Inactivation by sequence-specific methylations of adenovirus promoters in a cell-free transcription system.

摘要: Studies on the biochemical mechanism of promoter inhibition or inactivation by sequence-specific methylations necessitated development a cell-free transcription system that responded to in vitro methylations. Such systems were hitherto not available. In nuclear extracts from HeLa cells, activities two adenovirus type 2 promoters nonmethylated and methylated forms compared. The late E2A at three 5'-CCGG-3' (HpaII) sequences nucleotides -215, +6, +24, major nucleotide -52 sequence -13 5'-GCGC-3' (HhaI) exhibited strikingly lower than did constructs no activity all. designated positions calculated relative cap sites promoters. Upon transcription, methylation pattern was shown be stable. For inhibitory effects elicited, circular templates had used, DNA titers critical levels for each extract, high protein concentrations maintained. When template mixture active inhibited inactivated. Activity different could assessed simultaneously same assay due differences lengths between products Thus proven specific promoter. We are currently pursuing hypothesis cellular factors crucial recognizing somehow participate their inactivation.