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At least 811 records · Page 45Linked to original sources

PubMed1991

Structural and functional analysis of the mouse mdr1b gene promoter.

The overproduction of P-glycoprotein, an integral membrane protein thought to function as a drug efflux pump, is the hallmark of the multidrug resistance phenotype. In murine multidrug resistant J774.2 cell lines, distinct mdr genes, mdr1a and mdr1b, encode unique P-glycoprotein isoforms. To examine the transcriptional regulation of the mdr1b gene, its promoter was isolated and characterized. The transcription initiation site was mapped by primer extension, and the 5'-flanking region was sequenced. Several potential regulatory elements were identified in this region. A transient expression vector was constructed by fusion of 540 base pairs of 5'-flanking sequence and part of the first untranslated exon to the chloramphenicol acetyltransferase (CAT) gene. When transfected into monkey kidney COS-1, rat pituitary GH3 or T47D human breast cells, the mdr1b 5'-flanking sequences were capable of driving CAT expression. Transient transfection studies using deletion subclones of the mdr1b-CAT construct were done to locate potential cis-acting sequences. The studies indicate the presence of cis-acting elements in the 5'-flanking region of the mdr1b gene. The implications of these findings for expression and regulation of the mdr1b gene are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Unexpected characteristics of the IFN-gamma reporters in nontransformed T cells.

Analysis of the IFN-gamma promoter has primarily been conducted by transient expression of reporter constructs in transformed cells. However, the activity of cis elements may differ when expressed transiently compared with their activity within native chromatin. Furthermore, the transcription factors and signaling mechanisms in transformed cells may differ from those in normal T cells. To analyze IFN-gamma promoter regulation in normal T cells, we developed a novel retroviral bottom-strand reporter system to allow the chromatin integration of promoter regions in primary developing T cells. As controls, both the IL-2 and IL-4 promoters were inducible in this system, with the IL-4 reporter having Th2-specific activity. Strikingly, the IFN-gamma promoter exhibited constitutive activity in both Th1 and Th2 subsets, in contrast to the behavior of the endogenous IFN-gamma gene, which is inducible only in Th1 cells. In mapping this activity, we found that the AP-1/GM-CSF site in the distal promoter element is the most critical element for the constitutive activity. Transgenic reporter lines for the IFN-gamma promoter confirmed the constitutive behavior of the isolated IFN-gamma promoter. This constitutive activity was resistant to inhibition by cyclosporin A and was independent of Stat4 and p38 mitogen-activated protein kinase. These results suggest that IFN-gamma promoter regulation may require cis elements residing either downstream or >3.4 kb upstream of the transcriptional start site, involving repression of constitutive activity.

Animals↗

PubMed2006

Chromium (VI) inhibits heme oxygenase-1 expression in vivo and in arsenic-exposed human airway epithelial cells.

Inhaled hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms. One hypothesis for this lung pathogenesis is that Cr(VI) silences induction of cytoprotective genes, such as heme oxygenase-1 (HO-1), whose total lung mRNA levels were reduced 21 days after nasal instillation of potassium dichromate in C57BL/6 mice. To investigate the mechanisms for this inhibition, Cr(VI) effects on basal and arsenic (As(III))-induced HO-1 expression were examined in cultured human bronchial epithelial (BEAS-2B) cells. An effect of Cr(VI) on the low basal HO-1 mRNA and protein levels in BEAS-2B cells was not detectible. In contrast, Cr(VI) added to the cells before As(III), but not simultaneously with As(III), attenuated As(III)-induced HO-1 expression. Transient transfection with luciferase reporter gene constructs controlled by the full length ho-1 promoter or deletion mutants demonstrated that this inhibition occurred in the E1 enhancer region containing critical antioxidant response elements (ARE). Cr(VI) pretreatment inhibited As(III)-induced activity of a transiently expressed reporter construct regulated by three ARE tandem repeats. The mechanism for this Cr(VI)-attenuated transactivation appeared to be Cr(VI) reduction of the nuclear levels of the transcription factor Nrf2 and As(III)-stimulated Nrf2 transcriptional complex binding to the ARE cis element. Finally, exposing cells to Cr(VI) prior to co-exposure with As(III) synergized for apoptosis and loss of membrane integrity. These data suggest that Cr(VI) silences induction of ARE-driven genes required for protection from secondary insults. The data also have important implications for understanding the toxic mechanisms of low level, mixed metal exposures in the lung.

Animals↗

PubMed2001

Ectopic expression of the coxsackievirus and adenovirus receptor increases susceptibility to adenoviral infection in the human cervical cancer cell line, SiHa.

The expression level of the coxsackievirus and adenovirus receptor (CAR) gene in human cervical cancer cell lines (Hela, Caski, HT-3, and SiHa) appears to be correlated with their susceptibility to adenoviral vector-based gene transfer. Hela, Caski, and HT-3 cells, which express the CAR molecule on the cell surface, showed a higher susceptibility to infection of AdCMVGFP than SiHa cells with no detectable level of CAR expression. Transient expression of the CAR gene in SiHa cells dramatically enhanced the susceptibility to adenoviral infection. Furthermore, SiHa-CAR, a stable transfectant which expresses the CAR gene showed a highly increased susceptibility to adenoviral infection in contrast to SiHa. These results demonstrate that the low susceptibility of SiHa to adenoviral infection is closely related to its loss of the CAR gene expression. In addition, the low infection efficacy can be overcome by the ectopic expression of the CAR gene. These results also give insight into a possible application of the CAR gene to adenovirus-mediated gene delivery.

Adenoviridae↗

PubMed1998

Mapping of ORF121, a factor that activates baculovirus early gene expression.

The protein product of the 39k gene of Autographa californica nuclear polyhedrosis virus is thought to be important for viral replication because of its association with the virogenic stroma and its role in activation of late gene expression Transient expression assays showed that addition of a DNA fragment encoding a 58-amino-acid polypeptide increased expression of a 39k reporter plasmid. This stimulation was dependent on cotransfection of a plasmid encoding IE1. Cotransfection of this gene, orf121, also stimulated ie1 expression, and the activation of ie1 was even more dramatic in the presence of IE1. These data suggested that ORF-121 stimulated 39k expression by upregulation of IE1 expression. Activation of 39k by ORF121 and the viral transcription factor IE2 was additive, while activation by ORF-121 and the apoptotic suppressor P35 was synergistic. Cotransfection of p39cat and pIE1 with plasmids encoding ORF121, IE2, and P35 stimulated 39cat expression more than 100-fold compared to cells transfected with only p39cat and pIE1. These data suggest that IE2 and ORF121 work by similar mechanisms and indirectly activate p39cat by increasing IE1 expression, while P35 increases 39cat expression by a different mechanism.

Amino Acid Sequence↗

PubMed2001

The fusion protein of Peste des petits ruminants virus mediates biological fusion in the absence of hemagglutinin-neuraminidase protein.

To study the process of membrane fusion in Morbilliviruses, the fusion (F) glycoproteins of Peste des petits ruminants virus (PPRV) and Rinderpest virus (RPV) were expressed transiently in mammalian cells. The recombinant F proteins were found to be localized at the surface of transfected cells. The fusion activity, as evident from cell fusion assays and lysis of chicken erythrocytes, documented that transiently expressed PPRV F glycoprotein induces cell fusion in the absence of homotypic hemagglutinin-neuraminidase (HN) attachment glycoprotein. The coexpression of homotypic HN increased the extent of fusion by twofold, while the efficiency of fusion was found to be substantially enhanced. In contrast, in RPV F-expressing cells, fusion was detected only when homotypic hemagglutinin (H) or heterotypic HN protein was coexpressed. This differs from the strict type-specific requirement for the attachment protein as in the fusion process of most of the paramyxoviruses. Further, we demonstrate by fluorescence transfer experiments that while PPRV F brings about both hemifusion and complete fusion on its own, RPV F induces only hemifusion while it brings about complete fusion in the presence of homotypic or heterotypic attachment protein.

Animals↗

PubMed2001

Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 ORF50 gene product contains a potent C-terminal activation domain which activates gene expression via a specific target sequence.

The ART (Activator of Replication and Transcription) protein of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8 (HHV-8), is encoded by the ORF50 gene. It is expressed as an immediate-early gene and plays a crucial role in the transition between latency and productive infection. HHV-8 ART is a transcriptional transactivator which can up-regulate viral gene expression. Transient expression assays showed that ART strongly activated ORF57 and K8 promoter-directed gene expression in both CV-1 and BJAB cells. The ART target site was mapped to a 40-bp region compassing nt 81904 to 81943 on the ORF57 promoter. When linked upstream to a heterologous SV40 promoter, this region by itself was able to confer ART responsiveness. This 40-bp segment contains a 16-bp consensus sequence which is also found in the K8 promoter region located between nt 74769 to 74784. Deletion of the fragment including this 16-bp consensus abrogated the ART responsiveness of the K8 promoter. The role of this 16-bp consensus in ART transactivation was further supported by site-directed mutagenesis. Mutations of the conserved nucleotides within the 16-bp consensus in the ORF57 promoter dramatically impaired its responsiveness to ART. Fusion protein analysis with chimeric proteins containing the DNA binding domain of yeast transactivator Gal4 (residues 1 to 147) and different ART segments defined an acidic C-terminal region (amino acids [aa] 527 to 634) as a potent activator. Deletions of this activation domain in the ART protein resulted in a decrease or loss of its ability to activate ORF57 and K8 promoters containing the ART responsive element in transfected cells. How the ART activation domain activates ORF57 and K8 gene expression through the 16-bp consensus sequence remains to be determined.

Basic-Leucine Zipper Transcription Factors↗

PubMed1996

Relationship between perinatal appearance of cellular retinol-binding protein, type II and retinal reductase activity in chick liver.

To explore a role of the transiently appearing cellular retinol-binding protein, type II (CRBP(II)) in perinatal chick liver, we have examined whether the relationships exist among the perinatal changes in hepatic CRBP(II) protein and mRNA levels, retinal reductase activity and beta-carotene levels in liver and serum. Northern blot analysis for hepatic CRBP(II) revealed a transient expression of CRBP(II) mRNA around hatching. The protein of CRBP(II) was also expressed transiently and the highest levels of CRBP(II) were found in the livers 1-3 days after birth. The retinal reductase activity was very low at embryonic age, but its activity rapidly rose at hatching, peaking at 1 day after birth, followed by a gradual decrease to a lower level in 7-day-old chicks. This perinatal pattern of the retinal reductase activities was similar to the pattern of transient appearance of the hepatic CRBP(II), and was also paralleled to the developmental changes in serum and liver beta-carotene concentrations. These findings suggest that hepatic CRBP(II) transiently appearing during the perinatal period may involve in metabolizing hepatic beta-carotene, directing the retinal to the retinal reductase and leading further to the subsequent esterification of the converted retinol.

Alcohol Oxidoreductases↗

PubMed1990

The short arm of chromosome 11 likely is involved in the regulation of the human papillomavirus type 16 early enhancer-promoter and in the suppression of the transforming activity of the viral DNA.

Loci on the short arm of chromosome 11 between 11p11 and 11p15 likely are involved directly or indirectly in the regulation of the HPV-16 enhancer-promoter strength and this may contribute to the control of the level of viral early gene expression. Transient expression assays have shown that the HPV-16 enhancer-promoter functions much stronger in fibroblasts in which this region of chromosome 11 (del-11 cells) is deleted than in normal diploid human embryonic fibroblasts. The differential regulation of the HPV-16 long control region may be due either to the presence or activity of a cellular transcription factor which up-regulates HPV-16 early gene expression in del-11 cells or to a factor which down-regulates expression in diploid cells. Since the HPV-16 enhancer containing fragment (nt 7003 to nt 57) in combination with the SV40 promoter functions equally well in del-11 cells as in diploid cells a target of this factor likely is located in the putative HPV-16 early promoter region. The relative resistance of diploid human embryonic fibroblasts to HPV-16 induced transformation could be explained by the inactivity of the HPV-16 early promoter as these cells could be transformed by HPV-16 DNA constructs in which the early gene expression was driven from a heterologous enhancer-promoter. These results may indicate that loci on the short arm could suppress HPV-16-induced transformation by down-regulating the activity of the viral early promoter.

Cells, Cultured↗

PubMed1992

Molecular mechanisms of visna virus Tat: identification of the targets for transcriptional activation and evidence for a post-transcriptional effect.

Visna virus is a pathogenic lentivirus of sheep that is distantly related to the primate lentiviruses, including the human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 in cell culture requires the expression of a virus-encoded protein, Tat, which is a potent trans-activator of viral gene expression. Visna virus encodes an analogous Tat protein that greatly increases gene expression directed by the visna viral LTR. This report uses a stable vero cell line that constitutively expresses visna virus Tat to investigate the molecular mechanism of action of Tat on viral gene expression. Transient expression assays, using the visna virus LTR to drive transcription of the bacterial gene for chloramphenicol acetyltransferase (CAT), demonstrate that Tat trans-activates gene expression by increasing steady-state mRNA levels. The increase in steady-state mRNA levels is sufficient to account for the increase in protein observed and is due, in part, to an increase in the rate of transcription initiation. Tat mediates the accumulation of mRNA through AP-4 and AP-1 binding sites located in the U3 region of the LTR. Deletion of the upstream AP-1 and AP-4 binding sites results in a residual low level of trans-activation by Tat. Further experiments, using LTRs with R-U5 sequences deleted to +10, demonstrate AP-1 and AP-4 mediated responses to TAT at the RNA level, but no increase was observed in CAT protein.

Base Sequence↗

PubMed1995

Constitutively active Gq-alpha stimulates prolactin promoter activity via a pathway involving Raf activity.

We have investigated the ability of a constitutively active Gq-alpha mutant, Q209L-alpha q, to regulate target gene expression. Transient expression in GH3 pituitary cells of a rat proximal prolactin promoter-chloramphenicol acetyltransferase construct (-187)PRL-CAT, was stimulated by co-expression of Q209L alpha q, but not by wild-type alpha q. Q209L-alpha q stimulated expression of constructs driven by promoters for either rat prolactin or growth hormone, but not of a control construct driven by the thymidine kinase promoter. Thus, transcriptional effects of alpha q are specific both for the activated state of this G-alpha subunit and the promoter examined. Since both the prolactin and growth hormone promoters are activated by the pituitary cell-specific transcription factor Pit-1, we examined whether a Pit-1 binding site could direct a response to Q209L-alpha q. Two copies of prolactin promoter Pit-1 binding site 1P conferred upon a heterologous metallothionein promoter a response to Q209L-alpha q, implying an involvement of this site in the transcriptional action of Q209L-alpha q on the prolactin promoter. The phorbol ester activator of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate, stimulated (-187)PRL-CAT activity, but opposed the action of Q209L-alpha q on activity of this PRL-CAT construct. Q209L-alpha q stimulation of (-187)PRL-CAT activity was inhibited by co-expression of a dominant negative Raf mutant, Raf-C4, but not by a point mutant of Raf-C4 with reduced inhibitory properties. These results imply that activated alpha q subunits can stimulate prolactin promoter activity via a pathway that involves a Pit-1 DNA binding site(s), is opposed by protein kinase C, and is mediated by a pathway in which Raf-1 kinase plays a role.

Animals↗

PubMed1991

Tottering and leaner mutations perturb transient developmental expression of tyrosine hydroxylase in embryologically distinct Purkinje cells.

The mouse mutants tottering and leaner exhibit neurologic disorders associated, in part, with global noradrenergic hyperinnervation. Therefore, the expression of tyrosine hydroxylase (TH) mRNA and protein was examined in mutant and control mice. TH expression was normal in the major catecholaminergic nuclei. However, TH was expressed in vermal Purkinje cells of adult mutant but not control mice. TH expression in the Purkinje cells of both mutants was first observed on P21 and persisted throughout adulthood; in contrast, Purkinje cells of normal mice expressed TH transiently during development from P21 to P35. Thus, tottering and leaner mice are deficient in suppressing the normal transient expression of TH in developing Purkinje cells, suggesting that the protein encoded by the tg locus may play a crucial role in neuronal development.

Animals↗

PubMed2003

An intronic endogenous retrovirus-like sequence attenuates human haptoglobin-related gene expression in an orientation-dependent manner.

The human haptoglobin-related gene (HPR) gene codes for a haptoglobin-related protein (Hpr), a component of trypanosome lytic factor which circulates in plasma in small quantities. Except for the presence of a retrovirus-like element, RTVL-Ia, in intron 1, HPR is 92% identical in sequence to the closely linked human haptoglobin gene (HP) gene coding for haptoglobin. We have explored experimentally in tissue culture and in vivo in mice and in humans the influence of the retroviral-like sequence type Ia (RTVL-Ia) element on HPR expression. Transient expression in HepG2 cells of plasmids carrying the HPR promoter joined by a shortened version of intron 1 to the chloramphenicol acetyltransferase (CAT) vector showed that fragments containing the 5' long terminal repeat (LTR) had no significant effect. In contrast, a gag-pol related part and a pol-env-3'LTR related part of RTVL-Ia decreased expression to 20% and 40% of that in their absence but only when they were in naturally occurring orientation. The latter fragment that contains sequences reminiscent of elements essential for retrovirus viability, such as a splicing acceptor site, TATA box and polyA addition signal sequence, was further tested in site-specific transgenic mice. Similar to in vitro experiment, insertion of this fragment into an HPR transgene in mice reduced HPR expression to 50% compared to a transgene without the insert, but none of the viral sequence motifs appear to explain this effect. Instead, we found within the fragment two cryptic splicing donor sites whose products were present in transgenic mouse and in human liver RNA. Our data suggest that a combination of multiple small effects of RTVL-Ia including aberrant splicing accounts for the low (6%) expression of the present-day HPR relative to HP.

Alternative Splicing↗

PubMed1998

Interaction of DNA-binding proteins with the 5'-flanking region of a cytokinin-responsive cucumber hydroxypyruvate reductase gene.

Transcription of the cucumber hpr-A gene is responsive to cytokinin and light. To investigate the molecular basis for transcriptional regulation by cytokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (-382 to -67) contains sequences necessary for cytokinin responsiveness of the luciferase reporter gene. Band shift assays detected cytokinin-enhanced and -reduced protein binding sites in a 97 bp fragment (-382 to -285) upstream of the hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5'-AAATGACGAAAATGC-3', that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5'-AAGATTGATTGAG-3', of unknown function. Two-dimensional band shift analysis of the cytokinin-responsive DNA protein complex revealed the presence of six DNA protein interactions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share identical signal transduction pathways in regulating hpr-A gene expression.

Alcohol Oxidoreductases↗

PubMed2004

Induction of hTERT expression and phosphorylation by estrogen via Akt cascade in human ovarian cancer cell lines.

We examined the mechanism by which estrogen regulates telomerase activity in Caov-3 human ovarian cancer cell lines, which express ER, to determine whether the regulation affects the expression and/or phosphorylation of the telomerase catalytic subunit (hTERT). 17beta-Estradiol (E(2)) induced telomerase activity and hTERT expression. Transient expression assays using luciferase reporter plasmids containing various fragments of hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the E(2)-induced activation of the hTERT promoter. Either pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or transfection with a dominant-negative Akt attenuated the E(2)-induced activation of the hTERT promoter. In addition, estrogen induced the phosphorylation of IkappaB inhibitor protein via the Akt cascade, and cotransfection with a dominant-negative subunit of NFkappaB attenuated the response of the ERE-deleted hTERT promoter to E(2). Moreover, E(2) induced the phosphorylation of hTERT, the association of 14-3-3 protein and NFkappaB with hTERT, and nuclear accumulation of hTERT in an Akt-dependent manner. These results indicate that E(2) induces telomerase activity not only by transcriptional regulation of hTERT via an ERE-dependent mechanism and a PI3K/Akt/NFkappaB cascade, but also by post-transcriptional regulation via Akt-dependent phosphorylation of hTERT. Thus, the phosphorylation of Akt is a key event in the induction of telomerase activity by E(2) in human ovarian cancer cells.

14-3-3 Proteins↗

PubMed2001

Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Cre-dependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.

Animals↗

PubMed1993

In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment.

The particle bombardment method of gene transfer provides an alternative approach for analysis of in vivo promoter activity and transgene expression. Transient expression of the firefly luciferase gene from five viral and five cellular promoters was assessed after in vivo gene transfer using this method. The relative strengths of these promoters were quantitatively determined in five different rat tissues: skin epidermis, dermis, muscle, liver, and pancreas. Cytomegalovirus immediate early enhancer/promoter activity was consistently the highest in each tissue, whereas other promoters displayed tissue-specific preferences. In liver, the mouse phosphoenolpyruvate carboxykinase and metallothionein promoters were stimulated in vivo by inducing agents at 1 and 5 days posttransfection. In dermis, sustained luciferase activity was observed for over 1.5 years after gene delivery. In vivo transgene expression was also detected in bombarded mouse, rabbit, and rhesus monkey tissues. These results suggest that particle bombardment provides an effective system for studies of in vivo gene transfer and gene therapy.

Animals↗

PubMed1993

Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo.

Heat shock protein 90 (hsp90) is associated with many steroid receptors in tissue homogenates. It is widely accepted that hsp90 regulates the binding of the receptor to the corresponding gene regulatory element. However there is no unequivocal evidence that steroid receptor-hsp90 complexes are present in the intact cells. We demonstrate here the absence of progesterone receptor (PR)-hsp90 complexes in intact target cell nuclei, using immunohistochemical and biochemical methods to determine the location and composition of the nonliganded (aporeceptor) and liganded (holoreceptor) PR complexes. In the chicken oviduct cells, both apo- and holoreceptors were nuclear, while hsp90 was exclusively cytoplasmic. When expressed transiently in HeLa cells, hsp90 was detected in the cytoplasm and PR was detected in the nucleus. Their location or staining intensity was not affected when they were coexpressed in the same cells. To confirm that the sensitivity of the immunohistochemical detection of hsp90 and PR did not differ significantly, a chimeric hsp90-PR was transiently expressed in HeLa cells. Both hsp90 and PR antigens of the chimera were detected in nuclei with the same intensity. In homogenates of the same tissue samples that were used for immunohistochemistry, the PR was complexed with hsp90. Hsp90-PR complexes were formed in vitro when immature bursa of Fabricius, known to contain high levels of hsp90, was homogenized in the presence of hsp90-free aporeceptor, while holoreceptor did not associate with hsp90. Our data show that nuclear PR is not complexed with hsp90 in vivo and suggest that the 8S-PR may be an in vitro artifact generated during tissue processing.

Animals↗