RH(D) ANTIGEN EXPRESSION AND ISOLATION OF A NEW RH(D) CDNA ISOFORM INHUMAN ERYTHROLEUKEMIC K562 CELLS

Human erythroleukemic K562 cells are known to have several erythroid p roperties. K562 cells possess Rh mRNAs, but expression of Rh proteins has not previously been reported. We immunoprecipitated Rh protein fro m K562 cell lysate using rabbit anti-Rh and detected Rh(D) antigens on K562 cells using fluorescence-activated cell sorting (FACS). These re sults suggest that K562 cells will be useful as an expression model fo r most Rh antigens. We also cloned a new Rh(D) cDNA isoform (RhK562-II ), from a K562 cDNA library using polymerase chain reaction (PCR) with 5' and 3' end oligonucleotides of the published Rh(e/E) antigen encod ing cDNA sequence as primers. Sequence analysis showed that RhK562-II is composed of 951 nucleotides (316 amino acids), identical to the fir st 939 nucleotides (exons 1 to 6) of one of the Rh(D) cDNAs (RhXIII), except for nucleotide 654 (C --> G exchange). However, this exchange i s the same as that of another published Rh(D) cDNA (RhPII cDNA). RhK56 2-II is deprived of exons 7 and 8 (nucleotides 940 to 1,153), followed by an identical sequence up to the 3' end of the open-reading frame o f the RhXIII cDNA, which causes a frameshift mutation and produces a p remature stop codon. In vitro expression of RhK562-II using the transc ription and translation rabbit reticulocyte lysate system produced two major Rh-related proteins (30 kD and 25 kD), which were immunoprecipi tated by rabbit polyclonal anti-Rh and separated on sodium dodecyl sul fate-polyacrylamide gel electrophoresis (SDS-PAGE). (C) 1994 by The Am erican Society of Hematology.