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 constructed a plasmid that contains a small piece of DNA with two vaccinia promoters running in opposite directions--a promoter from a late gene encoding an 11 K polypeptide (P11) and a promoter from an early gene encoding 25K (P25). These promoters were isolated from the Tian Tan strain of vaccinia virus and were flanked by the thymidine kinase (TK) sequence of the same virus. Genes encoding the hepatitis B virus surface antigen (HBsAg) and the Escherichia coli beta-galactosidase (LacZ) were inserted downstream of the 11 K and 25 K promoters respectively so that coexpression plasmids were constructed. Recombinant vaccinia viruses were selected directly by picking blue plaques formed under overlaying agarose medium containing X-gal. HBsAg was expressed to high level by these recombinant viruses. These recombinant viruses showed reduced virulence on rabbit skin and induced anti-HBs after intradermal inoculation of rabbits.
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PMID:Selection of recombinant vaccinia viruses (Tian Tan strain) expressing hepatitis B virus surface antigen by using beta-galactosidase as a marker. 211 Nov 42

A cDNA clone representing the genome of structural proteins of Japanese encephalitis virus (JEV) was inserted into the thymidine kinase gene of vaccinia virus strains LC16mO and WR under the control of a strong early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. Indirect immunofluorescence and fluorescence-activated flow cytometric analysis revealed that the recombinant vaccinia viruses expressed JEV E protein on the membrane surface, as well as in the cytoplasm, of recombinant-infected cells. In addition, the E protein expressed from the JEV recombinants reacted to nine different characteristic monoclonal antibodies, some of which have hemagglutination-inhibiting and JEV-neutralizing activities. Radioimmunoprecipitation analysis demonstrated that two major proteins expressed in recombinant-infected cells were processed and glycosylated as the authentic PreM and E glycoproteins of JEV. Inoculation of rabbits with the infectious recombinant vaccinia virus resulted in rapid production of antiserum specific for the PreM and E glycoproteins of JEV. This antiserum had both hemagglutination-inhibiting and virus-neutralizing activities against JEV. Furthermore, mice vaccinated with the recombinant also produced JEV-neutralizing antibodies and were resistant to challenge with JEV.
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PMID:Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus. 215 44

Interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes. Three transcriptional control elements within 300 base pairs of the 5'-flanking region of the rat glucagon gene interact with regulatory cellular proteins and direct transcription only in glucagon-producing islet cells. Two islet cell-specific enhancer-like elements (G2, G3) act together with the glucagon promoter (including the G1 element), which confers A cell specificity of glucagon gene expression. In the present study, the G3 element was analyzed in detail by protein binding and in vivo and in vitro transcription assays. Mutational analyses showed that the sequence of the G3 element comprises two distinct protein-binding domains: a more upstream domain A (5'-CGCCTGA-3'), and a more downstream domain B (5'GATTGAAGGGTGTA-3'). Binding of proteins to these two domains is mutually exclusive. Domain A, but not domain B, is responsible for both functional protein binding and the enhancement of transcription from the glucagon or thymidine kinase gene promoter chloramphenicol acetyltransferase reporter gene transfected in vivo into glucagon-producing islet cells (InR1-G9) and transcribed in vitro in a HeLa cell-free transcription system. In islet cell extracts, the Southwestern blot technique labeled a protein of 45 kDa binding to domain A within G3. We conclude that although the G3 sequence contains two protein-binding motifs, the organization of the G3 enhancer-like element is not bipartite. The islet cell specificity of the G3 element is conferred by a tissue-specific transcription factor or protein complex interacting with domain A of G3. This protein or protein complex recognizes different DNA sequences and provides promoter as well as enhancer activity because it binds also to the apparently unrelated sequence of the G1 promoter element.
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PMID:A pancreatic islet cell-specific enhancer-like element in the glucagon gene contains two domains binding distinct cellular proteins. 216 Apr 64

The respective pretreatment prognostic impacts of the following markers were evaluated in 125 patients with small cell lung cancer (SCLC): lactic dehydrogenase (LDH), serum thymidine kinase (S-TK), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and tissue polypeptide antigen (TPA). More traditional clinical and serologic markers were also evaluated. Univariate analysis showed that all of the biochemical markers mentioned above, the Karnofsky index (KI) and the patient's sex were related to both the stage of disease (limited/extensive disease: LD/ED) and to survival. The strongest marker for the clinical stage was S-TK, whereas TPA showed the strongest relationship with survival. Multivariate analyses produced a model consisting of S-TK, CEA, NSE, and the patient's sex for determining the clinical stage. To compare the prognostic capacity of easily determined biochemical and simple clinical variables to the more resource-demanding variable of the clinical stage, three multivariate analyses in relation to survival were performed: (1) biochemical markers and simple clinical variables; (2) LD/ED and simple clinical variables; and (3) all available variables. The model obtained from the first analysis included TPA, KI, age, and the patient's sex; the model from the second analyses included LD/ED, patient's age, and KI; and the model from the third analysis, TPA, KI, age, sex, and LD/ED. Indices based on these three multivariate models were calculated for each patient and the prognostic capacity of these indices was compared. Pretreatment serum marker levels also had the capacity to predict both the grade and the duration of the response to therapy.
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PMID:Clinical and serologic markers of stage and prognosis in small cell lung cancer. A multivariate analysis. 216 41

The carboxyl-terminal one-third of human topoisomerase II polypeptide expressed in Escherichia coli was used as antigen to generate polyclonal antibodies in rabbits. With the use of antiserum, DNA topoisomerase II levels of phytohemagglutinin-stimulated human lymphocytes were measured by immunoblotting. Our results showed that the increase in intracellular topoisomerase II level paralleled the entry of cells into proliferation. We also found that the increase in the topoisomerase II level resulted from an increase in the amount of topoisomerase II mRNA. The time course study indicated that the appearance of topoisomerase II mRNA was first observed at 36 h after phytohemagglutinin stimulation. The maximal level of topoisomerase II mRNA was seen at 45 h after stimulation. The same RNA blot was rehybridized with a thymidine kinase probe. The maximal level of thymidine kinase mRNA was observed at 39 h after phytohemagglutinin stimulation. In a comparison of the time course of topoisomerase II gene expression with that of [3H]thymidine incorporation and thymidine kinase gene expression, it was found that the expression of the topoisomerase II gene was later than the onset of DNA replication. Thus, this study suggests that topoisomerase I, which is constantly expressed throughout the cell cycle, might participate in the initiation of DNA replication, while topoisomerase II is involved in solving the DNA topological problems accompanying DNA strand separation during DNA replication.
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PMID:Induction of topoisomerase II gene expression in human lymphocytes upon phytohemagglutinin stimulation. 216 62

The equine herpesvirus 4 (EHV-4) gene glycoprotein H (gH) gene homologue was localized by virtue of the conserved genomic position of this gene throughout members of the herpesvirus family. The gene maps immediately downstream of the thymidine kinase gene at approximately 0.49 to 0.51 map units within genomic fragment BamH1 C. The EHV-4 gH primary translation product is predicted to be a polypeptide of Mr 94,100, 855 amino acids long, which possesses features characteristic of a membrane glycoprotein, namely an N-terminal signal sequence, a large hydrophilic domain containing 11 putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison to other herpesvirus glycoproteins revealed identities of 85%, 26% and 32% with the gH counterparts of the alphaherpesviruses EHV-1, herpes simplex virus 1 and varicella-zoster virus, respectively, and of 17% and 18% with those of human cytomegalovirus, herpesvirus saimiri and Epstein-Barr virus. The EHV-4 gH exhibits features previously reported to be conserved throughout the gH polypeptides of herpesviruses of all three subgroups. A region of direct repeat elements and a possible origin of DNA replication are located immediately downstream of the gH gene.
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PMID:The nucleotide sequence of an equine herpesvirus 4 gene homologue of the herpes simplex virus 1 glycoprotein H gene. 216 33

Bovine respiratory syncytial (BRS) virus causes a severe lower respiratory tract disease in calves similar to the disease in children caused by human respiratory syncytial (HRS) virus. While there is antigenic cross-reactivity among the other major viral structural proteins, the major glycoprotein, G, of BRS virus and that of HRS virus are antigenically distinct. The G glycoprotein has been implicated as the attachment protein for HRS virus. We have carried out a molecular comparison of the glycoprotein G of BRS virus with the HRS virus counterparts. cDNA clones corresponding to the BRS virus G glycoprotein mRNA were isolated and analyzed by dideoxynucleotide sequencing. The BRS virus G mRNA contained 838 nucleotides exclusive of poly(A) and had a major open reading frame coding for a polypeptide of 257 amino acid residues. The deduced amino acid sequence of the BRS virus G polypeptide showed only 29 to 30% amino acid identity with the G protein of either the subgroup A or B HRS virus. However, despite this low level of identity, there were strong similarities in the predicted hydropathy profiles of the BRS virus and HRS virus G proteins. A cDNA molecule containing the complete BRS virus G major open reading frame was inserted into the thymidine kinase gene of vaccinia virus by homologous recombination, and a recombinant virus containing the BRS virus G protein gene was isolated. This recombinant virus expressed the BRS virus G protein, as demonstrated by Western immunoblot analysis and immunofluorescence of infected cells. The BRS virus G protein expressed from the recombinant vector was transported to and expressed on the surface of infected cells. Antisera to the BRS virus G protein made by using the recombinant vector to immunize animals recognized the BRS virus attachment protein but not the HRS virus G protein and vice versa, confirming the lack of antigenic cross-reactivity between the BRS and HRS virus attachment proteins. On the basis of the data presented here, we conclude that BRS virus should be classified within the genus Pneumovirus in a group separate from HRS virus and that it is no more closely related to HRS virus subgroup A than it is to HRS virus subgroup B.
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PMID:Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus. 221 24

In a previous study, we isolated from the non-T, non-B acute lymphoblastic leukemia cell line, HOON, a molecular complex comprised of a minimum of two polypeptide chains linked by disulfide bonds and expressing two distinct epitopes, 44D7 and 44H7. The DNA from the HOON cell lines has been co-transfected with the herpes simplex thymidine kinase gene into mouse L cells. By using a flow cytometer, a stable thymidine kinase-positive cell population expressing both 44H7 and 44D7 antigens of the human leukemic cells has been generated by repeated sorting of cells reactive with monoclonal antibody 44H7. After three sequential sorting experiments, cloned cell lines were established, and a subclone designated 3D5 has been additionally characterized. Greater than 90% of the 3D5 cells stained positively with monoclonal antibodies 44H7 and 44D7 and with 4F2, which reacts with an epitope identical or spatially related to that seen by 44D7. The ratio of antigenic density 4F2:44D7 calculated from the relative fluorescence indices was similar on the HOON leukemic cells and on the 3D5 transfectant cells. However, 3D5, which was selected with antibody 44H7, expressed a higher ratio of 44H7:44D7 than did the HOON cells. The molecular complex carrying these epitopes was isolated from a 3D5 cell extract on a column of 44D7-IgG-Sepharose and was additionally purified by immunoprecipitation. Although several polypeptide chains were present in the immunoprecipitates, the major polypeptide band had an apparent m.w. of 127,000 under nonreducing conditions. After reduction, three bands of apparent m.w. 91,000, 38,000, and 33,000 appeared. The presence of the 91,000 and 38,000 subunits linked by disulfide bonds was also observed for the 44D7 antigen isolated from HOON cells, whereas the polypeptide of apparent m.w. 33,000 was only seen in immunoprecipitates of the transfectant cell extracts. Because the antigen expressed in the transfectants is associated with a multimeric complex containing disulfide-linked subunits, it is possible that only the gene encoding one of the polypeptide chains, namely that carrying the epitopes, was in fact transfected. This HOON gene product could be one subunit associating with murine subunits encoded by genes of the L cell. Alternatively, the antigenic complex may be the product of closely linked genes transfected together, or of a single human gene that is modified post-translationally to create a disulfide-linked complex.
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PMID:Mouse L cells express a molecular complex carrying the human epitopes recognized by monoclonal antibodies 44D7 and 44H7 after DNA-mediated gene transfer. 242 3

Complementary DNA clones corresponding to the mouse uterus estrogen receptor mRNA have been isolated and characterized. Nucleotide sequence analysis predicts that full-length cDNA has the potential to code for a polypeptide of 599 amino acids, and comparison with the protein sequences of the rat, human, and chicken estrogen receptors reveals overall homologies of 97%, 88% and 77%, respectively. Genomic clones for the mouse estrogen receptor have been isolated from a cosmid library and used in conjunction with the cDNA clones to study the expression of the receptor in vivo by RNase mapping, primer extension, and Northern blotting. These analyses demonstrate that transcription initiates at multiple sites which span a region of at least 62 base pairs and that the estrogen receptor is encoded by mRNA of approximately 6.5 kilobases in size. There are 10 major starts in total, one of which is situated 31 nucleotides downstream from a TATA box-like motif and coincides with the start of the cDNA clone pMOR8. The ability of the cDNA clone to produce a functional protein was verified by transfection into COS-1 cells which lack endogenous estrogen receptor. The mouse estrogen receptor, in a SV40-based expression vector, was cotransfected with a chimeric marker plasmid consisting of an estrogen response element from the vitellogenin A2 gene linked to the thymidine kinase promoter and the chloramphenicol acetyl transferase gene. In the presence of estradiol chloramphenicol acetyl transferase activity is stimulated by up to 80-fold, while tamoxifen and 4-hydroxytamoxifen act primarily as antiestrogens in this in vitro assay.
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PMID:Structural organization and expression of the mouse estrogen receptor. 248 14

KG111 is a mutant of herpes simplex virus (HSV), strain KOS, that exhibits temperature-dependent drug resistance. For example, it is almost as resistant as a thymidine kinase (tk)-deficient virus at 39 degrees, but is relatively sensitive to acyclovir at 34 degrees, Using marker transfer techniques, we have mapped the mutation conferring temperature-dependent drug resistance in KG111 to the 5' portion of the tk gene. Sequencing of this region revealed an amber mutation at codon 44, which lies between the first and second methionine codons of the tk polypeptide. This mutation is identical to that found in TK4, an HSV mutant derived from Cl 101 (L. Haarr et al., 1985, J. Virol. 56, 512-519). Analyses of immunoprecipitated tk proteins from KG111- and TK4-infected cells showed that KG111 and TK4 do not synthesize full-length tk polypeptides, but instead produce a truncated form of the protein. Small amounts of a similar truncated tk polypeptide are also produced in wild-type-infected cells and are thought to arise from initiation at a downstream AUG. The relative amounts and size of the mutant tk proteins compared with those of the wild-type are consistent with the amber mutation eliminating translation of full-length polypeptide and causing a four- to fivefold increase in the utilization of downstream AUG codons for initiation. The truncated polypeptides specified by KG111 and TK4 are less stable than the full-length polypeptide at 39 degrees, which may contribute to the conditional drug-resistant phenotype. On the other hand, the truncated polypeptides normally expressed by wild-type virus at low levels and the more highly expressed truncated tk polypeptides from a deletion mutant are relatively stable at 39 degrees. These results suggest that stability of the truncated tk polypeptide is influenced by the amount of tk present.
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PMID:Effect of an amber mutation in the herpes simplex virus thymidine kinase gene on polypeptide synthesis and stability. 253 79


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