臺灣博碩士論文加值系統

中文摘要
基因的不穩定現象是腫瘤形成的一個重要機轉，而此不穩定的特性包含有兩種不同的程度表現，分別是核苷酸與染色體兩個程度的變化。BUB基因是屬於細胞分裂監測點的一部份，而監測點的功能主要是監測細胞分裂時，染色體是否正確連結到紡錘體(spindle) 上。人類的BUB基因(即hBUB 基因)已被發現與一些腫瘤細胞的染色體不穩定性有相關。我們分析92個AML檢體以及5個血癌細胞株，藉由單股構形多形性分析法(SSCP)及自動化核酸定序法(Auto—Sequencing)分析hBUB1基因，觀察有無變異情形，及利用反轉錄聚合媒;連鎖反應法(RT-PCR)分析hBUB1基因的表現程度。結果DNA部分，在Raji細胞株及兩個AML檢體中，發現在exon10的剪接點上游第八個核苷酸有單一核苷酸多形性(single nucleotide polymor- phism)現象，但其不影響轉錄。而RT-PCR的分析，發現有些AML檢體其hBUB1基因部份或整體沒有表現，此外亦偵測到四個異常的轉錄。經由南方墨點法及其相對位置的剪接點之核苷酸分析，並無突變發現，因此不正常轉錄乃是因多樣性剪接 (alternative splicing)所造成。我們的研究結果指出hBUB1基因的突變在白血病檢體中是不常見的，而hBUB1基因可能是經由轉錄後修飾作用（post-tran scriptional modification）而影響其細胞分裂監測點的監測功能。

SUMMARY
Genetic instability is a key mechanism of tumorigenesis, and the instability exists at two distinct levels, the nucleotide and the chromosome levels. The BUB gene is a component of the mitotic checkpoint that monitors proper assembly of mitotic spindles and alteration of hBUB1 has been found to be associated with chromosomal instability in some tumor cell lines. We analyzed the coding region of hBUB1 by single strand conformational polymorphism (SSCP), complete sequence analysis and the expression of the hBUB1 gene by reverse transcription polymermase chain reaction (RT-PCR) in 92 AML specimens and 5 hematopoietic cell lines. A single nucleotide polymorphism at 8bp upstream of spicing site of exon10 was observed in Raji cell line and two AML specimens without a resultant change in the expression of hBUB1. Expression of partial or full coding region of hBUB1 gene were absent in some AML cases by RT-PCR analysis. In addition to four aberrant transcripts of hBUB1 gene were found in AML specimens .None mutation was found in the relevant region of genomic DNA by Southern blotting and sequence analysis by RT-PCR analysis, suggesting the abnormal transcripts represent the results of alternative splicing. Our study indicated that mutation of hBUB1 gene is a rare event in leukemia specimens, and hBUB1 gene may interfere the function of the mitotic checkpoint through the post-transcriptional modification.

目錄
中文摘要..........................1
英文摘要..........................2
第一章 序論.......................4
第二章 實驗材料與方法.............14
第三章 實驗結果...................33
第四章 討論.......................51
第五章 參考文獻...................57
第六章 附錄.......................62

參考文獻
1.Bishop,J.M.(1987) The molecular genetics of cancer. Science
235：305-311.
2.Vogelstein,B.,Fearon,E.R.,Hamilton,S.R.,Kern,S.E.,Presinger,A.
C.,Leppert,M.,Nakamura,Y.,White,R.,Smits,A.M.& Bos,J.L.
(1988) Genetic alteration during colorectal-tumor development.
New England Journal of Medicine 319：525-532.
3.Knudson, A. G. (1989) Genetics of human cancer. Annual
Reviews of Genetics 20：231-251.
4.Lengauer,C., Kinzler,K.W. and Voglstein,B. (1998) Genetic
instability in human cancers. Nature 396：643-649.
5.Paul Nurse, Yoshio Masui & Leland Hartwell (1998)
Understanding the cell cycle. Nature medicine 4：1103-1106.
6.Miyagawa,K. (1998) Genetic instability and cancer.
International Journal of Hematology 67：3-14.
7.Elledge,S.J. (1996) Cell Cycle checkpoints：Preventing an
identity crisis. Science 274：1664-1672.
8.Winey,M. (1996) Keeping the centrosome cycle on track
Curr.Biol. 6：962-964.
9.Sancar,A. (1996) DNA excision repair. Annu.Rev.Biochem. 65：
43-81.
10.Kraemer,K.H., Lee,M.M. & Scotto,J. (1984) DNA repair
protects against cutaneous and internal neoplasia：evidence
from Xeroderma Pigmentosum. Carcinogenesis 5：511-514.