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)

Cytoplasmic hybrid (cybrid) cell lines were formed by fusing whole cells of rat yolk sac tumor cell line (EST-II) with cytoplasts of mouse fibroblastic cell line (B-82cap), a variant of mouse L cell line that is deficient in thymidine kinase (TK-) and resistant to chloramphenicol (capr). The cybrid cell line with the nucleus of EST-II and cytoplasma of B-82cap was successfully obtained using double selection with HAT and chloramphenicol-containing medium. The cybrid's ability to synthesize proteins such as albumin, alpha-fetoprotein, gamma-EST, and gamma-GTP was found to be approximately one-fourth that of the nuclear donor, EST-II, at a early time of growth in culture, but this was followed by a gradual increase during the period of observation. The nude mice undergoing subcutaneous transplantation of 1 X 10(6) cells of EST-II and cybrid were killed by the tumor growth, with the survival time being 56 +/- 11 and 105 +/- 25 days, respectively. The histologic findings of cybrid tumor closely resembled those of the nuclear donor, EST-II. These facts suggest that factors from both the nucleus and cytoplasma will be able to affect the gene expression of the cybrid cell line.
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PMID:Protein synthesis and tumorigenicity of the cytoplasmic hybrid between rat yolk sac tumor and mouse fibroblastic cell line. 658 79

Co-microinjection of single linearized molecules of plasmids containing the human beta-globin gene (pRK1) and the herpes simplex virus (HSV) type I thymidine kinase gene (pX1) into the mouse TK-L cell nucleus results in covalent linkage between these (or derived) molecules within the nucleus as revealed by Southern blotting, plasmid rescue, and recovery of plasmid-derived DNA from a Charon 4A phage library of cellular DNA. The microinjected DNA is predominantly found as high molecular weight DNA as determined by Hirt fractionation. Southern blotting data and recombinants from the Charon 4A library suggest that the plasmid DNA is in the form of a head-to-tail linear concatamer of up to 80 copies. Passage of these microinjected cells in selective medium (HAT) results in coordinate amplification of both plasmids, which are maintained in an approx. 3:1 molar ratio of pRK1 to pX1-derived molecules. Hybridization in situ shows the DNA to be integrated on a translocation chromosome, t(4;4). Integration does not appear to be site-specific, since plasmid DNA from another microinjected cell line, C2B, appears on a different translocation chromosome, t(8?;14). Plasmid rescue experiments confirm a previous finding that passage of pBR322 DNA through eukaryotic cells may result in deletions of normally stable plasmid DNA upon subsequent transformation of E. coli. These deletions appear to occur in the bacteria, and originate in a 128 bp region between the Sal I and Hae II sites of pBR322.
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PMID:Fate and structure of DNA microinjected into mouse TK-L cells. 673 47

A 0.9-kilobase DNA fragment from the genome of Moloney murine leukemia virus, including the viral long terminal repeat, was covalently linked to the herpes simplex virus I thymidine kinase (tk) gene whose promoter was previously removed. The hybrid DNA structure was introduced into the chromosome of tk- mouse cells at single copy numbers, via transfection procedures. Cells expressing the newly introduced tk gene were identified by the HAT selection procedure and analyzed for tk- and moloney murine leukemia virus-specific DNA and RNA sequences by blot hybridization procedures. Expression of the tk gene is dependent on function(s) provided in cis by the viral DNA fragment. Vectors derived from this region are termed rGag (rG) vectors.
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PMID:Construction of a mammalian transducing vector from the genome of Moloney murine leukemia virus. 717 18

Mouse teratocarcinoma cells (OTT6050) deficient for thymidine kinase were fused with rat hepatoma cells ( Fu5AH ) deficient for hypoxanthine phosphoribosyltransferase using inactivated Sendai virus. The hybrid cells were selected and cultured in the presence of HAT medium. A clonally established hybrid cell line ( As3 ), which in addition to its mouse genome contains several rat chromosomes, expresses rat specific enzyme variants and produces large primarily undifferentiated tumors, with some hepatoma characteristics in athymic nude mice. To reveal the in vivo developmental potential of these cells and to determine whether, under different experimental conditions, they are capable of participating in tissue differentiation, the As3 cells were injected into mouse blastocysts from the C57BL/6 strain. The experimental blastocysts were then transferred into the uteri of pseudopregnant foster mothers to allow further development. From a total of 212 blastocysts transplanted, 61 fetuses developed and were analysed for As3 contributions between the 10th and 18th day of gestation. Four fetuses at day 18 showed hybrid cell participation in their livers and a few organs of only endo-mesodermal origin, as judged from the presence of rat-specific enzyme variants. The enzymes were organ-specifically expressed (e.g., lactate dehydrogenase) or appeared newly during in situ differentiation while being absent in the original hybrid cells (e.g., glycerol-3-phosphate dehydrogenase). During short in vitro culture of the chimaeric organs, it was possible to select for the hybrid cells which reverted to an enzyme pattern simiar to but not identical with the As3 cell line and different to that observed in situ.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue preference and differentiation of malignant rat x mouse hybrid cells in chimaeric mouse fetuses. 718 53

PEG-mediated fusion between mouse Cl1d cells and primary Chinese hamster spleen cells produced interspecific hybrids which slowly and nonrandomly segregated Chinese hamster chromosomes. Cytogenetic and isozyme analysis (31 loci) of HAT and BrdU selected hybrid clones and subclones and of members of a hybrid clone panel retaining different combinations of Chinese hamster chromosomes enabled provisional assignment of the following enzyme loci on Chinese hamster chromosomes: thymidine kinase, galactokinase, and acid phosphatase-1 to chromosome 7; galactose-1-phosphate uridyltransferase to chromosome 2; and adenosine kinase, esterase D, glutathione reductase, glyoxalase, nucleoside phosphorylase, peptidases B and S, and phosphoglucomutase (PGM) 2 to chromosome 1. Assignments of PGM1, 6-phosphogluconate dehydrogenase, and enolase 1 to chromosome 2 were confirmed, and a chromosome 2 deletion (q23-q33) enabled the provisional assignment of PGM1 to that region. The assignments provide markers for the study of the genetic consequences of chromosomal rearrangements in Chinese hamster cell lines and support the concept of conservation of mammalian autosomal linkage groups.
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PMID:Confirmational, provisional, and/or regional assignment of 15 enzyme loci onto Chinese hamster autosomes 1, 2, and 7. 732 47

We describe a new retroviral vector system pSXLC/pHa that utilizes a putative internal ribosome entry site (IRES) from encephalomyocarditis virus downstream from a multicloning site to co-express drug-selectable markers with a second non-selectable cDNA in a eukaryotic expression vector. The positive drug-selectable marker, MDR1, and the positive-negative marker, herpes simplex virus thymidine kinase (HSV-TK), were successfully introduced and expressed in the pSXLC/pHa system. The pSXLC-MDR and pSXLC-TK vectors contain the drug-selectable genes under translational control of the IRES and multiple cloning sites upstream for insertion of second cDNAs which can be co-expressed in this system. The inserts of these pSXLC plasmids were designed for easy transfer to the pHa retrovirus vector which has a strong promoter from Harvey murine sarcoma virus. The IRES-MDR-carrying retroviral vector, pHa-MCS-IRES-MDR, conferred resistance to vincristine and adriamycin. The IRES-TK-containing vector, pHa-MCS-IRES-TK conferred HAT-resistance in TK-deficient cells and the transfectants showed hypersensitivity to ganciclovir. These "flexible" vectors should be useful for co-expression of genes for selectable gene transfer and for positive-negative (suicide) selections in vitro and in vivo.
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PMID:Efficient expression of drug-selectable genes in retroviral vectors under control of an internal ribosome entry site. 776 14

After transfection of amplification-promoting DNA elements into mammalian cells, homogeneously staining regions (HSRs) are formed by high copy numbers of transfected DNA arranged in head-to-tail polymers. Here, we wanted to evaluate the stability of this type of HSR during prolonged cultivation of transfected cells in selective medium. Thymidine kinase-deficient mouse L cells were transfected with pAPR4tk DNA harboring the amplification-promoting element 4 (APR4) linked to the gene for thymidine kinase (TK) or, alternatively, transfected with a DNA construct (pARP4t-PA) carrying, in addition, the expression cassette for human tissue-type plasminogen activator (t-PA). After transfection, one or two HSRs per cell were formed that disintegrated spontaneously after 25-40 wk of continuous cultivation in the presence of selective HAT (hypoxanthine-aminopterin-thymidine) medium. Unexpectedly, plasmid DNA reinserted into a plethora of new chromosomal sites, as revealed by in situ hybridization and Southern blot analysis. Coincidently, secretion of t-PA decreased to 10-20% of its original level. After transfection of pAPR4tk DNA lacking the t-PA expression cassette, HSR decay and reintegration of plasmid constructs into multiple chromosomal sites were also observed, whereas the ptk vector without an amplification-promoting DNA element did not form an HSR after transfection. We conclude that, in contrast to the pattern of known structures with head-to-tail arrangements, the HSR formed by amplification-promoting DNA elements represents a novel type of HSR that disintegrates by transposition into a plethora of new chromosomal integration sites. This process is mediated by the amplification-promoting DNA element itself and can be observed even when selective pressure is maintained.
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PMID:A novel type of unstable homogeneously staining region with a head-to-tail arrangement: spontaneous decay and reintegration of DNA elements into a plethora of new chromosomal sites. 795 55

We report here the assignment of TK1, the gene for thymidine kinase to Syrian hamster (Mesocricetus auratus) chromosome 9 (MAU9) by complementation mapping. Syrian hamster chromosomes derived from a wild type (TK+) subline of BHK cells were introduced via microcell-mediated chromosome transfer into B82 mouse cells deficient in thymidine kinase (TK-), a defect that prevents their growth in HAT culture media. Hybrid clones were selected in HAT media. Chromosome analyses of the microcell hybrids showed that the thymidine kinase deficiency of B82 cells was corrected by MAU9. Therefore, we assigned TK1 to MAU9. Previously, TK1 was assigned to mouse chromosome 11 (MMU11), rat chromosome 10 (RNO10), Chinese hamster chromosome 7 (CGR7), and human chromosome 17 (HSA17). The striking banding homology of MAU9 with RNO10, MMU11, CGR7 and HSA17 provides additional support for the assignment of TK1 to MAU9. To our knowledge, this is the first report of gene assignment to a specific Syrian hamster chromosome using the somatic cell hybridization technique.
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PMID:Assignment of TK1 encoding thymidine kinase to Syrian hamster chromosome 9 by microcell-mediated chromosome transfer. 812 17

A human x Chinese hamster (CH) somatic cell hybrid subclone deficient in HPRT and containing only human chromosome 18 was irradiated with 7000 rad and fused to a thymidine kinase deficient CH cell line. Radiation-rescued hybrid cell lines, selected in HAT medium, were analyzed for human DNA with human interspersed-repeat sequence primers. Size and number of human chromosome fragments retained in a subset of hybrids were determined by FISH. A panel of 98 radiation hybrids (RH) was selected and analyzed for 90 chromosome 18-specific STSs by PCR, and for the D18Z1 centromeric marker by Southern blotting. STSs were developed from previously mapped RFLP loci and from published sequences. In addition, 32 novel STSs were generated from an 18-specific lambda library and from 18-specific YACs previously localized to chromosome bands by FISH. Marker retention frequency varied from 8-65% with an average of 24%. In selected RH the STS typing data were correlated with the chromosome 18 regions retained using 'reverse FISH' of IRS-PCR products from the RH to normal metaphase chromosomes. The order and intermarker distances of loci were determined using two-point and multipoint maximum likelihood methods. The resulting RH map covers most of chromosome 18 with four groups of tightly linked markers and three regions of loosely linked markers, one around the centromere and two on the long arm. More than a third of the markers are polymorphic and allow integration with the linkage map. This RH map provides a framework for establishing a clone contig of the entire chromosome 18.
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PMID:A radiation hybrid map of human chromosome 18. 812 19

Mouse x rat somatic cell hybrids were generated by fusing mouse cell lines that are heterozygous for reciprocal translocations involving the T42H and T9Ad breakpoints on mouse chromosome 11 (MMU11) to a thymidine kinase-negative (Tk-) rat cell line, RT2Tk-. Selection in HAT medium with geneticin disulfate (G418) resulted in some hybrid clones retaining only one derivative translocation chromosome with that part of MMU11 carrying the Tk-1 locus. Southern blot and PCR analyses of these hybrids were used to map the two breakpoints and 30 markers relative to them. The T42H breakpoint has been localized between Mpo and the Cola-1/Hox-2 cluster of loci and is proximal to the T9Ad breakpoint. The T9Ad breakpoint is proximal to the distal loci Tk-1, Gaa, D11Jkn1, and P4hb. The positions of 14 loci (Hox-2, Cola-1, Rara, Phb, Erba, Rnula-1, D11Pas1, Gfap, D11Mit13, D11Mit11, D11Mit12, Myla, Empb3 and Gh) have been further refined by their localization between the two breakpoints in band D. This study therefore improves the correlation of the genetic and physical maps of MMU11 and extends the known homology between MMU11 and human chromosome 17 (HSA17) by the assignment of three additional HSA17 markers, the profilin gene, Pfn, an anonymous marker, D17s28h, and the Crk oncogene, to above the T42H breakpoint; and the prohibitin gene, Phb, to between the T42H and T9Ad breakpoints in band D on MMU11.
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PMID:Somatic cell hybrid mapping on mouse chromosome 11 (MMU11): assignment of markers relative to two breakpoints in band D. 844 98


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