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)

Bacteriophage P22 which are incapable of making functional tail protein can be propagated by the addition of purified mature tail protein trimers to either liquid or solidified medium. This unique in vitro complementation condition has allowed us to isolate 74 absolute lethal tail protein mutants of P22 after hydroxylamine mutagenesis. These phage mutants have an absolute requirement for purified P22 tail protein to be present in a soft agar overlay in order to form plaques and do not grow on any nonsense suppressing strains of Salmonella typhimurium. In order to genetically map and physically locate these mutations we have constructed two complementary sets of fine structure deletion mapping strains using a collection of Tn1 insertions in gene 9, the structural gene for the tail protein. Fourteen bacteriophage P22 strains carrying unique Tn1 transposon insertions (Ap phage) in gene 9 have been crossed with Ap phage carrying Tn1 insertions in gene 20. Phage carrying deletions that arose from homologous recombination between the Tn1 elements were isolated as P22 lysogens. The deletion prophage were shown to be missing all genetic information bracketed by the parental Tn1 elements and thus form a set of deletions into gene 9 from the 5' end of the gene. From the frequency of production of these deletion phage the orientation of the Tn1 insertions in gene 9 could be deduced. The genetic end points of the deletions in gene 9 and thus the order of Tn1 insertions were determined by marker rescue experiments using the original Ap phage. The genetic end points of the deletions in gene 20 were determined in similar experiments using nonsense mutations in gene 20. To locate the physical end points of these deletions in gene 9, DNA containing the Tn1 element has been cloned from each of the original Ap phage into plasmids. The precise point of insertion of Tn1 into gene 9 was determined by restriction enzyme mapping and DNA sequencing of the relevant portions of each of these plasmids. In vitro deletion of different 3' gene 9 sequences in the plasmid clones was accomplished through the use of unique restriction endonuclease sites in Tn1. The resulting plasmids form a set of deletions extending into the 3' end of the gene which are complementary compared to the deletion lysogens.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fine structure genetic and physical map of the phage P22 tail protein gene. 253 51

Twenty-seven new mutations in the structural gene for the Salmonella typhimurium bacteriophage P22 tailspike protein have been isolated, mapped using a powerful plasmid-based genetic system and their DNA sequence changes determined. The mutations were generated by hydroxylamine treatment of the cloned gene on a plasmid expression vector. Assaying the activity of the tailspike protein produced from this plasmid and screening for plasmid mutants were accomplished by the in situ complementation of P22 capsids imbedded in soft agar to produce infectious phage. Deletion mutations in the cloned gene have been constructed by a two step procedure involving oligonucleotide linker insertion and in vitro deletion by restriction endonuclease digestion. The deletions, whose physical endpoints were determined by DNA sequencing, define 12 genetic and physical intervals into which the new mutations were mapped by marker rescue experiments. These deletions were transferred to phage P22 by recombination and used to map mutations carried on plasmids. Following mapping, the nucleotide change for each of the mutations was determined by DNA sequencing. The majority were absolute missense mutations although both amber and ochre nonsense mutations were also identified in the protein coding portion of the gene. The suppression pattern of the nonsense mutations was determined on several nonsense suppressors. Four of the mutations cause severely depressed levels of tailspike protein expression from both the cloned gene on the plasmid expression vector and from P22 phage carrying these mutations. These mutations were identified as nucleotide changes in what is probably the P22 late operon transcription terminator which immediately follows the tailspike protein coding sequence.
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PMID:The isolation and sequence of missense and nonsense mutations in the cloned bacteriophage P22 tailspike protein gene. 256 56

EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers.
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PMID:Clustering of null mutations in the EcoRI endonuclease. 283 17

DNA from the Streptomyces plasmid pIJ702 was mutagenized in vitro using hydroxylamine and transformed into Streptomyces lividans. One plasmid with temperature-sensitive replication (pMT660) and one plasmid with a temperature-sensitive tyrosinase (pMT661) were isolated. The plasmid pMT661 contains a novel PstI restriction endonuclease site within the tyrosinase gene.
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PMID:Temperature-sensitive mutants of the Streptomyces plasmid pIJ702. 299 43

The endogenous poly(ADP-ribosyl)--nonhistone protein conjugates were isolated from dimethyl-sulfate-treated rat hepatoma AH 7974 cells using aminophenylboronic-acid--agarose chromatography. Seven major components could be discerned on dodecyl sulfate gels (molecular mass 43, 60, 66, 86, 100, 110 and 170 kDa) while control cells indicated only slight staining at above 200 kDa. The most abundant conjugate formed in response to alkylation damage was further purified using preparative gel electrophoresis and identified on the basis of its intrinsic enzymic activity as automodified poly(APD-ribose) synthase. In addition, topoisomerase I activity was found associated with a 60-kDa peptide. ADP-ribosylated endonuclease and actin were not detect-able. The purified conjugate fraction contained maximally 8.8 nmol/mg ADP-ribose and 7.9 nmol/mg oligo(ADP-ribose) with a mean chain length of 2.3 residues. The modifying (ADP-ribosyl)n groups were attached to its acceptors by a hydroxylamine-insensitive bond and had practically no effect on the DNA affinity of either poly(ADP-ribose) synthase or topoisomerase I.
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PMID:Poly(ADP-ribose) synthase is the major endogenous nonhistone acceptor for poly(ADP-ribose) in alkylated rat hepatoma cells. 312 14

The work was aimed at studying the capability of chemical mutagens, such as hydroxylamine (HA), dimethyl sulfate (DMS) and nitrosomethylurea (NMU), to produce mutants of Serratia marcescens with an elevated synthesis of endonuclease. HA and DMS did not induce these mutations at tested concentrations. NMU caused such mutations resulting in a sharp rise of endonuclease production. The mutagen was added to the medium at different periods of synchronized DNA replication to generate a selective effect on certain genes located in the replication sites. A 15-minute period 40 min after the beginning of the lag-phase was shown to be most sensitive to NMU treatment for producing mutants with a high yield of endonuclease. A mutant of S. marcescens was obtained, which synthesized endonuclease 40-100 times as effective as the parent strain did. The ability of S. marcescens endonuclease to hydrolyze RNA and DNA and to inhibit the reproduction of RNA- and DNA-containing viruses is of practical importance.
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PMID:[Isolation of Serratia marcescens mutants superproducers of endonuclease by exposure to nitrosomethylurea in a synchronized culture]. 632 16

Colicin N was identified as the 39,000-molecular-weight protein encoded by the 4,900-base-pair, multiple copy number, amplifiable plasmid ColN -284. Its production was controlled by the SOS regulatory circuit and by catabolite repression. Colicin accumulated intracellularly to ca. 10(6) molecules per cell after growth for 2 to 3 h in medium containing 0.5 microgram of mitomycin C per ml and was then released as the cells underwent partial lysis. Strains carrying pColN -284 and its derivatives exhibited low-level immunity to colicin N and were fully sensitive to all other colicins tested. Regions of the plasmid responsible for colicin N activity (cna), for mitomycin-induced lysis ( cnl ), and for colicin N immunity ( cni ) were localized and characterized by cloning, transposon Tn5 and hydroxylamine mutagenesis, and restriction endonuclease deletion and mapping analysis. The results are discussed in terms of both the organization of the cna, cnl , and cni genes and the respective role of cnl expression and colicin N production in mitomycin sensitivity, colicin export, and induced partial lysis of ColN + cells.
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PMID:Genetic analysis of ColN plasmid determinants for colicin production, release, and immunity. 632 9

Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antisense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E. coli carrying the aadA gene construct, and transformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.
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PMID:Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease. 790 39

The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.
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PMID:Dominant negative mutator mutations in the mutL gene of Escherichia coli. 869 87

BsoBI is a type II restriction enzyme found in Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5'-CYCGRG-3' (Y=C or T; R=A or G) and cleaves between the first and second base to generate a four-base 5' extension. The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695-699]. Here we report the overexpression of BsoBI restriction endonuclease gene in E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive expression of BsoBI from pRRS is lethal to E. coli in the absence of the cognate methylase. The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.
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PMID:Overexpression of BsoBI restriction endonuclease in E. coli, purification of the recombinant BsoBI, and identification of catalytic residues of BsoBI by random mutagenesis. 909 56


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