| Abstract: | Viral RNA (vRNA) from avian myeloblastosis virus or DNA from virus-infected and uninfected cells was hybridized with 3H]DNA complementary to viral RNA (3H]cDNA) under conditions of 3H]cDNA excess. When 3H]cDNA was used to drive the hybridization reaction with vRNA, a rate constant of 33.2 liters/mol-s was obtained. The same rate constant was obtained when vRNA excess was used as the driver. The specific activities of the 3H]DNA probe, estimated from kinetic measurements of the hybridization reaction and from the amount of 3H]cDNA in hybrid form at equilibrium, were 9.1 and 8.6 cpm/pg, respectively. DNA isolated from uninfected cells contained five or six copies of proviral DNA per cell genome. DNA isolated from erythrocytes infected with avian myeloblastosis virus had an additional five or six viral genes added to the cell genome, and the virus-infected target cell (myeloblasts) contained about 15 additional copies of proviral DNA per cell. The use of excess 3H]cDNA probe is an easy and accurate method to quantify the frequency of proviral DNA sequences in cell DNA and to measure a small amount (40 to 200 pg) of vRNA. Probe excess hybridization offers a number of advantages over other procedures and these are discussed. |
