The overall objective of this project is to elucidate the regulation and genetics of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in the pathway of polyamine biosynthesis. We have selected mouse cells that overproduce the enzyme as a result of gene amplification, and we have used the overproducing cells to generate ODC cDNA clones. We now propose the following experiments. ODC activity can be made to change markedly in response to hormones, polyamines, tumor promotors, oncogenic transformation, and changes in cell growth state. We will determine the molecular mechanisms of that regulation. mRNA levels will be determined and the contribution of transcription rate and mRNA turnover to those levels measured. Protein stability and regulable alterations of that stability will be examined. Post-translational modifications that affect enzyme activity will be examined. Regulation of endogenous ODC will be studied in wild-type and mutant S49 mouse lymphoma cells (response to polyamines, cyclic AMP analogues), PC12 rat pheochromocytoma cells (response to cyclic AMP analogues and nerve growth factor), and mouse 3T3 fibroblasts (response to tumor promotors and epidermal growth factor). Using recombinant DNA techniques, expression vectors containing the ODC gene or portions of that gene will be constructed, recipient cells transformed, and expression of those constructs assessed. ODC in mouse constitutes a multi-gene family. We will determine whether these genes are clustered, establish a chromosomal assignment for the active gene or genes and find out whether pseudo-genes exist. Somatic mutants that are altered in ODC regulation will be generated. S49 cell mutants that overproduce ODC will be further charcterized genetically. Revertants of those mutants will be selected in an effort to obtain suppressor regulatory mutations. Mutants that are deficient for ODC expression will be sought. (B)
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