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)

We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.
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PMID:Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines. 241 16

Mouse macrophages grown from spleen cells were found to be very sensitive to the interferon (IFN) activity against herpes simplex virus type 1 (HSV-1). Therefore we have used these cells to investigate the level at which IFN blocks the replication of HSV-1. IFN treatment resulted in a strong inhibition of the induction of HSV DNA polymerase and other beta proteins. RNA hybridization experiments revealed that the amount of mRNA for the beta protein thymidine kinase was strongly reduced in IFN treated HSV-1 infected cells. Analysis of the effect of IFN on expression of the alpha genes indicated a strong inhibition of alpha protein synthesis. In contrast the synthesis of mRNA of the alpha protein ICP 4 was only moderately inhibited. The results indicate that IFN primarily acts on the translation of HSV alpha proteins.
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PMID:Synthesis of herpes simplex virus proteins and nucleic acids in interferon-treated macrophages. 242 81

Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally reduced to values of 40%, 58.5%, and 54.9%, respectively, at postsuperinfection hour (PSIH) 36. Values of these EHV-1 expressions were subsequently increased at PSIH 48. However, thymidine kinase activity was reduced to a maximum of 67.3% reduction at PSIH 48. In dually infected lymphocyte cultures, the EHV-1 titer, EHV-1 infective centers, EHV-1 enzyme-linked immunosorbent assay antigen titer, and thymidine kinase activity were maximally reduced to values of 77.4%, 78.7%, 98.3%, and 72.9%, respectively, at PSIH 24. These reductions of EHV-1 expressions were completely abrogated at PSIH 48 to 72. In both cell culture systems, a marked interference of EHV-1 by EHV-2 was observed; this was transient in the lymphocyte cultures, but was more prolonged in ED cell cultures. This interference appeared not to be interferon mediated. The multiplication of EHV-2 in the dually infected ED cell cultures appeared unaffected.
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PMID:In vitro interference between equine herpesvirus types 1 and 2. 242 20

Cotransformation with a plasmid containing a thymidine kinase gene (pTK2) and a plasmid encoding human IFN-gamma (pTG11) has been used to establish murine L cell lines expressing human IFN-gamma. The HuIFN-gamma gene was present in 30% of the tk+ cell lines and some of these secreted low levels of IFN into the culture medium. Two of the clones obtained after transformation were selected for detailed analysis. Clone 1-12 constitutively secreted very low levels of HuIFN-gamma in the culture medium. This antiviral activity was characterized by its species specificity and antigenicity as authentic human IFN-gamma In contrast, clone 3-47 produced a HuIFN-gamma activity which could only be detected intracellularly. This clone was resistant to infection both by Vesicular stomatitis (VSV) and Mengo viruses and contained increased levels of enzymes known to be induced by interferon. Our results suggest that clone 3-47 produces a non-secreted HuIFN-gamma like molecule which is able to trigger an antiviral state in the murine cell independent of the interaction with a specific IFN-gamma surface receptor.
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PMID:Expression of extracellular and intracellular human IFN-gamma in mouse L cells transformed with the human IFN-gamma cDNA gene. 242 24

The molecular mechanism of interferon action on vaccinia virus-specific immediate early protein synthesis was studied in interferon-treated chick cells. In line with previous observations, the synthesis of total vaccinia WR virus-specific mRNA, thymidine kinase (TK) mRNA, and several other early mRNAs was detectable by short [3H]uridine pulses. Under conditions of over 90% inhibition of poxvirus-specific TK induction, accumulation of TK mRNA was strongly inhibited. Northern blot analysis revealed strong degradation of residual TK mRNA prepared from interferon-treated chick embryo fibroblasts (CEF). Blot hybridization analysis using total vaccinia DNA and restriction fragment N as probes demonstrated a generally reduced steady-state amount of vaccinia virus-specific early mRNAs in interferon-treated CEF. When CEF were infected with a recombinant vaccinia virus strain into the TK gene of which the chloramphenicol acetyltransferase gene had been inserted, CAT activity was far lower in interferon-treated than in untreated CEF. We conclude that signals that specify rapid breakdown of viral TK mRNA in interferon-treated CEF are located in the regions flanking the coding sequences of the viral TK gene.
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PMID:Reduced steady-state levels of vaccinia virus-specific early mRNAs in interferon-treated chick embryo fibroblasts. 243 97

Human fibroblasts were induced to secrete interferon (IFN) by treatment with the double-stranded RNA poly(inosinic).poly(cytidylic) acid or by infection with Newcastle disease virus. Treatment with 0.1-1 microM dexamethasone reduced the amount of IFN secreted by approximately 40-70%, respectively. A similar decrease in secretion of human IFN-beta was detected in dexamethasone-treated murine C127 cells that carry an IFN expression vector. These cells transcribe constitutively human IFN-beta under the control of a viral thymidine kinase promotor. Secretion of murine IFN induced by double-stranded RNA was also reduced in dexamethasone-treated C127 cells. The amount of IFN-beta mRNA present in fibroblasts and C127 cells was measured by hybridization to complementary RNA. Treatment with dexamethasone markedly reduced the level of IFN-beta mRNA present in both cells. The time course of this decrease was measured in C127 cells; 50 and 80% loss of IFN mRNA was observed after approximately 7.5 and 12 h, respectively. Murine IFN mRNA was also decreased in dexamethasone-treated C127 cells induced with double-stranded RNA. However, the rate of transcription of human IFN mRNA measured by run-on assays in isolated nuclei of dexamethasone-treated C127 cells was found to be comparable to that of control untreated cells. The finding that dexamethasone reduces the level of IFN mRNA transcribed under the control of both its own promotor and an unrelated promotor, together with the observation that dexamethasone does not apparently alter the rate of transcription of this mRNA, suggest that glucocorticoids may regulate IFN production by decreasing the level of its mRNA.
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PMID:The glucocorticoid dexamethasone inhibits synthesis of interferon by decreasing the level of its mRNA. 245 6

Two methods, the colorimetric method (neutral red dye uptake), and DNA hybridization using a HSV thymidine kinase gene probe (TK) have been used to examine the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to two antiviral drugs, acyclovir (ACV) and alpha-interferon (alpha-IFN). Using the colorimetric method, HSV isolates had ED50s ranging from 0.03 +/- 0.02 micrograms/ml to 0.164 +/- 0.03 micrograms/ml for ACV and 6.3 +/- 5.2 IU/ml to 55.0 +/- 11.4 IU/ml for alpha-IFN. With the DNA hybridization method, ED50s ranged from 0.033 +/- 0.012 micrograms/ml to 0.190 +/- 0.031 micrograms/ml for ACV and 8.5 +/- 5.0 IU/ml to 43.5 +/- 6.0 IU/ml for alpha-IFN. Two strains of HSV-1 were found to be resistant to very high concentrations of ACV (greater than 50.0 micrograms/ml). The values obtained by the two methods showed good correlation (r = 0.724, P = 0.002). Furthermore, our results demonstrate that the two methods are reproducible, reliable and the dye uptake assay is suitable for use in a diagnostic virology laboratory.
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PMID:Comparison of two methods in the determination of the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to acyclovir and alpha-interferon. 254 87

A 14 bp interferon (IFN)-stimulated response element (ISRE) from 6-16, a human gene regulated by alpha-IFN, confers IFN inducibility on a heterologous thymidine kinase promoter. A 39 bp double-stranded oligonucleotide corresponding to a 5' region of 6-16 which includes the ISRE competes for factors required for gene expression by alpha-IFN in transfected cells and a single base change (A-11 to C) within the ISRE (GGGAAAATGAAACT) abolishes this competition. Band-shift assays performed with whole-cell extracts and the 39 bp oligonucleotide reveal specific complexes formed by rapidly induced and constitutive factors, both of which fail to bind to the A-11 to C oligonucleotide. A detailed footprinting analysis reveals that these two types of factors bind to overlapping sites within the ISRE, but in very different ways. These data were used to design oligonucleotides which decreased the formation of the inducible complex without affecting the constitutive one. Changes at the 5' margin of the ISRE and upstream of it markedly decrease formation of the induced but not the constitutive complex and also abolish the ability of the 39 bp sequence to function as an inducible enhancer with the thymidine kinase promoter. Thus, induction of 6-16 transcription in IFN-treated cells is likely to be stimulated by binding of the induced factor to the ISRE and upstream sequences, while the subsequent suppression of transcription may involve competition for the ISRE by the other class of factors.
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PMID:Overlapping sites for constitutive and induced DNA binding factors involved in interferon-stimulated transcription. 272 2

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.
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PMID:Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene. 283 Apr 97

The expression of the gene for the murine tissue inhibitor of metalloproteinases (TIMP) is induced in response to viruses, growth factors, and phorbol esters. In this report we show that the accumulation of TIMP mRNA after Newcastle disease virus induction is caused by transcriptional activation of the gene. Comparison of the sequences of cDNA and genomic clones along with RNase protection and primer extension analyses revealed that the murine TIMP gene possesses multiple cap sites and that the exon 1 consists exclusively of 5'-noncoding sequences. We observed that DNA regions analogous to those found upstream of the virus-inducible interferon genes are present within intron 1 of the TIMP gene. To investigate the possible role of TIMP intron 1 in gene expression, we used a functional assay based on the transfection of plasmids in which the DNA segment to be tested is placed in proximity to a marker gene driven by the heterologous herpes simplex virus thymidine kinase promoter. Our results indicate that TIMP intron 1 contains DNA sequence elements capable of modulating the activity of a heterologous promoter in two different ways: (i) by enhancing constitutive expression and (ii) by conferring virus inducibility. These results suggest that intron 1 may be involved in the transcriptional regulation of TIMP gene expression.
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PMID:Presence of transcription regulatory elements within an intron of the virus-inducible murine TIMP gene. 285 Apr 84


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