Molecular cloning and characterization of avian sarcoma virus circular DNA molecules

Abstract

Supercoiled DNA molecules were used for the molecular cloning of full-length avian sarcoma virus (ASV) DNA. Viral DNA produced by the Schmidt-Ruppin A (SR-A) strain of ASV was isolated from acutely infected transformed quail cells. Supercoiled DNA was separated from linear and open circular DNA by acid phenol extraction, opened into a full-length linear form by cleavage with the restriction endonuclease SacI, and cloned into lambda gtWES x lambda B. Four different cloned viral DNA molecules were isolated: SRA-1 contains two copies of the 330-base pair terminal redundancy normally found at each end of the linear DNA molecules, but harbors a 63-base pair deletion that spans the site at which the two copies of the terminal redundancy are joined in circular DNA molecules; SRA-2 contains two complete copies of the terminal redundancy; SRA-3 probably contains only one copy of the terminal redundancy but in all other respects appears to be similar to SRA-2; SRA-4 contains a 2,500-base pair deletion that removes all of the src gene (the gene responsible for transformation by ASVs) plus additional nucleotides adjacent to the src gene whose precise locations have not been determined. Transfection of chicken embryo fibroblasts by either SRA-1 or SRA-2 resulted both in the appearance of transformed cells and in the production of infectious virus. These results demonstrate that the cloned DNA molecules are functionally identical to viral DNA produced in vivo; therefore, molecular cloning did not cause any major alterations of the DNA. The infectivity of SRA-1 DNA indicates that the 63 base pairs missing from that molecule are not required for the initiation of viral RNA synthesis, even though the deletion is located in a copy of the terminal redundancy thought to carry a promoter for RNA synthesis. This suggests that the deletion does not remove any sequences required for the initiation of transcription.