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)

Mouse teratocarcinoma cells from the OTT6050 ascites tumor were established in tissue culture and selected for 5-bromodeoxyuridine (BrdUrd) resistance. The embryonal carcinoma cells grew without a feeder layer, remained deficient for thymidine kinase (EC 2.7.1.75), and differentiated like the original tumor into various tissues after subcutaneous injection into 129 mice. We fused the BrdUrd-resistant mouse teratocarcinoma cells with HT1080-6TG human diploid fibrosarcoma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and selected for hybrid cells in hypoxanthine/aminopterin/thymidine medium. The resulting hybrid cells segregated human chromosomes quickly and retained one to three human chromosomes including chromosome 17 that carries the human genes for thymidine kinase and galactokinase (EC 2.7.1.6). Single hybrid cells from five independent clones containing human chromosome 17 were injected into mouse blastocysts bearing several genetic markers that affect the coat color phenotype and strain-specific enzyme variants in order to detect tissue differentiation derived from the injected cells. After the injection of single hybrid cells into a total of 103 experimental blastocysts that had been surgically transferred to pseudopregnant foster mothers, 49 mice were born and 2 of them clearly revealed coat mosaicism. In 2 of 17 mice thus far analyzed, the injected hybrid cells proved to be capable of participating substantially in development of seven different organs. However, human gene products have not yet been detected unequivocally in those tissues and weak human-specific galactokinase activity could be recovered only from two mosaic tissues. Our results demonstrate that, after in vitro culture and selection, at least some of the human-mouse hybrid cells still retain their in vivo potential to differentiate and become functionally integrated in the living organism. It now seems feasible to cycle mouse teratocarcinoma cells carrying human genetic material through mice via blastocyst injection to study human gene expression during differentiation.
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PMID:Chimeric mice derived from human-mouse hybrid cells. 20 75

Retinoic acid (RA) receptors (RARs) are ligand-inducible transcription factors that bind to specific DNA sequences associated with the regulatory regions of RA-regulatable genes. Since RA has been implicated as an important factor both in normal development and in teratological studies, one would like to have a model system that detects the presence of activated receptors during development. We have constructed a recombinant reporter gene that has three copies of the RA response element (RARE) from the RAR beta-2 promoter 5' to the herpes simplex virus thymidine kinase promoter; this regulatory region is coupled to the bacterial beta-galactosidase reporter gene. This construct was RA inducible in transient transfection assays in F9 embryonal carcinoma cells. Transgenic embryos with this reporter gene construct exhibited restricted and reproducible patterns of beta-galactosidase activity during embryogenesis, beginning between gestational ages day 7.5 and 8.5. At day 8.5, beta-galactosidase activity was detected in the closed neurotube and somites. Day 8.5 embryos, from pregnant females fed RA 14 hr earlier, exhibited a greater intensity and distribution of beta-galactosidase activity. Similarly, at later stages of gestation, maternal RA exposure resulted in enhanced embryonic beta-galactosidase expression. This type of transgenic indicator mouse should be useful in detailing the role of activated RARs during embryonic development.
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PMID:Transgenic indicator mice for studying activated retinoic acid receptors during development. 131 86

The mouse forms of human keratins 18 and 8 (K18 and K8) are the first members of the large intermediate filament gene family to be expressed during embryogenesis. To identify potential regulatory elements of the human K18 gene, various recombinant constructions were expressed in cultured cells. An enhancer element was found in the first intron that functions on both the K18 and thymidine kinase promoters in differentiated cells. In F9 embryonal carcinoma cells, the level of expression was low in the presence or absence of the first intron. Cotransfection of F9 cells with K18 constructs that include the first intron and increasing amounts of an expression vector of c-jun results in a modest increase in the reporter gene expression. Cotransfection of the same construct with increasing amount of the mouse c-fos gene results in activation of the reporter gene by as much as 15-fold, with a near linear response to the amount of c-fos gene added. Site-specific mutagenesis of a putative AP-1 site within the intron abolishes trans-activation by c-fos in F9 cells. Furthermore, induction of c-fos in a derivative of F9 cells results in increased expression of the endogenous mouse form of K18. Cotransfection with c-jun or c-fos expression vectors had little effect on the expression of the K18 reporter construct in a parietal endodermal cell line already expressing the endogenous mouse gene. These results identify an enhancer within the first intron of K18 that may interact directly with c-jun and c-fos via a conserved AP-1-binding site. K18 expression in undifferentiated F9 cells may be limited by the low levels of c-jun and c-fos.
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PMID:Activation of an intron enhancer within the keratin 18 gene by expression of c-fos and c-jun in undifferentiated F9 embryonal carcinoma cells. 169 35

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex (MHC) class I genes. The binding occurs through the GG(T/A)CA motif present also in many other genes. The role of H-2RIIBP in developmental regulation of MHC class I genes has been studied in undifferentiated N-Tera2 embryonal carcinoma cells by transient cotransfection of an expressible H-2RIIBP plasmid and a chloramphenicol acetyltransferase reporter gene linked to the MHC class I promoter. Transfection of the expression plasmid led to production of H-2RIIBP transcripts and enhanced MHC class I promoter activity in cells that were treated with retinoic acid but not yet differentiated. Retinoic acid concentrations required for transactivation overlapped with those capable of inducing morphological differentiation and expression of endogenous MHC class I genes in these cells. This enhancement was mediated by region II, as a heterologous thymidine kinase promoter driven by region II also served as a target for H-2RIIBP transactivation. Deletion of the bulk of the DNA-binding domain or the ligand-binding domain of H-2RIIBP, but not of the N-terminal domain, abolished transactivation, indicating that the former two domains are critical for the enhancement. Moreover, H-2RIIBP transactivation exhibited a strict cell-type restriction. As observed in other cell lines, N-Tera2 cells that had undergone differentiation failed to elicit transactivation, suggesting that H-2RIIBP acts in concert with a cofactor expressed in undifferentiated N-Tera2 cells that requires retinoic acid for its function. These results suggest that H-2RIIBP can function as a developmentally specific transcription factor for MHC class I genes.
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PMID:Retinoic acid-dependent transactivation of major histocompatibility complex class I promoters by the nuclear hormone receptor H-2RIIBP in undifferentiated embryonal carcinoma cells. 173 9

Undifferentiated mouse embryonal carcinoma (EC) cells are capable of transactivating the adenovirus EIIa promoter in the absence of its normal transactivator, E1A protein, suggesting that EC cells contain an E1A-like activity. In an effort to identify where this activity appears during normal mouse development, mouse oocytes and preimplantation embryos were injected with plasmids containing the EIIa promoter coupled to various reporter genes. These expression vectors were fully active in human 293 cells where E1A is present, but were inactive in differentiated fibroblast cell lines unless cotransfected with the E1A gene. In mouse oocytes and preimplantation embryos, EIIa promoter activity in the absence of adenovirus E1A protein was equivalent to or greater than activity of the HSV thymidine kinase promoter coupled to a strong enhancer. Coinjection of the E1A gene failed to stimulate EIIa activity further, perhaps because c-myc protein, which has been reported to transactivate this promoter, was already present at high levels in mouse oocytes. Activation of the EIIa promoter in the absence of E1A was unique to mouse oocytes and preimplantation embryos because gene expression from an EIIa promoter introduced into transgenic mice was observed only in the adult ovary, and particularly in the oocytes. In addition, post-implantation transgenic embryos failed to express the E1A-activatable reporter gene, thereby indicating that expression from the EIIa promoter is restricted to the relatively undifferentiated stages of oogenesis and preimplantation development. These data suggest that cellular promoters of the class that can be transactivated by E1A may serve uniquely to initiate transcription of genes that are needed for preimplantation development.
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PMID:Transactivation of the adenovirus EIIa promoter in the absence of adenovirus E1A protein is restricted to mouse oocytes and preimplantation embryos. 253 79

Expression vectors have been constructed for a region of the human retinoic acid receptor-alpha (hRAR-alpha) and transferred into F9 embryonal carcinoma (EC) cells. When the vectors are overexpressed in F9 cells, clones can be selected for resistance to retinoic acid-induced differentiation. This effect is obtained even when the hRAR-alpha region is expressed as a beta-galactosidase fusion protein. Using the beta-galactosidase component of the fusion protein as a marker, overexpression of the fusion protein has been correlated with the retinoic acid-resistance effect. The clones resistant to retinoic acid no longer exhibit the normal retinoic acid induction of endo B cytokeratin, laminin B-1, and tissue plasminogen activator mRNAs observed with normal F9 cells. Retinoic acid induction of type IV alpha-1 collagen and Hox-1.3 RNAs is observed with these clones. When transfected with a thyroid receptor DNA-binding sequence (TRE)/thymidine kinase promoter/luciferase construct, the retinoic acid-resistant clones do not yield the same retinoic acid-induced level of luciferase obtained with F9 cells. It is hypothesized that the RAR vectors are interfering with endogenous RAR(s) in a dominant-negative manner to inhibit retinoic acid-induced differentiation of F9 EC cells.
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PMID:Retinoic acid receptor expression vector inhibits differentiation of F9 embryonal carcinoma cells. 255 44

Microinjection of the firefly luciferase gene coupled to a thymidine kinase (tk) promoter provided a quantitative assay to evaluate the requirements for gene expression in individual mouse oocytes and embryos. Polyoma virus (PyV) enhancers had no effect on the level of gene expression or competition for transcription factors as long as the DNA remained either in the oocyte germinal vesicle or the pronuclei of one-cell embryos. Expression of injected genes could be observed in pronuclei because the signal that normally triggers zygotic gene expression in two-cell embryos still occurred in one-cell embryos arrested in S phase. However, when the tk promoter was injected into zygotic nuclei of two-cell embryos, enhancers increased the number of embryos that expressed luciferase as well as the level of luciferase activity per embryo. PyV enhancer mutation F101, selected for growth in mouse embryonal carcinoma F9 cells, stimulated expression in developing two-cell embryos about seven times better than the wild-type PyV enhancer and competed effectively for factors required for transcription. These results were consistent with the fact that enhancers are required to activate the PyV origin of DNA replication in developing two-cell embryos but not in one-cell embryos. The maximum levels of gene expression in oocytes, one-cell embryos, and developing two-cell embryos (1:67:21) were inversely related to the extent of chromatin assembly, but the need for enhancers was independent of chromatin assembly. Therefore, it appears that the need for enhancers to activate promoters or origins of replication results from some negative regulatory factor that first appears as a component of zygotic nuclear structure.
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PMID:The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus. 255 65

A series of replication-defective retroviral vectors were assessed for their ability to efficiently transfer functional genes into undifferentiated cells. In these vectors (designated handicapped because of a deletion of enhancer and promoter sequences in the viral long terminal repeat) transcription of inserted genes is under the control of internal promoters. Although a composite promoter composed of a mutant polyoma virus enhancer (PyF441) coupled to the herpes simplex virus thymidine kinase promoter was anticipated to function efficiently, it was found to be significantly inferior to the mouse X-chromosome phosphoglycerate kinase (pgk-1) promoter, in its ability to express the selectable neomycin phosphotransferase gene in mouse embryonal carcinoma cells. The pHMB vector, which contains the pgk-1 promoter, was shown to confer the drug-resistant phenotype at high frequencies to F9 and P19 cells. This vector might prove to be of general utility for efficient gene expression in other developmental contexts.
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PMID:Comparative analysis of retroviral vector expression in mouse embryonal carcinoma cells. 256 Feb 17

Mouse embryonal carcinoma (EC) cells do not express the major H-2 class I transplantation antigens. The latter, however, become detectable upon in vitro differentiation of EC cells. Neither class I H-2 genes nor the gene coding for beta-2 microglobulin (beta 2m) is transcribed in EC cells. We have constructed two hybrid plasmids containing the 5' flanking region of an H-2Kb gene followed by the coding regions of either the herpes simplex virus thymidine kinase (H-2 tk) or the chloramphenicol acetyl transferase (H-2 CAT) genes. Upon transfer into EC cells, the H-2 tk hybrid gene is expressed in F9 tk- cell lines which thus acquire a stable tk+ phenotype. When such transformed clones are induced to differentiate in vitro, tk activity shows a moderate increase, which reflects an increase in transcription of the hybrid gene. In transient transformation experiments, EC cells were found to express the H-2 CAT hybrid gene as well. We conclude that the 2 kilobase pair region of the H-2Kb gene which we used contains an active promoter region, but does not include all the elements required for the correct regulation of the H-2Kb gene.
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PMID:Functional analysis of the mouse H-2Kb gene promoter in embryonal carcinoma cells. 298 66

The transient expression vector pSV2CAT, which carries the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of the SV40 early promoter, was used to transfect the murine embryonal carcinoma cell line F9 at various times during the retinoic acid-induced differentiation of these cells. Expression of the CAT gene under SV40 promoter control was found to increase markedly on F9 cell differentiation, measured relative to expression from the thymidine kinase promoter in the same cells. A series of constructs was prepared to identify the features of the SV40 early promoter required for transcription in differentiated and undifferentiated cells, as well as the factors limiting transcription in each case. The increased transcription seen on F9 cell differentiation was not observed when cells were transfected with molecules lacking a functional enhancer. It appears that as embryonal carcinoma cells differentiate, increased SV40 transcription results from enhancer sequence activation. In both differentiated and undifferentiated cell types the level of transcription was found to be limited by the availability and/or activity of cellular factors necessary for enhancer function.
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PMID:SV40 enhancer activation during retinoic acid-induced differentiation of F9 embryonal carcinoma cells. 300 73


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