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)

A linked cell-free system has been developed which is capable of transcribing and translating mamalian viral DNA, and its characteristics and requirements are outlined. In this system, simian virus 40 (SV40) DNA Form I (supercoiled) directed the synthesis of discrete polypeptides up to 85,000 daltons in size. One of these products was indistingusihable from authentic major virus capsid protein VPI, as judged by mobility on sodium dodecyl sulfate/polyacrylamide gels, antibody predipitation, and peptide analyses. The cell-free products larger than VPI comprised a number of polypeptides ranging in molecular weight from 50,000 to 85,000. These polypeptides demonstrated no immunological relationship whatsoever to the structural protein VPI. However, two of these products, along with one of approximately 25,000 dlatons, were precipitated with antiserum to SV40 tumor antigen. Linear SV40 DNA generated by the cleavage of Form I DNA with the restriction endonuclease EcoR1 was an efficient template in this system and also directed the synthesis of a polypeptide migrating with VPI on polyacrylamide gels. The potential of this system for defining a functional map of a DNA genome is discussed.
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PMID:Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system. 16 82

A method is described for mapping regions of eukaryotic viral DNA coding for specific proteins, utilizing a linked transcription-translation cell-free system primed with DNA fragments generated by restriction endonucleases. Three simian virus 40 (SV40) DNA fragments derived from that region of the DNA expressed late in lytic infection were purified. They were: Hpa I-A (0.76-0.175 map units), Bgl I-EcoRI-B (0.672-0), and Hpa II-EcoRI-B (0.735-0). (Fragments are named from the cleaving restriction endonuclease and electrophoretic mobility. End positions on the conventional map are in clockwise order.) These fragments efficiently stimulated the incorporation of [3H]UTP and [35S]methionine into trichloroacetic-acid-insoluble material in the linkec system. The location of the region of DNA coding for the viral structural proteins VPI, VP2 and VP3 was determined from the spectrum of polypeptide synthesis directed by the individual intact fragments and their specific endonucleolytic digests. The polypeptides synthesized in the cell-free system were characterized on urea-sodium dodecyl sulfate polyacrylamide gradient gels and by two-dimensional tryptic peptide analysis. ..
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PMID:Direct biochemical mapping of eukaryotic viral DNA by means of a linked transcription-translation cell-free system. 18 8

The DNA of the major capsid protein VP1 of the human polyomavirus JC virus (JCV), Taiwan-3 strain, was generated from the urine of an autoimmune disease patient by polymerase chain reaction (PRC). The VP1 DNA was cloned into a prokaryotic expression vector, pGEX-4T-1, for expression in E. coli. The nucleotide sequences and the deduced amino acid sequences were determined and compared with the JC virus prototype, Mad-1. Thirty nucleotides were different between these two strains. Six of the altered nucleotides affected amino acid coding and ten of them caused changes in endonuclease recognition sites. The recombinant VPI protein was purified and used to raise monospecific antiserum in rabbit. Recombinant JCV VP1 protein and its monospecific antiserum are important clinical reagents and could possibly be developed as a subunit vaccine and as a serological diagnostic antigen in the future. In addition, the region between amino acid residues 40 and 80 of JCV VP1 is predicted to be an antigenic epitope on the basis of its hydropathy plot and comparison with the VP1 sequences of SV40 and BK virus.
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PMID:Production of the antigen and the antibody of the JC virus major capsid protein VP1. 879 46

Clostridium difficile causes pseudomembranous colitis and antibiotic-associated diarrhea. The definitive diagnosis of C. difficile infection is finally accomplished by the isolation of toxigenic C. difficile. However, only a small number of Japanese clinical laboratories are able to reach a definitive diagnosis of C. difficile infection, probably because simple reliable assays for toxins in the isolates are not available. In this study, we examined the compatibility of a polymerase chain reaction (PCR) assay and tissue culture assay to identify toxigenic C. difficile, in toxigenic and nontoxigenic C. difficile isolates from Japanese patients and healthy carriers. The specificity of PCR primers was demonstrated by restriction endonuclease digestion and seminested PCR in C. difficile VPI 10463 strain. No PCR product was amplified in the eight other clostridial species used to check the specificity of the PCR assay. The detection limit was 10(3) cells. Both toxin A and toxin B genes (the genes encoding the major virulence factors of C. difficile) were detected in 58 toxigenic C. difficile isolates, which showed a wide range of cytotoxic activity in tissue culture assays. Neither of the toxin genes was carried by 40 nontoxigenic strains of C. difficile. The results of this study strongly suggest that a definitive diagnosis of C. difficile infection can be accomplished by PCR detection of the toxin genes rather than by tissue culture assay of isolates.
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PMID:Laboratory diagnosis of toxigenic Clostridium difficile by polymerase chain reaction: presence of toxin genes and their stable expression in toxigenic isolates from Japanese individuals. 1020 9

A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3' end of tcdA demonstrated identical 1. 8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.
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PMID:Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease. 1099 43