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At least 541 records · Page 30Linked to original sources
PubMed1986

Requirement of A-A-U-A-A-A and adjacent downstream sequences for SV40 early polyadenylation.

RNA mapping experiments and chloramphenicol acetyltransferase assays were used to analyze polyadenylation in COS cells of transcripts from derivatives of pSV2-neo and pSV2-cat, in which the SV40 early poly(A) signal has been modified. Neither the sequence A-A-U-A-A-A nor the sequences located immediately downstream from it in the SV40 early gene appear to function by themselves as a poly(A) signal. When combined, however, these two elements form a poly(A) signal whose efficiency and specificity closely resemble those of the wild type signal. The addition of six nucleotides between the A-A-U-A-A-A sequence and the poly(A) site has no detectable effect on the efficiency or site of polyadenylation. Deletion of the 60 nucleotides immediately upstream from the hexanucleotide also has no detectable effect on polyadenylation. Therefore, A-A-U-A-A-A and sequences downstream from it appear to be sufficient for SV40 early polyadenylation.

Animals↗
PubMed1987

The human beta-globin gene contains a downstream developmental specific enhancer.

The human beta-globin gene is part of a multigene family and is expressed specifically in adult human erythroid tissue (for review, 1). When the human beta-globin is introduced into fertilized mouse eggs, it is first activated in foetal liver and remains expressed in adult erythroid tissues. It therefore mimicks the pattern of expression of its murine counterpart. It has previously been shown in tissue culture and transgenic mice that sequences downstream from the beta-globin promoter are involved in this regulation. We now show that at least part of these sequences are located 0.5-1.2kb downstream from the poly A addition site and constitute a transcriptional enhancer element that is erythroid and developmental specific.

Animals↗
PubMed1988

Upstream and downstream transcriptional control signals in the yeast retrotransposon, TY.

The yeast retrotransposon, Ty, shares many structural and functional features with retroviral proviruses. These include production of a terminally redundant major transcript. There are also two less abundant transcripts of 5.0 kb and 2.2 kb. Ty transcription is regulated by cell-type, that is it is reduced 5-20 fold in a/alpha diploids as compared to haploids. However control of expression of Ty is not well understood. By deletion analysis we have identified regions of the element which are involved in the activation and regulation of transcription. These signals are found both upstream and downstream of the mRNA start site. The downstream signals are within the region encoding the major Ty proteins. This organisation of transcriptional control signals is discussed with reference to the organisation of control signals in other yeast genes and in retroviral proviruses and other retro-elements.

Base Sequence↗
PubMed1990

A highly conserved enhancer downstream of the human MLC1/3 locus is a target for multiple myogenic determination factors.

A potent muscle-specific enhancer element, originally described in the rat myosin light chain (MLC) 1/3 locus located downstream of the coding region, is found in an analogous position in the human MLC1/3 gene. When linked to a CAT reporter gene and transfected into muscle or non-muscle cells, the human MLC enhancer directs high levels of muscle-specific gene expression from homologous or heterologous promoters, irrespective of position or orientation relative to the CAT transcription unit. A significant degree of sequence homology (over 85%) in the 3'-flanking regions of the two MLC genes is restricted to a 200 bp sequence which lies approximately 1.5 kb downstream of the polyadenylation site in both species. The human enhancer sequence includes binding sites for human myogenic determination factors containing a common basic helix-loop-helix motif, and it can be trans-activated to varying degrees in non-muscle cells by these factors. This study establishes the MLC enhancer as an evolutionarily conserved, integral component of the MLC1/3 locus which constitutes a novel target for the action of myogenic determination factors.

Animals↗
PubMed1991

The promoter of the endo A cytokeratin gene is activated by a 3' downstream enhancer.

Mouse cytokeratin EndoA is an intermediate filament subunit of the type II cytokeratin class which initiates expression in trophectoderm cells of blastocyst during embryogenesis. To identify the regulatory elements of the endo A gene, we constructed a series of CAT expression vectors and transfected them into PYS-2 cells. We found an enhancer element locating 1 kb downstream from the endo A gene which acts on both the endo A and SV40 promoters. This enhancer consists of six direct repeated sequences with homology to the PEA3 motif in polyoma virus alpha enhancer core. In undifferentiated F9 embryonal carcinoma cells, expression of the construct containing the enhancer was not detected. These results indicate that one of the regulatory mechanisms of endo A gene expression is the 3' downstream enhancer.

Animals↗
PubMed1991

Cooperation between upstream and downstream elements of the adenovirus major late promoter for maximal late phase-specific transcription.

Transcription from the adenovirus major late promoter (MLP) is greatly stimulated during lytic infection, after replication of the viral DNA has started. This replication-dependent activation has previously been shown to be mediated by a positive regulatory cellular protein(s). Binding of this factor(s) to sequence elements (DE1 and DE2), located between positions +76 and +124, with respect to the MLP transcriptional startsite, is detected only after the onset of DNA replication. Using a cell-free transcription system which mimics the late phase induction of the MLP and DNA binding assays, we now present evidence showing that maximal stimulation also depends on the MLP upstream element (UE), without involving increased DNA binding activity of the corresponding factor (UEF) during the lytic cycle. Our results indicate that the upstream and downstream elements act cooperatively on transcription efficiency, although no direct interactions between the cognate factors could be demonstrated. These observations strongly suggest that the elevated rate of transcription originating at the MLP startsite, late in infection, results from the simultaneous action of factors bound at the upstream and downstream elements onto a common target within the basal transcription machinery.

Adenoviruses, Human↗
PubMed1993

The importance of downstream delta-factor binding elements for the activity of the rpL32 promoter.

A salient feature of mammalian ribosomal protein genes is the location of promoter elements downstream, as well as upstream, of the transcriptional start point. Previous functional studies of the mouse rpL32 gene (Chung and Perry, Mol. Cell. Biol. 9, 2075; 1989) indicated that the first intron of this gene contains such an element. We show here that this element encompasses a binding site for a zinc finger nuclear protein known as delta (YY-1, muE1, UCRBP). The intronic delta site (delta i) is located 32 bp downstream of another delta site in the first exon (delta e). Transfection experiments with genes containing deleterious mutations in one or both delta sites or having alterations in the spacing between the sites indicate that the two delta elements function independently and contribute additively to the overall strength of the rpL32 promoter. Moreover, the contribution of the delta i element is the same whether it is oriented parallel or antiparallel to the delta e element. Together, the two delta elements raise the expression level about 10-fold over that attained by the upstream and initiator portions of the promoter. The positive role of the delta factor in rpL32 expression contrasts strikingly with its repressive role in various other genes.

Animals↗
PubMed1995

Increasing binding of a transcription factor immediately downstream of the cap site of a cytomegalovirus gene represses expression.

A closely related family of ubiquitous DNA binding proteins, called MDBP, binds with high affinity to two 14 base pair (bp) sites within the human cytomegalovirus immediate early gene 1 (CMV IE1) enhancer and with low affinity to one site beginning 5 bp downstream of the CMV IE1 transcription start point (+5 site). Unlike several cap position downstream MDBP sites in mammalian genes, these MDBP sites do not require cytosine methylation for optimal binding. Mutation of one of the enhancer MDBP sites to prevent MDBP recognition modestly increased the function of a neighboring CREB binding site in a transient transfection assay in the context of one promoter construct. A much larger effect on reporter gene expression (a 10-fold reduction) was seen when the low affinity MDBP recognition sequence at position +5 was converted to a high affinity site in a plasmid containing the CMV IE1 promoter upstream of the reporter gene. Evidence that the increased binding of MDBP at the mutant site is largely responsible for the observed results was provided by transfection experiments with this high affinity MDBP +5 site re-mutated to a non-binding site and by in vitro transcription assay.

Base Sequence↗
PubMed1995

Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences.

We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied. We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency. The sequence of the G+C stem-loop itself also influences termination. Moreover, the dA template stretch at the 3' end of the terminator plays a major role in termination efficiency, but base pairing between the A and U tract of the transcript does not. From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner. The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.

Base Composition↗
PubMed1997

Effects of heterologous downstream sequences on the activity of the HIV-1 promoter and its response to Tat.

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.

Animals↗
PubMed2002

Contribution of downstream promoter elements to transcriptional regulation of the rice tungro bacilliform virus promoter.

Downstream sequences influence activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells. We previously identified a DNA element located between positions +50 and +90 relative to the transcription start site to which rice nuclear proteins bind. In this study, using DNA UV crosslinking assays, we show that two rice nuclear proteins bind specifically to this DNA element. We demonstrate that the DNA element enhances RTBV promoter activity in a copy number-dependent manner when transferred to a position upstream of the promoter. In addition, using electrophoretic mobility shift assays, we show that at least two novel nuclear proteins from rice cell suspension cultures bind to a subregion (from +50 to +59) of the DNA element and that a protein from rice root, but not shoot, nuclear extracts interacts with a perfect palindromic sequence motif located within the sequence +45 to +59. Furthermore, a position-dependent GAGA motif, present in three copies within downstream promoter sequences from +1 to +50, is involved in the regulation of RTBV promoter activity.

Badnavirus↗
PubMed2002

Glucose-inducible expression of rrg1+ in Schizosaccharomyces pombe: post-transcriptional regulation of mRNA stability mediated by the downstream region of the poly(A) site.

rrg1+(rapid response to glucose) has been isolated previously as a UV-inducible gene in Schizosaccharomyces pombe, designated as uvi22+. However, it was revealed that the transcript level of this gene was regulated by glucose, not by DNA-damaging agents. Glucose depletion led to a rapid decrease in the level of rrg1+ mRNA, by approximately 50% within 30 min. This effect was readily reversed upon re-introduction of glucose within 1 h. High concentrations (4 and 8%) of glucose showed similar effects on increasing the rrg1+ mRNA level compared with 2% glucose, while a low concentration (0.1%) was not effective in raising the rrg1+ mRNA level. In addition, sucrose and fructose could increase rrg1+ mRNA level. Interestingly, the rapid decline in mRNA level seen upon glucose deprivation resulted from precipitous reduction of mRNA half-life. Serial and internal deletions within the 3'-flanking region of rrg1+ revealed that a 210-nt region downstream of the distal poly(A) site was critical for glucose-regulated expression. Moreover, this downstream region participated in 3'-end formation of mRNA. Taken together, this is the first report on glucose-inducible expression regulated post-transcriptionally by control of mRNA stability in S.pombe.

3' Flanking Region↗
PubMed2006

The Saccharomyces cerevisiae RNA polymerase III recruitment factor subunits Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box.

Association of the TATA-binding protein (TBP) with its cognate site within eukaryotic promoters is key to accurate and efficient transcriptional initiation. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB. Previous data have suggested that the structure or dynamics of the TBP-DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. We show here, using the altered specificity TBP mutant TBPm3 and an iterative in vitro selection assay, that entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene. We suggest that the selected T*A base pair step at the downstream end of the 8 bp TBP site may provide a DNA flexure that promotes TFIIIB-DNA complex formation.

Base Sequence↗
PubMed1998

Downstream of phosphatidylinositol-3 kinase, a multifunctional signaling molecule, and its regulation in cell responses.

Phosphatidylinositol (PI)-3 kinase is an enzyme that phosphorylates the D-3 position of PI and its derivatives. It is activated immediately after growth factor or differentiation factor stimulation, suggesting that PI-3 kinase is involved in signal transduction of the stimulation. PI-3 kinase appears to play various important roles including signaling to the nucleus, vesicle transport, and rearrangement of the cytoskeleton since many cell responses which require these events are affected by inhibition or activation of PI-3 kinase. To understand how PI-3 kinase can act in such multiple ways, it is important to identify the factors downstream of PI-3 kinase. In this review, we discuss the factors downstream of PI-3 kinase and the methods used to identify them. Recent studies revealed that some proteins involved in vesicle transport or in rearrangement of the cytoskeleton are regulated by the phospholipids generated by PI-3 kinase, implying the mechanism by which PI-3 kinase regulates these cell responses.

Animals↗
PubMed2003

Cyclic adenosine monophosphate (cAMP) stimulation of the kit ligand promoter in sertoli cells requires an Sp1-binding region, a canonical TATA box, and a cAMP-induced factor binding to an immediately downstream GC-rich element.

Expression of Kit ligand (KL) mRNA is induced in primary prepuberal Sertoli cells by FSH and by other agents that increase cAMP levels. The cAMP effect is exerted at the transcriptional level and appears to be cell type specific, since it is not observed in other KL-expressing primary cells or cell lines. Deletion analysis of the 5'-flanking region of the mouse KL gene shows that the proximal promoter sequence between -88 and +8 from the transcriptional start site is necessary and sufficient to obtain the full cAMP responsiveness of the promoter in primary mouse Sertoli cells. In the -88/+8 promoter region, several cis-acting elements play a role in cAMP response. The -88/-56 sequence is necessary for full induction of the gene, since its removal causes a drastic decrease in cAMP responsiveness; however, cAMP-stimulated expression is still observed with the minimal promoter region between -55 and +8. A more detailed mutational analysis of the minimal promoter region shows that mutations in the canonical TATA box sequence and in an immediately downstream GC-rich element completely abolish cAMP responsiveness. DNA-binding experiments show that transcription factor Sp1 binds to the -88/-56 fragment of the KL proximal promoter in both control and cAMP-stimulated cells, whereas a new cAMP-induced complex is observed when the -55/+8 minimal promoter region is used as probe. The canonical TATA box sequence is essential for formation of the latter complex. We also show that the binding of an unknown nuclear factor (different from Sp1, Egr-1, Rnf6, and AP-2) to a GC-rich element between -19 and +8 increases after cAMP treatment, and this effect seems to be specific of primary Sertoli cells. Thus, cAMP-induced transcription from the KL gene promoter in primary mouse Sertoli cells is mediated by a complex interaction among a Sp1-binding region, factors recognizing the canonical TATA box sequence, and a not yet identified cAMP-induced factor binding a GC-rich sequence just downstream from it.

5' Flanking Region↗
PubMed2002

Identification of ras and its downstream signaling elements and their potential role in hamster sperm motility.

Ras, a member of the small G-protein family, regulates multiple signaling pathways in somatic cells. The objectives of the present study included the characterization and localization of Ras and the identification of its downstream effectors in hamster spermatozoa. Immunoblot analysis with a pan-Ras monoclonal antibody localized Ras to the particulate fraction of sonicated testicular and caput and cauda epididymal spermatozoa. However, Ras was present in both the particulate and soluble fractions of spermatocytes and round spermatids, suggesting that its membrane recruitment is completed during spermiogenesis. Immunoblots of plasma membrane fractions demonstrated that hamster spermatozoa express both N-Ras and K-Ras. Indirect immunofluorescence with pan-Ras antibody localized Ras to the flagellum. Immunoblot analysis of sperm plasma membrane fractions demonstrated the presence of phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C zeta (PKCzeta), the downstream targets of Ras, and coimmunoprecipitation analysis demonstrated their interaction with Ras. Inhibitors of PI3-kinase (wortmannin and 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and PKCzeta (staurosporine) inhibited the hyperactivation of sperm motility during capacitation in a dose-dependent manner, indicating that both PI3-kinase and PKCzeta are associated with development of this motility pattern. The interaction of Ras with both PI3-kinase and PKCzeta suggests that Ras may regulate several signaling pathways in spermatozoa.

Animals↗
PubMed2006

Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of alpha7 nicotinic receptor in oral keratinocytes.

The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and tobacco-derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly alpha7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into alpha7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of alpha7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real-time polymerase chain reaction (PCR) and In-cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT-3. These effects could be completely blocked or significantly (P<0.05) diminished if the cells were pretreated with the alpha7 antagonist alpha-bungarotoxin (alphaBTX) or transfected with anti-alpha7 small interfering RNA (siRNA-alpha7). The use of pathway inhibitors revealed that signaling through the Ras/Raf-1/MEK1/ERK steps mediated alpha7-dependent up-regulation of STAT-3. Targeted mutation of the alpha7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT-3 caused by ADSS or pure nicotine was mediated by janus-activated kinase (JAK)-2. Activation of JAK-2/STAT-3 pathway could be prevented by alphaBTX or siRNA-alpha7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from alpha7, which proceeds via two complementary pathways. The Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT-3, whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Elucidation of this novel mechanism of nicotine-dependent nuclear transactivation of STAT-3 identifies oral alpha7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco-related periodontal disease and progression of head and neck cancer by receptor inhibitors.

Animals↗
PubMed1999

Screening level dose assessment of aquatic biota downstream of the Marcoule nuclear complex in southern France.

Aquatic biota in the Rhone River downstream of the Marcoule nuclear complex in France are exposed to natural sources of radiation and to radioactivity released from the Marcoule complex. A simple conservative screening level model was used to estimate the range of concentrations in aquatic media (water, sediments, and aquatic organisms) of both artificial and natural radionuclides and the consequent absorbed (whole body) dose rates for aquatic organisms. Five categories of aquatic organisms were studied, namely, submerged aquatic plants (phanerogam), non-bottom-feeding fish, bottom-feeding fish, mollusca, and fish-eating birds. The analysis was based on the radionuclide concentrations reported in four consecutive annual radioecological monitoring reports published by French agencies with nuclear regulatory responsibilities. The results of this assessment were used to determine, qualitatively, the magnitude of any potential health impacts on each of the five categories of aquatic organisms studied. The range of dose rate estimates ranged over three orders of magnitude, with maximum dose rates estimated to be in the order of 1 to 10 microGy h(-1). These maximum dose rates are a factor 40 or more below the international guideline intended to ensure the protection of aquatic populations (about 400 microGy h(-1)), and a factor ten or more below the level which may trigger the need for a more detailed evaluation of potential ecological consequences to the exposed populations (about 100 microGy h(-1)). As a result, chronic levels of radioactivity, artificial and natural, measured in aquatic media downstream of Marcoule are unlikely to result in adverse health impacts on the categories and species of aquatic organisms studied. Thus, based on the screening level analysis discussed in this paper, a more detailed evaluation of the dose rates does not appear to be warranted.

Alpha Particles↗