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Molecular Cloning of the Endogenous Rat C-type Helper Virus DNA Sequence: Structural Organization and Functional Analysis of Some Restricted DNA Fragments

¹ Laboratory of Cell Biology
and² Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20205, U.S.A.
and³ Department of Pathology, USC School of Medicine, Los Angeles, California 99033, U.S.A.

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 BamHI site of the vector pBR322, cultured in an Escherichia coli RR1 host. After screening 5750 clones for ampicillin resistance and tetracycline sensitivity and testing by colony hybridization using ³²P-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, EcoRI, BamHI, PstI, KpnI, TaqI, PvuII and SmaI 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 TaqI and PstI 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 in vitro, confirming that the RHHV genomic DNA was successfully cloned with little fidelity loss or scrambling of the genetic information.

Keywords: DNA cloning, rat retrovirus, restriction map, hybrid-select translation

Received 9 February 1982; accepted 4 June 1982.