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)

High-molecular-weight DNA is known to collapse into very compact particles in a salt solution containing polymers like poly(ethylene oxide) [(EO)n] or polyacrylate. The biological relevance of this phenomenon is suggested by our recent finding that high concentrations of the highly acidic internal peptides found in the mature T4 bacteriophage head, as well as poly(glutamic acid) and poly(aspartic acid), can collapse DNA in a similar manner. The structure of DNAs collapsed by various methods has been studied with electron microscope. We find (EO)n collapses T4 or T7 bacteriophage DNA into compact particles only slightly larger than the size of the T4 and T7 head, respectively. In contrast, polylysine collapses DNA into different types of structures. Double-stranded DNA collapsed with (EO)n is cut by the single-strand specific Neurospora crassa endonuclease (EC 3.1.4.21) into small fragments. Extensive digestion only occurs above the critical concentration of polymer required for DNA collapse, demonstrating the (EO)n-collapsed DNA contains enzyme-vulnerable regions (probably at each fold), which are preferentially attacked. The size of the DNA fragments produced by limit-digestion with the nuclease ranges between 200 and 400 base pairs when DNA is collapsed by (EO)n. Only fragments of DNA which are larger than 600 base pairs are cut by the endonuclease in (EO)n-containing solution.
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PMID:Characterization of DNA condensates induced by poly(ethylene oxide) and polylysine. 106 Jan 8

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strains so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich in glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that the Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.
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PMID:The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages. 136

Initial amplification and sequencing of a 366-bp fragment of the cytochrome b gene by a conserved primer pair (MVZ 03 and MVZ 04) revealed a nonfunctional copy of the gene with two deletions (one of which is 17 bp in length and the other of which is 3 bp in length) in Chroeomys jelskii, a South American akodontine rodent. By means of an alternative primer to MVZ 03--namely, MVZ 05--from the region of the tRNA for glutamic acid, a functional copy of cytochrome b was subsequently amplified. Both primer pairs amplify functional sequence when applied to purified mitochondrial DNA (mtDNA). Restriction-endonuclease digestion of purified mtDNA from C. jelskii did not reveal any additional sets of bands that would suggest heteroplasmy in the mitochondrial genome. When probed with both functional and nonfunctional gene fragments, MboI restriction digests revealed the same pattern, providing further evidence that the nonfunctional copy must be located in the nucleus. Observed differences in the mitochondrial and nuclear sequences from two populations are consistent with a faster rate of change in mtDNA than in nuclear DNA.
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PMID:Mitochondrial DNA-like sequence in the nuclear genome of an akodontine rodent. 156 Jul 58

An analog of the alpha-human atrial natriuretic polypeptide (alpha-hANP) gene, articulated with a peptidase inhibitor SQ20881 at its N-terminal and two prolines at the C-terminal was expressed in E. coli by cloning the reconstituted plasmid pRHL-1 in vivo. This gene analog, RH-1, comprising 154 base pairs in total, was designed to contain an equivalent of the alpha-hANP gene, capping the peptidase inhibitor SQ20881 at its 5' end with a glutamic acid codon GAA to facilitate enzymatic cleavage of the expressed end product by endoproteinase Glu-C, wedging in two proline codons CCG & CCG before the double terminal codons TGA TAG at the 3' end to retard hydrolysis of the expressed product by exopeptidase, and adding 3 restriction sites to both ends. Synthesis of the RH-1 gene was effected enzymatically by joining in predicted order the ten segments of oligodeoxynucleotides which had been chemically synthesized by the solid-phase phosphite-triester method. The synthetic gene was cloned into vector M13mp18. Phage bearing the gene analog was identified by dot blotting and restriction endonuclease mapping. Nucleotide sequence of the gene was determined by the dideoxynucleotide chain termination method.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[A study of protein engineering for human cardionatrin. I. Synthesis, cloning and expression of a gene analog of human atrial natriuretic polypeptide in E. coli]. 214 Jul 17

The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and arginine 200, and major groove base moieties are the molecular determinants of specificity. We have investigated residue 144 using aspartate and glutamine substitutions introduced by site-directed mutagenesis. Substitution with glutamine results in a null phenotype (at least a 2000-fold reduction in activity). On the other hand, the aspartic acid mutant (ED144) retained in vivo activity. Substrate binding and catalytic studies were done with purified ED144 enzyme. The affinity of the ED144 enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about 50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme, the ED144 enzyme dissociates after the first strand cleavage. Partitioning between cleavage and dissociation at the first and second cleavage steps for the ED144 enzyme is extremely salt-sensitive. The altered partitioning results largely from a destabilization of the enzyme-DNA complex, particularly the enzyme-nicked DNA complex, with only small changes in the respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they appear to act cooperatively with other specificity contacts to stabilize the enzyme-DNA complex.
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PMID:Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements. 225 11

Virulent strains of Bacillus anthracis contain two large plasmids. pXO1 encodes the three component protein exotoxin and pXO2 is necessary for synthesis of the poly-D-glutamic acid capsule. A procedure for the isolation of these plasmids which yields high quantities of pure DNA is described. Restriction endonuclease analysis of these plasmids shows that they are not related. pXO1 is 174 kilobase pairs and pXO2 is 95 kilobase pairs. From their bouyant densities and melting temperatures we also determined their GC contents. pXO1 contains 31.1% GC base pairs and pXO2 is 31.4% GC. Both of these values are close to the GC content of B. anthracis genomic DNA which is 32.2%.
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PMID:Purification and physical analysis of Bacillus anthracis plasmids pXO1 and pXO2. 312 34

A mouse L-cell line, designated 111-OB3, is described which is resistant to two drugs, chloramphenicol and oligomycin. The cells contain two types of mitochondrial DNA molecules, in roughly equal proportions, which differ in that one is cleaved by endonuclease EcoRI at a novel site within the coding sequence for subunit 6 of the mitochondrial ATPase (ATPase-6). Sequence analysis reveals that the cleavage site was created by a single transversion which predicts a replacement of valine in the wild-type ATPase-6 by glutamic acid. The replacement occurs in a hydrophobic amino acid sequence which is highly conserved in mouse, human, and bovine proteins. The position of the replacement is similar to a substitution observed in one class of yeast mutants resistant to oligomycin. Both of the mitochondrial DNA molecules in 111-OB3 also have a single nucleotide change in the gene encoding the large (16S) rRNA. These observations are consistent with the hypothesis that oligomycin resistance in mammalian cells can be cytoplasmically determined and can result from alterations in ATPase-6. The appearance of the mutation before selection in oligomycin suggests a model for the origin of mitochondrial mutations in mammalian cells.
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PMID:Sequence analysis of mitochondrial DNA in a mouse cell line resistant to chloramphenicol and oligomycin. 622 6

The VirD2 polypeptide from Agrobacterium tumefaciens, in the presence of VirD1, introduces a site- and strand-specific nick at the T-DNA borders. A similar reaction at the origin of transfer (oriT) of plasmids is essential for plasmid transfer by bacterial conjugation. A comparison of protein sequences of VirD2 and its functional homologs in bacterial conjugation and in rolling circle replication revealed that they share a conserved 14 residue segment, HxDxxx(P/u)HuHuuux [residues 126-139 of VirD2; Ilyina, T.V. and Koonin, E.V. (1992) Nucleic Acids Res. 20, 3279-3285]. A mutational approach was used to test the role of these residues in the endonuclease activity of VirD2. The results demonstrated that the two invariant histidine residues (H133 and H135) are essential for activity. Mutations at three sites, histidine 126, aspartic acid 128 and aspartic acid 130, that are conserved in a subfamily of the plasmid mobilization proteins, led to the loss of VirD2 activity. Aspartic acid at position 130, could be substituted with glutamic acid and to a much lesser extent, with tyrosine. In contrast, another conserved residue, asparagine 139, tolerated many different amino acid substitutions. The non-conserved residues, arginine 129, proline 132 and leucine 134, were also found to be important for function. Isolation of null mutations that map throughout this conserved domain confirm the hypothesis that this region is essential for function.
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PMID:Mutational analysis of a conserved motif of Agrobacterium tumefaciens VirD2. 747 69

The PI-SceI endonuclease from yeast belongs to a protein family whose members contain two conserved dodecapeptide motifs within their primary sequences. The function of two acidic residues within these motifs, Asp218 and Asp326, was examined by substituting alanine, asparagine, and glutamic acid residues at these positions. All of the purified mutant proteins bind to the PI-SceI recognition site with the same affinity and specificity as the wild-type enzyme. By contrast, substituting alanine or asparagine amino acids at the two positions completely eliminates strand cleavage of substrate DNA, whereas substitution with glutamic acid markedly reduces the cleavage activity. Experiments using nicked substrates demonstrate that the wild-type enzyme shows no strand preference during cleavage. These results are consistent with a model in which both acidic residues are part of a single catalytic center that cleaves both DNA strands. Furthermore, substrate binding by wild-type PI-SceI stimulates hydroxyl radical or hydroxide ion attack at the cleavage site while binding by the alanine-substituted proteins either stimulates this attack significantly less or protects the DNA at this position. These finding are discussed in terms of possible reaction mechanisms for PI-SceI-mediated endonucleolytic cleavage.
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PMID:Substitutions in conserved dodecapeptide motifs that uncouple the DNA binding and DNA cleavage activities of PI-SceI endonuclease. 789 Jul 14

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.
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PMID:DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect. 792 64


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