Murine genomic DNA sequences replicating autonomously in mouse L cells

Abstract

Plasmids that replicate autonomously in mouse L cells were constructed by inserting random genomic DNA fragments from Ltk- cells into a plasmid containing the HSV-1 thymidine kinase gene with a truncated low-efficiency promoter. HAT resistance was used as a selective marker. The presence of free plasmids in the DNA of transformants was demonstrated by hybridization with a specific plasmid probe, by electron microscopic visualization of circular DNA, and by recovering these plasmids by E. coli transformation. Nineteen different DNA fragments were isolated. They were characterized as murine autonomously replicating sequences by Mbol restriction endonuclease sensitivity, by bromodeoxyuridine substitution, by copy number determination, and by segregation analysis. Sequence analysis of the inserts of nine plasmids revealed a conserved element of 12 bp (CTCATGAGAGGCCAA) in five out of nine autonomously replicating sequences.