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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Xenopus homolog of the transcription factor C/EBP (CCAAT/enhancer core binding protein), cloned from an adult Xenopus liver cDNA library, encodes a protein whose sequence is 67% homologous to that of rat C/EBP at the amino acid level, with virtually identical sequence of the basic-zipper region at the carboxyl terminus. As determined by gel electrophoretic mobility shift assays, the protein synthesized from xC/EBP cDNA bound specifically to the consensus binding site for C/EBP-like proteins. Northern blotting and RNase protection revealed a single species of xC/EBP mRNA of 2.7 kb which was most abundant in adult Xenopus liver, with smaller amounts in spleen, kidney, oviduct and brain and undetectable in heart and skeletal muscle. Although a small amount of this transcript could be detected in unfertilized eggs and early embryos, its accumulation rose sharply at the onset of metamorphosis (stage 55/56), and continued to increase through metamorphic climax to reach its highest level in stage 66 froglet liver, but thereafter declining in adult liver. In situ hybridization revealed a uniform pattern of distribution of xC/EBP mRNA in the liver and fat body throughout metamorphosis. Towards the end of metamorphosis, high levels of xC/EBP mRNA were detected in epithelial cells of the digestive tract. However, the spatial pattern of cells expressing the transcript changed markedly in the developing kidney. Our results suggest that xC/EBP may be involved as a transcription factor in the establishment of the adult phenotype during post-embryonic development of Xenopus.
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PMID:Characterization and developmental expression of Xenopus C/EBP gene. 152 39

To characterize the mechanisms determining tissue-specific ceruloplasmin gene expression during development, the rat ceruloplasmin gene was isolated in a series of overlapping phage clones. The 5'-flanking region was characterized and the transcription initiation site was identified by primer extension and RNase protection. Nucleotide sequence analysis of this region revealed a typical eukaryotic promotor structure, but no obvious homology with cis-acting elements previously characterized as determining tissue-specific gene expression. Transient expression of chimeric ceruloplasmin-reporter gene constructs containing up to 5200 base pairs (bp) of the 5'-flanking region revealed that sequences 732 bp upstream of the start nucleotide were sufficient to confer hepatocyte-specific expression. The region from -393 to -348 was determined by deletion analysis to contain a positive-acting element, and includes sequence partially homologous to the rat albumin D site. Mobility shift analysis revealed that this region specifically binds a heat-labile nuclear protein from rat liver and from newborn but not adult rat lung. Binding to this region was competed by oligonucleotides corresponding to the albumin D site, but not by oligonucleotides corresponding to binding sites for the hepatocyte transcription factors HNF-1, HNF-3, HNF-4, and C/EBP. These data indicate that ceruloplasmin gene expression is determined in part by a cis-acting region 393 bp upstream of the transcription start site, which binds a previously uncharacterized nuclear protein. The tissue distribution of this nuclear protein suggests that it plays a role in directing ceruloplasmin gene expression in lung and liver during development.
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PMID:Structural and functional analysis of the 5'-flanking region of the rat ceruloplasmin gene. 173 Jun 11

The 5'-flanking region of the gene coding for the alpha chain of human fibrinogen was isolated, sequenced, and characterized. The principal site of transcription initiation was determined by primer extension analysis and the RNase protection assay and shown to be at an adenine residue located 55 nucleotides upstream from the initiator methionine codon, or 13,399 nucleotides down-stream from the polyadenylation site of the gene coding for the gamma chain. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the alpha chain gene fused to the chloramphenicol acetyltransferase reporter gene showed that the promoter was liver-specific and inducible by interleukin 6 (IL-6). The shortest DNA fragment with significant promoter activity and full response to IL-6 stimulation encompassed the region from -217 to +1 base pairs (bp). Although six potential IL-6 responsive sequences homologous to the type II IL-6 responsive element were present, a single sequence of CTGGGA localized from -122 to -127 bp was shown to be a functional element in IL-6 induction. A hepatocyte nuclear factor 1 (HNF-1) binding site, present from -47 to -59 bp, in combination with other upstream elements, was essential for liver-specific expression of the gene. A functional CCAAT/enhancer binding protein site (C/EBP, -134 to -142 bp) was also identified within 217 bp from the transcription initiation site. An additional positive element (-1393 to -1133 bp) and a negative element (-1133 to -749 bp) were also found in the upstream region of the alpha-fibrinogen gene.
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PMID:Characterization of the 5'-flanking region of the gene for the alpha chain of human fibrinogen. 749 35

The cystic fibrosis transmembrane conductance regulator (CFTR) gene in man is controlled by a tightly regulated and weak promoter. The architecture of the CFTR promoter suggests regulatory characteristics that are consistent with the absence of a TATA-like sequence, including the ability to initiate RNA transcription at numerous positions. Detailed investigation of the most proximal region of the human CFTR gene promoter through deletion and mutational analysis reveals that expression is contingent on the conservation of the inverted CCAAT sequence. Basal expression of CFTR transcription and cAMP-mediated transcriptional regulation require the presence of an imperfect and inverted CCAAT element recognized as 5'-AATTGGAAGCAAAT-3', located between 132 and 119 nucleotides upstream of the translational start site. RNA isolated from a transfected pancreatic cell line carrying integrated wild-type and mutant CFTR-directed transgenes was used to map the 5' termini of the transgenic transcripts. Analysis of the transcript termini by ribonuclease protection analysis reflects the direct association of the conserved inverted CCAAT sequence in promoting transcript initiation. Because of the requirement for the inverted CCAAT sequence for promoting transcription of CFTR, the involvement of CCAAT-binding factors is suspected in the regulation of CFTR gene transcription. To test this, we used electrophoretic mobility shift assays to demonstrate that the majority of the binding to the inverted CCAAT element, between -135 and -116, was easily competed for by binding to cognate nucleotide sequences for CCAAT-enhancer binding protein (C/EBP). An antibody specific for the C/EBP-related protein, C/EBP delta, detected C/EBP delta as part of a nuclear protein complex bound to the inverted CCAAT sequence of the CFTR gene. Also, the detection of specific activating transcription factor/cyclic-AMP response element binding protein antigens by antibody supershift analysis of nuclear complexes suggest that species of this family of transcription factors could be involved in the formation of complexes with C/EBP delta within the CFTR gene inverted CCAAT-like element. These studies raise the possibility of interactions between individual members of the C/EBP and activating transcription factor/cyclic-AMP response element binding protein families potentially contribute to the tight transcriptional control rendered by the CFTR gene promoter.
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PMID:Transcription of cystic fibrosis transmembrane conductance regulator requires a CCAAT-like element for both basal and cAMP-mediated regulation. 749 10

The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451-462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CAAT box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.
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PMID:Characterization of the promoter region of the human MDR3 P-glycoprotein gene. 789 60

HNF1 and C/EBP-related proteins are transcription factors important for the activation of albumin gene expression. Fusion of mouse fibroblast (L) cells with rat pancreatic cells (AR42J) unexpectedly activated mouse albumin gene expression in the hybrid cells. In addition, several liver-specific genes such as tyrosine amino-transferase (TAT) and phosphoenolpyruvate carboxy-kinase (PEPCK) were also activated in the fibroblast-pancreatic hybrids. RNase protection assays using rat HNF1 riboprobes showed that AR42J cells and fibroblast-pancreatic hybrids expressed rat HNF1 transcripts. However, mouse or rat C/EBP alpha transcripts were not expressed in the fibroblast-pancreatic hybrids by RNase protection assays. Transfection of HNF1 expression vector alone was able to activate an albumin promoter (-1 kb, 5' flanking) promoted GPT construct in L cells, but not in HeLa cells, suggesting that different factors in L cells might interact with HNF1 to mediate activation. These results showed the global activation of liver-specific genes (including albumin, TAT, and PEPCK) in somatic cell hybrids. The presence of HNF1 in the hybrids may play a causal role. The absence of C/EBP alpha in the hybrids suggested its non-essential role in the activation of liver-specific gene expression. The other mechanisms responsible for the activation were also discussed.
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PMID:Activation of albumin and other liver-specific gene expression in fibroblast-pancreatic cell hybrids: different roles of transcription factors. 810 76

Primer extension and RNase protection analyses of the rat beta 2-adrenergic receptor (beta 2AR) gene identify two transcription start points at -64 and -220 nt, respectively. Transient transfections of putative promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragments -36 to -100 (PI) and -186 to -312 (P2) are sufficient to promote transcription, whereas -911 to -1122 contains a negative regulatory element(s). RNase protection analysis of the 3' untranslated region (3'-UTR) indicates the presence of two transcripts with 3'-UTR of 111 and 604 nt exclusive of the poly(A+) tails. Northern blots of beta 2AR mRNA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promoters as well as different polyadenylation signals. DNase I footprinting and DNA mobility shift assays (DMSA) using rat liver nuclear extracts identify a number of transcription factors binding to sequence elements within or upstream from P1 and P2, including Spl, CRE, CPl, AP-2, NF-1, NF-kappa B, and C/EBP. Supershift assays using antibodies against C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -933 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfection of C/EBP alpha but not C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or primary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta 2 AR mRNA levels and rate of transcription in the remnant liver. Thus, derepression via C/EBP alpha is likely involved in the up-regulation of beta 2AR in the regenerating rat liver.
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PMID:DNA elements and protein factors involved in the transcription of the beta 2-adrenergic receptor gene in rat liver. The negative regulatory role of C/EBP alpha. 885 51

The rat angiotensin II type 2 receptor (AT2-R) gene was isolated, and cis-regulatory regions in its 5'-flanking area were analyzed. Primer extension and RNase protection analyses revealed a single transcriptional initiation site at the position 24 bp downstream of the TATA box. The 5'-flanking region of AT2-R contained several cis-regulatory elements, such as AP-1, AP-2, C/EBP, NF-1, NF-IL6, NF-kappa B, and glucocorticoid- and cAMP-responsive elements (CRE). The treatment of PC12 cells with dibutyryl cAMP caused a marked decrease (90%) in the AT2-R mRNA level, which was blocked by the inhibitor of protein kinase A and did not require new protein synthesis. The protein level was also reduced 84% after a 24-h exposure to cAMP and the binding affinity was unchanged. The half-life of the AT2-R mRNA decreased -66% by cAMP as compared with control (18.4 +/- 0.4 h). Deletion and mutation analyses of the 5'-flanking region (1.2 Kb) revealed that there were one negative (-1,199 to -739) and two positive cis-regulatory regions (-739 to -436 and -59 to +45), and that the CRE motif located at -426 repressed (-23%) the promoter activity of the rat AT2-R gene. The region between -59 and +45 containing TATA box and AP-2 site accounted for 70% of the promoter activity. These findings indicate that the promoter activity of the rat AT2-R gene is modulated by several cis-regulatory regions and that cAMP markedly downregulates the expression of the AT2-R mainly by inducing AT2-R mRNA destabilization rather than CRE-mediated inhibition of the gene transcription. Thus, humoral factors that transduce cAMP as an intracellular signal may modulate AT2-R-mediated function of Ang II by reducing AT2-R expression.
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PMID:Down-regulation by cAMP of angiotensin II type 2 receptor gene expression in PC12 cells. 898 58

Interleukin 1 receptor antagonist (IL-1Ra) levels are elevated in the blood of patients with a variety of infectious, immune, or traumatic conditions. To examine whether IL1Ra is produced by liver cells with characteristics resembling an acute-phase protein, human primary hepatocytes isolated from liver biopsies and HepG2 hepatoma cells were stimulated with IL-1beta, IL-6, and TNFalpha. IL-1Ra was present in the supernatants of both cells, with production significantly enhanced by IL-1beta, and by the combination of IL-1beta and IL-6. The term IL-1Ra refers to two different proteins encoded by the same gene, but generated by alternative splicing of two different first exons. One isoform is secreted (17-kD sIL-1Ra), and the other isoform remains in the cytoplasm (18-kD icIL-1Ra). By Western blot analysis, the supernatants of human hepatoma (HepG2) cells contained only sIL-1Ra, whereas the lysates contained a novel smaller molecular mass isoform of 16 kD. RT-PCR and ribonuclease protection assay with RNA from HepG2 cells showed that only sIL-1Ra mRNA was expressed, and confirmed the inducing effect of IL-1beta and IL-6. Transfection studies were performed using constructs containing the promoters of either sIL-1Ra or icIL-1Ra coupled to the luciferase reporter gene. The sIL-1Ra promoter was active in HepG2 cells stimulated by IL-1beta and/or IL-6, whereas the icIL-1Ra promoter was inactive. Mutation of binding sites for transcription factors NF-kappaB and/or C/EBP within the proximal sIL-1Ra promoter led to significant decreases in response to IL-1beta and IL-6 in comparison to the wild-type promoter. Electromobility gel shift assays confirmed the presence of NF-kappaB and C/EBP binding sites within the sIL-1Ra promoter, and indicated a significant increase in the binding activities of nuclear proteins from HepG2 cells treated with IL-1beta and IL-6. In summary, sIL-1Ra, but not icIL-1Ra, is produced by hepatocytes, and is regulated by proinflammatory cytokines as an acute-phase protein. In addition, NF-kappaB and C/EBP family members are likely to play important roles in the full expression of IL-1Ra by hepatocytes during inflammatory conditions.
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PMID:Interleukin 1 receptor antagonist (IL-1Ra) is an acute-phase protein. 918

We describe the genomic organization and the functional promoter of the monocyte specific gene Dif-2, the human homologue to genes in mouse (gly96) and rat (PRG1), that is downregulated during cell differentiation. The Dif-2 gene consists of two exons and a single intron of 112 bp in length. RNase protection assay indicates one major transcription start site. Sequence analysis reveals several consensus sequences for transcription factors including NF-kappa B, C/EBP, SP1, and the lack of a classical TATA-box. To demonstrate promoter activity, DNA fragments of the Dif-2 5'-flanking region were ligated upstream to the luciferase gene and transfected into HepG2 and HeLa cells. A minimal promoter element between nt -158 and nt +74 containing NF-kappa B and SP1 binding sites was shown to be sufficient for basal activity. These transcription factor binding sites, which are conserved between Dif-2, gly96, and PRG1 promoter regions, indicate a significant role for Dif-2 expression and may explain LPS and C2-ceramide sensitivity. The Dif-2 gene was mapped to chromosome 6p21.3 using in situ hybridization technique.
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PMID:Genomic organization, promoter cloning, and chromosomal localization of the Dif-2 gene. 958 70


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