Isolation of cloned cDNA encoding mammalian ornithine decarboxylase.

Abstract

During the growth of mammalian cells, the level of ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase), the 1st enzyme in polyamine biosynthesis, undergoes rapid changes. As an initial step in the study of possible genetic mechanisms involved in these changes, c[complementary]DNA clones encoding OrnDCase were isolated. To obtain RNA enriched for OrnDCase messenger, mouse myeloma cells that overproduce OrnDCase were selected in the presence of the OrnDCase inhibitor, difluoromethylornithine. A pBR322 cDNA library was prepared from poly(A)+ RNA isolated from difluoromethylornithine-resistant cells, and the library was probed with [32P]cDNA representing mRNA sequences from resistant or parental (sensitive) cells. All clones hybridizing preferentially to the resistant cell probe shared nucleotide sequences. A representative clone containing 1.1 kilobases of cDNA was shown to encode OrnDCase sequences by in vitro translation of hybrid-selected mRNA followed by precipitation of hybrid-selected mRNA followed by precipitation of the translation products with anti-OrnDCase antiserum. Using this cDNA clone as a probe, mouse DNA yielded several restriction fragments that react with the OrnDCase cDNA. In the difluoromethylornithine-resistant myeloma cells, one of these DNA segments is amplified, and the level of OrnDCase mRNA is greatly increased compared with that in parental plasmacytoma cells. The level of OrnDCase mRNA is also increased in cultured 3T3 [mouse fibroblast] cells stimulated with serum and in mouse kidneys after administration of androgen, indicating that OrnDCase gene transcription and/or mRNA stability are regulated during cell growth.