Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase.

摘要: The 5'-flanking DNA of the mouse RII beta subunit cAMP-dependent protein kinase gene was characterized by transient transfection beta-CAT constructs into neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) gel mobility shift footprinting assays. minimal promoter composed two adjacent functional elements. A 3'-element which supported enhanced CAT activity located between base pairs (bp) -267/-168 from translation initiation start site. plasmids containing these sequences showed 12- 16-fold increased in NB2a CHO cells, respectively, compared to basic vector. Plasmids 20 additional bp 5' fragment 2-fold more than shorter while nearly same for both constructs. only this 20-bp 9- 13-fold respectively. core lacked classical TATA sequences, but contained 3 copies Sp1 consensus sequence. Gel assays using 32P-labeled -291/-49 nuclear extracts displayed several retarded bands gels suggesting complex formation with DNA-binding factors. Unlabeled blocked appearance all bands. Competition an oligonucleotide corresponding site effectively slowly migrating did not affect major rapidly DNase I analysis purified confirmed that could bind sites. Methylation interference mutational one faster result factor binding sequence Additional tissue-specific nuclear-binding were detected upstream promoter. Our data suggest can initiate transcription around sites there are factor-binding distal