Specific protein binding to a conserved region of the ornithine decarboxylase mRNA 5'-untranslated region.

An RNA gel retardation assay was used to identify one or more cellular protein(s) (ornithine decarboxylase mRNA 5'-UTR binding protein (ODCBP)) that bind specifically to a conserved region of the 5'-untranslated region (5'-UTR) of rat ornithine decarboxylase (ODC) mRNA. Ultraviolet light cross-linking demonstrated that this protein has an apparent Mr = 58,000 in mammalian cells. Treatment with the oxidizing agent diamide prevented binding of the ODCBP to ODC mRNA; addition of beta-mercaptoethanol reversed this inhibition and permitted mRNA.ODCBP complex formation. Cytoplasmic extracts from a variety of animal cells and tissues demonstrated similar binding activities; however, there was marked tissue-specific expression of the protein in the rat, with brain, heart, lung, and testis containing large amounts, and kidney, spleen, and skeletal muscle expressing negligible amounts. Binding was completely prevented by several mutations within a highly conserved heptanucleotide region (CCAU/ACUC) that was within 61 bases of the initiation codon in ODC mRNAs from mammals, Xenopus, and Caenorhabditis elegans; mutations 5' and 3' of the conserved heptanucleotide domain had no effect on binding activity. Binding was not affected by manipulation of cellular polyamine levels or by treatment of cells with agents that stimulate ODC biosynthesis. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 5'-UTR of ODC mRNA from many animal species; functional consequences of this binding remain to be determined.

Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure and phosphorylation of eucaryotic initiation factors eIF-4B and eIF-4E.

We investigated the possibility that insulin could stimulate translation of ornithine decarboxylase (ODC) mRNA in a murine fibroblast cell line that expresses large numbers of human insulin receptors (HIR 3.5 cells). Within 3 h after exposure to 70 nM insulin, ODC enzyme activity increased approximately 50-fold and mRNA accumulation 3-fold in the HIR 3.5 cells but not in normal fibroblasts. Pretreatment of cells with cycloheximide completely inhibited insulin-stimulated ODC expression; actinomycin D partially inhibited this effect. To determine the influence of the 5' untranslated region (5'UTR) of ODC mRNA on insulin-regulated ODC expression, plasmids were constructed which contained sequences from the 5'UTR of a rat ODC mRNA interposed between the ferritin promoter and the coding region of the human growth hormone gene. These constructions were then expressed transiently in HIR 3.5 cells. Insulin stimulated a 2-4-fold change in growth hormone accumulation in the medium of cells transiently expressing plasmids containing the entire 5'UTR of ODC mRNA or just the 5'-most 115 bases, a G/C-rich conserved sequence predicted to form a stem-loop structure and shown previously to be responsible for constitutive inhibition of translation. There was a direct correlation between the extent of insulin stimulation and the predicted secondary structure of the added 5'UTR sequences. To determine whether this effect might be due to insulin activation of initiation factors responsible for melting mRNA secondary structure, we examined the effect of insulin on the phosphorylation states of two such factors, eucaryotic initiation factors eIF-4B and eIF-4E. Insulin stimulated the phosphorylation of both initiation factors; this stimulation was evident at 15 min and maximal by 60 min. These results suggest a potential general mechanism by which insulin could preferentially stimulate translation of mRNAs whose 5'UTRs exhibit significant secondary structure by activating initiation factors involved in melting such secondary structures.

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells.

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.

Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a highly inducible protein whose expression involves a complex and variable array of regulatory mechanisms. We investigated the influence of the 5'-untranslated region (5'UTR) of the rat ODC mRNA on translation of the mRNA in a cell-free system and in cultured mammalian cells. ODC mRNA containing the full-length 5'UTR was translated in reticulocyte lysates at approximately 5% of the rate of mRNA containing no ODC 5' leader sequences. The complete 5'UTR inhibited expression of a heterologous gene product, human growth hormone, to the same extent in cultured mammalian cells. Furthermore, the 5'-most 130 bases of the rat ODC 5'UTR, a conserved G/C-rich region predicted to form a stable stem-loop structure (delta G = -68 kcal/mol), repressed translation to the same extent as the entire 5'UTR, both in the lysates and in intact cells. The 3'-most 160 bases of the 5'UTR, containing a small upstream open reading frame, decreased expression by 50-65% both in vitro and in intact cells, compared with controls lacking any ODC 5'UTR sequences. Mutation of the initiation codon AUG beginning this upstream open reading frame to GCG restored expression to rates equivalent to those seen in constructions containing no ODC 5'UTR sequences. We conclude that the rat ODC mRNA 5'UTR can inhibit translation of ODC mRNA both in vitro and in vivo, and that the predicted stem-loop structure at the 5' end of the 5'UTR is both necessary and sufficient for this inhibition.

High level, cell-specific expression of ornithine decarboxylase transcripts in rat genitourinary tissues.

We evaluated transcript levels for the rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC), in rat tissues by Northern blotting and in situ hybridization histochemistry, using a rat cDNA probe. ODC transcripts were expressed at a high level, relative to levels in other tissues, in the kidney and testis of the adult rat; maximal levels of transcripts in these tissues occurred after sexual maturation had taken place, i.e. between 20 and 150 days of age. In situ hybridization histochemistry revealed high level expression in the kidney, testis, prostate, and seminal vesicles of the male rat; this high level expression was limited to certain cell types: kidney, S3 cells of the proximal convoluted tubule; prostate and seminal vesicles, glandular or luminal epithelial cells; and testis, early spermatogenic cells. High level expression of ODC mRNA disappeared from the prostate and seminal vesicle epithelial cells after castration and reappeared with testosterone treatment; in contrast, levels of kidney ODC mRNA were essentially unchanged by castration and were similar in male and female adult rats. We conclude that high level ODC mRNA expression occurs in specific cell types in the adult rat, where it appears to be regulated by both androgen-dependent and independent mechanisms.