Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro , P labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide (EB) staining. As a result, although there are more unspecific bands in the EB staining assay than 32P labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.
The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery.To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100,we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection(ChIP-GLAS).From this assay,we determined that a set of promoter fragments,including several factors in the transforming growth factor beta(TGF-β)signaling pathway,exhibited interaction with p100.The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines.
