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)

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.
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PMID:African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases. 133 84

We describe a new method for obtaining DNA fragments starting at a desired point where there is no recognition sequence for any known restriction endonuclease. A single-stranded DNA containing the fragment of interest is annealed to a synthetic oligonucleotide hybridizing at the 5' end of the required fragment. Then, a partially double-stranded DNA is synthesized using the Klenow fragment of DNA polymerase I in the presence of the four deoxynucleoside triphosphates. The remaining single-stranded regions are removed by digestion with a single-strand nuclease, and the resulting 5' blunt-ended fragment is finally released by digestion with a restriction endonuclease at any site downstream its 3' end. The usefulness of the method was exemplified here by insertion of an epidermal growth factor-like African swine fever virus gene immediately downstream of the ribosome binding site of an expression vector.
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PMID:A general method to cleave a known DNA sequence at any site. 166 37

Restriction endonuclease maps of the variable DNA regions of African swine fever virus field isolates from the Iberian peninsula showed that the changes in length are located in the terminal-inverted repetitions and in unique sequences close to the DNA ends. Analysis of nine clones derived from the spleen of an infected pig revealed the existence of frequent length changes within the inverted terminal repetitions. In each clone, changes occurred symmetrically at both terminal-inverted repetitions, suggesting the existence of a terminal-inverted repetition transposition or correction mechanism. Large deletions in unique sequences were detected more frequently in the region located from 8 to 20 kb from the left DNA end. The analysis of this DNA segment from a virulent African swine fever virus isolated in Lisbon (LIS57) showed that this virus strain contains about 8 kb more DNA sequence than the prototype avirulent virus strain (BA71). Hybridization of the additional sequences from LIS57 virus with DNA from different virus field isolates revealed that this DNA region is highly variable in vivo and that it contains several repeated sequences. DNA sequences present around the deletion end points in the variable regions indicate that the deletion process may take place by both homologous and nonhomologous recombination.
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PMID:Genetic variation of African swine fever virus: variable regions near the ends of the viral DNA. 281 84

An African Swine Fever virus (ASFV) isolated in an 1983 outbreak of the disease in Piemonte, Italy, was related by restriction endonuclease analysis of the viral genome to ASFV strains isolated in the Dominican Republic (1978), Haiti (1981) and Cameroon (1982).
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PMID:Genome analysis of African swine fever virus isolated in Italy in 1983. 282 72

Infection of Vero cells with African swine fever (ASF) virus resulted in a marked increase of DNase active on single-stranded DNA (ss-DNase). No increase was observed for double-stranded DNA-specific nuclease activity. In contrast to uninfected cell ss-DNase, which has a pH optimum at pH range 8.5-9, virus-induced ss-DNase is most active at pH 7. Differences in sensitivity to several ions and other modifications of the reaction mixture and considerable difference in reaction kinetics suggest that the increase in nuclease activity is due to a new virus-induced enzyme. This is strengthened by the fact that anti-ASF virus antiserum inhibits the activity of ss-DNase from infected cells but not from uninfected cells. Exclusion chromatography of the digests shows that virus-specific ss-DNase is exclusively or predominantly an endonuclease. The increase in nuclease activity of infected cells is proportional to the multiplicity of infection. Virus-specific ss-DNase is synthesized at late times after infection and its synthesis is dependent on viral DNA replication since it is not induced when infected cells are treated with cytosine arabinoside. Most of ss-DNase activity in infected cells is associated to an insoluble cytoplasmic fraction, presumably virosomes. The enzyme can also be detected in partially stripped purified virions which hydrolyze 6.9 ng DNA per microgram viral protein.
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PMID:Single-stranded deoxyribonucleic acid nuclease induced by African swine fever virus and associated to the virion. 377 99

DNA from African swine fever (ASF) virus was isolated and was characterized by two restriction enzymes, SmaI and EcoRI. Although both enzymes can distinguish Vero cell-adapted ASF isolates by characteristic restriction endonuclease cleavage patterns, all ASF isolates examined exhibited a high degree of similarity, as measured by co-migration of most of the DNA fragments. The molecular weight of ASF DNA, based on size estimates of DNA fragments from cleavage patterns, ranged from 93 x 10(6) to 100 x 10(6). Virus genome heterogeneity was observed in uncloned, cell culture-adapted ASF isolates as well as in a plaque-purified virus after serial passage in Vero cells. In contrast to the rather minor differences in restriction pattern among the Vero cell-adapted isolates, a major alteration in restriction endonuclease cleavage sites was observed during adaptation of the wild-type virus to cell culture.
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PMID:African swine fever virus DNA: restriction endonuclease cleavage patterns of wild-type, Vero cell-adapted and plaque-purified virus. 629 85

African swine fever virus DNA (about 170 kbp) was cleaved with the restriction endonuclease EcoRI and most of the resulting 31 fragments were cloned in either the phage vector lambda WES lambda B or the plasmid pBR325. Three fragments were not cloned in those vectors, the largest fragment EcoRI-A (21.2 kbp) and the two crosslinked terminal fragments, EcoRI-K' and D'. Endonuclease SalI cut fragment EcoRI-A into three pieces which were cloned in plasmid pBR322. The two terminal EcoRI fragments were cloned after removal of the crosslinks with nuclease S1 and addition of EcoRI linkers to the fragment ends. The complete library of the cloned fragments accounted for about 98% of ASF virus genome, the missing sequences being those removed by the nuclease S1 in the process of cloning the terminal fragments.
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PMID:Molecular cloning of African swine fever virus DNA. 632 51

Plasmid vectors designed to facilitate the genetic manipulation of African swine fever virus (ASFV) are described. Our results demonstrate that the beta-glucuronidase enzyme (GUS) can be used to follow gene expression in ASFV-infected cells. Infectious plaques formed by ASFV expressing GUS are visually detectable, thus providing a simple and highly sensitive method for the selection of ASFV recombinants. These and previous results have allowed us to construct two chimeric gene cassettes that constitute the basic tools for the generation of vectors to carry out the deletion of multiple target sequences from the ASFV genome. These cassettes, formed by: (a) a virus promoter; (b) the coding sequence of a reporter gene, either Lac Z or gusA; and (c) a strong signal for the 3' end formation of ASFV mRNAs, can be easily isolated by endonuclease restriction from their corresponding plasmid vectors. A general insertion/coexpression plasmid vector, pEPV2, has also been constructed. pEPV2 facilitates the insertion of foreign genes, together with the Lac Z reporter, into the thymidine kinase locus of ASFV. The functionality of pEPV2 has been tested by generating a recombinant ASFV expressing the luciferase gene. The vectors presented in this report constitute the first reported set of tools for the genetic manipulation of ASFV.
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PMID:Vectors for the genetic manipulation of African swine fever virus. 761 41

This work provides a novel, highly sensitive, hot start PCR method for rapid and specific detection of African swine fever virus (ASFV) that can be used as a routine diagnostic test for ASFV in surveillance, control, and eradication programs. A confirmatory test of the specificity of this method based on restriction endonuclease analysis was also developed.
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PMID:Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. 1295 85

The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.
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PMID:A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples. 1536 58


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