Promoter sequence of fibroin gene assigned by in vitro transcription system

Abstract

We have shown that the silk fibroin gene from Bombyx mori is faithfully transcribed in an in vitro transcription system of the HeLa cell extract prepared by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Using this system and a series of deletion mutants of fibroin gene, we have assigned the promoter sequence of fibroin gene. The 5' boundary of the promoter is around nucleotide position -29, indicating that most of the T-A-T-A-A-A-A sequence (-30 to -24) is essential for the promoter function, where the transcription initiation point of fibroin gene is assigned as nucleotide position +1 [Tsuda, M., Ohshima, Y. & Suzuki, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 4872-4876]. The 3' boundary is around nucleotide position +6. However, to support the efficient, faithful transcription, some additional (more than 26 but less than 41) nucleotides of nonspecific origin are required at the 5' side of -29. Functions ascribed to the promoter region are discussed.