Zaret K S, Liu J K, DiPersio C M
Section of Biochemistry, Brown University, Providence, RI 02912.
Proc Natl Acad Sci U S A. 1990 Jul;87(14):5469-73. doi: 10.1073/pnas.87.14.5469.
A method based on the polymerase chain reaction is described for constructing a clustered set of basepair changes, deletions, or insertions at any site on a DNA fragment. Advantages of the procedure are that virtually every product has the desired sequence alteration and that only a single round of polymerase chain reaction is required. We used this method to demonstrate that the binding of a specific liver nuclear protein, which we call eH-TF, is essential for the function of the enhancer of the mouse albumin gene. The eH-TF binding activity is hepatocyte-specific; it binds to a functional region of the albumin promoter and is distinct from other albumin promoter factors, and part of the eH-TF binding sequence, TGTTTGC, occurs in functional regulatory sites of other liver-specific genes.
描述了一种基于聚合酶链反应的方法，用于在DNA片段的任何位点构建一组聚集的碱基对变化、缺失或插入。该方法的优点是几乎每个产物都具有所需的序列改变，并且只需要一轮聚合酶链反应。我们使用这种方法证明了一种特定肝核蛋白（我们称之为eH-TF）的结合对于小鼠白蛋白基因增强子的功能至关重要。eH-TF结合活性是肝细胞特异性的；它与白蛋白启动子的功能区域结合，并且与其他白蛋白启动子因子不同，eH-TF结合序列TGTTTGC的一部分存在于其他肝脏特异性基因的功能调节位点中。
