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)

An intragenic enhancer in the pol gene of human immunodeficiency virus type 1 has previously been identified (Verdin et al., Proc. Natl. Acad. Sci. USA 87:4874-4878, 1990). This element is composed of two subdomains both exhibiting phorbol ester-inducible enhancing activity on the viral thymidine kinase promoter in HeLa cells. Examination of the nucleotide sequence of one of these domains (nucleotides 4079 to 4342, HXB2 isolate) revealed the presence of three short DNA regions highly homologous to the recognition site for cellular transcription factor AP-1. Two short oligonucleotides containing these AP-1 sites each functioned as a phorbol ester-inducible enhancer when cloned upstream of the thymidine kinase promoter and transfected into HeLa cells. Gel mobility shift assays and competition experiments using the same two oligonucleotides demonstrated that they bound affinity-purified AP-1 or AP-1 present in uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced HeLa nuclear extracts. Footprinting experiments confirmed that all three predicted sites bound purified AP-1. These results suggest that the AP-1 factor could play a role in the transcriptional regulation of human immunodeficiency virus type 1 gene expression.
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PMID:The intragenic enhancer of human immunodeficiency virus type 1 contains functional AP-1 binding sites. 194 59

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by cis-acting DNA elements located in the viral long terminal repeats, by viral transregulatory proteins, and by cellular transcription factors acting in concert to modulate the degree of viral expression. We demonstrate that a DNA fragment corresponding to the central portion of the HIV-1 genome exhibits enhancer activity when cloned upstream of the thymidine kinase promoter of herpes simplex virus. This enhancer is inducible by phorbol 12-myristate 13-acetate in HeLa cells and is independent of its position and orientation with respect to the promoter. We have mapped the activity of the enhancer to two independent domains encompassing nucleotides 4079-4342 (end of the pol gene) and nucleotides 4781-6026 (vif gene and first coding exon of tat). This intragenic enhancer and its subdomains demonstrate cellular specificity because they are only active in specific cell lines. The presence of similar intragenic enhancer elements in other retroviruses suggests that they might be a conserved feature of this family of viruses.
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PMID:Identification and characterization of an enhancer in the coding region of the genome of human immunodeficiency virus type 1. 235 55

The acyclovir-induced herpes simplex virus Type 1 (HSV-1) strain, R9C2, a double mutant in thymidine kinase (TK) and DNA polymerase (DNA pol), and its parental strain SC16 were compared for their effects on ocular pathology and systemic immunity after unilateral inoculation into the anterior chamber (AC) of BALB/c mouse eyes. Although AC-injected R9C2 produced no retinal necrosis (0/18 eyes), this mutant induced active suppression (33-87%) of anti-HSV delayed type hypersensitivity similar to that induced by another HSV strain, KOS. AC-injected parental strain, SC16, caused fatal disease within 7-10 days, and induced bilateral retinal necrosis and suppression of DTH in 100% of the mice. Preimmunization with R9C2 protected mice in a dose-dependent fashion from the pathologic and lethal effects of AC-injected parental virus. These data suggest that the immunogenicity of the TK and DNA pol double mutant remains intact despite the decreased ocular and systemic pathogenicity observed after intracameral inoculation.
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PMID:Immunogenicity versus pathogenicity after anterior chamber inoculation of an acyclovir-induced double mutant of HSV-1. 282 59

Chimeric plasmids in which the herpes thymidine kinase (tk) gene replaced portions of the Rous sarcoma viral genome were used to assess the relationship between viral transcription and that of an exogenous gene located within the viral genome. The entire tk gene and portions of the gene were positioned in both orientations within the viral gag and pol genomic region (which serves as intron for viral env mRNA). Microinjection assays then determined the amount of viral genomic transcription by quantitation of the amount of viral env mRNA produced. Separate injections also assayed for the presence of tk mRNA. Both mRNAs were produced unless the 3' region of the tk gene was present within the viral genome and in the same transcriptional sense. In this case viral env mRNA production was nearly abolished.
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PMID:The effects of transcriptional regulatory sequences introduced into a retroviral genome. 301 46

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U; azidothymidine [AZT]) had potent bactericidal activity against many members of the family Enterobacteriaceae, including strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, and Enterobacter aerogenes. AZT also had bactericidal activity against Vibrio cholerae and the fish pathogen Vibrio anguillarum. AZT had no activity against Pseudomonas aeruginosa, gram-positive bacteria, anaerobic bacteria, Mycobacterium tuberculosis, nontuberculosis mycobacteria, or most fungal pathogens. Several lines of evidence indicated that AZT must be activated to the nucleotide level to inhibit cellular metabolism: AZT was a substrate for E. coli thymidine kinase; spontaneously arising AZT-resistant mutants of E. coli ML-30 and S. typhimurium were deficient in thymidine kinase; and intact E. coli ML-30 cells converted [3H]AZT to its mono-, di-, and triphosphate metabolites. Of the phosphorylated metabolites, AZT-5'-triphosphate was the most potent inhibitor of replicative DNA synthesis in toluene-permeabilized E. coli pol A mutant cells. AZT-treated E. coli cultures grown in minimal medium contained highly elongated cells consistent with the inhibition of DNA synthesis. AZT-triphosphate was a specific DNA chain terminator in the in vitro DNA polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I. Thus, DNA chain termination may explain the lethal properties of this compound against susceptible microorganisms.
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PMID:Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U). 355 32

We have used site-directed mutagenesis of cloned Moloney murine leukemia virus (MuLV) DNA to define a function encoded in the 3' region of the viral pol gene and required for efficient integration of viral DNA. One mutant, MuLV-SF1, contained a single base substitution (C to T at base 4950) that resulted in an arginine to cysteine change in a region highly conserved among retroviruses. Mutant DNA, introduced into rat cells by cotransfection with a herpes simplex virus thymidine kinase gene (HSV tk), directed production of virus particles with reverse transcriptase activity. Infection of cells with these particles led to synthesis of full-length linear and circular forms of unintegrated viral DNA; however, integrated viral DNA was decreased at least by a factor of 10 when examined by DNA hybridization, and the mutant particles were less efficient then wild-type virus at establishing an infection by a factor of at least 300. Pseudotypes formed with the proteins of MuLV-SF1 and the genome of a replication defective marker MuLV, carrying the HSV tk gene, were less effective by at least a factor of 100 in producing tk+ colonies than pseudotypes formed with proteins encoded by wild-type virus. When the MuLV-SF1 pseudotypes did produce tk+ cells, most of the proviruses were integrated aberrantly. We conclude that the MuLV-SF1 pol gene is defective for a function that is required for normal integrative recombination and dissociable from DNA synthesis.
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PMID:A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. 620 50

We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.
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PMID:Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. 631 91

Adult T cell leukemia/lymphoma (ATL) is derived from CD4+ T cells and has a poor prognosis because of its resistance to chemotherapy. To evaluate the effectiveness of gene therapy for ATL, the effect of ganciclovir on ATL cell lines transfected with the thymidine kinase gene of herpes simplex virus type 1 (HSV-TK) was analyzed. To transfer the HSV-TK gene to ATL cells, a human immunodeficiency virus (HIV) vector that has specific infectivity to CD4+ cells was used. HSV-TK was inserted into the long terminal repeats of HIV-1 and driven by the SL3 promoter HXBSL3TK. HXBSL3TK was co-transfected with HXBCAT as a reporter into MT2 or HUT102 cells by DEAE-dextran. The cells were incubated with ganciclovir, and chloramphenicol acetyltransferase (CAT) activity was analyzed. The CAT activity of the MT2 cells and HUT102 cells transfected with HXBSL3TK decreased dose-dependently with ganciclovir. HXBSL3TK was also co-transfected into COS cells with an HIV-1 packaging vector that has gag, pol, and env driven by a cytomegalovirus promoter. The supernatant was transferred to MT2 cells or Raji cells and incubated with ganciclovir. Ninety percent of the MT2 cells transduced by HXBSL3TK and incubated with ganciclovir were killed, but Raji cells were not killed. In addition, HXBTK that expresses the HSV-TK gene and Tat gene driven by the LTR of HIV-1 was constructed. HXBTK had a higher expression of the HSV-TK gene and higher sensitivity to ganciclovir than did HXBSL3TK.
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PMID:Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1. 895 10

The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.
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PMID:The E249K mutator mutant of DNA polymerase beta extends mispaired termini. 1058 71

Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1 alpha (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro, as well as transgene amplification and persistence in vivo. Retrovirus titers of >10(5) infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10- to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin.
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PMID:Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses. 1097 74


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