Insertional mutagenesis of the Abelson murine leukemia virus genome: identification of mutants with altered kinase activity and defective transformation ability.
A library of Abelson murine leukemia virus (A-MuLV) proviral DNAs with 12- or 6-base-pair (bp) insertional mutations was constructed. The 29 mutations characterized spanned the entire protein-coding region of the provirus. We tested the effects of these mutations both on the kinase activity of the gag-abl fusion protein encoded by the provirus and on the ability of the provirus to transform NIH 3T3 fibroblasts. To simplify assessment of the mutant kinases, we expressed the A-MuLV-encoded kinase in the bacterial expression vector pATH2, resulting in production of a trpE-gag-abl fusion protein in Escherichia coli. We used an immunoprecipitation kinase assay to measure both autophosphorylation and artificial substrate phosphorylation by the mutant kinases. To assay transformation ability of the mutant proviruses, we transfected NIH 3T3 fibroblasts with the mutants and with helper virus (Moloney MuLV) by the DEAE-dextran method. Our analysis of these A-MuLV insertional mutants allows the division of the protein-coding region of the provirus into four domains: domain A (proviral bp 1068 to 1685), in which insertions have no effect on the bacterially expressed kinase, but diminish both kinase activity and transformation efficiency in fibroblasts; domain B (bp 1750 to 2078), in which insertions have no effect on the provirus; domain C (bp 2181 to 2878), the critical kinase domain, in which 12-bp or even 6-bp insertions completely inactivate the A-MuLV kinase and result in transformation-defective proviruses; and domain D (bp 2956 to 4610), the large C-terminal domain in which mutations are silent.
Rees-Jones
R W
RW
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Goff
S P
SP
D000011:Abelson murine leukemia virus / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D005347:Fibroblasts; D015683:Gene Products, gag; D005814:Genes, Viral; D009052:Leukemia Virus, Murine / Q000235:genetics*; D051379:Mice; D009154:Mutation; D009857:Oncogenes; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000502:physiology; D011533:Proviruses / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology
D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins; D012191:Retroviridae Proteins; D014764:Viral Proteins; D011505:Protein-Tyrosine Kinases