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 patient who used contact lenses and had a history of blunt trauma developed vaccinia keratouveitis after accidental ocular autoinoculation from a recent vaccination site. Corneal and conjunctival cultures were taken for bacteria, fungi, Acanthamoeba, and viruses. Viral-like cytopathic effects became evident in tissue culture within three days. Immunofluorescence studies were negative for varicella-zoster virus, herpes simplex virus, adenovirus, measles, mumps, parainfluenza, and influenza. Pox viral particles were identified in the infected tissue cultures by electron microscopy. The Hind III restriction endonuclease profile of the viral DNA isolate was similar to the Lister strain of vaccinia virus. Ocular vaccinia may manifest as a masquerade syndrome and may mimic signs of herpes simplex virus, varicella-zoster virus, and Acanthamoeba infection. Although vaccination with vaccinia is currently limited to a few populations throughout the world, vaccinia must still be considered in the differential diagnosis of infectious keratouveitis.
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PMID:Vaccinia keratouveitis manifesting as a masquerade syndrome. 815 30

DNA from isolates of monkeypox virus, when digested with the endonuclease PstI, gave fragment-size profiles which correlated with the geographic area from which the isolate originated. Although some of the differences were located subterminally in the genome, others mapped to the central conserved region. Further differentiation of the viral genomes was sought by analysis of a short region within the central conserved part of the genome that appeared to be a partially deleted counterpart of an intact 1024 bp open reading frame (ORF) present in variola and vaccinia virus genomes. We reasoned that this region would not be conserved by functional selection and would therefore be likely to show more variation between isolates of monkeypox virus. The deletions found in monkeypox virus isolates from Liberia and from Benin were almost the same as that which we had previously found in the Denmark strain. A much shortened ORF, potentially coding for a product of 133 amino acids, was retained in all three West African isolates, but three Zairean isolates each showed an identical series of small insertions and deletions which effectively abolish the ORF. Three deletions, present in all isolates, must pre-date the geographical separation of monkeypox virus lineages; other, presumably more recent, changes differ between the Zairean and West African isolates. In contrast, the base similarity was found to be more than 99% when all the monkeypox virus sequences were appropriately aligned. This, in a disrupted and presumably nonfunctional gene also indicates that the changes described are recent. It is suggested that insertions and deletions occur regularly during poxvirus DNA replication, but are preserved only in sequences that are not required for continued transmission in the natural host.
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PMID:Evidence for recent genetic variation in monkeypox viruses. 820 96

The m1 species of reovirus RNA, which encodes the minor protein component mu 2, possesses two initiation codons, one "strong" according to Kozak rules and preceded by 13 residues (IC1), the other "weak" and located 49 codons downstream of the first (IC2). In reovirus-infected cells only IC2 is used, but initiation from IC1 can be activated, and efficiency of initiation from either initiation codon modulated over a wide range, by coupling unrelated sequences to either or both ends of m1 RNA. For example, when the M1 genome segment is cloned into the thymidine kinase gene of vaccinia virus in such a way that various "irrelevant" stretches of nucleotides comprising restriction endonuclease cleavage sites or promoter remnants are coupled to the 5' end of m1 RNA, translation of the resultant transcripts is also initiated at IC2, with frequencies controlled by the nature of the attached sequences. However, in rabbit reticulocyte lysates these same transcripts are translated from IC1 as well as from IC2, and transcripts in which m1 RNA is preceded by long sequences of encephalomyocarditis virus RNA (from the T7 polymerase-controlled pTM1 vector) are translated exclusively from IC1. By contrast, m1 RNA itself is translated only from IC2. It appears that the most important factor that controls the extent to which translation is initiated from IC1 and IC2 is their "availability," which is likely to be a function of the extent to which the regions on either side of them interact with each other (and also, to a lesser extent, with the 3' untranslated region) either directly or via interaction with host cell proteins. The effects described here are of considerable potential significance when genetic material is rearranged as a result of translocations, insertions, deletions, and amplifications--that is, when sequences that are normally separated are brought into apposition.
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PMID:Translation of reovirus RNA species m1 can initiate at either of the first two in-frame initiation codons. 841 36

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities.
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PMID:Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus. 843 22

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.
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PMID:A poxvirus-encoded uracil DNA glycosylase is essential for virus viability. 847 56

Restriction endonuclease analysis of the DNA extracted orf virus strain NZ2, which had been serially passaged in primary bovine testis cells, revealed a population of variants that had over-grown the wild-type virus. At least three distinct mutant forms were identified in which the right end of the genome had been duplicated and translocated to the left end, accompanied by deletions of sequences at the left end. Sequencing of a single variant isolated from the heterogeneous population revealed that recombination had occurred between non-homologous sequences. In this case, 6.6 kb of DNA at the left end of the genome had been replaced by 19.3 kb from the right end. The transposition resulted in the deletion at the left end of 3.3 kb of DNA encoding three genes and the terminal sequences of a fourth gene. The three genes completely deleted were a homologue of dUTPase, a gene that encodes a protein containing ankyrin-like repeats and a homologue of the 5K gene of the vaccinia virus WR strain. Experimental inoculation of sheep showed that the genes are also non-essential in vivo, but that the size of the lesion was reduced, compared with that induced by the wild-type, and resolved more rapidly.
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PMID:Genomic analysis of a transposition-deletion variant of orf virus reveals a 3.3 kbp region of non-essential DNA. 884 2

We investigated the feasibility of using vaccinia virus (VAC) recombinants containing large multigene fragments of orf virus DNA to identify protective antigens of orf virus (OV). Sixteen OV strain NZ2 DNA fragments with an average size of 11.4 kb were recombined into VAC strain Lister. Each fragment was mapped relative to OV restriction endonuclease maps but was otherwise uncharacterized. Together the recombinants represent 95% of the OV genome in an overlapping manner. Immunofluorescence showed all 16 constructs expressed products recognized by OV antiserum and radioimmune precipitation with the same antiserum allowed the localization of the major antigens of OV to specific recombinants. These data indicated the approximate genomic locations of the genes encoding the OV major antigens and showed that their expression was authentic rather than resulting from read through from VAC sequences adjacent to the site of recombination. Vaccination of OV-naive sheep with the recombinant library provided protection against a subsequent challenge with virulent OV. These data confirm the feasibility of the proposed strategy.
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PMID:A novel strategy for determining protective antigens of the parapoxvirus, orf virus. 912 61

The genome of Molluscum contagiosum virus (MCV) has a high G + C content, which largely differs from those of vaccinia virus (VAC) and other characterized poxviruses. This has precluded the use of DNA hybridization for the identification of MCV genes and the further establishment of the virus genetic map. To circumvent this problem, we have partially sequenced clones containing virus restriction endonuclease fragments, which were derived by either single or double-digestion of genomic DNA from the subtype I of MCV. The DNA sequences were translated and used to search protein data bases. This analysis resulted in the finding of high-scoring matches to data base entries, including forty-five VAC genes. In addition, MCV-specific sequences that encoded protein domains of known function (i.e. DNA J domain) were found. The locations of MCV clones were inferred from the presumed colinearity of both MCV and VAC genomes, and further confirmed by PCR technology. The data presented here led to the construction of a partial genetic map of MCVI, which revealed that the order and orientation of a large number of MCV genes were equivalent to those of their VAC homologues. The conserved gene arrangement was apparently disrupted in the terminal regions, where MCV sequences showing homologies with the VAC counterparts were not found.
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PMID:A random DNA sequencing, computer-based approach for the generation of a gene map of molluscum contagiosum virus. 920 57

The current members of the genus parapoxvirus are orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpoxvirus (PCPV) and parapoxvirus of red deer in New Zealand (PVNZ). BPSV and PCPV are maintained in cattle while ORFV is maintained in sheep and goats, but all three are zoonoses. Only the recently reported PVNZ has yet to be recorded as infecting humans. Tentative members of the genus are camel contagious ecthyma virus, chamois contagious ecthyma virus and sealpoxvirus. The separation of the parapoxviruses into 4 distinct groups has been based on natural host range, pathology and, more recently, on restriction endonuclease and DNA/DNA hybridisation analyses. The latter studies have shown that the parapoxviruses share extensive homology between central regions of their genomes, but much lower levels of relatedness within the genome termini. The high G + C content of parapoxvirus DNA is in contrast to most other poxviruses and suggests that a significant genetic divergence from other genera of this family has occurred. DNA sequencing of portions of the genome of ORFV, the type species of the genus, has allowed a detailed comparison with the fully sequenced genome of the orthopoxvirus, vaccinia virus (VACV). These studies have provided a genetic map of ORFV and revealed a central core of 88 kbp within which the genomic content was strikingly similar to that of VACV. This conservation is not maintained in the genome termini where insertions, deletions and translocations have occurred. The characterisation of specific ORFV genes may lead to the construction of attenuated vaccine strains in which genes such as those with the potential to interfere with the immune response of the host have been deleted. The current ORFV vaccines are living unattenuated virus and vaccination lesions produce virus which contaminates the environment in a manner similar to natural infection. The virus in scab material is relatively resistant to inactivation and this virus both perpetuates the disease in sheep and provides the most likely source of human infections. A vaccine which immunises animals without perpetuating the disease could be the best way of reducing the incidence of ORFV infection of humans. It is likely that protection against infection by ORFV is cell mediated and will require the endogenous production of relevant antigens. We have recently constructed a series of VACV recombinants each of which contains a large multigene fragment of ORFV DNA. Together the recombinants represent essentially all of the ORFV genome in an overlapping manner. Vaccination of sheep with the recombinant library provided protection against challenge with virulent ORFV. Further studies with this library may enable dominant protective antigens of ORFV to be identified and lead to their incorporation into a subunit vaccine.
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PMID:Molecular genetic analyses of parapoxviruses pathogenic for humans. 941 23

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.
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PMID:Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease. 942 5


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