The objective of the research proposed is to determine, at the nucleotide sequence level, the nature of a temperature-sensitive (for transformation) mutant of wild-type MuSV-349 virus designated ts110. Ts110 MuSV-infected cells and rescue of ts110 MuSV virions contain two viral RNAs which are deleted relative to wild-type MuSV-349 RNA. We will determine the number of indicated proviruses from which these RNAs arise. These ts110 provirus(es) will be molecularly cloned and sequenced to yield details on the extent of the mutation. Similarly, ts110 MuSV viral RNAs will be analyzed by S-1 nuclease mapping techniques as well as primer extension techniques to provide details on the positions of the deletion boundaries and the nucleotide sequence of these boundaries. If, as is our present interpretation, the smaller (3.5 kb) ts110 RNA arises by splicing of the larger (4.0 kb) transcript, these analyses will yield crucial data on the position of the proposed splice and whether it in fact is bounded by splice donor and acceptor sequences. Since the deletion(s) appear to include approximately the first 100 nucleotides in the mos gene, the S-1 and primer extension data should provide convincing data on this point also. There are revertants of ts110 MuSV available (infected cells transformed at both temperatures) in which a single 4.0 kb viral RNA is found and which produce a novel 100kd gag-mos fusion protein. Cloning of these proviruses, as well as S-1 and primer extension analysis will be performed and should prove instructive in our interpretation of the original mutation. Finally, we intend to study the biological activity of the MuSVts110 RNAs and cloned ts110 MuSV DNAs by microinjection into animal cells.
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