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Nucleic Acids Research, 1993, Vol. 21, No. 24 5554-5560
© 1993

MOLECULAR BIOLOGY

Isolation and cloning of putative mouse DNA replication initiation sites: binding to nuclear protein factors

Daniella Dimitrova, Lyubomir Vassilev1, Boyka Anachkova2 and George Russev2,*

International Center for Genetic Engineering (ICGEB) Pa'driciano 99, 1-34012 Trieste, Italy 1epartment of Cell and Developmental Biology, Roche Research Center Nutley, NJ 07110, USA 2nstitute of Molecular Biology, Bulgarian Academy of Sciences Acad. G.Bonchev str. bid. 21, 1113 Sofia, Bulgaria

*To whom correspondence should be addressed

Received September 23, 1993. Revised November 1, 1993. Accepted November 1, 1993.

By using an original two-step technique (trioxsalen crosslinking&2.urule;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 singlestrand 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&2.urule;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.


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