In vitro characterization of the fibroin gene promoter by the use of single-base substitution mutants.

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Abstract
A highly efficient method for segment-directed mutagenesis has been developed. The method relies on the deamination by sodium nitrite of the bases in the separated strands of a small DNA restriction fragment. The mutagen-treated strands produce transition mutations by the following sequence: (i) hybridization with the complementary strand of the wild-type DNA that had been cloned into a phage fl vector, (ii) repair synthesis in vitro, and (iii) transfection of Escherichia coli. Using this method, we have isolated 14 single-point mutants within a 31-base-pair stretch of the fibroin gene (from the T-A-T-A box at the nucleotide position -30 to the cap site at +1). In vitro transcription experiments with the HeLa cell or the silk gland cell extract show that single-base transitions at the T-A-T-A box (T to C at -30, A to G at -29, and T to C at -28) and at the -20 region (G to A at -21, T to C at -20, and A to G at -17) result in decreased promoter activities, whereas those at the cap site and the -10 regions have no effect. The initiation site of transcription is the same for five "down" (reduced activity) mutants (T to C at -30, T to C at -28, G to A at -21, T to C at -20, and A to G at -17), the cap site mutant (A to G at +1), and the wild-type genes--position +1. However, the A-to-G transition at -29 (the second base of the T-A-T-A box) induces an additional transcription start from position +4. Functions of the T-A-T-A box and the -20 regions are discussed.