Previous studies by Guntaka et al. have shown that the unintegrated DNA intermediates of avian RNA tumor virus replication can be readily isolated from cultures of the quail tumor line QT-6 at 1 day after infection. The intermediates include double-stranded linear and covalently closed circular DNA species. Using the analysis procedure of Southern together with previously obtained information regarding the sites of action of certain restriction endonucleases on avian sarcoma virus DNA, we have further characterized the viral DNA intermediates. Evidence is presented that, relative to the RNA genome, most of the linear species possess a direct terminal sequence redundancy equivalent to 0.5 x 106 ± 0.3 x 106 daltons of double-stranded DNA. Some of the circular forms also possess a sequence redundancy of 0.21 x 106 ± 0.03 x 106 daltons.

The state of integration of avian sarcoma virus DNA in the genomes of transformed chicken, duck, and quail fibroblasts was deduced by means of restriction enzyme digestion of total cell DNA, gel electrophoresis, and subsequent analysis by the procedure of Southern. The cells used in these studies were either mass-infected cultures or clones of infected cells selected by their ability to form colonies in agar. For both mass-infected cultures and clones of cells of all three species, we found that integration occurred at a specific site on the viral genome but appeared to occur at many sites on the cell genome. At least some of the integrated viral DNA existed as intact nonpermuted species flanked by direct terminal repeats of at least 0.134 megadalton (217 base pairs). For each of 12 transformed quail clones studied, it was possible to detect, after digestion with Kpn I, unique junctions between viral and cellular DNA. That is, at our level of analysis, the integration site on the cell genome for each clone was different. However, within each of the 17 chicken and 9 duck clones of transformed cells, a heterogeneity presumably occurred during the outgrowth of the cell clone population, in that we could not readily detect identifiable cell-virus junction frag

Full-size single-stranded DNA transcripts of the avian RNA tumor virus genome were isolated from the products of the endogenous reaction of detergentdisrupted avian sarcoma virus particles. These transcripts were converted with Escherichia coli DNA polymerase I and 32P-labeled nucleoside triphosphates into labeled double-stranded DNA. The latter DNA was used to map the sites of action of seven restriction enzymes (Pvu I, Hpa I, Kpn I, Xba I, EcoRI, HindIll, and Xho I) on the genome of three strains of avian sarcoma virus (Prague B, Prague C, and Bratislava 77).

Using chromatography on benzoylated naphthoylated DEAE-cellulose, we found that greater than 99.5% of the unintegrated linear viral DNA species detected in quail embryo cells infected with Rous sarcoma virus contained single-stranded regions, even at 16 h after infection. These regions were distributed across the genome and, on average, were primarily of plus-strand DNA. Within most of the linear viral DNA species, the minus strand was interpreted as being of genome size with two copies of the large terminal redundancy, LTR. In contrast, the plus strands in the linear viral DNA species were exclusively subgenomic.

Quail embryo fibroblasts were infected at low multiplicity with avian sarcoma virus, and transformed cells were selected by their ability to form colonies in agar. Five clones that failed to produce focus-forming virus were examined for (i) intactness of the integrated proviral DNA, (ii) intracellular viral RNA production, (iii) intracellular viral antigen production, (iv) production of virus particles, and (v) rescue of a functional src gene and of parental host range determinants by superinfection with Rous-associated virus-60, an avian leukosis virus of subgroup E. Deletions in the integrated viral DNA were apparent in three of the five nonproducer clones. In one clone producing focus-forming virus, analysis of the integrated viral DNA revealed an insertion in the region of the genome that codes for src.