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 cystic fibrosis transmembrane conductance regulator (CFTR) gene is highly conserved within vertebrate species. Its pattern of expression in vivo seems to be tightly regulated both developmentally and in a tissue-specific manner, but shows differences with species. To identify transcriptional regulatory elements in the CFTR promoter region, we have used a combined approach based both on the analysis of the chromatin structure in vivo in rat tissues and on evolutionary clues (i.e. phylogenetic footprinting). In CFTR-expressing tissues, 15 DNase I-hypersensitive sites were identified within a 36 kb region encompassing exon 1. Eleven of them are clustered in a 3.5 kb region that exhibits eleven phylogenetic footprints observed when comparing sequences from eight mammalian species representing four orders (Primates, Artiodactylia, Lagomorpha and Rodentia). Comparison of the two sets of data allows the identification of two types of regulatory elements. Some are conserved between species, such as a non-consensus cAMP response element (CRE) and a PMA-responsive element (TRE) located respectively at positions -0.1 and -1.3 kb relative to ATG. Some are species-specific elements such as a 300 bp purine.pyrimidine (Pu.Py) stretch that is present only in rodents. Analysis of protein/DNA interactions in vitro with rat tissue protein extracts on the conserved elements revealed that the TRE site binds a specific heterodimeric complex composed of Fra-2, Jun D and a protein immunologically related to Jun/CRE-binding protein in the duodenum, whereas the CRE-like site binds ATF-1 ubiquitously. Functional analysis in Caco-2 cells showed that the CRE-like site supports a high basal transcriptional activity but is not able by itself to induce a response to cAMP, whereas the TRE site acts as a weak transactivator stimulated by PMA. Lastly, we found that the rodent-specific Pu.Py stretch confers nuclease S1 hypersensitivity under conditions of acidic pH and supercoiling. This indicates a non-B DNA conformation and thus reinforces the biological significance of non-random Pu.Py strand asymmetry in the regulation of transcription. Thus the tight transcriptional regulation of CFTR expression involves the combination of multiple regulatory elements that act in the chromatin environment in vivo. Some of them are conserved throughout evolution, such as the CRE-like element, which is clearly involved in the basal level of transcription; others are species-specific.
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PMID:Cross-species characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene reveals multiple levels of regulation. 958 39

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.
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PMID:Formation of single-stranded DNA during DNA transformation of Neisseria gonorrhoeae. 974 44

The multi-stranded DNA complexes formed by the oligonucleotides d(T15G4T2G4), Tel, and d(T15G15), TG, were examined by nuclease digestion and Raman spectroscopy. Both Tel and TG can aggregate to form structures consisting of multiple, parallel-oriented DNA strands with two independent structural domains. Overall, the structures of the TG and Tel aggregates appear similar. According to the Raman data, the majority of bases are in C2'-endo/anti conformation. The interaction of guanines at the 3'-ends in both complexes stabilizes the complexes and protects them from degradation by exonuclease III. The 5'-extensions remain single-stranded and the thymines are accessible to single-strand-specific nuclease digestion. The extent of enzymatic cleavage at the junction at the 5' end of the 15 thymines implies a conformational change between this part of the molecule and the guanine-rich region. The differential enzymatic sensitivity of the complexes suggests there are variations in backbone conformations between TG and Tel aggregates. TG aggregates were more resistant to digestion by DNase I, Mung Bean nuclease, and S1 nuclease than Tel complexes. It is proposed that the lower DNase I sensitivity may be partly due to the more stable backbone exhibited by TG than Tel complexes. Structural uniformity along the guanine core of TG is suggested, as there is no indication of structural discontinuities or protected sites in the guanine-rich regions of TG aggregates. The lower extent of digestion by Mung Bean nuclease at the 3' end implies that these bases are inaccessible to the enzyme. This suggests that there is minimal fraying at the ends, which is consistent with the extreme thermal stability of the TG aggregates.
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PMID:Probing the structure of multi-stranded guanine-rich DNA complexes by Raman spectroscopy and enzymatic degradation. 1037 Oct 18

BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M(1) and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barB upstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barB translational start codon. The BARE located on the barA upstream region (BARE-3) was found 101 to 76 bp upstream of the barA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barB transcription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.
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PMID:Characterization of binding sequences for butyrolactone autoregulator receptors in streptomycetes. 1043 81

FarA of Streptomyces lavendulae FRI-5 is a specific receptor protein for IM-2, a butyrolactone autoregulator that controls the production of a blue pigment and the nucleoside antibiotics showdomycin and minimycin. Gel shift assays demonstrated that FarA binds to the farA upstream region and that this binding is abolished in the presence of IM-2. The FarA binding sequence was localized by DNase I footprinting to a 28-bp sequence located approximately 70 bp upstream of the farA translational start site. High-resolution S1 nuclease mapping of farA transcripts revealed a putative transcription start site, located at an A residue positioned 64 bp upstream from the farA translation start codon and 4 bp downstream from an Escherichia coli sigma(70)-like -10 recognition region. The FarA-binding sequence overlaps this -10 region and contains the farA transcription initiation site, suggesting that FarA acts as a repressor that, in the absence of IM-2, represses transcription of farA.
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PMID:In vitro analysis of the butyrolactone autoregulator receptor protein (FarA) of Streptomyces lavendulae FRI-5 reveals that FarA acts as a DNA-binding transcriptional regulator that controls its own synthesis. 1043 82

Hepatopancreatic parvovirus (HPV) causes disease in several species of penaeid shrimp. Heavy infections may result in poor growth and reduced production for shrimp farmers. From one southern Thai shrimp pond with a high prevalence of HPV infection, 790 shrimp were sampled randomly and the hepatopancreas (HP) removed. Most HP were preserved in liquid nitrogen. However, every 10th HP (79 total) was divided into 2 parts appropriately fixed for examination by transmission electron microscopy (TEM) and light microscopy. Based on light microscopy, the prevalence of HPV infection in the pond was approximately 30% and its presence was confirmed by TEM of parallel samples. The virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in liquid nitrogen. Negative staining of the purified viral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts revealed the presence of 2 fragments, one very intense (5.8 kb) and the other weak (4.2 kb). The larger fragment was degraded by DNase I and S1 nuclease, indicating single-stranded DNA (ssDNA) characteristic of the viral family Parvoviridae. The smaller fragment was degraded by DNase I but not by S1 nuclease, indicating that it comprised double-stranded DNA. A genomic DNA library of the 5.8 kb ssDNA was constructed in pUC18 and a clone containing a 659 bp fragment specific and sensitive for HPV was selected for sequencing. Based on this sequence, an HPV-specific primer set was designed to yield a 156 bp amplicon by polymerase chain reaction (PCR) amplification. The expected 156 bp amplicon was obtained only in the presence of HPV DNA template (at as little as 1 fg purified DNA) and not with nucleic acid templates extracted from healthy shrimp tissue or other shrimp pathogens. It is hoped that this PCR assay will be useful to shrimp aquaculturists for early detection and screening of shrimp larvae, parental broodstock or other possible carriers of HPV in the shrimp cultivation system.
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PMID:Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand. 1062 52

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function sigma factor belonging to a subgroup of the primary sigma(70) family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named sigma(AdsA). Transcription of adsA depended on A-factor and AdpA, since adsA was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that sigma(AdsA) is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomyces species examined by Southern hybridization suggests a common role in morphogenesis in this genus.
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PMID:An A-factor-dependent extracytoplasmic function sigma factor (sigma(AdsA)) that is essential for morphological development in Streptomyces griseus. 1091 94

Expression of human estrogen receptor-alpha (ERalpha) involves the activity from several promoters that give rise to alternate untranslated 5' exons. However, the genomic locations of the alternate 5' exons have not been reported previously. We have developed a contig map of the human ERalpha gene that includes all of the known alternate 5' exons. By using S1 nuclease and 5'- rapid amplification of cDNA ends, the cap sites for the alternate ERalpha transcripts E and H were identified. DNase I-hypersensitive sites specific to ERalpha-positive cells were associated with each of the cap sites. A DNase I-hypersensitive site, HS1, was localized to binding sites for AP2 in the untranslated region of exon 1 and was invariably present in the chromatin structure of ERalpha-positive cells. Overexpression of AP2gamma in human mammary epithelial cells generated the HS1-hypersensitive site. The ERalpha promoter was induced by AP2gamma in mammary epithelial cells, and trans-activation was dependent upon the region of the promoter containing the HS1 site. These results demonstrate that AP2gamma trans-activates the ERalpha gene in hormone-responsive tumors by inducing changes in the chromatin structure of the ERalpha promoter. These data are further evidence for a critical role for AP2 in the oncogenesis of hormone-responsive breast cancers.
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PMID:Genomic structure of the promoters of the human estrogen receptor-alpha gene demonstrate changes in chromatin structure induced by AP2gamma. 1127 55

The human beta-globin genes are regulated by the locus control region (LCR), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5' to the genes. Various functional studies indicate that the LCR confers high-level, position-independent, and copy number-dependent expression to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconstituted in an erythroid cell-specific manner on chromatin-assembled LCR templates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid proteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The generation of LCR HS sites in the absence of chromatin assembly leads to the formation of S1- and KMnO(4)-sensitive regions in HS2 and HS3. These sites are also sensitive to S1 nuclease in erythroid cells in vivo, suggesting a distorted DNA structure in the LCR core enhancer elements. Finally, we show that RNA polymerase II initiates transcription in the HS2 and HS3 core enhancer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidirectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and recruit polymerase II transcription complexes. These results further our understanding of the structural basis for LCR function and provide an explanation for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.
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PMID:Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly. 1128 43

BldD is a transcription factor required for aerial hyphae formation in the filamentous bacterium Streptomyces coelicolor. Three targets of BldD regulation were discovered by a number of means, including examination of bld gene interdependence, selective enrichment of chromosomal DNA fragments bound by BldD and searching the promoter regions of known developmental genes for matches to a previously characterized BldD binding site. The three BldD targets identified were the developmental sigma factor genes, whiG and bldN, and a previously uncharacterized gene, designated bdtA, encoding a putative transcription factor. In each target gene, the sequences bound by BldD were characterized by electrophoretic mobility shift and DNase I footprinting assays, and their alignment suggested AGTgA (n)m TCACc as a consensus BldD operator. The in vivo effect of mutation in bldD on the expression of these three target genes was assessed using S1 nuclease protection assays. In each case, target gene expression was upregulated during early colony development in the bldD background, suggesting that, in the wild type, BldD acts to repress premature expression of whiG, bldN and bdtA during vegetative growth.
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PMID:BldD is a direct regulator of key developmental genes in Streptomyces coelicolor A3(2). 1129 92


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