Evolutionary relationship of the members of the sulphur-rich hordein family revealed by common antigenic determinants

Abstract

Five monoclonal antibodies raised against an enriched C hordein fraction have been characterized in detail and were found to be specific for the members of the sulphur-rich hordein family. Two antibodies specific for B hordein polypeptides were identified, one of which reacted predominantly with CNBr cleavage class III polypeptides. γ1 hordein was recognized by two antibodies, of which one also reacted with γ2 hordein and several members of the CNBr cleavage class II B hordein polypeptides. One antibody recognized γ3 hordein but cross-reacted at higher antibody concentration with almost all of the B and C hordein polypeptides. The specificity of the monoclonal antibodies was confirmed by Western blotting of one- or two-dimensionally separated hordein from the B hordein-deficient mutant hor2ca and its wild-type Carlsberg II and the γ3 hordein-deficient genotype Nevsky. The identification of the γ hordein-specific monoclonal antibodies was further supported by immune precipitation of in-vitro transcribed and translated γ2 hordein, and hor2ca and Carlsberg II mRNA translation products. The monoclonal antibodies were used to screen for mutants in γ hordein synthesis. Two mutants, one deficient in γ 1 hordein synthesis and a second in γ 2 or closely related B hordein polypeptides were identified. A model is proposed for the evolution of the sulphur-rich hordein loci Hor5 and Hor2.