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

A mouse embryonal carcinoma cell line isolated for resistance to the adenine analogue 2,6-diaminopurine (DAP) was found to have near-wild-type levels of adenine phosphoribosyltransferase (APRT) activity in a cell-free assay. This DAP-resistant (DAPr) cell line, termed H29D1, also exhibited near-wild-type levels of adenine accumulation and the ability to grow in medium containing azaserine and adenine. Growth in this medium requires high levels of intracellular APRT activity. Using the polymerase chain reaction (PCR) and the dideoxy chain termination sequencing technique, an A-->G transition was discovered in exon 3 of the aprt gene in H29D1. This mutation resulted in an Arg-to-Gln change at amino acid 87 of the APRT protein that, in turn, resulted in a decreased affinity for adenine. An increased sensitivity of APRT to inhibition by AMP was observed when comparing H29D1 to P19, the parental cell line. Using a transgene containing the A-->G mutation, we demonstrated that this mutation is responsible for the biochemical and cellular phenotypes observed for the H29D1 cell line. The approach used in this study provides a definitive method for linking a mutation to a specific cellular phenotype.
 ...
PMID:Molecular and biochemical elucidation of a cellular phenotype characterized by adenine analogue resistance in the presence of high levels of adenine phosphoribosyltransferase activity. 129 76

A series of clones displaying high frequency "switching" phenotypes for expression of the adenine phosphoribosyltransferase (aprt) gene were previously isolated from the P19 mouse embryonal carcinoma stem cell line. Most clones contained only one aprt allele. We report here the characterization of each of these clones with regards to enzymatic activity, mRNA steady state levels, DNA methylation, and chromatin conformation. When clones were selected for resistance to the purine analog 2,6-diaminopurine, which requires markedly reduced levels of APRT enzymatic activity, two distinct classes were observed. The first class was associated with reduced or undetectable levels of aprt mRNA, hypermethylation of the 5' CpG island, and a closed chromatin conformation within this region. When clones of this class were selected for reacquisition of APRT enzymatic activity they were found to have increased mRNA levels, a hypomethylated CpG island, and an open chromatin conformation. In contrast, the second class of clones displayed wild-type levels of mRNA, CpG island hypomethylation, and an open chromatin conformation regardless of whether they were selected for the presence or absence of APRT enzymatic activity. The implications of these results for general mechanisms of epigenetic change in somatic cells and the possibility that expression of the mouse aprt gene may be developmentally regulated are discussed.
 ...
PMID:At least two distinct epigenetic mechanisms are correlated with high-frequency "switching" for APRT phenotypic expression in mouse embryonal carcinoma stem cells. 149 18

A series of clones displaying a high-frequency "switching" phenotype for expression of the adenine phosphoribosyltransferase (aprt) gene was previously isolated from the P19 mouse embryonal carcinoma stem cell line. In a subset of these clones, loss of aprt expression was correlated with increased DNA methylation, a nuclease-resistant chromatin conformation, and loss of RNA transcription; reactivation was associated with a reversal of these parameters. In this report, the role of DNA methylation in transcriptional inactivation was studied in the H22D3 clone. The cells of this clone contain a single inactive aprt allele that is methylated. Mass cultures of H22D3 were treated with 2-deoxy-5'-azacytidine (5aCdr) and found to reactivate aprt at frequencies ranging from 60 to 90%. Treated cultures were then assayed over time for aprt mRNA, chromatin conformation, and DNA methylation of the aprt gene. These studies demonstrated that 5aCdr treatment resulted in promoter region-specific hemidemethylation and chromatin relaxation starting at 12 h. This was followed by the appearance of RNA transcripts at 18 h and increasing levels of APRT enzymatic activity at 36 h after treatment. Complete demethylation occurred significantly later. Experiments in which cells were treated with 5aCdr for varying periods of time demonstrated that a single round of analog incorporation was sufficient for transcriptional reactivation of aprt in H22D3.
 ...
PMID:Hemidemethylation is sufficient for chromatin relaxation and transcriptional activation of methylated aprt gene in mouse P19 embryonal carcinoma cell line. 768 84

A 2.1-kilobase pair region located just upstream of the mouse aprt (adenine phosphoribosyltransferase) gene has a methylation pattern that is conserved in mouse tissues and culture cell lines. This upstream region includes four HpaII/MspI sites. Two of these sites are fully methylated, one is partially methylated, and one is unmethylated. Transfection experiments have demonstrated that the conserved methylation pattern can be reproduced in a mouse embryonal carcinoma stem cell line via de novo methylation (Turker, M.S., Mummaneni, P., and Bishop, P.L. (1991) Somat. Cell Mol. Genet. 17, 151-157). To examine the molecular basis of the conserved methylation pattern, a plasmid-based deletion analysis was conducted by removing and rearranging specific portions of the upstream region. Unmethylated versions of these plasmid constructs were then transfected into the mouse stem cell line and the methylation status of the remaining HpaII/MspI sites determined with a Southern blot analysis. By using this approach, a cis-acting sequence within the upstream region of approximately 0.8 kilobase pairs was identified which appears responsible for the conserved methylation pattern. We use the term "de novo methylation center" to denote this sequence. Based on the results obtained, a model is offered to explain the formation of the conserved methylation pattern in the upstream region.
 ...
PMID:A cis-acting element accounts for a conserved methylation pattern upstream of the mouse adenine phosphoribosyltransferase gene. 841 60

A mouse embryonal carcinoma cell line hemizygous for the adenine phosphoribosyltransferase gene (aprt) was exposed to ultraviolet light (UV) or to the alkylating agent, ethyl methanesulfonate (EMS). Thirty eight cell lines retaining the aprt gene were isolated by selecting for resistance to 2,6-diaminopurine (DAP), an adenine analogue which selects against aprt activity. Of these, six cell lines distinguished by significant levels of aprt enzymatic activity after selection in DAP, were found to carry mutations in the aprt gene affecting the apparent Km of the enzyme for adenine in every cell line, and the apparent Km for phosphoribosylpyrophosphate in two of the six cell lines. The results indicate that the ability of these cells to survive in the presence of toxic adenine analogues while maintaining significant levels of aprt enzyme activity may be due to a reduced affinity for the adenine analogue, DAP. This biochemical analysis along with results obtained from sequencing the aprt gene from 31 DAP resistant cell lines with no detectable aprt activity were used to implicate certain amino acids within aprt in substrate binding. It was also determined that, in contrast to UV, EMS did not appear to exhibit any strand bias in the distribution of mutations.
 ...
PMID:A role for certain mouse Aprt sequences in resistance to toxic adenine analogs. 921 1

DNA demethylation is used to establish and maintain an unmethylated state. The molecular mechanisms to induce DNA demethylation at a particular genomic locus remain unclear. The mouse H19/insulin-like growth factor 2 (Igf2) imprinted control region (ICR) is a methylation state-sensitive insulator that regulates transcriptional activation of both genes. The unmethylated state of the ICR established in female germ cells is maintained during development, resisting the wave of genome-wide de novo methylation. We previously demonstrated that a DNA fragment (fragment b) derived from this ICR-induced DNA demethylation when it was transfected into undifferentiated mouse embryonal carcinoma cell lines. Moreover, two octamer motifs within fragment b were necessary to induce this DNA demethylation. Here, we demonstrated that both octamer motifs and their flanking sequences constitute Sox-Oct motifs (SO1 and SO2) and that the SO1 region, which requires at least four additional elements, including the SO2 region, contributes significantly to the induction of high-frequency DNA demethylation as a Sox-Oct motif. Moreover, RNAi-mediated inhibition of Oct3/4 expression in P19 cells resulted in a reduced DNA demethylation frequency of fragment b but not of the adenine phosphoribosyltransferase gene CpG island. The Sox motif of SO1 could function as a sensor for a hypermethylated state of the ICR to repress demethylation activity. These results indicate that Sox-Oct motifs in the ICR determine the cell type, DNA region, and allele specificity of DNA demethylation. We propose a link between the mechanisms for maintenance of the unmethylated state of the H19/Igf2 ICR and the undifferentiated cell-specific induction of DNA demethylation.
 ...
PMID:Induction of DNA demethylation depending on two sets of Sox2 and adjacent Oct3/4 binding sites (Sox-Oct motifs) within the mouse H19/insulin-like growth factor 2 (Igf2) imprinted control region. 2311 43