Sakura H, Maekawa T, Imamoto F, Yasuda K, Ishii S
Laboratory of Molecular Genetics, Tsukuba Life Science Center, Ibaraki, Japan.
Gene. 1988 Dec 20;73(2):499-507. doi: 10.1016/0378-1119(88)90514-8.
Two cDNAs encoding new DNA-binding proteins (Dbps) have been cloned using a human placenta lambda gt11 recombinant cDNA library and DNA fragments as probes. Hybrid proteins expressed by the lambda gt11 cDNA library were blotted onto nitrocellulose filters, and incubated with three different radio-labeled DNA probes containing the human epidermal growth factor (EGF) receptor enhancer or the human c-erbB-2 promoter. Two kinds of clones, named dbpA and dbpB, showed high affinities for the DNA probes. The comparison of the nucleotide and the deduced amino acid (aa) sequences between these two cDNAs indicated that 100 of 109 aa located in the central region of these two Dbps were identical. The dbpA and dbpB-coded proteins also had an affinity for other cDNA probes such as the human c-ski gene, but not for poly(dI-dC).poly(dI-dC), suggesting that the sequence(s) recognized by the dbpA and dbpB-coded proteins may occur frequently, or that these proteins bind to DNA non-specifically in a different manner from that of histones. A simple method, described in this paper, can be used to isolate cDNA clones encoding Dbps. Strategies used for the detection of sequence-specific and non-specific Dbps are discussed.
利用人胎盘λgt11重组cDNA文库和DNA片段作为探针，克隆出了两个编码新的DNA结合蛋白（Dbps）的cDNA。将λgt11 cDNA文库表达的杂交蛋白印迹到硝酸纤维素滤膜上，并用三种不同的放射性标记的DNA探针进行孵育，这些探针包含人表皮生长因子（EGF）受体增强子或人c-erbB-2启动子。两种名为dbpA和dbpB的克隆对DNA探针显示出高亲和力。这两个cDNA之间的核苷酸和推导氨基酸（aa）序列比较表明，位于这两种Dbps中心区域的109个aa中有100个是相同的。dbpA和dbpB编码的蛋白质对其他cDNA探针如人c-ski基因也有亲和力，但对聚（dI-dC）·聚（dI-dC）没有亲和力，这表明dbpA和dbpB编码的蛋白质识别的序列可能频繁出现，或者这些蛋白质以与组蛋白不同的方式非特异性地结合DNA。本文描述的一种简单方法可用于分离编码Dbps的cDNA克隆。讨论了用于检测序列特异性和非特异性Dbps的策略。
