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At least 595 records · Page 33Linked to original sources
PubMed1988

Multiple forms of the human gene-specific transcription factor USF. II. DNA binding properties and transcriptional activity of the purified HeLa USF.

The gene-specific upstream stimulatory transcription factor (USF) is required for maximal expression of the adenovirus major late promoter in vivo as well as in vitro. We have examined the DNA binding and transcriptional properties of USF purified to near-homogeneity from HeLa cell nuclei (Sawadogo, M., Van Dyke, M. W., Gregor, P. D., and Roeder, R. G. (1988) J. Biol. Chem. 263, 11985-11993). The 44-and 43,000-dalton forms of USF displayed identical affinities for the major late promoter upstream sequence. Specific binding parameters were greatly influenced by neighboring sequences, but not by the topological state of the DNA. The dissociation rate was highly dependent upon the concentration of competitor DNA, indicating that USF can efficiently transfer from one binding site to another by passing through a doubly bound intermediate state (direct transfer mechanism). Transcription stimulation by purified USF showed titration curves identical to those observed with cruder preparations of the transcription factor. However, the overall stimulation observed at saturating USF concentration was significantly lower with the purified protein. By contrast, interaction with TATA box-binding RNA polymerase II transcription factor D was observed with both USF-containing fractions. This could suggest the existence of two different mechanisms for upstream sequence-dependent transcription stimulation, where one critical component (or some necessary modification of the upstream factor itself) may be missing in reactions reconstituted with purified USF.

Adenoviridae↗
PubMed1994

Cooperative binding of GA-binding protein transcription factors to duplicated transcription initiation region repeats of the cytochrome c oxidase subunit IV gene.

Transcriptional activity of the TATA-less cytochrome c oxidase subunit IV (COXIV) gene promoter depends upon two tandemly repeated sequence elements, each mapping immediately downstream of major loci of transcriptional initiation. In this paper, we demonstrate that binding of the GA-binding protein (GABP) to ets sequence motifs within each repeated unit is required for transcriptional activation of the COXIV promoter. High affinity binding of GABP to the COXIV promoter required both the DNA-binding GABP alpha subunit and the non-DNA-binding GABP beta subunit. Binding of the heteromeric GABP complex to sequences containing two GABP binding sites was shown to have a 10-20-fold greater affinity than to DNA sequences with a single site. GABP binding was necessary for promoter function of a 33-base pair fragment of the COXIV initiation region in transfected 3T3 or COS cells. Binding of GABP to the COXIV initiation region was also required for maximal transcriptional stimulation by an upstream Sp1 binding site. The initiation region was demonstrated to direct accurate transcriptional initiation in vitro, and mutations to the GABP binding sites affected not only transcriptional activity but also initiation site selection. These results indicate that the initiation region repeats of the COXIV promoter may function as GABP-dependent initiator motifs that position mRNA start sites in the absence of a TATA box or other promoter elements.

Animals↗
PubMed1993

Identification of a transcriptional initiator element in the cytochrome c oxidase subunit Vb promoter which binds to transcription factors NF-E1 (YY-1, delta) and Sp1.

We have mapped the basal promoter activity of the mouse cytochrome c oxidase (COX) subunit Vb gene to the -17 to +20 region which contains two putative ets binding sites flanking an NF-E1 site fused to an Sp1 site. A 17-nucleotide sequence flanking the major transcription start site (-8 to +9), referred to as 17Inr (initiator sequence) was able to drive CAT activity in 3T3 cells to a level comparable to the construct containing sequences -17 to +20. This suggests that the 17Inr sequence contains the initiator activity. The 17Inr contains a pyrimidine-rich sequence, commencing with a CA that corresponds to the major transcription start site. Primer extension of RNA from transfected cells demonstrated that transcription initiation with the 17Inr template occurs at a site identical to the endogenous gene. DNA-protein binding by gel mobility shift and methylation interference analyses indicated that the pyrimidine-rich sequence immediately flanking the transcription start site consists of an NF-E1 factor binding motif with an overlapping upstream Sp1 binding site. A 13-nucleotide sequence, 13Inr (-4 to +9), which retains the NF-E1 binding activity but does not bind Sp1, was able to promote chloramphenicol acetyltransferase gene expression at levels similar to the 17Inr sequence, suggesting that NF-E1 factor binding is critical for initiator function. Finally, using an in vitro transcription system from Drosophila embryos we demonstrate that NF-E1 is necessary for transcription activation of both the 17Inr and the 13Inr initiator templates. Thus NF-E1 binding appears to be important for basal promoter function of the mouse COXVb gene.

3T3 Cells↗
PubMed1998

Mutations in and monoclonal antibody binding to evolutionary hypervariable region of Escherichia coli RNA polymerase beta' subunit inhibit transcript cleavage and transcript elongation.

A 190 amino acid-long region centered around position 1050 of the 1407-amino acid-long beta' subunit of Escherichia coli RNA polymerase (RNAP) is absent from homologues in eukaryotes, archaea and many bacteria. In chloroplasts, the corresponding region can be more than 900 amino acids long. The role of this hypervariable region was studied by deletion mutagenesis of the cloned E. coli rpoC, encoding beta'. Long deletions mimicking beta' from Gram-positive bacteria failed to assemble into RNAP. Mutants with short, 40-60-amino acid-long deletions spanning beta' residues 941-1130 assembled into active RNAP in vitro. These mutant enzymes were defective in the transcript cleavage reaction and had dramatically reduced transcription elongation rates at subsaturating substrate concentrations due to prolonged pausing at sites of transcriptional arrest. Binding of a monoclonal antibody, Pyn1, to the hypervariable region inhibited transcription elongation and intrinsic transcript cleavage and, to a lesser degree, GreB-induced transcript cleavage, but did not interfere with GreB binding to RNAP. We propose that mutations in and antibody binding to the hypervariable, functionally dispensable region of beta' inhibit transcript cleavage and elongation by distorting the flanking conserved segment G in the active center.

Amino Acid Sequence↗
PubMed2005

Allele-specific transcript quantification detects haplotypic variation in the levels of the SDF-1 transcripts.

It has been suggested that SDF1-G801A, a single nucleotide polymorphism (SNP) in the 3' untranslated region (UTR) of the SDF1 gene, is associated with susceptibility to diseases such as AIDS and type-I diabetes. However, experimental studies examining the effect of SDF1-G801A on SDF-1 expression have not supported its functional importance. In this study, to examine whether other polymorphisms have a cis-acting effect on SDF1 expression, we carried out haplotype analyses of the SDF1 gene and the allele-specific transcript quantification utilizing Epstein-Barr virus-transformed lymphoblastoid cell lines with heterozygous genotype for SDF1-G801A. Haplotype-based analyses on the proportion of the allele-specific transcripts revealed the presence of haplotypes associated with a decreased amount of the transcripts. In addition, we observed haplotypic variation in response to dibutyl cyclic AMP and tetradecanoyl phorbol acetate that enhances the levels of SDF-1 transcripts probably through activation of transcription factors. Showing evidence that polymorphisms other than the SDF1-G801A have a cis-acting effect on expression of SDF-1 transcripts, the results of this study contribute to the interpretation of previous disease-association studies and to the selection of SNP markers for future studies. As shown in this study, allele-specific transcript quantification coupled with haplotype analyses can be an effective tool for detecting cis-acting polymorphisms in expressional regulation.

Alleles↗
PubMed1997

Spliced human endogenous retroviral HERV-H env transcripts in T-cell leukaemia cell lines and normal leukocytes: alternative splicing pattern of HERV-H transcripts.

The majority of human endogenous retroviral HERV-H elements in the human genome have large deletions in pol and lack most of env, 5-10% are more or less complete with a potentially immunosuppressive transmembrane protein-encoding env region. Spliced HERV-H env transcripts were detected in T-cell leukaemia cell lines and lymphocytes from healthy blood donors by using RT-PCR. The transcripts all contained a splice donor in the leader region downstream from the primer-binding site and a previously unreported splice acceptor in the integrase-encoding region of pol, absent in the HERV-H deletion elements. In singly spliced transcripts the leader and integrase regions were joined directly whereas in multiply spliced transcripts they were joined with an alternative exon from the protease-encoding region located between the two regions. env transcripts from three different HERV-H elements were identified: one element similar to a HERV-H consensus sequence was primarily amplified from the T-cell leukaemia cell lines and two other more defective elements were amplified from normal lymphocytes. One of these elements was shown to be a reintegrated spliced transcript where the protease and integrase regions were joined, removing most of pol but leaving gag intact. Other spliced transcripts, joining the protease region and the 3'-LTR, were also amplified. The fact that HERV-H elements with an intact env splice acceptor also use the splice sites in the protease-encoding region suggests that this unusual multiple splice pattern could have a biological function in the intact HERV-H.

Alternative Splicing↗
PubMed1997

A Transcriptionally Active State Is Required for Post-Transcriptional Silencing (Cosuppression) of Nitrate Reductase Host Genes and Transgenes.

Using tobacco nitrate reductase cosuppression as a model system of post-transcriptional gene silencing, we analyzed the influence of DNA and RNA dosages both together and independently. For this purpose, zero, one, two, or four active or transcriptionally silenced copies of a cauliflower mosaic virus 35S-Nia2 transgene were combined by transformation and subsequent crosses with zero, one, two, three, or four active, disrupted, or transcriptionally repressed copies of the wild-type host Nia genes. The analysis of the corresponding transgenic lines revealed that (1) the percentage of isogenic plants that are affected by cosuppression depends directly upon the relative dosage of both host gene and transgene; (2) transcriptional silencing of the 35S-Nia transgene impedes cosuppression; and (3) the absence of host gene transcription reduces the frequency of cosuppression or delays its triggering. Taken together, these results indicate that transgene DNA per se is not sufficient to trigger post-transcriptional cosuppression of nitrate reductase host genes and transgenes. The requirement for a transcriptionally active state is discussed with respect to both the RNA dosage and the DNA-DNA pairing hypotheses.

Journal Article↗
PubMed1998

Expression kinetics and mapping of the thymidine kinase transcript and an immediate-early transcript from channel catfish virus.

Three transcripts from the terminal repeat of the channel catfish virus (CCV; also known as ictalurid herpesvirus 1) genome were mapped by S1 nuclease and primer extension analyses as well as by cDNA sequencing. These transcripts, TR3, TR5/6, and TR6, are encoded by open reading frame (ORF) 3, ORFs 5 and 6, and ORF 6, respectively, and correspond to those previously identified by sequence analysis (A. J. Davison, Virology 186:9-14, 1992). ORF 5 has previously been determined to encode thymidine kinase, but ORF 3 and ORF 6 encode proteins of unknown function. Although all three transcripts accumulate to high levels in cells infected in the presence of cycloheximide, kinetic analysis demonstrates that TR5/6 and TR6 are either early or late transcripts that leak through the cycloheximide block. In addition, two transcripts from the terminal repeat of the CCV genome that were mapped previously and were thought to be immediate-early in character, TR8a/9 and TR9, exhibit kinetics characteristic of early or late transcripts. TR3 is an immediate-early transcript that appears to have a very short half-life. In the 3' untranslated region of TR3, there are three copies of an AU-rich element which has previously been shown to be involved in destabilization of the oncogene c-fos and granulocyte/macrophage colony-stimulating factor mRNAs. mRNA destabilization may represent another mechanism by which herpesviruses regulate the rapid switch in expression from immediate-early genes to early genes during the transition to the early phase of infection.

Animals↗
PubMed2004

Panel of genes transcriptionally up-regulated in squamous cell carcinoma of the cervix identified by representational difference analysis, confirmed by macroarray, and validated by real-time quantitative reverse transcription-PCR.

BACKGROUND: The Pap smear is currently the most widely used method of screening for squamous cell carcinoma of the cervix (SCCC). Because it is based on cell morphology, it is subject to variability in interpretation. Sensitive molecular markers capable of differentiating cancerous samples from noncancerous ones would be beneficial in this regard. METHODS: We performed representational difference analysis (RDA) using paired, noncancerous (normal) and cancerous (disease) tissues taken from the same specimen obtained from a single patient with a confirmed diagnosis of SCCC. Linearly amplified cDNA from normal and diseased tissues of the original patient and seven others were hybridized to DNA macroarrays containing the candidate gene transcript fragments. Real-time quantitative reverse transcription-PCR was used to validate the macroarray results. RESULTS: RDA identified a candidate pool of 65 transcript fragments up-regulated in diseased tissue compared with normal tissue. Forty-one transcripts were found to be up-regulated in diseased compared with normal tissue in at least one half the patients by macroarray hybridization. Eleven of those genes were selected for real-time quantitative reverse transcription-PCR analysis, and all were confirmed as transcriptionally up-regulated in cancer compared with normal tissue in at least one half the patients. CONCLUSIONS: RDA using tissues from a single patient identified gene fragments confirmed to be transcriptionally up-regulated in SCCC both in the original patient and in seven others. The confirmed genes have a variety of functions and also have the potential to serve as diagnostic or prognostic markers.

Carcinoma, Squamous Cell↗
PubMed1993

Studies on the link between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in lymph-fistula rats: evidence for both transcriptional and post-transcriptional mechanisms for down-regulation of the two enzymes by bile acids.

The mechanism of down-regulation of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase by cholic acid (CA) and chenodeoxycholic acid (CDCA) was investigated in rats with a lymph fistula. In accordance with the contention that bile acids are able to down-regulate HMG-CoA reductase by a mechanism independent of their effect on cholesterol absorption, inclusion of the above bile acids in diet (1%, wt/wt) reduced the HMG-CoA reductase specific activity by about 80%. The levels of mRNA were reduced by about 50% and the transcription rate (nuclear run-on) by 27% and 35%, respectively. Thus, HMG-CoA reductase appears to be regulated at both a transcriptional and a post-transcriptional level by bile acids. The cholesterol 7 alpha-hydroxylase activity as well as the corresponding mRNA levels were both reduced by about 50% by the same treatments. The transcription rate of the cholesterol 7 alpha-hydroxylase was not significantly affected by the treatment with CDCA, whereas the CA treatment reduced the transcription rate by about 24%. It is evident that bile acids regulate these two enzymes by different mechanisms in this model, in spite of the fact that the levels of mRNA were affected to about the same degree. The most obvious difference was the marked suppression of HMG-CoA reductase activity by cholic acid with relatively modest effects on transcription rate and level of mRNA. The latter finding is interesting in view of recent reports that dietary cholesterol also seems to down-regulate HMG-CoA reductase predominantly by a post-transcriptional mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Multiple transcripts of human endothelin-A receptor gene detected by reverse transcription and the polymerase chain reaction.

To elucidate the regulatory mechanism of gene expression of the human endothelin-A receptor (hET-AR), we characterized the hET-AR transcripts using reverse transcription (RT) and polymerase chain reaction (PCR) analysis in a variety of human tissues. The RT-PCR using a set of primers in exons 2 and 5 showed two lower-molecular-weight transcripts in addition to the expected fragment. PCR cloning of these two novel transcripts revealed that these transcripts contain a 199-base pair (bp) and a 327-bp deletion compared with the previously described hET-AR cDNA, respectively. Comparison of their sequences with that of the hET-AR gene showed that the lacking sequences exactly correspond to exon 4 and exons 3 and 4, respectively, suggesting that these lower-molecular-weight ET-AR transcripts may result from alternative RNA splicing. Therefore, we isolated the cDNAs of novel transcripts of hET-AR that might be generated by alternative RNA splicing. These results suggest that the alternative RNA splicing might contribute to the regulation of ET-AR gene expression.

Base Sequence↗
PubMed1993

Human transcription factor USF stimulates transcription through the initiator elements of the HIV-1 and the Ad-ML promoters.

Earlier in vitro studies identified USF as a cellular factor which activates the adenovirus major late (Ad-ML) promoter by binding to an E-box motif located at position -60 with respect to the cap site. Purified USF contains 44 and 43 kDa polypeptides, and the latter was found (by cDNA cloning) to be a helix-loop-helix protein. In this report, we demonstrate a 25-to 30-fold stimulation of transcription via an upstream binding site by ectopic expression of the 43 kDa form of USF (USF43) in transient transfection assays. More recent data have also revealed alternate interactions of USF43 at pyrimidine-rich (consensus YYAYTCYY) initiator (Inr) elements present in a variety of core promoters. In agreement with this observation, we show here that USF43 can recognize the initiator elements of the HIV-1 promoter, as well as those in the Ad-ML promoter, and that ectopic expression of USF43 can stimulate markedly the corresponding core promoters (TATA and initiator elements) when analyzed in transient co-transfection assays. Mutations in either Inr 1 or Inr 2 reduced the USF43-dependent transcription activity in vivo. In addition, in vitro transcription assays showed that mutations in either or both of the Inr 1 and Inr 2 sequences of the HIV-1 and Ad-ML promoters could affect transcription efficiency, but not the position of the transcriptional start site. These results indicate that USF43 can stimulate transcription through initiator elements in two viral promoters, although the exact mechanism and physiological significance of this effect remain unclear.

Base Sequence↗
PubMed1994

A TATA sequence-dependent transcriptional repressor activity associated with mammalian transcription factor IIA.

In the process of characterizing cellular proteins that modulate basal transcription by RNA polymerase II, we identified a novel repressor activity specific for promoters containing consensus TATA boxes. This activity strongly represses TATA-binding protein (TBP)-dependent transcription initiation from core promoter elements containing a consensus TATA sequence, but activates TBP-dependent transcription from core promoter elements lacking a consensus TATA sequence. Purification of this activity to near homogeneity from rat liver nuclear extracts led to the surprising discovery that it co-purifies closely with mammalian transcription factor IIA (TFIIA). The close association of TATA sequence-dependent transcriptional repressor activity with TFIIA adds a new and unexpected dimension to the already complex picture of this factor's function in transcription by RNA polymerase II.

Animals↗
PubMed1997

Alternate COX-2 transcripts are differentially regulated: implications for post-transcriptional control.

Prostaglandin (PG) synthesis during inflammation occurs mainly via the transcriptionally regulated cyclooxygenase, COX-2. In pulmonary type II A549 cells, Northern analysis identified multiple IL-1 beta-inducible COX-2 mRNA transcripts. Amplification of 3'-cDNA ends by anchored PCR revealed products corresponding to the predominant 4.5 and 2.7 kb transcripts. Sequence analysis of amplification products indicated that these transcripts arose by alternate consensus and non-consensus polyadenylation site usage. The predominant 4.5 kb transcript showed a half-life in excess of two hours that was further stabilized by IL-1 beta. In addition, the COX-2 3'-untranslated region (UTR), which contains 22 copies of the putative RNA instability motif, AUUUA, when cloned downstream of a constitutively expressed luciferase gene, was found to confer partial IL-1 beta responsiveness in LA-4 cells. Finally, in vivo in LPS-treated rats, differential expression of similar COX-2 mRNA isoforms was also observed. Taken together these data suggest a functional role for post-transcriptional mechanisms, including alternate polyadenylation, in the control of COX-2.

Animals↗
PubMed1994

Irradiation increases expression of GM-CSF in human fibroblasts by transcriptional and post-transcriptional regulation.

Fibroblasts produce a variety of cytokines including granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF is pivotal for proliferation and function of myeloid cells. In this report, we describe the regulation of GM-CSF gene by irradiation in human fibroblasts. We found that fibroblasts constitutively produced GM-CSF; irradiation markedly increased the production of GM-CSF. The increase in GM-CSF transcripts by irradiation was both time- and dose-dependent. Moreover, irradiation increased GM-CSF mRNA in cells with prolonged exposure to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). WI38 fibroblasts constitutively produce low levels of IL-1. Induction of GM-CSF mRNA by irradiation was partially blocked by anti-IL-1 antibodies. On the other hand, inhibition of prostaglandin synthesis did not affect induction of GM-CSF RNA. Transcriptional run-on analysis showed that irradiation increased the rate of GM-CSF transcription. Stability studies of GM-CSF mRNA in these cells showed that half-life (t1/2) increased from < 20 min in unirradiated cells to > 100 min in irradiated cells. These findings suggest that the increase in GM-CSF mRNA observed after irradiation is regulated by transcriptional and post-transcriptional mechanisms. Our results indicate that induction of GM-CSF gene by irradiation requires de novo protein synthesis and increased levels of GM-CSF transcripts also occur through a pathway distinct from protein kinase C activation.

Dose-Response Relationship, Radiation↗
PubMed2002

The basic-helix-loop-helix transcription factor HAND2 directly regulates transcription of the atrial naturetic peptide gene.

The HAND basic Helix-Loop-Helix (bHLH) transcription factors are essential for the development of heart and extra embryonic structures. Although essential for embryonic development, the molecular pathways in which HAND factors participate are poorly understood. In efforts to identify downstream transcriptional targets, we have determined that HAND2 regulates the transcription of the Atrial Naturetic Peptide (ANP) gene. Results show that ANP expression is reduced in HAND2 null mice. Transactivation assays show significant transcriptional upregulation of ANP by HAND2 and cotransfection experiments using HAND2 and E12 suggest that an E-protein/HAND heterodimer is the likely trans -acting complex. The required cis -elements reside within a 258bp proximal region that contains three evolutionarily conserved Ebox consensus sites. Surprisingly, mutations in these three sites suggest HAND2 activity is DNA-binding independent. In addition, HAND2 and the homeobox factor Nkx2.5 exhibit transcriptional synergy in the regulation of ANP. Taken together, this data shows that HAND2 is an upstream transcriptional regulator of ANP expression, and furthermore HAND2 can synergistically interact with Nkx2.5, showing a functional relationship between HAND2 and Nkx2.5 supporting the genetic observation, that mice null for both HAND2 and Nkx2.5 lack ventricle specification.

Animals↗
PubMed1993

Transcriptional and post-transcriptional regulation of the human IGF-II gene expression.

The human insulin-like growth factor II (IGF-II) gene consists of nine exons and has four promoters (P1-4). The promoters exhibit a tissue-specific and developmental stage-dependent expression pattern. In fetal liver promoters P2-4 are expressed, but after birth these promoters are shut off and another promoter, P1, is activated. We have investigated some properties of the human promoters P1 and P3 and identified a number of sequence elements, that are recognized by transcription factors. Promoter P1 is stimulated by the liver-enriched transcription factors C/EBP and LAP, whereas in the proximal region of P3 we have identified several elements that are recognized by transcription factors, including krox20/egr2 and krox24/erg1. Besides transcriptional regulation of expression also regulation at the post-transcriptional level occurs. We have found that the IGF-II mRNAs are subjected to site-specific endonucleolytic cleavage yielding a labile 5' specific fragment and a stable polyadenylated 3' specific cleavage product of 1.8 kb. Two widely separated sequence elements within the last exon were identified that are able to interact and yield a double-stranded stem structure. It is likely that this structure is essential for post-transcriptional cleavage of IGF-II mRNAs.

Animals↗
PubMed1990

The chloroplast psbK operon from mustard (Sinapis alba L.): multiple transcripts during seedling development and evidence for divergent overlapping transcription.

The mustard chloroplasts genes psbK and psbI are co-transcribed, giving rise to precursor transcripts of several size classes, which are processed to the monocistronic mature RNAs. The psbK and psbI coding regions are flanked by the two tRNA genes trnS-GCU and trnQ-UUG on the opposite DNA strand. Transcript mapping indicates that the (primary) psbK-psbI transcript overlaps the complete trnS-GCU and trnQ-UUG transcripts. The transcription start site of the psbK operon appears to overlap that of the rps16 gene. During seedling development, the psbK and psbI precursors and mature transcripts all become detectable between 30 and 48 h after sowing and then remain at constant levels without much difference either in light or in darkness.

Base Sequence↗