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At least 91 records · Page 5Linked to original sources
PubMed1986

Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration.

Human cells expressing adenovirus E1A proteins transcribe transfected tRNA and adenovirus VAI genes at greater than 10-fold higher levels than uninfected HeLa cells. Here we show that the increased transcription observed in vivo is reflected in the in vitro transcriptional activity of cell extracts. Depletion of E1A protein from these extracts by immunoprecipitation with a monoclonal antibody did not diminish the activity, suggesting that E1A proteins do not stimulate transcription directly. Fractionation of the extracts by chromatography on phosphocellulose suggests that the higher activity of extracts of adenovirus-infected cells was due to increased activity of the transcription factor (TF) which is the limiting component required for specific initiation of tRNA and VAI transcription in extracts of uninfected HeLa cells, i.e. TFIIIC. Template commitment titrations further suggest that the increased TFIIIC activity was due to an increase in the concentration of active TFIIIC. On the basis of these results and recent genetic analyses of early adenovirus promoters, we suggest that E1A proteins stimulate transcription of adenovirus genes indirectly by increasing the effective in vivo concentration of the limiting cellular transcription factors required for their transcription.

Adenovirus Early Proteins↗
PubMed1991

The tissue-specific mammalian transcription factor, Pit-1, activates transcription in Saccharomyces cerevisiae.

Pit-1 is a tissue-specific transcription factor which binds to specific DNA sequences within 5' flanking regions of the PRL and GH genes and activates the transcription of these genes. Previous studies have shown that expression of Pit-1 is necessary to activate transcription from the PRL or GH promoters in heterologous mammalian cells. In the present study the ability of Pit-1 expression vectors to activate expression of reporter genes in Saccharomyces cerevisiae was examined. The test system used Pit-1 expression vectors and an indicator plasmid containing multiple copies of a Pit1-binding site as a replacement for the upstream activator sequence of the CYC1 promoter. Significant activation of indicator plasmid expression was detected only in the presence of functional Pit-1 expression vectors. In both mammalian and yeast cells, amino-terminal deletions of the Pit-1 coding sequence produced similar and gradual loss of transcriptional activation. This finding indicates that similar or identical regions of Pit-1 are required for transcriptional activation in mammalian and yeast cells. Although synthetic DNA elements containing multiple copies of a single Pit-1-binding site were sufficient to permit Pit-1-mediated transcriptional activation in both yeast and mammalian cells, DNA fragments representing the proximal region or distal enhancer region of the PRL gene were transcriptionally active only in mammalian cells. These studies establish the ability of Pit-1 to stimulate transcription in the absence of other tissue-specific factors and provide a system for further genetic studies of Pit-1 structure/function relationships as well as evaluation of target sequences necessary for Pit-1 action.

Animals↗
PubMed1992

Two distinct yeast transcriptional activators require the function of the GCN5 protein to promote normal levels of transcription.

When yeast cells are grown under conditions of amino acid limitation, transcription of amino acid biosynthetic genes is increased through the action of the GCN4 transcriptional regulator. gcn5 mutant strains exhibit poor growth under such conditions. We have established that GCN4 requires the function of GCN5 in order to promote normal levels of transcriptional activation. In addition, we have shown that GCN5 is also required for the activity of the HAP2--HAP3--HAP4 transcriptional activation complex, which mediates the transcription of genes involved in respiratory functions. Thus, GCN5 is a new member of the recently revealed general class of transcriptional regulators that collaborate with certain specific DNA binding activators to promote high levels of transcription. We have cloned and sequenced the GCN5 gene. The deduced GCN5 protein contains a region conserved in other yeast, Drosophila and human proteins, all members of this new class of transcriptional activators.

Amino Acid Sequence↗
PubMed1988

Transcription analysis of a human U4C gene: involvement of transcription factors novel to snRNA gene expression.

We have investigated the promoter requirements for in vivo transcription of a human U4C snRNA gene following transfection into HeLa cells. Two elements required for maximal U4C transcription were identified. The first, located upstream of -50, provides a basal level of transcription 2-3% of the full activity, and probably corresponds to the previously identified snRNA gene proximal element. The distal element, centered around -220, acts as a transcriptional enhancer and contains motifs for three previously recognized transcription factors: the octamer-binding protein, NF-A, which binds to motifs in the distal elements of other snRNA genes, and two factors not previously shown to be involved in snRNA gene transcription, cAMP response element binding protein (CREB) and AP-2. The octamer and putative AP-2 motifs are required for maximal transcription of the U4C gene. Specific binding of NF-A and CREB to the motifs in the distal element has been shown in vitro by DNase I and DMS methylation protection footprint competition analyses using HeLa nuclear extracts. The presence of a binding motif for the inducible factor CREB, together with the transcriptional requirement for the putative AP-2 motif, suggests a means by which expression of snRNA genes might be regulated.

DNA-Binding Proteins↗
PubMed1985

Regulation of transcription of the repA1 gene in the replication control region of IncFII plasmid NR1 by gene dosage of the repA2 transcription repressor protein.

Transcription of the repA1 gene of the IncFII plasmid NR1 is initiated at two promoters in the replication control region. Transcription from the upstream promoter is constitutive at a low level, whereas transcription from the downstream promoter is regulated. The 5' end of the constitutively synthesized transcript also encodes the transcription repressor protein for the regulated downstream promoter. Therefore, the level of the repressor protein in the cell is gene dosage dependent. Using both lac gene fusions and quantitative hybridization methods, we have determined the in vivo relationship between the rate of transcription from the regulated promoter and the repressor protein concentration as a function of gene dosage. At the wild-type copy number of NR1, transcription from the regulated promoter is 96% repressed, but substantial derepression occurs when the copy number falls below the normal value. At or above the normal plasmid copy number, the basal level of repA1 mRNA is provided by transcription from the constitutive upstream promoter.

Adenosine Triphosphatases↗
PubMed1992

Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor.

The DNA-binding proteins which recognize the regulatory sequence elements of the hepatitis B virus (HBV) major surface antigen promoter were examined by gel retardation analysis, using nuclear extracts from the human hepatoma cell line Huh7. Using this assay, we identified four regions (B, D, E, and F) of the promoter that interact with the same or similar transcription factor(s). In addition, the recognition sequence for the Sp1 transcription factor bound the same or similar transcription factor(s) present in Huh7 cell nuclear extracts, and this binding was inhibited by the four major surface antigen promoter elements, B, D, E, and F. Purified Sp1 transcription factor was shown to bind to three (B, D, and F) of the major surface antigen promoter regulatory sequence elements by DNase I footprinting. Using transient transfection assays with Drosophila Schneider line 2 cells, we found that transcription from the major surface antigen promoter was transactivated by exogenously expressed Sp1, whereas transcription from the other three HBV promoters was not. Deletion analysis of the major surface antigen promoter demonstrated that the promoter region between -35 and +157 was sufficient to confer Sp1 responsiveness. This promoter region includes one of the regulatory elements footprinted by the purified Sp1 transcription factor. The function of the B, D, E, and F promoter elements was further examined by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. This finding demonstrates that the HBV major surface antigen promoter contains four functional Sp1 binding sites which probably contribute to the level of expression from this promoter during viral infection.

Antigens, Viral↗
PubMed1992

Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor.

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.

Base Sequence↗
PubMed1990

A transcriptional terminator between enhancer and promoter does not affect remote transcriptional control.

Enhancers stimulate transcription of RNA polymerase II-transcribed genes in an orientation-independent manner and over long distances. This stimulation is known to be associated with an increased polymerase density over the linked gene. However, many aspects of the exact mechanism of remote gene control remain to be elucidated. Based on some reports on RNA polymerase I transcription, we wanted to test whether RNA polymerase II enters at the enhancer and from there proceeds towards the promoter while synthesizing unstable transcripts ("scanning/readthrough transcription" model). For this, we have inserted a complete terminator region from the mouse beta-globinmaj gene between the SV40 enhancer and the rabbit beta-globin promoter. In contrast to what the model predicts, insertion of the terminator had no affect on remote enhancer action. Furthermore, we have determined the RNA polymerase density over the spacer DNA between enhancer and promoter, and over the reporter gene, by means of the so-called run-on transcription assay. We find very low transcription of the spacer, but high transcription of the globin reporter gene. Thus, our data are not consistent with a scanning/readthrough transcription mechanism where RNA polymerase II would move from the enhancer to the promoter while transcribing the intervening spacer DNA. These and other findings are compatible with a model where enhancer and promoter are brought into close proximity, perhaps with concomitant looping out of the intervening DNA.

Animals↗
PubMed1989

Induction of heat shock protein transcripts and B2 transcripts by various stresses in Chinese hamster cells.

We have investigated the induction of known hsp (heat shock protein) RNA and other heat shock (HS) inducible transcripts in Chinese hamster cells by various stresses including DNA damaging agents. cDNA clones coding for at least 14 different HS-inducible transcripts were isolated. By DNA sequence analysis and homology with cDNA clones of other species, some of these cDNA clones were identified as coding for hsp27, hsp89 alpha, hsp89 beta, two different hsp70s, ubiquitin, and the HS-inducible RNA polymerase III transcript B2. In addition, hsp-related cDNA clones, hsp60 and four with hsp70 homology, were isolated which coded for transcripts which were not induced by HS or other stresses in two different Chinese hamster cell lines. After HS or treatment with the HS-mimetic agent ethanol, there was coordinate induction of all 14 transcripts. With severe HS treatments which produced substantial cytotoxicity, the increase in all transcripts except B2 RNA was delayed and, in some cases, suppressed. The only DNA damaging agent, which induced many HS-inducible transcripts, was high-dose methylmethane sulfonate (MMS). However, induction by MMS was not coordinate for all transcripts as it was for HS, and B2 RNA was not induced. hsp27 RNA induction differed from the others in several respects including induction by irradiation and other agents which produce high levels of DNA damage repaired by nucleotide excision repair. The implications of these findings in cellular events such as cytotoxicity, thermotolerance, and regulation of stress responses will be discussed.

Animals↗
PubMed1979

Transcription maps of mtDNAs of two strains of saccharomyces: transcription of strain-specific insertions; Complex RNA maturation and splicing.

We have developed a two-dimensional method for simultaneously mapping on the yeast mtDNA genome all the transcripts representing more than 0.01% of mtRNA. In two yeast strains, Saccharomyces carlsbergensis NCYC-74 and Saccharomyces cerevisiae KL14-4A, about 25 discrete transcripts were found apart from tRNAs. The mtDNAs of these strains differ by the absence (NCYC-74) or presence (KL 14-4A) of various large insertions located within genetically active regions. The transcripts can all be related to known loci on the genetic map. In nearly all cases the RNAs are much longer than required to specify the known protein product of the locus concerned. The organization of the transcripts is similar in the two strains except at the positions of the large insertions (500-3300 bp) in the oxi-3 and cob loci. The sequences of these insertions are present in RNA species larger than 25S, but are absent from smaller transcripts of the same regions. This is probably due to splicing, since the coding sequences for most of these smaller transcripts are noncontiguous. The smaller transcripts of other loci also seem to arise from processing of larger RNA species. The oxi-3 locus, containing the structural gene for cytochrome c oxidase subunit l, is transcribed in a very complex fashion that suggests differential splicing into partially overlapping transcripts. This may indicate that oxi-3 has additional genetic functions, including possible control of the biosynthesis of cytochrome c oxidase holoenzyme or its assembly into the mitochondrial inner membrane. As in the case of the eucaryote nucleus, the regulation of mitochondrial gene expression seems to occur more at the level of RNA processing than has been recognized thus far.

Chromosome Mapping↗
PubMed1991

Effects of protein synthesis inhibition on the transcription and transcript stability of Dictyostelium prespore genes.

The in vivo accumulation of several prespore transcripts of Dictyostelium discoideum has previously been shown to depend upon concomitant protein synthesis (Ratner, D.I., Pentz, W.H. and Pelletier, D.A. (1989) Biochim. Biophys. Acta 1008, 71-78). Measurements of in vivo mRNA decay and nuclear run-on transcription assays have now been used to learn whether protein synthesis is required primarily for mRNA synthesis or transcript stability. The translational inhibitors cycloheximide and pactamycin stabilized existing prespore transcripts, despite their effect upon mRNA accumulation. Transcriptional assays, performed at intervals throughout the developmental cycle, demonstrated that temporal changes in the abundance of several cell-specific transcripts correlated closely with changes in their rates of synthesis. Finally, blocking protein synthesis strongly inhibited the transcription of the prespore genes examined. These results imply that one or more developmentally regulated, labile proteins are needed for the activation of prespore gene transcription.

Cycloheximide↗
PubMed1988

Transcriptional analysis of multisite drug-DNA dissociation kinetics: delayed termination of transcription by actinomycin D.

An in vitro transcription assay was used to measure the relative occupancy, sequence specificity, and dissociation kinetics of six actinomycin D binding sites on DNA during conditions of active transcription of the DNA from the lac UV5 promoter. Five of the sites contained a GpC sequence, with three of these having a common AGCT sequence that differed by up to an order of magnitude in affinity for the drug, as indicated by their relative occupany and dissociation kinetics. Positive cooperativity was observed by higher occupancy and slower dissociation kinetics for neighboring GpC sites on a different DNA fragment (UV5-lambda PL). Termination of transcription was observed at some drug binding sites, while complete drug-induced termination of transcription was seen 7-10 nucleotides downstream of two drug sites. This delayed termination was minimized when ITP was incorporated into the transcripts and suggests that a time delay is required to enable stable RNA hairpin helices to form. A model is presented of the role of RNA hairpin helices in delayed, drug-induced termination of transcription. The classical picture of DNA-binding drugs as inhibitors of transcription now appears too simplistic as it does not accommodate this phenomenon. It will be important to gain a greater understanding of the mechanism of this phenomenon of drug-induced termination of transcription, as there are many implications for the design of DNA-acting drugs.

Bacterial Proteins↗
PubMed1977

Transcription of yeast DNA by homologous RNA polymerases I and II: selective transcription of ribosomal genes by RNA polymerase I.

Purified yeast DNA was transcribed by homologous RNA polymerases I and II and Escherichia coli RNA polymerase. Transcripts synthesized in vitro were analyzed by molecular hybridization with complementary DNA (cDNA) synthesized from yeast poly(A)-containing mRNA with viral reverse transcriptase and ribosomal DNA labeled in vitro by nick translation with E. coli DNA polymerase I. RNA synthesized by polymerase I and II in the presence of Mn2+ contained sequences complementary to cDNA and rDNA at a frequency consistent with random transcription of the template. Similarly, E. coli RNA polymerase synthesized an apparently random transcript in the presence of either Mn2+ or Mg2+. In contrast to these results, RNA polymerase I but not polymerase II transcripts were markedly enriched in sequences complementary to rDNA when transcription was carried out in the presence of Mg2+. The observed enrichment was 15-30-fold higher than observed for polymerase II or E. coli polymerase transcripts and is consistent with the transcript being comprised of 6-10% ribosomal sequences. These data strongly suggest that RNA polymerase I plays a critical role in selective transcription of ribosomal cistrons.

DNA-Directed RNA Polymerases↗
PubMed1990

The c-ets proto-oncogenes encode transcription factors that cooperate with c-Fos and c-Jun for transcriptional activation.

Cell transformation by oncogenes leads to changes in gene expression. A key event in this process seems to be activation of the transcription factors AP-1 and PEA 3. Their synergistic activities are required for efficient activation of transcription from different promoters by many different oncogenes, serum growth factors and the tumour promoter TPA. We show here that the products of the ets-1 and -2 proto-oncogenes, whose biological function was previously unknown, are transcription factors that activate transcription through the PEA 3 motif. The p68c-ets-1 protein specifically binds to DNA and contains a transcriptional activation domain. The ets-like gene family therefore seems to encode a new family of transcription factors, apparently unrelated to other transcription factors. The p68c-ets-1 protein cooperates with c-Fos and c-Jun (components of AP-1) for activation of transcription from the oncogene-responsive domain of the polyoma enhancer, indicating that combined activity of all three oncoproteins could be involved in the response of cells to growth stimuli.

Base Sequence↗
PubMed1985

Translation activates the paused transcription complex and restores transcription of the trp operon leader region.

It has been proposed that RNA polymerase pausing in the leader region of the tryptophan (trp) operon of Escherichia coli is responsible for the synchronization of transcription and translation essential to attenuation control. In this report we use an in vitro coupled transcription/translation system to study the effect of trp leader peptide synthesis on RNA polymerase pausing in the trp leader region. Wild-type and translation-defective trp leader templates of E. coli and Serratia marcescens were employed, and pause RNA synthesis and paused complex release (activation) were quantified relative to synthesis of the terminated leader transcript. It was observed that pausing in the trp leader region was prolonged when translation of the leader transcript was reduced by mutations in the leader region or by addition of the translation inhibitor kasugamycin or chloramphenicol. Experiments with S-30 extracts from a mutant strain that is inefficient in translating the tryptophan codons in the leader transcript indicated that ribosome movement to these codons also releases the paused transcription complex. These findings indicate that the paused trp leader transcription complex resumes transcription when released by ribosome movement over the leader peptide coding region. This release would facilitate the coupling of transcription and translation essential to attenuation control.

Amino Acid Sequence↗
PubMed1985

Transcriptional and post-transcriptional regulation of storage protein gene expression in sulfur-deficient pea seeds.

We have used in vitro transcription in isolated pea nuclei to determine whether the expression of genes for the seed storage proteins legumin and vicilin is regulated transcriptionally or post-transcriptionally in response to changing sulfur status in the developing seed. During the first 48 h of recovery from S deficiency, transcription of legumin genes increased by two-fold whereas the level of legumin mRNA increased 20-fold in the same period. Vicilin gene transcription decreased during recovery, consistent with a gradual decrease in mRNA levels. We conclude that regulation of legumin mRNA levels in response to changing S status is post-transcriptional, whereas that of vicilin mRNA is transcriptional. In contrast, during normal plant development the expression of both of these gene families appears to be under transcriptional control.

Gene Expression Regulation↗
PubMed1988

Expression of herpes simplex virus type 1 (HSV-1) latency-associated transcripts and transcripts affected by the deletion in avirulent mutant HFEM: evidence for a new class of HSV-1 genes.

During latent herpes simplex virus type 1 (HSV-1) infection in the trigeminal ganglia of mice, three virus-specific transcripts, 2.0, 1.5, and 1.45 kilobases (kb), are detectable by Northern (RNA) blot analysis, but only the 2.0-kb transcript can be detected in HSV-1-infected tissue culture cells (J.G. Spivack and N. W. Fraser, J. Virol. 61:3842-3847, 1987). Since these latency-associated genes map to a diploid region of the genome, transcription from the deletion mutant HFEM, which contains only one complete copy of these genes, was investigated to determine the effect of gene dosage. The 4.1-kb HFEM deletion is located between the alpha genes ICP0 and ICP27. ICP0 mRNA and the 2.0-kb latency-associated transcript were present at normal levels during HFEM infection, but ICP27 mRNA and 0.9- and 1.1-kb transcripts that map near the deletion were not readily detectable. The levels of expression of one or more of these genes might be an important determinant of HSV-1 virulence in animal hosts. ICP27 mRNA accumulated when protein synthesis was inhibited before HFEM infection, implying that the deletion may affect ICP27 regulatory rather than coding elements. Expression of the 2.0-kb latency-associated transcript was characterized in infected CV-1 cells with metabolic inhibitors and strand-specific probes. On the basis of metabolic inhibitor studies, the gene encoding the 2.0-kb latency-associated transcript is not an alpha gene. During HSV-1 replication in infected tissue culture cells, the beta and gamma genes require the prior expression of alpha gene products. However, the latency-associated RNAs are expressed in the absence of detectable levels of alpha transcripts in latently infected mice. Thus, this latency-associated gene family appear to be regulated quite differently than alpha, beta, or gamma genes. For these reasons, and because the latency-associated genes may perform latent rather than replicative functions, we propose that they should be considered members of a new HSV-1 gene class, the lambda genes.

Cell Line↗