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)

Manganous superoxide dismutase (MnSOD) gene expression is stimulated by endotoxin, tumor necrosis factor, and interleukin-1, agents thought to cause cellular damage through intracellular generation of reactive oxygen species. To study the molecular mechanisms underlying the induction of MnSOD mRNA by these stimuli, we cloned a bovine MnSOD cDNA and used it to isolate the promoter region of the bovine MnSOD gene. A 14 kb genomic DNA fragment (lambda BS1) containing the first and second exons and 5' flanking region of the gene was characterized. The transcription start site was determined by primer extension and S1 nuclease protection assays and found to be 88 bp upstream of the translation initiation codon. The sequence of approximately 1 kb of DNA upstream of the start site was determined and examined for potential regulatory elements. DNA immediately upstream of the transcription start site was GC-rich and contained two AP-2 and eight Sp-1 consensus sequences. It did not contain either a CCAAT or TATA box. A 956 bp fragment of this DNA fragment was transcriptionally active when fused to a luciferase reporter gene and transfected into both bovine pulmonary artery endothelial and hamster insulinoma tumor cells. Transfection analysis of three additional deletion mutants, whose 5' end-points were -317, -182, and -70 bp, respectively, showed a step-like reduction in transfection efficiency, suggesting the presence of regulatory elements throughout this DNA fragment that contribute to transcriptional activity of the MnSOD promoter. Despite the high homology of the bovine MnSOD cDNA to other mammalian MnSODs, the promoter sequences of bovine and rat MnSOD genes showed a virtual lack of similarity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification and functional characterization of the bovine manganous superoxide dismutase promoter. 829 76

The 5'-flanking region of the human neurotrophin-3 (NT-3) gene was isolated from a human placental genomic library using the oligonucleotide corresponding to the 5'-noncoding region of the NT-3 cDNA as a probe. A 3.8 kbp genomic fragment containing the 5'-flanking region, the first exon and a portion of the first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 nuclease mapping, was located 27 bp downstream from the TATA-like sequence. Several plasmids, in which the NT-3 promoter regions were fused to the chloramphenicol acetyltransferase (CAT) gene, were constructed. Transient expression in human glioma Hs683 cells demonstrated that a fragment of about 0.1 kbp from the transcriptional initiation site was sufficient for promoter activity. While, in human plasma cell leukemia ARH77 cells, in which NT-3 mRNA was not detected, the region upstream from -65 functioned to silence CAT activity. It is suggested that this region contains the transcriptional regulatory element for the specific expression of the NT-3 gene.
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PMID:Identification of the functional regulatory region of the neurotrophin-3 gene promoter. 838 96

The 80 kDa diacylglycerol kinase (DGK) is abundantly expressed in oligodendrocytes and lymphocytes but not to a detectable extent in other cells such as neurons and hepatocytes. As an initial attempt to delineate the mechanism of the transcriptional control of the DGK gene, we have cloned from a human genomic library a 22 kb genomic fragment. The genomic clone consists of the 5'-flanking region and 17 exons coding for approx. 53% of the total exons of human DGK, including those encoding EF-hand and zinc-finger regions. The translation initiation site is located in the second exon. S1 nuclease mapping and primer extension analysis of the human DGK mRNA identified a major transcription initiation site (position +1) at 264 bp upstream from the initiator ATG. In the 5'-flanking sequence we detected a single GC box at -35 but no canonical TATA and CAAT sequences. However, the sequence starting from the cap site (AGTTCCTGCCA) is very similar to the initiator element that specifies the transcription initiation site of some housekeeping genes. In addition, the 5'-upstream region contains several putative cis-elements. Jurkat and HepG2 cells were transfected with various 5'-deletion mutants of the upstream region fused to the structural gene of chloramphenicol acetyltransferase (CAT). The CAT assay revealed that among constructs containing up to 3.4 kb of the 5'-flanking region, a fragment of 263 bp from the transcription initiation site contains a basic promoter that is active in both types of cells. Moreover, the region between -263 and -850 contains a negative element that is active in HepG2 but not in Jurkat cells. This negative element may, at least in part, be responsible for the cell type-specific expression of the DGK gene.
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PMID:Isolation and characterization of the human diacylglycerol kinase gene. 839 13

The human insulin-like growth factor II (IGF-II) gene contains four promoters (P1, P2, P3 and P4). In order to determine the mechanism by which the P4 promoter is controlled, the human IGF-II P4 promoter was analyzed in cell lines. DNA sequence analysis of the human IGF-II P4 promoter gene showed that the P4 promoter region contains a TATA-like sequence and several G+C rich regions which are essential for transcription. Analysis of the transcription initiation site by S1 nuclease mapping revealed two transcription start sites; both are located immediately behind TATA-like sequence. To determine the location of sites that may be important for the function of the human IGF-II P4 promoter, we constructed chimeric genes of the human IGF-II P4 promoter fused to the coding region for chloramphenicol acetyltransferase (CAT). These constructs were transfected into HepG2, PLC/PRF/5, G401 and A549 cells, and were examined for CAT activity. All transfected cells showed a similar profile of CAT activity. Sequences responsible for putative enhancer and silencer regions were identified and the 5' flanking sequences of the human IGF-II P4 promoter contain negative regulatory regions (-213 to -174). The 53-base pair fragment located between 111 and 59 base pairs upstream of the start site contains positive regulatory activity. Gel mobility shift assay showed that Sp1 and another proteins might be involved in positive regulation of the human IGF-II P4 promoter.
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PMID:Characterization of the P4 promoter region of the human insulin-like growth factor II gene. 840 33

We have previously shown that in vivo the steady-state level of c-myc mRNA in different quiescent organs and its induction in the early stages of hepatic regeneration and after inhibition of protein synthesis are mainly controlled by post-transcriptional mechanisms. In order to localize the target sequences for these mechanisms, transgenic lines expressing various versions of the human c-myc proto-oncogene have been constructed. To avoid all possible transcriptional controls due to the c-myc 5' regulatory region, the c-myc genomic sequences were fused to MHC H-2Kb class I regulatory sequences, which have previously been shown to be able to drive reporter gene expression in most adult tissues. The transgenes contained either all human c-myc genomic sequences or were deleted for one of the sequences which have been shown in in vitro experiments to play a role in c-myc mRNA stabilization, in particular exon 1, intron 1 and the 3' non-coding region. Several independent transgenic lines were derived for each construct. Using S1 nuclease protection analysis, we have monitored H-2K, mouse c-myc and transgene mRNA expression in several quiescent adult organs, at the start of liver regeneration and after inhibition of protein synthesis in each transgenic line. Our results indicate that the 5' non-coding sequences, including exon 1 and intron 1, and the 3' untranslated region are all dispensable in the different aspects of c-myc post-transcriptional regulation.
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PMID:The 5' and 3' non-coding sequences of the c-myc gene, required in vitro for its post-transcriptional regulation, are dispensable in vivo. 851 Sep 35

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in length. The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids. The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution. Southern hybridization analysis showed that the nucS gene existed as a single copy in the A. oryzae chromosome. When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A. oryzae, the yield of secreted nuclease S1 increased about 100-fold compared with the recipient strain.
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PMID:Cloning, characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae. 859 44

The overlapping tandem promoters of the streptokinase gene, P1 and P2, identified previously by S1 nuclease transcript mapping were functionally dissected by mutagenesis of their -10 regions and fused transcriptionally with or without the 202-bp upstream region (USR) to the luciferase reporter gene (luc) from Photinus pyralis to analyze the contribution of the different sequence elements to promoter activity in Escherichia coli and the homologous Streptococcus equisimilis strain H46A. In E. coli, virtually the entire promoter activity derived from the upstream promoter P1. In S. equisimilis, luc expression increased in the following order of the involved sequence elements: P2 approximately equal to P2 + USR < P1 < P1 + P2 < P1 + USR < P1 + P2 + USR. This shows that (1) in the homologous system, P1 and P2 alone are extremely weak, (2) in the USR-less arrangement, only the combined core promoters have substantial activity, and (3) the USR stimulates only P1 and the combination of P1 + P2. Thus, the tandem promoters presumably function by mutual contributary action and their full activity strongly depends on the AT-rich and statically bent upstream region. The distinctive feature determining the strength of P1 in both hosts appears to be its extended -10 region which matches the consensus TRTGN established for strong S. pneumoniae and Bacillus subtilis promoters.
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PMID:Structural dissection and functional analysis of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A. 880 48

17alpha-Hydroxylase cytochrome P450 (P450(17alpha)) is the enzyme which synthesizes C19 steroids in a two-step reaction in which 17alpha-OH pregnenolone is an intermediate. In the bovine and human adult female, 17alpha-hydroxylase is expressed in adrenocortical cells where 17alpha-OH pregnenolone and 17alpha-OH progesterone are precursors of cortisol, and in theca cells of the ovary where these intermediates are precursors of C19 steroids. In both adrenal cortex and theca, 17alpha-hydroxylase gene expression is stimulated by cyclic AMP (cAMP). The aim of this study was to determine the mechanism regulating 17alpha-hydroxylase gene expression in the bovine ovary. Our results indicate that the bovine 17alpha-hydroxylase gene is regulated in a tissue-specific fashion. Primer extension and S1 nuclease protection assays reveal that the start site of transcription in the theca is identical to that in the adrenal. Transfection studies employing beta-globin reporter gene constructs fused to successive deletions of the 5' regulatory region of the bovine 17alpha-hydroxylase gene indicate that sequences between -80 and -37 basepairs (bp) (CRS2) confer cAMP-regulated transcription in bovine theca cells in culture. These results are in contrast to similar studies conducted in bovine adrenocortical cells, which indicate that the major cAMP response element (referred to as CRS1) is located at -243 to -225 bp. The Ad4 element (AGGTCA, -42 to -37 bp) within CRS2, which has been shown to be involved in cAMP responsiveness in other steroidogenic P450 genes, cannot by itself confer cAMP-regulated reporter gene expression in bovine cells. These results indicate that in the cow, 17alpha-hydroxylase gene expression is regulated in a tissue-specific fashion, and that this regulation may be conferred, at least in part, by the use of tissue-specific cis-acting elements in the bovine 17alpha-hydroxylase gene.
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PMID:17alpha-Hydroxylase gene expression in the bovine ovary: mechanisms regulating expression differ from those in adrenal cells. 900 34

The vitamin D receptor (VDR) is known to mediate the pleiotropic biological actions of 1,25-dihydroxyvitamin D3 through its ability to modulate the expression of target genes. The regulation of this ligand-activated cellular transcription factor is reported to occur at both transcriptional and posttranslational levels. To begin to address the molecular basis by which the VDR gene is regulated transcriptionally, we report here an initial characterization of the human VDR gene and its promoter. We isolated several overlapping A-phage and cosmid clones that cover more than 100 kb of human DNA and contained the entire VDR gene. The gene is comprised of 11 exons that, together with intervening introns, span approximately 75 kb. The noncoding 5'-end of the gene includes exons 1A, 1B, and 1C. Eight additional exons (exons 2-9) encode the structural portion of the VDR gene product. While primer extension and S1 nuclease-mapping studies reveal several common transcriptional start sites, three unique mRNA species are produced as a result of the differential splicing of exons 1B and 1C. The DNA sequence lying upstream of exon 1A is GC rich and does not contain an apparent TATA box. Several potential binding sites for the transcription factor SP1 and other activators are evident. Fusion of DNA fragments containing putative promoter sequences upstream of the luciferase structural gene followed by transient transfection of these plasmids into several mammalian cell lines resulted in significant reporter activity. Due to the size and complexity of the 5'-end of the VDR gene, we examined the activity of a DNA fragment surrounding exon 1C. An intron fragment 3' of exon 1C conferred retinoic acid responsivity when fused to a reporter gene plasmid, suggesting a molecular mechanism for the previously observed ability of retinoic acid to induce the VDR. The recovery of the gene for the human VDR will enable further studies on the transcriptional regulation of this gene.
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PMID:Structural organization of the human vitamin D receptor chromosomal gene and its promoter. 921 63

The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli.
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PMID:Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli. 935 35


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