An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs

Abstract

Abstract A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of Eco RI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intra molecularly rather than inter molecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to Eco RI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the Eco RI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the Eco RI restriction endonuclease cleavage site in the polyoma genome.