Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A somatic cell hybrid line, called M11-X, was developed in order to study the expression and regulation of the human beta-like globin genes in a mouse erythroid environment. M11-X cells were obtained by fusing the human fibroblast cell line GM3552 (which contains the translocation chromosome t(11;X) that carries the human beta-like globin genes) with hypoxanthine phosphoribosyltransferase (HPRT) -negative tetraploid (2S) mouse erythroleukemia (MEL) cells. After induction with 5 mM hexamethylene bisacetamide (HMBA), these cells contain approximately 300-600 copies per cell of correctly initiated, processed, and terminated human beta-globin mRNA; however, neither human epsilon- nor gamma-globin mRNAs were detected. Carboxymethylcellulose chromatography followed by SDS-polyacrylamide gel electrophoresis and Western blotting revealed that normal human beta-globin protein was also present. These results suggest that the human beta-globin gene, when present in mouse erythroid cells, can be transcribed and its mRNA translated into normal products, but at a much lower level than the mouse beta-globin genes. Analysis of the frequency of cytosine methylation near the human gamma-globin genes indicated that these genes are heavily methylated in M11-X cells. The inability to express the human gamma-globin genes of these cells might be accounted for, at least in part, by DNA methylation.
 ...
PMID:Human globin gene expression in hybrid 2S MEL X human fibroblast cells. 658 92

'In-out' gene targeting using a hypoxanthine phosphoribosyltransferase (HPRT) minigene was applied to generate two new alleles in the gene (Apob) coding for apolipoprotein B (apo B) in murine embryonic stem (ES) cells. Homologous integration of the targeting vector during the 'in step' disrupted the Apob gene leading to an allele encoding apo B81, having a 19% carboxyl-terminal truncation. All six targeted cells obtained had more than one insert at the locus, and the chromosomal target sequence in four of them was changed during the recombination. These results suggest that concatenation of the targeting vector prior to insertion was needed to generate sufficient gene product to yield the HPRT+ phenotype, and that recombination between the concatenated DNA and endogenous DNA was a gene replacement more frequently than a simple insertion. The 'out step' recombination event which occurs between sequences duplicated in the 'in step', was planned to replace the sequences encoding the putative LDL receptor-binding domains of apo B100 with sequences encoding human beta-globin peptides (designated apo B100-beta). 6-Thioguanine (6-TG) resistant colonies were obtained from all the 'in-step' cell lines tested at frequencies of 10(-5) to 10(-4), but the frequency of physical loss of the HPRT sequences accompanied by retention of the modified Apob sequence was variable, indicating that mechanisms other than a simple excision are responsible for the generation of 6-TG resistance. Mice from the 'in-step' produce apo B81 and display characteristics of familial hypobetalipoproteinemia; some homozygotes develop hydrocephaly or exencephaly. Mice from the 'out-step' produce apo B100-beta and secrete lipoprotein particles containing the modified protein; their phenotypic changes are subtle, suggesting the lack of the putative LDL receptor-binding domains is not sufficient to increase the steady-state level of apo B100-beta particles above that of apo B100 particles in control mice.
 ...
PMID:Two distinct apolipoprotein B alleles in mice generated by a single 'in-out' targeting. 892 9

To test the hypothesis that mitochondrial DNA (mtDNA) is more prone to reactive oxygen species (ROS) damage than nuclear DNA, a continuous flux of hydrogen peroxide (H2O2) was produced with the glucose/glucose oxidase system. Using a horse radish peroxidase (HRPO)-based colorimetric assay to detect H2O2, glucose oxidase (GO; 12 mU/ml) produced 95 microM of H2O2 in 1 h, whereas only 46 microM of hydrogen peroxide accumulated in the presence of SV40-transformed human fibroblasts ( approximately 1 x 10(6). DNA damage was assessed in the mitochondira and three nuclear regions using a quantitative PCR assay. GO (12 mU/ml) resulted in more damage to the mitochondrial DNA (2.250 +/- 0.045 lesions/10 kb) than in any one of three nuclear targets, which included the non-expressed beta-globin locus (0.436 +/- 0.029 lesions/10 kb); and the active DNA polymerase b gene (0.442 +/- 0.037 lesions/10 kb); and the active hprt gene (0.310 +/- 0.025). Damage to the mtDNA occurred within 15 min of GO treatment, whereas nuclear damage did not appear until after 30 min, and reached a maximum after 60 min. Repair of mitochondrial damage after a 15 min GO (6 mU/ml) treatment was examined. Mitochondria repaired 50% of the damage after 1 h, and by 6 h all the damage was repaired. Higher doses of GO-generated H202, or more extended treatment periods, lead to mitochondrial DNA damage which was not repaired. Mitochondrial function was monitored using the MTT (3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. A 15 min treatment with 6 mU/ml of GO decreased mitochondrial activity to 80% of the control; the activity recovered completely within 1 h after damage. These data show that GO-generated H202 causes acute damage to mtDNA and function, and demonstrate that this organelle is an important site for the cellular toxicity of ROS.
 ...
PMID:Preferential mitochondrial DNA injury caused by glucose oxidase as a steady generator of hydrogen peroxide in human fibroblasts. 944 35

Retinal pigment epithelial cell dysfunction mediated by reactive oxygen intermediates has been suggested as a possible cause of age-related macular degeneration. To test the hypothesis that retinal pigment cells are susceptible to genetic damage mediated by reactive oxygen intermediates, retinal pigment epithelial cells were treated with 50 micrometers-200 micrometers of hydrogen peroxide in vitro. Damage to mitochondrial DNA and three nuclear loci were assessed using quantitative polymerase chain reaction. Hydrogen peroxide treatment of retinal pigment epithelial cells resulted in significantly increased mitochondrial DNA damage. Significant mitochondrial DNA damage occurred rapidly and was not completely repaired within 3 hr post-treatment. By contrast, no DNA damage was observed in three different nuclear loci (beta-globin gene cluster, hprt, and beta- polymerase genes). Hydrogen peroxide treatment of retinal pigment epithelial cells also resulted in decreased mitochondrial redox function compared to controls, consistent with increased mitochondrial DNA damage. Consequently, retinal pigment epithelial cell mitochondrial DNA appears susceptible to hydrogen peroxide mediated damage in vitro, and thus, may serve as a catalyst in the initial events leading to retinal pigment epithelial cell dysfunction in vivo.
 ...
PMID:Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells. 1037 40

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.
 ...
PMID:Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA. 1088 49

Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.
 ...
PMID:Multiple splicing defects in an intronic false exon. 1093 19

Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H2O2; 0-5 mM) or iron (as Fe(II)SO4, 0-500 microM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, beta-pol and beta-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H2O2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H2O2. The mitochondrial genome of human spermatozoa was significantly (P<0.001) more susceptible to H2O2-induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage.
 ...
PMID:Quantitative analysis of gene-specific DNA damage in human spermatozoa. 1294 17