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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)

When a functional murine adenine phosphoribosyltransferase (aprt) gene linked to bovine papilloma virus (BPV) DNA is transfected into Aprt- L cells, the cells are rendered Aprt+ and the aprt gene persists as an episome. Cotransfection with two BPV vectors, one containing the 5' half of the aprt gene and the other the 3' half of the gene, that share about 300 bp of common sequence in intron 2, produces Aprt+ cells with functional aprt as an episome. Southern blot analysis of low molecular weight DNA derived from Hirt extracts revealed the regeneration of a diagnostic SmaI fragment, consistent with establishment of an episome with functional aprt that was reconstituted as a consequence of recombination. To establish cells with an episomal target for recombination, BPV vectors containing a G418 resistance marker and either the 5' half or 3' half of aprt were transfected into Aprt- L cells. Stably transfected cells, selected by their growth in G418, were in turn transfected with DNA containing the other half of the aprt gene. Following selection of Aprt+ cells, Southern blot and polymerase chain reaction (PCR) analysis of low molecular weight DNA confirmed the presence of a complete episomal aprt gene. The region of DNA shared by the episomal aprt fragment and the transfected aprt half was sequenced after PCR amplification of the reconstituted, episomal gene and was found to be wild type. The region of overlap that serves as the substrate for recombination lies entirely within an intron and can, therefore, tolerate nucleotide substitutions and deletions. The absence of such errors in the sequences examined is consistent with recombination events that are not error prone.
Mol Gen Genet 1992 Mar
PMID:Reconstitution of an episomal mouse aprt gene as a consequence of recombination. 131 48

In some biological systems ionizing radiation appears to induce large deletions and rearrangements, while in others point mutations predominate the mutational spectrum. Moreover, while the point mutations are often randomly distributed, some systems exhibit "hot spots." Retroviral shuttle vectors are particularly useful for investigating the basis of these differences since the genetic target can be conveniently analyzed in a variety of host backgrounds and genomic locations. We have studied the mutational specificity of X-rays in a Chinese hamster ovary cell line (CHO) containing a stably integrated retroviral shuttle vector, carrying the CHO aprt cDNA as the genetic target. Cells were irradiated with 7 Gy using a 180 kVp X-ray source. The predominant mutation (87% of all APRT mutants), as determined by Southern analysis, was the complete deletion of the shuttle vector construct. In addition, 23 APRT mutants, carrying an apparently intact shuttle vector, were characterized at the sequence level: 5 were transitions, 9 were transversions, 3 were small deletions or insertions, 4 were frameshifts, and 2 were small rearrangements. Although the type and the location of the point mutations characterized appeared largely random, small deletions, insertions, and frameshifts were frequently associated with direct sequence repeats.
Environ Mol Mutagen 1992
PMID:Investigation of the mutagenic specificity of X-rays using a retroviral shuttle vector in CHO cells. 133 May 46

Expression plasmids containing the human alpha 1-antitrypsin (alpha 1 AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT- HPRT- rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes in cis, as only three of 20 clones tested were affected for expression of alpha 1AT mRNA. In contrast, double selection yielded predominantly trans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal alpha 1AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1 trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.
Somat Cell Mol Genet 1992 Jul
PMID:Direct selection of hepatoma cell variants deficient in alpha 1-antitrypsin gene expression. 133 97

Five purine auxotrophic mutants of Lactococcus lactis were isolated. L. lactis was capable of converting adenine, guanine and hypoxanthine to AMP, GMP and IMP, respectively, indicating the existence of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) activities. A 1.3 kb DNA fragment from L. lactis was cloned by complementation of the hpt mutation in Escherichia coli. Introduction of this fragment into L. lactis resulted in an increase in HGPRT activity. In vitro transcription and translation analysis showed that the fragment coded for a polypeptide with M(r) of 22,000. The nucleotide sequence of this hpt gene was determined.
Mol Gen Genet 1992 Nov
PMID:Isolation of purine auxotrophic mutants of Lactococcus lactis and characterization of the gene hpt encoding hypoxanthine guanine phosphoribosyltransferase. 146 8

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.
Somat Cell Mol Genet 1992 May
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

Growth factors induce the sequential expression of cellular genes whose products are thought to mediate long-term responses to the growth factors. In mouse 3T3 fibroblastic cells, the first genes to be expressed (immediate-early genes) are activated within minutes after the addition of platelet-derived growth factor, fibroblast growth factor, or serum. By cDNA cloning, we have identified genes that are activated after a delay of a few hours and several hours prior to serum-induced DNA replication. Activation of these delayed early response genes requires new protein synthesis, presumably the synthesis of immediate-early transcription factors described previously. Partial or complete sequencing of 13 different delayed early cDNAs, representing about 40% of the 650 primary cDNA isolates, revealed that 8 were related to known gene sequences and 5 were not. Among the former are cDNAs encoding nonhistone chromosomal proteins [HMGI(Y) and HMGI-C], adenine phosphoribosyltransferase (APRT), a protein related to human macrophage migration inhibitory factor (MIF), a protein of the major intrinsic protein (MIP) family homologous to the integral membrane protein of human erythrocytes, and cyclin CYL1. In 3T3 cells, the delayed early gene response to growth factors appears to be at least as complex as the immediate-early gene response previously described.
Mol Cell Biol 1992 Sep
PMID:Growth factor-induced delayed early response genes. 150 93

An intact cDNA from Arabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced. The cDNA is 729 nucleotides in length and predicts a protein of Mr 27,140. The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to the Escherichia coli protein. Construction of a molecular tree of the known APRT amino acid sequences indicates the A. thaliana and E. coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping. Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.
Plant Mol Biol 1992 Feb
PMID:A complete cDNA for adenine phosphoribosyltransferase from Arabidopsis thaliana. 155 43

We have determined the nucleotide sequence surrounding a BclI restriction fragment length variation near the aprt gene of CHO cells. By BclI digestion of the PCR-amplified DNA from a variety of APRT-deficient mutants derived from CHO cells, we were able to infer the following. First, all three heterozygotes of class II, which are known to undergo the second mutational step via a large deletion event occurring at high frequency, are mutant at the chromosome Z4-linked allele, and wild type at the Z7 allele. Second, both class-III heterozygotes, which mutate to the APRT- phenotype at low frequency, are mutant at the Z7 allele, wild type at the Z4 allele. A total of 12 class-I lines, defined as having already undergone a deletion event and yielding fully APRT- mutants at low frequency were all found to have lost the Z7-linked allele. We conclude that the Z7-linked allele is substantially more susceptible to mutation by the large deletion event than is the Z4-linked allele. This supports a hypothesis we advanced earlier to explain the existence of the class-III heterozygotes but does not support previous work suggesting that a chromosomal inversion break-point junction near the Z4-linked aprt allele is responsible for the high frequency deletion event.
Somat Cell Mol Genet 1991 May
PMID:Analysis of second-step mutations of class II and class III CHO aprt heterozygotes: chromosomal differences in deletion frequencies. 167 90

Morphologically differentiated cell lines were previously isolated from a mouse teratocarcinoma stem cell line exhibiting an unstable heterozygous deficiency for adenine phosphoribosyltransferase (APRT) expression. In this study, the methylation sensitive and insensitive isoschizomer restriction endonucleases HpaII and MspI, respectively, were used to demonstrate that the aprt gene in the heterozygous deficient stem cell line was hypomethylated. Loss of APRT activity in this stem cell line was not associated with DNA methylation change. However, differentiation of this stem cell line was associated with hypermethylation of three consecutive HpaII/MspI sites that were located in the second intron and the third exon of the aprt gene. A total of 15 independent APRT homozygous deficient cell lines were isolated from three differentiated heterozygous deficient cell lines, and in all 15 cell lines this differentiation-related methylation pattern was altered. Two classes of alterations were noted: (1) hypomethylation of a site located in the second intron or (2) the apparent spreading of methylation to downstream methylation sites. The CpG-rich promoter region remained hypomethylated in the APRT homozygous deficient differentiated cell lines and a methylation change affecting a specific CpG site upstream of the promoter region was noted in only two of the 15 homozygous deficient cell lines. It is proposed that methylation of the mouse aprt gene may be involved in controlling phenotypic expression in the differentiated cell lines, but not in the stem cell line they were derived from.
Somat Cell Mol Genet 1990 Jul
PMID:Methylation of mouse adenine phosphoribosyltransferase gene is altered upon cellular differentiation and loss of phenotypic expression. 169 89

We have analyzed the adenine phosphoribosyltransferase (APRT) enzyme from Chinese hamster ovary cells through the study of mutants that are able to grow in the presence of the toxic adenine analogue 8-azaadenine. The distribution of the amino acid alterations was analyzed in terms of the binding regions for the purine and phosphoribosylpyrophosphate substrates and a comparison was made with mutants known in human APRT and human, mouse and hamster hypoxanthine-guanine phosphoribosyltransferase. A number of mutants were found to cluster in several regions of the amino acid sequence. Residual enzyme activity with adenine was determined and this was correlated with substrate binding regions. A model of the secondary structure features is proposed.
J Mol Biol 1991 Sep 05
PMID:Mutational analysis of the structure and function of the adenine phosphoribosyltransferase enzyme of Chinese hamster. 171 94


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