STRUCTURE, ORGANIZATION AND EXPRESSION OF THE MOUSE ORNITHINE DECARBOXYLASE ANTIZYME GENE

Ornithine decarboxylase antizyme is a protein that participates in the regulation of cellular polyamine levels. In this study we have isolat ed and sequenced the mouse gene encoding antizyme protein. Transfectio n of various cell lines with a 5.5 kb genomic fragment containing the antizyme locus resulted in the production of a 29 kDa antizyme protein , confirming that this locus contained a functional gene. Comparison o f the mouse gene with the corresponding rat gene [Miyazaki, Matsufuji and Hayashi, (1992) Gene 113, 191-197] revealed an identical exon/intr on organization and high level of nucleotide sequence conservation tha t was 89% for the entire transcription unit. Protein-coding regions of the two genes exhibited 97% nucleotide sequence identity and there we re only four amino acid differences between the 227-residue antizyme p rotein sequences of the mouse and rat. The promoter of the antizyme ge ne was functional in mouse (N2A and NIH/3T3) and hamster (CHO) cell li nes. The presence of 0.l mM spermidine in culture medium increased the amount of immunoreactive antizyme protein in cells transfected with t he antizyme gene or antizyme cDNA, possibly owing to facilitated frame shifting in the translation of antizyme mRNA. Recombinant antizyme pro tein was also produced in Escherichia coli and used to raise specific polyclonal antibodies in rabbits and to devise immunological methods f or the measurement of antizyme concentration.