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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The expression of Herpes Simplex Virus 1 (HSV-1) glycoprotein C (gC), a well defined herpesvirus late gene, was studied by linking the promoter-regulatory region of this gene to the coding sequences for the bacterial enzyme, beta-galactosidase (beta-gal). A chimeric gene, containing the beta-gal gene under the control of gC sequences from -1350 to +30 relative to the mRNA start site, was inserted by homologous recombination into the thymidine kinase (TK) locus of the HSV-1 genome. Selection of the TK- recombinant virus by plaque assay was facilitated by addition of a beta-gal indicator to the agarose overlay. Recombinant virus containing the gC promoter-beta-gal chimeric gene faithfully expressed beta-gal as a viral late gene, as shown by the absence of beta-gal expression when viral DNA replication was inhibited with phosphonoacetic acid. In contrast, the inhibition of viral DNA replication had no effect on the expression of beta-gal when the beta-gal gene was under the control of the early HSV-1 TK promoter in a separate recombinant virus. Analysis of recombinant viruses containing 5' to 3' deletions in the gC regulatory region revealed no apparent difference in beta-gal expression as deletions extended from -1350 to -109 base-pairs (bp) before the RNA start site, demonstrating that sequences between -109 and +30 are sufficient for regulated gC expression in the viral genome. Analysis of the mRNA made by these recombinant viruses confirmed the results of the beta-gal assays, and demonstrated that the transcriptional start sites of the gC promoter-beta-gal chimeric genes were the same as the start site of the gC gene.
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PMID:The use of beta-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1 glycoprotein C gene in recombinant herpesviruses. 284 20

The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base-pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S-components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis-acting sites for the inversion of herpes simplex virus DNA sequences.
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PMID:Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene. 300 70

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. However, the mechanism by which alpha 4 regulates gene expression remained unknown. We report that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from infected-cell lysates as detected by a gel electrophoresis binding assay. The protein(s) exhibits sequence specificity since autologous DNA fragments but not heterologous DNA fragments, synthetic polydeoxynucleotide chains, or salmon sperm DNA competitively displace the DNA probe from the complexes. Murine monoclonal antibody to alpha 4 protein added before or after DNA-protein complex formation further retarded the electrophoretic mobility of the complexes whereas monoclonal antibody to alpha 0, alpha 27, or to a viral glycoprotein had no effect. Complexes consisting of the promoter-regulatory domain of the beta-class thymidine kinase gene and infected cell proteins were low in abundance and could be detected only in the presence of antibody to alpha 4 protein. The alpha 4 protein, therefore, forms stable complexes with promoter-regulatory domains of alpha genes and of selected other herpes simplex virus type 1 genes either alone or in combination with other proteins.
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PMID:Alpha 4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter-regulatory domains of alpha genes and of selected other viral genes. 301 Feb 85

The map position of the coding sequence of glycoprotein H of herpes simplex virus type 1 was determined by marker transfer studies in which DNA fragments cloned from a virus resistant to neutralisation by an anti-gH monoclonal antibody were used to transfer antibody resistance to wild type virus DNA following cotransfection. The gH coding sequence was mapped to the BglII "m" fragment of HSV-1 DNA (map coordinates 0.27-0.312), confirming the map position previously determined by intertypic recombinant analysis (Buckmaster et al., 1984). The complete nucleotide sequence of the BglII "m" fragment revealed two large open reading frames in addition to the thymidine kinase gene. The open reading frame lying immediately 3' of the thymidine kinase gene has a predicted translation product with the features of a large glycoprotein. This open reading frame translates to an amino acid sequence of 90,323 mol wt with a signal peptide, a membrane anchor sequence, a large external domain containing potential N-glycosylation sites, and a charged C- terminal cytoplasmic domain. We suppose that this amino acid sequence corresponds to gH of HSV-1, and A. Davison (personal communication) has noted the existence of homologous glycoproteins predicted from the nucleotide sequences of Varicella-zoster virus and Epstein-Barr virus. The properties of monoclonal antibody LP11, directed against gH show remarkable similarities to the properties for gD antibodies. LP11 efficiently neutralizes virus infectivity, blocks cell fusion by syncytial virus strains, and inhibits the formation of plaques when added to cell monolayers after infection. These similarities in antibody activity imply functional relatedness between gH and gD of herpes simplex virus.
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PMID:The properties and sequence of glycoprotein H of herpes simplex virus type 1. 301 91

Expression of the early and late genes of herpes simplex virus type 1 (HSV-1) during infection of tissue culture cells requires the prior expression of the immediate early (IE) genes. The requirement for the product of IE gene 3, Vmw175, for the activation of early promoters has been revealed by studies with temperature-sensitive virus mutants. Recent experiments using transfection assays have shown that both Vmw175 and the product of IE gene 1, Vmw110, are involved in the transactivation of a variety of HSV-1 early promoters. This paper describes experiments which compared the activation of two early promoters [those of the glycoprotein gD and thymidine kinase (tk) genes] with that of a member of a later class of genes (the major capsid protein, VP5). Plasmids containing these promoters linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into HeLa cells with plasmids containing one or more HSV-1 IE genes. Promoter activity was estimated by measurement of CAT activity in extracts of transfected cells. The gD and tk promoters were activated by both Vmw175 and Vmw110, and the combination of these two IE gene products resulted in very high levels of activation. Addition of further IE gene products did not result in any significant increase in the activation seen with the combination of Vmw175 and Vmw110. In contrast, the activation of the VP5 promoter brought about by the combination of Vmw175 and Vmw110 was relatively slight, but was increased further when plasmids containing IE gene 2, encoding Vmw63, were included in the transfection. These data suggest that Vmw63, like Vmw175 and Vmw110, is also involved in the activation of transcription from HSV-1 promoters. The effect of Vmw63 may be limited to the activation of a subset of HSV-1 genes.
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PMID:The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2 and 3 can activate HSV-1 gene expression in trans. 302 36

Pseudorabies virus (PRV) glycoprotein gX accumulates in the medium of infected cells. In an attempt to study the function of gX, two viruses were constructed that lacked a functional gX gene. One virus, PRV delta GX1, was derived by insertion of the herpes simplex virus thymidine kinase gene into the gX-coding region. The other virus, PRV delta GXTK-, was derived by subsequent deletion of the inserted herpes simplex virus thymidine kinase gene. Both viruses replicated in cell cultures but produced no gX. Furthermore, PRV delta GX1 was capable of killing mice with a 50% lethal dose of less than 100 PFU.
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PMID:Replication and virulence of pseudorabies virus mutants lacking glycoprotein gX. 302 2

The cis-acting DNA sequences required for regulated expression of a herpes simplex virus type 1 (HSV-1) late (gamma 2) gene were studied by using viruses containing specific deletions in the 5' transcribed noncoding and upstream regions of the HSV-1 glycoprotein C (gC) gene, a model gamma 2 gene. Nine mutant viruses which had variable 5' and 3' deletions within bases -569 to +124 relative to the 5' terminus of the gC mRNA were isolated. The mutants were isolated by a simple in situ hybridization screening procedure not requiring any prior selective pressure for or against expression of the gC gene. Analysis of RNA extracted from cells infected with individual mutants showed that the DNA sequences required for regulated expression of this gamma 2 gene lay within bases -34 to +124. This 158-base-pair fragment was sufficient to confer accurate and quantitative expression of gC mRNA and to maintain the stringent requirement on viral DNA replication for expression of this gene. Moreover, it was found that sequences located between -34 and +14 contained signals essential for expression of gC. To determine whether the -34 to +124 sequences would function as a gamma 2 promoter when moved to another region of the HSV-1 genome, the 158-base-pair fragment was substituted for the normal thymidine kinase promoter-regulatory sequences in the thymidine-kinase gene locus. Transcription of this chimeric gene was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis. The only recognizable consensus sequence upstream of the transcription initiation site for this gene was the TATAAA sequence at -30.
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PMID:Transcriptional control signals of a herpes simplex virus type 1 late (gamma 2) gene lie within bases -34 to +124 relative to the 5' terminus of the mRNA. 302 6

Herpes simplex virus type 1 (HSV-1) has a broad host range but the KOS strain of HSV-1 did not replicate efficiently in murine embryonal carcinoma (EC) cells. The yield of infectious HSV-1 from EC cells was 100- to 1000-fold lower than that from fibroblast cell lines of mouse, monkey or human origin. The thymidine kinase (TK) gene of HSV-1 is expressed early during the infectious cycle. The levels of TK mRNA and of TK activity in infected EC cells were only two- to threefold lower than levels from infected fibroblast cells. Infected EC cells supported replication of about half as much HSV-1 DNA as did fibroblast cells. The reduced yield of infectious virus was consistent with a paucity of virions in infected EC cells examined by electron microscopy, suggesting a major block late during the HSV-1 infectious cycle. We isolated a variant strain of HSV-1, called KOSEC, which replicated as efficiently in EC cells as in mouse fibroblasts. KOSEC infected EC and fibroblast cells, synthesized more TK mRNA, more TK enzyme, and more HSV-1 DNA than did the same cells infected with the KOS stain. Both HSV-1 strains induced similar levels of synthesis of gD, an early viral glycoprotein. By co-infection of EC cells with the KOS and KOSEC virus, both the elevated virus yield and the elevated TK synthesis seen in KOSEC-infected cells appeared to be recessive. Apparently a viral mutation that affects expression of some early viral functions can also overcome the EC cell restriction to HSV-1 replication.
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PMID:Restricted replication of herpes simplex virus type 1 in murine embryonal carcinoma cells. 302 90

Using both selection enrichment and site-directed mutagenesis, a herpes simplex virus type 1 (HSV-1) strain 17 genome lacking all four XbaI sites has been generated. The site at 0.45 map units which lies within the gene coding for a polypeptide of 28,000 molecular weight was removed by selection enrichment, while the site at 0.29 map units which lies within the gene coding for glycoprotein H was removed by site-directed mutagenesis. The parental virus from which these two XbaI sites were deleted had previously had the sites at 0.07 and 0.63 map units removed through selection enrichment. The variant devoid of XbaI sites (X4) showed normal growth characteristics; its phenotype was normal apart from the absence of the thymidine kinase protein, which is believed to be unrelated to the loss of XbaI sites.
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PMID:Generation of a herpes simplex virus type 1 variant devoid of XbaI sites. 303 32

Transcription of the type 1 herpes simplex virus (HSV-1) genome in trigeminal ganglia of latently infected mice was studied using in situ hybridization. Probes representative of each temporal gene class were used to determine the regions of the genome that encode the transcripts present in latently infected cells. Probes encoding HSV-1 sequences of the five immediate early genes and representative early (thymidine kinase), early-late (major capsid protein), and late (glycoprotein C) genes were used in these experiments. Of the probes tested, only those encoding the immediate early gene product infected-cell polypeptide (ICP) 0 hybridized to RNA in latently infected tissues. Probes containing the other immediate early genes (ICP4, ICP22, ICP27, and ICP47) and the representative early, early-late, and late genes did not hybridize. Two probes covering approximately equal to 30% of the HSV-1 genome and encoding over 20 early and late transcripts also did not hybridize to RNA in latently infected tissues. These results, with probes spanning greater than 60% of the HSV-1 genome, suggest that transcription of the HSV-1 genome is restricted to one region in latently infected mouse trigeminal ganglia.
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PMID:RNA from an immediate early region of the type 1 herpes simplex virus genome is present in the trigeminal ganglia of latently infected mice. 303 40


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