Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We here present a general method to detect alkylation damage in specific genomic regions. Cells are treated with nitrogen mustard or dimethyl sulfate; the DNA is extracted and restricted, and the parental DNA is separated. Strand breaks are created at sites of N-alkylpurines by neutral depurination followed by alkaline hydrolysis. The DNA is then separated on alkaline agarose gels and transferred, and gene fragments are detected after hybridization with specific probes. Using this approach, we have examined damage formation and repair in the active genes dihydrofolate reductase and adenosine phosphoribosyltransferase, in a fragment containing the inactive c-fos gene and in a nontranscribed region downstream from the dihydrofolate reductase gene in Chinese hamster ovary cells. We find variations in the formation of nitrogen mustard adducts in these different regions. Nitrogen mustard adducts are preferentially repaired from the active genes as compared to the inactive gene and the noncoding region. However, we find no preferential damage or repair in these regions of the N7-methylpurines after dimethyl sulfate damage. Thus, there are significant differences in the repair mechanisms for two alkylating agents; this may implicate that there are important differences in the structural alterations in chromatin invoked by these agents. As a comparison to the studies of adduct levels in specific genomic regions, we have examined the overall genome, average adduct formation, and repair by these agents in the hamster cells. We used alkaline sucrose gradient sedimentation, and also a novel approach: quantitation of the DNA smears stained by ethidium bromide in the alkaline gels (used in the gene-selective repair analysis). Both these techniques gave similar data for adduct formation and repair; there was less initial damage formation and repair in the average genome than in specific genomic regions.
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PMID:Heterogeneity of nitrogen mustard-induced DNA damage and repair at the level of the gene in Chinese hamster ovary cells. 238 Jan 93

Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Parnas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphoribosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemistry and metabolism of Giardia. 265 35

CC-1065 is a potent antitumor antibiotic which bonds to duplex DNA specifically; the biological effects of the drug are presumably the consequences of its DNA interactions. In order to investigate the factors which may affect drug-DNA bonding in cells, a method using a thermal-alkaline treatment to induce phosphodiester bond breakage at the drug-DNA bonding sites and Southern DNA transfer-hybridization to quantify drug-DNA bonding at defined sequences in drug-treated cultured mammalian cells was developed. We have found that in vivo, in cultured Chinese hamster ovary (CHO) cells, CC-1065 bonds twice as efficiently in the highly amplified dihydrofolate reductase (DHFR) gene domains as in the nonamplified adenine phosphoribosyltransferase (APRT) gene domain. However, in vitro, in purified CHO cellular DNA, CC-1065 bonds equally to both the DHFR and APRT genes. We observed a significant degree of "gene-specific" preferential repair for drug-DNA adducts in the amplified DHFR gene domains, and it appears that this "gene-specific" repair reflects "transcribed-strand specific" repair. These results suggest that DNA amplification may affect drug-DNA adduct formation and transcription may affect its repair.
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PMID:Formation and repair of antitumor antibiotic CC-1065-induced DNA adducts in the adenine phosphoribosyltransferase and amplified dihydrofolate reductase genes of Chinese hamster ovary cells. 811 38

Using Uvr proteins we have quantified benzo(a)pyrene diol epoxide (BPDE)-DNA adduct formation and repair at the dihydrofolate reductase (DHFR) and adenine phosphoribosyltransferase (APRT) genes in two Chinese hamster ovary cell lines: B-11 cells, which are 50-fold amplified for DHFR, and AT3-2 cells, which are diploid for DHFR. We have found that: 1) BPDE-DNA adduct formation in different regions of the DHFR gene is proportional to the concentration of BPDE. 2) There is no significant difference in the repair of BPDE-DNA adducts between the coding and noncoding regions in either amplified or nonamplified DHFR gene domains. 3) Repair in the nonamplified DHFR gene is more efficient (30-40%) than in the amplified DHFR genes. 4) There are no significant differences of repair in the transcribed or nontranscribed strands of the DHFR gene. 5) BPDE-DNA adduct formation and repair in the APRT gene in B-11 and AT3-2 cells are the same. These results contrast those for the repair of cyclobutane pyrimidine dimers, which occurs preferentially in the transcribed strand of the DHFR gene and in which gene amplification appears to play no role.
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PMID:Repair of benzo(a)pyrene diol epoxide- and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells. 817 87

Using a strategy based on reverse transcription and the polymerase chain reaction, we have determined the order of splicing of the four introns of the endogenous adenine phosphoribosyltransferase (aprt) gene in Chinese hamster ovary cells. The method involves a pairwise comparison of molecules that retain one intron and have either retained or spliced another intron(s). A highly preferred order of removal was found: intron 3 > 2 > 4 = 1. This order did not represent a linear progression from one end of the transcript to the other, nor did it correlate with the conformity of the splice site sequences to the consensus sequences or to the calculated energy of duplex formation with U1 small nuclear RNA. By using actinomycin D to inhibit RNA synthesis, the in vivo rate of the first step in splicing was estimated for all four introns; a half-life of 6 min was found for introns 2, 3, and 4. Intron 1 was spliced more slowly, with a 12-min half-life. A substantial amount of RNA that retained intron 1 as the sole intron was exported to the cytoplasm. In the course of these experiments, we also determined that intron 3, but not intron 4, is spliced before 3'-end formation is complete, probably on nascent transcripts. This result is consistent with the idea that polyadenylation is required for splicing of the 3'-most intron. We applied a similar strategy to determine the last intron to be spliced in a very large transcript, that of the endogenous dihydrofolate reductase (dhfr) gene in Chinese hamster ovary cells (25 kb). Here again, intron 1 was the last intron to be spliced.
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PMID:Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA. 841 21

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.
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PMID:New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel. 968 51

The constitutive splicing factor ASF/SF2 has been shown to affect the choice between alternative splice sites by favoring the proximal as opposed to the distal choice. HnRNP A1 antagonizes ASF/SF2 by promoting the distal choice for competing 5' splice sites. We have tested the in vivo effects of these proteins on alternative 3' splice site choices. Cotransfection of a dihydrofolate reductase-calcitonin chimeric construct togetherwith a plasmid specifying the SR protein ASF/SF2 into cells of several mammalian lines increased use of a proximal 3' splice site, resulting in the inclusion of a terminal calcitonin exon. This stimulation of 3' proximal splicing was antagonized by cotransfection with an hnRNP A1 plasmid. This effect of hnRNP A1 in promoting distal splicing was also seen in an hnRNP A1-deficient MEL cell line. A similar effect of hnRNP A1 was demonstrated with mutant hamster adenine phosphoribosyltransferase (aprt) transcripts that are normally constitutively spliced, suggesting that hnRNP A1 may be a general inhibitor of proximal splicing. Intron size also influenced splice site choice in mutant aprt transcripts, with larger introns favoring proximal splicing. These results support the idea that the ratios of particular but general splicing factors and hnRNPs play a role in alternative splicing.
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PMID:Control of 3' splice site choice in vivo by ASF/SF2 and hnRNP A1. 992 47

DNA damage is preferentially repaired in the transcribed strand of many active genes. Although the concept of DNA repair coupled with transcription has been widely accepted, its mechanisms remain elusive. We recently reported that in Chinese hamster ovary cells while ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in the transcribed strand of dihydrofolate reductase gene, CPDs are efficiently repaired in both strands of adenine phosphoribosyltransferase (APRT) locus, in either a transcribed or nontranscribed APRT gene (1). These results suggested that the transcription dependence of repair may depend on genomic context. To test this hypothesis, we constructed transfectant cell lines containing a single, actively transcribed APRT gene, integrated at different genomic sites. Mapping of CPD repair in the integrated APRT genes in three transfectant cell lines revealed two distinct repair patterns, either preferential repair of CPDs in the transcribed strand or very poor repair in both strands. Similar kinetics of micrococcal nuclease digestion were seen for all three transfectant APRT gene domains and endogenous APRT locus. Our results suggest that both the efficiency and strand-specificity of repair of an actively transcribed gene are profoundly affected by genomic context but do not reflect changes in first order nucleosomal structure.
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PMID:Transcription-coupled DNA repair is genomic context-dependent. 1182 23

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