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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We utilized a plasmid vector host cell reactivation assay to probe the biological functioning of DNA expression vectors and their encoded genes. We studied the effect of ultraviolet radiation or acid-heat treatment on the transient expression of genes transfected into normal human cells and into DNA repair deficient (xeroderma pigmentosum) cells and modification of gene expression by sodium butyrate. U.v. inactivation of transient expression of the bacterial gpt gene contained in a non-replicating expression vector plasmid, pSV2catSVgpt, was much greater in three xeroderma pigmentosum lines than in the four other human cell lines tested. In contrast, treatment of pSV2catSVgpt with acid and heat to produce apurinic sites resulted in a similar slope of the inactivation curve of the bacterial cat gene in the repair deficient and repair proficient cells. Thus, u.v. damage of DNA expression vectors was subject to repair by the normal host cells, but acid-heat treatment resulted in damage (apurinic sites) that was handled in a similar manner by excision repair deficient and excision repair proficient human cells. In both normal and xeroderma pigmentosum cells sodium butyrate treatment of cells resulted in a greater stimulation of chloramphenicol acetyltransferase expression with u.v. damaged than with undamaged plasmid. This assay thus permits examination of the effects of defined types of DNA damage on plasmid expression and study of its modulation by cellular repair activities.
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PMID:Host cell reactivation by human cells of DNA expression vectors damaged by ultraviolet radiation or by acid-heat treatment. 346 38

In this paper we report both transient and stable complementation of pyrimidine dimer repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4, coding for endonuclease V, a dimer-specific DNA glycosylase. Cotransfection with pRSVdenV in SV40-transformed XP12RO(M1) cells (complementation group A) restored transient expression of an indicator plasmid (pRSVcat) bearing a UV-inactivated chloramphenicol acetyltransferase (cat) gene. In addition, XP12RO(M1) clones stably transformed by pRSVdenV-SVgpt expressed transient chloramphenicol acetyltransferase activity when transfected with UV-inactivated pRSVcat plasmid. These clones also showed partial restoration of colony forming ability and excision repair synthesis after UV irradiation. Immunofluorescence, using an endonuclease V polyclonal antibody, showed the presence of the phage glycosylase in stably transformed xeroderma pigmentosum cells. The cotransfection assay affords a rapid, sensitive procedure to screen for functional cloned DNA repair genes and to test mutant cells for the deficiency of specific steps in DNA repair, such as incision.
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PMID:Transient and stable complementation of ultraviolet repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4. 356 13

Transfected recombinant DNA with regulatory elements such as eukaryotic promoter and termination sites is transiently expressed in human fibroblast cells. Utilizing an expression vector containing the simian virus 40 (SV 40) early control region followed by the E. coli chloramphenicol acetyltransferase (CAT) gene, we investigated the ability of normal, Xeroderma pigmentosum and Cockayne Syndrome cells to repair UV lesions in transfected DNA. Fibroblasts from Xeroderma pigmentosum patients which cannot excise pyrimidine cyclobutane dimers were unable to restore expression of UV irradiated CAT gene. An UV dose inducing one thymine cyclobutane dimer in the transcribed strand of the CAT gene blocked its transcription in these repair deficient cells. Normal cell were able to repair the lesions in transfected DNA during an incubation period of about 40 h and in this way could overcome the UV block. In several fibroblast cell lines from patients suffering from Cockayne Syndrome expression of UV damaged CAT gene was restored significantly less than in normal fibroblasts, indicating that Cockayne Syndrome is associated with a UV repair defect.
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PMID:Transient expression of a plasmid gene, a tool to study DNA repair in human cells: defect of DNA repair in Cockayne syndrome; one thymine cyclobutane dimer is sufficient to block transcription. 395 12

In order to conveniently measure cellular DNA repair in immortalized and primary human cells we have combined the features of high cellular infectivity of adenovirus (Ad) with that of host-cell reactivation (HCR) of ultraviolet light (UV)-damaged reporter genes. We show that Ads having either the cat (chloramphenicol acetyltransferase) or seap (secreted alkaline phosphatase) reporter gene under control of a strong constitutive promoter can be used to measure relative levels of DNA repair by HCR. Most importantly, the SEAP assay allows for a convenient, inexpensive, and sensitive colorimetric microtiter assay. Only a few steps are involved and it is possible to process many samples simultaneously in a relatively short time, which is not as easily done with other reporter gene assays. Furthermore, we show that co-infection of UV-damaged SEAP Ad with an Ad carrying a prokaryotic repair gene significantly increased the HCR levels in xeroderma pigmentosum cells. The Ad gene delivery system, and the SEAP assay in particular, should simplify existing HCR assays considerably. By using non-lytic Ad as a vehicle it should be possible to quantitatively introduce normal or dominant negative mutant DNA repair genes into bulk cell populations for DNA repair studies.
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PMID:Host-cell reactivation of reporter genes introduced into cells by adenovirus as a convenient way to measure cellular DNA repair. 752

Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in the tumorigenesis of UV-induced skin cancer. Different wavelengths of UV radiation cause different kinds of DNA damage and mutations. UVB (290-320 nm) generates pyrimidine dimers by direct excitation of the DNA molecule. UVA (320-400 nm) can damage the DNA only indirectly through a photosensitized reaction. This indirect action is mediated mainly by singlet oxygen, which generates purine base modifications, and has been implicated in the carcinogenic effects of UVA. In order to study the processing of directly and indirectly UV-induced DNA damage in human cells, we first treated the replicating plasmid pRSVcat with up to 10 kJ/m2 UVB or with the photosensitizer methylene blue plus visible light (which generates singlet oxygen) in vitro. Then, the damaged plasmid was transfected into normal or repair deficient xeroderma pigmentosum complementation group A (XP-A) cells. DNA repair was assessed by measuring activity of reactivated chloramphenicol acetyltransferase (CAT) enzyme, encoded by the plasmid's cat gene, in cell extracts after 3 days. While XP-A cells exhibited a significantly reduced repair of UVB-induced DNA damage, they showed a normal repair of singlet oxygen-induced DNA damage. This indicates a differential DNA repair pathway for directly and indirectly UV-induced DNA damage in human cells. Irradiation of the plasmid with UVA alone did not result in a genotoxic effect. Only in conjunction with a cell extract, which provides all candidate cellular photosensitizers, did we find a reduced CAT activity after transfection. This indicates that the genotoxicity of UVA is mediated by a cellular photosensitizer.
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PMID:Processing of directly and indirectly ultraviolet-induced DNA damage in human cells. 759 99

Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.
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PMID:Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes. 776 57

To determine the contribution of a human DNA repair gene, ERCC2 (XPD), to mutagenesis in human cells, two ERCC2 (XPD)-transformed xeroderma pigmentosum complementation group D (XPD) cell lines with increased UV survival compared to XP6BE(SV40), the original XPD line, were studied: D6BE-ER2-2 with slightly increased UV survival; and D6BE-ER2-9 with normal UV survival. ERCC2 (XPD) antibody-reactive protein levels were elevated 4.8-fold in D6BE-ER2-2 and 17.6-fold in D6BE-ER2-9 relative to XP6BE(SV40). DNA repair ability was assessed by measuring the ability of the cells to restore expression to UV-treated plasmids. Transfection of pRSVcat exposed to 1000 J/m2 UV resulted in 0.3% chloramphenicol acetyltransferase activity in XP6BE(SV40) cells but 20-80% in D6BE-ER2-2, D6BE-ER2-9, and repair-proficient cells compared to untreated control plasmids. The UV hypersensitivity of the mutagenesis shuttle vector pSP189 in XP6BE(SV40) cells was partially corrected and the UV hypermutability and excess of G:C-->A:T mutations of pSP189 fell to the normal range in D6BE-ER2-2 and D6BE-ER2-9 cells. However, the frequency of plasmids recovered with multiple base substitution mutations was significantly reduced with XP6BE(SV40) cells and remained low in D6BE-ER2-2 and D6BE-ER2-9 cells, when compared with the normal fibroblasts. The human DNA excision repair gene, ERCC2 (XPD), substantially corrected the plasmid UV hypersensitivity and UV hypermutability of xeroderma pigmentosum complementation group D cells; however, the dose response relationship varied for different end points.
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PMID:The human DNA repair gene, ERCC2 (XPD), corrects ultraviolet hypersensitivity and ultraviolet hypermutability of a shuttle vector replicated in xeroderma pigmentosum group D cells. 803 4

Trichothiodystrophy (TTD) is a rare genetic disease with heterogeneous clinical features associated with specific deficiencies in nucleotide excision repair. Patients have brittle hair due to a reduced content of cysteine-rich matrix proteins. About 50% of the cases reported in the literature are photosensitive. In these patients an altered cellular response to UV, due to a specific deficiency in nucleotide excision repair, has been observed. The majority of repair-defective TTD patients have been assigned by complementation analysis to group D of xeroderma pigmentosum (XP). Recently, the human excision repair gene ERCC2 has been shown to correct the UV sensitivity of XP-D fibroblasts. In this work we describe the effect of ERCC2 on the DNA repair deficient phenotype of XP-D and on two repair-defective TTD cell strains (TTD1VI and TTD2VI) assigned by complementation analysis to group D of XP. ERCC2 cDNA, cloned into a mammalian expression vector, was introduced into TTD and XP fibroblasts via DNA-mediated transfection or microneedle injection. UV sensitivity and cellular DNA repair properties, including unscheduled DNA synthesis and reactivation of a UV-irradiated plasmid containing the chloramphenicol acetyltransferase reporter gene (pRSVCat), were corrected to wild-type levels in both TTD and XP-D cells. These data show that a functional ERCC2 gene is sufficient to reestablish a wild-type DNA repair phenotype in TTD1VI and TTD2VI cells, confirming the genetic relationship between TTD and XP-D. Furthermore, our findings suggest that mutations at the ERCC2 locus are responsible for causing a similar phenotype in TTD and XP-D cells in response to UV irradiation, but produce quite different clinical symptoms.
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PMID:Correction by the ERCC2 gene of UV sensitivity and repair deficiency phenotype in a subset of trichothiodystrophy cells. 805 25

Using a DNA-protein binding assay, we have previously identified and characterized a UV-damaged DNA recognition protein (UVDRP) from HeLa cells [(1991) Nucleic Acids Res. 19, 6413-6418]. In this report, the photoreactivating activity of UVDRP from the yeast, Saccharomyces cerevisiae, and HeLa cells was investigated. Although yeast and human cells are evolutionarily different from each other, both UVDRPs were conserved in the sense of their biochemical characteristics except that the yeast UVDRP also exhibited an enzymatic photoreactivating activity. A mammalian expression vector plasmid DNA carrying the bacterial chloramphenicol acetyltransferase (CAT) gene was UV irradiated in vitro followed immediately by exposure to photoreactivating light, and its transient expression in repair-deficient xeroderma pigmentosum (XP) cells was investigated. More than 80% of the CAT activity was inhibited by UV irradiation, which was partially restored (> 60%) by a partially purified yeast photolyase. In contrast, HeLa cell extracts did not express a photoreactivatable recovery from UV-induced inhibition of the CAT activity tested in the same system. This study has demonstrated the potential use of the DNA-mobility shift assay to investigate enzymatic photoreactivation, and has indicated the absence of the repair mechanism in human cells.
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PMID:Lack of DNA enzymatic photoreactivation in HeLa cell-free extracts. 828 3

Ultraviolet (UV) irradiation of human cells induced expression of a stably maintained fusion gene consisting of the human immunodeficiency virus long terminal repeat promoter controlling the bacterial chloramphenicol acetyltransferase gene. Two experiments demonstrated that DNA damage can initiate induction: UV induction was greater in DNA repair-deficient cells from a xeroderma pigmentosum patient than in repair-proficient cells, and transfection of UV-irradiated DNA into unirradiated cells activated gene expression. Increased repair of cyclobutane pyrimidine dimers by T4 endonuclease V abrogated viral gene activation, suggesting that dimers in DNA are one signal leading to increased gene expression. This signal was spread from UV-irradiated cells to unirradiated cells by co-cultivation, implicating the release of soluble factors. Irradiation of cells from DNA repair-deficiency diseases resulted in greater release of soluble factors than irradiation of cells from unaffected individuals. These results suggest that UV-induced cyclobutane pyrimidine dimers can activate the human immunodeficiency virus promoter at least in part by a signal-transduction pathway that includes secretion of soluble mediators.
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PMID:Cyclobutane pyrimidine dimers in UV-DNA induce release of soluble mediators that activate the human immunodeficiency virus promoter. 838 27


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