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 examined the toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea (ENU) in three Epstein-Barr virus-transformed human lymphoblastoid cell lines, each with a different DNA repair phenotype. One cell line lacks O6-alkylguanine-DNA alkyltransferase (AGT) activity; another, derived from a patient with xeroderma pigmentosum, complementation group A, lacks nucleotide exicision repair (NER) capability, and the third is competent in both repair functions. ENU-induced toxicity and mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase locus were increased to a similar degree relative to the repair-competent cells in both AGT-deficient and NER-deficient cells. We determined the mutational spectra for ENU by identifying DNA sequence changes at the hypoxanthine-guanine phosphoribosyltransferase locus in at least 26 clones resistant to 6-thioguanine from each cell line. Of the characterized mutations, 89% were single-base pair substitutions. Transitions and transversions were found at AT and GC base pairs in all three cell lines. The biggest difference within the spectra was in the rate of transitions at GC base pairs. Compared to the repair-competent cell line, this mutation was elevated about 8-fold in the AGT-deficient cells and about 3-fold in the NER-deficient cells. We conclude that both AGT and NER play an important role in protecting human cells from the toxic and mutagenic effects of ENU. Furthermore, the mutational spectra suggest that both of these repair systems participate in the repair of O6-ethylguanine adducts.
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PMID:Toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea in human cell lines with different DNA repair phenotypes. 165 49

Human cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this problems. The method relies on the use of a recently developed episomal Epstein-Barr-virus-derived cDNA expression vector (Belt et al. (1989) Gene 84, 407-417). The cloning of hypoxanthine phosphoribosyltransferase (HPRT) cDNA, corresponding to a low abundant mRNA in wild type cells is used as a model system. Size fractionated poly (A)+ RNA from wild type cells, which resulted in an approximately 10 fold enrichment in HPRT mRNA, was used to construct a cDNA library of 25,000 independent clones in the pECV25 vector. An HPRT deficient human cell line was transfected and subsequently selected with hygromycin B for DNA uptake. In a small scale experiment only 7000 hygromycin BR transfectants were sufficient to isolate 2 independent HATR clones which were shown to replicate episomes harbouring HPRT cDNA. The first insert had a 5' untranslated region (UTR) and a 3' UTR perfectly in agreement with published data. The second cDNA clone harboured an unusually long 5' UTR and a shorter 3' UTR due to alternative polyadenylation of the HPRT transcript which has not been previously recognized.
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PMID:Efficient cDNA cloning by direct phenotypic correction of a mutant human cell line (HPRT-) using an Epstein-Barr virus-derived cDNA expression vector. 165 80

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.
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PMID:Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells. 184 3

Molecular analysis of an unusual patient with the Lesch-Nyhan syndrome has suggested that the mutation is due to a partial HPRT gene duplication. We now report the cloning and sequencing of the mutant HPRT cDNA which shows the precise duplication of exons 2 and 3. This mutation is the result of an internal duplication of 16-20 kilobases of the gene. The structure of the mutant gene suggests that the duplication was not generated by a single unequal crossing-over event between two normal HPRT alleles. Growth of Epstein-Barr virus-transformed lymphoblasts from this patient in selective medium has permitted isolation of spontaneous HPRT+ revertants of this mutation. The reversion event involves a second major HPRT gene rearrangement where most or all of the duplicated portion of the mutant gene is deleted. The original mutation therefore has the potential for spontaneous somatic reversion. This may explain the relatively mild symptoms of the Lesch-Nyhan syndrome exhibited by this patient.
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PMID:Spontaneous reversion of novel Lesch-Nyhan mutation by HPRT gene rearrangement. 283 25

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.
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PMID:Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells. 284 88

Three new kinds of recombinant DNA constructs were used to transfer cloned human class I HLA genes (A2 and B8) into unique HLA mutant lymphoblastoid cells: pHeBo(x): a class I gene, "x," in plasmid vector pHeBo, which contains a hygromycin resistance gene and Epstein-Barr virus oriP element that sustains extrachromosomal replication; pHPT(x): gene x in a vector with a hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene; pHPTe(x): gene x in a vector with the HPRT gene and oriP element. Cell surface class I antigen expression was strong in transferents made with class I-deficient lymphoblastoid cell line mutants .144 (A-null), .53 (B-null), and .184 (A-null, B-null). Transferents expressing HLA-A2 were recognized specifically by HLA-A2-specific cytotoxic T lymphocytes. When introduced on either of the vectors with the Epstein-Barr virus oriP element, the class I gene replicated extrachromosomally and was lost at rates of 0.2 to 0.3 per cell division. When introduced with vector pHPT (lacking Epstein-Barr virus oriP), the B8 gene was inserted at different chromosomal locations. Introduction of the HLA-B8 gene failed to restore antigen expression by HLA-B-null mutant .174, providing evidence that, unlike mutants exemplified by .53, .144, and .184, some HLA antigen loss mutants are deficient in a trans-acting function needed for class I antigen expression. Of more general interest, the results obtained with HLA class I genes in vectors that replicate extrachromosomally suggest ways of relating genic expression to chromatin structure and function and of attempting to clone functional human centromeres.
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PMID:Transfer of cloned human class I major histocompatibility complex genes into HLA mutant human lymphoblastoid cells. 302 67

The hereditary dysplastic nevus syndrome (DNS) is a well-characterized disorder in which affected individuals have increased numbers of premalignant (dysplastic) nevi and a markedly increased risk of developing cutaneous melanoma. Seeking evidence of a systemic disorder in DNS, we examined the effect of ultraviolet radiation on cultured lymphoid cells. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS had similar survival values following treatment with 2.3 to 9.0 J of 254-nm ultraviolet radiation per m2 as did lines from control individuals. Mutagenesis at the hypoxanthineguanine phosphoribosyltransferase locus was assessed by measuring the induction of resistance to thioguanine using a microtiter well assay. Three lymphoblastoid cell lines from patients with hereditary DNS and melanoma had a 2- to 3-fold greater frequency of induced mutants per clonable cell than three normal lines following exposure to 4.5 to 9.0 J of ultraviolet radiation per m2. Expanded clones of mutated DNS lymphoblastoid cell lines had less than 6% of normal hypoxanthine-guanine phosphoribosyltransferase activity. Inhibition and recovery of DNA synthesis following ultraviolet exposure were similar in 2 DNS and 2 normal lines. Repair by DNS lines of ultraviolet-induced DNA damage was in the normal range as measured by alkaline elution. Thus, hereditary DNS exhibits in vitro hypermutability which may reflect increased susceptibility to ultraviolet-induced somatic mutations in vivo. This abnormality may be related to the increased melanoma susceptibility of patients with hereditary DNS.
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PMID:Hereditary dysplastic nevus syndrome: lymphoid cell ultraviolet hypermutability in association with increased melanoma susceptibility. 394 Jun 25

A mutant of the Jurkat human T lymphoblastoid cell line deficient in hypoxanthine phosphoribosyltransferase, and resistant to ouabain, was fused with peripheral blood T lymphocytes primed in vitro with Epstein Barr virus- (EBV) transformed autologous B lymphocytes. After selection of somatic cell hybrids and cloning, hybridoma cell lines were obtained that reacted with autologous EBV-infected B lymphocytes, as detected by the release of interleukin 2 into the culture medium. The hybridomas did not react with i) EBV-uninfected autologous or allogeneic B lymphocytes, ii) three out of four allogeneic EBV-transformed cell lines, or iii) two established EBV-negative B cell lines. These functional hybridomas may ultimately prove useful in dissecting the means by which human T lymphocytes recognize and regulate EBV infection in vivo.
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PMID:Human T cell hybridomas specific for Epstein Barr virus-infected B lymphocytes. 629 1

Epstein-Barr virus (EBV)-negative, Burkitt-like lymphoma-derived cells were transformed with a transducing vector (pSV2-gpt) containing the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase (XGPRT) and with a derivative of PSV2-gpt that carries the genes for the EBV-associated small RNAs on the EcoRI J fragment of B95-8 EBV DNA inserted at the unique EcoRI site (pJ-gpt). Cells transformed with PSV2-gpt and pJ-gpt express the E. coli gpt gene to approximately the same extent, judged by determinations of the XGPRT activity of cell extracts. Blot hybridisation experiments with restriction endonuclease-cleaved DNA from the transformants have revealed the presence of vector DNA sequences in the cells, at least some of which are most probably integrated into high mol. wt. chromosomal DNA. Northern blot hybridisation analysis of cytoplasmic RNA from pJ-gpt-transformed cells revealed the presence of an EcoRI J DNA complementary RNA species of the same size as the EBV DNA-encoded small RNAs found in EBV-transformed cells.
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PMID:Transfer of the Epstein-Barr virus genes coding for small RNAs to human lymphoid cells with a vector carrying a dominant selectable marker. 631 18

Primary nasopharyngeal carcinoma (NPC) cells were fused to hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-defective cells derived from adenoid tissues using Sendai virus. Some of the fused cells developed into epithelial-like hybrid cells in a selective HAT medium. The hybrid cells (NPC-KT) were Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive cells. There have been no reports on the establishment of EBNA-positive epithelial cell lines derived from NPC. Thus, the epithelial-like hybrid cells might serve as an in vitro model for studying the biologic activity of NPC-associated EBV.
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PMID:Establishment of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive nasopharyngeal carcinoma hybrid cell line (NPC-KT). 631 11


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