Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5' non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo.
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PMID:Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities. 1548 85

Matrix metalloproteinase-2 (MMP-2) plays important roles in cancer development and aggression. Our previous studies revealed a strong association between the MMP-2 -1306C/T polymorphism and risk of several cancers. A novel -735C/T polymorphism in MMP-2 promoter has been identified but the function is undefined. This study examined our hypothesis that these two polymorphisms might have functional relevance and impact on risk of esophageal squamous cell carcinoma in the context of haplotype. Genotypes and haplotypes were analyzed in 527 cases and 777 controls and odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The function of the polymorphisms was examined by electrophoretic mobility shift assays, luciferase gene expression assays, and reverse transcriptase-PCR analyses. It was found that the -735C-->T transition disrupts an Sp1 site and displays a lower promoter activity. The C(-1306)-C(-735) haplotype had 7-fold increased luciferase expression and 3.7-fold increased MMP-2 mRNA levels in esophageal tissues compared with the T(-1306)-T(-735) haplotype. A case-control analysis revealed a 1.52-fold (95% CI = 1.17-1.96) or 1.30-fold (95% CI = 1.04-1.63) excess risk of developing esophageal squamous cell carcinoma for the -1306CC or -735CC genotype carriers compared with noncarriers, respectively. A greater association was observed between elevated risk of developing esophageal squamous cell carcinoma and C(-1306) or C(-735) allele containing haplotypes, with the risk being highest for the C(-1306)-C(-735) haplotype compared with the T(-1306)-T(-735) haplotype (OR = 6.53; 95% CI = 2.78-15.33). The C(-1306)-C(-735) haplotype was also associated with increased risk for distant metastasis of esophageal squamous cell carcinoma (OR = 3.34; 95% CI = 1.16-9.63). These findings suggest that the C(-1306)-C(-735) haplotype in the MMP-2 promoter contributes to risk of the occurrence and metastasis of esophageal squamous cell carcinoma by increasing expression of MMP-2.
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PMID:Functional haplotypes in the promoter of matrix metalloproteinase-2 predict risk of the occurrence and metastasis of esophageal cancer. 1549 91

Telomerase, the enzyme which maintains the ends of linear chromosomes in eukaryotic cells is found in murine embryonic stem cells; however, its activity is downregulated during in vitro differentiation. Previous work has indicated that this is due to the transcriptional downregulation of murine reverse transcriptase unit (mTert) of telomerase. To investigate the factors that cause the transcriptional repression of mTert we defined a 300 bp region which is essential for its transcription and performed site directed mutagenesis and electrophoretic mobility shift assays. This analysis indicated that Sp1, Sp3 and c-Myc bind to the GC-boxes and E-boxes, respectively, within the promoter and help activate the transcription of mTert gene. We also identified a novel binding sequence, found repeated within the mTert core region, which when mutated caused increased mTert expression. Yeast one hybrid screening combined with electrophoretic mobility shift assays indicated that the nuclear protein Zap3 binds to this site and its overexpression leads to the downregulation of mTert during differentiation. This suggests that regulation of mTert transcription is a complex process which depends on a quantitative balance between transcription factors that cause activation or repression of this gene. Overexpression of Zap3 in murine embryonic stem cells results in reduction in telomerase activity and telomere length as well as reduced proliferative capacity and limited ability to contribute to the development of haematopoietic cells upon differentiation.
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PMID:A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonic stem cell differentiation. 1551 42

Telomerase activity is observed in approximately 90% of human cancer including esophageal squamous cell cancer. Normal somatic cells do not display telomerase activity on a regular basis. The major mechanism to regulate telomerase activity in human cells is the transcriptional control of the catalytic subunit, the human reverse transcriptase gene hTERT. However, the manner in which telomerase activity is regulated during malignant transformation and whether this regulation is influenced by single genetic alterations important in this process are not well understood. In this study we investigated the transcriptional regulation and activity of human telomerase in a cellular model representing important known genetic alterations observed in esophageal cancer. We characterized the respective cells with regard to their telomere biology and telomerase expression, transcriptional regulation using promoter--as well as electrophoretic mobility shift assay--analyses and their promoter methylation status. We could demonstrate that telomerase expression and subsequent activity are differentially regulated in the progression from normal esophageal epithelial cells to genetically defined esophageal cells harboring a specific genetic alteration frequently found in esophageal cancer and compared those changes with esophageal cancer cells. Whereas primary esophageal cells are mainly regulated by Sp1, in cells harboring a genetic alteration as cyclin D1 overexpression other transcription factors like E2F and c-myc as well as promoter methylation influence hTERT transcription. This model demonstrates that the transcriptional regulation of telomerase is influenced by a given genetic alteration important in esophageal cancer, and therefore provides new insight in telomerase regulation during carcinogenesis.
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PMID:Differential transcriptional regulation of human telomerase in a cellular model representing important genetic alterations in esophageal squamous carcinogenesis. 1595 20

The enzyme telomerase is essential for maintaining the ends of linear chromosomes. It plays an important role in cell proliferation, differentiation, tumorigenesis and aging. Telomerase is composed of an RNA subunit (TR) and a reverse transcriptase catalytic subunit (TERT). We report here the cloning and characterization of the gene encoding the TERT subunit from a teleost fish, Fugu rubripes. This is the first fish TERT gene to be cloned. The fugu TERT (fTERT) gene comprises of 16 exons and 15 introns similar to the human TERT (hTERT), and encodes a 1074 amino acid protein. The fTERT protein showed 33% to 35% sequence identity to other vertebrate TERTs, and contained all the signature motifs of the TERT family. Analysis of the promoter region of fTERT showed the presence of several transcription factor binding sites (E2F-1, E-box, ER, Sp1 and USF sites) in common with the hTERT promoter, and whose binding factors are known to regulate hTERT. The fTERT gene is expressed in a variety of tissues, with high expression detected in the gill, testis, and ovary. fTERT expression was detected in an immortalized fugu eye-derived cell line. The level of expression was found to be higher in actively dividing cells and reduced at quiescence, suggesting cell cycle regulation of TERT and possibly telomerase activity, in this cell line.
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PMID:Cloning and expression of the reverse transcriptase component of pufferfish (Fugu rubripes) telomerase. 1596 Dec 61

Telomerase biology is complicated by studies that show that telomere expression and telomere biology differs between species, and that existing animal models do not closely resemble the human situation. We have previously reported a description of telomere/telomerase biology in the dog and have suggested this as an alternative model system. To further elucidate telomerase biology in this species we have cloned and characterised the canine reverse transcriptase (dogTERT) promoter. We demonstrate that core promoter activity is contained within a region extending approximately 300 bp upstream of the ATG codon. Transient transfections in telomerase-positive canine cell lines and telomerase negative fibroblasts showed that the promoter is only active in telomerase positive cell lines. Sequence analysis demonstrated that the 5' regulatory region is GC-rich and contains no TATA or CAAT box, similar to the human TERT promoter. Motif searches revealed the presence of multiple transcription factor binding sites common to both the human and canine TERT promoters, including a single E-box, Sp1, AP1, MZF-2 and ER/Sp1 binding sites. These findings suggest that the dogTERT gene shares similar transcriptional control to hTERT. Identification of the core promoter necessary for activity may allow the use of naturally occurring cancers in dogs as a preclinical testing ground for telomerase targeted therapies in human cancer patients.
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PMID:The canine telomerase catalytic subunit (dogTERT): characterisation of the gene promoter and identification of proximal core sequences necessary for specific transcriptional activity in canine telomerase positive cell lines. 1605 48

While early transposon (ETn) endogenous retrovirus (ERV)-like elements are known to be active insertional mutagens in the mouse, little is known about their transcriptional regulation. ETns are transcribed during early mouse embryogenesis in embryonic stem (ES) and embryonic carcinoma (EC) cell lines. Despite their lack of coding potential, some ETns remain transposition competent through their use of reverse transcriptase encoded by a related group of ERVs-MusD elements. In this study, we have confirmed high expression levels of ETn and MusD elements in ES and EC cells and have demonstrated an increase in the copy number of ETnII elements in the EC P19 cell line. Using transient transfections, we have shown that ETnII and MusD LTRs are much more active as promoters in P19 cells than in NIH 3T3 cells, indicating that genomic context and methylation are not the only factors determining endogenous transcriptional activity of ETns. Three sites in the 5' part of the long terminal repeat (LTR) were demonstrated to bind Sp1 and Sp3 transcription factors and were found to be important for high LTR promoter activity in P19 cells, suggesting that as yet unidentified Sp binding partners are involved in the regulation of ETn activity in undifferentiated cells. Finally, we found multiple transcription start sites within the ETn LTR and have shown that the LTR retains significant promoter activity in the absence of its noncanonical TATA box. These findings lend insight into the transcriptional regulation of this family of mobile mouse retrotransposons.
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PMID:Transcriptional regulation of early transposon elements, an active family of mouse long terminal repeat retrotransposons. 1625 22

Telomerase is essential for immortalization of most human cancer cells. Expression of the core telomerase RNA (hTR) and reverse transcriptase (hTERT) subunits is mainly regulated by transcription. However, hTR transcriptional regulation remains poorly understood. We previously showed that the core hTR promoter is activated by Sp1 and is repressed by Sp3. Here, we show that the mitogen-activated protein kinase kinase kinase 1 (MEKK1)/c-Jun-NH(2)-kinase (JNK) pathway represses hTR expression by a mechanism that involves Sp1 and Sp3. Promoter activity was induced by the JNK inhibitor SP600125 and was repressed by activated MEKK1. Repression by MEKK1 was blocked by SP600125 or enhanced by coexpression of wild-type but not phosphoacceptor mutated JNK. SP600125 treatment also increased levels of endogenous hTR. Mutations in the hTR promoter Sp1/Sp3 binding sites attenuated SP600125-mediated promoter induction, whereas coexpression of MEKK1 with Sp3 enhanced hTR promoter repression. Chromatin immunoprecipitation showed that levels of immunoreactive Sp1 associated with the hTR promoter were low in comparison with Sp3 in control cells but increased after JNK inhibition with a reciprocal decrease in Sp3 levels. No corresponding changes in Sp1/Sp3 protein levels were detected. Thus, JNK represses hTR promoter activity and expression, apparently by enhancing repression through Sp3.
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PMID:Transcriptional repression of telomerase RNA gene expression by c-Jun-NH2-kinase and Sp1/Sp3. 1645 90

Thioredoxin is a redox-active protein that plays multiple roles in regulating cell growth, cell signalling and apoptosis. Here, we have demonstrated that a complex mechanism involving multiple regulatory elements is involved in the tBHQ [tert-butylhydroquinone or 2,5-di-(t-butyl)-1,4-hydroquinone]-mediated activation of the thioredoxin gene. Luciferase assays, utilizing various wild-type and mutated thioredoxin promoter fragments, revealed roles for the ORE (oxidative stress responsive element), ARE (antioxidant responsive element), three Sp1 (specificity protein 1)-binding sites and the TATA box in the activation of the thioredoxin gene by tBHQ. The ORE required the presence of the ARE to elicit its response, whereas the independent removal of three Sp1-binding sites and the TATA box also decreased activation of the thioredoxin gene, with mutation of the TATA box having the greatest effect. Real-time RT (reverse transcriptase)-PCR analysis also revealed varying roles for two TSSs (transcription start sites) in the activation of the thioredoxin gene by tBHQ. Transcription was initiated from both TSSs; however, different response rates and fold inductions were observed. Together, these results suggest that the thioredoxin gene is controlled by a novel arrangement of two overlapping core promoter regions, one containing a TATA box and the other TATA-less. Altering the intracellular levels of thioredoxin in a breast cancer cell line also influenced the induction of thioredoxin transcription in response to tBHQ. Stable transfections with a redox-inactive thioredoxin mutant produced 3.6 times higher induction levels of thioredoxin transcription compared with control cells, indicating an intrinsic form of control of promoter activity by the thioredoxin system itself.
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PMID:The tert-butylhydroquinone-mediated activation of the human thioredoxin gene reveals a novel promoter structure. 1671 25

Calreticulin (CRT) is a resident protein of the endoplasmic reticulum where it serves as a calcium modulator and chaperone to newly synthesized glycoproteins. In mammals, CRT is a structurally conserved 46 kDa protein that demonstrates anomalous migration at 60 kDa on SDS polyacrylamide gels and can be up-regulated by A23187 and thapsigargin due to the endoplasmic reticulum stress elements (ERSE) in the promoter region of its gene. CRT has numerous proposed functions and has been localized to the surface of PHA-stimulated T lymphocytes. CRT has been identified in mammals, plants and more recently from rainbow trout. Here, we report the cloning of the CRT proximal promoter from rainbow trout which includes elements typical of genes transcribed by RNA polymerase II including a TATA box, an Sp1 binding site, CCAAT boxes and the conservation of promoter stress elements (ERSE) demonstrated to be responsible for calcium modulation in mammals. This report demonstrates that the anomalous 60 kDa gel migration of mammalian CRT is conserved in rainbow trout and that CRT exists primarily as a dimer or oligomer in all tissues tested, excluding muscle and sperm in which it exists as a single polypeptide. Although it contains a potential N-glycosylation site, rainbow trout CRT is not subject to N-type glycosylation. Through the use of reverse transcriptase (RT) PCR along with western blotting, in both primary cultured leukocytes and the macrophage cell line RTS11, this report demonstrates that, unlike mammals, rainbow trout CRT is not strongly up-regulated by the calcium homeostasis antagonists, A23187 and thapsigargin, but is present on the cell surface of PHA-stimulated leukocytes. Taken together, this data suggests that CRT may have an alternative mode of regulation or function in fish.
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PMID:Calreticulin in rainbow trout: a limited response to endoplasmic reticulum (ER) stress. 1749 Sep 7


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