The long-term objectives of this research are the elucidation of the regulation of hemoglobin synthesis in normal and abnormal hematologic conditions; emphasis is placed on the translational control of the synthesis of globin and other proteins in erythroid cells by heme and eIF- 2alpha kinases and on the relationship of this control to erythroid cell development and maturation.
The specific aims i nclude elucidation of: the structure-function relations of heme-regulated and double-stranded RNA activated eIF-2alpha kinase in the human hematologic disorders characterized by heme-deficiency. This research is relevant to eukaryotic cells generally and specifically it is significant for understanding the human anemias in which heme-deficiency occurs: iron-lack anemia, one of the most prevalent world-wide disorders, the anemia of lead poisoning, and the sideroblastic anemias. The experimental design and methods involve: the study of protein synthesis in situ in rabbit reticulocytes and their lysates and in vitro in components of these cells; the purification of the heme-regulated eIF-2alpha kinase; the study of the effects of heme and other metalloporphyrins on the eIF-2alpha kinase; and the cloning of the cDNAs of the eIF-2alpha kinases, the determination of their amino acid sequences and their ATP binding sites; the preparation of polyclonal and monoclonal antibodies to the eIF-2alpha kinases and the determination by immunofluorescence of the presence of the eIF-2alpha kinases in erythroid cells at different stages of development in normal man and in the heme- deficiency clinical disorders.
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