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Results for “regulatory element transcripts”

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PubMed1990

[Molecular cloning and molecular biological studies of human thymidylate synthase gene].

Thymidylate synthase (TS) provides the only de novo source of thymidylate for deoxyribonucleic acid (DNA) synthesis and is a key target of cancer chemotherapeutic agents. Expression of its activity is strongly dependent on the cell cycle. Therefore, it is important for understanding the abnormal growth of cancer cells as well as for cancer chemotherapy to elucidate the structure of a TS gene and the regulatory mechanism of expression of its gene at the molecular level. From this point of view, genomic DNA segments partially coding for human TS were cloned from the mouse cell transformant obtained by gene transfer. By using them as a probe, functional cDNA and genomic DNA (approx. 18 kb) clones for human TS were isolated, and the entire nucleotide sequence of these cloned DNAs including all the introns determined. On the basis of the sequence data, we revealed an amino acid sequence of human TS, and organization and several structural features of its gene were revealed. Then, multiple transcription initiation sites and transcriptional regulatory elements of the TS gene were identified. Furthermore, the importance of the post-transcriptional regulation in the cell-cycle dependent expression of the gene was shown. The results obtained so far have opened the way to elucidate the molecular mechanism regulating the TS gene expression entirely.

Amino Acid Sequence↗

PubMed1991

Importance of SV40 early 5' distal sequences in directing heterologous gene expression.

The chloramphenicol acetyltransferase-encoding reporter gene (cat) is used extensively in assessing the ability of transcriptional regulatory elements (TRE) to direct gene expression in eukaryotic cells. Two commonly utilized plasmids contain the cat coding sequences under the transcriptional control of the Rous sarcoma virus LTR (pRSVcat) or simian virus 40 early (SV40E) promoter (pSV2cat). In the present study, we have recloned the RSV-LTR and SV40E TRE into a pUC18 vector. Direct comparison of these TRE in different plasmid vectors, as well as reevaluation of their relative level of cat expression revealed: (1) a small but significant increases in SV40E-directed reporter gene expression was observed when the TRE was inserted into the pUC18 vector; and (2) a significant increase in SV40E-directed gene expression was realized by inclusion of the 69-bp 5' of the sequences present in pSV2cat. These distal sequences are required for maximal activity of the SV40 TRE in the cell lines tested.

Animals↗

PubMed1993

Regulation of expression of transgenes in developing fish.

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

Actins↗

PubMed2001

Retrolyc1 subfamilies defined by different U3 LTR regulatory regions in the Lycopersicon genus.

Retrolycl, a Ty1/copia-like element, was originally isolated from the Lycopersicon peruvianum genome and shown to be present also in other Lycopersicon species. It shares extensive similarities with Tntl, except in its U3 regulatory region. In order to evaluate Retrolycl diversity, we analyzed partial sequences including both coding domains and the U3 regulatory region in four different species of the Lycopersicon genus. Two Retrolycl subfamilies defined by different U3 regions were identified. RetrolyclA is most abundant in L. peruvianum and L. hirsutum, while Retrolyc1B is distributed in all four species studied here. The RetrolyclA U3 region contains tandemly repeated elements of 53 bp. Transient expression analysis suggests that Retrolyc1A is a transcriptionally active family, and that the repeated motifs found in its U3 region are important transcriptional regulatory elements.

Base Sequence↗

PubMed1993

Overexpression of Myc suppresses CCAAT transcription factor/nuclear factor 1-dependent promoters in vivo.

Overexpression of Myc in cells can suppress the transcription of specific genes. Because several of these genes have common transcriptional regulatory elements, we investigated the possibility that this effect of Myc is mediated through a specific transcription factor. In vitro DNA-binding assays detect only one form of CCAAT transcription factor/nuclear factor 1 (CTF/NF-1) in quiescent 3T3-L1 cells. By contrast, quiescent 3T3-L1 cells that stably overexpress either c-Myc or N-Myc contain at least three forms of CTF/NF-1. Biochemical characterization of the various CTF/NF-1 forms showed that they have the same native molecular weight but differ in charge density. The more negatively charged CTF/NF-1 forms present in Myc-overexpressing cells are converted into that found in normal cells by treatment with acid phosphatase, suggesting that they represent a more phosphorylated form of the CTF/NF-1 protein. The various CTF/NF-1 forms have a similar DNA-binding affinity. Transfection experiments demonstrated that transcription from CTF/NF-1-dependent promoters is specifically suppressed in cells that stably overexpress c-Myc. This effect requires CTF/NF-1 binding. CTF/NF-1-dependent promoter activity is also suppressed in 3T3-L1 cells during active growth (relative to the quiescent state). Interestingly, actively growing 3T3-L1 cells contain forms of CTF/NF-1 similar to those in quiescent cells that stably overexpress c-Myc. Thus, the CTF/NF-1 forms present in cells that express high amounts of c-Myc correlate with a lower transcription rate of CTF/NF-1-dependent promoters in vivo. Our results provide a basis for the suppression of specific gene transcription by c-Myc.

3T3 Cells↗

PubMed2006

First exons and introns--a survey of GC content and gene structure in the human genome.

Most transcriptional regulatory elements are located in non-coding DNA. In particular, some first introns play a vital role in transcriptional control and splicing. The length and GC-content of first exons and introns in complex organisms suggests that these structural units are likely to be important functional elements in large genomes. Hence, in this paper we perform a systematic comparison of exon-intron structure and GC content on all known genes in the human genome. Our in-silico analysis found that the GC content of introns and exons varies significantly depending on their length. On average, the first intron of a gene is significantly longer than other introns in the same gene. Our results also show that first introns and exons are more GC rich than last and internal. This study provides insight into the structure of eukaryotic genes. These results confirm and expand the previously identified regulatory potential of first exons and introns.

Base Composition↗

PubMed1991

Characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene.

To identify the transcription regulatory elements of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, DNA fragments located in the 5'-upstream region were fused with the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and transfected into various cell lines to test for promoter activity. The results of these studies suggested that there were at least two positive and one negative cisacting elements involved in CFTR transcription initiation. One of them was a proximal, positive element delimited by the 5' deletion constructs -226 base parts upstream of the transcription start site. This minimal promoter sequence (-226 to +98) alone seemed to be sufficient to direct cell-specific CAT expression. The sequences immediately upstream of -227, on the other hand, appeared to contain a negative regulatory element; inclusion of this sequence with the proximal element (e.g. a construct containing sequences -345 to +98) rendered the CFTR promoter inactive. This negative regulatory element could also suppress the activity of a heterologous promoter. In addition, the DNA transfection study suggested the existence of another positive regulatory element outside the CFTR promoter region examined, as the inability of this region (e.g. -658 to +98) to function in a CAT assay could be overcome by the presence of a viral enhancer element.

Amino Acid Sequence↗

PubMed1991

Developmental regulation of a serum response element binding activity in amphibian embryos.

As part of our studies of transcriptional control during early development in vertebrates, we have examined embryos of the amphibian Xenopus laevis for the presence of sequence-specific DNA-binding proteins, using gel electrophoresis mobility-shift assays. Our analysis has focused on sequence elements in the cytoskeletal actin gene, whose embryonic transcription is initially activated at the gastrula stage, approximately 16 hours after fertilization. We detect activities capable of specific binding to two known transcriptional regulatory elements, the serum response element and the GC-box, located in the 5'-flanking region of the cytoskeletal actin gene. Binding activity specific for a region downstream of the transcriptional startsite is also detected, in a region which may be involved in controlling developmental activation of this gene. Serum response element-binding activity, as well as the downstream binding activity, is enriched in extracts from gastrula and neurula stage embryos, compared to egg extracts, suggesting that increased levels of one or both of these activities might play a role in developmentally timed transcriptional activation of the cytoskeletal actin gene in the embryo.

Actins↗

PubMed2004

[Retroviral inheritance in man].

The data provided by the sequencing of the human genome showed that retroviral-like elements constituted approximately 8 % of the euchromatin. The origin of these elements, their propagation leading to an organization in families, their genetic structure and the identification of the fonctional domains of the components of these elements are described. Placenta is used as a model to illustrate the physiological involvement of HERVs. Transcriptional regulatory element (LTR) functions and the putative implication of retroviral proteins in resistance to infection, immunosuppression, and cellular differentiation are clarified. The data implicating the envelope encoded by the ERVWE1 locus of the HERV-W family in the fusion process, leading to syncytiotrophoblast formation, is analysed. The putative pathological effect of HERVs is illustrated by the expression of the HERV-K superfamily in cancer. More precisely, the association between the Rec regulatory protein encoded by HERV-K(HML-2) and testicular tumorigenesis is developed. Whether HERVs are triggers or markers in other physiopathological contexts is discussed. To conclude, the dual benefit-hazard underlying the acquisition/propagation of HERVs is examined with respect to species evolution, considering the multicopy trait of HERV families and the mainly multi-factorial aspect of autoimmune diseases and cancers.

Endogenous Retroviruses↗

PubMed1995

ATF-1CREB heterodimer is involved in constitutive expression of the housekeeping Na,K-ATPase alpha 1 subunit gene.

Na,K-ATPase alpha 1 subunit is an essential protein for cell growth and homeostasis. The gene coding for the protein is expressed in various types of tissues. We previously demonstrated that the transcription regulatory element of the gene (ARE) is located in the position -102 to -61 from the transcription initiation site. To identify the minimal regions that are essential for the constitutive expression, the sequences of the ARE were analyzed in detail by in vitro transcription assays using nuclear extracts from rat kidney, brain and liver. The analyses of various mutations in the promoter demonstrated that the proximal region of the ARE is required for the efficient transcription in every nuclear extract. The factors binding to this region in these nuclear extracts exhibited identical mobility in gel retardation assays. The ATF/CRE core motif is indicated to be important for the factor binding and for the promoter function in all nuclear extracts. The common binding factor in the nuclear extracts was revealed to be an ATF-1/CREB heterodimer by gel retardation assays using specific antibodies. We conclude that the ATF-1/CREB heterodimer is involved in the constitutive expression of the Na,K-ATPase alpha 1 subunit gene.

Activating Transcription Factor 1↗

PubMed1996

Regulatory elements of the mb-1 gene encoding the Ig-alpha component of the human B-cell antigen receptor.

The mb-1 gene encodes the Ig-alpha component of the B-cell antigen receptor. It is specifically expressed in pre-B and mature B cells but not in plasma cells losing membrane Ig (mIg) expression. We looked for transcriptional regulatory elements within a 12 kb genomic fragment. A strong promoter activity was found in a 591 bp fragment harboring consensus binding sites for known transcription factors including Ets, EBF/BlyF, LyF1/micro B and Spl. It was able to drive transcription of a reporter gene in the absence of any additional enhancer and was mostly active in B lymphocytes not in plasma cells or T cells. Although no fragment from the mb-1 gene displayed enhancer activity in combination with either the SV40, a Ig VH or a Ig VL promoter, a 1078 bp fragment corresponding to the 5' part of the gene behaved as a strong enhancer in either orientation in constructs driven by the mb-1 promoter itself. Deletions within this fragment allowed to delineate shorter sequences with enhancer activity upstream the first exon. The tissue-restricted, promoter-restricted and stage-specific activity of this 5' flanking region suggests that it is the main regulatory element of the mb-1 gene.

Animals↗

PubMed2002

The Opitz syndrome gene Mid1 is transcribed from a human endogenous retroviral promoter.

Human endogenous retroviruses (HERVs) and other long terminal repeat (LTR)-containing elements comprise a significant portion (8%) of the human genome and are likely vestiges of retroviral infections during primate evolution. Many of the HERVs present in human DNA have retained functional promoter, enhancer, and polyadenylation signals, and these regulatory sequences have the potential to modify the expression of nearby genes. To identify retroviral elements that contribute to the transcription of human genes, we screened sequence databases for chimeric (viral-cellular) transcripts. These searches revealed a fusion transcript containing the LTR of an HERV-E element linked to the Opitz syndrome gene Mid1. We confirmed the authenticity of the chimeric transcript by 5' rapid amplification of cDNA ends (RACE) and established that the Mid1 mRNA isoform was transcribed from a retroviral LTR. The identification of a retroviral first exon suggested the existence of alternative promoters for Mid1 because nonretroviral (native) 5' untranslated regions (UTRs) had been reported previously for this gene. Although Mid1 transcripts could be detected in all tissues tested, quantitative real-time reverse transcription-polymerase chain reaction indicated that the retroviral promoter contributes significantly to the level of Mid1 transcripts in placenta and embryonic kidney, where chimeric mRNAs were found to represent 25% and 22% of overall Mid1 mRNAs, respectively. Transient transfection studies supported a role for the LTR as a strong tissue-specific promoter in placental and embryonic kidney cell lines and suggested a function for the LTR as an enhancer. These findings provide further evidence that some endogenous retroviruses have evolved a biological function by contributing transcriptional regulatory elements to cellular genes.

Base Sequence↗

PubMed1997

Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes. A possible role for the DNA-binding protein Puralpha.

Nicotinic acetylcholine receptors constitute a multigene family (alpha2-alpha9, beta2-beta4) expressed in discrete temporal and spatial patterns within the nervous system. The receptors are critical for proper signal transmission between neurons and their targets. The molecular mechanisms underlying receptor gene expression have not been completely elucidated but clearly involve regulation at the level of transcription. We previously identified a novel 19-base pair (bp) transcriptional regulatory element in the promoter region of the rat beta4 subunit gene. This 19-bp element interacts specifically with DNA-binding proteins enriched in nuclear extracts prepared from adult rat brain. Using a combination of cellulose-phosphate, DNA-cellulose, and DNA sequence-specific affinity chromatographies, we purified the 19-bp element binding activity approximately 19,000-fold. Analysis by denaturing gel electrophoresis revealed the presence of four polypeptides in the most purified fraction, ranging in molecular masses between 31 and 114 kDa. Peptide sequence analysis revealed that one of the polypeptides is the bovine homologue of the transcriptional regulatory factor, Puralpha. Electrophoretic mobility shift assays indicated that Puralpha interacts directly and specifically with the 19-bp element. In addition, mobility shift assays using an anti-Puralpha monoclonal antibody revealed the presence of Puralpha, or an immunologically related protein, in nuclear extracts prepared from brain tissue. We hypothesize that the interaction between Puralpha and the 19-bp element is critical for proper expression of the beta4 subunit gene.

Amino Acid Sequence↗

PubMed2002

Enhancement of antibody responses to an HIV-2 DNA envelope vaccine using an expression vector containing a constitutive transport element.

Because immune responses to DNA vaccines in humans remains suboptimal, strategies need to be devised to facilitate expression of the vaccine in vivo. One method to improve response to a DNA vaccine is to construct plasmid vectors with leader sequences and post-transcriptional elements that facilitate export of transcribed RNA. In this study, we sought to determine if a mammalian expression vector (pND-14) containing a tissue plasminogen activator (TPA) leader sequence and a constitutive transport element (CTE) from simian retrovirus was superior to other mammalian expression vectors containing a post-transcriptional regulatory element (PRE) from hepatitis B virus (pCMV-link) or a minimal mammalian expression vector (pVAX1). Toward this objective, we evaluated protein expression of the HIV-2 envelope gene (gp140) in vitro and immune responses in immunized mice. We found that pVAX1 produced three- to fourfold lower levels of gp140 in vitro (5 ng/ml) in contrast to the pCMV-link and pND-14 vectors. When we immunized groups of mice intradermally with two of the HIV-2 gp140 DNA vaccine constructs, we found that pND-14 induced higher levels of envelope-specific systemic and mucosal antibodies than pCMV-link. We conclude that expression vectors for DNA vaccines should contain TPA and CTE sequences to facilitate immune responses.

AIDS Vaccines↗

PubMed2001

Sp1 and ETS family transcription factors regulate the mouse Mta2 gene expression.

Dynamic changes in chromatin structure through nucleosome remodelling and core histone tail acetylation play important roles in transcriptional regulation. The purification and functional characterization of a nucleosome remodelling and histone deacetylase complex, NuRD, has suggested that nucleosome remodelling and core histone tail modification are potentially linked processes. MTA2, a component of the NuRD complex, plays an important role in regulating histone deacetylase activity of the NuRD complex. Similar to the candidate metastasis associated protein MTA1, an elevated level of MTA2 correlates with cellular proliferation. To understand the regulation of Mta2 transcription, we characterized the mouse Mta2 gene and its transcriptional regulatory elements. We found that MTA2 is encoded by 18 exons that span 10 kb. Primer extension analysis identified a major transcriptional start site locates 259 base pairs upstream of the ATG translational start codon. Transient transfection studies localized its promoter, lacking a canonical TATA box, to within 60 base pairs upstream of the transcriptional start site. Gel-mobility-shift and mutagenesis studies revealed that Sp1 and ETS elements play important roles in regulating Mta2 transcription. Information concerning the regulation of the Mta2 gene expression will be useful in understanding the regulation of NuRD histone deacetylase activity, which in turn will help in our general understanding of the transcriptional repression mechanism.

3T3 Cells↗

PubMed2002

Insulators prevent transcriptional interference between two promoters in a double gene construct for transgenesis.

In transgenesis, the expression of two transgenes is often subject to mutual interference by each of the two expression cassettes when they are driven by different transcriptional regulatory elements in a single construct. To study this problem, we constructed vectors consisting of two expression units, one contains a strong ubiquitous promoter and the other contains a tissue-specific transcriptional element. The expression pattern of each transgene was examined in transfected cell lines and also in transgenic mice. In both cases, two expression units in a single construct were expressed in an independent manner and were controlled by their respective regulatory element only if we placed insulators at both ends of one expression unit. These results indicate that usage of insulators is a valuable tool for transfection of double gene constructs in transgenesis.

Animals↗

PubMed1986

Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus.

The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (rI1) gene when placed within 2.0 kilobases or less of the rI1 gene cap site. Inclusion of 4.0 kilobases of upstream rI1 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the rI1 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human beta-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent beta-globin transcription by a factor of 5 to 15. This rI1 "silencer" element functions in a manner relatively independent of position and orientation and requires a cis-dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rI1 silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains.

Animals↗

PubMed1991

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

Animals↗