Interaction of composite protein complex with the fibroin enhancer sequence.
To characterize proteins that bind to the upstream region of the fibroin gene, we used an electrophoretic mobility assay and a DNase I footprinting assay. A crude nuclear extract from the posterior silk glands forms a sequence-specific complex with the upstream region, -234 to -66, which includes a signal for tissue-specific transcriptional enhancement. This specific complex is composed of at least two factors of proteinous nature and the DNA. The composite proteins specifically protect an approximately 13-base pair (-167 to -155) region in a DNase I footprinting assay. This region contains an interesting repeated sequence, AATTTAATTT. Crude nuclear extracts from the middle silk glands and the ovarian tissues, but not a HeLa whole cell extract, also give the band complexes of similar electrophoretic mobility to the one formed in the posterior silk gland extract. However, under a higher EDTA condition, these complexes are more unstable than the one constructed in the posterior silk gland extract. These results suggest a possibility that these proteinous molecules in the complex, although apparently ubiquitous in Bombyx cells, might be specifically modified in the posterior silk gland cells to play an important role in the specific expression of the fibroin gene.
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D012831:Bombyx / Q000032:analysis / Q000235:genetics; D002467:Cell Nucleus / Q000032:analysis; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D005033:Ethylmaleimide / Q000494:pharmacology; D005088:Exocrine Glands / Q000032:analysis; D005260:Female; D005349:Fibroins / Q000235:genetics*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D010053:Ovary / Q000032:analysis; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014357:Trypsin / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004247:DNA; D005349:Fibroins; D004492:Edetic Acid; D003850:Deoxyribonuclease I; D014357:Trypsin; D005033:Ethylmaleimide