Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae.

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a "sensitive" region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions -27 to +37 but not in the remaining 3' distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5' cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.     

Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro.

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.     

Control of laminin synthesis during differentiation of F9 embryonal carcinoma cells

Molecular clones complementary to the mRNA species for the A, B1 and B2 chains of murine laminin were identified by hybrid-selection and in vitro translation. Northern blot analysis demonstrated that the three clones, p59 (A), p2 (B1) and p16 (B2) hybridized to mRNA species 9.8, 6.0, and 8.0 kb in length, respectively. The three clones were used as probes to monitor the steady-state levels of laminin mRNA species during differentiation of F9 embryonal carcinoma cells induced by treatment with retinoic acid and dibutyryl cyclic AMP. The steady-state levels of the three mRNA species appeared to increase in a coordinate manner. Undetectable levels at the beginning of induction were followed by a dramatic increase in the levels of the three mRNA species between 48 and 72 h. The kinetics parallel the increase in laminin synthesis and the striking morphological changes previously reported.     

Coupling of GCN4 mRNA translational activation with decreased rates of polypeptide chain initiation

The steady-state translational activation of the GCN4 mRNA is based upon an increase in the rate of ribosome initiation at the protein coding AUG following translation of the 5' most proximal open reading frame located in its untranslated region. Such an increase is effected when the cellular amount of the GCN2 protein kinase is increased or when the function of the GCD1 gene product is defective. Here, we report conditions that result in a dramatic transient increase in the rate of GCN4 protein synthesis, which also requires the prior translation of the 5' most proximal open reading frame but is independent of the GCN2 protein. This activation of GCN4 mRNA translation coincides with a decrease in the rate of total cellular protein synthesis. We also observed low rates of protein synthesis in the gcd1 strain and in strains that overexpress the GCN2 protein kinase. The process in protein synthesis that is affected is formation of 43S preinitiation complexes. These results reveal the existence of a coupling between this process in translational initiation and the mechanism that activates translation of GCN4 mRNA.     

Expression of antioxidant enzymes in rat lungs after inhalation of asbestos or silica.

Several studies indicate that active oxygen species play an important role in the development of pulmonary disease (asbestosis and silicosis) after exposure to mineral dust. The present study was conducted to determine if inhaled fibrogenic minerals induced changes in gene expression and activities of antioxidant enzymes (AOE) in rat lung. Two different fibrogenic minerals were compared, crocidolite, an amphibole asbestos fiber, and cristobalite, a crystalline silicon dioxide particle. Steady-state mRNA levels, immunoreactive protein, and activities of selected AOE were measured in lungs 1-10 days after initiation of exposure and at 14 days after cessation of a 10-day exposure period. Exposure to asbestos resulted in significant increases in steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) at 3 and 9 days and of glutathione peroxidase at 6 and 9 days. An increase in steady-state mRNA levels of copper, zinc-containing superoxide dismutase (CuZnSOD), was observed at 6 days. Exposure to asbestos also resulted in overall increased enzyme activities of catalase, glutathione peroxidase and total superoxide dismutase in lung. In contrast, silica caused a dramatic increase in steady-state levels of MnSOD mRNA at all time periods and an increase in glutathione peroxidase mRNA levels at 9 days. Activities of AOE remained unchanged in silica-exposed lungs. In both models, increases in gene expression of MnSOD correlated with increased amounts of MnSOD immunoreactive protein in lung and the pattern and extent of inflammation. These data indicate that the profiles of AOE are dissimilar during the development of experimental asbestosis or silicosis and suggest different mechanisms of lung defense in response to these minerals.     

Translational fusions with fragments of the trpE gene improve the expression of a poorly expressed heterologous gene in Escherichia coli

A series of plasmids expressing fusions between the trpE gene product, anthranilate synthase component I and the major immunogen (VP1) of foot and mouth disease virus were constructed such that increasing amounts of the 3' end of trpE were deleted. Deletions removing up to 70% of trpE had little effect on the quantity of fusion protein expressed, while the number of molecules appeared to increase. Larger deletions led to a steady decrease in both the quantity of fusion protein produced and in the number of molecules. This is consistent with trpE being responsible for the high levels of expression of VP1 by its gene product stabilizing VP1 against proteolytic degradation. Some out-of-frame deletion mutants were also produced. All deletion mutants in one wrong reading frame expressed low levels of two VP1-containing polypeptides. This observation is interpreted as being due to re-initiation of translation at a site inside the VP1 sequence which is activated by local termination of translation.     

Glucose-induced translational control of proinsulin biosynthesis is proportional to preproinsulin mRNA levels in islet beta-cells but not regulated via a positive feedback of secreted insulin

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (/=10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the beta-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.