| The mammalian SWI/SNF-like BAF complex and the transcription factor NF1/CTF play very important roles in regulating gene transcription. It is well documented that they jointly take part in gene transcription that led by RNA Polymerase II.However, whether BAF complex and NF1/CTF are also involved in the formation of RNA polymerase II-mediated transcription initiation complex is still uncertain, and a number of the currently proposed models still lack the support of experimental data. In this dissertation, Jurkat cells,Hela cells and mouse spleen T lymphocytes were chosen as experimental materials to answer the question. With the aid of various techniques such as ELISA, immuno-co-precipitation, indirect immunofluoresent co-localization, double-labeling immunoelectron microscopy and so on, the relationships of BAF complex with NF1/CTF and RNA polymerase II were careful observed and analyzed. The results are shown as follows:1,Western blot analysis confirmed that BAF complex and NF1/CTF exist in the nuclei of self-proliferative Jurkat cells. Further, in the mouse spleen T lymphocytes activation system, it was found that, without activation by ConA, there were no or extremely low level of expression of BAF complex and NF1/CTF in the spleen T lymphocytes. After activated by ConA, the mouse spleen T lymphocytes started expressing BAF complex and NF1/CTF. The longer the stimulation, the higher theexpression level of BAF complex and NF1/CTF in the cells. This data indicated the expression of BAF complex and NF1/CTF is closely related to cell activation and gene transcription activities.2,Immuno-co-precipitation showed that in the activated mouse spleen T lymphocytes and self-proliferative Jurkat cells, BRG1, NF1/CTF and RNA Polymerase II were closely bound together, and this was not found in un-activated cells. In Hela cells, by using anti-BRGl antibodies, anti-RNA Polymerase II antibodies and anti-NFl/CTF antibodies, the core subunit BRG1 of BAF complex and RNA Polymerase large subunit were found well immunofluorescently co-localized, while NF1/CTF and RNA Polymerase large subunit were poorly co-localized. BRG1 and NF1/CTF were also well co-localized. In order to further reveal the relationships of BAF complex with NF1/CTF and RNA Polymerase II large subunit at the ultra-microscopic level, we performed the double-labeling immunoelectron microscopy experiment with Hela cells. After the statistical analysis on the distances between different particles representing BAF complex, NF1/CTF and Polymerase II large subunit, respectively, we further proved that the BAF complex is closely associated to NF1/CTF and RNA polymerase II large subunit. But NF1/CTF and RNA polymerase II are weakly associated.3,by immunofluoresent microscopy, we also found that BRG1 did not clearly co-localize with RNA Polymerase II small subunit B6, but partially co-localized with RNA Polymerase II small subunit B8; NF1/CTF clearly co-localized with RNA Polymerase II small subunit B6 and partially with B8. These data implies that BRG1 may be partially associated to B8 and NF1/CTF may be involved in the formation of transcription initiation complex through associating to BRG1 and B6. Through double-labeling immunoelectron microscopy, the same patternwas found as that of the above immunofluoresent microscopic data.4,it has been showed that actin is a subunit of BAF complex. By using immuno-co-precipitation, immunofluoresent co-localization, double-labeling immunoelectron microscopy, we thus examined the relationships of actin with BRGl, NFl/CTF and RNA Polymerase II. Our results showed that, in the activated mouse spleen T lymphocytes and self-proliferative Jurkat cells, actin bound to BRGl, NFl/CTF and RNA Polymerase II during gene transcription. Whereas, in unactivated mouse spleen T lymthocytes, no binding could be found. Actin was closely associated to BAF complex and RNA Polymerase II, but not so closely to NFl/CTF.In general, we suppose that in transcriptionally active cells, BAF complex, NF1/CTF, RNA Polymerase II an...
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