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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To initially determine the effect that base-pair mismatch has on homologous recombination in mammalian cells, we have studied genetic recombination between thymidine kinase (tk) gene sequences from herpes simplex virus 1 and 2. These tk genes are approximately 81% homologous at the nucleotide level. We observed that, in mouse LTK- cells, intrachromosomal recombination between type 1 and type 2 tk sequences is reduced by a factor of at least 1000 relative to the rate of intrachromosomal recombination between homologous type 1 tk sequences. In sharp contrast, the rate of intermolecular or intramolecular extrachromosomal recombination between the heterologous tk sequences introduced by calcium phosphate or microinjection was reduced only by a factor of 3 to 15 compared with extrachromosomal homologous tk crosses. Our results suggest differences between the mechanisms of extrachromosomal and intrachromosomal recombination in mammalian cells.
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PMID:Differential effects of base-pair mismatch on intrachromosomal versus extrachromosomal recombination in mouse cells. 303 44

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, we have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic Rm of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of Mr = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity. 1) The Mr = 24,000 polypeptide co-migrates with thymidine kinase activity in electrophoretic and sedimentation analyses. 2) A subunit Mr = 25,504 is predicted by the nucleotide sequence of a recently isolated cDNA clone that encodes HeLa thymidine kinase. 3) Mouse LTK- cells transformed with this clone express a cytosolic thymidine kinase activity, as well as a novel Mr = 24,000 polypeptide detectable with immune serum raised against the purified human enzyme.
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PMID:Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells. 333 3

The cDNA for mouse thymidine kinase (TK) was isolated from a cDNA library in lambda-gt11 and sequenced. It was used as a probe to follow the time course of TK mRNA expression in growth stimulated mouse fibroblasts. Linked to the HSV-TK promoter the cDNA was able to transform LTK-cells to the TK+ phenotype. The transformed cells expressed the TK mRNA and enzyme activity in a growth dependent fashion suggesting that the regulatory element is localized on the cDNA.
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PMID:Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA. 382 14

Chromosome-mediated gene transfer (CMGT) of the human genes for hypoxanthine phosphoribosyl transferase (HPRT) and cytosol thymidine kinase (TK1) into HPRT deficient mouse A9 cells or TK deficient Swiss mouse 3T3TK- cells was found to occur at frequencies at least one order of magnitude higher than DNA-mediated gene transfer (DMGT). The frequency of CMGT into 3T3TK- cells was reduced by more than an order of magnitude by a posttreatment of the recipient cells with dimethyl sulphoxide (DMSO). After CMGT, expression of the non-selected genes coding for galactokinase (GALK) and acid alpha-glucosidase (GAA), both syntenic with TK1, was observed in a number of transformants. From the pattern of cotransfer, a tentative gene ordering of CENTROMERE-GALK-TK1-GAA on human chromosome 17 was deduced. Chromosome-mediated cotransfer of X-linked human phosphoglycerate kinase (PGK) with HPRT was observed in two out of 33 A9 transformants analysed. DNA-mediated cotransfer of a syntenic gene was only observed for GALK, cotransferred with TK1 in two out of 18 TK+ transformants of mouse LTK- cells. Therefore, with murine cells as recipients of human donor genetic material, CMGT results in a higher frequency of transfer and a higher incidence of cotransfer of syntenic genes than DMGT using cellular DNA in the same cell system.
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PMID:Cotransfer of syntenic human genes into mouse cells using isolated metaphase chromosomes or cellular DNA. 388 35

A recombinant lambda phage containing mouse mammary tumor virus (MMTV) proviral DNA was isolated from a gene library constructed from GR mouse liver DNA. Restriction enzyme analyses reveal that the cloned molecule contains a copy of one of the GR endogenous MMTV proviruses flanked on both sides by 2--3 kb of mouse genomic DNA. In this report we have examined the expression of the cloned MMTV provirus after cotransfection with the herpes thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase,, EC 2.7.1.21) gene and integration into mouse LTK- cells. Nine individual TK+ transformants were selected, and all were found to contain MMTV-transfected DNA. One of the TK+ transformants was chosen for further study. Total poly(A)-containing RNA was isolated from the cells, and liquid hybridization analyses with MMTV cDNA showed that it contained 0.02% MMTV-specific RNA. The sizes of the MMTV-specific species were determined and found to correspond to the 35S and 24S mRNAs synthesized in MMTV-infected cells. Glucocorticoid hormones have been shown to increase the concentration of MMTV RNA in virus-infected cultured cells. Therefore, we tested the effect of dexamethasone on the concentration of MMTV-specific RNA in cells transfected with the MMTV proviral DNA. The amount of MMTV-specific poly(A)-containing RNA found in the cells grown in the presence of hormone was 0.17%. Therefore, dexamethasone causes an 8-fold increase in the amount of MMTV-specific RNA in mouse cells containing several copies of a cloned and transfected MMTV proviral gene.
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PMID:Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells. 626 58

A recombinant plasmid based on pBR322 has been constructed which carries the replicator proximal early region of SV40 DNA, including the viral origin of replication (ORI). It lacks a major part of the tumour antigen 3'-coding region, the large T-antigen termination codon and the polyadenylation site. The recombinant plasmid was transferred together with the herpes simplex virus thymidine kinase gene, as a selectable marker into mouse LTK- cells. Integration and expression of the cloned SV40 gene fragment in TK+ transformants could be demonstrated by DNA restriction and blot hybridization and by immunofluorescence techniques.
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PMID:Production of a T-antigen-related protein in mammalian cells after stable transformation with a cloned SV40 gene fragment. 628 Nov 54

Recently we have described that the Herpes simplex virus (HSV)-induced thymidine kinase (TK) induces AMP- and ADP-dThd-5'-phosphotransferase activities. We now demonstrate the heterogeneity of the described activities in isoelectric focusing experiments and polyacrylamide gel electrophoresis. A TK--mutant of HSV type 1 fails to induce these activities. The activities of the type 1 enzyme complex was neutralized by an anti-HSV-serum. The TK-enzyme complex expressed in LTK--cells transformed to a TK+-phenotype by sheared HSV-1 DNA was compared with the wild type TK complex in isoelectric focusing experiments. Additionally we demonstrate that the HSV type 1 enzyme complex has thymidylate kinase activity, while the type 2 TK complex did not exhibit thymidylate kinase activity. Feedback regulation mechanisms by dTMP, dTDP and dTTP were investigated using partially purified enzyme preparations of HSV types 1 and 2 infected TK--cells.
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PMID:Analysis of the TK enzyme complex induced by HSV types 1 and 2 by means of isoelectric focusing and polyacrylamide gel electrophoresis. 628 60

The recombinant plasmid p102 based on pBR322 carrying approximately equal to 50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK- cells. TK+ cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA . DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed.
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PMID:Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells. 628 10

All cells that harbor the Epstein-Barr virus (EBV) genome contain a neoantigen in the nucleus (EBNA). By transfection we located a segment of the genome that encodes or induces an antigen serologically related to EBNA. The responsible genes are found in the 3.4-megaldalton BamHI fragment K of EBV DNA, specifically in the left 1.9 megadaltons represented by HindIII fragment I1. Mouse LTK- cells were cotransformed with recombinant plasmids, containing the herpes simplex virus thymidine kinase gene and either EcoRI fragment B or BamHI fragment of K of EBV DNA. The TK+ cells surviving in selective medium were cloned. About 50% of the clones expressed the neoantigen in every nucleus. These mouse cells were used as antigens in immunofluorescence tests. Antibody to the nuclear antigen was found in 30 human sera known to contain antibody to EBNA; it was not detected in 18 sera that did not have antibody to EBNA. Mouse cells expressing EBNA as the result of acquisition of cloned EBV DNA fragments should prove useful in the characterization of the structure of this antigen and as reagents for the diagnosis of EBV infections.
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PMID:Stable expression in mouse cells of nuclear neoantigen after transfer of a 3.4-megadalton cloned fragment of Epstein-Barr virus DNA. 629 Oct 59

To observe the effects of polyoma virus DNA on the expression of the herpes simplex virus (HSV) thymidine kinase (TK) gene early after transfer into TK-deficient mouse cells and the subsequent development of stable TK-positive transformants, we constructed a series of recombinant plasmids containing the herpes simplex virus TK gene joined with various segments of the polyoma virus genome and microinjected them into the nuclei or cytoplasm of LTK-A cells (TK(-), APRT(-)). The frequency of nucleus-injected cells expressing TK after 1 day, measured by autoradiography of cells incubated with [(3)H]thymidine, increased approximately 30-fold when the plasmids contained the polyoma virus origin of replication. The origin includes sequences with homology to the simian virus 40 origin of replication and adjoining sequences, including a recently defined transcription-enhancing sequence. After microinjection of a single origin-containing plasmid molecule per cell, TK expression was detected in approximately 50% of the injected cells. When a larger number of origin-containing plasmid molecules were injected per cell, all cells showed early TK activity. When the entire polyoma virus early region was present, neighboring uninjected cells became TK positive. When plasmids were injected into the cell cytoplasm, approximately 400 times as many molecules per cell were needed to cause early TK activity. The frequency of stable transformation observed 2 weeks after nuclear injection of 10 to 20 polyoma virus origin-containing plasmid molecules per cell was at least 2 orders of magnitude greater than with plasmids containing the TK gene alone. The greatest enhancement of stable TK transformation was obtained with plasmids containing the origin alone, when the maximum frequency of stable transformation was 5%. The addition of the coding regions for the small and medium T antigens or the entire early region significantly decreased TK transformation frequency in a copy-dependent fashion. The timing of stabilization of TK-positive transformation was analyzed by releasing hypoxanthine-aminopterin-thymidine selection pressure at various times after microinjection, culturing the cells in nonselective medium, and assaying for TK activity. Stabilization was found to occur between 3 and 6 days after nuclear injection. Cells injected with a plasmid containing the origin and the early region were examined for expression of the large T antigen with polyoma virus antitumor serum and immunofluorescent staining. The expression of the large T antigen was clearly associated with a cytopathic effect. TK-positive clones observed 2 weeks after injection of the plasmid were uniformly T antigen negative. Cytotoxicity may be the result of plasmid replication and toxic levels of T antigen or TK. In addition, expression of the large T antigen may block stabilization by preventing the integration of origin-containing plasmid molecules.
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PMID:Expression and stabilization of microinjected plasmids containing the herpes simplex virus thymidine kinase gene and polyoma virus DNA in mouse cells. 630 96


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