Jiang W D, Philippsen P
Institut für Mikrobiologie und Molekularbiologie, Universität Giessen, Federal Republic of Germany.
Mol Cell Biol. 1989 Dec;9(12):5585-93. doi: 10.1128/mcb.9.12.5585-5593.1989.
The DNA subregions CDEI and CDEIII of Saccharomyces cerevisiae centromeres are highly conserved, and both are binding sites for proteins. We describe here the purfication of a CDEI-specific binding protein using biotin-labeled synthetic CDEI DNA coupled to streptavidin agarose. The binding properties of this 64-kilodalton (kDa) protein were characterized by competition assays and by methylation interference assays. DNA fragments with single base-pair changes at positions 7 and 8 of CDEI were less efficient competitors than fragments with nonmutated CDEI. Mutations at these positions have previously been shown to decrease centromere activity in vivo. Methylation of guanosines at either side of the 8-base-pair CDEI sequence did not interfere with binding, whereas methylation of any of the four guanosines within CDEI prevented binding. A smaller CDEI-specific binding protein of 37 kDa was also purified and characterized. It is most likely a degradation product of the 64-kDa protein.
酿酒酵母着丝粒的DNA亚区域CDEI和CDEIII高度保守，且二者均为蛋白质的结合位点。我们在此描述了一种使用与链霉亲和素琼脂糖偶联的生物素标记合成CDEI DNA来纯化CDEI特异性结合蛋白的方法。通过竞争分析和甲基化干扰分析对这种64千道尔顿（kDa）蛋白的结合特性进行了表征。在CDEI的第7和第8位具有单碱基对变化的DNA片段作为竞争者的效率低于具有未突变CDEI的片段。先前已表明这些位置的突变会降低体内着丝粒活性。8碱基对CDEI序列两侧的鸟苷甲基化不会干扰结合，而CDEI内四个鸟苷中任何一个的甲基化都会阻止结合。还纯化并表征了一种较小的37 kDa的CDEI特异性结合蛋白。它很可能是64 kDa蛋白的降解产物。
