Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.2.7 (adenine phosphoribosyltransferase)
 692 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.
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PMID:Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells. 179 88

We previously showed that ultraviolet (UV) irradiation of cotransfected plasmid DNA molecules stimulated genetic transformation that depended on intermolecular homologous recombination in Chinese hamster ovary (CHO) cells. Repair-proficient cells and an excision repair complementation class 1 (ERCC1) UV-sensitive DNA repair-deficient mutant responded similarly to UV stimulation in cotransfections with plasmids containing linker insertion-disrupted copies of the herpes simplex virus thymidine kinase (HSV-TK) gene. In this study, we cotransfected homologous DNA molecules containing nonoverlapping deletions of the hamster adenine phosphoribosyltransferase (APRT) gene into APRT-deficient CHO ERCC1 (UVL-10) and ERCC2 (UVL-1) excision-repair mutants and parental repair-proficient CHO cells. UV damage in cotransfected circular plasmid molecules stimulated transformation in repair-proficient cells and an ERCC1 mutant, but not in an ERCC2 mutant. Linearization of plasmids prior to cotransfection greatly enhanced transformation frequencies in all three cell lines, but UV stimulation using linear recombination substrates was no longer evident. Our results suggest (i) that the ERCC1 gene defect in CHO UVL-10 cells does not affect UV stimulation of homology-dependent extra-chromosomal recombination, and (ii) that a CHO cell ERCC2 excision-repair mutant, although recombination proficient, may exhibit altered recombination in response to UV damage.
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PMID:Ultraviolet stimulation of intermolecular homologous recombination in Chinese hamster ovary cells. 179 89

An adenovirus-5 recombinant virus Adapt1 carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene was constructed by insertion of a 2.5-kb fragment containing the complete CHO aprt structural gene linked to a Moloney murine sarcoma virus (MSV) promoter into the E3 region of adenovirus-5. The CHO aprt gene was in the opposite orientation to the adenovirus E3 promoter. Mouse Lapt- tk- (LAT) cells expressed the CHO aprt gene when infected with the virus, even at low MOI (O.1). APRT activity was detectable from approximately 20 h postinfection. At a low frequency, LAT cells were transformed to aprt+, and four stable transductants were selected in adenine, azaserine (AA) medium. Such cells expressed APRT at approximately 50% wild-type activity and the enzyme was shown to be CHO APRT by starch gel electrophoresis. DNA was isolated from the transductants and probed with CHO aprt-specific DNA and with viral DNA probes. The results indicated that the CHO aprt gene was integrated into the LAT cells at a site other than mouse aprt. Although neighboring viral sequences were integrated and maintained in the transductants, viral sequences further upstream and downstream of the aprt gene were absent.
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PMID:Transduction of the CHO aprt gene into mouse L cells using an adeno-5/APRT recombinant virus. 188 32

A plasmid was constructed by fusion of a selectable mammalian gene, hamster adenine phosphoribosyltransferase (APRT), to the Zn(2+)-inducible sheep metallothionein I (MT I) promoter. This plasmid was used to produce stable Chinese hamster ovary (CHO) cell transformants by electroporation to study the effects of induced gene expression on DNA-mediated transformation. The sheep MT Ia promoter was chosen for these experiments because it regulates gene expression differently than murine MT promoters, exhibiting low basal levels of gene expression in uninduced conditions. We have shown that in the absence of Zn2+, there is very low expression of a sheep MT I-APRT fusion gene in stable CHO cells transformants; induction of APRT mRNA and enzyme activity by Zn2+ produced a "threshold" response, from low basal levels to high induced levels, in Zn2+ responsive stable transformant clones. In electroporation experiments, transformation frequencies were unaffected by Zn2+ treatments during a preselection period, but the presence of Zn2+ during selection increased the recovery of stable transformant clones 8- to 10-fold. All stable transformants analyzed displayed Zn(2+)-inducible APRT enzyme activity. Our results indicate that stable mammalian cell transformants with inducible genes under regulation of the sheep MT I promoter should be useful, because of low basal and high induced expression, for studies in which modulation of transcriptional activity is required.
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PMID:Characterization of Chinese hamster ovary cells stably transformed by a plasmid with an inducible APRT gene. 192 58

Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.
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PMID:A polymerase chain reaction-based method to detect cisplatin adducts in specific genes. 195 80

Loss of heterozygosity at previously heterozygous loci may occur by one of several possible mechanisms and account for a large fraction of all mutations occurring at such loci. In order to investigate loss of heterozygosity events, we have chosen the aprt locus of Chinese hamster ovary (CHO) cells as our model since it is readily available in either heterozygous or hemizygous form. Cloning and sequencing of the two heterozygous aprt alleles from the CHO derivative D423 identified a single polymorphic site, which does not create a restriction fragment length polymorphism. In order to evaluate the loss of heterozygosity events at this locus, we devised a method that creates an artificial restriction fragment length polymorphism in one of these two alleles as a direct consequence of enzymatic amplification. Restriction enzyme digestion of the amplified sequences can then conveniently identify the genotype of the DNA sample. This same methodology also provides for the selective cloning of only one allele of a heterozygous pair into a plasmid vector for subsequent DNA sequence analysis, and can be easily adapted to other situations requiring the analysis of single base changes at a particular position within known sequences. Using this technique, we have determined that 16/37 (43%) spontaneous APRT- mutants had undergone a loss of heterozygosity event.
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PMID:Loss of heterozygosity in mammalian cell mutagenesis: molecular analysis of spontaneous mutations at the aprt locus in CHO cells. 197 45

A system for studying mutational specificity at a heterozygous locus in Chinese hamster ovary (CHO) cells is described. The strategy employed is based on restriction fragment analysis and DNA sequencing of enzymatically amplified mutant adenine phosphoribosyltransferase (aprt) alleles. We have demonstrated the usefulness of this approach through the characterization of a collection of aprt- mutants with respect to the role played by loss of heterozygosity events in ultraviolet light (UV) induced mutagenesis. A similar strategy has also been applied to speculate on the identity of the premutational lesion responsible for a UV-induced mutational hotspot at the aprt locus.
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PMID:The nature of ultraviolet light-induced mutations at the heterozygous aprt locus in Chinese hamster ovary cells. 197 78

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.
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PMID:Nucleotide pools and mutagenic effects of alkylating agents in wild-type and APRT-deficient Friend erythroleukaemia cells. 198 59

An accurate diagnosis of heterozygotes for autosomal recessive disorders with unknown mutations can be difficult. Using a unique phenomenon occurring in vivo, we designed a method for the diagnosis of heterozygotes for adenine phosphoribosyltransferase (APRT) deficiency which makes way for a qualitative distinction between normal and heterozygous subjects. We cultured peripheral blood mononuclear cells with 2,6-diaminopurine, an APRT-dependent cytotoxin, to search for in vivo mutational cells. Fifteen putative heterozygotes examined were found to possess such mutant cells at rather high frequencies; thus, a false negative diagnosis is unlikely. The analysis of genomic DNA in 82 resistant clones from two of the heterozygotes clarified that 64 (78%) had lost the germinally intact alleles. Thirteen members of APRT-deficient families were examined; eight proved to be heterozygotes. Among 425 individuals from two separate residential areas of Japan, two heterozygotes were found. The authenticity of the heterozygosity was validated by two separate methods for the two heterozygotes; hence, a false positive diagnosis can be ruled out. Our data showed a calculated heterozygote frequency of 0.47% (95% confidence limits; 0.05%-1.7%), a value compatible with that (1.2%) calculated from data concerning the incidence of 2,8-dihydroxyadenine urolithiasis. This novel genetic approach for identifying heterozygotes is now being tested to search for other enzyme deficiencies in humans.
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PMID:Diagnosis of heterozygous states for adenine phosphoribosyltransferase deficiency based on detection of in vivo somatic mutants in blood T cells: application to screening of heterozygotes. 199 41

A region upstream of the mouse adenine phosphoribosyltransferase (aprt) gene has a well characterized methylation pattern for HpaII/MspI sites. When an unmethylated plasmid construct containing this region was transfected into P19 mouse teratocarcinoma stem cells appropriate de novo methylation was observed. However, de novo methylation was significantly reduced when this plasmid was introduced into a differentiated derivative of the P19 stem cell line. Finally, a position effect for de novo methylation was shown by demonstrating methylation of a normally unmethylated HpaII/MspI site when it was placed in this upstream region. This system should prove useful for elucidating DNA signals for de novo methylation and changes in DNA methyltransferase activities that occur during cellular differentiation.
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PMID:Region- and cell type-specific de novo DNA methylation in cultured mammalian cells. 201 93


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