On the Regulation of Ornithine Decarboxylase

The polyamines putrescine, spermidine and spermine have been shown to be involved in cell growth and differentiation, and are considered to be crucial components of the living cell. The synthesis of polyamines in the cell is strictly regulated. The first and rate-limiting step in the biosynthesis of polyamines is catalysed by the enzyme ornithine decarboxylase (ODC). Mammalian ODC has a very fast turnover, and is highly regulated at several levels, including translational mechanisms. Translational control of ODC has been shown to be important in the polyamine-mediated feedback regulation, as well as in the hypotonic induction of the enzyme. This control has been suggested to be dependent on the very long 5' and 3' untranslated regions of ODC mRNA. In the present thesis, the importance of these mRNA regions in the translational regulation of ODC exerted by polyamines or occurring after hypotonic shock were studied. A series of ODC cDNA constructs with various truncations in the 5' UTR, coding region and 3' UTR were used. These constructs were expressed in different cellular expression systems, and the effects of polyamines and hypotonicity, respectively, were analysed. The results showed that the polyamine-mediated regulation of ODC synthesis was neither affected by the 5' UTR nor the 3' UTR of the ODC mRNA, but instead appeared to be dependent on the ODC mRNA sequence coding for the C-terminal degradation domain of the enzyme. The hypotonic induction of ODC, on the other hand, was highly dependent on the 3' UTR, but not on the 5' UTR, which demonstrates the first more specific role for the 3' UTR in the regulation of ODC. This also indicates that the mechanisms by which hypotonicity and polyamines affect the translation of ODC mRNA are unrelated. In order to gain more knowledge about the mechanisms involved in the regulation of ODC, the ODC gene from the trypanosomal parasite Crithidia fasciculata, which is not subjected to polyamine-mediated feedback regulation of ODC, was cloned and characterised. C. fasciculata ODC was found to lack the sequence corresponding to the degradation domain of mammalian ODC, although it is a metabolically unstable protein even when expressed in mammalian cells, suggesting that C. fasciculata ODC contains other signals important for the rapid degradation of the enzyme