To study the mechanism by which the kidney hormone, erythropoietin, regulates normal and abnormal erythropoiesis, we have isolated and sequenced the genomic and cDNA genes for the human erythropoietin receptor (ER). The transcription initiation site was determined to be at approximately -134 bases upstream of the first codon. Based on the sequence information obtained from the genomic clone, primers were designed which allowed the construction by polymerase chain reaction (PCR) techniques of a human ER cDNA clone containing from -10 bases upstream of the translation start site to 27 bases past the translation stop site. With the possession of the cDNA clone which allowed the construction of a competitive DNA template, we were able to develop a PCR assay for the quantitation of the human ER in both tissue culture cells and peripheral blood samples. The number of ER transcripts per nucleated cell from the peripheral blood of two normal individuals was determined to be on the order of magnitude of 10-3 copies per cell. We believe that expression of the ER gene is indicative of an erythroid progenitor cell and that the small number of ER transcripts observed in these samples is consistent with the expectation that only a very minor population of erythropoietin responsive progenitor cells exists in the peripheral blood of normal individuals. The cloning and sequencing of the genomic and cDNA ER genes has allowed the development of a method for quantitation of low levels of ER mRNA from small amounts of starting material. This approach is being tested as a new method for detection and monitoring of erythroid progenitors in patients with sickle cell anemia who are under erythropoietin and hydroxyurea therapy. In addition, transcriptional regulatory sequences and receptor function are being studied by deletional and site directed mutagenesis.
