The goal of the proposed research is determination of the role of diadenosine 5 feet, 5 '''-P4-tetraphosphate (Ap4A) in the cell cycle and differentiation of the acellular slime mold, Physarum polycephalum. Intracellular levels of Ap4A have been previously shown to correlate with the degree of cellular proliferation, and Ap4A stimulates DNA replication in quiescent culture cells. it has been previously proposed that AP4A is a regulatory nucleotide. Ap4A may modulate traverse of the cell cycle and transitions from undifferentiated, vegetative plasmodia to differentiate states of spherules and spores in the diploid phase of Physarum. Cellular levels of Ap4A will be measured in microplasmodia, in macroplasmodia during the synchronous cell cycle, and during differentiation to spores and spherules. Ap4A will be quantiated byhplc after partial purification of cell extracts. The enzymology of Ap4A synthesis and degradation will be studied. Diadenosine tetraphosphate pyrophosphohydrolase will be purified and kinetically characterized, especially with regard to activators and inhibitors. The possible existence of a specific anabolic enzyme for the synthesis of Ap4A will be examined. Specific tRNA synthetases which catalyze the synthesis of Ap4A will be determined. The enzymic activities will be assayed in the same cell populations in which levels of Ap4A are measured to determine the enzymologic basis for changes in AKP4A level. The intracellular levels of Ap4A will be manipulated by use of enzymes, antibodies, and modifiers which will be added to the media or microinjected. Perturbation of proliferation, non-terminal differentiation, or irreversible differentiation will establish a functional role for Ap4A. Changes in DNA replication, measured biochemically and autoradiographically, in response to altered levels of Ap4A will be measured to determine if Ap4A has a role in DNA replication. The limits of our understanding of the process of disease are set by our knowledge of the underlying normal processes. The proposed research should help elucidate one component in the mechanism of normal growth and differentiation. Eventually, a full understanding of these normal processes will help in elucidating the mechanism of transformation and abnormal cell growth.
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