臺灣博碩士論文加值系統

摘要 細胞分裂時，爲了確認紡綞體(spindle)結構的完整而使染色體(chromosome)可以正確的分開，有一個檢查點，我們稱爲細胞分裂紡綞體檢查點(mitotic spindle checkpoint)。不論何種物種，簡單至酵母菌，複雜至人類，在此檢查點中都有個必備的基因MAD1(mitotic arrest defective)。我們利用酵母菌二雜交技術（yeast 2-hybrid）在人類的肝互補去氧核醣核酸庫(liver cDNA library)中選殖與此檢查點相關的基因，以人類的MAD1爲餌，我們釣到另一個基因LMO7，先前的文獻指出，LMO7含有LIM區塊(domain)，且在肝細胞中有替代剪接(alternative splicing)的現象，在試管內蛋白質結合分析(in vitro binding assay)中，以人工在大腸桿菌中表達的GST-HsMAD1融合蛋白質可以直接和同樣方式表達的LMO7 C298蛋白質片段結合，也可以抓下HeLa細胞萃取物中的LMO7。我們建構HsMAD1和LMO7的各式刪除變異(deletion mutants) ，利用酵母菌二雜交技術來分析，發現HsMAD1經由其上320到480之間的胺基酸與LMO7結合，另一方面，LMO7則藉由其C端298個胺基酸來與HsMAD1結合。為了觀察LMO7在細胞週期中的角色，我們也利用螢光染色(immunofluorecence staining)來觀察LMO7在細胞中的位置，LMO7在細胞核中顯現密集的點狀分布，而在細胞質是位於肌動蛋白絲(actin filaments)上，至於LMO7在細胞核上的小點，經由後續的分析發現並非染色體中間的著絲點(centromere)或末端的端粒(telomere)。轉殖感染(transfection)僅含有LMO7 C298蛋白質片段到HeLa細胞中，讓其與細胞內原有的LMO7競爭，發現在LMO7不正常下，雖然細胞處在微質管(microtubule)被破壞的情形下，但細胞仍無法有效的停止在細胞分裂期進行修復，總和以上，我們的研究顯示LMO7是第一個被發現與肌動蛋白絲結合的蛋白質也同時參與在人類紡垂體檢查點中。

Abstract Mitotic spindle checkpoint can ensure precise chromosome separation during mitosis progression. Mad1 (mitotic arrest defective) is conserved between human and yeast and plays a key role in spindle assembly checkpoint. To clone and characterize the genes associated with mitotic spindle checkpoint, we have used Human MAD1 (HsMAD1) as a bait to perform yeast two-hybrid screening from human liver cDNA library. We have cloned LMO7, a gene containing a tissue-specific altermatively spliced LIM domain. By in vitro binding assays, bacterially expressed GST-HsMAD1 could interact with bacterially expressed LMO7 C298 and coprecipitate with LMO7 from HeLa cell extracts. We have constructed several deletion mutants of both HsMAD1 and LMO7 to perform yeast two-hybrid analysis and found that the region from amino acids 320 to 480 in HsMAD1 was required to interact with LMO7 and LMO7 C-terminal 298 amino acids to interact with HsMAD1. Immunofluorescence staining showed that LMO7 localized to nucleus as dots and was associated with actin filaments but not with microtubules in cytoplasm. LMO7 was not localized to centromere and telomere in nucleus. Overexpression of dominant-negative mutant LMO7 disrupted checkpoint arrest in human cells during microtubule depolymerization. We conclude that LMO7 was the first discovered actin-associated protein required for the spindle checkpoint.

致謝..............................................................................................i English abstract...........................................................................ii 中文摘要 ………………………………………………….….iii Table of Contents………………………………………………iv List of Tables and Figures……………………………………...v Introduction.................................................................................1 Materials and Methods................................................................4 Results.........................................................................................8 Discussion.................................................................................12 Reference...................................................................................14 Figures……………………………………………………...…17

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