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)

Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive diseases with clinical and cellular hypersensitivity to UV radiation, differ in ability to repair UV DNA photoproducts in their overall genome: normal repair in CS, defective repair in XP. In order to characterize a DNA repair defect in an active gene in CS, we measured the capacity of cells from patients with CS and XP to reactivate 2 major types of UV-induced DNA damage, photoreactivatable (i.e., cyclobutane pyrimidine dimers) and non-photoreactivatable (primarily pyrimidine-(6-4)pyrimidone photoproducts), in the actively transcribing chloramphenicol acetyltransferase (cat) gene of the plasmid expression vector pRSV-cat. Epstein-Barr virus-transformed lymphoblast lines from 4 normal persons and from 3 patients with CS and from two with XP were transiently transfected with the plasmid, and the cat activity in cell extracts was determined. When the cells were transfected with UV-irradiated plasmid, expression was abnormally decreased in both the CS and XP cells. When the cyclobutane pyrimidine dimers in the UV-irradiated plasmid were removed by photoreactivation prior to transfection, cat expression in the CS, but not in the XP, lines reached normal levels. These data imply that both the XP and CS cells are unable to repair normally the cyclobutane pyrimidine dimer photoproducts which block transcription of cat. However, the CS, but not XP, cells can repair normally the other UV-induced photoproducts which block transcription. The ability of CS, but not XP, cells to repair these non-dimer photoproducts indicates that the active gene repair mechanism treats the cyclobutane pyrimidine dimer differently from the non-dimer photoproducts.
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PMID:Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells. 171

Accumulation of gadd153 mRNA is strongly stimulated in mammalian cells by treatments which arrest growth or damage DNA (A. J. Fornace, Jr. et al., Mol. Cell. Biol., 9: 4196-4203, 1989). In previous studies, we demonstrated that the increased expression of gadd153 following treatment with several DNA-damaging agents was mediated transcriptionally (J. D. Luethy et al., J. Biol. Chem., 265: 16521-16526, 1990). To better define the specificity of this response, we have established a sensitive reporter system in which we have stably integrated a chimeric gene containing the gadd153 promoter linked to the coding region of the chloramphenicol acetyltransferase (CAT) gene into the genome of HeLa cells. Transcriptional activation from the gadd153 promoter was monitored by determining levels of CAT activity in cellular lysates prepared from gadd153CAT/HeLa cells treated with a variety of agents. The gadd153 promoter was strongly activated by a broad spectrum of genotoxic agents including UV-mimetic agents, DNA-cross-linking and alkylating agents, DNA intercalators, and topoisomerase inhibitors. Of the DNA-damaging agents tested, only X-irradiation and bleomycin treatments failed to induce gadd153 promoter activity. Agents which inhibit replication and cell division and agents which otherwise result in cytotoxicity or growth arrest also had little influence on gadd153 promoter activity. Expression of the gadd153CAT chimeric gene in xeroderma pigmentosum Group A cells, which are deficient in nucleotide excision DNA repair of pyrimidine dimers, was maximally induced at UV doses at least 6-fold lower than those required for similar induction in repair-proficient HeLa cells. However, the methyl methanesulfonate-induced gadd153 promoter activities were similar in both cell lines. Novobiocin pretreatment inhibited both UV- and methyl methanesulfonate-induced gadd153CAT expression. Collectively, these data indicate that: (a) the gadd153 promoter is activated rapidly and specifically by DNA damage; (b) the altered DNA structure is the inducing signal for the activation of the signal transduction pathway responsible for enhanced gadd153 expression; and (c) regulation of gadd153 by growth arrest is distinct from that of DNA damage. Thus, the gadd153CAT/HeLa cells are a useful model for examining the molecular mechanisms associated with the response to DNA damage and provide a reporter system for the screening of potential genotoxic agents.
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PMID:Activation of the gadd153 promoter by genotoxic agents: a rapid and specific response to DNA damage. 172 86

A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.
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PMID:Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. 193 49

We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D37 greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.
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PMID:Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells. 204 39

UV-irradiated plasmids (pNPV B and pBSCATSV) were transfected into RBCF-1 cells derived from a goldfish (Carassius auratus) and into a xeroderma pigmentosum (group A) cell line, XP20SSV. The frequency of stable neor transformation by pNPV B decreased in a dose-dependent manner. However, in spite of large differences in UV sensitivity detected in the colony formation assay, the dose-response curves of RBCF-1 cells and XP20SSV cells were almost the same. The photorecovery (PR) of transforming activity of UV-irradiated plasmids was confirmed in RBCF-1 cells but its extent was much smaller than that observed in the survival assay. The expression of the transfected cat (chloramphenicol acetyltransferase; CAT) gene after 24-h incubation in the dark was much more sensitive to UV irradiation when compared with the stable transformation assay. The extent of PR of cat gene expression in RBCF-1 cells was high and comparable with that of the survival assay. The CAT value of RBCF-1 cells transfected with UV-irradiated plasmids relative to that of unirradiated controls increased as incubation time in the dark after transfection became longer. This suggests that the UV lesions on the plasmids transfected in the RBCF-1 cells were repaired in the dark. The cat gene expression of UV-irradiated plasmids in XP20SSV was very low and independent of incubation time after transfection.
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PMID:The repair of UV-irradiated plasmids transfected into cultured fish cells. 236 98

The activity of the chloramphenicol acetyltransferase (cat) gene after transfection into human fibroblasts has been measured following treatment of the plasmid pRSVcat with either restriction enzymes or ultraviolet light. Restriction enzymes producing single cuts in the plasmid inactivated the expression of the cat gene whether the enzymes cut the plasmid inside the coding region of the gene or several kilobases away from the gene. Ultraviolet light produced a dose-dependent inactivation of the gene. The inactivation curve was steeper if the recipient cell strain was derived from a patient with xeroderma pigmentosum. The findings with this transient expression system contrast with previously reported results of experiments using plasmids which transform cells stably by integrating into the cellular genomic DNA.
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PMID:Effect of DNA damage on the expression of the chloramphenicol acetyltransferase gene after transfection into diploid human fibroblasts. 298 42

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).
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PMID:Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line. 299 46

We have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. We studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines (D0 = 56 J X m-2) than in the other human cell lines tested (normal, ataxia-telangiectasia, Lesch-Nyhan, retinoblastoma)(D0 = 680 J X m-2)(D0 is the dose that reduces the percentage of CAT activity by 63% along the exponential portion of the dose-response curve). The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.
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PMID:One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. 299 75

Fibroblasts from a patient with xeroderma pigmentosum complementation group D were treated with Simian virus 40 to establish a transformed cell line suitable for studies of DNA-mediated gene transfer. After progressing through 2 crises, a stable line, XP6Be(SV40), was established and cultured for more than 1 year. This line retains the characteristic xeroderma pigmentosum ultraviolet hypersensitivity and is able to complement a SV40-transformed group A line when fused and assayed for ultraviolet radiation inhibition of colony-forming ability. XP6Be(SV40) expressed high levels of transfected chloramphenicol acetyltransferase activity (0.1 nmole X mg-1 X min-1) in a transient expression assay, showed stable expression of transfected gpt or neo genes (frequency 1-20 X 10(-5)), and permitted replication of the mutagenesis shuttle vector plasmid, pZ189. Ultraviolet treatment (500 J X m-2) of pZ189 prior to replication in XP6Be(SV40) resulted in a large reduction in plasmid yield (5% survival) and a 60-fold increase in the mutation frequency, reflecting the reduced ability of these cells to repair ultraviolet-damaged transfecting DNA. This cell line provides the opportunity to utilize transfection studies in cells with the xeroderma pigmentosum group D defect in excision repair.
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PMID:An SV40-transformed xeroderma pigmentosum group D cell line: establishment, ultraviolet sensitivity, transfection efficiency and plasmid mutation induction. 302 95

To determine whether DNA excision repair is enhanced in mammalian cells in response to DNA damage, as it is in bacteria as part of the SOS response, we used an expression vector-host cell reactivation assay to measure cellular DNA repair capacity. When UV-damaged chloramphenicol acetyltransferase (CAT) vector DNA was introduced into monkey cells (CV-1), the level of CAT activity was inversely related to the UV fluence due to inhibition of CAT gene expression by UV photoproducts. When CV-1 cells were treated with either UV radiation or mitomycin C, 24-48 h before transfection, CAT expression from the UV-irradiated plasmid was increased. This increase also occurred in a line of normal human cells, but not in repair-deficient human xeroderma pigmentosum cells. We confirmed that this increase in CAT expression was due to repair, and not to production of damage-free templates by recombination; the frequency of generation of supF+ recombinants after transfection with UV-irradiated pZ189 vectors carrying different point mutations in the supF gene did not significantly increase in carcinogen-treated CV-1 cells. From these results we conclude that carcinogen treatment enhances the excision-repair capacity of normal mammalian cells.
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PMID:Enhancement of DNA repair capacity of mammalian cells by carcinogen treatment. 313 2


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