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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinds and locations of mutations in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 75 independent mutants, derived from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated normal human fibroblasts, were characterized by direct sequencing of mRNA-polymerase chain reaction (mRNA-PCR)-amplified cDNA. Treatment of human cells with low (6 or 8 microM) or high (10 or 12 microM) doses of MNNG resulted in 35-fold or 150-fold average increases in mutation frequency, respectively. A high frequency of mutants lacking a complete exon was observed in both groups. Further characterization of half of these mutants by DNA-PCR amplification of intron-exon boundaries showed that they contained base substitutions. The kinds of base substitutions differed distinctly between these two groups. In the low dose group, a broad mutational spectrum was observed: ten out of the 31 base substitutions were A.T to G.C transitions, six contained G.C to A.T transitions, and the other 15 exhibited transversions. In contrast, the majority (84%) of base substitutions among the high dose group were G.C to A.T transitions; the others (16%) were transversions. All of the 32 G.C to A.T transitions were located on the non-transcribed strand, assuming that the causative premutational lesion was O6-methylguanine. These results indicate preferential repair of lesions located on the transcribed strand. In addition, G.C to A.T and A.T to G.C transitions preferentially occurred at positions with guanine and thymine at the adjacent 5' position, respectively.
J Mol Biol 1991 Sep 20
PMID:Novel mutational spectrum induced by N-methyl-N'-nitro-N-nitrosoguanidine in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human fibroblasts. 192 Apr 27

We have isolated and characterized 47 ultraviolet light-induced hprt mutants from a simian virus 40-transformed excision-repair-deficient xeroderma pigmentosum cell line (complementation group A). Twenty-one independent mutations were found, of which the majority were point mutations. Eleven of these were identified as base changes, nine of which could be attributed to ultraviolet damage on the transcribed DNA strand. Both transitions and transversions were found among the single base changes. A large proportion of the mutations (13/21) resulted in aberrant splicing of the hprt gene, suggesting that the target size for mutations resulting in aberrant splicing must be quite large. A small number of spontaneous mutations were identified, most of which were large deletions. Our data provide a spectrum for the intrinsic mutations resulting from ultraviolet damage in human cells in the absence of repair.
J Mol Biol 1991 Jan 20
PMID:Molecular analysis of ultraviolet-induced mutations in a xeroderma pigmentosum cell line. 199 58

We have introduced a 4-bp insertion into the hypoxanthine phosphoribosyltransferase (HPRT) gene of a mouse embryonic stem (ES) cell line by using an "in-out" targeting procedure. During the in step, a homologous integration reaction, we targeted a correcting plasmid to a partially deleted hprt- locus by using an integrating vector that carried a 4-bp insertion in the region of DNA homologous to the target locus. HPRT+ recombinants were isolated by direct selection in hypoxanthine-aminopterin-thymidine (HAT) medium. The HATr cell lines were then grown in medium containing 6-thioguanine (6-TG) to select for hprt- revertants resulting from the excision of the integrated vector sequences. The revertants were examined by Southern blot hybridization to determine the accuracy of this out reaction and the frequency of retaining the 4-bp modification in the genome. Of the 6-TGr colonies examined, 88% had accurately excised the integrated vector sequences; 19 of 20 accurate revertants retained the 4-bp insertion in the resulting hprt- gene. We suggest a scheme for making the in-out targeting procedure generally useful to modify the mammalian genome.
Mol Cell Biol 1991 Mar
PMID:Testing an "in-out" targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. 199 1

We have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 microgram/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and beta-actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA-primed lymphocytes/well in 96-well round-bottom microtiter plates containing a medium supplemented with interleukin-2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 x 10(3) cells/well; 90 Gy) and irradiated autologous feeder cells (5 x 10(4) cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0-150 mg/kg of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, or 0-50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG) and these contained 5 x 10(3) irradiated TK6 cells and 5 x 10(4) primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose-dependent increase in TGr cells. In addition, the frequency of ENU-induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.
Environ Mol Mutagen 1991
PMID:Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide. 202 92

The frequency of 6-thioguanine resistant (TGr) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations.
Environ Mol Mutagen 1991
PMID:Cytosine arabinoside enhancement of gamma irradiation induced mutations in human T-lymphocytes. 205 Jan 30

The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population. The proportion of MTX-resistant cells among the survivors increased logarithmically with dose, up to a 1000-fold increase over unirradiated cells at 1000 cGy, the highest dose tested. The induced frequency of MTX resistance after X-irradiation was greater than the induced frequency of 8-azaguanine resistance, which indicates deletion of the hypoxanthine phosphoribosyltransferase gene. Inhibition of poly(ADP-ribose) polymerase by the addition of 3-aminobenzamide before irradiation increased both cell killing and MTX resistance. Metaphase spreads of chromosomes from EMT-6 cells that had been irradiated and subjected to stepwise increases in MTX concentration showed numerous double minutes. Pulsed-field gel electrophoresis of the DNA from cells containing radiation-induced double minutes showed that many copies of the dhfr gene were present on circular DNA molecules of 10(6), 2 x 10(6), and 3 x 10(6) base pairs. These results suggest a relationship between the induction of chromosome aberrations and the induction of gene amplification.
Somat Cell Mol Genet 1990 Sep
PMID:X-ray induction of methotrexate resistance due to dhfr gene amplification. 212 27

The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT- Chinese hamster V79 cells. Several gpt- cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. Each cell line exhibits a characteristic spontaneous mutation frequency (10(-5) to 10(-2)) in 6-thioguanine (6TG) selection. While spontaneous mutagenesis to gpt- occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency (approximately 3 x 10(-5)), was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and beta-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The gpt locus of the g12 transfectants, however, is two to three times more sensitive to UV and 2.5-4.5 times more sensitive to X-ray mutagenesis than the endogenous hprt of wild-type V79 cells. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus. Future studies with these transgenic cells and other transgenic lines are planned to compare the mutability and repair of the same gene (gpt) at different integration sites in mammalian cells.
Environ Mol Mutagen 1990
PMID:Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis. 219 84

The abundant production of antisense hypoxanthine phosphoribosyltransferase (HPRT) RNA in NIH-3T3, COS, or HeLa cells leads to an inhibition of HPRT synthesis. HPRT enzyme levels in cells transfected with mouse HPRT antisense RNA expression vectors are reduced to less than 1% of parental cell activity, resulting in resistance to 6-thioguanine (6TG). The expression of antisense HPRT RNA leads to a marked reduction in the steady-state levels of endogenous HPRT mRNA. Furthermore, we demonstrate that intron-specific antisense RNA, complementary to sequences adjacent to splice donor or acceptor sites of the first intron of the mouse HPRT gene, are effective in depressing endogenous HPRT levels. These studies suggest that antisense RNA can inhibit gene expression in the nucleus, possibly by perturbing nuclear RNA processing.
Somat Cell Mol Genet 1990 Jul
PMID:Antisense RNA inhibition of HPRT synthesis. 221 24

Purines and purine nucleotides were found to affect transcription of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in whole nuclei isolated from intestinal mucosa of adult rats fed a purine- and purine nucleotide-free diet. Nuclear run-on transcription assays, performed on whole nuclei from different tissues and cell types, identified an intestine-specific decrease in the overall incorporation of [alpha-32P]UTP in HPRT transcripts from intestinal epithelial cell nuclei when exogenous purines or purine nucleotides were omitted from either the diet or culture medium. Using a 990-base-pair genomic fragment that contains the 5'-flanking region from the HPRT gene, we generated plasmid constructs with deletions, transfected the DNA into various cell types, and assayed for chloramphenicol acetyltransferase (CAT) reporter activity in vitro. We determined that an element upstream from the putative transcriptional start site is necessary to maintain the regulatory response to purine and nucleotide levels in cultured intestinal epithelial cells. These results were tissue and cell type specific and suggest that in the absence of exogenous purines, the presence of specific factors influences transcriptional initiation of HPRT. This information provides evidence for a mechanism by which the intestinal epithelium, which has been reported to lack constitutive levels of de novo purine nucleotide biosynthetic activity, could maintain and regulate the salvage of purines and nucleotides necessary for its high rate of cell and protein turnover during fluctuating nutritional and physiological conditions. Furthermore, this information may provide more insight into regulation of the broad class of genes recognized by their lack of TATA and CCAAT box consensus sequences within the region proximal to the promoter.
Mol Cell Biol 1990 Aug
PMID:A regulatory element is characterized by purine-mediated and cell-type-specific gene transcription. 237 Aug 69

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.
Mol Cell Biol 1985 Oct
PMID:X chromosome reactivation in mouse embryonal carcinoma cells. 242 74


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