Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus

Abstract

Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly cloned at its unique EcoRI site in [Escherichia coli plasmid] pBR322, for 3 nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9 to 1.0 kilobase pair region in the 5'' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat and chicken cells, 1-3 bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to 1-3 bands in bat and chicken DNA. Earlier work showed that the 5'' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein. Two subgenomic HindIII clones whose shared HindIII site mapped within the 5'' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions were used. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these 2 nontransforming DNA to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5'' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.