Cattle were divided into three groups according to the red cell-protein 4.2 (P4.2) phenotypes P4.276, P4.275 and P4.276/75, whose red cells contained Mr 76000 (P4.2/76), 75000 (P4.2/75) and both 76000 and 75000 isoforms respectively. To elucidate the molecular basis that underlies the diversity of P4.2, the gene structures of bovine P4.2/76 and P4.2/75 were investigated. Two P4.2 cDNA clones were isolated from bone-marrow cDNAs of the animal with the P4.276/75 phenotype. These were identical in size (2.2 kb), encoding major erythroid P4.2 with 687 amino acids, but were different in three nucleotides, resulting in changes of amino acids at the 599th, 601st and 627th residues. Analysis of genomic DNA from the three phenotypes demonstrated that these two clones were derived from gene transcripts by which P4.2/76 and/or P4.2/75 were produced. In vitrotranscription and translation of P4.2/76 and P4.2/75 cDNAs indeed generated P4.2/76 and P4.2/75 identical in size to the red-cell proteins. These findings demonstrated that polymorphism of the P4.2 gene at codons 599, 601 and 627 of P4.2 cDNA was the cause of the molecular diversity of bovine red-cell P4.2. Although distinct electrophoretic mobilities suggested a structural difference in the two isoforms, this polymorphism appeared to have little effect at least on P4.2 association with band 3, since no significant difference was observed in the amount of P4.2 relative to total membrane proteins despite the phenotype difference for P4.2.
The nucleotide sequences reported in this paper have been deposited in GenBank under the accession numbers AF030029 and AF030030.