Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A shuttle vector system was developed to quantitate and analyze ionizing radiation-induced mutation in mammalian host cells, COS-1 and CV-1. The shuttle vector pSV2-lacY, which was constructed to detect both point mutations and deletions, was irradiated in vitro with 60Co gamma rays before introduction into unirradiated host cells. The plasmid was then isolated and reintroduced into HB101 (lacY-) bacterial host cells for identification of mutated lacY marker genes. Gamma-irradiation produced a decrease of the survival (recovery) and an increase of mutation of the shuttle vector. The mutated shuttle vector molecules were examined for structural changes by means of restriction endonuclease digestion and agarose gel electrophoresis. A dose dependent increase was observed in the percentages of gross alteration events of total mutations in mammalian host. This system will be useful for studies of ionizing radiation-induced mutagenesis.
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PMID:A shuttle vector system for studying ionizing radiation-induced mutagenesis in mammalian cells. 836 Aug 57

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.
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PMID:Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). 842 92

The two known receptors mediating the actions of cholecystokinin (CCK) and gastrin, CCK type A (CCKAR) and CCK type B (CCKBR) receptors, are G protein-coupled receptors having approximately 50% amino acid homology. Both the CCKAR and CCKBR have high affinity for sulfated CCK peptides, while only the CCKBR has high affinity for gastrin peptides. To determine the structural basis for the selectivity of the CCKBR for gastrin, we first constructed a series of CCKB/AR chimeras in which restriction endonuclease-defined segments of the CCKBR were replaced with the corresponding segments of the CCKAR. Chimeras transiently expressed in COS-1 cells were screened for the selective loss of gastrin affinity according to the displacement of 125I-labeled Bolton-Hunter-CCK-8 binding by gastrin-17-I and CCK-8. The sequence spanning from transmembrane domain III (TM III) to TM V was the only segment that resulted in the selective loss of gastrin affinity. This segment could account for 100 of the expected 300-fold lower affinity of gastrin-17-I observed for the control CCKAR compared to the control CCKBR. Using site-directed mutagenesis in this segment of the CCKBR, we identified a sequence of 5 amino acids in the second extracellular loop responsible for this 100-fold selective loss in gastrin affinity. 125I-labeled Bolton-Hunter-CCK-8 binding displacement by L365,260 (a CCKBR selective antagonist) was unaffected by the changes in these 5 amino acids. These results present for the first time the identification of the amino acid sequence of the CCKBR conferring the majority of the selectivity for gastrin.
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PMID:A segment of five amino acids in the second extracellular loop of the cholecystokinin-B receptor is essential for selectivity of the peptide agonist gastrin. 866 21

Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-mediated redox activity and is important in regulation of cellular processes, including proliferation, apoptosis, and gene expression. The activity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such factor is nuclear factor (NF)-kappa B, whose DNA-binding activity is markedly augmented by TRX treatment in vitro. Similarly, the DNA-binding activity of activator protein 1 (AP-1) is modified by a DNA repair enzyme, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox-active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myristate 13 acetate efficiently translocated TRX into the HeLa cell nucleus where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX was transported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an in vitro diamide-induced cross-linking study and an in vivo mammalian two-hybrid assay we proved that TRX can associate directly with Ref-1 in the nucleus; also, we demonstrated the requirement of cysteine residues in the TRX catalytic center for the potentiation of AP-1 activity. This report presents an example of a cascade in cellular redox regulation.
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PMID:AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. 910 29

Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.
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PMID:Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage. 961 Dec 36

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5' splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor-like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the triangle upEGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.
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PMID:Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency. 968 Mar 60

A plasmid DNA encoding bacterial beta-galactosidase gene was encapsulated in poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres. Plasmid DNA extracted from PLGA microspheres retained both structural and functional integrity as evidenced by its restriction endonuclease digestion pattern and its ability to transfect COS-1 cells in vitro. PLGA microspheres protected plasmid DNA from digestion by deoxyribonuclease I (DNase I) in vitro. The encapsulation efficiency of plasmid DNA and its release rate depended on the molecular mass of PLGA. Lastly, J-774A macrophages phagocytosed PLGA microspheres loaded with plasmid DNA. Co-encapsulated monophosphoryl lipid A increased the rate of phagocytosis. These results suggest that biodegradable PLGA microspheres can deliver intact and functional plasmid DNA at controlled rates. Thus, PLGA microspheres may be used to jointly deliver genes and other biologically active molecules, e.g., immunomodulators, to antigen presenting cells.
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PMID:Encapsulation of plasmid DNA in biodegradable poly(D, L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. 986 34

Kell, a 93 kDa type II membrane glycoprotein, and XK, a 444 amino acid multi-pass membrane protein, are blood group proteins that exist as a disulfide-bonded complex on human red cells. The mechanism of Kell/XK assembly was studied in transfected COS cells co-expressing Kell and XK proteins. Time course studies combined with endonuclease-H treatment and cell fractionation showed that Kell and XK are assembled in the endoplasmic reticulum. At later times the Kell component of the complex was not cleaved by endonuclease-H, indicating N-linked oligosaccharide processing and transport of the complex to a Golgi and/or a post-Golgi cell fraction. Surface-labeling of transfected COS cells, expressing both Kell and XK, demonstrated that the Kell/XK complex travels to the plasma membrane. XK expressed in the absence of Kell was also transported to the cell surface indicating that linkage of Kell and XK is not obligatory for cell surface expression.
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PMID:Intracellular assembly of Kell and XK blood group proteins. 1055 84

The complete S1 gene from mouse hepatitis virus (MHV) was amplified by RT-PCR and cloned into the pMD18-T vector. After confirmed by the restriction endonuclease analysis and PCR amplification, the positive clone of S1 gene was sequenced and then was transferred into eukaryotic expressing vector pVAXI. The recombinant plasmid pVAX1-S1 was transfected into COS-7 cells. The expressed S1 protein was successfully detected with indirect immunofluorescent assay. Finally, The recombinant plasmid pVAX1-S1 was transformed by electroporation into attenuated Salmonella typhimurium strain SL7207 and confirmed by PCR and Salmonella agglutination test. The recombinant was named as SL7207(pVAX1-S1). 6-week-old BALB/c mice were inoculated orally with SL7207 (pVAX1-S1) at dosage of 5 x 10 (8) CFU, 1 x 10(9) CFU and 2 x 10(9) CFU respectively. The immunized mice showed no clinic symptom. The results suggested that SL7207 (pVAX1-S1) was safe for mice after oral immunization at dosage of 2 x 10(9) CFU or below. BALB/c mice were immunized orally with SL7207 harboring recombinant plasmid at the dosage of 109 and boosted two weeks later with the same dose, for a total of three times. The recombinant Salmonella SL7207 ( pVAX1-S1 ) could induce significant humoral immune response in mice compared with the control (P < 0.05 or 0.01) at 2 w post-boosting and 2 w post-three immunization. The antibodies against MHV were also detected in small intestinal mucosal samples from immunized mice at 2 w post-three immunization. These results indicated that recombinant SL7207(pVAX1-S1) induced both systemic and local mucosal immunity.
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PMID:[Immunogenicity of S1 gene DNA vaccine of mouse hepatitis virus delivered by attenuated Salmonella typhimurium]. 1743 39

Total RNA was extracted from periodic Brugia malayi Specific primers were designed on the basis of known sequences of paramyosin gene from B. malayi (BmPmy). The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli (E. coli) strain DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The right gene fragments encoding BmPmy in positive clones for prokaryotic and eukaryotic expression plasmids were digested with restrictive endonuclease, and were subcloned into pcDNA3.1(+). The recombinant eukaryotic plasmid (pcDNA3.1-BmPmy) was then transfected into COS-7 cells. The transient expression of BmPmy was examined with RT-PCR. BmPmy mRNA was highly expressed in transfected COS-7 cells.
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PMID:[Construction of eukaryotic expression plasmids with paramyosin gene of periodic Brugia malayi]. 1803 90


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