Berent S L, Sevall J S
Biochemistry. 1984 Jun 19;23(13):2977-83. doi: 10.1021/bi00308a020.
Cloned DNA containing the first nine exons of the rat albumin gene was digested with EcoRI and HindIII, and the resulting fragments were used to screen for regions with relatively high affinity for protein. Of three restriction fragments preferentially bound, the fragment containing the first two exons of the albumin gene was consistently bound over others by heat-stable protein extracted from liver nuclei with 0.35-1.0 M NaCl. Proteins extracted with lower and higher ionic strength buffers bound the DNA fragments, but with little specificity. The DNA fragment that was preferentially bound consistently by the 1.0 M nuclear extract was subcloned into pBR325 and was used to isolate the specific DNA-binding activity. After purification, histone H1 was the polypeptide with preferential DNA-binding activity. Histone H1 has a high-affinity binding site in the 5' end of the rat albumin gene within 440 5'-flanking base pairs and the first two exons of the gene.
用EcoRI和HindIII消化含有大鼠白蛋白基因前九个外显子的克隆DNA，所得片段用于筛选与蛋白质具有相对高亲和力的区域。在优先结合的三个限制性片段中，含有白蛋白基因前两个外显子的片段始终被用0.35 - 1.0 M NaCl从肝细胞核中提取的热稳定蛋白优先于其他片段结合。用较低和较高离子强度缓冲液提取的蛋白质能结合DNA片段，但特异性较差。被1.0 M核提取物始终优先结合的DNA片段被亚克隆到pBR325中，并用于分离特异性DNA结合活性。纯化后，组蛋白H1是具有优先DNA结合活性的多肽。组蛋白H1在大鼠白蛋白基因5'端440个5'侧翼碱基对和该基因的前两个外显子内有一个高亲和力结合位点。
