Natural fibroin genes purified without using cloning procedures from fibroin-producing and -nonproducing tissues...

Natural fibroin genes were purified from total DNA extracted from the fibroin-producer cells (posterior silk gland) and -nonproducer cells (middle silk gland or pupa) by two cycles of CsCl/actinomycin D centrifugation followed by sucrose density gradient centrifugation. Purity of the final samples was greater than 14%. DNA sequences of these natural genes between positions -171 and +104 were identical and showed no sign of base modification as assayed by the method of Maxam and Gilbert. The determined sequence includes the promoter and a major part of the modulator. When assayed in an in vitro transcription system prepared from middle silk gland, template activities of the purified natural fibroin genes from the producer and the nonproducer were indistinguishable from that of cloned fibroin DNA. Digestion and blotting of total genomic DNAs with several restriction enzymes that recognize methylation changes on DNA revealed no difference of hybridization pattern of fibroin DNAs in a region from -650 to +326 between the producer and nonproducer. Thus, it is unlikely that the differential transcription of the fibroin gene is controlled by a change of base modification in the regions of transcription signals.