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 previously reported a family with generalized resistance to thyroid hormone (GRTH) which had a point mutation with codon 448 CCT (proline) being converted to ACT (threonine) in the thyroid hormone receptor (TR) beta. To characterize functional properties of the mutant TR beta, transient expression studies were performed in COS cells. A double stranded oligonucleotide encompassing thyroid hormone response element (TRE) derived from the rat GH gene was synthesized. We constructed chloramphenicol acetyl transferase (CAT) plasmid containing the thymidine kinase promoter under the control of the rat GH TRE. T3 induction of CAT activity by the mutant TR beta was significantly reduced as compared with that of the normal TR beta. This was observed in the presence of 0.5-50 nM T3, but not at 500 nM T3. When the normal and mutant TR beta were cotransfected, the mutant TR beta inhibited gene activation regulated by the normal TR beta. However, a high molar excess was necessary to significantly inhibit the function of the normal receptor. Additionally, the binding of in vitro synthesized mutant TR beta to TRE was preserved.
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PMID:Transcriptional activity of a mutant thyroid hormone receptor beta in a family with generalized resistance to thyroid hormone. 130 92

Treatment of HSV-infected cells with 5-10 mM beta-hydroxynorvaline (Hnv), a threonine analog, specifically affects herpesvirus DNA replication: both the rate of and total DNA synthesis are reduced, the former approximately 15-fold by Hnv (6 h post-infection) and the latter by 12-fold (between 3 and 12 h post-infection). The effect on DNA replication was due to inhibition of HSV-1 thymidine kinase (TK) and DNA polymerase (DP) activities; the former is reduced by 75% and whereas DP returns to baseline levels (when compared to untreated and/or uninfected cells). Host cell TK and DP activities are unaffected. It is suggested that beta-hydroxynorvaline is incorporated into these enzyme(s), either close to or at the active site thus perturbing viral DNA synthesis. beta-Hydroxynorvaline should have unique utility as a targeted antiviral compound, acting on both membrane-mediated phenomena (fusion, penetration and attachment) and DNA replication.
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PMID:Inhibition of herpesvirus-induced thymidine kinase and DNA polymerase by beta-hydroxynorvaline. 282 76

The thymidine kinase encoded by herpes simplex virus type 1 contains an amino acid sequence homologous to a consensus sequence related to the ATP-binding site in many proteins. We have used site-directed mutagenesis to investigate the importance of the five highly conserved amino acids within this segment. When any one of the three glycines was changed to valine the corresponding mutant enzyme was inactive. The mutation of lysine 63 to isoleucine destroyed the enzymatic activity. When threonine 64 was changed to alanine the mutant enzyme lost its activity. However, when this threonine was changed to serine the enzyme was still active but with different apparent Michaelis constants (Km) for thymidine and ATP. The wild-type thymidine kinase has apparent Km's of 0.5 and 20 microM for thymidine and ATP, respectively, while the mutant enzyme displayed Km's of 2.3 and 60 microM for thymidine and ATP. These results indicate that this homologous segment is essential for the function of the thymidine kinase and is involved in the substrate binding domain of the enzyme.
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PMID:Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. 283 27

In the course of studies on the a sequences located at the termini of and at the junction between the L and S components of herpes simplex virus 1 DNA, J. Chou and B. Roizman (J. Virol. 57:629-637, 1986) noted that the a sequence acted as a gamma 1 promoter when fused to the structural sequence of the thymidine kinase gene, the b inverted repeat sequences located in the L component next to the a sequences contained an open reading frame predicted to encode the protein of 358 amino acids with a molecular weight of 37,054, and the transcription of an RNA homologous to the open reading frame initiated within the a sequence. The nucleotide sequence of the open reading frame predicted the presence of the triplet Ala-Thr-Pro repeated 10 times. To verify the existence of the predicted gene, designated gamma 134.5, a synthetic peptide consisting of the triplet Ala-Thr-Pro repeated 10 times was synthesized and used to raise antibodies in rabbits. The results were as follows. The antiserum to the peptide reacted with a 43,500-apparent-molecular-weight protein present in lysates of cells infected with herpes simplex virus 1 but not present in mock-infected or herpes simplex virus 2-infected cells. We genetically engineered a recombinant virus containing a single copy of a truncated gene. Concordant with predictions, the antibody reacted with a faster-migrating protein in cells infected with this recombinant. The gamma 134.5 gene product was soluble, and it accumulated primarily in the cytoplasm late in infection. The overlap of the domain of the gamma 134.5 gene with the a sequence raises the possibility that it acts in trans on the a sequence and is associated with one of the functions currently ascribed to the a sequences.
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PMID:Identification by antibody to a synthetic peptide of a protein specified by a diploid gene located in the terminal repeats of the L component of herpes simplex virus genome. 300 91

In order to estimate the effects of protein and amino acids on regenerating liver, the induction of enzymes involved in synthesis of DNA was studied in rats fed protein free diet. In the regenerating livers of rats of the protein free diet, increase of liver weight and DNA content were stopped 48 hours after hepatectomy, and induction of DNA synthesizing enzymes such as dCMP deaminase, ribonucleotide reductase, and thymidine kinase were depressed and shortened. On the other hand, induction of protein or RNA synthesizing enzymes such as polyamine, ornithine decarboxylase, and tyrosine aminotransferase were not depressed by protein deprivation. The results indicate that protein deprivation inhibits the DNA synthesizing enzymes specifically, and regenerating liver cells can not enter S phase of cell cycle. When rats were maintained solely by total parenteral nutrition after hepatectomy, amino acids were essential for induction of DNA synthesizing enzymes. In particular, induction of these enzymes were regulated by 7 amino acids include Val, Leu, Ile, Met, Trp, Phe, and Thr, and most of these plasma amino acid levels were depressed after hepatectomy. By administration of amino acids for 12 hours just after hepatectomy, the DNA synthesizing enzymes were almost normally induced. This suggests that amino acids administration just after hepatectomy is effective to induce the DNA synthesizing enzymes for hepatic regeneration.
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PMID:[The effects of protein and amino acids on DNA synthesis in regenerating liver]. 308 37

The 545-residue Cln2 protein, like the other G1 cyclins of Saccharomyces cerevisiae, is a very unstable protein. This instability is thought to play a critical role in regulating cell cycle progression. The carboxyl-terminal domains of Cln2 and the other G1 cyclins contain sequences rich in Pro, Glu (and Asp), Ser, and Thr (so-called PEST motifs) that have been postulated to make up the signals that are responsible for the rapid degradation of these and other unstable proteins. To test this hypothesis, the carboxyl-terminal 178 residues of Cln2 were fused to the C terminus of a reporter enzyme, a truncated form of human thymidine kinase (hTK delta 40). The resulting chimeric protein (hTK delta 40-Cln2) retained thymidine kinase activity but was markedly less stable than hTK, hTK delta 40, or an hTK-beta-galactosidase fusion protein, as judged by enzyme assay, immunoblotting with anti-hTK antibodies, pulse-chase analysis of the radiolabeled polypeptides, and ability to support the growth of a thymidylate auxotroph (cdc21 mutant) on thymidine-containing medium. Thus, the presence of the Cln2 PEST domain was sufficient to destabilize a heterologous protein. Furthermore, the half-life of hTK delta 40-Cln2 was similar to that of authentic Cln2, and the rate of degradation of neither protein was detectably enhanced by treatments known to cause G1 arrest, including exposure of MATa haploids to alpha-factor mating pheromone and shifting cdc28ts and cdc34ts mutants to the restrictive temperature. These results suggest that the major signals responsible for Cln2 instability are confined to its C-terminal third. Because hTK delta 40-Cln2 and Cln2 were expressed from heterologous promoters yet their half-lives both in asynchronous cultures and when arrested at various cell cycle stages were always similar, the Cln2 PEST domain contains a signal for rapid protein turnover that is constitutively active and operative throughout the cell cycle. Removal of the 37 codons that encode the most prominent PEST-like segment from either hTK delta 40-Cln2 or Cln2 decreased the turnover rate of the resulting proteins, as expected; however, an hTK delta 40 chimera containing only this 37-residue segment was not detectably destabilized, suggesting that this PEST sequence, when removed from its normal context, is not a self-contained determinant of protein instability.
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PMID:G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover. 796 35

The mechanism responsible for the decreased sensitivity of a clinical herpes simplex virus type 1 (HSV-1) isolate, HSV-145, to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was examined. Measurements of 50% inhibitory doses of several drugs demonstrated that although HSV-145 was sensitive to phosphonoacetic acid, adenine arabinoside and acyclovir, its sensitivity to BVDU and 5-(2-chloroethyl)-2'-deoxyuridine was significantly less than that normally observed for HSV-1. Analysis of the thymidylate kinase (TMP-K) activity of HSV-145 thymidine kinase (TK) demonstrated a decreased level of TMP-K activity when compared to HSV-1 TK. The TMP-K activity of HSV-145 resembled that observed for HSV-2 and the TK-deficient strain HSV-1 TK-7. When the nucleotide sequence of the HSV-145 TK gene was compared to that of the HSV-1 strains C1(101) and SC16 a single nucleotide substitution (G changed to A at base position 502) was detected which would result in the substitution of threonine at amino acid position 168 for alanine. The substitution is the same as that for the laboratory-derived BVDU-resistant virus HSV-1 SC16B3. Collectively, these studies highlight the importance of amino acid conservation at position 168 of the HSV-1 TK in conferring efficient TMP-K activity and BVDU sensitivity.
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PMID:Analysis of the thymidine kinase of a herpes simplex virus type 1 isolate that exhibits resistance to (E)-5-(2-bromovinyl)-2'-deoxyuridine. 802 3

The present work has examined the effects of okadaic acid, an inhibitor of serine/threonine protein phosphatase, PP-1 and PP-2A, on the regulation of EGR-1 gene expression in normal peripheral blood T- and Jurkat cells. The results demonstrate that okadaic acid treatment is associated with a transient induction of EGR-1 gene expression which was detectable by 30 min to 1 h and peaked at 3-6 h. EGR-1 mRNA was superinduced in cells treated with both okadaic acid and the protein synthesis inhibitor cycloheximide. The half-life of EGR-1 mRNA was similar in both control and okadaic acid-treated cells. In contrast, treatment with both okadaic acid and cycloheximide prolonged the half-life of EGR-1 transcripts. Nuclear run-on assays demonstrated that induction of EGR-1 gene expression by okadaic acid is controlled at least in part by a transcriptional mechanism. Transient expression assays with EGR-1 promotor fragments linked to the chloramphenicol acetyltransferase gene demonstrate that okadaic acid-induced EGR-1 transcription is conferred by the 5' most distal CArG box, CC (AT)6GG, in the EGR-1 promoter. Moreover, chloramphenicol acetyltransferase activity was induced by okadaic acid when the 5' most distal CArG element was linked to the heterologous herpes simplex virus thymidine kinase promoter, and not induced with a similar heterologous construct containing a mutated CArG sequence. These studies demonstrate that okadaic acid regulates EGR-1 gene expression at the transcriptional level via the CArG element and suggest that PP-1 and PP-2A play a role in T-cell activation.
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PMID:Involvement of serum response element in okadaic acid-induced EGR-1 transcription in human T-cells. 817 32

The molecular mechanisms for signaling by receptor serine/threonine kinases are incompletely understood. To test the potential involvement of p21 H-Ras proteins in signal transduction for type beta transforming growth factors (TGF beta), TGF beta-responsive and constitutive reporter genes were cotransfected into cardiac myocytes and mink lung epithelial cells, with dominant inhibitory (Asn-17) or activated (Arg-12) Ras expression vectors. Asn-17 Ras inhibited both TGF beta-dependent and basal expression of inducible promoters (skeletal alpha-actin and plasminogen activator inhibitor-1), with equivalent dose-response relations. All seven reporter constructs were comparably sensitive to down-regulation by Asn-17 Ras, including those driven by nominally constitutive viral control regions or a TATA-less initiator element. All constructs were up-regulated by Arg-12 Ras more variably. Wild-type Ras had intermediate effects and could rescue a minimal thymidine kinase promoter from inhibition by dominant negative Ras. Thus, a Ras-dependent event is required for efficient expression of an unexpectedly global or inclusive set of genes.
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PMID:p21 Ras as a governor of global gene expression. 819 82

Earlier studies have shown that the thymidine kinase-negative baby hamster kidney (BHKTK-) cell lines expressing constitutively the herpes simplex virus 1 (HSV-1) glycoprotein D (gD), designated BJ, restrict infection by HSV-1 at the level of virus entry. U10, a HSV-1 mutant not restricted by the BJ cells, carried the substitution of proline for Leu25 in the gD gene, suggesting that gD encodes a specialized domain which precludes virus entry into cells expressing gD. Analyses of a new series of 36 unrestricted viral mutants showed the following. (i) Only two mutants contained mutations at a site which did not overlap with the previously reported mutation. A representative of a previously mapped mutant and one of the two new mutants were examined in detail. Thus, in the gD of mutant U30 Ala185 was replaced by threonine, whereas in gD of U21, Ala185 and Leu25 were replaced with threonine and proline, respectively. U30 and U21 multiplied better than the wild-type parent virus in the parental BHKTK- cells. (ii) Transfer of the gD gene from U21 or U30 to wild-type parent virus or to the gD- virus FgD beta yielded recombinants which, while capable of infecting BJ cells, were considerably less efficient than the parent unrestricted mutants, suggesting that the latter contained additional mutations which were responsible in part for the unrestricted phenotype. Conversely, marker rescue of mutant viruses with wild-type gD reduced but did not abrogate entirely the unrestricted phenotype. (iii) Mutations in gD which conferred the unrestricted phenotype were not random. (iv) gD plays a role in the restriction, inasmuch as preincubation of cells expressing gD with antibodies to gD abolished restriction. (v) In mutant R5000, the gD substitution Ser140 to Asn was capable of overcoming a restriction of a BHKTK- clonal line which does not express gD but conferred very low ability to replicate on BJ cells. We conclude that (a) uncloned stocks of BHKTK- cells exhibit a low level restriction to infection with wild-type virus, (b) clonal lines of BHKTK- cells which vary with respect to the stringency of restriction express either allelic genes differing in the properties of their products or products of different genes, and (c) both the restricted and unrestricted phenotypes reflect the interactions of gD with these cellular products. The implications of these conclusions with respect to the restriction imposed on BHK cells by the expression of gD are discussed.
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PMID:Mapping of herpes simplex virus 1 genes with mutations which overcome host restrictions to infection. 820 98


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