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)

Chinese hamster ovary (CHO) cells were subjected to electroporation in the presence of 5-methyl deoxycytidine-triphosphate. This treatment increases by 10 to 100-fold the frequency of cells lacking thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, or adenine phosphoribosyltransferase. The inactivation of the genes coding for these enzymes is thought to occur following the direct incorporation of the methylated nucleotide triphosphate into DNA. The enzyme-deficient clones were stable, but almost all were reactivated at high frequency by the demethylating agent 5-azacytidine, to produce derivatives with enzyme activity. The results indicate that there is a direct relationship between DNA methylation and gene silencing.
Somat Cell Mol Genet 1991 Nov
PMID:Gene silencing in mammalian cells by uptake of 5-methyl deoxycytidine-5'-triphosphate. 172 91

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.
Mol Carcinog 1991
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.
Mol Carcinog 1991
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.
Somat Cell Mol Genet 1991 Jul
PMID:Transduction of the CHO aprt gene into mouse L cells using an adeno-5/APRT recombinant virus. 188 32

A BclI restriction-fragment-length variation (RFLV) has been identified 7 kb downstream of the 3' end of the adenine phosphoribosyltransferase (aprt) gene in the Chinese hamster CHO cell line. This RFLV generates 19 kb and 17 kb + 2 kb BclI fragments. Three other cell lines derived from different Chinese hamsters are homozygous for this RFLV. The 17 kb + 2 kb allele was mapped to the Z4 chromosome based upon the absence of this allele in the CHO-derived hemizygote AA8-5, whose functional allele is known to map to chromosome Z7. The class 2 aprt+/-line D416 loses the 19-kb allele after undergoing the high-frequency (HF) deletion event, indicating that the HF deletion event occurs on the Z7 chromosome. On the other hand, deletion events in the class 3 heterozygote D423 (which do not spontaneously occur at HF) resulted in loss of the 17 kb + 2 kb allele. This is consistent with a previously proposed model holding that the two aprt alleles in the CHO cell line have different inherent probabilities of undergoing a large deletion event.
Somat Cell Mol Genet 1990 May
PMID:Loss of alleles in aprt mutants of CHO cells demonstrated by BclI restriction-fragment-length variation. 197 16

Spontaneous deletion mutants can be isolated from CHO cell lines heterozygous at the adenine phosphoribosyltransferase locus at frequencies up to 10(-4), i.e., about 100-fold higher than spontaneous point mutations. Indirect evidence has suggested that most deletions were in the megabase range. We have fully characterized one such mutant, Del 155, and shown that it resulted from an illegitimate recombination that took place between overlapping tetranucleotides. Comparisons with sequences of other deletions at various loci revealed a number of similarities, most striking of which was a CHI-like motif found within 6 bp of the upstream breakpoint of Del 155 and breakpoints of 8/21 previously described short deletions at the CHO aprt locus. Homology also existed between the downstream breakpoint of Del 155 and breakpoints of retinoblastoma gene deletions (3/6 cases) and also a 20-bp stretch of an Alu sequence in which breakpoints at the low-density lipoprotein receptor locus have been shown to cluster. The magnitude of the deletion event in Del 155 was assessed by pulsed field (PF) gel analysis and found to be at least 2100 kb long. PF analysis also indicated that the downstream breakpoint was near a region of structural differences between the two chromosomes carrying apart. These structural differences were probably not implicated in the mechanism of the high frequency event, since no indication of breakpoint clustering among a large collection of mutants was found either by conventional or PF electrophoretic analyses.
Somat Cell Mol Genet 1991 Jan
PMID:A very large spontaneous deletion at aprt locus in CHO cells: sequence similarities with small aprt deletions. 199 42

We have used four gene probes specific for mouse chromosome 8, including adenine phosphoribosyltransferase (aprt), to demonstrate that the P19 teratocarcinoma stem cell line contains two distinct chromosome 8 homologs. One represents the common laboratory mouse C3H (Mus musculus domesticus) homolog while the second homolog was presumably contributed by a feral Mus musculus musculus animal. Six cell lines with APRT heterozygous deficiencies were isolated from P19 subclones. A molecular analysis of these heterozygotes demonstrated that three arose by deletion of the Mus musculus musculus aprt allele and three arose by aprt gene inactivation. APRT homozygous deficient cell lines were isolated from both classes of heterozygote; most contained little or no detectable APRT activity. When the heterozygous deficiency was due to deletion of the Mus musculus musculus aprt allele, the most frequent event yielding homozygous deficient cell lines was associated with loss of heterozygosity for all tested markers on the Mus musculus domesticus homolog indicating chromosome loss. In contrast, when the initial event resulting in APRT heterozygous deficiency was gene inactivation, homozygotes arose predominantly from gene deletion or a second inactivation event. These results suggest a potential relationship between the first- and second-step events resulting in APRT deficiencies.
Somat Cell Mol Genet 1991 Mar
PMID:Molecular analysis of APRT deficiency in mouse P19 teratocarcinoma stem cell line. 201 91

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.
Somat Cell Mol Genet 1991 Mar
PMID:Region- and cell type-specific de novo DNA methylation in cultured mammalian cells. 201 93

We analyzed the nature of spontaneous mutations at the autosomal locus coding for adenine phosphoribosyltransferase in the human colorectal carcinoma cell line SW620 to establish whether distinctive mutational pathways exist that might underlie the more complex genome rearrangements arising in tumor cells. Point mutations occur at a low rate in aprt hemizygotes derived from SW620, largely as a result of base substitutions at G.C base pairs to yield transversions and transitions. However, a novel pathway is evident in the form of multiple dispersed mutations in which two errors, separated by as much as 1,800 bp, fall in the same mutant gene. Such mutations could be the result of error-prone DNA synthesis occurring during normal replication or during long-patch excision-repair of spontaneously arising DNA lesions. This process could also contribute to the chromosomal instability evident in these tumor cells.
Mol Cell Biol 1991 Jun
PMID:Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line. 203 24

We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT+ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt- "pop-out" recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to "pop-out" of integrated plasmid/gpt gene sequences occur at a rate of approximately 6.3 x 10(-6) per cell generation. Depending on the site of crossover, such "pop-out" events result in either replacement or restoration of the original APRT target gene sequence.
Somat Cell Mol Genet 1990 Sep
PMID:Targeted gene replacement at the endogenous APRT locus in CHO cells. 223 39


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