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

The isolation and characterization of a restriction endonuclease from Bacillus cereus IOC 243 are described. The enzyme recognizes the palindromic sequence 5'-G(met-A,A)TC-3' as determined by PEI chromatography of pancreatic DNase, snake venom phosphodiesterase digestion products of labelled fragments, analysis of restriction digests from normal and N6-methyladenine-free DNA and direct sequence analysis of cloned fragments. The staggered cleavage products with 5' -terminal pGATC extensions are efficiently labelled with polynucleotide kinase and are easily cloned into BamHI sites. The enzyme, denoted Bce243, is thus an isoschizomer of Sau3AI. Its use and potential advantages in substituting Sau3AI. are discussed.
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PMID:An Sau3 AI restriction endonuclease isoschizomer from Bacillus cereus. 608 3

A new method for isolating DNA from agarose gels is described. The method involves the simultaneous transfer of all DNA-fragments from an agarose slab gel onto DEAE-cellulose paper and the elution of the individual fragments from the paper with 1 M NaCl. DNA isolated from agarose gels in this way is susceptible to cleavage with several restriction endonucleases, and can be labeled in vitro with E coli DNA-polymerase I, T4 DNA-polymerase and T4 polynucleotide kinase. We have used the method to construct restriction endonuclease maps of adenovirus type 16 DNA.
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PMID:Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA. 625 42

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.
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PMID:A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase. 626 Apr 93

Endonuclease V of bacteriophage T4 has been purified to physical homogeneity from T4D-infected Escherichia coli 1100. The enzyme, whose molecular weight is 16,000, possessed two distinct catalytic activities, a pyrimidine dimer-DNA glycosylase and an apurinic/apyrimidinic endonuclease. They acted on UV-irradiated poly(dA) . poly(dT) in a sequential manner; the glycosylase cleaved the N-glycosyl bond between the 5'-pyrimidine of a dimer and the corresponding sugar and then the endonuclease hydrolyzed a phosphodiester bond on the 3'-side of the apyrimidinic site. The 5'-termini thus generated were phosphorylated by T4 polynucleotide kinase only after they had been subjected to direct photoreversal and then treated with alkaline phosphatase. By using two phage mutants, uvs-5 and uvs-13, it was shown that occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities. Suppression of the mutation of uvs-5 rendered both activities partially active. When the mutation of uvs-13 was suppressed, a mutant form of enzyme that possessed only a glycosylase activity was produced. This suggests that there are two distinct domains in a single enzyme, each of which corresponds to one of the activities.
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PMID:Purification and characterization of normal and mutant forms of T4 endonuclease V. 627 6

The influence of polyamines on various enzymes involved in the excision repair pathway of DNA, such as UV endonuclease, DNA polymerase I, DNA ligase and polynucleotide kinase, and two AP-endonucleases, were studied. The polymerizing activities of DNA polymerase I and polynucleotide kinase were found to be markedly affected by polyamines. In the former enzyme the effect can be attributed to the stabilization of the correct bihelical structure at the 3' end and in the latter case polyamines stabilize the polynucleotide kinase protein itself in the correct oligomeric structure. The effect of polyamines on the hydrolysis of apurinic and apyrimidinic sites in DNA and nucleosome particles were also investigated. Spermine and spermidine were found to be the most efficient polyamines in causing such hydrolysis both in the free DNA and in the nucleosome particles.
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PMID:Effect of polyamines on enzymes involved in DNA repair. 627 78

We describe the construction and characterization of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits. Poly(A)-RNA isolated from rat livers was enriched for glutathione S-transferase mRNA activity and used as templates to synthesize double stranded cDNA. The double stranded cDNAs were annealed to pBR322 through terminal deoxynucleotidyl transferase generated GC-tails followed by transformation into E. coli. Several candidate clones were selected by colony hybridization using polynucleotide kinase labeled liver and testis poly(A)-RNA probes. These candidate clones were further characterized by hybrid-selected translation of mRNA followed by immunoprecipitation and SDS gel electrophoresis. The positive clone, pGTR112 was mapped with restriction endonuclease analysis and sequenced by the chemical method of Maxam and Gilbert. The largest upen reading frame contains 142 amino acids very rich in Arg and Lys residues. The C-terminal residue phenylalanine of this open reading frame is consistent with what was reported for one of the ligandin subunits by Bhargava et al., (J. Biol. Chem. 253, 4116-4119, 1978). Among the 352 nucleotides covered by both pGTR112 and pGST94 described by Kalinyak and Taylor (J. Biol. Chem. 257, 523-530, 1982), there are only 9 nucleotide differences resulting in four changes of amino acid sequences.
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PMID:Cloning and sequence analysis of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits. 629 39

The fate of host tRNAs during T4 bacteriophage infection was investigated with Escherichia coli CTr5x, the only known host strain that is restrictive to RNA ligase and polynucleotide kinase mutants. Three CTr5x tRNA species were cleaved during infection. One was leucine tRNA1, which was cleaved in the extra arm, as reported elsewhere for E. coli B infected with bacteriophage T2 or T4. The other two were specific to E. coli CTr5x and were not cleaved in various other hosts. One of the cleaved CTr5x-specific tRNAs had an anticodon sequence of the E. coli B "major" isoleucine tRNA but otherwise little sequence homology. Both CTr5x-specific tRNAs were cleaved by a distinct T4-induced endonuclease, other than that of leucine tRNA1, because the CTr5x-specific cleavages (i) were induced later in infection, (ii) persisted with a T4 mutant deficient in leucine tRNA1 endonuclease, and (iii) occurred in the anticodon loop. The specific manifestation of the anticodon-directed endonuclease activity in T4-infected E. coli CTr5x suggests roles for RNA ligase and polynucleotide kinase in processing of host tRNA species.
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PMID:Bacteriophage T4-induced anticodon-loop nuclease detected in a host strain restrictive to RNA ligase mutants. 629 15

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.
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PMID:An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII. 630 60

To determine the relative importance of the 2',5'-phosphodiester bond of 2-5A in its binding to and activation of the 2-5A-dependent ribonuclease (RNase L, RNase F), a number of phosphodiester linkage isomers of 2-5A were prepared. These isomers were obtained either by lead ion-catalyzed polymerization of adenosine 5'-phosphorimidazolidate or by T4 polynucleotide kinase-catalyzed 5'-phosphorylation of adenylyl(3' leads to 5')adenylyl(3' leads to 5')adenosine followed by reaction of the corresponding phosphorimidazolidates with tri(n-butylammonium)pyrophosphate. The following 2-5A isomers thus were prepared: ppp5'A2'p5'A3'p5'A, ppp5'A3'p5'A2'p5'A, ppp5'A3'p5'A3'p5'A("3-5A"), ppp5'A2'p5'A3'p5'A2'p5'A,and ppp5'A3'p5'A2'p5'-A2'p5'A. The ability of these isomeric 2-5As to interact with the 2-5A-dependent endonuclease was ascertained by three different criteria: (i) ability to prevent the protein synthesis inhibitory effects of 2-5A, (ii) activity as an inhibitor of translation in encephalomyocarditis RNA-programmed L cell extracts, and (iii) ability to prevent binding of the radiolabeled probe, ppp5'A2'p5'A2'p5'A2'p5'A3'[32P]p5'Cp, to the endonuclease of L cell extracts. In certain experiments, degradation of oligonucleotide was minimized or eliminated by altering assay conditions, providing alternate phosphodiesterase substrates, or by using purified endoribonuclease of Ehrlich ascites cells. By all criteria, replacement of 2',5'-bond by a 3',5'-bond led to a substantial decrease in biological activity. Generally, replacement of just one 2',5'-phosphodiester bond with a 3',5'-linkage led to at least a one order of magnitude loss of activity. In accord with this trend, ppp5'A3'p5'A3'p5'A(3-5A) was greater than 10,000 less active than 2-5A in binding to the endonuclease or as an inhibitor of protein synthesis.
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PMID:Biological activities of phosphodiester linkage isomers of 2-5A. 663 Feb 22

We examined the structure of the frog virus 3 (FV 3) genome by using electron microscopic and biochemical techniques. The linear FV 3 DNA molecules (Mr approximately 100 x 10(6) formed circles when partially degraded with bacteriophage lambda 5'-exonuclease and annealed, but not when the annealing was done without prior exonuclease digestion. The results suggest that the DNA molecules contain direct terminal repeats. The repeated region composed about 4% of the genome. Complete denaturation of native FV 3 DNA molecules followed by renaturation produced duplex circles each bearing two single-stranded tails at different points along the circumference. The tails presumably represent the terminal repeats. The formation of duplex circles suggests that the FV 3 genome is circularly permuted. This is further borne out by (i) failure to identify a specific restriction endonuclease fragment containing the label when the molecular ends were radiolabeled by using the polynucleotide kinase procedure, and (ii) similarity in the restriction patterns of virion DNA and large concatemeric replicating viral DNA as revealed by endonucleolytic cleavage of both DNAs with HindIII. From the above data, we conclude that the FV3 genome is both circularly permuted and terminally redundant--unique features for an animal virus.
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PMID:The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant. 695 82


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