Total mRNA Synthesis Research Articles

The regulation of glutathione peroxidase gene expression by oxygen tension in cultured human cardiomyocytes

In earlier studies we have shown that the activity of the antioxidant enzyme glutathione peroxidase is regulated by oxygen tension in cultured tetralogy of Fallot (TOF) ventricular myocytes and in the ventricles of TOF patients having corrective cardiac surgery. The present study was undertaken to determine the mechanism of this regulation. Northern and slot blot analysis was performed using RNA isolated from TOF myocytes cultured at oxygen tensions of 150 and 40 mmHg for 3, 7, 14, 21, and 28 days. As was found for enzyme activities, glutathione peroxidase mRNA levels were lower in the cells cultured at a pO 2 of 40 mmHg than at 150 mm Hg and could be elevated with an increase in oxygen tension. These results were standardized against house-keeping gene hexosaminidase B which showed no difference in mRNA levels between the two oxygen tensions throughout the time course. Nuclear run-off assays indicated that glutathione peroxidase was regulated by oxygen tension at the transcriptional level, while hexosaminidase B and total mRNA synthesis levels remained unchanged.

Effects of UV, 4NQO and TPA on gene expression in cultured human epidermal keratinocytes

In an approach to study effects of UV light on gene expression in human epidermal keratinocytes, a cDNA library was constructed from poly(A)RNA isolated after UV irradiation from cultured keratinocytes. The cDNA library was differentially screened with labelled cDNA probes synthesized on poly(A)RNA isolated from UV irradiated or nonirradiated keratinocytes. Forty clones were selected and subjected to further analysis, 31 of them are described in this report. Whereas total mRNA synthesis is reduced after UV irradiation or treatment with 4-NQO Northern blot analysis revealed that there is an at least relative increase in the level of mRNAs corresponding to the majority of the isolated cDNA clones. Among these 15 were identified as corresponding to mRNAs for 50K and 56K keratins and for 50K- and 46K-related keratin. In addition, clones were found corresponding to the proteinase inhibitor cystatin A and to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of keratinocytes with the tumor promoter TPA had no effect on the mRNA level for most of the clones except those corresponding to keratins. Our results indicate that in keratinocytes UV irradiation leads to a relative increase in the level of some mRNAs.

The effect of phorbol esters and diacylglycerol on expression of the phosphoenolpyruvate carboxykinase (GTP) gene in rat hepatoma H4IIE cells.

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on expression of the P-enolpyruvate carboxykinase gene was studied in rat hepatoma H4IIE cells. Like insulin, PMA provokes a concentration and time-dependent decrease of mRNA coding for that enzyme that is due to an inhibition of P-enolpyruvate carboxykinase gene transcription. This effect of PMA is rapid, reversible, specific for phorbol esters known to be active in other systems, and it does not require on-going protein synthesis. PMA overrides the stimulatory effects cAMP and glucocorticoid analogs have on the transcription of this gene. A synthetic diacylglycerol, sn-1,2-dioctanoylglycerol, also inhibits P-enolpyruvate carboxykinase gene transcription. These effects of PMA and synthetic diacylglycerol are specific, since neither affected total mRNA synthesis. We conclude that diacylglycerol and phorbol esters, specific stimulators of protein kinase C, inhibit the transcription of P-enolpyruvate carboxykinase gene in H4IIE cells. The findings support the hypothesis that diacylglycerols generated in the plasma membrane can act as an intracellular signal that regulates specific gene expression.

Preferential synthesis of differentiation related cytoplasmic proteins in low-serum adapted differentiated glioma cells

Rat glioma (C6) cells can be induced to morphological and biochemical differentiation by culturing in serum-deprived conditions. The induced differentiated cells were found to preferentially synthesize some distinct cytoplasmic proteins, and the rate of synthesis quantitatively correlated well with the degree of morphological differentiation, whereas the total mRNA synthesis was decreased in the differentiated cells. These findings suggest that reduced total mRNA syntheses and prefereential translation of differentiation-related mRNAs are parts of the biochemical events during the differentiation process of neuroglia.

Mechanism of host defense suppression induced by viral infection: mode of action of inosiplex as an antiviral agent.

The mechanism of influenza virus (INFV)-induced immunosuppression and the mode of inosiplex action against INFV infection were studied. INFV suppressed both anti-lipopolysaccharide and anti-sheep erythrocyte antibody production in mice. INFV infection caused viral mRNA synthesis and increased total RNA synthesis in lymphocytes, but total mRNA synthesis was decreased. The translational ability of INFV-infected lymphocytes was also suppressed. Thus, INFV seemed to cause suppression of both mRNA synthesis and the translational ability of lymphocytes, resulting in suppression of lymphocyte functions. Inosiplex potentiated antibody production against sheep erythrocytes but not against lipopolysaccharide in normal and INFV-infected mice. Adamantanamine did not produce such a potentiating effect. The lymphocytes obtained from INFV-immunized and inosiplex-treated mice conferred resistance against INFV infection. This resistance was partially inhibited by anti-Thy 1.2 antibody treatment of the lymphocytes. In an adoptive cell transfer system, inosiplex treatment of T-cell donors potentiated antibody production when a non-immunosuppressive carrier (human serum albumin) was used. When an immunosuppressive carrier (INFV) was used, inosiplex treatment of either B-cell donors or T-cell donors increased antibody production. Direct introduction of inosiplex into lymphocytes by a cell fusion technique stimulated anti-sheep erythrocyte antibody production more effectively than the addition of inosiplex to cultures. Inosiplex increased total RNA and total mRNA syntheses in phytohemagglutinin-treated lymphocytes. In INFV-infected lymphocytes, inosiplex decreased syntheses of total RNA, total mRNA, and viral mRNA and restored translational ability. From these results, we concluded that inosiplex penetrates into lymphocytes and suppresses viral RNA synthesis and that it supports lymphocyte functions by promoting RNA synthesis and translational ability, both of which are necessary for hosts.

Regulation of ribosome production in Escherichia coli: Synthesis and stability of ribosomal RNA and of ribosomal protein messenger RNA at different growth rates

Four parameters related to ribosome production have been determined in Escherichia coli growing exponentially at rates between 0·1 and 1·4 generations per hour at 30 °C. 1. (1) The fraction of the total transcript which is ribosomal RNA was measured by hybridization of pulse-labeled total cellular RNA to DNA from a transducing λ-phage carrying a complete set of rRNA genes (λ-d ilv5). The relative rate of rRNA transcription increases from 0·27 to 0·67 over the range of growth rates studied. 2. (2) The fraction of the total transcript from approximately 25% of the ribosomal protein genes was measured by hybridization of pulse-labeled RNA to DNA from a transducing phage which carries 14 of the ribosomal protein genes (λ-d spc1). The relative rate of r-protein messenger RNA does not vary with the growth rate. 3. (3) The fraction of the total transcript which accumulates in mature ribosomes and transfer RNA was calculated from the rate of stable RNA accumulation and the total rate of RNA synthesis, measured by short-term kinetics of uptake of [ 3H]adenine, corrected for the specific activity of the ATP pool. The relative rate of stable RNA accumulation increases from 0·07 to 0·5 over the range of growth rates studied. 4. (4) The amount of r-protein mRNA relative to the amount of total cellular RNA was determined by hybridization of increasing amounts of pulse-labeled RNA to limiting amounts of λ-d spc1 DNA. The amount of r-protein mRNA per ribosome is closely proportional to α r, the relative rate of r-protein synthesis. Correlations of the different parameters permit the following conclusions. The transcription of rRNA and r-protein mRNA does not vary in concert as a function of the growth rate. It indicates that different mechanisms are responsible for the control of these two types of ribosomal operons. The rate of transcription of rRNA is always greater than the rate of accumulation of rRNA. The difference is particularly pronounced at low growth rates; at μ = 0·1 generation per hour, only 30% of the newly made rRNA is incorporated into mature ribosomes and the rest is broken down. At high growth rates about 10% of the newly made rRNA is broken down. An approximate estimate of total mRNA transcription can be made from determinations of the relative rate of rRNA synthesis; total mRNA constitutes a decreasing fraction of the total transcript varying from 0·8 to 0·25 between μ = 0·1 and 1·4. After the calculation of the relative rate of total mRNA transcription, the fraction of the total mRNA which is r-protein mRNA can be drived. The result is that the rate of r-protein mRNA syntheis relative to total mRNA synthesis is nearly proportional to α r, also the amount of r-protein mRNA varies in proportion to α r, indicating that the rate of r-protein synthesis is regulated at the level of transcription in the same way as synthesis of bulk protein.

The effect of antibiotics on the in vivo synthesis of messenger ribonucleic acid from the lactose operon of Escherichia coli.

Lac transcription is rapidly reduced to 3–5% of the original value in E. coli cells treated with antibiotics which block protein synthesis in general. Chloramphenicol (CM) exerts the most rapid, streptomycin (Sm) an intermediate, puromycin a slower effect. Cyclic AMP relieves about 15% of the CM-, 20% of the Sm-, and 40–60% of the puromycin effect indicating an increasing role of catabolite repression in the reduction of lac transcription. The remaining effects are due to the arrest of ribosomes on the nascent newly synthesized mRNA (CM and Sm) and probably the release of ribosomes from the RNA after termination of peptide synthesis (puromycin). In both cases mRNA unprotected by ribosomes is synthesized which either becomes rapidly degraded or hinders the further progression of RNA polymerase. Experiments with the polarity suppressor suA+ argue for the rapid degradation. Both, CM and Sm, reduce the total mRNA synthesis of E. coli to only 70%, while lac transcription is reduced to 15–20% (in the absence of catabolite repression). A possible reason for this difference is discussed.

Dissociation of Lac Messenger Ribonucleic Acid Transcription from Translation During Recovery from Inhibition of Protein Synthesis

Studies were made of the synthesis of Lac messenger ribonucleic acid (mRNA) by Escherichia coli in the absence of protein synthesis and of the coupling of transcription of lac operon to translation. Lac mRNA was not synthesized in the presence of chloramphenicol, and its synthesis steadily decreased during K(+) deprivation and treatment with puromycin. Since under these conditions total mRNA synthesis is not inhibited it is suggested that the control of Lac mRNA is distinct from that which regulates total mRNA synthesis. Lac mRNA synthesized during recovery from K(+) starvation or from chloramphenicol inhibition is not translated into functional enzyme, suggesting translational control over beta-galactosidase synthesis.