Assumed initiation site of fibroin gene transcription

To map the initiation site of fibroin gene transcription on cloned genomic fibroin DNA (pFb29), we looked for precursors of fibroin mRNA, first by applying the hybridization mapping method to the RNA from the posterior silk gland of Bombyx mori. The 6.2-kilobase (kb) DNA fragment used for the assay, from 5.7 kb upstream to 0.5 kb downstream from the cap site, was end-labeled at the Xho I site (position +514) within the intervening sequence of the fibroin gene. By analyzing the labeled DNA protected by the RNA from nuclease S1 treatment, we have shown that the intervening sequence is transcribed together with the surrounding gene sequences in the posterior silk gland (producer) but not in the middle silk gland (nonproducer). Large transcripts that could include entire intervening (1 kb) and fibroin mRNA (16 kb) sequences were found. The 5' end of such transcripts was located around or near the cap locus. Synthesis and analysis of the DNA complementary to the transcripts primed within the intervening sequence region also confirmed that the 5' end of the transcripts is near the cap locus. These results strengthen the possibility that the cap site is the transcription initiation site of fibroin gene.