Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants.
HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.
D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics; D005805:Genes, MHC Class I; D006680:HLA Antigens / Q000235:genetics* / Q000276:immunology; D015789:HLA-A2 Antigen; D015791:HLA-A3 Antigen; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D011995:Recombination, Genetic
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006680:HLA Antigens; D015789:HLA-A2 Antigen; D015791:HLA-A3 Antigen