A study of the coregulation and tissue specificity of XG and MIC2 gene expression in eukaryotic cells

CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantita tive polymorphism coregulated with the polymorphism of the XG blood group g ene. As a preliminary study of this phenomenon, human XG and CD99 recombina nt proteins were expressed in murine RAG cells and analyzed by flow cytomet ry, Both proteins were expressed independently and at a similar level in si ngle and double transfectants. Immunoprecipitation and Western blot analysi s, using the murine monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd (CD99), respectively. A putative 28-kd intracellula r precursor of CD99 was also detected, as was a 26-kd species after neurami nidase treatment of CD99-expressing cells. No evidence of association or co mplex formation between XG and CD99 proteins could be proven, either on tra nsfected RAG cells or on human erythrocytes, These results were confirmed u sing somatic hybrids between single transfectants, These findings suggest t hat the phenotypic relationship between XG and CD99 is mostly regulated at the transcriptional level, but they do not formally exclude some posttransc riptional effect. Studies on the tissue specificity of XG expression showed that surface expression of the XG protein could not be restored in somatic hybrids between B-lymphoblastoid cell lines from Xg(a +) persons and fibro blasts (RAG) or erythroid (MEL) cells. RT-PCR analysis of the transcripts r evealed the existence of an XG mRNA in each cell line, suggesting that the tissue-specific regulation of cell surface XG expression occurs either at a quantitative transcriptional level or is a posttranscriptional event. By N orthern blot analysis, XG transcripts were detected in erythroid tissues an d several nonerythroid tissues. (C) 2000 by The American Society of Hematol ogy.