Characterizationn of FACTp140

• Main Authors: Wan-Ting Chaung, 莊婉婷
• Other Authors: Sheng-Chung Lee
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/07606465789945607247

碩士 === 國立臺灣大學 === 分子醫學研究所 === 89 === 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.