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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human KB cell or alpha folate receptor (alpha hFR) is a membrane glycoprotein of 42 kDa that participates in the internalization of folates and antifolates. Seven independent alpha hFR cDNA isoforms have been reported that contain unique 5' termini but share a common open reading frame (ORF). To investigate the molecular basis of these heterogeneous 5' sequences, we determined the sequence of the alpha hFR gene from two clones isolated from a human lymphocyte lambda DASH genomic library. The gene is composed of seven exons that span 6.8 kb. The ORF is encoded by exons 4 through 7 while the reported 5' termini of the cDNA isoforms (including two novel cDNAs designated KB2 and KB4) are encoded by exons 1 through 4. Using RNase protection assays, we demonstrate that transcripts corresponding to the KB1 and KB4 cDNAs originate from promoters upstream from exon 1 and exon 4, designated P1 and P4, respectively, and that these mRNA isoforms are the most abundant transcripts expressed in KB cells and selected normal tissues (including kidney, lung, and cerebellum). We observed a heterogeneous start site within exon 1 from the P1 promoter while transcripts from the P4 promoter originate from a single site. In addition, we detected tissue specificity for the P1 and P4 promoter utilization. Transcripts originating from the P1 promoter are the most abundant transcripts expressed by human cerebellum and kidney. In contrast, transcripts from the P4 promoter are the most abundant transcripts expressed by human KB cells and lung. Total RNA from KB cells also protects a 66 bp fragment of an exon 3 riboprobe that is consistent with an alternatively spliced transcript. To examine the functional activity of the predicted P1 and P4 promoters, alpha hFR promoter-CAT chimeric plasmids were constructed using sequences flanking exon 1 and exon 4. We observed a 7.5- and 10-fold increase in CAT activity in HeLa cells transiently transfected with the P1 and P4 promoter constructs, respectively. These data demonstrate that a single gene encodes the divergent 5' termini of the alpha hFR cDNAs and that the alpha hFR transcripts are transcribed from two promoters that are activated in a tissue-specific manner.
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PMID:The divergent 5' termini of the alpha human folate receptor (hFR) mRNAs originate from two tissue-specific promoters and alternative splicing: characterization of the alpha hFR gene structure. 906 95

Cis-acting regions regulating transcription of the alpha1(VI) collagen chain have been investigated in vitro by transfection of promoter-CAT (where CAT is chloramphenicol acetyltransferase) constructs in different types of cultured cells and in vivo in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomic and cDNA sequences in which small deletions of the collagenous domain had been engineered. 215 bp of 5'-flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transgenic lines, indicating that this fragment contains the basal promoter, but not activator sequences. Constructs with 0.6 and 1.4 kb of the 5'-flanking region produced significantly higher CAT activity in transfected cells and were expressed in tissues of about 30% of transgenic lines. Although CAT activity was totally unrelated to the pattern of expression of the alpha1(VI) mRNA, these results suggest the presence of an activator(s) between -0.2 and -0.6 kb from the transcription start site. When the promoter size was increased to 5.4 or 6.5 kb, CAT activity was stimulated severalfold relative to the construct p1.4CAT and p4.0CAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimulation was, however, not observed in C2C12 myoblasts. Transgenic mice generated with 6.5CAT construct or minigenes, containing 6.2 kb of promoter, exhibited very high levels of expression, which was similar to the relative amount alpha1(VI) mRNA in the majority of tissues, with the exception of lung, adrenal gland and uterus. CAT activity in tissues was 100-1000-fold higher than that measured in transgenic mice with shorter promoter (0.6 or 1.4 kb). Since expression of minigenes was determined by RNase protection assay, the levels of mRNA per transgene copy were compared to those of the chromosomal gene and found to be always less than one quarter. These data suggest that the region -4.0/-5.4 contains an important activator(s) sequence which induces transcription in several, but not all, type VI collagen-producing tissues. Finally, analysis with the longest promoter fragment (7.5 kb) revealed a complex effect of the region -6.5/-7.5 on alpha1(VI) chain transcription. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblasts and activating in chondroblasts in vitro, whereas transgenic animals generated with 7.5CAT construct produced a pattern of expression comparable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreased. However, the ratio of transgene/chromosomal gene expression was not constant, but varied in a way dependent on the tissue. This observation suggests that the fragment studied contains key sequences for the age-dependent regulation of the alpha1(VI) gene. No phenotypic alterations were induced by the presence of mutations in the minigenes.
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PMID:Tissue-specific expression of promoter regions of the alpha1(VI) collagen gene in cell cultures and transgenic mice. 924 27

The human dopamine transporter (hDAT) is a member of the subfamily of monoamine transporters which show a common topological structure and possess significant amino acid sequence homology. We isolated and characterized the hDAT gene including about 1 kb of 5'-flanking region. The hDAT gene spans over 64 kb, consisting of 15 exons separated by 14 introns. The intron-exon structure of the hDAT gene is most similar to that of the human noradrenaline transporter (hNAT) gene. Promoter sequence analysis demonstrated a 'TATA'-less, 'CAT'-less and G+C-rich structure. Two E box and several Sp-1-binding sites exist in the promoter region. These structural features are similar to that of the human D1A dopamine receptor gene and the human monoamine oxidase A gene. The transcription start site was determined by both 5'-RACE and RNase protection assay. We determined the 5' end of the mRNA by identifying the 5'-terminal cap-G residue in 5'-RACE and RNase protection assay.
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PMID:Structure and organization of the gene encoding human dopamine transporter. 930 Aug 14

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.
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PMID:Decreased Stability of Transforming Growth Factor beta Type II Receptor mRNA in RER+ Human Colon Carcinoma Cells 939 99

Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell growth and tumor progression. Previous work has shown that loss of functional TGF-beta type II receptor (RII) due to a frameshift mutation in the 5' half of the RII gene leads to TGF-beta resistance in a highly progressed, RER+ human colon carcinoma cell line designated HCT116. Expression of this mutated RII gene was highly repressed in RER+ cell lines such as HCT116 and RKO, as analyzed by RNase protection assays. Nuclear run-on and RII promoter-reporter (CAT) assays showed that the transcriptional levels of the RII gene in these RER+ cells were not reduced, compared to RII-expressing cells. However, the half-lives of the RII mRNA, as analyzed by RNase protection assays following actinomycin D treatment, were significantly decreased. This suggested that the decreased expression of the RII gene mutant was due to decreased mRNA stability. Furthermore, RII mRNA from HCT116 transfected with wild-type RII had a longer half-life than the endogenous mutated RII mRNA. A dominant negative RII mutant, which encodes a similarly truncated RII protein as HCT116 but lacks the extensive 3' untranslated region of RII mRNA, gave the same half-life as endogenous wild-type RII mRNA. We conclude that the frameshift mutation which results in a premature stop codon in the 5' half of the mRNA transcript accounts for the reduced RII mRNA levels in RER+ cells.
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PMID:Decreased stability of transforming growth factor beta type II receptor mRNA in RER+ human colon carcinoma cells. 940 53

Water channel aquaporin-1 (AQP1) is expressed in erythrocytes and various epithelia and endothelia. To study AQP1 gene regulation, human cell lines were screened for inducible AQP1 expression. Human erythroleukemia HEL cells showed AQP1 transcript expression on RNase protection assay. After butyrate-induced erythroid differentiation, AQP1 transcript expression increased strongly, producing water-permeable cells with plasma membrane localization of immunoreactive AQP1. In addition, a clonal subline of K562 cells [K562(S)] showed strong butyrate-induced expression of functional AQP1. A 1.8-kb DNA fragment of the 5' flanking region of the human AQP1 gene was isolated, sequenced, and analyzed functionally by the CAT reporter assay. The AQP1 promoter contained TATA and CCAAT boxes; Sp1, AP1, AP2, and E-box elements; and erythrocyte-specific CACCC and Kruppel-like (CCCCACCCA) elements. AQP1 promoter activity was more than 24-fold higher in HEL and K562(S) cells than in nonerythroid (HeLa) cells, indicating the presence of erythroid-specific factors. In K562(S) cells, CAT activities for promoter fragments to bp +23 [relative to beta-gal and normalized to 100% for the plasmid CP-282 (bp -282 to +23)] were 22 (-1779), 73 (-1402), 61 (-1129), 31 (-789), 87 (-487), 100 (-282), 73 (-229), 52 (-152), and 60% (-79). After butyrate-induced differentiation, CAT activities were stimulated approximately 10-fold for constructs -229/+23 and longer, compared to approximately 5-fold for -152/+23 and -79/+23; glucocorticoids did not affect CAT activities. These results suggest a basis for erythroid-specific AQP1 expression and the presence of a butyrate-response sequence involved in inducible AQP1 regulation in erythroleukemia cells.
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PMID:Isolation of the human aquaporin-1 promoter and functional characterization in human erythroleukemia cell lines. 948 Jul 47

Carnitine acetyltransferase (CAT; EC 2.3.1.7) is localized in two subcellular organelles, peroxisomes and mitochondria, in an n-alkane-assimilating yeast, Candida tropicalis. The isozymes are synthesized from the first and second ATG codon of the open reading frame of one gene, CtCAT. Primer extension analysis and RNase protection assay (RPA) revealed that multiple transcription initiation sites were found upstream of the first ATG codon. 5' ends could not be detected between the first and second ATG codons. These results suggested that the peroxisomal CAT of C. tropicalis, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When CtCAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts and the protein products were similar to those observed in C. tropicalis. This suggested that the transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Using S. cerevisiae as the host cell, introduction of mutations into the sequence near the first AUG codon or a deletion in the region between the first and second AUG codons resulted in an increased ratio of translation from the first AUG codon, although initiation sites of transcription did not change. Moreover, replacing the 5' leader sequence to that of C. tropicalis isocitrate lyase promoter (UPR-ICL) eliminated the product initiating at the second AUG codon. The transcript from these cells was shorter than those detected from the native CtCAT-harboring cells. From these results, it was strongly suggested that peroxisomal and mitochondrial CAT isozymes occurred by the alternative initiation of translation mainly dependent on the structure and sequence context of the region from the 5' end to the second AUG codon, and not the insufficient length of the 5' leader.
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PMID:Peroxisomal and mitochondrial carnitine acetyltransferase isozymes of the n-alkane-assimilating yeast, Candida tropicalis, occurred by alternative initiation of translation from the transcripts of a single gene. 956 89

The M1 protein of influenza virus inhibits the in vitro transcriptase activity of ribonucleoprotein cores from virions. This inhibitory activity is thought to be relevant in vivo because accumulation of M1 at the late stages of viral replication may be the cue to halt viral mRNA production. A model influenza reporter genome was used to explore the effect of M1 on the activity of the influenza virus transcriptase complex within cultured cells. Expression of M1 in cells bearing the model influenza virus reporter genome was accompanied by a reduction of CAT gene expression to 12% of control levels. Quantification of RNA by ribonuclease protection assay revealed that the influenza reporter genome mRNA levels in M1-expressing cells were reduced by approximately 74% compared with those of cells expressing a control protein. These findings are consistent with the proposed model in which M1 is responsible for limiting viral transcription during late stages of infection. By expressing truncated forms of M1, the inhibitory activity was found to reside within the amino-terminal half of the M1 protein. Two independent inhibitory domains were identified in this region: one between amino acid residues 1-90 and the other spanning residues 91-127.
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PMID:The matrix 1 protein of influenza A virus inhibits the transcriptase activity of a model influenza reporter genome in vivo. 974 Jul 76

Cell cycle control of histone H4 gene transcription is mediated by the multipartite promoter domain H4-Site II, which supports transcriptional activation at the G1/S phase transition and modulates basal H4 gene transcription. Proliferation-specific transcription is determined by the integrated activities of three distinct promoter factors interacting with H4-Site II: the interferon regulatory factor IRF-2 (synonymous with HiNF-M), HiNF-D (a complex between the homeodomain protein CDP-cut and the cell cycle mediators CDC2, cyclin A and pRB), as well as HiNF-P/H4TF-2. However, the contribution of HiNF-D to the enhancement and/or suppression of H4 gene transcription at specific cell cycle stages remains to be established. We used a panel of synchronized HeLa S3 cell lines containing stably integrated H4 promoter/CAT reporter gene constructs with mutations in H4-Site II. The temporal regulation of CAT mRNA accumulation under the control of the H4 promoter was analyzed by RNase protection analysis. Our main finding is that mutation of the HiNF-D/CDP-cut binding site alters the timing of histone gene activation during the cell cycle. Furthermore, our data indicate that HiNF-P/H4TF-2 may functionally compensate for HiNF-M/IRF-2 at Site II to regulate histone H4 gene transcription in HeLa S3 cervical carcinoma cells during early S phase. We postulate that HiNF-D (CDP-cut/cyclin A/CDC2/pRB containing complex) promotes HiNF-M/IRF-2 (and/or HiNF-P/H4TF-2) dependent histone H4 gene activation at the G1/S phase transition and attenuates H4 gene transcription at later cell cycle stages. The mechanistic division in the gene regulatory functions of the three H4-Site II binding proteins may ensure that histone H4 gene expression is stringently coupled with the onset of S phase in response to growth factor/cytokine-induced cell cycle progression.
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PMID:HiNF-D (CDP-cut/CDC2/cyclin A/pRB-complex) influences the timing of IRF-2-dependent cell cycle activation of human histone H4 gene transcription at the G1/S phase transition. 980 53

Several lines of evidence indicate that the number of GnRH receptors (GnRH-R) and therefore, gonadotrope responsiveness to GnRH, is highly dependent upon the level of GnRH-R mRNA. To explore this aspect of regulation, we have isolated a 3.3 kb fragment encompassing the promoter region of the rat GnRH-R gene. Primer extension and RNase protection assays allowed the identification of five major transcriptional start sites located within the 110 bp region upstream of the translation start codon. Transfection experiments using the CAT reporter gene demonstrated that the 1261 bp 5' flanking region is required to direct high efficient expression in the gonadotrope-derived alphaT3-1 cell line thus contrasting with mouse in which the only 500 bp proximal sequence appeared to be sufficient. Another difference between rat and mouse was apparent in the 183 bp region of the rat promoter which induced a 3-fold stimulation of thymidine kinase promoter activity in both alphaT3-1 and CHO cells. Subsequent deletion analysis of the region residing between -1261 and -519 revealed the presence of multiple regulatory domains that contributed to the cell-specific activity. However, despite this efficiency in the context of the wild-type promoter, they failed to induce the activity of the minimal thymidine kinase (TK) promoter in the absence of the proximal 600 bp promoter region. Accordingly, a composite TK promoter containing the entire 1.2 kb promoter induced a 10-fold increase in the activity of the TK promoter in alphaT3-1 cells. Taken together these data suggest that distal regulatory regions are critical and require cooperation with proximal specific-promoter elements for activating basal R-GnRH gene expression in gonadotrope cells.
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PMID:Multiple elements in the distal part of the 1.2 kb 5'-flanking region of the rat GnRH receptor gene regulate gonadotrope-specific expression conferred by proximal domain. 986 30


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