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At least 793 records · Page 44Linked to original sources
PubMed1996

Ethylene control of E4 transcription during tomato fruit ripening involves two cooperative cis elements.

E4 gene transcription is controlled by ethylene during tomato fruit ripening. To define the ethylene-responsive promoter elements, we have tested the activity of mutations of the E4 promoter, and of chimeric genes in transient assay. Using a set of linker scan mutations of the region from -160 to -91, we determined that sequences located between -150 and -121 bp from the transcription start site are required for normal levels of ethylene-regulated transcription. However, E4 sequences from -193 to -40 were not able to confer ethylene-responsiveness to the minimal (-46) 35S promoter. The E4/E8 binding protein (E4/E8 BP) interacts with sequences in the 5'-flanking regions of both E4 and the coordinately regulated E8 gene, and its role in regulation of E4 transcription was investigated. The E4 binding site spans the E4 TATA box, and so mutations of this site were limited to those that did not disrupt the E4 TATA box. Mutations of this site which reduced affinity for the E4/E8 BP also resulted in reduced activity in transient assay, supporting a role for this element in normal regulation of the gene. Fusion of the 35S enhancer to E4 sequences from -85 to +65 did not result in an ethylene-responsive promoter, indicating that the E4/E8 BP-binding site is not sufficient for ethylene response. We conclude that at least two cis elements are required for ethylene-responsive transcription of the E4 gene during fruit ripening, one between -150 and -121 and the other between -40 and +65.

Base Sequence↗
PubMed2002

Transcriptional regulation of the interleukin-6 gene of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus).

Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus is linked to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), all of which are viewed as cytokine-driven malignancies. In particular, interleukin-6 (IL-6) has been found to promote the growth and proliferation of cells from KS and PEL. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]), which functions similarly to the cellular IL-6. Therefore, vIL-6 has been proposed to play an important role in tumor progression. Several groups have reported that vIL-6 is expressed from the HHV-8 genome at higher levels in PEL and MCD lesions than in KS lesions. However, it is not clear how vIL-6 expression is regulated. We characterized the transcription at the vIL-6 gene locus by Northern blot analysis and, in contrast to previous reports, we observed two distinct transcripts from induced PEL cell lines. This observation was confirmed by primer extension, as well as 5' and 3' rapid amplification of cDNA ends. Two transcription initiation sites and putative TATA boxes were mapped. A luciferase reporter system was used to show that each of the two putative TATA boxes contributed to vIL-6 promoter activity. Since virally encoded transcriptional activator Rta potently activates the viral lytic gene expression cascade, we examined the role of Rta in controlling vIL-6 gene expression and found that Rta activated the vIL-6 promoter. The Rta-responsive element was further mapped through a series of deletion constructs. Electrophoretic mobility shift assays demonstrated that Rta binds directly to the vIL-6 Rta-responsive element, and the core Rta-responsive element was mapped to a 26-bp region spanning from nucleotide 18315 to 18290 on the viral genome. We propose that the existence of two vIL-6 promoters offers opportunities for differential regulation of vIL-6 gene expression in different tissue types and may account for the variable vIL-6 levels observed in KS, PEL, and MCD.

Amino Acid Sequence↗
PubMed1994

RNA polymerase II promoter strength in vitro may be reduced by defects at initiation or promoter clearance.

We have measured the ability of three TATA box promoters, adenovirus 2 major late (Ad 2 ML), Ad 2 ML with a point mutation in the TATA box, and mouse beta-globin, to support abortive and productive RNA synthesis in vitro. We have also measured the ability of these promoters to direct the assembly of preinitiation complexes using a nuclease protection assay. The relative strengths in productive transcription, determined from the synthesis of RNAs 10 nucleotides or longer, were 12:6:1 for Ad 2 ML, mouse beta-globin, and the TATA mutant of Ad 2 ML. However, the TATA mutant was reduced only 4-fold in its ability to assemble preinitiation complexes, compared to Ad 2 ML. Complexes formed on the Ad 2 ML TATA mutant may therefore also be reduced in their ability to initiate transcription. The mouse beta-globin promoter directed assembly and initiation as well as Ad 2 ML, but the beta-globin transcription complexes were less able to clear the promoter, resulting in an increase in aborted transcripts at the expense of productive RNA synthesis. We have thus shown that the transcriptional strength of eukaryotic promoters may be determined not only at the step of transcription complex assembly but also at the level of promoter clearance and possibly at transcription initiation as well.

Adenoviruses, Human↗
PubMed1995

Repression of activator-mediated transcription by herpes simplex virus ICP4 via a mechanism involving interactions with the basal transcription factors TATA-binding protein and TFIIB.

Infected-cell polypeptide 4 (ICP4) of herpes simplex virus is both a transcriptional activator and a repressor. It has been previously demonstrated that both SP1-activated transcription and USF-activated transcription are repressed by ICP4 without affecting basal transcription (B. Gu, R. Rivera-Gonzalez, C. A. Smith, and N. A. DeLuca, Proc. Natl. Acad. Sci. USA 90:9528-9532, 1993; R. Rivera-Gonzalez, A. N. Imbalzano, B. Gu, and N.A. DeLuca, Virology 202:550-564, 1994). In this study, it was found that ICP4 repressed the activation function of two other activators, VP16 and ICP4 itself, in vitro. ICP4 inhibited transcription by interfering with the formation of transcription initiation complexes without affecting transcription elongation. Repression of activator function required that an ICP4 DNA binding site was present in one orientation within approximately 45 bp 3' to the TATA box. DNA binding by ICP4 was necessary but not sufficient for repression. ICP4 has been shown to form tripartite complexes cooperatively with the TATA box-binding protein and TFIIB on DNA containing an ICP4 binding site and a TATA box (C. A. Smith, P. Bates, R. Rivera-Gonzalez, B. Gu, and N. DeLuca, J. Virol. 67:4676-4687, 1993). A region of ICP4 that enables the molecule to form tripartite complexes was also required in addition to the DNA binding domain for efficient repression. Moreover, repression was observed only when the ICP4 binding site was in a position that resulted in the formation of tripartite complexes. Together, the data suggest that ICP4 represses transcription by binding to DNA in a precise way so that it may interact with the basal transcription complex and inhibit some general step involved in the function of activators. The steps or interactions involved in transcriptional activation that are inhibited by ICP4 are discussed.

Base Sequence↗
PubMed1991

Functional dissection in vitro of the human c-fos promoter.

Using internal and 5' deletions, the elements contributing to the in vitro activity of the human c-fos promoter have been identified. Wild type and mutant promoters were fused to the G-free cassette and tested, using HeLa nuclear and whole cell extracts, with the fos wild type promoter as the internal control. The proximal promoter domain, spanning from -124 to -58 in the fos promoter, is the primary determinant of activity. Two elements in this domain are important, the direct repeats and the -60 element, which contains overlapping MLTF/USF and CREB/ATF transcription factor binding sites. CREB/ATF appears to be dominant, since a canonical CRE functions well in place of the -60 element. Interestingly, the direct repeats appear to require the -60 element to exert their effect. Upstream elements do not Influence promoter activity in their normal position or adjacent to the TATA box, except the serum response element (SRE). Templates containing various lengths of the fos wild type SRE next to the TATA box are stimulated by adding purified serum response factor (SRF), while SRE mutants are not responsive. The stimulation is independent of small spacing differences between the SRE and TATA elements, and the CArG core of the fos SRE suffices to respond to added SRF in vitro.

Activating Transcription Factors↗
PubMed2002

The initiator element in a herpes simplex virus type 1 late-gene promoter enhances activation by ICP4, resulting in abundant late-gene expression.

The start site regions of late genes of herpes simplex virus type 1 are similar to the eukaryotic initiator sequence (Inr), have been shown to affect the levels of expression, and may also play a role in transcription activation by the viral activator ICP4. A series of linker-scanning mutations spanning the start site of transcription and several downstream mutations in the true late gC promoter were analyzed in reconstituted in vitro transcription reactions with and without ICP4, as well as in the context of the viral genome during infection. The nucleotide contacts previously found to be important for Inr function were also found to be important for optimal induction by ICP4. While the Inr had a substantial effect on the accumulation of gC RNA during infection, no other sequence downstream of the TATA box to +124 had a significant effect on levels of expression during infection. Therefore, these studies suggest that TATA box and the Inr are the only cis-acting elements required to achieve optimal expression of gC, and that the high levels of late-gene transcription may be largely due to the induction by ICP4, functioning through the Inr element.

Animals↗
PubMed1997

Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe.

Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.

Animals↗
PubMed1990

Isolation and characterization of the gene encoding the muscle-specific isozyme of human phosphoglycerate mutase.

The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.

Amino Acid Sequence↗
PubMed1997

Promoter analysis of a cysteine-rich Campoletis sonorensis polydnavirus gene.

Promoter activity of the Campoletis sonorensis polydnavirus (CsPDV) WHv1.6 gene was analysed by transient transfection assays in insect cell culture using constructs expressing the CAT gene. Deletions of the WHv1.6 gene promoter were used to define promoter regions important for expression. Progressive deletion of the regions upstream of the TATA box reduced the promoter activity, whereas deletions eliminating the TATA box abolished promoter activity. Cis-activating elements were detected up to 1 kb upstream of the WHv1.6 transcription initiation site (TIS). Promoter elements increasing transcription were detected between -444 and -550 bp and between -831 and -1035 bp relative to the TIS. Analysis of the 3' flanking sequences of the WHv1.6 gene indicated that the polyadenylation signals were the only important elements affecting expression in the constructs. Comparison of promoter regions of four cysteine-rich CsPDV genes revealed homologous sequences that may be important for transcriptional regulation of polydnavirus gene expression in parasitized Heliothis virescens larvae.

Animals↗
PubMed1996

Regulation of protamine gene expression in an in vitro homologous system.

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

Animals↗
PubMed1996

Interactions of progesterone-dependent endometrial nuclear factors with the promoter of the rabbit uteroglobin gene.

We have studied the in vitro interactions of a promoter fragment of the rabbit uteroglobin (UG) gene with endometrial nuclear factors from either control rabbits or progesterone-treated animals, a treatment that induces the transcription of the gene in the endometrium. Nuclear factors from liver, which does not express UG, were also compared. Using DNase I footprinting, several protected zones were observed on the promoter; some of these were common to all three nuclear extracts, whereas others seemed to be specific to progesterone treated endometrium. One of these footprints was over the TATA box region. A 28-bp synthetic oligonucleotide encompassing the sequence of that region was used as a probe in electrophoretic mobility shift assays (EMSA) and UV-crosslinking assays. In EMSA experiments, endometrial nuclear extracts, but not liver extracts, generated a major retarded complex that was strongly increased following progesterone stimulation. Competition experiments with unlabeled mutated versions of the probe showed that sequences 3' downstream from the TATA box (but not this motif) were responsible for the formation of the complex. UV-crosslinking experiments indicated that the probe interacted with two progesterone-dependent nuclear proteins of 55 and 60 kDa, different from the TATA box-binding protein. The results suggest that progesterone induction of the UG gene in the endometrium might be mediated by both general and tissue-specific nuclear factors.

Animals↗
PubMed2001

The overlap of Inr and TATA elements sets the use of alternative transcriptional start sites in the mouse galectin-1 gene promoter.

In the mouse gene encoding the protein galectin-1, transcription initiation at the +1 site is directed by a TATA box. Here we show that a consensus Inr element (TCCAGTT), which spans residues -34 to -28 and overlaps the TATA box, directs RNA initiation also from a previously uncharacterized site located at position -31. Upstream transcripts are polyadenylated and contribute to more than half of the galectin-1 mRNA population in all tissues analyzed. The promoter architecture is evolutionarily conserved to man, and galectin-1 mRNA size variants accumulate also in human HeLa cells. The 5' end terminus of the transcripts initiated at residue -31 is extremely GC-rich, and may fold into a relative stable hairpin which could influence translation and thus modulate the intracellular levels of galectin-1. The interval -63/+45 contains sufficient information to ensure RNA initiation from both -31 and +1 sites, and a Sp1 site spanning residues -57 to -48 is crucial for promoter functioning. The unusual overlap of core promoter elements suggests that RNA initiation from the -31 and the +1 sites may take place in a sequential manner.

3T3 Cells↗
PubMed1983

Transcription from the SV40 early-early and late-early overlapping promoters in the absence of DNA replication.

Transcription for a hybrid SV40 promoter-beta globin coding sequence recombinant initiates from both early-early (EE) and late-early (LE) SV40 start sites (EES and LES) in the absence of DNA replication. The 72-bp repeat is essential to potentiate the elements of the two overlapping EE and LE promoters (EEP and LEP). Two current models, which can account for the EE to LE shift in RNA chain initiation during the SV40 replication cycle, are that LE transcription is linked to replication and occurs on newly replicated DNA molecules or that there are two promoter elements, a stronger EEP and a weaker LEP, T antigen repressing the EEP late in infection. Our results support the second model. A 5'-TATTTAT-3' to 5'-TATCGAT-3' mutation in the putative SV40 TATA box decreases transcription from EES, increases transcription from LES, and inhibits DNA replication. Therefore, this element acts as a classical TATA box for transcription, and yet is also important for DNA replication.

Animals↗
PubMed1996

pHIVTATA-CAT, a versatile vector to study transcriptional regulatory elements in mammalian cells.

We have constructed a vector, pHIVTATA-CAT, that contains the Escherichia coli cat gene, encoding chloramphenicol acetyltransferase, under the control of a minimal promoter consisting of the HIV TATA box and the adenovirus major late promoter initiator element. Putative transcriptional elements can be inserted either directly upstream from the TATA box or downstream from the reporter gene in an enhancer position. Transcription can be monitored enzymatically or by RNase protection mapping. An analysis of mRNAs generated from pHIVTATA-CAT constructs revealed that transcription starts at the transcription start point and no read-through transcripts are generated.

Adenoviridae↗
PubMed2001

Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs.

To investigate the basis for enhancer-promoter specificity, we compared the ability of enhancers to activate transcription in vivo from core promoters containing either downstream promoter element (DPE) or TATA box motifs. To eliminate position effects, we generated and analyzed pairs of sister Drosophila lines that contain a DPE- or TATA-dependent reporter gene at precisely the same genomic position relative to each enhancer. These studies revealed transcriptional enhancers that are specific for promoters that contain either DPE or TATA box elements. Thus, the core promoter not only mediates the initiation of transcription, but also functions as a regulatory element.

Animals↗
PubMed1993

Structure and expression of the tobacco nuclear gene encoding the 33 kDa chloroplast ribonucleoprotein.

Tobacco chloroplasts contain a family of ribonucleoproteins (RNPs) which appear to be involved in mRNA processing and splicing in chloroplasts. We have characterized a new cDNA, 33k-6, potentially encoding a tobacco 33 kDa chloroplast RNP (cp33) homologue. This cDNA has a 78 bp insertion near the 3' end with respect to previously characterized cp33 cDNAs, leading to the creation of an alternative C-terminal sequence. The cp33 protein is encoded by a single-copy nuclear gene in Nicotiana sylvestris, which contains three introns. No typical TATA box is present in the upstream region of the gene. Multiple transcription start sites are often observed for promoters lacking TATA boxes, and have been suggested in the cp33 gene. Sequence comparison revealed that the 78 bp insertion in 33k-6 is derived from the third intron of the cp33 gene which is not removed during pre-mRNA splicing. Ribonuclease protection analysis showed that the processing of the third intron is slow compared to the other introns. A possible role for the partially spliced mRNA (cp33k-6) is discussed.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of promoter of the mouse mafB gene.

The MafB transcription factor plays a pivotal role in controlling the development and differentiation. The author reports the isolation and analysis of genomic clone of the mouse mafB gene. The gene lacks intron structure, at least, within its coding and 5'-untranslated sequences that are similar to the chicken mafB gene. RNA protection analysis determined one transcription initiation site of the gene at 389-bp upstream from the translation initiation site. Sequence analysis showed that the 5'-flanking region upstream to the ATG codon did not contain a conventional TATA box. A TATA-like sequence (5'-GATAAAA-3') and an inverted CCAAT-box (5'-ATTGG-3') were found to be located at nucleotide -31 and -86, referring to the transcription initiation site, respectively. Upstream to these sequences, there were several potential regulatory elements, including two GC-boxes (5'-GGGCGG-3': from -148 to -143; and from -123 to -118), and a palindromic sequence (5'-GTCAGCTGAC-3': from -164 to -155) which contained two halves-MARE (Maf recognition element), 5'-GCTGAC-3', and an E-box (5'-CAGCTG-3'). Promoter activity of the 5'-flanking region was analyzed by reporter transfection assay, which suggested that these segments were an important transcriptional activator. It was also suggested that MyoD transactivated the mouse mafB promoter and this gene was positively autoregulated by its product, MafB.

Animals↗
PubMed1994

Functional differences between mammalian transcription activation domains at the yeast GAL1 promoter.

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.

Animals↗