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 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

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.
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PMID:Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding. 753 47

The catalytic center of the restriction endonuclease (ENase) EcoRI is structurally homologous to that of EcoRV, BamHI and PvuII. Each of these ENases contains a short motif of three to four amino acid (aa) residues which are positioned in a similar orientation to the scissile phosphodiester bond. We have mutated these aa (Pro90, Asp91, Glu111 and Lys113) in EcoRI to determine their individual roles in catalysis. The replacement of Asp91 and Lys113, respectively, by conservative mutations (Ala91, Asn91, Ala113, Gln113, His113 and Leu113) resulted in a reduction of binding affinity and complete loss of cleavage activity. Only Lys113-->Arg substitution still allows to cleave DNA, albeit with a rate reduced by at least four orders of magnitude. Lys113 seems to stabilize the structure of the wild-type (wt) ENase since all five ENase variants with mutations at this position show a strongly enhanced tendency to aggregate. The Ala and Gln mutants of Glu111 bind the recognition sequence slightly stronger than wt EcoRI and cleave it with a low, but detectable rate. Only the Glu111-->Lys mutant, in which the charge is reversed, shows neither binding nor cleavage activity. Pro90 is not important for catalysis, because the Ala90 mutant cleaves DNA with an only slightly reduced rate. Under star conditions, however, this mutant is even more active than wt EcoRI. Therefore, the charged aa Asp91, Glu111 and Lys113 are essential for catalytic activity of the EcoRI ENase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis in the catalytic center of the restriction endonuclease EcoRI. 760 70

We report a factor VII (FVII) variant, FVIIShinjo, characterized by normal FVII antigen levels and variable procoagulant activity using tissue thromboplastin from different sources. Normal FVII activity is obtained using human placenta thromboplastin but low activity using rabbit or bovine brain thromboplastin. Exons 2-8 and the intron-exon junctions of the FVII genes of the propositus were amplified by PCR from DNA extracted from peripheral white blood cells, and screened by single-strand conformational polymorphism (SSCP) analysis. DNA fragments showing aberrant mobility were cloned and sequenced. We detected a single-point mutation, a homozygous G to A transition at nucleotide position 6,055 in exon 4, which results in the substitution of Arg 79 by Gln in the first EGF-like domain. This mutation results in a loss of a site for the restriction endonuclease MspI. The Msp I digestion pattern of the PCR-amplified exon 3+4 fragments from each member of the family was determined. The Msp I haplotypes were consistent with this G to A transition being associated with reduced FVII activity as detected using thromboplastins from various species. We conclude that the Arg 79 to Gln substitution in the first EGF-like domain of FVII identified in the propositus is responsible for the inherited FVII abnormality in this Japanese family. We postulate that one of the sites of interaction between FVII and tissue thromboplastin includes Arg 79 in the first EGF-like domain of factor VII.
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PMID:Factor VIIShinjo: a dysfunctional factor VII variant homozygous for the substitution Gln for Arg at position 79. 760 84

We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wild-type or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg140 is crucial for the structure of an enzymatically active LCAT protein.
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PMID:A single G to A nucleotide transition in exon IV of the lecithin: cholesterol acyltransferase (LCAT) gene results in an Arg140 to His substitution and causes LCAT-deficiency. 760 41

A dramatic loss of glutamate transport has been observed in sporadic amyotrophic lateral sclerosis and has been postulated to contribute to the disease. Experimentally, this hypothesis was corroborated by mimicking the chronic loss of glutamate transport in postnatal rat spinal cord organotypic cultures through the use of glutamate transport inhibitors. This system is characterized by a relatively selective slow loss of ventral horn motor neurons resulting from glutamate transport inhibition. In this study, spinal cord organotypic cultures were used to test various drugs to evaluate their neuroprotective properties against this slow glutamate-mediated neurotoxicity The most potent neuroprotectants were drugs that altered glutamate neurotransmission, including non-NMDA receptor antagonists (GYKI-52466, PD144216, and PD13997) and drugs that could block presynaptic release or synthesis (riluzole and gabapentin). In addition, some antioxidants (U83836E and N-t-butyl-alpha-phenylnitrone) and inhibitors of nitric oxide synthesis (NG-monomethyl-L-arginine acetate) were modestly neuroprotective. The calcium endonuclease inhibitor aurintricarboxylic acid and the calcium release inhibitor dantrolene also provided partial motor neuron protection. However, several antioxidants and calcium channel antagonists had no excitotoxic neuroprotectant activity. This system provides a preclinical screening method for the burgeoning number of drugs postulated for clinical trials in motor neuron disease and a model to evaluate the mechanisms of chronic glutamate toxicity.
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PMID:Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity. 761 20

Constitutively active nitric oxide synthases (NOS) are a unique class of NADPH-dependent, calcium/calmodulin-dependent enzymes that catalyze the conversion of L-arginine to nitric oxide and L-citrulline. However, little is known about the molecular similarities or differences between the two prototypical constitutive NOS enzymes, endothelial NOS (ECNOS) and brain NOS (bNOS). The aims of this study were to begin characterizing the gene structure and tissue distribution of messenger RNAs (mRNAs) for ECNOS and bNOS and to examine the immunological resemblance of the proteins by Western blotting. Full-length complementary DNAs (cDNAs) encoding bovine ECNOS and rat bNOS hybridized, under high stringency, to different-sized fragments of endonuclease-digested bovine, rat, and human genomic DNA. In addition, more than one fragment was detected with both cDNAs, suggesting that ECNOS and bNOS genes contained multiple introns. Tissue distribution of ECNOS mRNA (4.4 kb) and bNOS mRNA (9.5 kb) in the rat was detected by Northern blotting. Patterns among tissue extracts were strikingly different, with ECNOS mRNA being most abundant in aorta, heart, lung, kidney, adrenal gland, spinal cord, and urogenital tissues and bNOS mRNA most prominent in brain regions, intestine, stomach, spinal cord, adrenal gland, and aorta. Interestingly, ECNOS cDNA detected two equally abundant RNA transcripts (4.4 and 4.0 kb) in most brain regions tested, suggesting an alternative splicing of the ECNOS pre-mRNA. Western blotting, using an ECNOS monoclonal antibody, recognized ECNOS protein from native bovine endothelial cells, cultured bovine endothelial cells, and COS cells transfected with ECNOS cDNA but did not recognize purified bNOS.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Genomic analysis and expression patterns reveal distinct genes for endothelial and brain nitric oxide synthase. 768 5

The Apn1 DNA repair enzyme of Saccharomyces cerevisiae acts on abasic sites and oxygen radical damages. Apn1 is homologous to the repair endonuclease IV of Escherichia coli, but the yeast protein is approximately 80 residues longer at the C terminus. The Apn1 C terminus is rich in basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins. We show here by indirect immunofluorescence that Apn1 is localized to the yeast nucleus. Mutant Apn1 proteins were engineered with progressive deletions inward from the C terminus. Elimination of just the last 12 residues from Apn1 (to yield Apn355) did not alter the stability in yeast cells or the in vitro activity of the enzyme. Greater truncation of Apn1 produced proteins of apparently lower (Apn334) or much lower (Apn315 and Apn293) in vivo stability. Both Apn355 and Apn334 failed to concentrate in the yeast nucleus and remained in the cytoplasm. These delocalized derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a delta apn1 strain. Apn355 and Apn334 complemented repair-deficient E. coli as effectively as did wild-type Apn1. Resistance to these DNA-damaging agents in yeast was restored if Apn355 and Apn334 (but not Apn315 or Apn293) were overproduced approximately 20-fold, which suggests either weak active transport or passive diffusion of these derivatives into the nucleus. Replacement of the C-terminal 12 residues of Apn1 with the nuclear targeting sequence of SV40 T-antigen did not restore effective function or nuclear localization in yeast.
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PMID:Intracellular localization of the Apn1 DNA repair enzyme of Saccharomyces cerevisiae. Nuclear transport signals and biological role. 769 Jul 56

A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N-benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsin-like and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.
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PMID:Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing. 773 79

The objectives of this study were to elucidate the structural organization of the gene for human deoxyribonuclease I (DNase I) and to identify the mutation site underlying its classical genetic polymorphism. In order to determine the organization of this gene, we utilized a combination of direct polymerase chain reaction (PCR)-amplification of human genomic DNA and isolation of the overlapping clones from a cosmid human genomic library. Restriction endonuclease mapping, Southern blotting and DNA sequencing showed that the DNase I gene was approximately 3.2 kilobases long, it comprised 9 (I-IX) exons separated by eight introns and its complete sequence was determined. The first exon contained only the non-translated sequences of mRNA. In addition to several putative regulatory elements, TATA-like and CAAT-like sequences were observed in the region upstream of the translation initiation codon. These results provide information that will help to understand the expression and regulation of DNase I. The isoelectric focusing patterns of human DNase I showed that it exhibits classical genetic polymorphism (Kishi et al. 1989, 1990). A comparison of the entire translated sequences of the DNase I gene from two pairs of individuals with common DNase I phenotypes 1 and 2 revealed only one nucleotide residue difference in exon VIII, A for phenotype 1 and G for phenotype 2, thus producing Gln and Arg amino acid substitutions respectively at position 222 from the NH2-terminus of the mature enzyme. The predicted charge changes attributable to these amino acid substitutions are entirely consistent with the isoelectric focusing profiles of these two DNase I isozymes. We conclude that this substitution is solely responsible for the classical polymorphism of DNase I protein.
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PMID:Structure of the human deoxyribonuclease I (DNase I) gene: identification of the nucleotide substitution that generates its classical genetic polymorphism. 776 78


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