In vitro transcription of the plasma protein genes of Bombyx mori.

An efficient cell-free transcription system was developed from the extract of BmN cells established from an ovarian tissue of the silkworm, Bombyx mori. The cloned genes coding for major plasma proteins of B. mori including SP 1, SP 2 and 30K protein, were faithfully and efficiently transcribed in the extract prepared from BmN cells. The S1 nuclease mapping and primer extension analyses demonstrated that the transcription initiation site recognized in vitro is identical to that which functions in vivo. The transcription assay reconstituted from the fractionated BmN cell extract revealed that at least four protein factors are required for accurate transcription of the SP 1 and adenovirus major late genes. The results of in vitro transcription experiments employing a series of the 5' deleted mutant templates of the SP 1 gene indicated that partial deletion of the TATA box results in considerable loss of faithful transcript, while complete removal of the TATA-sequence totally abolishes the transcript. These observations suggest that the promoter element essential for transcription in cell-free systems is located in a region between nucleotide positions -44 and +16 of the SP 1 gene.