The mechanisms controlling the differential accumulation of human Hb A (alpha 2 beta 2) and Hb A2 (alpha 2 delta 2) during normal erythropoiesis (Hb A/Hb A2 approximately equal to 40/1) will be investigated in intact erythroid cells and also in model in vitro systems. Cloned cellular DNA fragments containing the human delta and beta globin genes and their surrounding DNA sequences will provide probes for the establishment of highly specific molecular hybridization assays to detect sequences complementary to the mRNA-coding regions and intervening sequences of each gene, as well as to intergenic sequences. The relative content of delta and beta globin mRNA in the steady state will be analyzed in nuclear and cytoplasmic RNAs from fractionated early and late erythroid cells, and in labeled RNA synthesized either in intact erythroid precursor cells (in short term primary bone marrow cultures or in longer term plasma clot cultures of stem cells) or in cell-free (wheat germ) or heterologous intact cell transcription systems programmed with the cloned human DNA fragments. Identification and kinetic analysis of mRNA precursor, of transcripts of intervening and flanking sequences, and of mature mRNAs will be accomplished by molecular hybridization using the various globin DNA probes. In addition, nuclei from human erythroid cells will be subjected to limited digestion with pancreatic DNase in order to compare the transcriptional configuration of the chromatin that contains each individual delta and beta globin gene subregion. Finally, the potential for enhanced expression of delta globin genes will be studied by similar hybridization techniques in cultured human-mouse erythroleukemic somatic cell hybrids known to contain transcriptionally active human chromosome no. 11, after exposure to agents known to alter the patterns of globin synthesis in cultured parent mouse erythroleukemia cells. Knowledge of the mechanisms that control the normal differential expression of human delta and beta globin genes may provide insight into therapeutic approaches to increase delta globin gene expression as a means to control the deleterious manifestations of inherited hemoglobinopathies affecting the beta chain such as sickle cell anemia and beta-thalassemia.
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