PHYSICAL MAPPING OF A LARGE-PLAQUE MUTATION OF ADENOVIRUS TYPE-2

A simple method based on cotransfection of overlapping DNA restriction fragments was developed for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0-59) and Ad5 SalI-A (map position 45-100). Analysis of the recombinant DNA by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45-59) between the Ad2 EcoRI-A fragment and the Ad5 SalI-A fragment. By using this method, several recombinants were constructed between a large-plaque (lp) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI and HindIII revealed that the lp mutation is located within the left 41% of Ad2 genome.