MOLECULAR CHARACTERIZATION OF GLYCOPHORIN-A TRANSCRIPTS IN HUMAN ERYTHROID-CELLS USING RT-PCR, ALLELE-SPECIFIC RESTRICTION, AND SEQUENCING

Glycophorin A (GPA) is an erythroid-lineage-specific membrane sialogly coprotein which occurs in two allelic forms, M and N, which form the a ntigens of the MN blood group. Purified cDNAs and RNAs isolated from p eripheral blood and erythroleukemia cell lines, HEL and K562, were use d to develop an RT-PCR technique for amplifying GPA gene transcripts ( GYPA). The relative expression of transcripts from the M and N alleles was determined using restriction analysis of these amplified products with four allele-specific restriction endonucleases. The use of this method permits the sensitive identification of GYPA transcripts in the se cells and confirms GPA protein expression in the erythroleukemia ce ll lines and the MN phenotypes of individuals determined by immunolabe ling with GPA allele-specific monoclonal antibodies. A novel restricti on pattern was obtained using peripheral blood RNA from two individual s with a rare inherited variant allele, GPA M(g). Sequencing of the cD NA obtained using this method revealed a single C to A transversion in the fourth codon in the mature GYPA N coding sequence is responsible for the difference between GYPA M(g) and GYPA N.