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)

Twenty-two infectious bursal disease virus (IBDV) strains were examined using the reverse transcriptase/polymerase chain reaction-restriction endonuclease (RT/PCR-RE) assay. A 394-bp fragment of the VP2 gene was amplified and tested for six different restriction enzyme sites. Although the EcoRII enzyme was used in previous RT/PCR-RE assays, results obtained using the isoschizomer BstNI were more consistent because its activity does not rely on multiple restriction sites. Ten different RT/PCR-RE profiles were observed. IBDV strains previously reported to be variant type viruses were BstNI and StyI negative except variant strain IN. The Bursine, Bursine-2, Bursine-Plus and Bio-Burs viruses were BstNI negative. The presence of a second enzyme site, StyI, was observed in these viruses and could be used to differentiate them from the known variant viruses, which were StyI negative. Nucleotide sequence data also indicated that these viruses were not identical to variant or classic type viruses. The base substitution observed in the BstNI site of Bio-Burs and Bursine-2 was responsible for changing the amino acid at position 222 to serine. The amino acid at this position has been reported to influence a neutralizing epitope on VP2. Three IBDV strains were examined after propagation in different hosts. The RE profiles of the STC and MD IBDV strains did not change after propagation in either BGM-70 cell culture or chicken bursas, whereas the Del-A RE profile changed at the Sau3AI site after adaptation to BGM-70 cell culture. This site has not been associated with antigenic or other phenotypic characteristics of IBDV.
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PMID:Molecular identification of infectious bursal disease virus strains. 908 25

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. In PAHENU1, the phenotype is mild. The Pahenu1 mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. In PAHENU2, the phenotype is severe. The Pahenu2 mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. In PAHENU2, the sequence information was used to devise a direct genotyping system based on the creation of a new Alw26I restriction endonuclease site.
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PMID:Characterization of mutations at the mouse phenylalanine hydroxylase locus. 911 79

The human leukemia cell line, HL60 is very sensitive to various apoptotic stimuli and p53-null. The death-related cysteine proteases of the caspases family play a central role in the execution phase of apoptosis, and we recently reported the importance of serine protease activation in camptothecin-induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CED-3-related cysteine proteases) and serine proteases in cell death induced by the topoisomerase I inhibitor, camptothecin, in HL60 cells and in a cell-free system. We found that CPP32 is activated during camptothecin-induced apoptosis, and that N-benzyloxycarbony-Val-Ala-Asp (O-methyl) -fluoromethyketone (Z-VAD-fmk), a cell permeable caspase inhibitor blocks all features of apoptosis: morphological changes, cleavage of caspase 3 (CPP32/Yama/Apopain) and poly(ADP-ribose) polymerase, lamin B degradation and DNA fragmentation. However, Z-VAD-fmk and two other ICE/CED-3 inhibitors, YVAD-CHO and DEVD-CHO, were inactive in a cell-free system reconstituted from nuclei of untreated HL60 cells and cytosol from camptothecin-treated cells, suggesting that caspases are not required for endonuclease activation or lamin B cleavage in the cell-free system. By contrast, the serine protease inhibitors, 3,4-dichloroisocoumarin (DCI) and L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone (TPCK), abolished the apoptosis-associated biochemical changes induced by camptothecin both in whole cells and in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32 and serine proteases are activated in camptothecin-induced apoptosis.
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PMID:Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases. 926 76

According to the crystal structure of Cfr10I restriction endonuclease the acidic residues D134, E71 and E204 are clustered together and presumably chelate metal ion(s) at the active site. Indeed, investigation of the DNA cleavage properties of substitutional mutants of Cfr10I D134A, E71Q, E71A and E204Q reveals that D134, E71 and E204 residues are essential for cleavage activity, supporting their active site function. Structural comparison indicates that the D134 residue of Cfr10I spatially overlaps with aspartate residues D91 and D74, from the invariant active site motifs 90PDX19EAK and 73PDX15DIK of EcoRI and EcoRV, respectively. However, structural studies in conjunction with mutational analyses suggest that the sequence motif 133PDX55KX13E corresponds to the active site of Cfr10I, but differs from canonical active site motifs of EcoRI and EcoRV. According to the crystal structure of Cfr10I the serine S188 residue from the 188SVK sequence motif is a spatial equivalent of the acidic residue from the (E/D)XK-part of the active site motif, which is conserved between EcoRI and EcoRV. Site-directed mutagenesis experiments of Cfr10I, however, revealed that the S188 was not so important for catalysis while the E204 residue located 2.8 A away indeed was essential for cleavage, suggesting that the glutamate E204 rather than the S188 residue contributes to the metal binding site in Cfr10I. In addition, model-building studies suggest that mutual interchange of the E204 and S188 residues should lead only to minor positional differences of the carboxylate residues of glutamate side-chains. The double mutant S188E/E204S was therefore prepared by site-directed mutagenesis where the active site motif 133PDX55KX13E of Cfr10I was changed to a canonical motif 133PDX53EVK, which is similar to that of EcoRI and EcoRV. Interestingly, the double mutant S188E/E204S of Cfr10I with redesigned active site structure, exhibited 10% of Wt cleavage activity in a gamma DNA cleavage assay. Thus, structure guided redesign of the catalytic/metal binding site of Cfr10I, provides novel experimental evidence to suggest that spatial rather than sequence conservation plays the dominant role in the formation of restriction enzyme active sites.
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PMID:Structure-based redesign of the catalytic/metal binding site of Cfr10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues. 964 51

To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp. KNK712 was mutagenized randomly to increase its thermostability. The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli. The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates. Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5 degrees C compared with those of the native enzyme. As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.
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PMID:Increase in thermostability of N-carbamyl-D-amino acid amidohydrolase on amino acid substitutions. 980 66

The amino acid residue Asn141 of the restriction endonuclease EcoRI was proposed to make three hydrogen bonds to both adenine residues within the recognition sequence -GAATTC-. We have mutated Asn141 to alanine, aspartate, serine, and tyrosine. Only the serine mutant is active under normal buffer conditions although 1000-fold less than wild-type EcoRI. The alanine and aspartate mutants can be activated by Mn2+. At acidic pH the latter mutant becomes even more active than the wild-type enzyme in the presence of Mn2+. We conclude that Asn141 is essential for DNA recognition and that serine can partly substitute it.
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PMID:Asn141 is essential for DNA recognition by EcoRI restriction endonuclease. 982 60

Mycoplasma infection may lead to various pathologies in a broad range of hosts. It has been shown that Mycoplasma may trigger cell death in cell cultures; however, the mechanism remains unknown. In the present paper we show that Mycoplasma infection of different lymphocyte and epithelial tumour cell lines leads to the inhibition of proliferation, and increased cell death, accompanied by DNA fragmentation and the morphological features of apoptosis. We also showed that this infection leads to an increased sensitivity of cells to various inducers of apoptosis targeting different signalling pathways. Finally, we show that increased apoptosis is associated with overexpression of an endonuclease produced by Mycoplasma. This endonuclease is recovered in the nuclear fraction of host cells, introduces mostly DSB and is active at neutral pH in the presence of divalent cations. Activation of this endonuclease is connected with limited proteolysis, which may be reproduced in vitro by snake venom serine proteinase.
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PMID:Mycoplasma infection can sensitize host cells to apoptosis through contribution of apoptotic-like endonuclease(s). 989 30

Microinjection of the restriction endonuclease HaeIII, which causes DNA double-strand breaks with blunt ends, induces nuclear accumulation of p53 protein in normal and xeroderma pigmentosum (XP) primary fibroblasts. In contrast, this induction of p53 accumulation is not observed in ataxia telangiectasia (AT) fibroblasts. HaeIII-induced p53 protein in normal fibroblasts is phosphorylated at serine 15, as determined by immunostaining with an antibody specific for phosphorylated serine 15 of p53. This phosphorylation correlates well with p53 accumulation. Treatment with lactacystin (an inhibitor of the proteasome) or heat shock leads to similar levels of p53 accumulation in normal and AT fibroblasts, but the p53 protein lacks a phosphorylated serine 15. Following microinjection of HaeIII into lactacystin-treated normal fibroblasts, lactacystin-induced p53 protein is phosphorylated at serine 15 and stabilized even in the presence of cycloheximide. However, neither stabilization nor phosphorylation at serine 15 is observed in AT fibroblasts under the same conditions. These results indicate the significance of serine 15 phosphorylation for p53 stabilization after DNA double-strand breaks and an absolute requirement for ATM in this phosphorylation process.
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PMID:Requirement of ATM in phosphorylation of the human p53 protein at serine 15 following DNA double-strand breaks. 1008 48

To continue elucidation of the biochemical and molecular pathways involved in the induction of apoptosis in granulosa cells (GC) of ovarian follicles destined for atresia, we characterized the occurrence and protease modulation of high and low molecular weight (MW) DNA fragmentation during rat GC death. Atresia of ovarian follicles, occurring either spontaneously in vivo or induced in vitro, was associated with both high MW and internucleosomal (low MW) DNA cleavage. Incubation of follicles in the presence of a putative irreversible and non-competitive inhibitor of caspase-1 (interleukin-1beta-converting enzyme or ICE), sodium aurothiomalate (SAM), completely prevented internucleosomal, but not high MW, DNA cleavage. As reported previously, morphological features of apoptosis (pyknosis, cellular condensation) and atresia (granulosa cell disorganization, oocyte pseudomaturation) remained detectable in SAM-treated follicles. The potential involvement of proteases in endonuclease activation was further analyzed in cell-free assays using nuclei from both GC (which autodigest their DNA) and HeLa cells (HC, which do not autodigest their DNA unless incubated with extracts prepared from other cell types). Crude cytoplasmic extracts prepared from GC induced both high MW and internucleosomal DNA cleavage in HC nuclei. The induction of low, but not high, MW DNA cleavage in HC nuclei by GC extracts was suppressed by pretreatment of the extracts with SAM or with any one of the serine protease inhibitors, dichloroisocoumarin (DCI), N-tosyl-L-leucylchloromethylketone (TLCK) or N-tosyl-L-phenylchloromethylketone (TPCK). Interestingly, SAM and DCI also prevented cation-induced low MW DNA fragmentation in GC nuclei; however, TLCK and TPCK were without effect. Our results support a role for cytoplasmic and nuclear serine proteases in the activation of the endonuclease(s) responsible for internucleosomal DNA cleavage during apoptosis.
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PMID:High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays. 1020 Apr 44

The restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation C134S was purified to homogeneity but appeared to be very unstable. In contrast, the C167S mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the C167S BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease.
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PMID:The amino acidic substitution of cysteine 167 by serine (C167S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability. 1038 8


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