Mapping of transforming region of the Harvey murine sarcoma virus genome by using insertion-deletion mutants constructed in vitro.

Abstract

Circular DNA intermediates of Harvey murine sarcoma virus (Ha-MuSV) were cloned in .lambda.gtWES.cntdot..lambda.B and were capable of transforming mouse [fibroblast] NIH 3T3 cells. By using the cloned Ha-MuSV DNA insert as a parental genome, a series of insertion-deletion mutants were constructed by inserting an octomer containing the SalI linker sequence (G-G-T-C-G-A-C-C) into various regions of the Ha-MuSV genome after partial digestion with HaeIII. After ligation into .lambda.gtWES.cntdot..lambda.B-SalI vector molecules, the mutant Ha-MuSV DNA were cloned. Fourteen insertion-deletion mutants were mapped by restriction enzyme digestion and their biological activites correlated with the locations of mutations. The mutants whose lesion mapped within 3.0 kilobases (kb) from the 3''-end of the Ha-MuSV genome retained full transforming ability. The mutants containing the SalI linker insertion at 0.4 or 1.5 kb from the 5''-end retained transforming ability, but the number of foci induced by the DNA in transfection assays was greatly reduced. A mutant containing a deletion of 1.5 kb at the 5''-end and a mutant with a deletion of the sequences between 1.0 and 1.5 kb from the 5''-end completely lost their transforming potential. A model for the transforming region of Ha-MuSV is discussed. Since Ha-MuSV sequences can be rescued from the mouse cells transformed by these mutants using Moloney murine leukemia virus as a helper virus, in vitro modified DNA may be converted into genuine mutant viruses.