李玉梅臺灣大學：生化科學研究所張孔仁Chang, Kung-JenKung-JenChang2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52765本實驗室在建立GCH dominant-negative (DN) 細胞株時，發現有些細胞株並沒有產生DN現象，在分析其核醣核酸表現量後，我們篩選到了一個基因在non-dominant negative (non-DN) 細胞株中有高量的表現，我們將其命名為BC1 (Brain coregulator 1)。 BC1是一個具有559個胺基酸序列的蛋白，大小約70kD，其C端約一百個胺基酸序列發現到有與YT521b等蛋白質相類似的保守序列 (conserve sequence) 稱之為YTH domain。 去年我們實驗室發現到BC1中的YTH domain具有水解三磷酸腺苷酸 (ATP) 的能力。本論文成功表現及純化全長蛋白並試驗其水解ATP的能力，同時並測試其N端刪除之突變蛋白以及YTH domain上定點突變之突變蛋白是否對水解ATP的能力有所影響，進一步推測BC1與三磷酸腺苷酸的作用區域為何。 此外我們發現到將BC1基因轉殖到細胞中，會形成小顆粒聚集細胞質的現象，由於細胞中顆粒的聚集及運送與tubulin有密切的關係，本篇論文也將探討BC1及tubulin之間的交互作用。Previously our lab established the GCH dominant-negative (DN) cell lines. We found some cell lines do not present DN phenomenon. After analyzing the amount of RNA expression by Northen blot, we select a gene that was highly expressed in non dominant negative (non-DN) cell line. We named this gene BC1 (Brain coregulator 1). BC1 is composed of 559 amino acid residues. Its molecular weight is about 70 kilodalton. In the C-terminus of BC1, we found a conserved region similar to YT521b protein, and it is now defined as YTH domain. In previous studies, we found YTH domain of BC1 had ATPase activity which could hydrolyze ATP. In this thesis, we expressed and purified full-length BC1 and performed ATPase assay. Next, we tested N-terminal deletion and site-specific mutagenesis in YTH domain to understand the effect in ATPase activity, and the binding region of BC1 to ATP. We also found when we transiently transfected EGFP-BC1 to cell, BC1 formed some small granule in cytosol. Cytosolic RNA-binding proteins are involved in RNA granule formation and transportation, which have closely relationship with tubulin, This thesis will investigate BC1 and the interaction with tubulin.English Abstract 1 Chinese Abstract 2 Abbreviation 3 1.Introduction 4 1.1 Discovery of BC1 4 1.2 ATP binding and ATPase activity 5 2.Materials and Methods 8 2.1 Plasmids 8 2.2 Site-specific mutagenesis 8 2.3 Protein expression 9 2.4 Protein purification 9 2.5 GST-pull down assay 10 2.6 SDS-PAGE and immunoblot analysis 11 2.7 Transient transfection 12 2.8 ATPase assay 12 2.9 Cell culture 13 2.10 Immunoflurosense 14 2.11 Binding of GST-fusion proteins to ATP-agarose 14 2.12 Immunoprecipitation (IP) assay 15 2.13 Hill coefficient calculate 15 3.Results 17 3.1 Molecular cloning of GST-BC1 and deletion mutant 17 3.2 Expression and purification of GST-BC1 and deletion mutants 17 3.3 Measurement of Steady-State ATPase activity of GST-BC1 18 3.4 ATPase activity was disrupted by deletion mutants 18 3.5 ATPase activity of site specific mutants of YTH domain of BC1 19 3.6 BC1 would form granules in cultured cells and is partially co-localized with tubulin 20 3.7 BC1 didn’t interact with tubulin in vitro and in vivo directly 21 4.Discussion 22 4.1 GST-BC1 has ATPase activity 22 4.2 Mutant constructs discussion 23 4.3 The relationship of BC1 and tubulin 24 Reference 25 Figure legend 28 Table legend 481106699 bytesapplication/pdfen-USATPaseBC1tubulingranuleotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52765/1/ntu-94-R92b46028-1.pdf