Deletions of specific regions of the simian sarcoma-associated virus genome are found in defective viruses and in the simian sarcoma virus

Abstract

Extrachromosomal DNA was isolated from tissue culture cells that were acutely infected with simian sarcoma virus (SSV) and its associated helper (simian sarcoma-associated virus [SSAV]). Two sizes of closed circular viral genomic DNA intermediates were isolated, cleaved at the single EcoRI site, and ligated to the Charon 21A phage lambda vector. Cloned molecules of the larger size all represented the full-length (9.0-kilobase [kb]) SSAV molecule. A heterogeneous group of clones was derived from the smaller DNA circles. These included the SSV genome and SSAV deletion mutants. When two SSV clones were compared with the helper, they contained the following three characteristic deletions: (i) a 250-base pair deletion in the gag gene about 1.0 kb from the 5' end of the genome; (ii) a 1.85-kb deletion in the pol gene; and (iii) a 1.9-kb deletion at the 3' end, which included part of the env gene. This latter deletion was the site of the onc gene substitution. Six other clones of the smaller molecules represented the following variants of the SSAV genome: (i) two clones of the entire genome containing only one long terminal repeat unit; (ii) one clone with the 1.85-kb deletion of the pol gene observed in SSSV; and (iii) three clones having a deletion of the 3' end of the SSAV genome. In each of the latter clones, the 5' border of the deletion was indistinguishable from the 5' border of the onc substitution in SSV. The fidelity of genetic deletions observed suggested that certain regions of the SSAV genome were deleted at a high frequency. In certain cases, these deletions may have been accompanied by a substitution of cellular sequences to generate SSV.

Molecular cloning of circular unintegrated DNA of two types of the SEATO strain of gibbon ape leukemia virus

“…Phage DNA was isolated from large-scale lysates by pelleting particles and banding twice in CsCl (4). Restriction enzyme analyses and hybridization conditions for Southern blots have been described previously, as have the conditions for heteroduplex analysis (4). [32P]cDNA was prepared by transcription of GaLV-S 70S RNA with avian myeloblastosis virus reverse transcriptase (Life Science, St. Petersburg, Fla.) with DNase-digested calf thymus DNA as a random primer (15).…”