Lafer E M, Sousa R J, Rich A
Dept. of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Mol Biol. 1988 Sep 20;203(2):511-6. doi: 10.1016/0022-2836(88)90017-4.
Z-DNA affinity adsorption of an Escherichia coli lysate in the presence of excess B-DNA results in a 1000-fold enrichment for three proteins with apparent molecular weights, on SDS/polyacrylamide gel electrophoresis, of 50,000, 90,000 and 100,000. When retention of these proteins on resins constructed with Z-DNA (Br-poly(dG-dC).poly(dG-dC)) was compared with retention on resins constructed with B-DNA or Br-B-DNA, it was found that approximately 100-fold more of the 50,000 Mr protein, 1000-fold more of the 90,000 Mr protein, and greater than 1000-fold more of the 100,000 Mr protein was retained on the Z-DNA resin. No difference in retention on the B-DNA versus brominated B-DNA resin was found, indicating that the increased retention on the Z-DNA resin was not due to bromination of the Z-DNA. This demonstration of Z-DNA-specific binding in vitro makes these proteins candidates for binding to Z-DNA in vivo. In an effort to determine the function of these proteins we have prepared monoclonal antibodies against each protein and isolated its respective gene. Western blot analysis of lysogens carrying these genes confirms their identity and shows that the complete coding region and promoter for each gene has been cloned.
在存在过量B-DNA的情况下，对大肠杆菌裂解物进行Z-DNA亲和吸附，可使三种蛋白质得到1000倍的富集，在SDS/聚丙烯酰胺凝胶电泳上，其表观分子量分别为50,000、90,000和100,000。当将这些蛋白质在由Z-DNA构建的树脂（溴化聚（dG-dC）·聚（dG-dC））上的保留情况与在由B-DNA或溴化B-DNA构建的树脂上的保留情况进行比较时，发现50,000 Mr蛋白质在Z-DNA树脂上的保留量大约多100倍，90,000 Mr蛋白质多1000倍，100,000 Mr蛋白质多1000倍以上。未发现B-DNA树脂与溴化B-DNA树脂在保留情况上有差异，这表明Z-DNA树脂上保留量的增加并非由于Z-DNA的溴化。这种体外Z-DNA特异性结合的证明使这些蛋白质成为体内与Z-DNA结合的候选物。为了确定这些蛋白质的功能，我们针对每种蛋白质制备了单克隆抗体并分离了其各自的基因。对携带这些基因的溶原菌进行的蛋白质印迹分析证实了它们的身份，并表明每个基因的完整编码区和启动子已被克隆。
