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Results for “GC Rich Sequence”

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At least 19 recordsLinked to original sources
PubMed1998

Silencing homologous RNA recombination hot spots with GC-rich sequences in brome mosaic virus.

It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799-3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar "homologous recombination silencing" effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.

Base Sequence↗
PubMed1999

A GC-rich sequence within the 5' untranslated region of human basonuclin mRNA inhibits its translation.

By the method of RNase protection, the 5' ends of the basonuclin mRNA were mapped to four sites distributed over 400 bases of the genomic sequence, a result implying four different basonuclin transcripts within the cell. Despite the heterogeneity at the 5' end, all four basonuclin mRNA shared the same translation initiation codon. However, only two transcripts contained, in their 5' untranslated region (UTR), a GC-rich sequence of approx. 180 bases. The ability of this GC-rich sequence to form a large and stable secondary structure was suggested by experimental results from primer extension, RNase resistance, and computer analysis of the sequence. In vitro study showed that translation of basonuclin RNA containing this putative structure could not be initiated efficiently from the first two AUGs, whereas those that lacked it could.

5' Untranslated Regions↗
PubMed1996

Molecular origins of selectivity in the interaction of amsacrine-4-carboxamide with GC-rich sequences in DNA.

To determine the molecular origins of the preferential binding of an antitumour amsacrine-4-carboxamide derivative to GC-rich sequences in DNA, we have used the polymerase chain reaction to synthesize a series of oligodeoxynucleotides in which the position of the purine 2-amino group is varied and then investigated the binding of the drug to normal and modified DNA molecules by means of DNase I footprinting. The results indicate that the 2-amino group of guanine represents an important but not unique element which directs selective binding of amsacrine-4-carboxamides to GC-rich sequences.

Amsacrine↗
PubMed2006

Involvement of the GC-rich sequence and specific proteins (Sp1/Sp3) in the basal transcription activity of neurogranin gene.

Neurogranin (Ng), a neuronal protein implicated in learning and memory, contains a TATA-less promoter. Analysis of 5'-deletion mutations and site-directed mutations of the mouse Ng promoter revealed that a 258bp 5'-flanking sequence (+3 to +260) conferred the basal transcription activity, and that the GC-rich sequence (+22 to +33) served as an important determinant of the promoter activity. Transient transfection of the Sp1 expression plasmid transactivated the reporter activity in neuroblastoma N2A cells while knocking down of endogenous Sp1 expression resulted in a 2.5-fold reduction of the reporter activity in HEK 293 cells. Exogenous expression of Sp3 in HEK 293 cells, however, repressed the reporter activity by 50%. Nevertheless, by gel shift assays, Sp1 and Sp3 were not found to be responsible for the protein-DNA complexes formed by the GC-rich sequence. Moreover, a nuclear factor from the mouse brain tissues was discovered to bind to multiple AT-rich regions in Ng promoter.

Base Composition↗
PubMed1994

Repression of transforming growth factor beta 1 promoter by the adenovirus oncogene E1A. Identification of a unique GC-rich sequence as a target for E1A repression.

The transforming growth factor beta 1 (TGF-beta 1) is a key regulator of proliferation and differentiation in a wide variety of cell types. It is a potent growth inhibitor for most epithelial, endothelial, lymphoid, and myeloid cells. In the present study, we showed that a DNA virus oncoprotein, E1A, strongly repressed the activity of the TGF-beta 1 promoter in a variety of cell lines. Interestingly, this repression was specific for 12 S E1A because 13 S E1A was much less active in this assay. Analysis of a series of E1A mutants showed that the repression was dependent on the amino terminus and the conserved region 1 of the E1A protein. To identify the target sequence for E1A repression in the TGF-beta 1 promoter, a series of mutant promoters were analyzed and a 10-base pair GC-rich sequence between -91 and -82 was found to be the major target for E1A repression of the promoter. Using chimeric reporter constructs, we provide evidence that the 10-base pair GC-rich sequence is sufficient to impart sequence-specific E1A repression to a heterologous promoter. Additionally, we suggest that the mechanism of E1A repression through this GC-rich element does not involve abrogation of the retinoblastoma control of the TGF-beta 1 promoter.

Adenovirus E1A Proteins↗
PubMed1991

Spermidine regulation of ornithine decarboxylase synthesis by a GC-rich sequence of the 5'-untranslated region.

The nucleotides in the 70-170 region upstream from the initiator AUG have been shown to be important in the strong stimulation of ornithine decarboxylase (ODC) synthesis by low spermidine concentrations and in the inhibition of ODC synthesis at high spermidine concentrations [Ito, K., et al. (1990) J. Biol. Chem. 265, 13036-13041]. In this region, a GC-rich sequence as well as a small open reading frame (MGQASQATVL) existed. In order to clarify which of these was of greater importance for the spermidine regulation of ODC synthesis, the synthesis was performed with various ODC mRNAs, possessing different sizes and nucleotide sequences in the 5'-untranslated region. The results show that a GC-rich sequence, but not a small potential leader peptide, plays an important role in the spermidine regulation of ODC synthesis.

Amino Acid Sequence↗
PubMed1997

HPV16 E6 oncoprotein stimulates the transforming growth factor-beta 1 promoter in fibroblasts through a specific GC-rich sequence.

Human papillomaviruses (HPV) have been etiologically linked to human cervical cancer. Transforming growth factor-beta 1 (TGF-beta 1) is a cytokine which is a potent growth inhibitor of most epithelial, endothelial, lymphoid, and myeloid cells, but is mitogenic for mesenchymal cells and bone cells. In this study, we analyzed the effects of HPV 16 oncoproteins E6 and E7 on the TGF-beta 1 promoter. The results showed that the HPV 16 E6 significantly induced (sixfold) the TGF-beta 1 promoter activity while HPV 16 E7 showed no significant effect. The E6 effect was cell type-specific and was observed only in the fibroblast cell lines, not in epithelial cells. Promoter analysis revealed that a 9-bp sequence, GGGGCGGGG, representing the consensus Sp1-binding site between -109 and -100 of the TGF-beta 1 promoter, was the major target for E6-mediated transactivation. Mutation analysis of the E6 polypeptide showed that the retention of amino acids between 123 and 136 of the HPV 16 E6 protein was critical for the transactivation of the TGF-beta 1 promoter. Previous studies have shown that the adenovirus 12S E1A oncoprotein represses the TGF-beta 1 promoter by targeting an adjacent (-90 to -81) but different GC-rich sequence (TGGGTGGGG). These studies provide evidence that variant GC-rich promoter elements are not functionally identical and are differentially regulated by the DNA virus oncoproteins.

3T3 Cells↗
PubMed2005

GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site.

5-lipoxygenase (5LO) catalyzes formation of leukotrienes, mediators with roles in several inflammatory disorders, including atherosclerosis. The human 5LO gene promoter contain multiple GC-boxes. The relevance of these for expression of 5LO in the human monocytic cell line Mono Mac 6 (MM6) was studied. A downregulating effect of the GC-box binding compound mithramycin indicated that GC-rich sequences in the 5LO gene promoter are important for expression of native 5LO. In DNase I footprinting, mithramycin and Sp1 protected known GC-boxes, but also a novel Sp1 binding site was found, comprising 20 bp upstream of the major transcription initiation site, beside an Initiator-like sequence. Mutation of this site reduced Sp1 binding and expression of reporter genes in MM6 cells, compatible with a function as basal promoter element for the TATA-less 5LO gene. When differentiation was induced by TGFbeta and vitamin D(3), 5LO expression became prominent, but expression levels of Sp1/3 and Egr-1 were the same as for control cells. Also, 5LO reporter gene activity in transiently transfected MM6 cells was insensitive to differentiation. Thus, the GC-rich part of the 5LO gene promoter, including a novel Sp1 site, appear important for basal (rather than upregulated) transcription of 5LO in MM6 cells.

Arachidonate 5-Lipoxygenase↗
PubMed1995

v-Raf activates transcription of growth-responsive promoters via GC-rich sequences that bind the transcription factor Sp1.

The serine/threonine kinase, Raf-1, is a component of intracellular signaling pathways that control responses to extracellular stimuli. Previously, we have shown that serum-induced transcription from the murine rep-3b and human mdr1 promoters is Raf-dependent and that the activated Raf kinase, v-Raf, induces transcription of mdr1 via a GC-rich element. We now demonstrate that GC-rich sequences in the rep-3b promoter are both necessary and sufficient for induction by v-Raf. The GC-rich, v-Raf-responsive elements of rep-3b and mdr1 bind the general transcription factor Sp1 in electromobility shift assays. Mutation of a minimal GC-rich element abolished inducibility by v-Raf and eliminated binding by the transcription factor Sp1. However, Sp1 binding activity following serum stimulation of quiescent NIH 3T3 cells was unchanged, suggesting that mitogenic signals may stimulate the transactivation potential of prebound Sp1.

3T3 Cells↗
PubMed1997

A short GC-rich sequence involved in deletion formation of cloned DNA in E. coli.

A spontaneous deletion probably caused by rec A independent recombination between short-direct repeat sequences was observed in pUC19 plasmid carrying a piece of Streptomyces genomic DNA after culturing in liquid medium. The deletion removed an unknown portion of the cloned DNA and the BamHI-EcoRI part of the multiple cloning site with an additional flanking 111 bp from the vector. At the junction a 13 bp GC-rich DNA sequence highly homologous to a known spontaneous deletion hotspot was found.

Base Composition↗
PubMed1996

Genomic structure and promoter activity of the mouse polysialic acid synthase gene (mST8Sia II). Brain-specific expression from a TATA-less GC-rich sequence.

The mouse ST8Sia II (mST8Sia II/STX) gene encodes a neural cell adhesion molecule-specific polysialic acid synthase whose expression is regulated during the developmental stages of mouse brain. To elucidate the molecular mechanism by which the expression is tissue-specifically and developmentally regulated, we isolated the complete genomic DNA and characterized the promoter of the gene for mST8Sia II. The gene encoding mST8Sia II was found to span about 80 kilobases and to be composed of six exons. Primer extension and S1 nuclease protection analyses revealed that the transcription started from 167 nucleotides upstream of the translational initiation site. Promoter analyses of the 5'-flanking region of the mST8Sia II gene using a luciferase gene reporter system revealed strong promoter activity in retinoic acid-induced differentiated P19 cells, which highly express the mST8Sia II gene. Deletion analyses demonstrated that the minimal promoter activity detected for the proximal region 325 base pairs upstream from the translational initiation codon (-158 to +167) could be modulated by various sequences within the 9. 5-kilobase 5'-flanking region. The minimal promoter was embedded in a GC-rich domain (74%, GC content), in which two Sp1 binding motifs as well as a long purine-rich region were found, but it lacked TATA and CAAT boxes. The positive regulatory region located between -159 and -659 contained two additional Sp1 binding motifs and a long pyrimidine-rich region. We also found that the minimal promoter region of the mST8Sia II gene was sufficient for expression of a reporter gene in mST8Sia II gene-expressing neural differentiated P19 cells but not in nonexpressing ones. Thus the TATA-less GC-rich minimal promoter region of mST8Sia II probably controls the cell type-specific expression of the mST8Sia II gene.

3T3 Cells↗
PubMed1995

DNA condensation by the rat spermatidal protein TP2 shows GC-rich sequence preference and is zinc dependent.

Transition protein-2 (TP2), isolated from rat testes, was recently shown to be a zinc metalloprotein. We have now carried out a detailed analysis of the DNA condensing properties of TP2 with various polynucleotides using circular dichroism spectroscopy. The condensation of the alternating copolymers by TP2 (incubated with 10 microM ZnSO4), namely, poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), was severalfold higher than condensation of either of the homoduplexes poly(dG).poly-(dC) and poly(dA).poly(dT) or rat oligonucleosomal DNA. Between the two alternating copolymers, poly(dG-dC).poly(dG-dC) was condensed 3.2-fold more effectively than poly(dA-dT).poly(dA-dT). Preincubation of TP2 with 5 mM EDTA significantly reduced its DNA-condensing property. Interestingly, condensation of the alternating copolymer poly(dI-dC).poly(dI-dC) by TP2 was much less as compared to that of poly(dG-dC).poly(dG-dC). The V8 protease-derived N-terminal fragment (88 aa) condensed poly(dA-dT).poly(dA-dT) to a very small extent but did not have any effect on poly(dG-dC).poly-(dG-dC). The C-terminal fragment (28 aa) was able to condense poly(dA-dT).poly(dA-dT) more effectively than poly(dG-dC).poly(dG-dC). These results suggest that TP2 in its zinc-coordinated form condenses GC-rich polynucleotides much more effectively than other types of polynucleotides. Neither the N-terminal two-thirds of TP2 which is the zinc-binding domain nor the C-terminal basic domain are as effective as intact TP2 in bringing about condensation of DNA.

Amino Acid Sequence↗
PubMed1991

The anaerobic responsive element contains two GC-rich sequences essential for binding a nuclear protein and hypoxic activation of the maize Adh1 promoter.

We have identified a protein (GCBP-1) in nuclear extracts from maize suspension cell cultures that binds to specific sequences within the Anaerobic Responsive Element (ARE) of the maize Adh1 promoter. Competition analyses show that the GCBP-1 binding activity distinguishes ARE sequence motifs from other enhancer elements or pUC19 sequences. The binding activities of several mutant ARE sequences define two regions of the ARE important for GCBP-1 binding in vitro, between nucleotides -135 to -131 and nucleotides -120 to -112 of the maize Adh1 promoter. Both regions are required for efficient GCBP-1 binding to occur in vitro. The minimum consensus binding site for GCBP-1 is 5'-GC(G/C)CC-3'. This sequence is similar to a part of the binding site of the human transcription factor Sp1 (1). We demonstrate that maize GCBP-1 and human Sp1 have similar recognition properties. Using ARE mutants in a transient assay in maize protoplasts we have shown that mutation of the GCBP-1 binding sites prevents significant hypoxic activation of the maize Adh1 promoter. These results suggest a direct role for GCBP-1 in the hypoxic activation of Adh1 gene expression. GCBP-1 is present in both uninduced and induced nuclei, indicating that inducible gene expression is not dependent upon synthesis of GCBP-1 and suggesting that post-translational modification of bound GCBP-1 may be important for enhanced transcription to occur.

Alcohol Dehydrogenase↗
PubMed1995

Human dipeptidyl peptidase IV gene promoter: tissue-specific regulation from a TATA-less GC-rich sequence characteristic of a housekeeping gene promoter.

The dipeptidyl peptidase IV gene encodes a plasma-membrane exopeptidase that is highly expressed in small intestine, lung and kidney. In order to better understand the mechanisms responsible for this tissue-specific expression we cloned, sequenced and functionally characterized the 5'-flanking region of the human dipeptidyl peptidase IV gene. The first 500 bases of the 5'-flanking sequence constituted an unmethylated CpG island, contained several Sp1-binding sites and lacked a consensus TATA box, all characteristics of gene promoters lacking tissue-specific expression. RNase-protection analysis using both small intestinal and Caco2 cell RNA indicated that the dipeptidyl peptidase IV transcript was initiated from no fewer than six major and 12 minor start sites. The 5'-flanking sequence also exhibited functional promoter activity in transient transfection experiments. Here, various lengths of the sequence were cloned upstream of a luciferase gene and introduced into cultured cells using lipofectin. A region located between bases -150 and -109 relative to the start of translation was found to be important for high-level promoter activity in both Caco2 and HepG2 cells. Moreover, Caco2 cells and HepG2 cells, which express high levels of dipeptidyl peptidase IV activity, exhibited much higher normalized luciferase activity after transfection than did 3T3, Jurkat or COS-7 cells, which have low enzyme levels. Sodium butyrate was found to increase both enzyme activity and normalized luciferase in HepG2 cells. Thus the dipeptidyl peptidase IV promoter possesses the ability to initiate transcription in a tissue-specific fashion in spite of having the sequence characteristics of a housekeeping gene promoter.

3T3 Cells↗
PubMed1998

IFN-gamma induction of the human monocyte chemoattractant protein (hMCP)-1 gene in astrocytoma cells: functional interaction between an IFN-gamma-activated site and a GC-rich element.

We characterized regulation of the human monocyte chemoattractant protein-1 (hMCP-1) gene by IFN-gamma in astrocytoma cells, because astroglial cells express chemokines in several central nervous system inflammatory states. It was found that IFN-gamma-induced hMCP-1 transcription was rapid, transient, and mediated by a 213-bp promoter-proximal regulatory region of the gene. Our studies on both in vitro and in vivo states of the hMCP-1 regulatory region established requirement of an IFN-gamma-activated site (GAS) and the presence of IFN-gamma-inducible GAS-binding activity involving at least STAT-1alpha for IFN-gamma-induced hMCP-1 expression. Unexpectedly, in vivo genomic footprinting of the proximal regulatory region of the IFN-gamma-induced gene revealed protection of a GC-rich sequence (GC box) with the same temporal pattern as that seen at the GAS; in vitro, this GC-rich element is associated with nuclear factor Sp1. These observations suggested a cooperative interaction between the GAS and the GC box element. Interestingly, site-specific mutations that abolished GC-box or GAS-element function produced clearly disparate results. Disruption of the GC box did not affect fold induction by IFN-gamma but reduced promoter-reporter expression by half. Conversely, GAS mutation abrogated induction but did not affect the magnitude of expression. These results establish the importance of the GAS element for induction of hMCP-1 and further our understanding of IFN-gamma-mediated transcriptional induction by providing the first evidence in vivo for inducible signaling to the GC box by this cytokine.

Astrocytoma↗
PubMed1994

Regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Localization of an intron EBV-responsive enhancer and characterization of its cognate GC-box binding factors.

EBV infection of human B lymphocytes induces expression of the low affinity IgE receptor, Fc epsilon RII/CD23. CD23 is constitutively expressed in EBV-immortalized B cells and may play an essential role in immortalization. We previously explored the regulation of CD23 by EBV, showing that induction results from transcriptional activation that is mediated, in part, by an EBV-responsive transcriptional regulatory element in the 5' region of CD23 (-229 to +305 relative to the type a promoter). We now report the localization of the regulatory element and characterization of its cognate DNA-binding proteins. Reporter gene assays in EBV-positive and -negative lines localized a functional EBV-responsive enhancer to a 37-bp fragment (+248 to +284) that contains a GC-rich sequence (GC box) within intron I of type a CD23. This fragment was shown by mobility shift assays to specifically bind nuclear protein(s) from EBV-positive lines, but not EBV-negative lines. Mutation of the GC box resulted in a loss of protein-binding activity, implicating involvement of a GC box-binding protein in the DNA/protein interaction. Supershift assays suggested that the ubiquitous GC box-binding transcription factor, Sp1, is not a part of the complex, and UV-crosslinking studies demonstrated that the DNA/protein complex contains at least two proteins that differ in size from other known GC box-binding proteins. Binding of these proteins to the enhancer element requires phosphorylation, because phosphatase treatment of nuclear extracts abolished formation of the DNA/protein complex. These studies reveal the presence of an EBV-responsive enhancer element in intron I of type a CD23 and implicate a GC box-binding transcription factor in the activation of CD23 by EBV.

Base Sequence↗