In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships.
Oligonucleotide-directed, site-specific mutagenesis has been employed to elucidate the role of individual amino acids on the expression and function of a MHC class I antigen. Two oligonucleotides were synthesized to introduce single amino acid substitutions in the murine H-2Ld gene. The highly conserved glycosylation site at amino acid position 86 was changed from asparagine to lysine to remove the carbohydrate moiety from the first external domain of the H-2 molecule, and the phenylalanine at position 116 was changed to tyrosine, replacing the Ld residue with the Kb type amino acid analogous to Kb mutants: bm5 and bm16 mutants derived from the Kb antigen have the Ld-type residue at this position. The mutant genes were constructed by annealing the mutagenic oligomers to the single stranded H-2Ld gene, followed by chain elongation reaction. The expected mutations were confirmed by DNA sequence determination. The mutant genes were introduced into mouse L cells by DNA-mediated gene transfer. Both mutant genes expressed the antigens on the cell surface, as detected by antibody binding; these antigens were reactive with the cytotoxic T cells specific for the H-2Ld antigen. Detailed examination with 16 monoclonal anti-H-2Ld antibodies revealed that the binding of some antibodies was significantly reduced in the glycosylation mutant, implying a certain contribution of the carbohydrates to the antigenic activity of some determinants. No detectable changes have been observed in the mutant of the substitution at position 116 by the parameters we tested.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D005434:Flow Cytometry; D005796:Genes; D005822:Genetic Vectors; D006183:H-2 Antigens / Q000235:genetics*; D007739:L Cells / Q000201:enzymology; D008285:Major Histocompatibility Complex; D051379:Mice; D009154:Mutation; D013329:Structure-Activity Relationship; D013937:Thymidine Kinase / Q000235:genetics
D006183:H-2 Antigens; D013937:Thymidine Kinase