Detection of transforming ras proteins containing leucine at position 61 by a new mouse monoclonal antibody, ras(53-69)Leu61.
A monoclonal antibody (mAb) was prepared after immunization of mice with a peptide that corresponds to amino acids 53 to 69 of a transforming ras protein. The amino acid sequence in this region is conserved among all members of the ras protooncogene family in rodent, rabbit, and human cells. The peptide used for immunization differs from the normal sequence by a single residue; Leu replaces Gln at a site corresponding to amino acid 61. A bacterial expression vector was constructed to synthesize H-ras transforming protein that contains this change (rasLeu61). In immunoblotting experiments, the affinity purified mAb, ras(53-69)Leu61, reacts specifically with the purified, bacterially produced rasLeu61 protein and does not react with bacterially produced normal H-ras protein. In immunoblotting experiments with cell lysates, the mAb recognizes the transforming protein in NIH3T3 cells transformed by the c-rasHLeu61 oncogene but fails to react with normal H-ras protein in the same cells or cells which produce 100 times more normal protein than NIH3T3. The mAb immunoprecipitates [35S]methionine-labeled H- and N-rasLeu61 proteins from transformed NIH3T3 cells under conditions in which the cells produce basal levels of the transforming protein, equivalent to the low amount of the normal protein ordinarily present in nontransformed NIH3T3 cells. The antibody fails to immunoprecipitate normal H-ras protein, even when present at high levels, or N-ras protein containing Lys as amino acid 61. Affinity purified mAb ras(53-69)Leu61 also recognizes the transforming ras protein specifically in immunohistochemical staining of tissue culture cells, and this staining is abolished by preincubating the antibody with the corresponding peptide. Staining was not observed with control NIH3T3 cells or cells that produce 100 times more normal H-ras protein than NIH3T3. However, in thin sections of normal human or rabbit skin the antibody reacted strongly with an unknown antigen, in cells of the basal layer of the epidermis, that is neither normal nor transforming ras protein. This new immunological reagent should be useful for the selective identification of Leu61 containing H-, K-, and N-ras transforming proteins in in vitro studies and analyses using rodent, rabbit, or human tissue culture cells. Its utility for direct staining of tissues may be limited to situations in which the presence of transforming protein can be verified by another method such as immunoblotting after gel electrophoresis.
Bizub
D
D
Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, PA 19111.
Fischberg-Bender
E
E
Heimer
E P
EP
Felix
A
A
Skalka
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D005260:Female; D011905:Genes, ras; D005822:Genetic Vectors; D005973:Glutamine; D015151:Immunoblotting; D007930:Leucine; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000032:analysis / Q000235:genetics*
D000911:Antibodies, Monoclonal; D005973:Glutamine; D015689:Oncogene Protein p21(ras); D007930:Leucine