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 nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector. The runaway derivative pBEU17 carries the promoter-proximal portion of the E. coli alanyl-tRNA synthetase gene and proved useful for expressing cloned genes lacking their native transcription initiation signals. The alaS promoter is located approximately 500 base pairs upstream of a single BamHI restriction endonuclease cleavage site utilized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product. After 5-h thermal induction of cells carrying the runaway recombinant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells. The high concentration of protein B2 in crude extracts of induced cells has enabled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations. Protein B2 produced from pBS1 is identical to the chromosomally encoded nrdB product of E. coli as regards molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity, tyrosine radical content, and structure of the binuclear iron center. Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical. They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine.
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PMID:Overproduction and purification of the B2 subunit of ribonucleotide reductase from Escherichia coli. 300 19

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced.
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PMID:Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase. 302 93

Mutants of Escherichia coli K-12 deficient in glutaredoxin were isolated and partially characterized. The mutants have detectable but significantly reduced glutaredoxin activity in assays of whole cells made permeable with ether as well as in assays of crude extracts coupled to ribonucleotide reductase. In vivo, the mutants appear to be deficient in both sulfate and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide reductase and adenosine 3'-phosphate 5'-phosphosulfate reductase. Complementation of the mutant phenotype by transformants was used to clone the wild-type glutaredoxin allele. The transformants had a high level of glutaredoxin activity and contained a plasmid with an insert that had a restriction endonuclease pattern identical to that predicted by the DNA sequence for glutaredoxin determined by Hoog et al. (J.-O. Hoog, H. von Bahr-Lindstrom, H. Jornvall, and A. Holmgren, Gene 43:13-21, 1986).
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PMID:Isolation and characterization of an Escherichia coli K-12 mutant deficient in glutaredoxin. 327 17

Restriction enzyme digests of DNA from 22 unselected isolates of EHV-1 were analysed by hybridization with cloned DNA fragments covering the genome. In addition to a small amount of inter-strain variation, heterogeneity within strains was observed, caused by loss of specific restriction endonuclease sites in the DNA of a proportion of the virus particles of any one stock. Fifteen strains demonstrated the same intra-strain variation involving loss of the BamHI L-M site which was shown to lie within coding sequence for the large subunit of ribonucleotide reductase. This particular mutation may therefore be selected for by passage in RK13 cells.
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PMID:Inter- and intra-strain genomic variation in equine herpesvirus type 1 isolates. 827 52

Analysis of 76 kb of newly sequenced DNA, located between map positions 182 and 258 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 175 open reading frames (ORFs) of 65 codons or longer. One hundred and five of these 175 ORFs were considered major ORFs. Twenty-one of the 105 major ORFs resembled proteins in databases including ribonucleotide reductase small subunit, RNase III, thioredoxin, glutaredoxin, protein disulfide isomerase, deoxynucleoside kinase, frog virus 3 ATPase, Acetobacter cellulose synthase, a bacteriophage encoded endonuclease, and two C-5 cytosine DNA methyltransferases. One of the ORFs was the PBCV-1 major capsid protein. The 105 major ORFs were evenly distributed along the genome. One set of ORFs was separated by 543 nucleotides whereas 75 of the ORFs were separated by fewer than 100 nucleotides. Nineteen of the 175 ORFs resembled other PBCV-1 ORFs, suggesting that they represent either gene duplications or gene families.
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PMID:Analysis of 76 kb of the chlorella virus PBCV-1 330-kb genome: map positions 182 to 258. 880 66

This report describes a simple and efficient system for construction of recombinant pseudorabies (Aujeszky's disease) virus (PrV) which is based on the use of a unique restriction site inserted into the viral genome. This system enables the recovery of genetically modified viruses without screening or selection for a specific phenotype, since practically all mature viral particles obtained carry the foreign sequences. To demonstrate, we introduced the tumour suppressor protein-53 (p53) gene into two different intergenic locations of PrV: the ribonucleotide reductase (rr) gene and the promoter of a putative latency gene (PLAT), located at the inverted repeat (IR) region of the viral genome. As a first step, we engineered a unique EcoRI recognition site into the rr gene or into both copies of PLAT with the help of marker transfer using the bacterial lacZ gene. Then, in both cases viral DNAs were cut with the restriction endonuclease EcoRI followed by treatment with calf intestinal phosphatase and used for cotransfection into porcine kidney cells with a plasmid containing the p53 gene flanked by viral DNAs homologous to the target region. As a result of this process, in most of the experiments, we obtained recombinant viruses without the background of parental viruses. Here we show that this method can be used for directional insertion of exogenous sequences into either the unique or the IR region of the PrV chromosome. In principle, this system should be applicable to the construction of recombinant derivatives of any viruses having infectious DNA.
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PMID:A restriction cleavage and transfection system for introducing foreign DNA sequences into the genome of a herpesvirus. 960 3

PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing endonucleases and provides some of the catalytic residues necessary for DNA cleavage activity. Site-specific mutagenesis studies showed that two acidic residues in the motifs, Asp149 and Glu250 in PI- Pfu I, and Asp156 and Asp249 in PI- Pfu II, were critical for catalysis. The third residues of the active site triads, as predicted from the structure of PI- Sce I, were Asn225 in PI- Pfu I and Lys224 in PI- Pfu II. Substitution of Asn225 in PI- Pfu I by Ala did not affect catalysis. The cleavage activity of PI- Pfu II was 50-fold decreased by the substitution of Ala for Lys224. The binding affinity of the mutant protein for the substrate DNA also decreased 6-fold. The Lys in PI- Pfu II may play a direct or indirect role in catalysis of the endonuclease activity.
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PMID:PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis. 1051 8

White spot syndrome virus (WSSV) is a taxonomically unclassified virus which causes a disease in shrimps worldwide. A 936 bp long open reading frame (ORF) was found on a 7.2 kb HindIII fragment of the DNA genome of WSSV located adjacent to the ribonucleotide reductase small subunit gene. This putative ORF showed homology to prokaryotic and eukaryotic endonucleases, which contain a non-specific endonuclease motif. Alignment with viral and eukaryotic endonuclease ORFs revealed that most catalytically and structurally important amino acid residues were present in the putative WSSV non-specific endonuclease gene. An unrooted parsimonous phylogenetic tree of non-specific endonucleases indicated that the WSSV ORF was located in a well bootstrap supported clade containing only arthopods, including one of WSSV's natural hosts, Penaeus japonicus. A similar conjunction was found for the only other viral homologue, present in Fowlpox virus, which was also found in a well bootstrap-supported clade with its natural host, Gallus gallus. This clustering of virus and host suggests that both WSSV and Fowlpox virus may have acquired their nuclease genes from their respective natural hosts. Because the motif for non-specific nucleases is found in only two viruses, this gene cannot be used to clarify the taxonomic position of WSSV. However, the presence of this type of nuclease rarely found in viruses adds a novel feature to WSSV.
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PMID:Identification and phylogeny of a non-specific endonuclease gene of white spot syndrome virus of shrimp. 1177 1

A bacterial ribonucleotide reductase gene was found to encode four inteins and three group II introns in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The 13,650-bp ribonucleotide reductase gene is divided into eight extein- or exon-coding sequences that together encode a 768-amino acid mature ribonucleotide reductase protein, with 83% of the gene sequence encoding introns and inteins. The four inteins are encoded on the second half of the gene, and each has conserved sequence motifs for a protein-splicing domain and an endonuclease domain. These four inteins, together with known inteins, define five intein insertion sites in ribonucleotide reductase homologues. Two of the insertion sites are 10 amino acids apart and next to key catalytic residues of the enzyme. Protein-splicing activities of all four inteins were demonstrated in Escherichia coli. The four inteins coexist with three group II introns encoded on the first half of the same gene, which suggests a breakdown of the presumed barrier against intron insertion in this bacterial conserved protein-coding gene.
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PMID:Four inteins and three group II introns encoded in a bacterial ribonucleotide reductase gene. 1297 59

In the present study, the existence of white spot syndrome virus (WSSV) in blue crab (Callinectes sapidus) collected from 3 different American coastal waters (New York, New Jersey, and Texas) was confirmed by 2-step diagnostic polymerase chain reaction and in situ hybridization analysis. When geographic isolates were also compared using a gene that encodes the WSSV ribonucleotide reductase large subunit RR1 (WSSV rr1), a C(1661)-to-T point mutation was found in the New Jersey WSSV isolated. This point mutation, which resulted in the creation of an additional RsaI endonuclease recognition site, was not found in the WSSV from the New York and Texas blue crab samples, or in the WSSV Taiwan isolate, or in any of the other WSSV geographical isolates for which data are available. WSSV rr1-specific RsaI amplified restriction fragment length polymorphism of an amplified 1156-bp fragment thus distinguished the New Jersey blue crab samples from the other WSSV isolates.
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PMID:Sequencing and amplified restriction fragment length polymorphism analysis of ribonucleotide reductase large subunit gene of the white spot syndrome virus in blue crab (Callinectes sapidus) from American Coastal Waters. 1496 79


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