臺灣博碩士論文加值系統

中文摘要
FACTp140在細胞中是與SSRP1形成異體單元蛋白錯合物（nuclear heterodimeric FACT complex），已知FACTp140-SSRP1是參與在細胞轉錄作用的延長過程（elongation of transcription）中的一個因子。已知FACTp140-SSRP1在yeast與frog之homologes會參與在DNA複製（DNA replication）的過程中。在本論文中製造了一株抗FACTp140的單株抗體，利用此單株抗體進行免疫染色，發現FACTp140在核內的分布除了均質的小點外，有部分的FACTp140會聚集在特定的區域中，且這些區域恰為DAPI淡染的部分。當細胞進入mitosis時，FACTp140會被排除在chromosome之外的細胞質中並均勻的分布。已知FACTp140與SSRP1會在細胞中結合並執行其功能，本論文更進一步定出SSRP1中1~205與FACTp140中477~633部分的胺基酸序列對其間的interaction是重要的。利用nuclear matrix的製備法，我發現大部分的FACTp140是屬於soluble fraction及chromatin fraction，只有少部分的FACTp140會在nuclear matrix的部分以點狀的方式分布。另外，利用此FACTp140的抗體所進行的免疫沉澱實驗中，除了已知的SSRP1外，更發現Mcm3以及Mcm6也會被沉澱出來。因為Mcm3和Mcm6都是參與在DNA複製始期的重要蛋白，所以推測哺乳類細胞核中的FACTp140可能也在此扮演了重要的角色。

Abstract
FACTp140 and SSRP1 form a nuclear heterodimeric FACT complex, which is a known chromatin-specific elongation factor required for transcription of chromatin templates (Orphanides et al., 1999). The homologues of FACT complex in yeast and frog, Spt16-Pob3 and DUF, have been implicated in DNA replication and transcrption (Wittmeyer et al., 1999; Okuhara et al., 1999; Mylynda and Tim, 2000). In this study, monoclonal antibodies specifically against FACTp140 were generated and used for Western blot analysis, indirect immunofluorescence, and immunoprecipitation experiments. Indirect immunofluorescence revealed that FACTp140 exhibited a finely grainy distribution throughout nucleoplasm of interphase nuclei and mostly excluded from heterochromatin. During mitosis, FACTp140 is apparently excluded from chromosome. Furthermore, in situ nuclear matrix isolation and indirect immunofluorescence showed most of FACTp140 was detergent-sensitive soluble form and/or associated with chromatin. Only a small fraction of FACTp140 is apparently associated with nuclear matrix. Using immunoprecipitation and mass spectrometry analysis, we identified SSRP1, Mcm3 and Mcm6 as FACTp140-interacting proteins. We further demonstrated amino acids 1~206 of SSRP1 and amino acids 470~633 of FACTp140 were responsible for the interactions between FACTp140 and SSRP1.

Table of contents
Abstract (Chinese) 7
Abstract (English) 8
Introduction 9
Materials and Methods 13
Plasmid constructs
Transformation and plasmid preparation
Antibody
Cell culture and transient transfection
Cell lysis, immunoprecipitation, and Western blot analysis
Protein sequencing
Cell fractionation
In situ nuclear matrix isolation and indirect immunofluorescence analysis
Results 19
Generation, characterization of monoclonal antibodies against FACTp140
Identification of FACTp140 containing multiprotein complex
Mapping the interaction domains between FACTp140 and SSRP1
Subcellular localization of FACTp140
Discussions 22
FACTp140 form complex with Mcm3 and Mcm6
Mapping the interaction domains between FACTp140 and SSRP1
Interrelationship of subcellular localization and functions of FACTp140
Concluding remarks 26
Reference 27
Figures 33
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.

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