Recently, we have identified and purified the integrated and proviral DNA sequences specific for two endogenous rat type C leukaemia helper viruses: WR-RaLV which originated from a fibrosarcoma induced in a feral rat and RHHV from the cell line HTC-H1 which originated from a Buffalo rat hepatoma. The rat leukaemia helper virus DNA sequences have previously been shown to be 8.4 to 8.8 kilobases (kb) in size. In this communication, we report the molecular cloning of the 8.8 kb DNA of RHHV by ligation at the HI site of the vector pBR322, cultured in an RR1 host. After screening 5750 clones for ampicillin resistance and tetracycline sensitivity and testing by colony hybridization using P-labelled RHHV cDNA, four clones were isolated, two of which carried the total 8.8 kb DNA. A detailed restriction endonuclease map of the cloned RHHV DNA was deduced by sequential digestions of either 3′- or 5′-labelled DNA. Of the 14 restriction enzymes tested, RI, HI, I, I, I, II and I gave informative cleavage patterns. At least two copies of long terminal repeated sequences (LTR) flanking the 3′ and 5′ termini of the proviral DNA were identified by I and I cleavages. LTR in the rat endogenous leukaemia helper virus DNA measured 780 ± 20 nucleotides in length. The genetic information encoded by the cloned DNA was also analysed by hybridization selection of RHHV mRNA, which was then used in cell-free protein synthesis in a rabbit reticulocyte lysate system. Essentially all major RaLV-specific proteins precipitable by anti-RaLV serum were synthesized , confirming that the RHHV genomic DNA was successfully cloned with little fidelity loss or scrambling of the genetic information.


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