| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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A point-mutation changing aspartate-233 to valine was introduced into mouse ODC cDNA and ligated to the pMSG expression vector under MMTV-LTR promotor. The effects of the mutation on the enzymatic activity and catalytic properties of ODC were studied by establishing C55.7 cell lines expressing either wild-type or mutated ODC. Colonies transfected with the pODC1.7 plasmid encoding wild-type ODC grew faster and were larger than those transfected with pODC1.7(D233V). Twenty-two of pODC1.7 transfectants and eight of pODC1.7(D233V) transfectants were assayed for ODC activity. Clone K2 of pODC1.7 and clone B7 of pODC1.7(D233V) transfectants were selected for further studies. The integration of the transfected constructs into genomes of K2 and B7 cells was confirmed both by the polymerase chain reaction and Southern analysis. K2 and B7 clones expressed ODC at relatively high level, but were still typical representative of their groups. ODC activity in B7 cells was 4.6-fold lower compared to that in K2 cells, although the level of ODC mRNA was 2.3-fold higher in the B7 cells. The D233Vmutation resulted in an increase in the Km values for the substrate L-ornithine and the cofactor PLP by about 20-fold. Similarly, the mutated enzyme had 15-fold higher Ki value for DFMO.