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)

Alignment of prokaryotic and vertebrate type II thymidine kinases (TK) (EC 2.7.1.21), such as that encoded by vaccinia virus (VVTK), reveals three conserved regions: designated domains I, III, and VII. Domains I and III of VVTK contain residues which closely resemble segments A (ATP) and B (Mg2+), respectively, of a Mg.ATP binding descriptor proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951). In support of this hypothesis, domain I of the VVTK enzyme has previously been identified as the ATP binding site (Black and Hruby, 1990b). With regard to Mg2+ binding, several features of the VVTK domain III suggest that it may be responsible for this activity: 1) sequence similarity to a magnesium binding motif proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951); 2) alignment of the predicted secondary structure of type II TK enzymes with other magnesium-binding enzymes such as adenylate kinase, EF-TU, and p21 reveals a conserved aspartic acid residue preceded by several hydrophobic residues with domain III; and 3) the conserved VVTK domain III aspartic acid residue (D82) aligns with D93 residue of adenylate kinase which is has been shown by NMR to participate in Mg2+ binding (Yan, H., and Tsai, M.-D., Biochemistry, in press). To directly examine the potential contribution of the conserved domain III D82 residue of VVTK in magnesium binding, site-directed mutagenesis was performed on positions D82 and G84 to generate four mutants, N82, L82, I82, and V84. Each mutant was analyzed for enzyme activity, divalent cation requirements, tetramer formation, and ATP binding ability. The results obtained were consistent with D82 playing a direct role in Mg2+ binding and suggest that while the aspartic acid does not appear to participate directly with ATP binding it may instead act to facilitate ATP hydrolysis by binding Mg2+ which aids to correctly position ATP for nucleophilic attack.
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PMID:Site-directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase. Evidence for a potential role in magnesium binding. 155 87

We have previously described the inhibition of glucocorticoid-dependent transcription from the mouse mammary tumor virus long terminal repeat promoter by products of the H-ras and v-mos oncogenes. We have studied the effects of conditional oncogenes on expression of glucocorticoid-dependent indicator genes. Expression of the glucocorticoid-dependent transcription of the tyrosine aminotransferase gene was monitored in FTO-2B rat hepatoma cells during Mr 21,000 protein (p21) H-ras induction. A strong transcriptional repression of the tyrosine aminotransferase gene followed p21 H-ras expression. The sequences in a glucocorticoid-dependent promoter which are responsible for the oncogene-mediated repression could be localized to the glucocorticoid response element; a construct in which a 15-base pair glucocorticoid response element was inserted 5' of the thymidine kinase promoter exhibited the oncogene-mediated repression of transcription. We observed a strong repression of glucocorticoid-dependent promoters and promoter constructs not only in the presence of p21 H-ras and p37 v-mos but also with p60 v-src. p57 v-myc, however, had no effect. Oncogene expression is not a sufficient prerequisite for an initial repression of glucocorticoid hormone-dependent gene transcription, since even in the presence of constitutively high levels of oncogene product a transient stimulation of glucocorticoid-dependent gene expression was found. Protein synthesis inhibition experiments revealed that no hormonally induced cellular protein is needed for the oncogene-mediated repression. It seemed reasonable that this phenomenon might reflect oncogene effects on the glucocorticoid receptor. We, therefore, made measurements of the glucocorticoid receptor protein. In the presence of glucocorticoid hormone the receptor translocated rapidly from the cytoplasm to the nucleus. In normal NIH 3T3 cells, after 24-h treatments the nuclear receptor levels had declined to about 50% of those determined at 2 h and in the presence of p21 H-ras they declined to 15%. The levels of cytoplasmic receptor were not affected by p21 H-ras expression.
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PMID:Oncogene mediated repression of glucocorticoid hormone response elements and glucocorticoid receptor levels. 256 9

The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells.
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PMID:Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product. 298 21

The nucleotide sequence of a 2549-bp DNA fragment containing the entire coding region of the marmoset herpesvirus (MarHV) thymidine kinase gene (tk) and the flanking sequences was determined by the dideoxynucleotide chain termination method. The MarHV thymidine kinase polypeptide predicted from the nucleotide sequence contained 376 amino acids and had a molecular weight of 41,281. The sequencing data also reveal that the coding portion of another MarHV gene probably begins only 292 nucleotides downstream from the stop codon of the MarHV tk gene. There was relatively little nucleotide sequence homology between the MarHV tk gene and that of the herpes simplex virus (HSV) types 1 and 2 tk genes. Comparisons of the predicted amino acid sequences of the MarHV thymidine kinase polypeptide with that of the HSV-1 and HSV-2 thymidine kinase polypeptides, however, revealed clear, but interrupted, homology within several regions of the polypeptide chains. Amino acid sequence homology was particularly striking at residues 10 to 27 of the MarHV thymidine kinase polypeptide and residues 49 to 66 of the HSV-1 and HSV-2 thymidine kinase polypeptides. These same amino acid residues exhibit noticeable sequence homology to the mitochondrial beta subunit ATPase, oncogene p21 protein, adenylate kinase, and to other nucleotide-binding proteins. It has been proposed that the indicated regions of homology are elements of a nucleotide-binding pocket in ATPase, p21, and adenylate kinase, raising the possibility that amino acid residues 15 to 25 of the MarHV thymidine kinase and 54 to 64 of the HSV-1 and HSV-2 enzymes are likewise parts of nucleotide-binding sites.
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PMID:Nucleotide sequence of the marmoset herpesvirus thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide. 633 Sep 76

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

alpha 1-Chimaerin mRNA, which encodes a neuron-specific GTPase-activating protein for the signal transduction molecule p21 Rac, is highly expressed in certain brain regions and neuronal cell lines. The promoter region of human alpha 1-chimaerin transcriptional unit contains no TATA box, Sp1-binding site or initiator motif. However, a CCAAT box located in the proximal promoter region is essential for promoter activity. We now describe a negative regulatory element in the 5' untranslated region of exon 1 of the human alpha 1-chimaerin gene. Deletion of this 70-bp region from the alpha 1-chimaerin minimal promoter increased the promoter activity 5- to 6-fold. The negative element can suppress heterologous thymidine kinase promoter activity in an orientation-independent manner when placed in its native position. However, its function is position-dependent. The presence of a putative factor in rat liver, HepG2 and SK-N-SH cell nuclear extracts but not in rat brain nuclear extract which interacts with this element suggests a possible role of the negative element in controlling the neuron-specific expression of alpha 1-chimaerin in vivo.
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PMID:The human neuronal alpha 1-chimaerin gene contains a position-dependent negative regulatory element in the first exon. 889 65

Gene transfer and antisense therapy offer novel approaches to the study and treatment of vascular diseases. The localized nature of vascular diseases like restenosis has made the application of genetic material an attractive therapeutic option. Viral and nonviral vectors have been developed to facilitate the entry of foreign DNA or RNA into cells. Vector improvement and production, demonstration of vector safety and demonstration of therapeutic efficacy are among the main present challenges. Various strategies have already been shown to be successful in preventing restenosis in animal models and include: the transfer of the herpes simplex virus thymidine kinase associated with ganciclovir: transfection of the cell cycle regulatory genes encoding for the active form of retinoblastoma gene product (Rb) or the cyclin-dependent kinase inhibitor p21, and antisense therapy. Therapeutic angiogenesis using gene transfer is a new strategy for the treatment of severe limb ischemia. Transfection of DNA encoding for the vascular endothelial growth factor has resulted in increasing collateral flow in animal models of peripheral ischemia. This approach is currently being investigated in a clinical trial in patients with distal ischemia. Other potential targets for genetic treatment in cardiovascular diseases include thrombosis, extracellular matrix synthesis and lipid metabolism.
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PMID:Gene and other biological therapies for vascular diseases. 910 54

Vascular gene transfer may be useful for the treatment of several cardiovascular diseases. It can also be used as an experimental tool to test the effects of various genes in a local vascular compartment. Promising therapeutic effects have been obtained in animal models of restenosis with the transfer of vascular endothelial growth factor, nitric oxide synthase, thymidine kinase, retinoblastoma, growth arrest homeobox gene, cyclin/cyclin-dependent kinase inhibitor (p21), and hirudin genes, and antisense oligonucleotides against transcription factors or cell cycle regulatory proteins. Vascular endothelial growth factor and fibroblast growth factor gene transfers have improved blood flow and capillary development in models of ischaemic limb and myocardium. First experiences of vascular endothelial growth factor gene transfer to human peripheral arteries have also been reported. However, further developments in gene delivery techniques and gene transfer vectors will be required before a full therapeutic potential of gene therapy in cardiovascular diseases can be evaluated.
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PMID:Vascular gene transfer. 918 44

In Lactobacillus acidophilus R-26, the synthesis of DNA precursor deoxynucleotides occurs exclusively by salvage of deoxynucleosides, beginning with phosphorylation by four deoxynucleoside kinases. Subunits bearing three of these activities are uniquely organized into two heterodimers, deoxyadenosine/deoxycytidine kinase (dAK/dCK) and deoxyadenosine/deoxyguanosine kinase (dAK/dGK), which, along with a distinct deoxythymidine kinase (TK), catalyze the parallel first committed steps of dNTP biosynthesis. Whereas TK is common to most prokaryotes (and eukaryotes), the other three activities that are the emphasis of this review are quite unusual in bacteria. Each activity is regulated in cis by its homologous end-product (dNTP) which is understood to act as a multisubstrate inhibitor capable of binding to both nucleoside and phosphate subsites. Conversely, the inactive dAK subunit is progressively activated by 1) association with a dGK or dCK subunit and 2) the conformationally driven heterotropic affect of dGuo or dCyd bound to the opposing subunit. Limited proteolysis has proven to be a powerful probe of conformational states. Further indication of conformational or structural differences between dAK and dGK (or dCK) is that the former follows an ordered kinetic path, while dGK or dCK exhibits rapid-equilibrium random kinetics. The multi-substrate behavior of end-product binding provides a convenient new diagnostic tool for distinguishing kinetic mechanisms. Tandem dak-dgk genes have been cloned from Lactobacillus DNA and expressed in Escherichia coli as dAK/dGK, utilizing the associated promoter. Sequence alignments reveal 65% identity in their DNA and 61% in their derived amino acid sequences. Encoded N-terminal sequences are identical for the first 18 residues, and both subunits share conserved sequences in common with adenylate kinase and viral TK. A more unusual conserved element, which appears to play a role in the activation of dAK, resembles the G2 loop of p21 ras. Remarkably, no homologous gene(s) for the dAK/dCK pair could be found. Comparisons of amino acid sequences, isoelectric pHs and subunit masses strongly indicated that native dCK and dGK are identical in sequence, except at their extreme N-termini (M-IVL for dCK and -TVIVL for dGK), suggesting that processing of a common precursor occurs in Lactobacillus. Accordingly, deletion of codons 2 and 3 from dgk resulted in the expression of dAK/dCK in the E. coli host; its kinetic properties are indistinguishable from those of native dAK/dCK. Subcloning the dgk or engineered dck gene resulted in expression of active dGK or dCK homodimers, each with a virtually unchanged Km toward its primary deoxynucleoside. However, in common with human dCK, dCK (or dGK) homodimer exhibits secondary activities with much larger Kms towards dAdo and dGuo (or dCyd). dCTP (or dGTP) is the best inhibitor of all three activities of the respective homodimer. Fully active heterodimers can be reconstituted simply by mixing a homodimer with independently expressed (inactive) dAK.
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PMID:Life on the salvage path: the deoxynucleoside kinase of Lactobacillus acidophilus R-26. 942 44

Our laboratory has developed two cellular models of human prostate cancer progression. The LNCaP prostate cancer progression model is based upon the well-known cellular interaction between human prostate or bone stromal cells and LNCaP cells in vivo. The marginally tumorigenic LNCaP cells acquired tumorigenic and metastatic potential upon cellular interaction with either prostate or bone fibroblasts. A subline termed C4-2 was observed to grow readily in castrated animals and acquired metastatic potential spreading from the primary tumor site to the lymph node, the seminal vesicles, and the axial skeleton, resulting in an intense osteoblastic reaction. The second model is ARCaP, where prostate cancer cells derived from the ascites fluid of a man with metastatic disease exhibited an Androgen- and estrogen-Repressed Prostate Cancer cell growth and tumor formation in either a hormone-deficient or a castrated environment. However, the growth of either the tumor cells in vitro or the tumors in vivo was suppressed by both estrogen and androgen. While the tumor cells expressed low levels of androgen receptor and prostate-specific antigen (PSA), they were highly metastatic when inoculated orthotopically. Distant metastases to a number of organs were detected, including the liver, lung, kidney, and bone. We have employed a human prostate cancer progression model as a system to study the efficacy of gene therapy. Results of the study show that whereas universal promoters, such as Cytomegalovirus (CMV) and Rous Sarcoma Virus (RSV) promoter-driven tumor suppressors (e.g. p53, p21, and p16), were effective in inhibiting prostate tumor growth, the advantages of driving the expression of therapeutic toxic genes using a tissue-specific promoter prostate-specific antigen (PSA) and a tumor--but not tissue-specific promoter, osteocalcin (OC), are preferred. In the case of the PSA promoter, we can achieve cell-kill in PSA-producing human prostate cancer cells. To circumvent the supporting role of bone stroma for prostate cancer epithelial growth, we have recently developed a novel concept where the expression of therapeutic toxic genes is driven by a tumor--but not a tissue-specific OC promoter. Osteocalcin-thymidine kinase (OC-TK) was found to efficiently eradicate the growth of osteosarcoma, prostate, and brain tumors both in vitro and in vivo. We observed that androgen-independent human prostate cancer cells lines expressed OC-TK at higher levels than androgen-dependent human prostate cancer cell lines. We have obtained data to suggest that Ad-OC-TK plus a pro-drug acyclovir (ACV) may be used as an effective therapy to treat prostate cancer bone metastasis in models where the growth of androgen-independent PC-3 and C4-2 tumors in the bone has occurred.
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PMID:Human prostate cancer progression models and therapeutic intervention. 943 28


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