title: Isolation and cloning of putative mouse DNA replication initiation sites: binding to nuclear protein factors.
abstract: 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.
authors.affiliation: International Center for Genetic Engineering (ICGEB), Trieste, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Dimitrova
authors.forename: L
authors.initials: L
authors.lastname: Vassilev
authors.forename: B
authors.initials: B
authors.lastname: Anachkova
authors.forename: G
authors.initials: G
authors.lastname: Russev
mesh_terms: D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids; D014307:Trioxsalen; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D014307:Trioxsalen