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)

Aldehyde dehydrogenase (ALDH1) is highly expressed in the dorsal cells of the undifferentiated retina, where it has been proposed to play a role in the formation of a retinoic acid gradient along the ventrodorsal axis. In contrast to the retina, ALDH1 levels increase with differentiation in the liver and remain elevated in the adult tissue. To understand the molecular basis for differential expression of ALDH1 during development, we characterized the ALDH1 transcripts expressed in chick retina and liver. By sequencing, primer extension, and S1 nuclease analysis, we show that retina ALDH1 mRNA has an additional 300 nucleotides of 5'-untranslated sequence resulting from the transcription of two 5' noncoding exons. There is a 24-29-kilobase pair (kb) gap between exons 1 and 2 and a 290-base pair gap between exons 2 and 3. Exon 3, which contains the ALDH1 start codon, represents the first exon of the liver transcript. Using a reporter gene assay, we have identified tissue-specific regulatory elements that govern ALDH1 expression in primary retina and liver cultures. Constructs with >1.6 kb of DNA flanking the 5'-end of exon 1 showed elevated activity in retinal cultures but only basal activity in liver cultures. In contrast, constructs with <1 kb of 5'-flanking DNA were active in both retina and liver cultures. Our results suggest that an important mechanism for the control of ALDH1 transcriptional activity is through the presence of inhibitory elements located 0.7-1.6 kb upstream of the ALDH1 gene. DNase I footprint analysis reveal four sites of protein-DNA interaction within this region, one of which is specific to the liver and corresponds to a NF-kappaB/Rel binding site.
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PMID:Differential regulation of the aldehyde dehydrogenase 1 gene in embryonic chick retina and liver. 1143 38

The Na, K-ATPase is formed by two major subunits (alpha and beta) encoded by a gene family of at least four alpha and three beta isoforms. These genes show distinctive expression patterns involving complex tissue-specific and developmental regulation, although the control mechanisms are not well understood. Here we study the role of chromatin structure in the tissue-specific expression of rat Na, K-ATPase beta2 isoform, which is mainly found in the central nervous system. We have examined the presence and characteristics of nuclease hypersensitive sites and the cytosine methylation patterns in the 5'-flanking region of the beta2 isoform gene from various nuclear preparations. Our results show that in this 5'-flanking region there is only one nuclease hypersensitive site. It is located upstream of the transcription initiation site and shows tissue-specific characteristics. Digestion with deoxyribonuclease I (DNase I), S1 nuclease and micrococcal nuclease yield patterns consistent with a triple-helix structure present only in the active state of the promoter. We also demonstrate that the 5'-flanking region of the beta2 gene co-localizes with a CpG island free of methylation in every tissue tested. The results presented here support a role for specific chromatin remodeling events in the regulation of the Na, K-ATPase beta2 gene expression. They also provide the basis for future studies of the transcription factors involved in the regulation of this gene.
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PMID:Chromatin structure analysis of the rat Na, K-ATPase beta2 gene 5'-flanking region. 1194 94

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3' to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the DeltaafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation of afsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues.
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PMID:afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2). 1195 95

We have developed a microtiter plate assay for the detection and screening of anti-DNA hydrolytic antibodies. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5'-end of the 3'-biotinylated DNA strands. The substrate binds specifically to the wells of streptavidin-coated microtiter plates where the reaction takes place. Uncleaved substrate retains the digoxigenin label, which is then detected with an enzyme-labeled anti-digoxigenin antibody. We first assessed the efficiency of this assay by measuring S1 nuclease and DNase I activities and the inhibitory effect of EDTA on the reaction. The ALONA procedure was then successfully applied to the screening of a high number of hybridoma clones derived from nonimmunized (NZB x NZW)F1 mice with spontaneous lupus erythematosus. We detected three potential catalytic antibodies and investigated their substrate specificity. Overall, our findings demonstrate the value of the ALONA method for high throughput screening of potential nucleases and catalytic antibodies. Although this assay was designed for the selection of catalysts active in DNA hydrolysis, it can be adapted to detect most types of substrate cleavage reaction.
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PMID:A method for the detection and screening of catalytic anti-DNA antibodies. 1237 59

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.
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PMID:Development of nonradioactive microtiter plate assays for nuclease activity. 1238 60

In the "Two-step fermentation" of Vitamin C synthesis, Gluconobacter oxydans SCB329 is responsible for the production of 2-keto-L-gulonic acid (2-KLG), which is an important precuror of vitamin C synthesis. The intact chromosome was prepared from logarithmic phase cells by agaraseembedded method and was analysized by restriction endonucleases and contour-clamped homogeneous electric field pulsed-field gel electrophoresis (PFGE). Spe I (5-ACTAGT) produced 24 fragments, ranging in size from 10 to 320 kilobases (kb). Xba I (5-TCTAGA) yielded 40 fragments (4 to 200 kb). A total genome size of approximately 2,700 kb was determined by summing the fragment length. Analysis of the entire genome of SCB329 by PFGE revealed that the genome of SCB329 consist of a chromosome which is 2,500 Kb in length and a large plasmid which is 245 kb. After linearization of the DNA by DNase I and S1 nuclease, in contrast with the band which can not be viewed, the band of chromosome and plasmid were appeared, this suggest that structure of the chromosome and the plasmid were circular.
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PMID:[Studies on the genome size and structure of Gluconobacter oxydans SCB329]. 1255 98

Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2). The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg(2+) stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg(2+) stimulon in E. coli.
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PMID:Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli. 1281 61

Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the sigma54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.
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PMID:Identification of a positive transcription regulatory element within the coding region of the nifLA operon in Azotobacter vinelandii. 1600 Jul 81

Here, we examined the effects of molecular crowding on the function, structure and stability of nucleases. We found that the hydrolysis of a 29-mer double-stranded DNA by the endonucleases DNase I and S1 nuclease was substantially enhanced by molecular crowding using polyethylene glycol (PEG); however, molecular crowding had little effect on hydrolysis by exo III and exo I exonucleases. Moreover, kinetic analysis showed that the maximum velocity for the reaction of DNase I at 25 degrees C was increased from 0.1 to 2.7 microM/min by molecular crowding with 20% (w/v) PEG, whereas that of exonuclease I at 37 degrees C decreased from 2.2 to 0.4 microM/min. In contrast, molecular crowding did not significantly affect the Michaelis constant of DNase I or exonuclease I. These results indicate that molecular crowding has different effects on the catalytic activities of exonucleases and endonucleases.
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PMID:Regulation of DNA nucleases by molecular crowding. 1756 1

Reaction conditions for numerous endonucleases are detailed in this unit along with discussions of potential applications. Specific enzymes include BAL 31 nuclease, S1 nuclease, mung bean nuclease, micrococcal nuclease and DNase I.
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PMID:Endonucleases. 1826 21


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