency in human is still not clear.
In this study, we focused on mouse mMYH gene. A radioactively labeled fragment of mMYH DNA was used as probe to screen a λphage library containing 129SV genome. The DNA of the positiveλphage clones was purified and digested with restriction enzyme for mapping. We then applied polymerase chain reaction（PCR）, cloned the PCR products, and analysed the DNA sequences. mMYH genomic DNA is 5.5 kb in full length and composed of 15 exons and 14 introns. From the no 3 λphage, we obtained a 2.8-kb DNA fragment of 5’ end and a 5-kb DNA fragment of 3’ end of mMYH. The information is beneficial for knock-out mouse preparation. Studying the effects of mMYH gene mutation in mouse could provide some hints of how hMYH function in human and the association between hMYH gene and diseases including cancers.

中文摘要………………………………………………………………………1
英文摘要………………………………………………………………………2
緒論……………………………………………………………………………3
實驗材料與方法…………………..………………………………………..10
1. 載體與菌種…………………………………………………...10
2.129SV老鼠基因體庫之噬菌體庫titer測試………………….10
3.聚合酵素連鎖反應…………………………………………….11
4.mMYH 探針（probe）製備………………………………………11
5.129SV老鼠基因體庫之噬菌體庫擴增………………………..12
6.mMYH 基因之篩選……………………………………………13
7.Recovery of the Plaque…………………………………………14
8.噬菌體DNA製備………………………………………………...14
9.南方墨點分析法……………………………………………………….15
10.噬菌體上夾帶的mMYH 基因片段相對於載體之方向的確定……...16
11.基因選殖……………………………………………………….16
12.自動定序反應………………………………………………….17
實驗結果…………………………………………………………………..18
討論………………………………………………………………………..24
圖表…………………………………………………………………………..28
參考文獻…………………………………………………………………..46
附錄………………………………………………………………………..50

Ames, B. N., Shigenaga, M. K., and Hagen, T. M., （1993） Oxidants, Antioxidants, and the Degenerative Diseases of Aging, Proc. Natl. Acad. Sci. USA 90, 7915-7922
Au, K. G.., Susanna, C., Miller, J. H., and Paul, M., （1989）Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispares, Proc. Natl. Acad. Sci. USA 86, 8877-8881
Boiteux, S., and Radicella, J. P., （1999）Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress, Biochimie 81, 59-67
Boldogh, I., Milligan, D., Lee, M. S., Bassett, H., Lloyd, R. S., and McCullough, A. K., （2001）hMYH cell cycle-dependent expression, subcellular localization and association with replication foci: evidence suggesting replication-coupled repair of adenine:8-oxoguanine mispairs , Nucleic Acids Res. 29, 2802-2809
Fishel, R., Lescoe, M. K., Rao, M. R., Copeland, N. G., Jenkins, N. A., Garber, J., Kane, M., and Kolodner, R., （1993） The Human Mutator Gene Homolog MSH2 and Its Association with Hereditary Nonpolyposis Colon Cancer, Cell 1993 75, 1027-1038
Jiricny, J., （1998）Eukaryotic mismatch repair: an update, Mutation Research 409, 107-121
Krokan, H. E. Standal, R. and Slupphaug, G.., （1997）DNA glycosylases in the base excision repair of DNA, Biochem. J. 325, 1-16
Leach, F. S., Nicolaides, N. C., Papadopoulos, N., Liu, B., Jen, J., Parsons, R., Peltomaki, P., Sistonen, P., Aaltonen, L. A., and Nystrom-Lahti, M., （1993）Mutations of a MutS Homolog In Hereditary Nonpolyposis Colorectal Cancer, Cell 75, 1215-1225
Lehmann, A. R., （1995）Nucleotide excision repair and the link with transcription, Trends Biochem. Sci. 20, 402-405
Lewin, B., （2000）gene VII
Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., and Darnell, J., （1999）4th ed. Molecular cell biology
Lu, A. L. and Chang, D. Y., （1988a） A Novel Nucleotide Excision Repair For the Conversion of an A/G Mismatch to C/G Base Pair In E. Coli, Cell 1988 54, 805-812
Lu, A. L. and Chang, D. Y., （1988b） Repair of Single Base-Pair Transversion Mismatches of Escherichia coli in Vitro: Correction of Certain A/G Mismatches Is Independent of dam Methylation and Host mutHLS Gene Functions , Genetics 118, 593-600
Manuel, R. C., Lloyd, R. S., （1997）Cloning, Overexpression, and Biochemical Characterization of the Catalytic Domain of MutY , Biochemistry 36(37) , 11140-11152
Michaels, M. L., Pham, L., Nghiem, Y., Cruz, C., and Miller, J. H., （1990）MutY, an adenine glycosylase active on G-A mispairs, has homology to endonuclease III, Nucleic Acids Res. 18(13),3841-3845
Michaels, M. L., and Miller, J. H., （1992a）The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine), J. Bacteriol. 174, 6321-6325
Michaels, M. L., Tchou, Julia, Grollman, A. P., Miller, J. H., （1992b） A repair system for 8-Oxo-7,8-dihydrodeoxyguanine, Biochemistry 31(45), 10964-10968
Michaels, M. L., Cruz, C., Grollman, A. P., and Miller, J. H., （1992c）Evidence that MutY and MutM Combine to Prevent Mutations by an Oxidatively Damaged Form of Guanine in DNA, Proc. Natl. Acad. Sci. USA 89, 7022-7025
Mol, C. D., Parikh, S. S., Putnam, C. D., Lo, T. P., and Tainer, J. A., （1999）DNA repair mechanisms for the recognition and removal of damaged DNA bases, Annu. Rev. Biomol. Struct. 28, 101-128
Moriya, M., and Grollman, A. P., （1993）Mutations in the mutY gene of Escherichia coli enhance the frequency of targeted G．C→T．A transversions induced by a single 8-oxoguanine residue in single-stranded DNA, Mol. Gen. Genet 239, 72-76
Nghiem, Y., Cabrera, M., Cupples, C. G., and Miller, J. H., (1998). The mutY gene: a mutator locus in Escherichia coli that generates G．C→T．A transversions, Proc. Natl. Acad. Sci. USA 85, 2709-2713
Nielsen, P. E., Mollegaard, N. E., and Jeppesen, C., （1990）DNA conformational analysis in solution by uranyl mediated photocleavage , Nucleic Acids Res. 18, 3847-3851
Pascucci, B., Stucki, M., Jonsson, Z. O., Dogliotti, E., and Hubscher, U., （1999）Long patch base excision repair with purified human proteins, The Journal of Biological Chemistry 274(47), 33696-33702
Slupska, M. M., Baikalov, C., Luther, W. M., Chiang, J. H., Wei, W. F., and Miller, J. H., （1996）Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage, J. Bacteriol. 178, 3885-3892
Slupska, M. M., Luther, W. M., Chiang, J. H., Yang, H., and Miller, J. H., （1999）
Functional Expression of hMYH, a Human Homolog of the Escherichia coli MutY Protein, J. Bacteriol. 181, 6210-6213
Sun, B., Latham, K. A., Dodson, M. L., and Lloyd, R. S., （1995） Studies on the Catalytic Mechanism of Five DNA Glycosylases, J. Biol. Chem. 270, 19501-19508
Tajiri, T., Maki, H., Sekiguchi, M., （1995）Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli, Mutation Research 336, 257-267
Thayer, M. M., Ahern, H., Xing, D., Cunningham, R. P., and Tainer, J. A., （1995） Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure, EMBO J. 14, 4108-4120
Tsai-Wu, J J., Liu, H., and Lu, A. L., （1992）Escherichia coli MutY Protein has Both N-Glycosylase and Apurinic/ Apyrimidinic Endonuclease Activities on A C and A G Mispairs, Proc. Natl. Acad. Sci. USA 89, 8779-8783
Tsai-Wu, J. J., and Lu, A. L., （1994）Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract, Mol Gen Genet 244, 444-450
Wilson III, D. M., and Thompson, L. H., （1997）Life without DNA repair, Proc. Natl. Acad. Sci. USA 94, 12754-12757
Williams, S. D., and David, S. S., （1998）Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA, Nucleic Acids Res. 26, 5123-5133
Wood, R. D., （1997） Nucleotide Excision Repair in Mammalian Cells, J. Biol. Chem. 272, 23465-23468
Zhang, Q. M., Ishikawa, N., Nakahara, T., and Yonei, S., （1998） Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-- >C:G transversions , Nucleic Acids Res. 1998 26, 4669-4675.
Zharkov, D. O., Grollman, A. P., （1998）MutY DNA Glycosylase: Base Release and Intermediate Complex Formation , Biochemistry 37(36), 12384-12394