Enhanced transcription of fibroin gene in vitro on covalently closed circular templates.

A modified method for S1 nuclease assay has been developed to quantitate an amount of radioactive transcripts in the presence of unlabeled homologous RNA. Using this method, we have assayed in vitro transcription of fibroin gene on circular and linear templates. In a posterior silk gland cell extract, covalently closed circular (ccc) DNA forms the superhelical state and supports three to 10 times more transcription than nicked circular or linear DNA does. The efficient transcription on ccc template is also observed in a middle silk gland cell extract but never seen in a HeLa cell extract. When the posterior silk gland cell extract is first incubated with pBR322 DNA and then challenged for the fibroin gene transcription on ccc DNA, the pBR322 DNA of the ccc form, but not of the linear one, inhibits the reaction. A deletion mutant carrying only 44 base pairs of the 5'-flanking sequence reserves the preference of the ccc template, but seven single-point mutants at the TATA box and -20 region abolish it. These results suggest that some components in the silk gland extract supercoil ccc DNA and stimulate the fibroin gene transcription in the absence of the upstream enhancing sequence.