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)

Evidence suggesting the presence in rat liver nuclear extracts of a new RNP complex of 70-110S has been provided [Hatzoglou, M., Adamtziki, E., Margaritis, L. and Sekeris, C.E (1985) Exp. Cell. Res. 157, 227-241]. Biochemical features unique to this RNP were its stability to salt and RNase digestion and the presence of a pair of polypeptides of 72/74 kDa. By producing antibodies against the 72/74 kDa polypeptides these proteins have been defined as integral components of the 70-110S RNP complex. They comprise two immunologically related polypeptides with an exclusively nucleoplasmic localization, giving a speckled pattern in a diffuse background, similar, but not identical, to the Sm antigen. The 70-110S RNP complex, referred to as large heterogeneous nuclear RNP (LH-nRNP), has a simple protein pattern that includes, in addition to the 72/74 kDa proteins, three stably associated polypeptides of apparent molecular size 110, 61 and 59 kDa. The bulk of its RNA component represents a discrete RNA population of 10-20S, belonging to a subset of the RNA detected within immunopurified HeLa hnRNP complexes. These RNA species are RNA polymerase II transcripts of greater stability relative to the bulk of hnRNA, containing oligo(A) or poly(A) sequences. Immunodepletion and/or antibody addition studies in HeLa splicing extracts using antibodies with specificity for the 72/74 kDa proteins revealed a rather strong inhibition of splicing activity, suggesting participation of the LH-nRNP complex in in vitro splicing.
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PMID:Two immunologically related polypeptides of 72/74 kDa specify a novel 70-100S heterogeneous nuclear RNP. 765 36

EBNA 1 is the only antigen expressed in both Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma cells. Previous studies showed that the mRNA of EBNA 1 in these two tumor tissues was initiated from a promoter located in the Bam HI F fragment (Fp) on the viral genome. Two regulatory elements located in the downstream Bam HI Q region include an EBNA 1 binding site and a positive regulatory region between the Fp and the EBNA1 binding site. This data strongly suggested that a cellular factor(s) may modulate the usage of the Fp. To locate the shortest responsible viral sequence, we constructed a series of luciferase gene and chloramphenicol acetyltransferase (CAT) gene plasmids that contained various portions of the Bam HI F/Q region. Plasmid DNA was then introduced into cells to examine the promoter activity of each construct. By this method, we identified a 186-bp fragment within the Bam HI Q region that possessed the highest activity. This promoter was designated as Qp and found to be orientation-dependent and down-regulated by EBNA 1 in both the type I BL cells and human epithelial cells. Furthermore, RNase protection assay showed that a transcription initiation site was located at nucleotide 62,416 of the EBV genome. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis further confirmed that the transcript was initiated from the Qp, and not the Fp. Therefore, our data suggested that a novel promoter, Qp, located within the Bam HI Q existed for the EBNA 1 expression in the latently infected type 1 BL cells. The biological significance of the selection of the Qp needs further investigation.
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PMID:Identification of a novel promoter located within the Bam HI Q region of the Epstein-Barr virus genome for the EBNA 1 gene. 766 54

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

The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis (amiB), DNA repair (mutL), tRNA modification (miaA) and proteolysis (hflX-hflK-hflC). We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon. RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutL and miaA. In addition, two internal promoters, PmiaA and P1hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites. PmiaA contains poor matches to the -10 and -35 regions of the sigma-70 RNA polymerase consensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences. The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes. As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both. The amounts of steady state level mutL-miaA cotranscript, PmiaA transcript and P1hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne+ parent. In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc+ parent. These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity. A model is presented for RNase E-mediated posttranscriptional regulation that may coordinate mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.
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PMID:Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation. 774 52

Spontaneous production of insulin-like growth factor-I (IGF-I) by inflammatory macrophages contributes to aberrant wound healing, but little is known about regulation of IGF-I synthesis in myeloid cells. The T cell-derived cytokine interferon-gamma (IFN gamma) inhibits several fibrogenic and angiogenic components of the wound-healing response. We have used metabolic labeling of primary colony stimulating factor-1 (CSF-1)-derived macrophages and a transformed macrophage cell line (PU5-1R) followed by immunoprecipitation to demonstrate that synthesis of the 17 kilodalton (kDa) prepro-IGF-I protein by these cells is substantially inhibited by IFN gamma. An exon 4 IGF-I/beta-actin riboprobe expression cassette was used in RNase protection assays to show that IFN gamma also reduces steady state levels of IGF-I mRNA in three different populations of macrophages in a time- and dose-dependent manner. This effect is specific for IFN gamma because neither the IFNs-alpha/beta nor lipopolysaccharide (LPS) affects expression of steady state IGF-I transcripts. Down-regulation of IGF-I mRNA by IFN gamma is dependent on de novo protein synthesis and is abrogated by coculture with cycloheximide. Nuclear run-on assays revealed that elongation of IGF-I transcripts is absent in fresh bone marrow cells but is induced several-fold after cells are cultured for 6 days with CSF-1. Treatment of these CSF-1-derived macrophages with IFN gamma for 6 h substantially inhibits synthesis of IGF-I mRNA. Studies on the decay of IGF-I mRNA in PU5-1R macrophages treated with an RNA polymerase inhibitor confirmed that the decline in IGF-I steady state mRNA in IFN gamma-treated cultures arises from an inhibition of transcription rather than from a reduction in mRNA stability. Since a variety of inflammatory mediators can induce expression of IGF-I in macrophages, inhibition of macrophage IGF-I synthesis by IFN gamma provides a mechanism by which leukocytes regulate levels of this growth factor in their microenvironment.
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PMID:Interferon-gamma inhibits macrophage insulin-like growth factor-I synthesis at the transcriptional level. 777 81

RNase protection studies reveal two stable RNAs (250 and 280 nucleotides) transcribed from the antisense strand of the human ribosomal protein gene RPS14's first intron. These transcripts, designated alpha-250 and alpha-280, map to overlapping segments of the intron's 5' sequence. Neither RNA encodes a polypeptide sequence, and both are expressed in all human cells and tissues examined. Although alpha-280 is detected among both the cells' nuclear and cytoplasmic RNAs, the great majority of alpha-250 is found in the cytoplasmic subcellular compartment. As judged by its resistance to high concentrations of alpha-amanitin, cell-free transcription of alpha-250 and alpha-280 appears to involve RNA polymerase I. Tissue culture transfection and cell-free transcription experiments demonstrate that alpha-250 and alpha-280 stimulate S14 mRNA transcription, whereas free ribosomal protein S14 inhibits it. Electrophoretic mobility shift experiments indicate specific binary molecular interactions between r-protein S14, its message and the antisense RNAs. In light of these data, we propose a model for fine regulation of human RPS14 transcription that involves RPS14 intron 1 antisense RNAs as positive effectors and S14 protein as a negative effector.
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PMID:Regulation of human RPS14 transcription by intronic antisense RNAs and ribosomal protein S14. 786 28

In previous studies we have shown that specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) particles that represent the splicing machinery in vivo. The lnRNP particles contain all U small nuclear ribonucleoproteins (snRNPs) required for splicing, as well as several proteins including non-snRNP splicing factors. Here we show that upon addition of EDTA to sucrose gradient-fractionated 200 S particles, part of their components (e.g. part of the U snRNPs) are no longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg2+ is required for the integrity of the 200 S lnRNP particles.
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PMID:Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles. 786 77

The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate. However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called algS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma E of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the wild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that algT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.
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PMID:Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. 796 21

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis.
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PMID:Accurate transcription initiation by RNA polymerase II from Candida utilis. 798 59

Within the hematopoietic lineage, the monoclonal antibody (MoAb) CD66 reacts with cells of the granulocyte lineage, but not with the majority of progenitor cells from human bone marrow. Our previous studies have shown that CD66 binds specifically to at least three carcinoembryonic antigen (CEA) superfamily members, ie, CEA itself, nonspecific cross-reacting antigen (NCA), and CGM1, but not to CGM6 (NCA-95). In this report, we show that CD66 will also identify the biliary glycoproteins (BGP). A full-length cDNA for the BGPc molecule (a cytoplasmic splice variant of BGPa) was isolated by expression cloning using the CD66 MoAbs. This protein has an identical extracellular and transmembrane sequence to BGPa with one N-terminal IgV like domain, three IgC-like extracellular domains (A1, B1, and A2), plus a transmembrane domain, but the cytoplasmic domain is spliced by 53 nucleotides. Reverse transcriptase-polymerase chain reaction experiments show that this splice variant can be detected in colonic carcinoma cell lines, in primary colonic adenocarcinomas, and in myeloid and B-cell lines to varying degrees. Quantitative analyses of BGPc RNA expression by RNase protection indicate that abundant levels occur only in the colonic, but not in the hematopoietic, cell lines tested. Studies presented here show that BGPc mediates homotypic adhesion and suggest that the cytoplasmic splicing does not alter the initial homotypic adhesion properties of BGPa.
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PMID:CD66 identifies the biliary glycoprotein (BGP) adhesion molecule: cloning, expression, and adhesion functions of the BGPc splice variant. 801 19


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