吳益群臺灣大學：分子與細胞生物學研究所劉如玲Liu, Ju-LingJu-LingLiu2007-11-252018-07-062007-11-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/49905多細胞生物體中，TLK(tousled-like kinase)是一種保守的絲氨酸/蘇氨酸激酶家族，之前研究顯示，人類的TLK在細胞週期中S期作用於染色質的組成(chromatin assembly)，以及果蠅的TLK在M期TLKs作用於染色體的濃縮。最近研究顯示，線蟲TLK-1在轉錄上扮演著重要角色。我們則發現tlk-1 (RNAi)突變體停留在約五十五個細胞時期，並具有大小不一致分怖的細胞核，由於非整倍體表型(aneuploidy)所造成。我們利用GFP 標示tubulin和組蛋白(histone) 的轉殖基因(transgenes)，結果顯示tlk-1 (RNAi)突變體在有絲分裂時具有DNA濃縮和分離的缺失。我們另外發現著絲點蛋白(holocentric proteins) HCP-1/CENP-F和HCP-3/CENP-A在tlk-1 (RNAi)突變體的mitotic DNA有著不正常分佈。此外，在yeast two-hybrids實驗中，TLK-1可與HCP-3和HCP-4 /CENP-C結合。我們推論線蟲TLK-1控制HCP-1和HCP-3正確地排列在mitotic染色體的外側 (poleward face)，是藉由與HCP-3和HCP-4的結合，進而影響在有絲分裂中染色體的濃縮與分裂。TLK, tousled-like kinase, is a family of conserved serine/ threonine kinase in multicellular organisms. Previous studies showed that human TLK functions in chromatin assembly at S phase and that Drosophila TLK acts in chromosome condensation at M phase during the cell cycle. Recent studies showed that C.elegans TLK-1 plays an essential role in transcription. We found that tlk-1 (RNAi) mutants arrested at about the 55-cell stage with uneven distribution of small and large nuclei due to aneuploidy. Using GFP to label tubulin and histone, we found that tlk-1 (RNAi) embryos have defects in DNA condensation and segregation during mitosis. We also found that holocentric proteins HCP-1/CENP-F and HCP-3/CENP-A are not properly localized to mitotic DNA in tlk-1 (RNAi) embryos. Furthermore, in the yeast-2 hybrid system TLK-1 interacts with HCP-3 and HCP-4/CENP-C. We propose that TLK-1 controls the proper localization of HCP-3 and HCP-1 to the poleward face of mitotic DNA, likely through direct interaction of TKL-1 with HCP-3 and HCP-4, and thereby functions in chromosome condensation and segregation during mitosis.Contents Abstract…………………..……………………………………………4 中文摘要…………………………………………………………………5 Introduction………………..…………………………………………6 Materials and Methods…………………...…………….…………10 Caenorhabditis elegans Strains……………………..………...10 RNA mediated interference (RNAi)……………………………...10 DAPI staining …………………………………………….………..11 Time-lapse and z-series stacks microscopy……….………...11 Immunostaining……………………………………………....…….12 TOPOCloning………. …………………………………….....…….13 Gateway System…………………………………………….........14 Yeast two-hybrid assay……………………………….…....……14 Results………………………………………………..………………16 tlk-1 (RNAi) mutant causes the early embryonic lethality with aneuploidy phenotype…….………………….……………..16 tlk-1 (RNAi) mutant exhibited defects in chromosome condensation and segregation……….......….……………….17 C. elegans TLK-1 affects centromeric protein assembling on chromosome…………………….....………….………………....18 C. elegans holocentric protein does not affect the TLK-1 localization............................................19 C. elegans TLK-1 physically interacts with HCP-3 and HCP-4 in yeast two-hybrid system……………………..……………..20 Discussion……………………......………………………………21 Chromosome condensation and segregation defects in tlk-1 (RNAi)mutants……………….………………………………………21 C. elegans TLK-1 affects centromeric protein assembling on chromosome………………………………………………………....22 The possible phosphorylation site of HCP-3 and the function of the phosphorylation…….………………………..23 Figures and Tables………….…………………………………...24 References……………….………………………………………...362431951 bytesapplication/pdfen-US著絲點TLK-1otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49905/1/ntu-94-R92b43004-1.pdf