Structural analysis of the fibroin gene at the 5' end and its surrounding regions

Abstract

Using the genomic clones of the fibroin gene with its flanking sequences, a detailed restriction map was prepared with particular attention given to the region neighboring the 5' end of the gene. About 60% of the fibroin mRNA has a complete cap (m7GpppAmUmCXG), and the remaining 40% has an unmethylated cap (GpppAPyXXG). The latter can be radioactively labeled by the use of vaccinia capping enzymes. Using the labeled mRNA, we mapped the 5' end of the gene by hybridization to restriction enzyme digests. DNA sequencing of this region revealed a sequence ATCAGCATCAG that corresponds to ApyXXG in tandem. The 5' end of mRNA with a complete cap was also located in these 11 nucleotides. Since the second ATCAG region was protected from S1 digestion following hybridization with total fibroin mRNA, we have tentatively assigned it as the sole 5' end of the transcription unit. About 700 bp were sequenced in the region from -800 + 1700 bp. The region of about 210 bp preceding the 5' end of the gene which may accommodate a promoter sequence is very AT-rich and contains clusters of A and T residues. This region contains statistically significant dyad symmetries, direct repeats and inverted repeats. At -30 to -24, the sequence TATAAAA was observed, and this is similar to a Pribnow-type sequence (TATAGATG). The sequence that codes for the gly-ala repetitious peptide characteristic of fibroin begins somewhere between 1340 and 1600 bp from the 5' end. An intervening sequence of about 1.1 kb that interrupts the gene about 70 bp from the 5' end was detected by R loop formation. Structures of the fibroin gene cloned from fibroin producing and nonproducing cells look the same, especially around the 5' end and the intervening region.