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)

In preparation for studies using gene transfer, we have identified transcriptional control elements which are active in primary rat hepatocytes. We used plasmids which were constructed so that the promoter or enhancer of interest initiated transcription of the bacterial chloramphenicol acetyltransferase (CAT) gene. Plasmids were introduced into primary rat hepatocytes in culture, into Hep G2 cells and other human and animal cell lines and into bone marrow stromal cells, and CAT activity was assayed after 48 hr. In primary rat hepatocytes, the highest CAT activity was obtained with plasmids carrying the Rous sarcoma virus long terminal repeat (pRSVCAT), or the SV40 early region promoter and enhancer (pSV2CAT). Hepatocytes carrying the murine cytomegalovirus immediate early promoter (pUCRNmCMVX/HCAT) also had appreciable CAT activity. No CAT activity was detected in rat hepatocytes carrying pSVOCAT (a promoterless construct), pUCRNtKCAT (herpes simplex thymidine kinase gene promoter), pLPVCAT (lymphocytotrophic papovavirus promoter) and pHBV1CAT (hepatitis B virus enhancer and core gene promoter). Therefore, for future studies of gene transfer in primary rat hepatocytes, the Rous sarcoma virus long terminal repeat or the SV40 early region promoter and enhancer can be effectively used to drive gene expression. Hep G2 cells carrying pHBV1CAT had high CAT activity. Hep G2 cells carrying pHBV2CAT (similar to pHBV1CAT, but with the hepatitis B virus sequences in reverse orientation with respect to the CAT sequences) and pHBV3CAT (similar to pHBV2CAT, but hepatitis B virus sequences are separated from the CAT sequences by about 700 bases) also expressed CAT activity, but not as strongly as with pHBV1CAT.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue-specific activity of heterologous viral promoters in primary rat hepatocytes and Hep G2 cells. 280 56

We investigated the role of sequences flanking the transcription initiation site of the delta 1-crystallin gene in transient transfection assays of primary embryonic chicken lens epithelial cells or fibroblasts. Varying lengths of the 5' flanking sequence of the delta 1-crystallin gene (containing some untranslated sequence from exon 1) were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene in the pSVOCAT plasmid. A plasmid carrying the bacterial beta-galactosidase gene driven by the Rous sarcoma virus (RSV) promoter was used as an internal control. Standardized results showed that the sequence located between -120 to -43 exhibited strong promoter activity; however, the promoter activity was markedly reduced (20-fold) when the upstream sequence between -603 and -120 was included in the construct. The delta 1-crystallin promoter displayed little lens preference. This upstream sequence did not reduce the activity of the Simian virus 40 (SV40) early promoter (with or without its enhancer) or the Herpes thymidine kinase promoter in transfection tests, indicating some specificity in its effect. Evidence for a delta 1-crystallin negative trans-acting factor was provided by competition experiments. Our data raise the possibility that expression of the delta 1-crystallin gene involves a negative cis-acting transcription element, a speculation which may deserve further attention in view of the gradual decrease in delta-crystallin synthesis in the developing lens.
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PMID:Evidence for positive and negative regulation in the promoter of the chicken delta 1-crystallin gene. 283 46

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down
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PMID:The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. 284 67

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

Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic Fo mouse by the "plasmid rescue" technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed.
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PMID:Rearrangements of microinjected recombinant DNA in the genome of transgenic mice. 301 83

We have used a protoplast fusion protocol to introduce the genes encoding neomycin phosphotransferase (neo) and chloramphenicol acetyltransferase (CAT) into murine and human T-lymphocyte lines. Plasmid constructs containing the neo gene under the control of the promoters from the Rous sarcoma virus long terminal repeat (RSV LTR), the SV40 early region, or the herpes simplex virus thymidine kinase gene (HSV TK) can stably transform each of three T-cell lines to G-418 resistance. The characteristic frequencies for different cell lines can differ by at least two orders of magnitude, although initial DNA uptake and transient expression are similar. In the two murine cell lines, low numbers of gene copies are retained in long-term transformants. Prior to integration, transient expression assays for cat or neo gene products reveal that the differences in intrinsic promoter strength of different constructs are further influenced by the coding sequences being transcribed. Thus, while transient expression of the neo protein is similar from both the Rous LTR and the SV40 early promoter, the Rous LTR directs synthesis of CAT protein at levels two orders of magnitude higher than those from the SV40 early promoter.
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PMID:Differential transient and long-term expression of DNA sequences introduced into T-lymphocyte lines. 302 36

Mouse fibroblast cell lines which secrete bovine (b) GH have been generated. This was accomplished by cotransforming mouse L cells (thymidine kinase-negative [TK-] and adenine phosphoribosyl transferase-negative [APRT-]) with DNA molecules encoding the Rous sarcoma virus-long-terminal repeat and bGH genes along with the herpes viral TK gene and the hamster APRT gene. One stable cell line, Pd lambda-pbGH 4-13, was found to secrete approximately 75 micrograms bGH per 24 h/5.0 X 10(6) cells. Media from this cell line were collected for purification of recombinant bGH (rbGH). Purification involved (NH4)2SO4 fractionation, ion-exchange chromatography, and gel filtration on Sephacryl S-200. The rbGH was characterized by bioassay, RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis. Results of these analyses were compared with those obtained with a highly purified pituitary bGH. In the rat tibia bioassay, rbGH was found to be as potent as pituitary bGH. Results from the RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis and Western blot analysis also suggested that the rbGH was similar to that of pituitary origin. Amino acid composition, partial (amino-terminal) sequence, and tryptic peptide maps were also found to be similar between the rbGH and pituitary bGH preparations. The amino terminus of the rbGH showed similar heterogeneity to that of the bGH of pituitary origin. We conclude that rbGH which was synthesized, processed, and secreted from transformed mouse fibroblasts possessed almost exactly the same physiochemical properties as pituitary bGH.
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PMID:Purification and physiochemical properties of a recombinant bovine growth hormone produced by cultured murine fibroblasts. 375

A continuous line of rodent cells carrying the genome of the Rous sarcoma virus was stably transformed by exposure to viable herpes simplex virus of either subtype 1 or 2. Transformation was accompanied by alteration of morphology, growth characteristics, acquisition of a thymidine kinase activity (EC 2.7.1.21) resembling the corresponding specific activity of the enzyme in the herpes simplex virus, and capacity to express continuously some antigens specific for herpes simplex virus.
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PMID:Transformation of cultured mammalian cells by viable herpes simplex virus subtypes 1 and 2. 435 29

Expression of mouse mammary tumor virus (MMTV) proviruses is transcriptionally regulated by glucocorticoid hormones. We have linked the MMTV long terminal repeat (LTR) to the coding region of the herpes simplex virus thymidine kinase gene and used this construction to characterize sequences within the LTR that are involved in glucocorticoid regulation. Our results show that 290 base pairs (bp) of the MMTV LTR, including 190 bp upstream from the start site for transcription, are sufficient to confer regulation on the downstream gene. Deletion of an additional 50 bp, leaving sequences from position -140 to +100, eliminates the response. However, the constitutive level of expression is maintained even after deletion of sequences upstream from position -80, indicating that sequences required for the hormone response can be distinguished from those required for basal expression. We also have shown, by making a 4-bp insertion or a 20-bp deletion around position -107, that the distance between the putative signal for hormone response and the start site of transcription can be varied without affecting regulation. Furthermore, replacement of MMTV sequences from position -59 to +100 with analogous sequences from the Rous sarcoma virus LTR does not change the regulation.
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PMID:A small region of the mouse mammary tumor virus long terminal repeat confers glucocorticoid hormone regulation on a linked heterologous gene. 631 May 97

Most patients with hepatocellular carcinoma have an elevated alpha-feto-protein (AFP) level. This high level of AFP expression is transcriptionally controlled by the 5'-flanking sequence of the AFP gene. Using the 5'-flanking sequence as a promoter for the herpes simplex virus thymidine kinase (HSV-TK) gene in an adenoviral vector (Av1AFPTK1), the therapeutic efficacy of adenovirus-mediated HSV-TK gene transduction, followed by ganciclovir (GCV) administration, was studied in tumors in athymic nude mice. Av1AFPTK1 transduction of two cell lines demonstrated HSV-TK enzyme activity only in the AFP-producing cells (HuH7) and not in the AFP nonproducing cells (SK-Hep-1). As expected, only transduced HuH7 cells were killed by GCV treatment. Transduction by an adenoviral vector harboring a Rous sarcoma virus promoter and HSV-TK gene (Av1TK1) showed enzymatic activity and GCV killing in both cell lines. All HuH7 tumors that were transduced with either Av1AFPTK1 or Av1TK1 completely regressed after GCV treatment. On the other hand, there was complete regression of SK-Hep-1 tumors only when treated with Av1TK1 and GCV and not when treated with Av1AFPTK1 and GCV. Thus, cell-specific killing was achieved by adenoviral vector containing AFP promoter for the HSV-TK gene and GCV treatment.
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PMID:Adenovirus-mediated gene therapy of hepatocellular carcinoma using cancer-specific gene expression. 758 89


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