ISOLATION AND CLONING OF PUTATIVE MOUSE DNA-REPLICATION INITIATION SITES - BINDING TO NUCLEAR-PROTEIN FACTORS
ISOLATION AND CLONING OF PUTATIVE MOUSE DNA-REPLICATION INITIATION SITES - BINDING TO NUCLEAR-PROTEIN FACTORS
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DOI：
10.1093/nar/21.24.5554
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发表时间：
1993-12-11
影响因子：
14.9
通讯作者：
RUSSEV, G
中科院分区：
文献类型：
--
作者：
DIMITROVA, D;VASSILEV, L;RUSSEV, G
通过使用原始的两步技术(三氧沙林交联/免疫沉淀)，我们能够以单链形式分离出富含假定复制起始序列(RIS)的一部分小鼠DNA。将分离纯化的单链片段体外双链克隆到pUC12中，构建限制性RIS文库。随机选择30个RIS插入物，使用来自分裂或静止的小鼠细胞的核提取物进行凝胶迁移率改变分析。在30个RIS片段中，有12个片段显示出与分裂细胞核提取液中存在的蛋白质的特异性结合，而静止细胞的提取液没有显示出抑制作用。对RIS12、RIS18和RIS30进行测序，发现它们富含A+T，并且含有不同的调控元件。通过两步法(肝素-琼脂糖层析/DNA亲和层析)，我们分离到与RIS12特异结合的蛋白因子。其表观分子质量分别为63和65kD，为双带。
By using an original two-step technique (trioxsalen crosslinking/immunoprecipitation) we were able to isolate in a single-stranded form a fraction of mouse DNA enriched in putative Replication Initiation Sequences (RIS). The isolated and purified single-strand fragments were made double-stranded in vitro and were cloned in pUC12 to prepare a confined RIS library. 30 randomly selected RIS inserts were subjected to gel mobility shift assay using nuclear extracts either from dividing, or from quiescent mouse cells. Twelve out of the 30 RIS fragments showed specific binding to proteins present in nuclear extract from dividing cells, while none were retarded by extracts from quiescent cells. RIS12, RIS18 and RIS30 were sequenced and it was found that they were A + T rich and contained different regulatory elements. By using a two step procedure (Heparin - sepharose chromatography/DNA affinity chromatography) we isolated the protein factor that specifically binds to RIS12. It appeared as a double band with apparent molecular masses of 63 and 65 kD.