Understanding the molecular events which underlie megakaryocyte (MK) formation has lagged behind that of erythroid and myeloid cell development. With the recent cloning of thrombopoietin (TPO), and demonstration of its critical role in MK and platelet production, we have the opportunity to gain fundamental new insights into this intriguing process. The long term goal of this research proposal is to identify the molecular mechanisms responsible for MK proliferation and differentiation. To this end we propose a research plan of three specific aims. First, we will study the structural features of TPO responsible for its biological activity. In addition to our previous methods used to study GM-CSF, IL-3 and SCF, we will employ a novel """"""""back mutant"""""""" screening procedure to rapidly detect sites on the molecule critical for receptor interaction. Second, we will generate and screen two cDNA libraries in an attempt to identify genes responsible for developmental progression of MK progenitor cells. Here, too, we will employ newly developed methods to purify MK and their progenitors, and then to screen sense and antisense libraries by both gain- of-function and loss-of-function strategies. Finally, studies of the third specific aim will investigate the genetic basis for polyploidy, the most remarkable and least understood feature of hematopoietic development. The availability of purified cell cycle-synchronized normal marrow MK will allow us to investigate several hypotheses of the physiologic basis for polyploidy, the uncoupling of DNA synthesis and mitosis. A better understanding of the molecular basis for these three processes, growth factor binding to receptor, the genetic mechanism(s) of lineage development, and the biochemical basis of polyploidy will advance our understanding of normal thrombopoiesis, and has important implications for both the therapeutic use of TPO and for the normal physiology of cell division and differentiation.
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