Polyamines are an essential component of all forms of life, and ornithine decarboxylase (ODC) is a key enzyme in their synthesis. The ubiquity of this enzyme, the rapidity and complexity of its regulation in response to multiple stimuli that later cell growth of differentiation and the essentiality of polyamines, demonstrable by pharmacological and genetic means, have led to the inference that regulation of ODC activity is both complex and worthy of serious consideration by biologists. In recent years biochemical, genetic, pharmacological and molecular biological studies have indicated that regulation of ODC expression depends on the level of the mRNA, on the efficiency of translation of the mRNA and on the stability of the enzyme. The following Aims are proposed: With respect to the gene: We will define the elements of the ODC gene necessary for transcriptional regulation by transfecting cultured cells with recombinant forms of the ODC gene and with recombinant constructs containing altered and fragmented parts of the gene. A recombinant construct containing ODC promoter-leader-bacterial beta galactosidase reporter will be transferred into cultured cells and will be used to form transgenic mice. Histochemical staining will be used to monitor regulation of expression mediated by transcription and by 5' untranslated leader sequences. To distinguish enough-is-enough from critical control models of ODC expression, transgenic mice will be created expressing ODC genes disabled for all known regulatory modes, in an effort to assure constitutive, high-level expression. If such global expression is incompatible with viability, the experiment will be performed with a tissue-specific promoter. With respect to protein: The structural determinants of intracellular ODC stability will be determined by mutational analysis. Progressive carboxy-terminal deletions will be carried out to establish the minimum degree of truncation required to confer stability. The carboxy-terminus of ODC will be appended to proteins that are stable to determine whether its presence confers lability. The stability and regulatory properties of ODC's cloned from lower eukaryotes will be determined. Methods will be developed to identify intermediates of degradation. An in vitro system for studying the biochemical mechanisms of intracellular ODC degradation will be established and utilized.
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