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

---

Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).
...
PMID:Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase. 137 51

Heparin-agarose chromatography was used to isolate a restriction endonuclease (ENase) from the cellulolytic Gram+ anaerobe, Ruminococcus albus 8. The enzyme, Ral8I, was eluted from the column using 230-310 mM Na+. However, the preparation was active only with DNA substrates that were not Dam-methylated. Moreover, the restriction fragment pattern generated from simian virus 40 (SV40) DNA was not consistent with the expected number of Dam-methylation sites. Alignment of the Dam-methylation sites in SV40 DNA indicated that Ral8I may actually recognize the asymmetric sequence, GGATC. This was confirmed by nucleotide (nt) sequence analysis and, further, Ral8I was found to cause cleavage of the DNA approx. 5 nt downstream from the recognition sequence. Ral8I can therefore be classified as a type-IIS restriction endonuclease and is an isoschizomer of AlwI, BinI and BthII.
...
PMID:Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5'-GGATC. 154 46

A 66 base-pair (bp) DNA template carrying a site-specifically placed psoralen cross-link downstream from a phage T7 promoter was constructed. This template can support transcription by T7 RNA polymerase. Transcription was blocked specifically at the psoralen cross-link. We studied the characteristics of elongation complexes, formed in this manner, by enzymatic and chemical footprinting and by a nitrocellulose filter-binding assay. The DNase I footprint of the elongation complex was quantified on a residue by residue basis. It was found that T7 RNA polymerase made the strongest contacts in the central region of the footprint whereas the leading and lagging edges of the polymerase were weakly bound to the DNA. Reducing the NaCl concentration in the transcription reaction resulted in the visualization of two T7 RNA polymerase molecules bound to the same template. A leading polymerase molecule, arrested at the psoralen cross-link, showed a much smaller DNase I footprint than a lagging polymerase molecule that was bound upstream. This upstream polymerase molecule occupied approximately one-half of the promoter region and therefore did not achieve complete promoter clearance. These experiments suggest that complete promoter clearance is required for a gross conformational change in the polymerase, consisting of a contraction in the size of its footprint to occur. DNase I footprinting also revealed that an elongation complex arrested at a psoralen cross-link undergoes several subtle changes in structure in a time-dependent manner and therefore can be considered to be in a state of dynamic flux. Methylation protection showed that some G residues in the top (non-coding) strand are protected against attack by dimethylsulfate, whereas the G residues on the bottom (coding) strand appear not to be protected from reaction with dimethylsulfate. We probed the transcribing complexes for single-stranded regions with T7 gene 3 endonuclease. From the pattern of sensitivity to T7 gene 3 endonuclease on the template strand, we conclude that the RNA-DNA hybrid in the elongation complex is about 7 bp. A nitrocellulose filter-binding assay showed that the elongation complex, consisting of a 36 (+1) nucleotide RNA, the 66 bp DNA template and the T7 RNA polymerase was stable for at least 30 minutes at high salt concentrations. Heparin caused the quantitative release of 36 (+1) RNA nucleotides within 30 seconds, but the DNA was not simultaneously released from the elongation complex under these conditions.
...
PMID:Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. I. Characterization of elongation complexes. 194 44

By chromatography on phosphocellulose and Heparin-Sepharose the modification methylase M.Sau3239I was detected and partly purified from cells of Streptomyces aureofaciens 3239. Methylation by this enzyme protects DNA from cleavage by the restriction endonuclease R.Sau3239I. The enzyme catalyzes methylation of adenine to N-6-methyladenine in the 5'-CTCGmAG-3' recognition sequence.
...
PMID:Modification methylase M.Sau3239I from Streptomyces aureofaciens 3239. 212 28

Restriction of protein intake in the diet has been shown to produce a change in transcription activity as determined in vitro. Conditions for a reduced transcription activity were investigated with selective digestion of nuclei with micrococcus nuclease (EC 3.1.4.7). Liver nuclei from young male rats fed a diet containing either 20 or 3% casein for 6 days were digested with 1.3 microgram of micrococcus nuclease protein/mg of nuclear DNA (specific enzyme activity 15,000 or 150 units/mg of protein). Part of the chromatin-bound RNA polymerase activity was transferred from the 2,000 x g to the 102,000 x g pellet. Independent of the type of endonuclease use, the specific activity of chromatin-bound and soluble RNA polymerase I plus III was similar in the two groups of rats. After protein restriction RNA polymerase II activity was significantly diminished in the 2,000 x g pellet, and was unchanged in the 102,000 x g pellet. Heparin-stimulated and soluble RNA polymerase II activities were significantly reduced. Number and length of RNA chains synthetized by chromatin-bound RNA polymerase I plus III remained unchanged by dietary treatment. After a low protein diet, RNA polymerase II in the absence and presence of heparin synthesized an unchanged number of RNA chains with reduced length. A selective digestion of chromatin with micrococcus nuclease is needed to show the reduction in RNA polymerase II activity after protein restriction.
...
PMID:Reduced transcription activity of rat liver chromatin after protein restriction and selective digestion of nuclei with micrococcus nuclease. 616 34

The effect of dimethylnitrosamine on functional activities of liver chromatin was studied in mice. After a single dose of dimethylnitrosamine injected i.v. (25 mg/kg body wt, 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcal nuclease (EC 3.1.4.7) to an acid-solubility of 2.5% of total DNA. Chromatin was fractionated into a 1,200 g pellet P1, 102,000 g pellet P2 and supernatant fraction S2. Chromatin-bound RNA polymerase I plus III activity decreased 15% in the P1 and 25% in the P2 fraction. No changes in activity were observed in the S2 fraction. Chromatin-bound RNA polymerase II activity decreased 19% in the P1, 49% in the P2 and 32% in the S2 fraction. Heparin stimulated RNA polymerase II activity decreased 10% in the P1 and 44% in the P2 fraction. Formation of initiation in nuclear lysates with RNA polymerase from Escherichia coli increased after administration of dimethylnitrosamine suggesting an increase in the number of sites available for the start of new RNA chains. The results show that limited digestion of nuclei with endonuclease cleaves chromatin regions which are more affected by dimethylnitrosamine than the total chromatin suggesting a non-random effect of the hepatotoxin on chromatin. Modifications of the DNA template by dimethylnitrosamine is indicated by the change in number of initiation complexes.
...
PMID:Non-random effect on RNA synthesis in liver chromatin by administration of dimethylnitrosamine to mice. 619 51

This work describes the purification and biochemical characterization of BstLVI restriction endonuclease, a thermostable isoschizomer of ClaI, from Bacillus stearothermophilus LV. The enzyme was purified by successive DEAE-cellulose, Affi-Gel Blue and Heparin-Sepharose CL-6B column chromatography. A molecular weight of 37,000 was determined for Bst LVI by gel filtration. As expected from thermophilic proteins, the enzyme showed a high stability towards heat and also to other known protein-denaturing agents.
...
PMID:Purification and characterisation of Bst LVI restriction endonuclease, a thermostable isoschizomer of ClaI from Bacillus stearothermophilus LV. 842 53

The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.
...
PMID:Cloning and expression of AatII restriction-modification system in Escherichia coli. 903 20

An endonuclease was isolated from 5 days old Agropyron elongatum 8x = Elytrigia turcica McGuire seedlings. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-cellulose and Heparin Sepharose column. The final preparation, named nuclease A, gave a single band after silver staining had followed SDS-electrophoresis that was identified with nuclease activities. The enzyme also showed a single band after activity staining on gel polymerized in the presence of heat denatured DNA (ssDNA)/RNA. The Mr of native enzyme was 36 and the enzyme's moiety consisted of one polypeptide chain. Nuclease A activity was stimulated in the presence of Zn(2+) and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA > or = RNA; hence it was classified as a plant nuclease type I (EC 3.1.30.2). Synthetic homopolyribonucleotides were hydrolyzed in the order polyU > polyI > or = polyA > polyG > polyC. Nuclease A nicked the supercoiled plasmid DNA while it was incapable of hydrolyzing dinucleoside monophosphates. With regard to nuclease A base linkage specificity towards a synthetic 5'-(32)P labeled deoxydecanucleotide [5'-(32)P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap downward arrow G bond and then to Gp downward arrow T, Cp downward arrow A and Gp downward arrow G bonds while it was incapable of hydrolyzing the Cp downward arrow C bond. The substrate's products of nuclease A were oligonucleotides with the monoesterified phosphate at the 3' position. Nuclease A may perform a crucial function in the metabolism of nucleic acids during seedling growth and could be used as a biochemical tool for analysis of nucleic acids structure.
...
PMID:Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings. 1559 99

TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin agarose, SP sephadex C50, and Mono-Q fast protein liquid chromatography and was found to be a homodimer of 40 kDa. Restriction mapping and run-off sequencing of TspMI-cleaved DNA ends depicted that it cleaved at 5'C/CCGGG3' to generate a four-base, 5'-CCGG overhang. The enzyme was sensitive to methylation of second and third cytosines in its recognition sequence. TspMI worked optimally at 60 degrees C with 6 mM Mg(2+), no Na(+)/K(+), and showed no star activity in the presence of 25% glycerol. The enzyme could efficiently digest the DNA labeled with a higher concentration of YOYO-I (one dye molecule to one nucleotide), making it a useful candidate for real-time imaging experiments. Single molecule interaction between TspMI and lambda DNA was studied using total internal reflection fluorescence microscopy. The enzyme survived 30 polymerase chain reaction (PCR) cycles in the presence of 10% glycerol and 0.5 M trehalose without any activity loss and, hence, is suitable for incorporation in restriction-endonuclease-mediated selective-PCR for various applications.
...
PMID:TspMI, a thermostable isoschizomer of XmaI (5'C/CCGGG3'): characterization and single molecule imaging with DNA. 1684 5


1 2 Next >>

---