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)

Previous studies have demonstrated that the selective toxicity of leucyl-leucine methyl ester (Leu-Leu-OMe) for cytotoxic lymphocytes and myeloid cells is dependent on intracellular conversion to membranolytic metabolites by the acyl transferase activity of the granule enzyme dipeptidyl peptidase I (DPPI) that is enriched in these cells. The mechanism of cell death remained unclear, however, and was the subject of the experiments reported here. When human U937, HL60, or THP-1 myeloid tumor cell lines or murine CTLL-2 cells were treated with Leu-Leu-OMe, early release of both cytosolic 51Cr and soluble [3H]TdR labeled DNA fragments was observed, whereas antibody + C treatment of these cells caused only 51Cr release. Killing of U937 or THP-1 cells by incubation with the lysosomotropic amino acid methyl ester, Phe-OMe also induced only 51Cr release without evidence of DNA fragmentation. Preincubation with Zn2+, a known inhibitor of endonuclease activity prevented Leu-Leu-OMe-induced 51Cr or [3H]TdR release from these cell lines, but had no effect on antibody + C or Phe-OMe-induced 51Cr release. Zn2+ also prevented Leu-Leu-OMe-mediated killing of normal human CD16+ NK cells. Zn2+ had no inhibitory effect on Leu-Leu-OMe uptake or intracellular conversion to (Leu-Leu)n-OMe metabolites by these cell lines. Moreover, Zn2+ did not inhibit 51Cr release from nonnucleated E or nucleated U937 targets induced by extracellular production of DPPI-generated metabolites of Leu-Leu-OMe. Thus, killing of cytotoxic lymphocytes and myeloid cells by Leu-Leu-OMe appears to be dependent on generation of metabolites with membranolytic properties, but cell death induced by this process does not involve simple lysis of the plasma membrane. Rather, intracellular production of DPPI generated (Leu-Leu)n-OMe metabolites appears to trigger, an additional Zn(2+)-sensitive process that is associated with induction of apoptosis in cells with cytolytic potential.
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PMID:Apoptosis is induced in cells with cytolytic potential by L-leucyl-L-leucine methyl ester. 160 38

Hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis are closely related, congenital disorders of the red blood cell usually associated with defective spectrin self-association and abnormal limited tryptic digestion of the N-terminal of domain of spectrin. Enhanced cleavage by trypsin of spectrin from affected individuals at arginyl residue 45* and lysyl residue 48* frequently yields increased amounts of an alpha 1/74-Kd fragment at the expense of the normal alpha 1/80-Kd parent fragment. Limited tryptic digestion of three unrelated individuals with HPP showed the alpha 1/74 defect. To ascertain the molecular defect responsible for the abnormality, the structure of exon 2 of the alpha-spectrin gene was examined. Genomic DNA from the subjects was amplified by the polymerase chain reaction using primers flanking exon 2. Restriction endonuclease digestion of amplified products showed the loss of the HindIII site at codons 47 and 48 in one allele of subject 1 and abolished the AhaII site at codons 27 and 28 in one allele of subjects 2 and 3. Nucleotide sequence analysis of subcloned amplified DNA from the HPP subjects showed three novel amino acid substitutions. In subject 1 (a black individual), a single base substitution (AAG----AGG) at codon position 48 changes amino acid residue lysine to arginine. In subject 2 (a white individual), a single base substitution (CGT----AGT) at codon 28 changes arginine to serine. In subject 3 (a black individual), a different base substitution at position 28 (CGT----CTT) changes arginine to leucine. These mutations occur at positions of the alpha l domain where other mutations have also been described, indicating that the normal residues at these positions play an important role in spectrin dimer self-association and thus, in membrane stability.
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PMID:Heterogeneity of the molecular basis of hereditary pyropoikilocytosis and hereditary elliptocytosis associated with increased levels of the spectrin alpha I/74-kilodalton tryptic peptide. 187 97

Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.
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PMID:Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli. 200 77

Endonuclease V, a pyrimidine dimer specific endonuclease in T4 bacteriophage, is able to scan DNA, recognize pyrimidine dimer photoproducts produced by exposure to ultraviolet light, and effectively incise DNA through a two-step mechanism at the damaged bases. The interaction of endonuclease V with nontarget DNA is thought to occur via electrostatic interactions between basic amino acids and the acidic phosphate DNA backbone. Arginine-3 was chosen as a potential candidate for involvement in this protein-nontarget DNA interaction and was extensively mutated to assess its role. The mutations include changes to Asp, Glu, Leu, and Lys and deleting it from the enzyme. Deletion of Arg-3 resulted in an enzyme that retained marginal levels of AP specificity, but no other detectable activity. Charge reversal to Glu-3 and Asp-3 results in proteins that exhibit AP-specific nicking and low levels of dimer-specific nicking. These enzymes are incapable of affecting cellular survival of repair-deficient Escherichia coli after irradiation. Mutations of Arg-3 to Lys-3 or Leu-3 also are unable to complement repair-deficient E. coli. However, these two proteins do exhibit a substantial level of in vitro dimer- and AP-specific nicking. The mechanism by which the Leu-3 and Lys-3 mutant enzymes locate pyrimidine dimers within a population of heavily irradiated plasmid DNA molecules appears to be significantly different from that for the wild-type enzyme. The wild-type endonuclease V processively incises all dimers on an individual plasmid prior to dissociation from that plasmid and subsequent reassociation with other plasmids, yet neither of these mutants exhibits any of the characteristics of this processive nicking activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis of the T4 endonuclease V gene: the role of arginine-3 in the target search. 269 Sep 47

Nonclassic steroid 21-hydroxylase deficiency is a frequent, relatively mild disorder of cortisol biosynthesis characterized by variable signs of postnatal androgen excess. It is inherited as an allelic variant of the CYP21B gene encoding the 21-hydroxylase enzyme. CYP21B is located in the HLA histocompatibility complex, and a nonclassic allele is often associated with characteristic HLA antigens: B14;DR1. A CYP21B gene from a HLA-B14;DR1 homozygous patient with nonclassic 21-hydroxylase deficiency was cloned and analyzed. Five deviations from the normal sequence of CYP21B were found, but only one appeared likely to affect the functional integrity of the protein: codon 281, GTG, encoding valine, was changed to TTG, leucine. An oligonucleotide probe was constructed corresponding to the mutant sequence surrounding codon 281 and hybridized with DNA samples digested with restriction endonuclease Taq I. Samples from 8 nonclassic 21-hydroxylase deficiency patients carrying HLA-B14;DR1 contained a hybridizing fragment 3700 base-pairs long, indicating presence of the val-281 mutation in the CYP21B gene. In contrast, unaffected individuals and one patient who lacked HLA-B14;DR1 showed no evidence of the val-281 mutation in CYP21B. We conclude that the codon 281 mutation is a consistent molecular genetic marker for nonclassic 21-hydroxylase deficiency associated with HLA-B14;DR1.
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PMID:Clinical and genetic characterization of nonclassic 21-hydroxylase deficiency. 278 81

The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene. Transformants were selected on the medium lacking leucine. This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease. The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases. The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours. DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB. The genetic control of the recombinational repair of plasmid DNA DSB has been studied.
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PMID:[A model system for the study of repair of DNA double-strand breaks in Saccharomyces cerevisiae]. 282 6

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to a proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAH cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM- phenotype. Together with the other mutations recently reported in the PAH gene, the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.
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PMID:Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene. 284 Sep 52

To search for a genetic marker for type 2 Gaucher's disease (acute neuronopathic form), we compared the nucleotide sequence of a cloned glucocerebrosidase gene from a patient with Gaucher's disease with a normal gene. We found only a single base substitution (T----C) in exon X. This mutation results in the substitution of proline for leucine in position number 444 and produces a new cleavage site for the NciI restriction endonuclease. We analyzed NciI enzymatic digests of genomic DNA from 20 patients with type 1, 5 with type 2, and 11 with type 3 Gaucher's disease, and 29 normal controls for a restriction-fragment-length polymorphism (RFLP). Four of 5 patients with type 2 disease and all 11 with type 3 disease had at least one allele with the mutation. Two of 5 patients with type 2 disease and 7 of 11 with type 3 were homozygous for this mutation. Only 4 of 20 patients with type 1 Gaucher's disease had the mutant allele and were heterozygous for it. None of the 29 normal controls had the mutant allele. The high frequency of this mutation (444leucine----proline) in patients with neuronopathic Gaucher's disease, detectable by the NciI RFLP, may be of value in the identification of patients who will have the neurologic sequelae of Gaucher's disease.
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PMID:A mutation in the human glucocerebrosidase gene in neuronopathic Gaucher's disease. 288 Feb 91

A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH. A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D. radiodurans was subcloned into the EcoRI site of pAT153. The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D. radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D. radiodurans to repair proficiency with DNA derived from the gene library. Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153. The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60. The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62. None of the uvs genes introduced into the UV-sensitive E. coli CSR603 (uvrA-) was able to complement its repair defect. The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70. It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D. radiodurans genome.
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PMID:Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans. 298 93

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.
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PMID:Primary structure of colicin M, an inhibitor of murein biosynthesis. 303 84


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