A homologous in vitro system to analyze transcription of a mouse immunoglobulin mu heavy-chain gene.
In order to investigate the molecular mechanisms of the regulation of immunoglobulin (Ig) gene transcription, a cell-free system was developed in which a cloned mouse Ig mu heavy-chain gene was transcribed using nuclear extracts prepared from a mouse B cell hybridoma line. To monitor transcription, an RNA.RNA hybridization assay was developed in which a 32P-labeled, SP6-synthesized RNA probe complementary to Ig mu RNA was hybridized to unlabeled RNA transcribed in the nuclear extract. Accurate initiation of transcription, which resulted in the protection of the RNA probe from digestion with nuclease S1, was detected by the separation of the products on denaturing polyacrylamide gels, followed by autoradiography. Using this assay, an in-vitro-synthesized RNA was detected. The 5' end of the in-vitro-transcribed Ig mu RNA maps exactly to the same position as the 5' end of the corresponding in vivo mRNA and its formation was sensitive to the addition of low levels of alpha-amanitin (1 microgram/ml), indicating transcription by RNA polymerase II. It was shown by competition experiments with oligonucleotides containing the 'decamer recognition site' that this sequence interacts with (a) decamer-binding factor(s) and plays a positive role in transcription. The competition effects of the decamer-containing oligonucleotide appeared to be restricted to the decamer motif present in the promoter region. No effects of the enhancer region were detectable in vitro. Little or no transcriptional activity was found in transcription experiments using the Ig mu promoter and nuclear extracts prepared from HeLa cells. This suggests that tissue-specific factors involved in Ig mu heavy-chain gene transcription are present in the mouse B cell extracts.
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D006825:Hybridomas; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D051379:Mice; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D012313:RNA / Q000032:analysis; D012319:RNA Polymerase II / Q000032:analysis; D014157:Transcription Factors / Q000032:analysis; D014158:Transcription, Genetic / Q000187:drug effects
D000546:Amanitins; D007148:Immunoglobulin mu-Chains; D014157:Transcription Factors; D012313:RNA; D012319:RNA Polymerase II