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 gene coding for the secreted Serratia marcescens endonuclease was fused with the mannopine synthase promoter of Agrobacterium tumefaciens Ti plasmid and transferred to Nicotiana tabacum SR1 plants. The promoter is leaf- and root-specific. The resulting transgenic plants demonstrated elevated nuclease activity. The level of the transgene product was determined in the transgenic lines.
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PMID:[Transgenic tobacco (Nicotiana tabacum SR1) plants expressing the gene coding for Serratia marcescens nuclease]. 1189 19

The human DNA fragmentation factor (DFF) is a heterodimer of 40 kD and 45 kD subunits. The 40 kD subunit (DFF40) has an intrinsic DNase activity responsible for the genomic DNA degradation into nucleosomal fragments during apoptosis. As an inhibitor for DFF40, the 45 kD subunit (DFF45) complexes with DFF40, inhibiting DNase activity until certain apoptosis signals are received. In cells undergoing apoptosis, the cleavage of DFF45 by activated caspase-3 frees DFF40from the complex and initiates the apoptosis-specific DNA fragmentation. In this report, the coding region of human DFF45 gene was amplified from the total RNA of HeLa cells by RT-PCR. The resulting 1 kb DNA fragment was cloned into the bacterial expression vector pET-28a(+) with a 6xhistidine tag fused to the N-terminus of DFF45, generating plasmid pET28a-DFF45, which was then used to transform E.coli BL21(DE3). Induced by IPTG, the recombinant DFF45 was expressed efficiently with a yield of 56.6% of total bacterial proteins. The product was purified to homogeneity through a nickel affinity column, followed by heat treatment, and approximately 4--6 mg of DFF was purified from 100 ml culture. Purified recombinant human DFF45, added into the apoptotic cell-free system of Xenopus egg extracts, could effectively inhibit both the digestion of lambdaDNA and the degradation of chromosomal DNA into nucleosomal fragments in the nuclei of chicken red blood cells. Our results demonstrated the existence of an apoptosis-specific endonuclease in this cell-free system, the activity of which could be inhibited by recombinant human DFF45.
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PMID:Recombinant Human DFF45 Inhibits Apoptosis-specific Endonuclease in a Cell-free System of Xenopus Egg Extracts. 1205 94

An erythromycin resistance plasmid, pAJ01 was isolated from Loctococcus lactis isolate C5 that was isolated from a healthy two-week-old chicken cecum. A 4 kb plasmid was transformed into plasmidless L. lactis MG1363 before a restriction endonuclease map was constructed. It was then fused with pUC19 to form pAJ02, which can replicate in Escherichia coli XLI-Blue as well as L. lactis MG1363. The plasmid was stably maintained in Lactococcus for more than 100 generations.
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PMID:Characterisation and molecular cloning of an erythromycin resistance plasmid of Lactococcus lactis isolated from chicken cecum. 1218 76

To investigate the influence of mitochondrial genes on stamen development of higher plants, protoplasts from three different, male-sterile tobacco cultivars were fused. The fused cells were cultured individually into calli, from which plants were regenerated. Cybrid plants were obtained that exhibited flowers with recombined biparental male-sterile morphology and with novel male-sterile stamens that differed from any types from sexual or somatic hybridizations described previously. The male-sterile morphologies of these cybrids and their parents support the hypothesis that nuclear-mitochondrial interaction occurs at several stages in tobacco floral development and that several mitochondrial genes are necessary for normal stamen and corolla development. Analysis by restriction endonuclease digestion of mitochondrial DNA of male-sterile cybrids and their parents revealed that the mitochondrial DNA of male-sterile cybrids with parental floral morphology was unchanged when compared with parental mitochondrial DNA. Cybrids that were morphologically similar to one parent's male-sterile phenotype had mitochondrial DNA almost identical to that parent, whereas cybrids with recombined biparental or novel male-sterile phenotypes contained mitochondrial DNA different from both male-sterile parents and from each other. A set of mitochondrial DNA fragments could be correlated with split corollas, a feature found in several tobacco male-sterile cultivars. DNA gel blot analysis using a number of mitochondrial genes confirmed the conclusions based on ethidium bromide staining of mitochondrial DNA restriction digests.
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PMID:Modifications of Mitochondrial DNA Cause Changes in Floral Development in Homeotic-like Mutants of Tobacco. 1232 12

The restriction endonuclease SmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T-->G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F(0)F(1)-ATPase. Our ultimate goal is to apply SmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed the SmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by the SmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targeted EcoRI induced a decrease in cytochrome c oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases.
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PMID:Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. 1237 91

In developing lymphocytes, the recombination activating gene endonuclease cleaves DNA between V, D, or J coding and recombination signal (RS) sequences to form hairpin coding and blunt RS ends, which are fused to form coding and RS joins. Nonhomologous end joining (NHEJ) factors repair DNA double strand breaks including those induced during VDJ recombination. Human radiosensitive severe combined immunodeficiency results from lack of Artemis function, an NHEJ factor with in vitro endonuclease/exonuclease activities. We inactivated Artemis in murine embryonic stem (ES) cells by targeted mutation. Artemis deficiency results in impaired VDJ coding, but not RS, end joining. In addition, Artemis-deficient ES cells are sensitive to a radiomimetic drug, but less sensitive to ionizing radiation. VDJ coding joins from Artemis-deficient ES cells, which surprisingly are distinct from the highly deleted joins consistently obtained from DNA-dependent protein kinase catalytic subunit-deficient ES cells, frequently lack deletions and often display large junctional palindromes, consistent with a hairpin coding end opening defect. Strikingly, Artemis-deficient ES cells have increased chromosomal instability including telomeric fusions. Thus, Artemis appears to be required for a subset of NHEJ reactions that require end processing. Moreover, Artemis functions as a genomic caretaker, most notably in prevention of translocations and telomeric fusions. As Artemis deficiency is compatible with human life, Artemis may also suppress genomic instability in humans.
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PMID:Defective DNA repair and increased genomic instability in Artemis-deficient murine cells. 1261 95

We purified and characterized both the methyltransferase and the endonuclease containing the HsdS delta 50 subunit (type I restriction endonucleases are composed of three subunits--HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutation hsdS delta 50 of the type IC R-M system EcoR 124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsDs delta 50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificity in vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme--a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTase delta 50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused the hsdM and hsdS delta 50 genes and showed that the HsdM-HsdS delta 50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.
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PMID:Characterization of an EcoR124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit, which results in a novel DNA specificity. 1287 41

L1 retrotransposons play an important role in mammalian genome shaping. In particular, they can transduce their 3'-flanking regions to new genomic loci or produce pseudogenes or retrotranscripts through reverse transcription of different kinds of cellular RNAs. Recently, we found in the human genome an unusual family of chimeric retrotranscripts composed of full-sized copies of U6 small nuclear RNAs fused at their 3' termini with 5'-truncated, 3'-poly(A)-tailed L1s. The chimeras were flanked by 11-21 bp long direct repeats, and contained near their 5' ends T2A4 hexanucleotide motifs, preferably recognized by L1 nicking endonuclease. These features suggest that the chimeras were formed using the L1 integration machinery. Here we report the identification of 81 chimeras consisting of fused DNA copies of different RNAs, including mRNAs of known human genes. Based on their structural features, the chimeras were subdivided into nine distinct families. 5' Parts of the chimeras usually originated from different nuclear RNAs, whereas their 3' parts represented cytoplasmic RNAs: mRNAs, including L1 mRNA and Alu RNA. Some of these chimeric retrotranscripts are expressed in a variety of human tissues. These findings suggest that RNA-RNA recombination during L1 reverse transcription followed by the integration of the recombinants into the host genome is a general event in genome evolution.
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PMID:The human genome contains many types of chimeric retrogenes generated through in vivo RNA recombination. 1288 97

Two variants (A and B) of human herpesvirus 6 (HHV-6) can be isolated from humans, with each variant having unique biological properties. HHV-6 variant typing is mainly done following amplification of viral genomic DNA followed by restriction endonuclease digestion. Our objective was to generate a monoclonal antibody (mAb) that would allow us to discriminate between variants A and B of HHV-6. BALB/c mice were immunized with a recombinant glutathione-S-transferase protein fused to the immediate-early (IE) 2 protein from HHV-6 variant A. Following splenocytes fusion, one IgG1 kappa light chain mAb (P6H8) was isolated and found to react specifically with variant A IE2 protein in immunofluorescence and western blot assays. The P6H8 antibody represents a useful tool for both fundamental research and clinical applications allowing for the discrimination of infections caused by HHV-6 variants A or B.
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PMID:Generation and characterization of a monoclonal antibody specific for human herpesvirus 6 variant A immediate-early 2 protein. 1452 67

A novel tri-primer polymerase chain reaction method (TP-PCR) was developed for the construction of a fused fpg gene, in which no endonuclease and ligase were used. Instead, two templates and three specifically designed primers were applied. Results showed that pheB and gfp genes, which encodes the catechol 2, 3-dioxygenase and the green fluorescent protein (GFP), respectively, were successfully fused into an fpg gene through the rapid TP-PCR system, indicating that TP-PCR method could be a useful tool for DNA fragment fusion in which no proper endonuclease sites were available.
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PMID:A novel tri-primer PCR method (TP-PCR) for rapid construction of fpg gene. 1496 27


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