Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conditions for reliable and efficient assay of mutations affecting the activity of HPRT (hypoxanthine phosphoribosyltransferase EC 2.4.2.8) and APRT (adenine phosphoribosyltransferase EC 2.4.2.7) have been determined for a strain of CHO (Chinese hamster ovary) cells that has been adapted for rapid growth both in suspension culture and in monolayer. To facilitate measurement of mutation at the aprt locus, clones were derived that are presumptively heterozygous at that locus. At a limiting concentration of 8 microgram/ml of azaadenine, 14/16 of the resistant clones picked and tested had approximately 1/2 of the APRT activity of the wild-type cells. One such clone, strain AA8, was chosen for further studies and found to be readily mutable to resistance to 80 microgram/ml azaadenine. Most of the highly resistant colonies isolated (21/24) had very low in vitro APRT activity. The optimal conditions for detection of TGr and AAr mutations were determined for two critical parameters, expression time and cell density. Cultures treated with mutagen either in monolayer or in suspension were allowed to express mutations in suspension. The expression of mutations induced by UV light, EMS, and ICR-191 was complete by 3 days for AAr and by 4-5 days for TGr. The time required to reach a maximal frequency of mutants was essentially independent of the type of mutagen and the level of survival after treatment. Induced mutation frequencies for both loci were notably stable during the time intervals examined. With respect to cell-density conditions, both markers were detected at frequencies that were independent of the cell inocula over the range of 1 x 10(5) to 1 x 10(6) cells per 100-mm petri dish (i.e. 1.6 x 10(3) to 1.6 x 10(4) cells/cm2) containing 20 ml of medium. These results were obtained with both mutagenized populations and with reconstructed mixtures obtained by adding drug-resistant cells to varying numbers of wild-type cells. The rapid expression of mutations for both markers, particularly AAr, combined with the advantage that large inocula can be plated for selection of mutants, make this CHO strain an attractive system for the simultaneous measurement of mutations at the autosomal aprt and X-linked hprt loci.
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PMID:Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells. 736 Jan 55

The mutational specificity of N-methylnitrosourea (MNU), nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), sodium azide (NaN3), 4-nitroquinoline oxide (4NQO), benzo[a]pyrene (BP), nitrofurantoin (NF), aflatoxin B1 (AFB1), adriamycin (ADM) and UVA-activated angelicin in Salmonella typhimurium strain TA100 has been examined using allele-specific oligonucleotide hybridization and DNA sequence analyses. These ten mutagens produced five unique classes of reversion spectra, distinct from spontaneous, or the previously characterized 5-azacytidine, ultraviolet light (UV), 8-methoxypsoralen plus UVA (PUVA) and 60Co-induced mutation spectra. For example, 90% of MNU and MNNG-induced mutations in strain TA100 revertants were G:C-->A:T transitions with the majority (82%) occurring in the first position of the CCC codon. In contrast, NaN3 preferentially induced G:C-->A:T transitions at the second codon position (78%). Although MMS, NQO, BP, NF, ADM and AFB1 induced primarily G:C-->T:A transversions (73-86%), these mutagens fall into two classes based on site preference: NF and AFB1 yielded almost exclusively position two transversions (69-78%) whereas ADM, NQO, BP and MMS exhibited a two-fold preference for site 2 over site 1 (on average 52% versus 22%). Angelicin photomutagenesis resulted in the recovery of G:C-->A:T and G:C-->T:A mutations at both codon positions in roughly equal proportions (approximately 20-25% each). Approximately 1% of the mutagen-induced revertants occurred via extragenic tRNA suppressor mutations, while 1% were multiple (usually tandem double) base substitutions. Ultraviolet mutagenesis experiments demonstrated that tandem base substitutions are promoted by pKM101-encoded mucAB gene products. A comparison of the mutagenic specificity derived for several carcinogens in hisG46 with the responses of several eukaryotic gene targets (e.g. HPRT, aprt, supF) revealed a high concordance between these targets. Thus, the Salmonella hisG46 locus provides a rapid, simple system for determining base substitution specificity and for studying mechanisms of mutagenesis.
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PMID:Salmonella typhimurium strain TA100 differentiates several classes of carcinogens and mutagens by base substitution specificity. 829 52

The TK6 human B lymphoblastoid cell line contains two easily and widely used selectable markers: the X-linked, hemizygous hprt locus, and the heterozygous tk locus on chromosome 17q. In this study, rare APRT heterozygotes were directly isolated from the TK6 population by clonal selection in cell culture medium supplemented with 5 micrograms/ml of 8-azaadenine. One of nine isolated heterozygotes, AZH1, was characterized extensively. APRT- mutants can be recovered from AZH1 at a mutation rate of 1.5 x 10(-7), similar to rates previously determined for the selection of TK- and HPRT- mutants from TK6. A unique sequence alteration was identified in the non-functional aprt allele at position 1930. A G:C to A:T transition at this site alters the canonical AG splice acceptor dinucleotide in exon 3, and also results in the destruction of a Stul recognition sequence. This polymorphism was used to analyze loss of heterozygosity in a set of 32 spontaneous APRT- mutants by restriction analysis following PCR amplification. Analysis of flanking microsatellite dinucleotide polymorphisms demonstrated that LOH occurring in spontaneous APRT- mutants is nearly always a multi-locus event extending at least 7.5 cM along chromosome 16q. This pattern of LOH among APRT- mutants differs from extensive LOH in spontaneous, normal-growth TK- mutants derived from TK6 cells (p < 0.0001), and suggests that cis-acting factors may be equally important in shaping the mutational spectrum as trans-acting factors such as cellular apoptotic capacity.
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PMID:Isolation of an APRT heterozygote from TK6 human lymphoblasts: predominance of multi-locus loss of heterozygosity among spontaneous APRT-mutants. 921 76

Spontaneous mutations and neocarzinostatin-induced mutations were investigated in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in exponentially growing Chinese hamster ovary cells. Neocarzinostatin (NCS) treatment caused an 4.5-fold increase in mutation frequency. Analysis by multiplex polymerase chain reaction and sequencing of hprt cDNA revealed that spontaneous mutations in this system were characterized by a relatively high rate of large deletions and double-base substitutions, and a low rate of splice mutations compared with data reported in fibroblastic cell lines. NCS hardly affected this spectrum of spontaneous mutations, which indicates the rare incidence of NCS-specific change in the exponential growth phase. This is in contrast to aprt gene mutations reported in plateau phase cells in which base substitutions occur preferentially at sites affected by NCS. These results suggest that differences in the loci assayed or in the processes involved in mammalian mutagenesis in the exponential growth phase and the plateau phase may be the source of the different results.
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PMID:Neocarzinostatin-induced mutations at the hprt locus in exponentially growing CHO cells, compared with spontaneous mutations. 963 64

Several alkylanilines with structures more complex than toluidines have been associated epidemiologically with human cancer. Their mechanism of action remains largely undetermined, and there is no reported evidence that it replicates that of multicyclic aromatic amines even though the principal metabolic pathways of P450-mediated hydroxylation and phase II conjugation are very similar. As a means to elucidate their mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt (+/-)) gene induced in several Chinese hamster ovary cell types by 2,6- and 3,5-dimethylaniline (2,6-DMA, 3,5-DMA) and their N- and ring-hydroxyl derivatives (N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP) were assessed. Dose-response relationships were determined in the parental AA8 cell line, its repair-deficient UV5 subclone and other repair-deficient 5P3NAT2 or -proficient 5P3NAT2R9 subclones engineered to express mouse cytochrome P4501A2 (CYP1A2) and human N-acetyltransferase (NAT2), and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Separately, treatment of AS52 cells with N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP, and 3,5-DMAP led to intracellular production of reactive oxygen species (ROS) for at least 24h after removal of the mutagens in every case. Using the comet assay, DNA strand breaks were observed in a dose-dependent manner in AS52 cells when treated with each of the four N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, and 3,5-DMAP derivatives. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action is likely to be through redox cycling of intracellularly bound aminophenol/quinone imine structures to generate ROS rather than through formation of covalent DNA adducts.
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PMID:Genotoxicity of 2,6- and 3,5-dimethylaniline in cultured mammalian cells: the role of reactive oxygen species. 2283 70