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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta-globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.
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PMID:Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences. 301 46

We have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream region is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of greater than or equal to 600 nucleotides. In addition, we demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them.
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PMID:Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo. 342 49

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.
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PMID:The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein. 759 22

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory ganglionic neurons of infected animals. Expression of latency-related (LR) gene products is controlled by a 980-bp fragment (LR promoter). DNA sequence analysis revealed that two major open reading frames (ORFs) are in the LR gene. Antibodies directed against both ORFs were generated in rabbits by using synthetic peptides. Antibody P2, which is directed to sequences near the amino terminus of ORF 2, recognized a 41-kDa protein in lytically infected cells, suggesting that ORF 2 encodes a protein. When the LR gene was inserted into a mammalian expression vector and subsequently transfected into COS-7 cells, a 41-kDa protein was detected by use of silver-stained sodium dodecyl sulfate-polyacrylamide gels and by the P2 antibody. In contrast, this protein was not detected in mock-transfected cells. Deletion of DNA sequences containing ORF 2 blocked synthesis of the 41-kDa protein in COS-7 cells. Reverse transcriptase-mediated PCRs indicated that splicing occurs near the C terminus of ORF 2. Further studies indicated that LR RNA was alternatively spliced in latently infected cattle and that a fraction of LR RNA was poly(A)+. Taken together, these studies suggested that a spliced LR transcript has the potential to encode a 41-kDa protein.
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PMID:Identification of gene products encoded by the latency-related gene of bovine herpesvirus 1. 763 78

Previous mutations associated with lecithin:cholesterol acyltransferase (LCAT) deficiency have been identified using genomic DNA. To facilitate mutation analysis, we used cDNA from cultured fibroblasts which were shown to express LCAT mRNA. Using reverse-transcriptase PCR, LCAT cDNA was obtained from a 13-year-old boy with complete LCAT deficiency, characterized by low HDL-C (3 mg/dl), nondetectable initial cholesterol esterification rate, LCAT activity, and minimal LCAT mass (0.16 vs. 5-7.5 micrograms/ml). Sequencing of LCAT cDNA clones identified two mutations. A novel frameshift mutations caused by deletion of cytosine at the third nucleotide position of amino acid 168 (exon 5) predicts a disrupted protein catalytic site by converting Ser181-->Ala and creates a Pvu-II restriction site prior to premature truncation at amino acid 238. A C-->T transition results in a substitution of methionine for threonine at amino acid position 321 and creates an Nla-III restriction site on the maternal allele. Expression studies of mutant LCAT cDNA confirmed the virtual absence of LCAT activity in transfected COS-1 cells. The molecular defect in a young male with complete LCAT deficiency has been identified using fibroblast cDNA.
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PMID:Lecithin: cholesterol acyltransferase deficiency: identification of two defective alleles in fibroblast cDNA. 765 65

Extracellular androgen-binding proteins (ABP) are thought to modulate the regulatory functions of androgens. These proteins, which are secreted by the testis (ABP) and liver [sex hormone-binding globulin (SHBG)], are encoded by the same gene. In a previous study, the rat ABP/SHBG gene was sequenced, and a promoter (P1) was identified by primer extension. This promoter regulates synthesis of the mRNA that encodes secreted testicular ABP and fetal liver SHBG. In this study, the P1 transcriptional start site in testis and fetal liver was confirmed by RNase protection assays. We also identified an alternate promoter (PA) in the ABP/SHBG gene located 15 kilobases up-stream from the previously characterized testicular promoter (P1). The PA region has the characteristics of a GC-rich housekeeping-type promoter. RNAase protection and primer walking experiments with RNA polymerase chain reaction identified a region where the major sites of transcription initiation occur. Promoter PA directs the synthesis of alternate ABP RNAs, which contain an alternate exon 1 (exon A) sequence. One alternate ABP RNA, which contains exons A and 2-8 sequences, is expressed in testis, fetal liver, and brain. This alternate ABP RNA encodes an ABP-like protein (46,000 daltons) with an altered N-terminal sequence without a secretory signal peptide. Expression of the ABP-like protein in COS cells revealed that it is not secreted and does not appear to bind dihydrotestosterone. Another similar alternate ABP RNA is missing exon 6 sequence and encodes a nonsecretory truncated protein (28,000 daltons) that does not bind androgen. The functions of the ABP-like proteins are not obvious, but their functions are clearly different from secreted ABP and SHBG.
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PMID:Identification of an alternate promoter in the rat androgen-binding protein/sex hormone-binding globulin gene that regulates synthesis of a messenger RNA encoding a protein with altered function. 768 53

Two distinct calcitonin (CT) receptor (CTR)-encoding cDNAs (designated GC-2 and GC-10) were cloned and characterized from giant cell tumor of bone (GCT). Both GC-2 and GC-10 differ structurally from the human ovarian cell CTR (o-hCTR) that we cloned previously, but differ from each other only by the presence (GC-10) or absence (GC-2) of a predicted 16-amino acid insert in the putative first intracellular domain. Expression of all three CTR isoforms in COS cells demonstrated that GC-2 has a lower binding affinity for salmon (s) CT (Kd approximately 15 nM) than GC-10 or o-hCTR (Kd approximately 1.5 nM). Maximal stimulatory concentrations of CT resulted in a mean accumulation of cAMP in GC-2 transfected cells that was greater than eight times higher than in cells transfected with GC-10 after normalizing for the number of receptor-expressing cells. The marked difference in maximal cAMP response was also apparent after normalizing for receptor number. GC-2 also demonstrated a more potent ligand-mediated cAMP response compared with GC-10 for both human (h) and sCT (the EC50 values for GC-2 were approximately 0.2 nM for sCT and approximately 2 nM for hCT; EC50 values for GC-10 were approximately 6 nM for sCT and approximately 25 nM for hCT). Reverse transcriptase PCR of GCT RNA indicated that GC-2 transcripts are more abundant than those encoding for GC-10. In situ hybridization on GCT tissue sections demonstrated CTR mRNA expression in osteoclast-like cells. We localized the human CTR gene to chromosome 7 in band q22. The distinct functional characteristics of GC-2 and GC-10, which differ in structure only in the first intracellular domain, indicate that the first intracellular domain of the CTR plays a previously unidentified role in modulating ligand binding and signal transduction via the G protein/adenylate cyclase system.
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PMID:Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction. 776 7

We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.
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PMID:Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase. 777 23

Using a combination of polymerase chain reaction and genomic library screening we have cloned a human gene for a subtype of the somatostatin (SST) receptor (SSTR) termed human SSTR5 (hSSTR5), which is located on chromosome 16. The predicted amino acid sequence of hSSTR5 displays 75% sequence identity with a recently identified rat SSTR [Mol. Pharmacol. 42:939-946 (1992)], suggesting that it is the human homologue of this receptor. hSSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays considerable sequence identity to hSSTR1 (42%), hSSTR2 (48%), hSSTR3 (47%), and hSSTR4 (46%). Membranes prepared from COS-7 cells transiently expressing the hSSTR5 gene bound 125I-Leu8,D-Trp22,Tyr25-SST-28 (125I-LTT-SST-28) with high affinity and in a saturable manner. SST-14, SST-28, and various synthetic SST peptide agonists produced dose-dependent inhibition of radioligand binding with the following rank order of potency: LTT-SST-28 > SST-28 > D-Trp8-SST-14 > SST-14 approximately RC-160 approximately BIM 23014 > MK-678 > SMS 201-995. hSSTR5 bound SST-28 with a 12.6-fold greater affinity (Ki = 0.19 nM), compared with SST-14 (Ki = 2.24 nM), indicating that the receptor is SST-28 selective. Addition of GTP, guanosine-5'-O-(3-thio)triphosphate, Na+ ions, or pertusis toxin greatly reduced 125I-LTT-SST-28 binding, thereby indicating that hSSTR5 is coupled to pertussis toxin-sensitive G proteins. Both SST-14 and SST-28 displayed dose-dependent inhibition of forskolin-stimulated cAMP accumulation, consistent with functional coupling of the receptor to adenylyl cyclase inhibition. Northern blot analysis of SSTR5 mRNA revealed a 2.4-kilobase transcript in normal rat pituitary and GH3 rat pituitary tumor cells and a 4.0-kilobase transcript in normal human pituitary. Reverse transcriptase polymerase chain reaction revealed expression of the hSSTR gene in fetal human pituitary and hypothalamus but not in human cerebral cortex. In situ hybridization of the rat pituitary showed that SSTR5 mRNA is selectively localized in the anterior lobe. SSTR5 mRNA was not expressed in four human pituitary tumors (somatotroph adenoma, prolactinoma, and chromophobe adenomas) or in a human insulinoma. Although hSSTR5 displays approximately 75% sequence identity with rat SSTR5, the two receptors display significantly different pharmacological profiles, especially with respect to their binding affinities for the SST analogue SMS 201-995.
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PMID:Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28. 790 5

Two hybrid proteins between Pseudomonas aeruginosa exotoxin A (PE) and poliovirus protease 2Apro have been generated. One hybrid protein contains the poliovirus 2Apro sequence replacing the region of PE corresponding to amino acids 413-607. The other hybrid contains in addition the transforming growth factor sequence. The two hybrid proteins were efficiently synthesized in E. coli cells using the inducible pET vectors. Both hybrid toxins cleaved p220 (eIF-4 gamma) when the recombinant plasmids were transfected in COS cells infected with recombinant vaccinia virus bearing the T7 RNA polymerase gene.
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PMID:Hybrid proteins between Pseudomonas exotoxin A and poliovirus protease 2Apro. 795 60


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