USE OF A MARKED ERYTHROPOIETIN GENE FOR INVESTIGATION OF ITS CIS-ACTING ELEMENTS

To examine the function of conserved noncoding regions in the erythrop oietin (Epo) gene, we have prepared clones and pools of Hep3B cells st ably transfected with a marked 4.1-kilobase Epo gene and deletions the reof, The marked transcripts had single base substitutions at three si tes in the coding portion of Exon 5, enabling them to be distinguished from endogenous Epo mRNA by ribonuclease protection and competitive p olymerase chain reaction. The basal expression and hypoxic induction o f the marked Epo gene that had no deletions were indistinguishable fro m that of the endogenous Epo gene. Likewise, deletion of conserved int ervening sequence 1 had minimal effect on hypoxic induction, In contra st, a 3'-deletion that included the conserved S'-enhancer element resu lted in a substantial, but not complete, suppression of hypoxic induct ion while a 3' deletion downstream of the enhancer resulted in enhance ment, A 188-base pair deletion of a conserved 3'-untranslated region i n Exon 5 had minimal effect on hypoxic induction, However, the truncat ed Epo mRNA had a markedly prolonged half life (15 h) in comparison to the endogenous Epo mRNA (2.0 h) or the marked full-length Epo mRNA (2 .1 h), Further deletions in the 3'-UTR showed that a relatively small region of approximately 50 bases is responsible for the relatively rap id turnover of Epo mRNA. These experiments provide information on cis- acting elements of the Epo gene that cannot be obtained from conventio nal reporter gene transfection experiments.