Drevet J R, Skeiky Y A, Iatrou K
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
J Biol Chem. 1994 Apr 8;269(14):10660-7.
To characterize a DNA-binding protein, BCFI, which regulates the expression of silkmoth chorion genes through binding to gene promoter elements identical to those recognized by the GATA family of transcription factors, we have carried out polymerase chain reaction amplifications of Bombyx mori genomic DNA using degenerate primers derived from the conserved DNA binding domain of mammalian GATA factors. Two single copy genes, BmGATA alpha and BmGATA beta, were identified, which encode sequences containing GATA-type zinc finger motifs. The BmGATA beta gene is expressed in follicular and Bm5 tissue culture cells, the two cell types that contain BCFI. No BmGATA alpha gene transcripts were detectable in the tissues that were tested. Upon overexpression in Escherichia coli, a peptide encompassing the BmGATA beta zinc finger motif was able to bind specifically to the BCFI recognition motif of the chorion gene promoters. A polyclonal antibody directed against the zinc finger domain of BmGATA beta was also used in gel retardation assays to confirm that factor BCFI is indeed encoded by the BmGATA beta gene. Conceptual translation of a complete cDNA clone encoding the BmGATA beta protein revealed that this protein has a size similar to that of an immunoreactive protein, presumably BCFI, which is present in follicular cell extracts.
为了鉴定一种DNA结合蛋白BCFI，该蛋白通过与家蚕绒毛膜基因启动子元件结合来调控基因表达，这些启动子元件与转录因子GATA家族识别的元件相同，我们使用了源自哺乳动物GATA因子保守DNA结合结构域的简并引物，对家蚕基因组DNA进行了聚合酶链反应扩增。鉴定出了两个单拷贝基因，BmGATAα和BmGATAβ，它们编码包含GATA型锌指基序的序列。BmGATAβ基因在卵泡细胞和Bm5组织培养细胞中表达，这两种细胞类型都含有BCFI。在所测试的组织中未检测到BmGATAα基因转录本。在大肠杆菌中过表达时，包含BmGATAβ锌指基序的肽能够特异性结合绒毛膜基因启动子的BCFI识别基序。一种针对BmGATAβ锌指结构域的多克隆抗体也用于凝胶阻滞分析，以确认因子BCFI确实由BmGATAβ基因编码。对编码BmGATAβ蛋白的完整cDNA克隆进行概念性翻译表明，该蛋白的大小与卵泡细胞提取物中存在的一种免疫反应性蛋白（可能是BCFI）相似。
