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)

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

Antisense oligonucleotides are ordinarily targeted to mRNA by double-stranded (Watson-Crick) base recognition but are seldom targeted by triple-stranded recognition. We report that certain all-purine methylphosphonate oligodeoxyribonucleotides (MPOs) form stable triple-stranded complexes with complementary (all-pyrimidine) RNA targets. Modified chloramphenicol acetyltransferase mRNA targets were prepared with complementary all-pyrimidine inserts (18-20 bp) located immediately 3' of the initiation codon. These modified chloramphenicol acetyltransferase mRNAs were used together with internal control (nontarget) mRNAs in a cell-free translation-arrest assay. Our data show that triple-strand-forming MPOs specifically inhibit protein synthesis in a concentration-dependent manner (> 90% at 1 microM). In addition, these MPOs specifically block reverse transcription in the region of their complementary polypyrimidine target sites.
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PMID:Triple-strand-forming methylphosphonate oligodeoxynucleotides targeted to mRNA efficiently block protein synthesis. 752 21

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

The 5'-terminal untranslated region (5' UTR) of the uncapped hepatitis A virus (HAV) RNA contains two pyrimidine-rich sequences; one about 20 nucleotides (nt) in length in the vicinity of the AUG initiation codon (nt 706-726), and a longer one (about 40 nt) encompassing nt 100 to 140. The latter includes a 13 nt 'core' sequence (positions 126-138 in the HM175 strain) which is 80% identical to the pyrimidine-rich tract of poliovirus type 1 RNA (Mahoney strain). Representative cDNAs of the entire 5' UTR of HAV RNA were inserted in the intercistronic region of the bi-cistronic plasmid pSV-GH/CAT between the genes coding for the human growth hormone (GH) and bacterial chloramphenicol acetyltransferase (CAT). When COS-7 cells were transfected with these constructs they transiently expressed CAT indicating that the 5' UTR of HAV was efficiently directing internal initiation of translation of the reporter gene. Under similar conditions the 5' UTR of poliovirus type 2 (Lansing strain) was 30% more efficient in directing the expression of the CAT gene. Removal of the 'core' sequence from the 5'-distal pyrimidine-rich stretch extending between nt 117 and 131 in the HAV 5' UTR reduced the CAT activity in the lysates of transfected cells by 40%, whereas point mutations engineered in this segment strongly decreased (80% inhibition) the HAV-driven expression of the reporter gene. Limited mutations systematically introduced in the reiterated (U)UUUCCC motifs of the 5'-distal pyrimidine-rich tract identified two major functional domains extending between nt 100-106 and 113-119. Substitutions in these hexanucleotides abrogated internal initiation of translation, whereas similar changes in the neighbouring domains (nt 107-112 and 120-126) had no effect on the expression of the reporter gene, suggesting that the 5'-most pyrimidine-rich tract is indeed part of the structure(s) recognized by ribosomes and associated factors at initiation of translation and that the hexanucleotides 100-106 and 113-119 constitute an important part of it. Although HAV replicates better at 33 degrees C than at 37 degrees C, incubation of transfected cultures at 33 degrees C delayed the expression and slightly reduced the level of CAT activity in the cell lysates, but the overall effect of the mutations remained unchanged.
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PMID:5' UTR of hepatitis A virus RNA: mutations in the 5'-most pyrimidine-rich tract reduce its ability to direct internal initiation of translation. 773 Aug 3

The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytes-macrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase [luc], and Salmonella typhimurium histidinol dehydrogenase [his]) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat. Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective. Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements. Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-kappa B. The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat. 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding gamma-actin or glucose 6-phosphate dehydrogenase. Thus, induction is selective and not an artifact induced by transfecting DNA into cells. In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP. Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels. 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-kappa B pathway in its time course and in its requirement for new protein synthesis. Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent. Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.
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PMID:Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors. 835 80

We have mapped the basal promoter activity of the mouse cytochrome c oxidase (COX) subunit Vb gene to the -17 to +20 region which contains two putative ets binding sites flanking an NF-E1 site fused to an Sp1 site. A 17-nucleotide sequence flanking the major transcription start site (-8 to +9), referred to as 17Inr (initiator sequence) was able to drive CAT activity in 3T3 cells to a level comparable to the construct containing sequences -17 to +20. This suggests that the 17Inr sequence contains the initiator activity. The 17Inr contains a pyrimidine-rich sequence, commencing with a CA that corresponds to the major transcription start site. Primer extension of RNA from transfected cells demonstrated that transcription initiation with the 17Inr template occurs at a site identical to the endogenous gene. DNA-protein binding by gel mobility shift and methylation interference analyses indicated that the pyrimidine-rich sequence immediately flanking the transcription start site consists of an NF-E1 factor binding motif with an overlapping upstream Sp1 binding site. A 13-nucleotide sequence, 13Inr (-4 to +9), which retains the NF-E1 binding activity but does not bind Sp1, was able to promote chloramphenicol acetyltransferase gene expression at levels similar to the 17Inr sequence, suggesting that NF-E1 factor binding is critical for initiator function. Finally, using an in vitro transcription system from Drosophila embryos we demonstrate that NF-E1 is necessary for transcription activation of both the 17Inr and the 13Inr initiator templates. Thus NF-E1 binding appears to be important for basal promoter function of the mouse COXVb gene.
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PMID:Identification of a transcriptional initiator element in the cytochrome c oxidase subunit Vb promoter which binds to transcription factors NF-E1 (YY-1, delta) and Sp1. 838 96

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

Serum amyloid A (SAA) is a plasma protein which has been associated with several diseases, including amyloidosis, arthritis, and atherosclerosis, and its abnormal expression, particularly in nonhepatic cells, is implicated in the pathogenesis of these diseases. Transfection and DNA-binding studies were performed to investigate the mechanism controlling cytokine-induced, nonhepatic expression of the SAA gene. We have identified a novel promoter, located between positions -280 and 224, that confers interleukin-6 (IL-6) inducibility to an SAA-chloramphenicol acetyltransferase reporter gene in both nonhepatic and hepatic cells. DNase I protection assays revealed, within this region, three homologous highly pyrimidine rich octanucleotide sequence motifs, termed SAA-activating sequences (SAS). Specific mutations within these three SAS motifs severely reduced IL-6-mediated induction of the reporter gene in transfected nonhepatic cells but not in liver cells. A nuclear factor activated by IL-6 in both hepatic and nonhepatic cells efficiently interacts with the SAS. The induction kinetics and cycloheximide sensitivity of this SAS-binding factor (SAF) suggested that de novo synthesis of this factor itself or an activator protein is essential. Loss of DNA-binding ability as a result of in vitro dephosphorylation, induction of SAA-chloramphenicol acetyltransferase reporter gene activity in the presence of genistein, a protein kinase inhibitor, further indicate that a phosphorylation step is necessary for the activation of SAF. Our results suggest that SAF is a key regulator of cytokine-mediated SAA gene expression in some nonhepatic cells.
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PMID:A novel cis-acting element is essential for cytokine-mediated transcriptional induction of the serum amyloid A gene in nonhepatic cells. 865 33

A human recombinant cDNA clone that encoded a zinc-finger protein (Myc-associated zinc-finger protein of human islet; MAZi) was cloned by screening a cDNA library prepared from human pancreatic islet cells. The encoded protein showed a high degree of homology to the Myc-associated zinc-finger protein MAZ (ZF87 or Pur-1). However, differences between the cDNAs for MAZi and MAZ were found in the length of the encoded polyalanine stretch and in the sequence of the 5'-end leader. MAZi transcripts were significantly more abundant in rat pancreatic islet carcinoma tissue than in normal rat islet cells. Moreover, MAZi protein bound specifically to the pyrimidine-rich strand of the CT-element of the c-myc gene in vitro and strongly induced the expression of chloramphenicol acetyltransferase (CAT) from a c-myc promoter/ CAT reporter construct in human pancreatic cells. Our results suggest that a distinct member of the MAZ family is expressed in human islet cells and enhances the transcriptional activity of the c-myc gene.
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PMID:Members of the MAZ family: a novel cDNA clone for MAZ from human pancreatic islet cells. 883 93


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