• Suppression of GATA-1 gene expression by a hammerhead ribozyme reduces hemoglobin synthesis in HEL cells

      Hu, Huaiyu; Department of Biochemistry and Biology (Augusta University, 1992-12)
      GATA-1 is a major erythroid trans-acting factor. To determine if GATA-1 is required for globin gene expression in HEL cells, we have used an antisense RNNribozyme strategy to suppress the expression of the gata-1. DNA coding for a hammerhead ribozyme with flanking sequences antisense to the GATA-1 mRNA was cloned into an eukaryotic expression vector pMAMneo-luc at a position within the 3'-untranslated region of the firefly luciferase gene. Following stable transfection into HEL cells, both luciferase assays and RNA-PCR were used to show that the luciferase mRNA is present. HEL cells possessing the antisense RNNribozyme showed morphological changes that may resemble macrophages. Latex uptake experiments showed that these cells indeed acquired more phagocytic activities than the_ controls suggesting that they resemble a macrophage phenotype. Northern blot analyses show a 50% reduction of GATA-1 mRNA in the HEL cells possessing the antisense RNNribozyme. Following induction with o-ALA, isoelectric focusing gel data showed a 50-90% reduction of fetal and embryonic hemoglobins in these cells. Cell growth was unaffected. Northern blot analyses also showed that the level of the chimeric luciferase mRNA was lower than that required for a stoichiometric antisense effect. Therefore, the hammerhead ribozyme may be acting catalytically. Our conclusion is that GATA-1 is requi_red for hemoglobin gene expression in HEL cells.