ISOLATION AND CLONING OF PUTATIVE MOUSE DNA-REPLICATION INITIATION SITES - BINDING TO NUCLEAR-PROTEIN FACTORS

By using an original two-step technique (trioxsalen crosslinking/immun oprecipitation) we were able to isolate in a single-stranded form a fr action of mouse DNA enriched in putative Replication Initiation Sequen ces (RIS). The isolated and purified single-strand fragments were made double-stranded in vitro and were cloned in pUC12 to prepare a confin ed 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. RIS1 2, RIS18 and RIS30 were sequenced and it was found that they were A T rich and contained different regulatory elements. By using a two ste p procedure (Heparin - sepharose chromatography/DNA affinity chromatog raphy) we isolated the protein factor that specifically binds to RIS12 . It appeared as a double band with apparent molecular masses of 63 an d 65 kD.