CLONING OF FRAGMENTS OF THE SEA URCHIN HISTONE GENE CLUSTER WITH SV40 DNA AS A VECTOR

Abstract

This chapter describes the cloning of fragments of the sea urchin histone gene cluster with SV40 DNA as a vector. DNA from the sea urchin histone gene cluster was digested with restriction enzymes. Selected fragments were linked at one end to portions of the SV40 genome through ligation of cohesive termini. Hybrid molecules formed consisted of the large Taq I-Pst I fragment of SV40 linked to a Pst I-Eco R1 fragment containing the start of the Hi histone gene, and the large Bam H1-Hae II fragment of SV40 linked to a Hae II-Eco R1 fragment containing the entire H2B histone gene. Permissive African green monkey kidney cells were coinfected with this DNA and with DNA from temperature sensitive mutants of SV40, tsA58 for insertions of sea urchin DNA into the late region of SV40, and tsBC11 for insertions into the early regions of SV40. Individual plaques appearing at the nonpermissive temperature were expanded and screened for the presence of virus containing DNA from the sea urchin histone gene cluster, using a filter hybridization assay developed for this purpose. Circularization of the hybrid linear DNA molecules occurred in vivo during the transfection procedure as evidenced by various restriction endonuclease analyses.