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PubMed1982

Chromatin structure of Xenopus rDNA transcription termination sites. Evidence for a two-step process of transcription termination.

The ultrastructure of Xenopus laevis (X.l.) 40S pre-rRNA transcription termination sites was investigated by electron microscopy. Amplified nucleolar chromatin was rapidly dispersed and processed for chromatin spread preparations. This type of preparation revealed that many rDNA termination sites appeared as 50 nm long segments of chromatin axis covered by a complex of three closely spaced RNA polymerase particles. Particle (Ptl) was characterized by the association with the terminal full-length pre-rRNP fibril; particles (Pt2) and (Pt3) are located downstream from (Ptl) and appear to be devoid of transcript fibrils. This particular structural arrangement as well as sequence homology analyses of 3' adjacent spacer rDNA segments indicate that transcript release and dissociation of polymerase particles are not necessarily coupled and termination of X.l. rDNA transcription may occur as a two step process. The structural data are correlated with homologies of DNA sequences at Xenopus rDNA transcription termination regions and are discussed with respect to sequence data of 3' termination sites of rRNA genes from other species.

Animals↗
PubMed2003

Regulation of human insulin gene transcription by the immunosuppressive drugs cyclosporin A and tacrolimus at concentrations that inhibit calcineurin activity and involving the transcription factor CREB.

Cyclosporin A and tacrolimus are important immunosuppressive drugs. They share a diabetogenic action as one of their most serious adverse effects. In a single study, tacrolimus (100 nM) inhibited human insulin gene transcription in the beta-cell line HIT. Using transfections of a human insulin-reporter gene into HIT cells, the present study shows that this inhibition is seen only at high concentrations of tacrolimus and is not caused by cyclosporin A. However, after stimulation by the major second messengers in the regulation of the insulin gene, cAMP and depolarization-induced calcium influx, both tacrolimus and cyclosporin A inhibited human insulin gene transcription in a concentration-dependent manner with IC(50) values of 1 nM and 30 nM, respectively. A further analysis offers a mechanism for this effect by revealing that the activation by cAMP and calcium of human insulin gene transcription is mediated by the transcription factor cAMP-responsive element binding protein (CREB) whose activity is inhibited by the immunosuppressants. These data demonstrate for the first time that cAMP- and calcium-induced activity of the human insulin gene is mediated by CREB and blocked by both tacrolimus and cyclosporin A at concentrations that inhibit calcineurin phosphatase activity. Since also the immunosuppressive effects of cyclosporin A and tacrolimus are thought to be secondary to inhibition of calcineurin, the present study suggests that inhibition of human insulin gene transcription by the immunosuppressants is clinically important and may contribute to their diabetogenic effect.

Calcineurin Inhibitors↗
PubMed1996

Preferential RNA editing at specific sites within transcripts of two plant mitochondrial genes does not depend on transcriptional context or nuclear genotype.

Transcripts of most plant mitochondrial protein-coding genes exhibit C-to-U RNA editing events. In Petunia, two co-transcribed genes, nad3 and rps12, exhibit transcripts which are not fully edited at all potential editing sites. We investigated the nad3/rps12 transcript population in four different genotypes. In one pair of genotypes, the nuclear genome is identical but the nad3/rps12 genes are in different transcriptional contexts. Both the nad3/ rps12 genes and the plant mitochondrial genomes are identical in a second pair of genotypes, but the nuclear background is derived from two different Petunia species. We found that the overall extent of editing varied greatly between genotypes and is affected by nuclear genotype but not by the global transcriptional context. Local sequence context around a particular site does affect editing frequency. In all genotypes, certain sites exhibit high editing frequency, but these sites do not share obvious primary sequence characteristics. In all genotypes examined, editing sites which do not affect the encoded amino acid are less frequently edited than sites which alter codons to non-synonymous forms. All these data indicate that an unidentified property of the sequences immediately surrounding a cytosine affect its selection as a target in the editing process.

Amino Acid Sequence↗
PubMed1997

Measurement of transcription factor c-fos and EGR-1 mRNA transcription levels in synovial tissue by quantitative RT-PCR.

The transcription factors Fos and EGR-1 are known to be involved in the regulation of the transcription of metalloproteinases and their specific inhibitors. Since the overexpression of metalloproteinases is responsible for the matrix degradation in rheumatoid arthritis (RA), exact analysis of transcription levels of c-fos and EGR-1 ex vivo may serve to monitor progression or remission of the disease activity in RA. Here we report on a method based on a quantitative reverse transcription polymerase chain reaction (RT-PCR) for rapid estimation of the transcription levels of the immediate early genes c-fos and EGR-1. Coamplification with suitable internal standards, easily generated by the use of hybrid primers, allows us to semiquantitatively measure c-fos and EGR-1 induction levels in low numbers of cultured cells or very small tissue samples obtained by synovial biopsy. The sensitivity of the method was 3.5 pg/ml for c-fos- and 10 pg/ml for EGR-1-specific cDNAs.

Arthritis, Rheumatoid↗
PubMed1997

Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing.

The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

Chromatin↗
PubMed1981

Specificity of chromatin transcription in vitro. Asymmetric transcription of the globin gene by Escherichia coli RNA polymerase.

The transcription of globin genes in mouse foetal liver chromatin and nuclei by exogenous Escherichia coli RNA polymerase is prone to artifacts due to RNA-dependent transcription of endogenous mRNA sequences. This is particularly evident when Mn2+ is used as divalent cation in the RNA transcription reaction. However, substitution or supplementation with Mg2+ eliminates this artifact and gives essentially asymmetric DNA-dependent transcription by the polymerase. In this paper we discuss a number of general criteria which can be applied to test the validity of specific gene transcription in vitro.

Animals↗
PubMed1986

Detection of a potential transcription control sequence on the cauliflower mosaic virus genome by dinucleotide primed "in vitro" transcription.

The three sites of selective dinucleotide-primed "in vitro" transcription initiation on a cloned cauliflower mosaic virus DNA fragment have been localised by S1 nuclease mapping. Two of these sites lie within a region which has been shown to be essential for transcription complex formation on the viral sequences, one corresponding to a nuclease S1 hypersensitive site and the other to an imperfect repeat 100bp downstream. These sequences show striking homology with known transcription control sequences. These observations and the effect of the sequences on "in vitro" transcription raise the possibility that they may be involved in control of transcription of the viral genome.

Base Sequence↗
PubMed1990

Transcriptional and post-transcriptional suppression of P450IIC11 and P450IIC12 by inflammation.

Induction of inflammation in rats by treatment with endotoxin or turpentine is known to suppress levels of hepatic mRNAs for P450IIC11 and P450IIC12. We report that transcription of CYP2C12 in female rats is not significantly reduced from control levels; suppression of this gene during inflammation appears to be mediated post-transcriptionally. In contrast, transcription of CYP2C11 in male rats is reduced to 23% and to 5% of control levels by turpentine and by endotoxin, respectively. Sex-specificity of CYP2C11 expression is also regulated transcriptionally, whereas sex-specificity of CYP2C12 expression appears to be regulated by a post-transcriptional mechanism.

Animals↗
PubMed1992

Transcriptional- and post-transcriptional-dependent regulation of glutathione S-transferase expression in rat hepatocytes as a function of culture conditions.

Transcriptional activity of the glutathione S-transferase (GST) alpha (subunits 1 and 2), mu (subunits 3 and 4) and pi (subunit 7) gene families has been analyzed using the nuclear 'run-on' technique on adult rat hepatocytes maintained for 4 days in conventional culture and for 4 and 12 days in co-culture with rat liver epithelial cells. Several medium conditions are included in this study, namely with or without fetal calf serum and with nicotinamide or dimethylsulphoxide. Hepatocytes co-cultured for 4 days maintain approximately 30-70% of the alpha gene family transcriptional activity, whatever the medium conditions, when compared to freshly isolated hepatocytes. A marked decrease is observed after 12 days of co-culture or when hepatocytes are maintained in conventional culture. The transcriptional activity of the mu gene family is maintained at 40-160% when hepatocytes are cultured with or without fetal calf serum, and is inducible by nicotinamide (approximately 4-fold) and dimethylsulphoxide (approximately 2-fold) in conventional culture and/or in co-culture. In contrast to freshly isolated hepatocytes, GST pi gene transcriptional activity is observed in conventional and co-cultured hepatocytes, irrespective of the medium conditions. Dimethylsulphoxide treatment however, represses the expression of GST 7 in vitro. These results demonstrate that the expression of GST alpha, mu and pi genes in conventional and co-cultured rat hepatocytes is controlled primarily at the level of transcription. It cannot be excluded, however, that dimethylsulphoxide stabilizes the GST mRNA levels in vitro.

Animals↗
PubMed1994

Transcription of the halophage phi H repressor gene is abolished by transcription from an inversely oriented lytic promoter.

The temperate phage phi H of the extremely halophilic archaebacterium Halobacterium salinarium encodes a repressor, Rep, which in the immune state represses the production of an early lytic transcript, denoted T4. Rep acts at the transcriptional level by blocking the promoter for T4. The promoter for the rep gene itself is positioned back to back to the promoter for T4, in a manner analogous to that of the cI/cro genes in bacteriophage lambda. Transcription of the rep gene does not occur when the phase is growing lytically. We show that this repressor of rep transcription during lytic growth is due to the transcription per se from the stronger, oppositely oriented promoter for T4, without the need of a phage gene product.

Bacteriophages↗
PubMed1995

Sodium butyrate inhibits expression of urokinase and its receptor mRNAs at both transcription and post-transcription levels in colon cancer cells.

The effects of butyrate on the modulation of urokinase plasminogen activator (uPA) and its receptor (uPAR) mRNAs were studied. While both mRNA levels were increased after stimulation by tumor necrosis factor alpha (TNF alpha), phorbol ester (PMA) and cycloheximide, they were inhibited by butyrate at 2.5 to 25 mM. Nuclear run-on transcription assays indicated that uPA mRNA was modulated by butyrate at the transcriptional level but the uPAR gene was regulated at both transcriptional and post-transcriptional levels in the presence or absence of TNF alpha. In the presence of PMA, however, butyrate acts at the post-transcriptional level on both genes.

Butyrates↗
PubMed1979

Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression.

The mechanism by which prolactin, a peptide hormone, regulates casein gene expression has been studied in mammary gland organ culture. After prolactin addition, a 2-4 fold increase in the rate of casein mRNA transcription was observed within 1 hr and maintained for at least 24 hr. This increased rate of transcription is not sufficient to account for the mass accumulation of casein mRNA. The half-life of casein mRNA is also increased 17-25 fold in the presence of prolactin. This change in casein mRNA half-life, coupled with a 2-4 fold increase in the rate of transcription, can account for the normal accumulation of casein mRNA observed after prolactin addition. This hormone-induced change in casein mRNA half-life appeared to be selective, since prolactin was found to exert only a slight effect (1-4 fold) on the half-life of poly(A) RNA determined under identical pulse-chase conditions. The hormonal regulation of casein gene expression thus does not app-ar to be an "all or none" process occurring only at the transcriptional or post-transcriptional levels, but rather may involve a coordinated response at several levels to permit the efficient expression of specialized differentiated functions.

Animals↗
PubMed1985

Transcripts and the putative RNA pregenome of duck hepatitis B virus: implications for reverse transcription.

Duck hepatitis B virus (DHBV) is a DNA virus that replicates by reverse transcription. We have examined transcripts of DHBV to elucidate mechanisms of gene expression and replication. Three major transcripts were characterized and related to the expression of the genes for the core antigen (DHBcAg), the surface antigen (DHBsAg), and the pre-S/DHBs protein, respectively. They are unspliced and overlap partially since they start at different promoter sites, but they terminate near a common polyadenylation signal. The messenger RNA related to DHBcAg expression is the only major transcript of more than genome length and therefore also represents the putative template for reverse transcription (pregenome). Its structure predicts a strategy of replication of hepatitis B viruses different from that of retroviruses.

Animals↗
PubMed1982

Leftward transcription in the Escherichia coli bio operon does not require products of the rightward transcript.

The amount of leftward transcription in the Escherichia coli bio operon, as measured by hybridization and by beta-galactosidase assays in lac-bio fusion strains, was determined in bacteria lysogenic for lambda bio phage carrying different amounts of DNA corresponding to rightward message, and in bacteria with polar or nonpolar bioB mutations. The positions of the bioB endpoints in relation to the pB promoter were determined by electron microscopy of heteroduplexes. Normal rates of leftward transcription were found in all cases, except that the shortest lambda bio (lambda bio showed a 2- to 3-fold increase in leftward transcription, which was not abolished by the presence of a wild-type bio operon in trans. These results indicate that no product of the rightward transcript is needed to turn on leftward transcription.

Bacteriophage lambda↗
PubMed1995

Upstream promoter elements are sufficient for selenocysteine tRNA[Ser]Sec gene transcription and to determine the transcription start point.

The TATA box, located upstream at about nt -30, and the proximal sequence element, located at about nt -60, are both essential and sufficient for basal level transcription of the Xenopus laevis (Xl) selenocysteine (Sec) tRNA[Ser]Sec gene as demonstrated by its microinjection into Xl oocytes. Point mutations within either of these regions abolish transcription, while deletion of the internal boxA element or insertion of 13 nt within the internal boxB element does not impair transcription. The latter mutations (within the internal regions) affect processing of the 3'-trailer sequence. Replacement of the tRNA[Ser]Sec coding sequence with an Escherichia coli M1 RNA gene resulted in expression of the E. coli gene governed by the upstream tRNA[Ser]Sec promoter elements. These studies demonstrate unequivocally that the upstream promoter elements are sufficient for the basal level of tRNA[Ser]Sec gene transcription. Primer extension studies with spacer mutants show that the internal elements do not play a role in selecting the transcription start point (tsp), but that selection of the tsp is determined by the region upstream from the gene. Further, studies with spacer mutants show that the distance between the TATA box and the tsp is quite likely the critical factor in selecting the position of tsp.

Animals↗
PubMed2004

5'-Heterogeneity of mouse Dda3 transcripts is attributed to differential initiation of transcription and alternative splicing.

We have previously shown that mouse Dda3 gene is a p53 and p73 transcriptional target whose expression suppresses tumor cell growth. Here, we report the identification of multiple variants of Dda3 transcripts with diverse 5' sequences through 5'] rapid amplification of cDNA ends (5'-RACE) and RT-PCR. Analysis by primer extension and RNase protection revealed that the 5'-heterogeneity was generated by transcription initiation at multiple sites in exon 1 and intron 1 and by alternative splicing. These transcripts, both coding and non-coding, exhibited distinct expression patterns in various adult tissues and were developmentally regulated. Furthermore, they were induced in a p53-dependent manner by various stress signals. These data demonstrated that differential initiation of transcription and alternative splicing both participate in the regulation of Dda3 gene expression.

Alternative Splicing↗
PubMed2006

hnulp1, a basic helix-loop-helix protein with a novel transcriptional repressive domain, inhibits transcriptional activity of serum response factor.

Many bHLH proteins are involved in cardiac development and cardiovascular diseases. Herein, we identified and characterized the human homologue (hnulp1) of mouse gene nulp1. The predicted protein contains a bHLH domain and a DUF654 domain in N-terminal and C-terminal, respectively. Northern blot analysis shows that a 2.3-kb transcript expressed broadly in early human embryonic and adult tissues, especially with a higher level in adult heart. hnulp1 is a transcription repressor when fused to GAL4 DNA-binding domain and co-transfected with VP-16, in which DUF654 motif represents the basal transcriptional repressive activity. Treatment of cells with trichostatin A can relieve this repression, suggesting that the DUF654 motif acts through increasing deacetylase activity at the GAL4-driven promoter. Overexpression of hnulp1 protein in COS-7 cells inhibits the transcriptional activity of serum response factor (SRF), suggesting that hnulp1 may act as a novel bHLH transcriptional repressor in SRF signaling pathway to mediate cellular functions.

Amino Acid Sequence↗
PubMed2006

The role of ETS transcription factors in transcription and development of mouse preimplantation embryos.

Embryonic transcription is a crucial process for the creation of new life. To clarify the mechanism of embryonic transcription, we investigated the expression and function of the erythroblast transformation specific (ETS) domain containing transcription factors (TFs) during preimplantation development in mice. The expression levels of several ETS TFs, i.e., etsrp71, elf3, and spic, increased after fertilization and remained at a high level until the blastocyst stage. To clarify the function of these TFs, we performed gene suppression using RNA interference, which revealed that they were involved in regulating development to the blastocyst stage. Furthermore, we found that suppression of ETS TFs affected the transcription of eIF-1A and oct3/4 genes whose expression is regulated by TATA-less promoters in the embryos. These results suggest that ETS TFs function in the regulation of transcription with TATA-less promoters in preimplantation embryos, which is essential in preimplantation development.

Animals↗