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:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed the organization, structure, and function of the murine T-cell receptor C alpha/C delta region. This region spans 94.6 kb of DNA and contains the C alpha and C delta genes, as well as the V delta 5, J delta 2, and 50 different J alpha gene segments. Within this sequence we have identified 15 new J alpha gene segments, 40 new 5' RNA splice signals, and 40 new DNA rearrangement signals for the J alpha gene segments. The murine C alpha/C delta sequence contains an exceptionally high level of coding sequence with over 5.7% of the total sequence found in the exons. This is much more than that found in the beta-globin locus and the HPRT locus. Using the sequence data obtained from the C alpha/C delta region, we have designed simple assays to test for J alpha gene segment transcription and to determine the level of polymorphism for simple repeat sequences among different inbred strains of mice using the polymerase chain reaction. Furthermore, comparisons of this 95 kb of sequence with the available sequence from homologous regions of other species have led to the identification of a highly conserved sequence that is present throughout vertebrates and in the mouse binds lymphocyte-specific nuclear proteins. Comparisons of a 10-kb region, which includes the C alpha gene in human and mouse, average 66% sequence similarity. These studies support the contention that large-scale DNA sequencing projects of homologous regions of mouse and human will provide powerful new tools for studying the biology and evolution of loci such as the T-cell receptor and for identifying and posing new questions about the functions of conserved sequences.
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PMID:Organization, structure, and function of 95 kb of DNA spanning the murine T-cell receptor C alpha/C delta region. 150 54

Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase gene). The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids. Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method. Several clones from the human beta-globin locus were isolated. These cosmids transform mouse L cells at high efficiency in both circular and linear form. The newly introduced genes are expressed accurately in L cells.
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PMID:The construction of cosmid libraries which can be used to transform eukaryotic cells. 629 12

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.
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PMID:Human globin gene expression in hybrid 2S MEL X human fibroblast cells. 658 92

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.
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PMID:Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy. 793 10

'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.
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PMID:Two distinct apolipoprotein B alleles in mice generated by a single 'in-out' targeting. 892 9

We have recently detected de-novo transcripts of the predominantly muscle-specific myotonin protein kinase gene in human preimplantation embryos from the 1-cell to the 4-cell stages. Others have shown de-novo transcripts of the Y-linked genes, ZFY and SRY, in the 1-cell zygote. In order to assess the significance of early transcription of these predominantly tissue-specific genes in preimplantation development, we have analysed individual human oocytes and preimplantation embryos for the presence of transcripts of two further tissue-specific genes, alpha-globin and beta-globin, and two house-keeping genes, HPRT and APRT. Reverse transcriptase polymerase chain reaction assays were developed to the required single cell sensitivity, using human red blood cells and fibroblasts, prior to their application to human oocytes and embryos. As expected, transcripts of the house-keeping genes, HPRT and APRT, were detected at all stages of preimplantation development. Transcripts of 'tissue-specific' alpha-globin were readily detected in preimplantation embryos from the 1-cell stage. However, transcripts of beta-globin were detected only rarely (in only one of the 11 embryos analysed). This difference may be due to the fact that alpha-globin contains a CpG island. A survey of the data on gene expression in early human development suggests that CpG-island-containing genes may be expressed in preimplantation embryos. Expression of these genes in gametes and early embryos may be involved in the survival of CpG islands in evolution.
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PMID:Transcription of tissue-specific genes in human preimplantation embryos. 940 90

Oxaliplatin is a clinical anticancer drug with a pharmacological profile distinct from that of cisplatin. Our studies compared site- and region-specificity of lesions induced by oxaliplatin and cisplatin in naked and intracellular DNA, respectively. Oxaliplatin adducts in naked Simian virus 40 (SV40 DNA) were mapped by repetitive primer extension. The sites of oxaliplatin adducts were nearly identical to the sites of cisplatin adducts and were focused in G clusters and GNG motifs probably reflecting intrastrand cross-links. Although alkaline agarose electrophoresis of specific SV40 fragments showed that oxaliplatin formed interstrand cross-links, the levels of this lesion type were low. Drug-induced lesions in discrete loci of cellular DNA were assessed by the polymerase chain reaction stop assay in human tumor A2780 cells. Oxaliplatin at 200 microM induced approximately 1300, approximately 1500, approximately 800, and approximately 300 lesions/10(6) bp in the human beta-globin, c-myc, and HPRT genes and in mitochondrial DNA, respectively. Cisplatin formed two to six times more lesions in the same regions. For both drugs, lesion frequencies seem to parallel the density of drug-binding motifs in the nuclear regions, whereas mitochondrial DNA was disproportionately less affected. Despite less potent induction of DNA lesions, oxaliplatin was more cytotoxic than cisplatin against A2780 cells. Because our findings clearly demonstrate that oxaliplatin forms covalent adducts with a similar sequence- and region-specificity to that of cisplatin, other properties of oxaliplatin adducts, factors other than DNA binding, or both determine the unique features of the mechanism of action of oxaliplatin.
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PMID:Sequence- and region-specificity of oxaliplatin adducts in naked and cellular DNA. 980 12

Bizelesin and adozelesin are DNA-reactive antitumor drugs that alkylate adenines at the 3' ends of their preferred binding sites [5'T(A/T)(4)A3'and 5'(A/T)(3)(-4)A3', respectively]. We used these drugs to examine the determinants for region-specific damage of human genomic DNA. The distribution of bizelesin binding motifs in several regions analyzed "in silico" correlated well with the experimentally determined lesions in these regions assessed by quantitative polymerase chain reaction (QPCR) stop assay. In contrast to the typically low motif density, clusters of potential bizelesin binding sites were found in the matrix-associated regions (MAR domains) of the c-myc and apolipoprotein B (apoB) genes. Accordingly, lesions induced by bizelesin in these domains (2.13 and 7.06 lesions kbp(-1) microM(-1), respectively) markedly exceeded lesions in bulk DNA (0.87 lesions kbp(-1) microM(-1)) or in regions with typically low motif density (e.g., 0.75 and 0.87 lesions kbp(-1) microM(-1) in a beta-globin gene and c-myc origin of replication regions, respectively). Consistent with the more frequent, less localized adozelesin motif, actual lesions induced by adozelesin exceeded by severalfold lesions by bizelesin in four selected regions (within the c-myc and HPRT loci). Whereas adozelesin is likely to affect similar regions as bizelesin, adozelesin's more promiscuous binding probably compromises its relative specificity for such targets. In contrast, findings for bizelesin provide for the first time a proof of principle that a small molecular weight drug can preferentially damage specific regions in cellular DNA. Targeting of critical repetitive sequences, such as AT-rich MAR domains, which allow for clustering of drug binding motif, can be the paradigm for region specificity of small molecular weight agents.
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PMID:Region-specific DNA damage by AT-specific DNA-reactive drugs is predicted by drug binding specificity. 1093 11

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5' nuclease PCR (TaqMan) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7kb beta-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using 32P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas 32P-postlabeling with nuclease P(1) enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B(1). Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing 32P-postlabeling assay with nuclease P(1) enrichment.
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PMID:Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan). 1173 68

Previous work has established that stably transfected human MCF7 cells over-expressing high mobility group A1 proteins (HMGA1) are deficient in global genomic repair (GGR) following exposure to either UV light or cisplatin. To investigate whether HMGA1 over-expression also interferes with gene-specific repair, we employed a rapid and convenient quantitative polymerase chain reaction assay for measuring repair in unique DNA sequences. Efficiency of UV-induced lesion removal was assessed for two genes in MCF7 cells either induced, or not, to over-express transgenic HMGA1 proteins: the constitutively active HPRT gene and the transcriptionally silent beta-globin gene. As controls, similar experiments were also performed in non-transgenic MCF7 cells that do not express detectable levels of HMGA1 and in normal human embryonic fibroblasts that naturally over-express HMGA1 proteins. Our results indicate that exposure of cells to a UV dose of 20 J/m2 produced an average of 0.21+/-0.03 and 0.19+/-0.02 lesions/kb in the HPRT and beta-globin genes, respectively, with no significant difference between HMGA1 over-expressing cells and non-expressing cells. On the other hand, analysis of repair following UV exposure revealed that, compared to controls, HMGA1 over-expressing cells take considerably longer to repair photo-lesions in both the active HPRT and the silent beta-globin loci, with non-expressing cells repairing 50% of lesions in HPRT 3-4 h faster than HMGA1 over-expressing cells. Interestingly, the delay in repair is even more prolonged in the silent beta-globin locus in HMGA1 over-expressing cells compared to control cells. To our knowledge, this is the first report of HMGA1 proteins inhibiting nucleotide excision repair (NER) within specific genes located in either transcriptionally active "open", or inactive "closed", chromatin domains. Furthermore, taken together with previous findings, these results suggest that HMGA1 over-expression interferes with repair processes common to both the GGR and transcription-coupled repair pathways.
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PMID:Gene-specific nucleotide excision repair is impaired in human cells expressing elevated levels of high mobility group A1 nonhistone proteins. 1754 Jun 22


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