The application is directed towards understanding the process of endomitosis, which is a hallmark of megakaryocytopoiesis and occurs also in other cells such as embryonic cells and vascular smooth muscle cells exposed to hypertension. The investigator has previously found that megakaryocytes do not continuously synthesize DNA but rather undergo a cell cycle consisting of Gap/S phases. She also demonstrated by antisense experiments that the G1 phase cyclin, cyclin D3, is essential for megakaryopoiesis in vitro. She has also cloned from megakaryocytes a CDNA for a protein which has high sequence homology to the family of Never in Mitosis (NIMA) kinases, which were originally identified by their ability to rescue mutant fungal cells arrested at entry to mitosis, and undergo repeated rounds of DNA synthesis. She has called this protein MegNIMA-like. She has data showing that both cyclin D3 and MegNIMA-like are regulated by thrombopoietin. The effect on CD3 is transcriptional. The goal of this proposal is to further the understanding of the regulation of these two genes in megakaryocytopoiesis.
The specific aims are: 1) To isolate the full-length MegNIMA-like CDNA and express and characterize the protein it encodes.2) To characterize CD3 genomic regulatory elements responsible for gene activation in megakaryocytes as well as the TPO-responsive DNA elements. 3) To determine the roles of MegNIMA-like, NIMA kinases and CD3 in megakaryocytopoiesis and thrombocytopoiesis in cultured cells and in vivo. The in vitro studies will be done with both rat megakaryocytes and with cell lines.
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