Characterization of the DNA of a Nonoccluded Baculovirus, Hz-1V

Abstract

The DNA of the nonoccluded baculovirus (Hz-1V) obtained from the IMC-Hz-1 cell line was characterized by physicochemical and restriction endonuclease techniques. Hz-1V DNA isolated from purified virus had buoyant densities of 1.58 and 1.54 g/ml in CsCl-ethidium bromide density gradients, which corresponded to supercoiled and to relaxed circular and linear DNA, respectively. Neutral CsCl equilibrium centrifugation indicated that the Hz-1V DNA had a buoyant density of 1.7024 g/ml, which corresponded to a guanine-plus-cytosine (G+C) content of 43%. Thermal denaturation indicated a high G+C domain(s) in the Hz-1V genomic DNA. The domain(s), which included about 11% of the total genomic DNA, exhibited a T m of 97°C. The remaining portion (89%) of the DNA had a T m of 86.5°C. The T m s corresponded to G+C contents of 42 and 67%, respectively. The mean genetic complexity of Hz-1V DNA determined by DNA reassociation kinetic analysis was found to be 152 × 10 6 . A possible rapidly reassociating component comprising approximately 13% of the genome was observed. The mean molecular weights from restriction endonuclease digests were 159 × 10 6 for both Hind III and Eco RI. Genomic heterogeneity was found in both the wild-type Hz-1V stock and in two plaque isolates. Of 12 single-plaque isolates, 3 basic restriction endonuclease DNA fragment patterns were observed. The molecular size estimates from electron microscopic contour lengths of uncloned viral DNA ranged from 70 to 158 megadaltons, and the mode was the 130- to 140-megadalton class.