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)

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

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.
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PMID:Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro. 19 79

The accessibility of extracellular and nuclear simian virus 40 (SV40-M and SV40-I, respectively) virion chromatin DNAs to micrococcal nuclease, DNase I, BglI, EcoRI, and RNA polymerase was examined. Our results support the following conclusions: (i) the intranucleosomal DNA of SV40-I chromatin, similar to the precursor 75S chromatin complex, is resistant to enzymatic activity; and (ii) SV40-M virion chromatin is modified in a manner which increases the accessibility of viral DNA to enzymes, and the distinction between nucleosomal DNA and linker DNA is absent. Micrococcal nuclease digestion of SV40-I virion chromatin gave a typical nucleosomal DNA ladder pattern with a repeat unit of 205 base pairs of DNA. SV40-I chromatin was sensitive to cleavage with endonuclease BglI, but not with EcoRI. When SV40-I virion chromatin was used as a template, the rate of incorporation of ribonucleoside triphosphates into RNA was 5% of that obtained with naked form SV40 form I DNA. Micrococcal nuclease digestion of SV40-M virion chromatin resulted in submonomeric DNA fragments of approximately 55 base pairs, but no larger repeating unit of DNA was observed. SV40-M virion chromatin was sensitive to cleavage with either BglI or EcoRI and was approximately 20% more susceptible to digestion with DNase I than was SV40-I virion chromatin. The transcriptional efficiency of the extracellular virion chromatin was almost equivalent to that of naked SV40 form I DNA and was 16-fold higher than the rate observed with nuclear virion chromatin. The increased transcriptional activity was dependent upon the presence of nonhistone viral protein VP1 or VP2 or both.
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PMID:Simian virus 40 maturation: chromatin modifications increase the accessibility of viral DNA to nuclease and RNA polymerase. 626 46

A reverse transcription-polymerase chain reaction (RT-PCR) amplification assay was developed to detect infectious bursal disease virus (IBDV) gene sequences in clinical samples, infected cell cultures and chicken embryos. Two pairs of primers were designed to amplify the 5'- and 3'-termini of segment A genes that partially code for the IBDV proteins VP2 and VP3, respectively. One primer pair specifies a 309-bp fragment, the other a 520-bp fragment. Direct RT-PCR analysis of 5 bursal samples of chickens derived from a suspected first outbreak of infectious bursal disease in New Zealand yielded the 309-bp and 520-bp by fragments. The identity of both amplified fragments was confirmed by restriction endonuclease analysis, chemiluminescence Southern blot hybridization and direct cycle sequencing. RT-PCR amplification of RNAs extracted from 4 out of 5 IBDV isolates propagated in Vero cells, chicken embryo fibroblasts and specific pathogen-free chicken embryos yielded IBDV-specific fragments of unpredicted small sizes.
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PMID:Detection of infectious bursal disease virus by reverse transcription-polymerase chain reaction amplification of the virus segment A gene. 767 88

Reverse transcription with polymerase chain reaction (PCR) followed by restriction endonuclease analysis detected genetic variations among serotype I isolates of infectious bursal disease virus (IBDV). Using a set of synthetic primers derived from the large genome segment of APHIS-IBDV, the hypervariable region (AccI-SpeI fragment) located in the VP2 gene was amplified. With all strains, a cDNA fragment of approximately 643 bp was amplified, indicating that there were no apparent deletions or insertions in this region among isolates. Fragments amplified from 9 isolates were digested with 14 restriction enzymes. Restriction fragment profiles generated by restriction enzymes NaeI, StuI, TaqI, and SacI, showed genetic variations among isolates. This study provided a simple and sensitive method for detection of genetic variations among isolates that are closely related serologically and could not be differentiated using current serologic methods.
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PMID:Detection of genetic variations in serotype I isolates of infectious bursal disease virus using polymerase chain reaction and restriction endonuclease analysis. 798 44

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

Bursa samples from the United States, Mexico, and Puerto Rico were tested for the presence of infectious bursal disease virus (IBDV) using the reverse transcriptase/polymerase chain reaction-restriction endonuclease (RT/PCR-RE) assay. This assay amplifies a 394-bp fragment of the IBDV VP2 gene. A total of 151 samples were tested. Each was from a different physical location or farm. Forty-eight of the samples were determined to contain IBDV using RT/PCR. The RE profiles on 44 of these positive samples were determined using the enzymes BstNI or EcoRII, StyI, DraI, SacI, Sau3AI or MboI, and TaqI. A majority of the samples (34) had RE profiles typical of variant IBDV strains. One of six samples from Mexico was positive for IBDV. This virus had an RE profile typical of variant strains of IBDV. Three of seven samples from Puerto Rico had RE profiles characteristic of variant viruses. Two samples from the United States had RE profiles characteristic of classic vaccine IBDV strains and nine samples had new RE profiles. Five of these new profiles were BstNI- and StyI-negative, indicating that these viruses may be antigenically related to variant types. Although the new RE pattern observed in the other four samples was BstNI- and StyI-positive, it was not typical of classic vaccine IBDV strains. One flock from the United States had a mixture of two RE profiles, a typical variant type profile and an unknown variant RE profile. Two-thirds of the positive samples from flocks where the age of the birds was reported were observed between 21 and 28 days of age. The results of these studies demonstrate that the RT/PCR-RE assay can be used to diagnose IBDV in chickens and that IBDV strains exist in commercially reared chickens that have RE patterns different than known IBDV strains. The molecular differences observed using the RT/PCR-RE test were in a region of the VP2 gene, which is known to code for important neutralizing epitopes and to be highly variable among IBDV strains. Although the results demonstrate RE patterns different than those observed in known classic and variant IBDV strains, the influence of these molecular differences on biological properties of the viruses requires further investigation.
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PMID:Detection of infectious bursal disease viruses in commercially reared chickens using the reverse transcriptase/polymerase chain reaction-restriction endonuclease assay. 908 30

The VP2 hypervariable region of infectious bursal disease virus (IBDV) from nine Mainland Chinese strains was amplified by reverse transcriptase/nested polymerase chain reaction and cloned into pGEM-T vector. The nine isolates, which were from the center (HN3), the north (Bj-1, B2/28, HD96), the east (JS-18 and AH-2), the northeast (D11-2, C4-2), and the west (Ts) of China, were sequenced and compared with each other and with six reference IBDV sequences. Clustering analysis separated the nine isolate into two groups. The six virulent isolates, propagated in bursae, formed the first group. They revealed only one to three amino acid changes from the very virulent (vv) European and Japanese isolates, suggesting that they might have the same origin as European and Japanese vvIBDV strains. On the basis of their distinct geographic origins, extensive dissemination of vvIBDV in China was indicated. (The other three chicken embryo fibroblast cell cultured isolates with mild pathogenicity were placed in the second group.) Their sequences correlated closely with those of the culture-adapted strains (Cu-1 (4) and Cj-801). None of the nine isolates showed very close sequence relationship with the antigenic variant strains from the USA. Although antigenic variants have been reported in China, the reverse transcriptase/polymerase chain reaction-restriction endonuclease analyses of the nine viruses tested herein were not similar to any U.S.A. variant strains on the basis of computer software analysis. Our results and conclusions agree with a previous molecular study of IBDV isolates from the south of China.
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PMID:Sequence analysis of the VP2 hypervariable region of nine infectious bursal disease virus isolates from mainland China. 987 46

An amino acid mutation at residue 284 (Ala to Thr) in the VP2 protein of infectious bursal disease viruses (IBDVs) has been correlated with the ability to replicate in cell culture. In this study, we designed a molecular test for this mutation. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to amplify a 743-bp region of the VP2 gene that contained the codon for amino acid 284. The restriction endonuclease NgoMIV was selected for this study because the first three nucleotides of its six-base recognition sequence are the codon responsible for the amino acid alanine at residue 284. The RT/PCR products from 10 known pathogenic and 16 vaccine strains of IBDV were examined for the presence or absence of the NgoMIV site. We also examined 189 field strains of IBDV for the NgoMIV site. All 10 known pathogenic IBDV strains contained the NgoMIV site, indicating they contained alanine at residue 284. None of the vaccine strains had the NgoMIV site, suggesting they had threonine or another amino acid at residue 284. The results suggest that the presence of this NgoMIV site can be used as a marker for the identification of wild-type (nonvaccine) IBDV strains. The RT/PCR products from 152 (80.4%) of the field strains had the NgoMIV site and thus have the potential to be wild-type pathogenic viruses. The RT/PCR products from 37 (19.6%) of the field strains were not cleaved by NgoMIV and thus are potentially attenuated vaccine strains. Molecular diagnostic assays have been used to place IBDV strains into genetically related groups. The identification of this genetic marker now makes it possible to identify viruses that are wild-type strains that have the potential to be pathogenic viruses.
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PMID:Use of a genetic marker for wild-type potentially pathogenic infectious bursal disease viruses. 1156 47

Infectious bursal disease (IBD) viruses detected in commercial flocks of different regions of Argentina were analyzed by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RFLP) of a VP2 gene fragment, followed by sequence analysis. Two out of eight IBD viruses presented an SspI restriction site, typical of the very virulent phenotype. Three IBD viruses presented a SacI restriction site, typical of classic virulent strains, and one isolate presented restriction sites for both enzymes. The Argentine IBD viruses showed three different molecular patterns by RFLP with the restriction endonuclease BstNI and five different patterns with MboI. By comparison of nucleotide and deduced amino acid sequences of the hypervariable region of the VP2 protein, four Argentine viruses were found to be closely related to Brazilian subclinical strains and two isolates were found to be related to vaccine IBDV strains in use in Argentina. Strain LD9569 was genetically characterized as a very virulent strain and was found to be closely related to international and regional vvIBDV strains. This is the first report on variability of IBDV strains circulating in Argentina.
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PMID:Characterization of infectious bursal disease viruses from Argentina. 1686 75


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