Isolation of recombinant DNA clones carrying complete integrated proviruses of Moloney murine leukemia virus

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Abstract
EcoRI DNA fragments from a Moloney murine leukemia virus (M-MuLV)-infected mouse fibroblast line (M-MuLV clone A9) were cloned in .lambda. phage Charon 4A cloning vector to derive clones containing integrated M-MuLV proviral DNA. A 10-16 Mdalton class of EcoRI fragments was chosen for cloning, based on its ability to induce XC-positive [rat sarcoma cell syncytium formation] virus upon transfection of NIH/3T3 cells, and its content of a 0.8 Mdalton viral KpnI fragment diagnostic for M-MuLV. Six recombinant DNA clones were isolated which contain a complete M-MuLV provirus, as judged by restriction endonuclease mapping and the fact that all of the clones gave rise to XC-positive, NB-tropic virus upon DNA infection in NIH/3T3 cells. The sizes of the inserts were 12.0 (for 3 clones) or 12.5 Mdaltons (for 3 clones). Restriction mapping indicated that these 6 clones represent 5 different M-MuLV proviral integrations into different cellular DNA sites.