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:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.
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PMID:Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters. 140 Mar 96

To analyze the mechanism of the cell type-specific expression of protein kinase C beta (PKC beta), we isolated the 5'-portion of the human gene for PKC beta and identified multiple positive and negative regulatory sequences that regulate its transcription. S1 nuclease mapping as well as primer extension analysis of the 5'-end of the PKC beta mRNA identified a putative transcriptional initiation site (position +1) 484 base pairs (bp) upstream of the first ATG codon. The 5'-upstream sequence contains a CCAAT sequence at position -110, but no TATA box. The transcriptional activities of various 5'-deletion mutants of the PKC beta gene upstream region, fused to the chloramphenicol acetyltransferase structural gene, were examined in terms of chloramphenicol acetyltransferase expression after transfection into three kinds of rodent cell lines: P19 and GH4C1, which are positive for the expression of PKC beta mRNA; and 3Y1, which is negative. Mutants containing a 5'-flanking sequence longer than 1.9 kilobases (kb) showed chloramphenicol acetyltransferase activities of the same order as the expression of the endogenous gene. This indicates that this region contains sequences regulating the cell-type specificity of PKC beta gene expression and that the specificity is determined at least partially at the level of transcription. The 1.9-kb sequence contains at least three positive elements: P1 (-56 to -234 bp), P2 (-234 to -411 bp), and PN (-1.4 to -1.9 kb). PN is active only in P19 cells, P1 in GH4C1 and P19 cells, and P2 in all three cell lines. In addition to these positive elements, there are negative elements: N1 (-411 to -674 bp), which is active in all three cell lines; and PN, which is active only in GH4C1 cells. These results suggest the presence of multiple trans-acting factors that act on these positive and negative cis-acting elements and regulate the cell type-specific expression of the PKC beta gene.
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PMID:Positive and negative regulation of the transcription of the human protein kinase C beta gene. 155 24

EGR2 is a human zinc finger encoding gene whose expression is induced with fos-like kinetics by diverse mitogens in several cell types. Since its cDNA sequence predicts a protein which contains zinc finger motifs, EGR2 may play a transcriptional regulatory role in cellular proliferation. The present study was undertaken to: 1) examine the genomic organization and 5' flanking sequence of EGR2 so as to identify upstream regulatory elements; 2) test whether these elements are functional in gel shift assays and by transient expression; and 3) examine whether pathways other than protein kinase C lead to serum induction of EGR2, and if they do, ask whether the different pathways converge on a serum response element. The EGR2 gene spans 4.3 kb and has one intron. The translation initiation site is located within the first exon. The transcription start site of EGR2 was determined by S1 nuclease and primer extension analysis and a TATA box was identified 28 bp upstream. Two putative serum response elements, designated CArG-1 and CArG-2 were identified in the 5' flanking sequence. By deletion analyses and mutagenesis, serum and PMA responsiveness of the cloned EGR2 promoter region was traced to the CArG-1 region in transient CAT assays performed in NIH 3T3 cells. Both protein kinase C dependent and independent pathways were found to converge on the CArG-1 box to induce the expression of EGR2.
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PMID:The serum and TPA responsive promoter and intron-exon structure of EGR2, a human early growth response gene encoding a zinc finger protein. 211 Oct 9

The nucleotide sequence of the long terminal repeats (LTRs) of retrovirus-transmissible mouse VL30 cDNA clones, NVL-1 and NVL-2 were determined and compared with that of the prototype NVL-3. Both shared the typical U3 R U5 structure together with unusual features of redundancy in the tRNAgly primer binding site and adjacent inverted repeat. NVL-1 and NVL-2 LTRs were almost identical and differed from the NVL-3 LTR in the U3 domain harbouring transcriptional regulatory determinants. S1 nuclease analysis of cellular and virus-encapsidated RNA suggested that NVL-1/2 and NVL-3 elements retrotranspose with comparable efficiency but that in contrast to transformation-regulated VL30 expression which affects all types of NVL element, only NVL-1/2 elements were found to be serum responsive. Both modes of VL30 regulation were found to be coupled through protein kinase C-independent pathways. Expression of N-ras transactivated U3 enhancer determinants in all classes of LTR. However the same region of NVL-1/2 LTR did not confer serum responsiveness implying that cis regulatory determinants of VL30 elements mediating growth factor responsiveness are at least in part dissociable from those responsible for cell transformation-regulated expression.
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PMID:Independent regulation of mouse VL30 retrotransposon expression in response to serum and oncogenic cell transformation. 215 38

Thrombomodulin is an endothelial membrane anticoagulant protein that is a cofactor for protein C activation. We have evaluated the expression of thrombomodulin in cultured mouse hemangioma cells before and after treatment with phorbol myristate acetate (PMA), an agent that stimulates protein kinase C. We also isolated a cDNA encoding 481 amino acids of mouse thrombomodulin and the entire 3'-untranslated portion of its mRNA. The deduced amino acid sequence of mouse thrombomodulin is similar to those determined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure thrombomodulin mRNA in hemangioma cells. The half-life for thrombomodulin mRNA was 8.9 +/- 1.8 h (S.D.) in cells treated with actinomycin D. Treatment with PMA had no effect on thrombomodulin mRNA levels. Thrombomodulin turnover was evaluated by immunoprecipitation of [35S]methionine-labeled thrombomodulin. The t1/2 was 19.8 +/- 3.9 h (S.D.); PMA treatment decreased the t1/2 to 10.9 +/- 1.1 h (S.D.) while increasing the rate of synthesis to a maximum of 190% of control. Protein C cofactor activity on hemangioma cells was reduced 35 +/- 4% by treatment with PMA within 30 min. This decrease was associated with a parallel decline in cell surface thrombomodulin antigen and with enhanced phosphorylation of thrombomodulin on serine residues. We conclude that thrombomodulin is phosphorylated in response to treatment of hemangioma cells with PMA which leads to decreased protein C cofactor activity and both increased degradation and synthesis of thrombomodulin.
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PMID:The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells. 284 23

The human colonic epithelial cell line HT29, and its clonal derivatives HT29-18 and HT-29-18-C1, differentiate in vitro. Differential screening of a subtraction cDNA library enriched for sequences unique to HT29-18-C1, a highly differentiated subclone of HT29-18, resulted in the isolation of a differentiation-dependent cDNA clone, A4. A full-length clone encoding A4 was obtained and sequenced to its entirety. It is 945 bp in length and contains an open reading frame (ORF) of 456 bp. The amino acid sequence deduced from the ORF reveals a polypeptide of 152 amino acids with a predicted molecular mass of 17,000 Da, a size confirmed by coupled in vitro transcription and translation directed by the full-length A4 cDNA. This polypeptide contains four potential membrane-spanning domains and consensus sequences for N-linked glycosylation as well as phosphorylation sites for protein kinase C and casein kinase II. Comparison of A4 to published DNA and protein sequences revealed no significant homology. Genomic Southern blot analysis suggests that the gene is present in a single copy within the human genome and is conserved in the rat. Northern blot analysis of RNA obtained from various rat tissues shows that the expression of the A4 gene is tissue-selective and is enriched in colonic mucosa. In situ hybridization using human intestinal tissues indicates that the expression of A4 follows a gradient along the crypt-to-villus axis with the most abundant message occurring in the lower half of the crypt. Furthermore, nuclear run-on assays suggest that the induction of the A4 gene during differentiation of HT29-18 is regulated at a transcriptional level. A clone was isolated from a human genomic library and found to contain all five exons of A4. S1 nuclease analysis localized the start site of transcription to an adenosine residue 91 nucleotides upstream from the ATG translation initiation codon. Examination of the immediate sequence 5' to the mRNA start site reveals no TATA box and multiple known enhancer sequences. A4 is also noted to share certain features with the gene encoding the cystic fibrosis transmembrane conductance regulator protein. They include a similar vertical distribution of expression along the intestinal epithelium, enhanced transcription upon differentiation of HT29-18, and multiple shared putative regulatory sequences in the promoter regions. Further characterization of the mechanisms regulating expression of the A4 gene could contribute to the understanding of mammalian intestinal differentiation.
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PMID:Isolation and characterization of a differentiation-dependent gene in the human colonic cell line HT29-18. 847 Aug 95

Microglia rapidly respond to lipoplysaccharide (LPS) by transformation from resting to active states and secretion of several neuro- and immuno-regulators including tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6). With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes. We have investigated LPS-mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alaninerich C kinase substrate) and MRP (MARCKS-related protein). Within 6 hours of addition, LPS induced a twofold increase in [3H]myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP. The differential effect of LPS on expression of MRP vs. MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40-fold increase in MRP mRNA levels (maximum at 4-6 hours), whereas a threefold increase was observed for MARCKS. TNF alpha and colony-stimulating factor 1 (CSF-1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription. CSF-1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS. Accordingly, these two substrates for PKC are differentially up-regulated by LPS, apparently independent of TNF alpha or CSF-1.
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PMID:Lipopolysaccharide stimulates differential expression of myristoylated protein kinase C substrates in murine microglia. 872 62

14-3-3 protein, a brain-specific protein, is thought to be a multifunctional protein involved in the activation of tyrosine and tryptophan hydroxylases, the inhibition or activation of protein kinase C, and the activation of signal transduction. The human 14-3-3 eta chain gene was isolated and its structure was determined. It is composed of two exons separated by one long intron (approximately 8 kb) and spans about 10 kb. A transcription initiation site was identified by a combination of S1 nuclease mapping, primer extension analysis, and RACE methods. In the 5'-flanking region, we found four GC box sequences, four anti-GC box sequences, a TATA box-like sequence, CAAT box-like sequences, a C/EBP element, two AP-2 sequences, an AP-3 sequence, an Oct-6-like sequence, six E boxes, and a CRE sequence. FISH with DNA probes of the human 14-3-3 eta chain gene mapped the 14-3-3 eta chain gene to chromosome 22q12.1-q13.1.
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PMID:Structural organization and chromosomal assignment of the human 14-3-3 eta chain gene (YWHAH). 881 17

Protein kinase C-eta (PKC-eta) is predominantly expressed in epithelial tissue, including lung, intestine, and skin. In skin, PKC-eta expression is limited to keratinocytes in the upper layers of the epidermis. To investigate regulation of cell type-specific expression of PKC-eta, we cloned the 5'-segment of the PKC-eta gene from a P1 genomic library. A 9.4-kilobase pair fragment encompassing the 5'-flanking region, first exon, and first intron, was localized on human chromosome 14 (14q22-23). Two major transcription initiation sites identified by reverse transcriptase polymerase chain reaction, primer extension, and S1 nuclease mapping, were located approximately 650 base pairs upstream from the translation start site. The human PKC-eta proximal promoter region lacks canonical TATA and CAAT boxes and GC-rich regions. A 1.6-kilobase pair 5'-flanking region displayed maximal promoter activity. This promoter was active in human keratinocytes but not human skin fibroblasts, in accord with endogenous PKC-eta gene expression. Stepwise 5' deletion analysis revealed the presence of adjacent regulatory regions containing silencer and enhancer elements located 1821-1702 base pairs and 1259-1189 base pairs upstream of the transcription initiation site. Deletion of the proximal PKC-eta promoter rendered the enhancer element inactive. Both the silencer and enhancer elements regulated heterologous promoters in keratinocytes but not fibroblasts. Electrophoretic mobility shift analysis demonstrated specific protein binding to Ets/heat shock factor and Ets/activator protein-1 consensus sequences in the enhancer and silencer regions, respectively. Mutations of the Ets/heat shock factor binding sites caused loss of functional enhancer activity. These data elucidate transcriptional regulation and tissue-specific expression of the PKC-eta gene.
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PMID:Cloning and characterization of the human protein kinase C-eta promoter. 1049 22

This study examines endothelin-induced modulation of extracellular matrix synthesis and remodeling by fibroblasts, and its potential role in the pathogenesis of systemic sclerosis (scleroderma). Endothelin-1 promoted fibroblast synthesis of collagen types I and III, but not fibronectin, by a mechanism dependent upon both ETA and ETB receptors. Conversely, endothelin-1 inhibited both protein expression of matrix metalloproteinase 1 and zymographic activity exclusively via ETA receptors. A dual regulatory role for endothelin-1 in transcriptional regulation was suggested by the ability of endothelin-1 to enhance steady-state levels of collagen mRNA and activate the proalpha2(I) collagen (Col1a2) promoter, but in contrast to reduce matrix metalloproteinase 1 transcript expression and suppress transcription of a human matrix metalloproteinase 1 promoter reporter construct in transient transfection assays. Although endothelin-1 significantly enhanced remodeling of three-dimensional collagen lattices populated by normal fibroblasts, this was not observed for lattices populated by systemic sclerosis fibroblasts. Promotion of matrix remodeling was dependent upon ETA receptor expression and was blocked by specific inhibitors of tyrosine kinases or protein kinase C. Reverse transcriptase polymerase chain reaction, S1 nuclease, and functional cell surface binding studies showed that normal and systemic sclerosis fibroblasts express both ETA and ETB receptors (predominantly ETA), but that ETA receptor mRNA levels and ETA binding sites on fibroblasts cultured from systemic sclerosis skin biopsies are reduced by almost 50%. Endothelin-1 is thus able to induce a fibrogenic phenotype in normal fibroblasts that is similar to that of lesional systemic sclerosis fibroblasts. Moreover, reduced responsiveness to exogenous endothelin-1 in systemic sclerosis suggests that downstream pathways may have already been activated in vivo. These data further implicate dysregulated endothelin-receptor pathways in fibroblasts in the pathogenesis of connective tissue fibrosis.
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PMID:Fibroblast matrix gene expression and connective tissue remodeling: role of endothelin-1. 1123 16


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