The ultimate objective is the elucidation of the molecular mechanism by which avian myeloblastosis virus (AMV) causes acute myeloblastic leukemia in chickens. An understanding of the mechanism of viral leukemogenesis in this experimental system should provode clues and guidelines for investigation of the etiology, pathology and therapy of human leukemia. Our working hypothesis is that the putative product of a chicken cellular sequence (luk gene?) inserted into the AMV genome is responsible for this chicken leukemia. We propose to identify and to characterize the nature of the AMV luk product and its function in leukemic cells. For this purpose we shall prepare antibodies against the leukemogenic molecule presumed to be either a protein or a modified protein, e.g., glycoprotein. The antibodies will be elicited against chemically synthesized pentadecapeptides corresponding to the C (or N) terminus of the putative luk protein deduced from its nucleotide sequence. The antibodies will be used to immunoprecipitate the luk product syntesized by cell-free translation of luk specific viral RNA or m-RNA, and to compare it with the molecules immunoprecipitated from leukemic cells. Attempts will also be made to synthesize the luk product in mammalian cells or bacteria by transfection or infection with the luk DNA sequenced cloned in plasmids or in viral vectors. To help dientify and characterize the cellular functions and target molecules affected by the luk product we will introduce a temperature-sensitive lesion in the cloned leukemogenic sequence by in vitro mutagenesis folowed by transfection and rescue with helper virus. We will also identify and characterize the nature and function of the normal chicken DNA sequences which are homologous to the AMV luk sequence and from which the latter is presumed to have originated. Comparison of the normal cellular product with the luk product should help to elucidate the leukemogenic process. The methodology will include transfection, DNA sequencing, DNA recombination of specific viral DNA fragments generated by restriction enzymes, cloning in plasmid or viral vectors, hybrid selection of RNA, in vitro protein synthesis with viral or messenger RNA, in vitro mutagenesis of DNA fragments, electron mciroscopic analysis of DNA heteroduplexes and DNA-RNA loops, and chemical analyses of proteins and glycoproteins.
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