Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The catabolite gene activator protein (CAP) and the lac repressor regulate the transcriptional activity of the lactose operon. An early step in the regulatory functions of these proteins is their binding to specific DNA sequences within the lac promoter-operator region. Using the gel electrophoresis mobility-shift technique, we have found that the ternary complex with CAP and repressor bound to their respective highest affinity sites is 4 to 11-fold more stable than is predicted from the affinities of the independently bound proteins. This favorable binding interaction is unexpected, because CAP and lac repressor exert opposing effects on lac operon transcription. Deoxyribonuclease I footprinting analyses show that interacting proteins remain bound to the sites occupied when the proteins bind singly. These sites have a center-to-center separation of 72 base-pairs (corresponding to 6.9 turns of a B-form DNA helix), and thus occupy the same "face" of the DNA cylinder. Such an orientation is compatible with models of the ternary complex in which DNA curvature facilitates the interaction of CAP and lac repressor.
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PMID:Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter. 216 65

Two Escherichia coli control regions have been compared in their ability to be unwound by RNA polymerase during formation of the transcriptionally active ("open") complex: the wild-type control region, consisting of two overlapping binding sites P1 and P2, both weakly transcribed, and an "up" P1 mutant, the strong lac L8UV5 promoter. The final complexes were characterized by their topological unwinding, by DNase I and orthophenanthroline footprints, as well as by methylation of unpaired cytosine residues. At the wild-type control region, the RNA polymerase footprint is weak, and single-strand formation is incomplete and slow. The same signals are strong, complete and quickly established at lac L8UV5; yet the final complexes were found to be equally unwound (by 1.7 turns) in the absence of nucleotide substrates as well as during an abortive initiation cycle. At the lac wild-type region, open complex formation occurs slowly enough to permit the measurement of the extent of a single-stranded region and of topological unwinding during the latency period. Not all the final species are active and unwinding appears to precede, in time, full open-complex formation. At the lac UV5 promoter the same conclusion was reached by a different method involving those changes in the various parameters that characterize open-complex formation monitored by an abortive initiation assay, conducted at increasing levels of template superhelicity. From both approaches we conclude that, at these promoters, the formation of the single-stranded region occurs at the expense of a negative change in linking number, initially stored in a closed intermediate, perhaps as negative writhing. Furthermore, abortive transcription assays indicate that the specific initiation efficiency of the species stored at both promoters, P1 and P2, on the wild-type template is increased as a whole with increasing superhelicity (conversion of inactive species to active ones, increased efficiency of active ones). We conclude that negative supercoiling is not an extra-regulatory element of the lactose system, allowing modulation of expression of the wild-type promoter to the profit of P1. Instead, P2 and P1, in the absence of active catabolite receptor protein (CRP-cAMP), appear to be equally weak and to be equally affected by negative supercoiling in the range of superhelical densities examined. The physiological importance of the P1-P2 competition in the regulation of expression in this region is thus questioned. The major effect of CRP-cAMP stimulation appears to be the direct activation at the P1 promoter.
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PMID:Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of Escherichia coli. 330 41

The lactose repressor of Escherichia coli (LacI) associates to a bidentate tetramer in solution and can simultaneously bind two operators to form a protein-mediated "looped complex". Studies have been conducted of the binding of LacI to two operators separated by approximately 11 helical turns of DNA. Quantitative DNase I footprint titration analysis of the stability of the LacI-mediated looped complex reveals that the Gibbs free energy of cyclization (delta Gzeroj) of the looped complex of 11.7 +/- 0.4 kcal/mol is invariant with temperature. van't Hoff analysis reveals a large and positive enthalpy of cyclization (delta H degrees = 12.3 +/- 2.4 kcal/mol) and an entropy that is small and positive (delta S degrees = 2.2 cal/deg). Quantitative DNase I footprint titration and kinetic dissociation studies were also conducted as a function of counter-ion type and concentration. Increasing concentrations of KCl or potassium glutamate destabilize the looped complex, a result completely accounted for by increases in the intrinsic DNA-binding free energies. While the value of delta Gzeroj is invariant with ion concentration, chloride is a positive regulator. The value of delta Gzeroj decreases by 1.5 kcal/mol upon substitution of chloride for glutamate. Measurements of delta Gzeroj conducted as a function of chloride concentration at constant ionic strength reveal that approximately one chloride ion per tetramer is bound upon looped complex formation. These results demonstrate specific allosteric regulation of the formation of the LacI-mediated looped complex by a mechanism distinct from the regulation of the constituent protein--DNA interactions.
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PMID:Modulation of the stability of a Lac repressor mediated looped complex by temperature and ions: allosteric regulation by chloride. 835 11

beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis.
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PMID:Transcriptional regulation of murine beta1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves both a housekeeping and a mammary gland-specific function. 861 93

Mapping of protein domains having a distinct function is essential to understanding the protein's structure-function relationship. We used a bacteriophage lambda surface expression vector, lambdafoo, in order to determine the minimal carbohydrate-binding domain of human galectin-3 (Gal-3). Gal-3 cDNA was randomly digested by DNase I and cloned into the phage vector. The library generated was screened by affinity selection using lactose immobilized on agarose beads. DNA sequence analysis of a set of isolated clones defined the minimal folding domain of Gal-3 required for lactose binding, which consisted of 136 amino-acid residues. Using the phage clones isolated, we also determined relative dissociation constants in solution between lactose and the minimal domain expressed on the phage surface. This technique does not require either purified or labeled proteins, and bacteriophage lambda surface display may, therefore, be useful for protein domain mapping and in vitro studies of various macromolecular interactions.
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PMID:Protein domain mapping by lambda phage display: the minimal lactose-binding domain of galectin-3. 1055 59

The physicochemical properties and gene transfer ability of lactose-polyethylene glycol-grafted poly-L-lysine (Lac-PEG-PLL) were investigated. A dye displacement assay showed that plasmid DNA self-assembled with Lac-PEG-PLL, and condensation began at a <1:1 charge ratio of plasmid DNA to polymer. In atomic force microscopy, spontaneously assembled Lac-PEG-PLL/DNA complexes revealed a compact structure, with a size of about 100-200 nm. Circular dichroism spectra of Lac-PEG-PLL/DNA complexes revealed that the secondary structure of DNA was altered by complex formation and was similar to that of the poly-L-lysine/DNA complex. Lac-PEG-PLL was shown to protect DNA against nuclease action in a DNase I protection assay. The cytotoxicity test demonstrated that the complex composed of plasmid DNA and Lac-PEG-PLL had little influence on the viability of HepG2 cells, especially in comparison with that of poly-L-lysine/DNA complexes. In conclusion, our copolymer, Lac-PEG-PLI, formed complexes with plasmid DNA (on average, 150 nm), gave little cytotoxicity, and showed increased efficiency of gene transfer into hepatoma cells in vitro. Lactose-polyethylene glycol was grafted to poly-L-lysine to be used as a gene carrier for hepatoma cell targeting and to improve the solubility of the polyplexes. The average size of the carrier/DNA complexes was about 150 nm. The complexes also proved to have high resistance against nuclease attack and little cytotoxicity. The polymer also delivered plasmid DNA efficiently into a HepG2 cell line. Lac-PEG-PLL was more efficient than Lipofectin or galactose-PEG-PLL in transfection efficiency.
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PMID:Characterization of a targeted gene carrier, lactose-polyethylene glycol-grafted poly-L-lysine and its complex with plasmid DNA. 1056 93

A genetic locus that is adjacent to the gene encoding the small acid-soluble protein SASP C-4 of Bacillus megaterium has been identified. This locus, designated fru, contains a beta-fructosidase gene (fruA), a gene encoding a hydrophobic protein that is closely related to non-PTS sugar permeases of the proton symport type (fruP), and a gene coding for a transcriptional regulator of the LacI/GalR family (fruR). The FruA protein can hydrolyze sucrose and raffinose, but not maltose, isomaltose, trehalose, melibiose or lactose. The transcription initiation site of fruP has been mapped and the fruP promoter identified. Gel mobility shift assays showed that the FruR protein can bind specifically to a DNA fragment containing the fruP promoter region. DNase I footprinting analysis has defined the FruR binding site. Disruption of fruR led to high-level constitutive expression of fruPA, but had no effect on expression from the fruR promoter itself, indicating that FruR acts as a repressor of fruPA expression, but does not autoregulate its own synthesis. Interestingly, expression of fruPA in B. megaterium was not induced by sucrose, raffinose, fructose or inulin, whereas the constitutive expression of fruPA in a fruR mutant was repressed by both glucose and sucrose. Possible physiological implications of these findings are discussed.
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PMID:Identification and characterization of the non-PTS fru locus of Bacillus megaterium ATCC 14581. 1239 98

The Gram-positive human pathogen Streptococcus pneumoniae possesses an unusually high number of gene clusters specific for carbohydrate utilization. This provides it with the ability to use a wide array of sugars, which may aid during infection and survival in different environmental conditions present in the host. In this study, the regulatory mechanism of transcription of a gene cluster, SPD0424-8, putatively encoding a cellobiose/lactose-specific phosphotransferase system is investigated. We demonstrate that this gene cluster is transcribed as one transcriptional unit directed by the promoter of the SPD0424 gene. Upstream of SPD0424, a gene was identified encoding a ROK-family transcriptional regulator (RokA: SPD0423). DNA microarray and transcriptional reporter analyses with a rokA mutant revealed that RokA acts as a transcriptional repressor of the SPD0424-8 operon. Furthermore, we identified a 25 bp AT-rich DNA operator site (5'-TATATTTAATTTATAAAAAATAAAA-3') in the promoter region of SPD0424, which was validated by promoter truncation studies, DNase I footprinting and electrophoretic mobility-shift assays. We tested a large range of different sugars for their effect on the expression of the SPD0424-8 operon, but only moderate variation in expression was observed in the conditions applied. Therefore, a co-factor for RokA-mediated transcriptional control could not be identified.
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PMID:Characterization of the ROK-family transcriptional regulator RokA of Streptococcus pneumoniae D39. 2308 33

Highly viscous mucus poses a big challenge for the delivery of particulates carrying therapeutics to patients with cystic fibrosis. In this study, surface modifying DNase I loaded particles using different excipients to achieve better lung deposition, higher enzyme stability or better biological activity had been exploited. For the purpose, controlled release microparticles (MP) were prepared by co-spray drying DNase I with the polymer poly-lactic-co-glycolic acid (PLGA) and the biocompatible lipid surfactant 1,2-dipalmitoyl-Sn-phosphatidyl choline (DPPC) using various hydrophilic excipients. The effect of the included modifiers on the particle morphology, size, zeta potential as well as enzyme encapsulation efficiency, biological activity and release had been evaluated. Powder aerosolisation performance and particle phagocytosis by murine macrophages were also investigated. The results showed that more than 80% of enzyme activity was recovered after MP preparation and that selected surface modifiers greatly increased the enzyme encapsulation efficiency. The particle morphology was greatly modified altering in turn the powders inhalation indices where dextran, ovalbumin and chitosan hydrochloride increased considerably the respirable fraction compared to the normal hydrophilic carriers lactose and PVP. Despite of the improved aerosolisation caused by chitosan hydrochloride, yet retardation of chitosan coated particles in artificial mucus samples discouraged its application. On the other hand, dextran and polyanions enhanced DNase I effect in reducing cystic fibrosis mucus viscosity. DPPC proved good ability to reduce particles phagocytic uptake even in the presence of the selected adjuvants. The prepared MP systems were biocompatible with lung epithelial cells. To conclude, controlled release DNase I loaded PLGA-MP with high inhalation indices and enhanced mucolytic activity on CF sputum could be obtained by surface modifying the particles with PGA or dextran.
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PMID:Inhalable DNase I microparticles engineered with biologically active excipients. 2393 40