Level Of mRNA Accumulation Research Articles

Transcriptional activation of 2 classes of genes during the hypersensitive reaction of tobacco leaves infiltrated with an incompatible isolate of the phytopathogenic bacterium Pseudomonas solanacearum

Fourteen cDNA clones whose corresponding mRNAs accumulate during the hypersensitive reaction (HR) of tobacco leaves infiltrated with an incompatible strain of the bacterial pathogen Pseudomonas solanacearum have been subdivided by sequence homologies into 6 families. Studies on the accumulation of the mRNAs encoded by these genes in compatible and incompatible plant-bacterial interactions have been carried out and indicate that the 6 cDNA clones can be subdivided into 2 groups. In one group corresponding to 3 cDNA clones, the maximal level of mRNA accumulation is similar in both types of interaction, whereas in the other group, maximal mRNA accumulation in leaves undergoing an HR is 3- to 7-fold higher than in leaves infiltrated with the compatible strain. Within each group, the timing and kinetics of accumulation of the corresponding mRNAs differ for each individual cDNA clone. Run-on experiments indicate that transcriptional activation of these genes plays a major role in the control of their expression. Genomic hybridizations have been performed and indicate that the mRNAs corresponding to the cDNA clones are encoded by multigene families (6 to 20 genes).

Translational regulation of herpes simplex virus DNA polymerase

Using as antigens fusion proteins expressed in bacteria, we have generated polyclonal antisera specific for the herpes simplex virus (HSV) type 1 DNA polymerase. A variety of immunologic, genetic, and biochemical assays were used to characterize these antisera and demonstrate their specificity for the HSV DNA polymerase. Using these antisera, measurements of the synthesis and accumulation of HSV DNA polymerase in infected Vero cells were made. Peak rates of polymerase synthesis were observed at 4 h postinfection, as much as 2 h before peak levels of polymerase mRNA accumulation. At all times examined, the HSV DNA polymerase polypeptide was found to be synthesized at a lower rate per mRNA than the viral thymidine kinase, with this difference being especially dramatic at later times. Infected-cell RNA isolated at 2 and 6 h postinfection directed the synthesis of similar amounts of polymerase polypeptide per polymerase transcript in rabbit reticulocyte lysates, indicating that polymerase transcripts are inherently as translatable at both times. An HSV mutant in which sequences including a short upstream open reading frame in the HSV DNA polymerase transcript were deleted specified polymerase mRNA whose translational efficiency was no more than marginally greater than that of the wild-type virus. These results demonstrate that polymerase expression is regulated by inefficient translation mediated by sequences other than the short upstream open reading frame and that this leads to an early shutoff of polymerase synthesis during HSV infection.

Mechanisms of differential regulation of interleukin-6 mRNA accumulation by tumor necrosis factor alpha and lymphotoxin during monocytic differentiation

In the present report we compare the capacity of two related cytokines, tumor necrosis factor (TNF) alpha and lymphotoxin (LT), to modulate mRNA levels of interleukin-6 (IL-6) in cells representing different stages of monocytic differentiation including the human leukemia cell lines HL 60, U 937, THP-1, MonoMac 1 and peripheral blood monocytes. We show that the capacity of TNF alpha and LT to induce IL-6 mRNA accumulation increases as monocytic differentiation proceeds with TNF alpha being more potent than LT, suggesting that alternate pathways may be used by differentiating cells to control expression of IL-6. In contrast, in monocytes which constitutively synthesize IL-6 transcripts, TNF alpha and LT treatment had opposite effects on levels of IL-6 mRNA accumulation. In these cells TNF alpha enhanced steady state levels of IL-6 transcripts due to mRNA stabilization, whereas LT shortened IL-6 mRNA half-life, most likely due to induction of a RNA destabilizer since LT-mediated downregulation of levels of IL-6 mRNA in monocytes could be prevented by inhibition of protein synthesis. Neither TNF alpha nor LT altered IL-6 mRNA accumulation by interfering with preexisting transcription factors since both TNF alpha and LT required de novo protein synthesis to exert their effects.

DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally

The full-length cDNA for a transcriptional activator, DBP, that binds to the D site of the albumin promoter has been cloned. DBP belongs to a family of related transcription factors including Fos, Jun, CREB, and C/EBP, which share a conserved basic domain. However, unlike most other members of this family, DBP does not contain a “leucine zipper” structure. Among several rat tissues tested, significant levels of its protein are only observed in liver; yet, with the exception of testis, DBP mRNA is present in all of the examined tissues. DBP as well as its mRNA accumulate to significant levels only in adult animals. During chemically induced liver regeneration, DBP expression is rapidly down-regulatel, suggesting that DBP may be involved in the proliferation control of hepatocytes. This cell growth-dependent expression of DBP, in contrast to its tissue specificity, appears to be controlled at the level of mRNA accumulation.

Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans

Genomic clones containing the entire crnA-niiA-niaD gene cluster of Aspergillus nidulans have been isolated, and the structures of the niiA and niaD genes have been determined by nucleotide sequence analysis. This gene cluster is required for the assimilation of nitrate in A. nidulans, and the three genes encode a product required for nitrate uptake and the enzymes, nitrite reductase and nitrate reductase, respectively. The putative coding sequences, as deduced by comparison to cDNA clones of both niiA and niaD, are interrupted by multiple small introns, and the two genes are divergently transcribed. Identification and characterization of specific mRNAs involved in nitrate assimilation indicates that only monocistronic transcripts are involved, and that the approximate sizes of these transcripts are 1.6 kb, 3.4 kb and 2.8 kb for crnA, niiA and niaD, respectively. The results also indicate that control of niiA and niaD gene expression is mediated by the levels of mRNA accumulation, in response to the source of nitrogen in the growth medium. Two types of transcripts for niiA were observed.

Flavonoids inhibit the expression of heat shock proteins.

Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetin and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents.

The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis.

Clones of Aspergillus nidulans genomic DNA spanning 20 kb have been isolated and shown by a combination of classical and molecular genetic means to represent the npeA locus, previously found to be one of four loci (npeA, npeB, npeC and npeD) involved in the synthesis of penicillin. As well as containing the gene encoding the second enzyme for penicillin biosynthesis, namely isopenicillin N synthetase (IPNS) (designated ipnA), our results show that these clones (pSTA200, pSTA201 and pSTA207) contain two more genes to form a cluster of three contiguous penicillin biosynthetic genes. Our evidence suggests that these genes encode delta (L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and acyl transferase (ACYT) (designated acvA and acyA respectively), the first and third enzymes required for penicillin biosynthesis, with the gene order being acvA-ipnA-acyA. Transcripts have been identified for the three genes and their approximate sizes determined--acvA 9.5 kb, ipnA 1.4 kb and acyA 1.6 kb. All three mRNA species are observed in cells grown in fermentation medium but not in cells grown in minimal medium, suggesting that the control of penicillin biosynthesis is, in part, at the level of mRNA accumulation. Finally our results show that acvA and ipnA genes are divergently transcribed, whilst acyA is transcribed in the same orientation as ipnA.

Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

Carbohydrate-binding Protein 35: Levels of transcription and mRNA accumulation in quiescent and proliferating cells

In previous studies, we observed proliferation-dependent expression and nuclear localization of the lectin, designated carbohydrate-binding protein 35 (CBP35), in 3T3 fibroblasts at the polypeptide level by Western blot and immunofluorescence analysis. In the present study, we have compared the expression of the CBP35 gene in quiescent and proliferative 3T3 cells at the levels of (a) accumulated mRNA by Northern blotting and (b) nuclear transcription by run-off assays. When serum-starved, quiescent cultures of 3T3 cells were stimulated by the addition of serum, there was an increase in the nuclear transcription of the CBP35 gene and in the accumulation of its mRNA early (1–3 h) in the activation process, well before the first wave of DNA synthesis. These increases were not dependent on de novo protein synthesis inasmuch as they occurred even in the presence of cycloheximide. There was also an elevated transcription rate and mRNA level in transformed cells when compared to their normal counterparts. Finally, the expression of CBP35 was compared between sparse, proliferating cultures of 3T3 cells and density-inhibited confluent monolayers of the same cells. Although the rate of transcription of the CBP35 gene was approximately the same in the two cultures, there was a higher level of CBP35 mRNA in the dense cells. Thus, it appears that posttranscriptional mechanisms may be involved in the accumulation of mRNA.

Production of lymphotoxin by isolated human tonsillar B lymphocytes and B lymphocyte cell lines.

The expression of lymphotoxin (LT) mRNA and cytokine in human tonsillar B cells and B cell lines was examined by Northern blots and cytotoxicity assays, respectively. In tonsillar B cells, phorbol myristate acetate (PMA) or Staphylococcus aureus Cowan l (SAC) alone induced low levels of LT mRNA accumulation. However, SAC and anti-mu were strongly synergistic with PMA in this induction. Peak LT mRNA expression in tonsillar B cells stimulated by PMA plus SAC occurred between 48 and 72 h and was approximately half as much as that in PMA plus anti-CD3-stimulated T cells. Cyclosporine A was not effective in inhibiting LT mRNA accumulation by stimulated tonsillar B cells. A number of B cell lines could also be stimulated by PMA to express LT mRNA. Peak accumulation of LT mRNA in the cell line RPMI 1788 stimulated with PMA peaked about 8 h. A23187 in combination with PMA caused this accumulation to increase slightly and to peak earlier. The cytotoxic effects in the supernatants of stimulated B cells were contributed mostly by LT. The results indicate that tonsillar B cells are important in LT production and that there are important differences in the stimulation requirements for LT production and in LT mRNA expression kinetics between tonsillar B cells and B cell lines.

Analysis of stress-induced gene expression in fish cell lines exposed to heavy metals and heat shock

We have examined the effect of heavy metals on the expression of two major groups of stress-induced proteins in fish cell lines: the 70 kDa heat-shock proteins (hsp70) and metallothioneins (MTs). The rainbow trout hepatoma (RTH) cell line synthesized the hsp70 protein in response to zinc and heat shock, while chinook salmon embryonic (CHSE) cells synthesized this protein in response to these inducers, as well as cadmium. The synthesis of this 70 kDa protein was correlated with the accumulation of hsp70 mRNA as measured by hybridization to a trout hsp70 gene probe. Heavy metals also induced the synthesis of MT in RTH cells. However, heat shock did not result in induction of MT and its mRNA. Unlike RTH cells, CHSE cells did not synthesiz MT following exposure or zinc. When these cells were treated with 5-azacytidine prior to heavy metal treatment, accumulation of MT mRNA was observed. Northern blot analysis of total RNA from 5-azacytidine treated CHSE cells, using a trout MT (tMT-B) cDNA probe, indicated that the time-course of induction and the maximal level of MT mRNA accumulation in response to cadmium and zinc paralleled that observed in RTH cells. Copper and dexamethasone were ineffective in inducing MT mRNA in 5-azacytidine-treated CHSE cells. These results indicate that MT is specifically induced in response to heavy metal treatment, whereas the synthesis of hsp70 appears to be a general stress response. Furthermore, MT is differentially regulated by heavy metals and dexamethasone in these cell lines and the expression of MT is cell-type-specific.

Regulation of the Aspergillus nidulans pectate lyase gene (pelA).

Aspergillus nidulans pectate lyase was purified from culture filtrates. The enzyme catalyzed a random eliminative cleavage reaction, had an apparent molecular weight of 40,000, and a pl of 4.2. Pectate lyase antisera were produced and used to identify pectate lyase clones in a cDNA expression library. Thirteen of 14 clones identified immunologically cross-hybridized. The identity of the single-copy pectate lyase gene, which we designated pelA, was confirmed in two ways. First, several cDNA clones expressed pectate lyase activity in Escherichia coli. Second, targeted mutation of the gene in A. nidulans resulted in complete loss of enzyme activity. pelA encodes a 1,300-nucleotide mRNA that was present in cells grown with polygalacturonic acid as carbon source but absent from cells grown with glucose or acetate as carbon source. Thus, pectate lyase expression is regulated at the level of mRNA accumulation.

Induction of chitinase and β-1,3-glucanase in tobacco plants infected with Pseudomonas tabaci and Phytophthora parasitica var. Nicotianae

We have measured by rocket immunoassay the glucan endo-1,3-β-glucosidase (β-1,3-glucanase, EC 3.2.1.39) and endochitinase (chitinase, EC 3.2.1.14) contents of Nicotiana tabacum cv. “Havana 425” plants following infection with the bacterial pathogen Pseudomonas tabaci and the fungal pathogen Phytophtorara parasitica var. nicotianae. Both enzymes were induced, relative to non-infected controls, in leaves infected with P. tabaci and in stems and leaves infected with P. parasitica. Increases in enzyme content were confined to infected parts of the plant indicating that this response is local rather than systemic. The β-1,3-glucanase- and chitinase-mRNA content of tissues was measured by RNA blot hybridization using the inserts of cDNA clones for the tobacco mRNAs as probes. P. tabaci and P. parasitica infection increased the mRNA content relative to controls indicating that both pathogens act at least in part at the level of mRNA accumulation. As judged by immunological tests and RNA hybridization, the β-1,3-glucanase and chitinase induced by P. tabaci and P. parasitica are the same basic isoforms known to be induced by tobacco mosaic virus. Therefore the induction appears to be a non-specific response of the host to pathogens.

Characterization of the calcium sensitivity of differentiation in SCC-13 human squamous carcinoma cells

The sensitivity to calcium of the human squamous carcinoma cell line, SCC-13, was demonstrated and characterized. Cultures grown to confluence in the presence of 0.2 to 2 mM calcium had approximately 10-fold higher levels of particulate transglutaminase activity and envelope competence than those grown in low calcium (0.025 to 0.05 mM) medium. Raising the calcium from 0.025 to 1.8 mM induced expression of this enzyme and of competence over the course of a week. Conversely, for cultures grown to confluence in 1.8 mM calcium, subsequent reduction of calcium to 0.025 mM resulted in a substantial decline in transglutaminase over a similar time period. Immunoprecipitable transglutaminase was clearly identifiable in cultures grown in 1.8 mM calcium-containing medium but not in those grown in low calcium medium or in the presence of retinoic acid, suggestive of regulation at the level of mRNA accumulation or translation rather than posttranslational modification.

Cellular and molecular regulation of tumor necrosis factor-alpha production by pentoxofylline

Tumor necrosis factor-alpha (TNF), a mononuclear phagocyte (MO)-derived peptide, is increasingly being recognized for its pleomorphic immunologic effects. A number of investigations have demonstrated that lipopolysaccharide (LPS) can induce TNF synthesis, yet mechanisms that regulate TNF expression at the cellular and molecular levels have not been fully elucidated. In this study, we present data demonstrating pentoxifylline, a methylxanthine, is efficacious in suppressing LPS-induced MO-derived TNF at the level of both TNF mRNA accumulation and TNF supernatant bioactivity. Pentoxifylline, at a dose of 1 x 10(-5)M, suppressed the production of both biologically active TNF and TNF mRNA expression by more than 50%. Furthermore, additional methylxanthines and dibutyryl cAMP have similar effects on TNF expression. These data support the mechanism for this suppressive effect is via the generation of intracellular cAMP.

Regulation of ornithine decarboxylase activity by IL-2 and cyclic AMP.

IL-2 regulates the expression and activation of the principal rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC). The apparent activation of ODC occurs by two independent steps. Rapid activation of enzyme occurs within the first 10 min of lymphokine treatment of cloned IL-2-dependent murine cell lines. The initial rapid rise in enzyme activity is insensitive to protein or mRNA synthesis inhibitors. The early rise in ODC activity is followed by a protracted increase in enzyme activity, apparent protein measured by [3H]difluoromethylornithine binding, and by accumulation of steady state ODC mRNA. Stable analogues of cAMP which inhibit IL-2-stimulated proliferation, suppressed only the mRNA-dependent increase in ODC activity. Phorbol esters were shown to increase steady state levels of ODC mRNA whereas cAMP analogue clearly inhibited the growth factor-induced increase in ODC mRNA. These studies show that IL-2 regulates the expression of ODC at multiple levels, including transcription and accumulation of steady state mRNA, and post-translational activation of an enzyme important for DNA synthesis. Furthermore, anti-proliferative signals such as cAMP may effect the regulation of ODC enzyme production at the level of mRNA accumulation and stability.

High level of c-fos mRNA accumulation is not obligatory for renewed cell proliferation

We have examined the expression of the c-fos gene and the formation of inositol phosphates with respect to the reentry of inducible C2 myoblasts into the cell cycle. GI arrested myoblasts were stimulated to proliferate by addition of fresh medium containing either 20% foetal calf serum (FCS) or 1.6 10(-6) M insulin and 7 microM Na+ vanadate. Our results show that renewed proliferation, which occurred in the presence of insulin + vanadate, was neither preceded by increased inositol phosphate formation, nor by high level of c-fos mRNA accumulation, while, as classically observed, FCS induced proliferation was. These results suggest that increased inositol phospholipids breakdown and transient accumulation of c-fos mRNA at a high level, are not obligatory for renewed cell proliferation.

A Discrete Element 3′ of Human Immunodeficiency Virus 1 (HIV-1) and HIV-2 mRNA Initiation Sites Mediates Transcriptional Activation by an HIV trans Activator

An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator.

An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.