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 complete nucleotide sequence of a 3.2-kilobase-pair chromosomal region containing the algP and algQ genes was determined. The algQ gene encodes an acidic 18-kilodalton polypeptide required for transcriptional activation of the algD gene. The algD gene product catalyzes a critical step in alginate biosynthesis, and its overproduction is necessary for the emergence of mucoid Pseudomonas aeruginosa during chronic infections in cystic fibrosis. A novel genetic element, algP, was identified immediately downstream of algQ. This gene appears to act synergistically with algQ. Unlike a biosynthetic gene, algD, and another regulatory gene, algR, which undergo transcriptional activation in mucoid cells, both algP and algQ are equally transcribed in mucoid and nonmucoid isogenic strains of P. aeruginosa. The promoter regions of algP and algQ were mapped by using S1 nuclease protection analysis. The algQ promoter was also analyzed and showed activity in an in vitro transcriptional runoff assay with major RNA polymerase species from P. aeruginosa and Escherichia coli. The putative algQ and algP promoter sequences, unlike algD and algR, resemble sigma 70-utilized promoters from E. coli and appeared constitutively transcribed at a low level in P. aeruginosa. The algP gene has an unusual DNA sequence, with multiple direct repeats organized in six highly conserved, tandemly arranged, 75-base-pair (bp) units. At a lower level, this sequence had 45 degenerate repeats of 12 bp overlapping with the 75-bp repeats and extending beyond the region of 75-bp repeats. The algP repeats appeared important for the function of the algQ-algP regulatory region in maintaining mucoidy.
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PMID:DNA sequence and expression analysis of algP and algQ, components of the multigene system transcriptionally regulating mucoidy in Pseudomonas aeruginosa: algP contains multiple direct repeats. 211 Jan 44

A new alginate regulatory gene, algQ, was identified in a chromosomal region which, when tandemly amplified, induces mucoidy in Pseudomonas aeruginosa. The algQ gene was found closely linked to the previously identified algR gene. Both algQ and algR were required for transcription of the key alginate biosynthetic gene, algD. In addition, expression of the algR gene was studied. The algR promoter was mapped by S1 nuclease and reverse transcription and found to be activated in mucoid cells. However, even in nonmucoid cells, transcription of algR was detectable at an approximately 50-fold-lower level, as opposed to the algD promoter, which was silent in the nonmucoid background. Transcription of both promoters was studied by using algR- and algD-specific oligonucleotides and total cellular RNA from fresh cystic fibrosis isolates of mucoid P. aeruginosa and their nonmucoid revertants. Identical patterns of activity were found in all strains: in mucoid cells, both algR and algD were activated. This finding indicated that common mechanisms were involved in the regulation of alginate gene expression. However, when the algR gene was cloned behind the tac promoter on a broad-host-range-controlled expression vector, induction of transcription with isopropropyl-beta-D-thiogalactopyranoside (IPTG) caused the appearance of a nonmucoid phenotype in previously mucoid cells. This effect was transient, since removal of the inducer (IPTG) made cells mucoid again. Since the algR gene product is homologous to transcriptional regulators from a class of environmentally responsive systems (known to have a second, sensory component), the algQ gene could be a candidate for the sensory component of the alginate system.
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PMID:Control of mucoidy in Pseudomonas aeruginosa: transcriptional regulation of algR and identification of the second regulatory gene, algQ. 254 50

We have found that preparations of DNA isolated from purulent sputum possess a novel activity which accelerates and stabilizes the binding of human leukocyte elastase to secretory leukoprotease inhibitor, a major endogenous antielastase in the respiratory tract. DNA in sputum is derived from the nuclear debris of disintegrated inflammatory leukocytes, and can attain concentrations ranging from 10(2) to 10(4) micrograms/ml, depending on the severity of pulmonary infection and inflammation. In the presence of 23 micrograms/ml DNA, a concentration lower than those found in most purulent sputa, the rate constant for association of secretory leukoprotease inhibitor with elastase is increased to 1.1 x 10(8) M-1s-1, 44-fold greater than that in the absence of DNA. The equilibrium dissociation constant for the enzyme-inhibitor complex drops to 0.7 pM, two orders of magnitude lower than that in the absence of DNA. The accelerating effect of DNA is further increased by thermal denaturation or by modification with exonuclease III, while it is significantly reduced by digestion with S1 nuclease or by binding of Escherichia coli single-stranded DNA binding protein. The results from these experiments indicate that the structural elements in sputum DNA that are responsible for the accelerating effect have the characteristics of single-stranded sites. Similar kinetic effects on elastase inhibition were also observed with human placental DNA and genomic DNAs from a variety of other species. These findings suggest that DNA in pulmonary secretions may participate in antielastase defense by promoting the binding of secretory leukoprotease inhibitor to leukocyte elastase. The results may have important implications for use of nuclease preparations in mucolytic therapy for cystic fibrosis.
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PMID:Accelerated binding of secretory leukoprotease inhibitor to human leukocyte elastase mediated by single-stranded sites in DNA from tracheobronchial mucus. 757 8

Alginate overproducition by mucoid Pseudomonas aeruginosa is a critical pathogenic determinant expressed by this organism during chronic infections in cystic fibrosis. Conversion to mucoidy and a subsequent loss of mucoid character can occur via different mutations in the algU mucA mucB gene cluster. The algU gene encodes a 22.2-kDa putative alternative sigma factor required for expression of the critical alginate biosynthetic gene algD. In this work, algU transcription was studied by S1 nuclease protection analysis. Transcription from the promoter proximal to the algU coding region was found to be dependent on AlgU. The -35 and -10 sequences of this newly mapped promoter showed strong similarity ot the promoters of two other critical alg genes: algD and algR. The proximal promoter of algR was also shown to depend on algU. Interestingly, the putative -35 and -10 regions of all three promoters displayed striking similarity to the consensus sequence of the sigma E-dependent promoters in Escherichia coli and Salmonella typhimurium. This 24-kDa sigma factor, controlling genes participating in resistance to high temperatures and oxidative stress, has been previously biochemically characterized, but the gene for sigma E remained unidentified. To examine whether AlgU is related to sigma E, the effect of algU inactivation on the sensitivity of P. aeruginosa to killing by heat and reactive oxygen intermediates was tested. Two isogenic pairs of algU+ and algU mutant strains were compared. The algU mutants, irrespective of the mucoid status of the parental strains, displayed increased sensitivity to killing by paraquat, known to generate intracellular superoxide radicals, and heat. Further lgobal homology searches revealed the presence of a previously unrecognized E. coli gene with the predicted gene product showing a striking 66% identity to AlgU. The corresponding gene from S. typhimurium was cloned and sequenced, and it is displayed one amino acid substitution relative to its E. coli equivalent. AlgU and its close homologs in E. coli and S. typhimurium may be functionally related.
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PMID:Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response. 796 22

To identify the intracellular barriers to efficient gene transfer, we studied the intracellular trafficking of biotinylated plasmid DNA complexed with either fluorescein-conjugated lactosylated or mannosylated polylysine by confocal microscopy. Both are known to be taken up by cystic fibrosis airway epithelial cells (SigmaCFTE29o- cells), but their gene transfer efficiencies differ markedly: lactosylated polylysine is the most efficient glycosylated polylysine while mannosylated polylysine is quite inefficient for gene transfer. Mannosylated complexes appeared to stay longer in endosomes labeled by anti-transferrin receptor antibody than lactosylated complexes (from 30 min to 3 h and from 10 min to 30 min, respectively). At 24 h, higher percentages of mannosylated than lactosylated complexes were localized inside lysosomes labeled by anti-LAMP-1 antibody (41.8 +/- 6.6% versus 19.8 +/- 5.2%, respectively, P < 0.05). Between 30 min and 8 h, complexes reached the nuclei labeled by anti-lamin A/C antibody and no difference was observed between the nuclear amounts of mannosylated and lactosylated complexes. However, as analyzed by a nuclease S1 transcription assay, initiation of transcription was prevented when plasmid DNA was complexed to mannosylated polylysine. Our results indicate that the major limiting steps for mannosylated versus lactosylated polylysine transfer of plasmid DNA are delayed exit from endosomes, high accumulation in lysosomes and limited transcription of the complexed plasmid DNA.
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PMID:Intracellular rate-limiting steps of gene transfer using glycosylated polylysines in cystic fibrosis airway epithelial cells. 1210 30

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was approximately 3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 +/- 0.85 micromol of HCN/mg of protein versus 2.4 +/- 0.40 micromol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains. Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.
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PMID:The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa. 1546 37

Polyethylenimine (PEI) is one of the most potent non-viral vectors. We have developed a lactosylated PEI (Lac-PEI) to enhance cell-specific transfection and have shown that Lac-PEI is more efficient than unsubstituted PEI for gene transfer into immortalized cystic fibrosis airway epithelial SigmaCFTE29o-cells. As both intact PEI/plasmid and Lac-PEI/plasmid complexes are found in the cell nucleus, we have investigated the transcription efficiency of the plasmid complexed with PEI or Lac-PEI, according to the polymer nitrogen/DNA phosphate (N/P) ratio (from 0 to 20). The initiation of transgene transcription was analyzed in an acellular nuclease S1 transcription assay. For both PEI and Lac-PEI complexes, transcription efficiency varied with the N/P ratio of the complexes. Transcription inhibition was observed when plasmid DNA was either loosely (N/P<5) or tightly condensed (N/P>15). For an N/P ratio of 5 and up to 15, transcription of the complexed plasmid was as efficient as that of the free plasmid. Similar results were observed when gene expression was studied after nuclear microinjection of the complexes into SigmaCFTE29o-cells. Our study shows that condensation of DNA influences the accessibility of the plasmid to the transcription machinery. Interestingly, the charge ratios that allow the most efficient transcription are those usually known to be the most efficient for gene transfer in vitro and in vivo.
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PMID:Transcription of plasmid DNA: influence of plasmid DNA/polyethylenimine complex formation. 1608 68