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)

Conditions were characterized for maximizing the uptake of exogenous mammalian cell DNA by hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster lung cells. Recipient cell cultures in an exponential growth phase were found to be more competent in taking up DNA than stationary cultures. Polyornithine enhanced the uptake of exogenous DNA more reproducibly and to a greater extent than did any of the other facilitators tested (DEAE-dextran, CaCl2, latex spheres, spermine, polylysine and polyarginine). Maximal DNA incorporation occurred when polyornithine and DNA were mixed together prior to inoculation. About 25-30% of the DNA inoculum became deoxyribonuclease-resistant in a typical experiment utilizing polyornithine as the facilitator. Both homologous and heterologous exogenous DNAs rapidly became associated with recipient cell nuclei: approximately 95% of the deoxyribonuclease-resistant donor DNA was nuclear-associated 15 min after inoculation.
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PMID:Optimal conditions for uptake of exogenous DNA by Chinese hamster lung cells deficient in hypoxanthine-guanine phosphoribosyltransferase. 116 8

The human androgen-receptor gene (HUMARA; GenBank) contains a highly polymorphic trinucleotide repeat in the first exon. We have found that the methylation of HpaII and HhaI sites less than 100 bp away from this polymorphic short tandem repeat (STR) correlates with X inactivation. The close proximity of the restriction-enzyme sites to the STR allows the development of a PCR assay that distinguishes between the maternal and paternal alleles and identifies their methylation status. The accuracy of this assay was tested on (a) DNA from hamster/human hybrid cell lines containing either an active or inactive human X chromosome; (b) DNA from normal males and females; and (c) DNA from females showing nonrandom patterns of X inactivation. Data obtained using this assay correlated substantially with those obtained using the PGK, HPRT, and M27 beta probes, which detect X inactivation patterns by Southern blot analysis. In order to demonstrate one application of this assay, we examined X inactivation patterns in the B lymphocytes of potential and obligate carriers of X-linked agammaglobulinemia.
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PMID:Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. 128 84

Constitutional loss or inactivation of one copy of a tumor-suppressor gene, as exemplified by hereditary retinoblastoma, increases the propensity for malignancies by reducing the number of events necessary for the complete loss of the negative regulatory function. We developed a selectable mutation assay employing a human lymphoblastoid cell line (LCL) derived from a heterozygous carrier of 2,8-dihydroxyadenine urolithiasis, adenine phosphoribosyltransferase (APRT) deficiency, for dissecting the second step in loss-of-function mutations and for determining the potential of physical and chemical agents for producing such mutations. The mode of mutational events arising in the wild-type allele of the functionally heterozygous APRT gene resembled that reported for tumor-suppressor genes in malignancies in that mitotic non-disjunctions or recombinations as well as deletions prevailed. Ultraviolet light (UV) was much less efficient in inducing these types of mutations than ionizing radiation. A group of autosomal recessive cancer-prone diseases, including xeroderma pigmentosum (XP), has been characterized as being more susceptible to genomic insults, owing to some defects in DNA processing, such as replication, repair, or recombination. This increased genomic instability may accelerate the gain-of-function mutation at a proto-oncogene and/or the loss-of-function mutation at a tumor-suppressor gene. XP complementation group A (XP-A) LCLs were extremely sensitive to UV-mutagenesis at the hypoxanthine phosphoribosyltransferase (HPRT) locus even at equicytotoxic doses. Some unique mechanism may operate in UV-mutagenesis in XP-A. We have succeeded for the first time in rendering XP-A cells tumorigenic in athymic mice by applying multiple exposures to UV and subsequent treatment with TPA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Molecular bases for hereditary cancer-prone diseases. 129 55

The Lesch-Nyhan disease is caused by an almost complete lack of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Partial HPRT-deficiency, associated with less severe phenotype, has also been identified. We have characterized mutations occurring in HPRT cDNA isolated from patients with HPRT-deficiency with an emphasis on examining the more unusual partial variants of HPRT-deficiency. HPRT cDNA was amplified by PCR, cloned and analyzed by automated DNA sequence analysis. Twenty-two, unrelated individuals with HPRT deficiency were studied including eight classic Lesch-Nyhan patients and fourteen patients representing the different groups of partial HPRT deficiency. We found a diverse pattern of mutations with point mutations accounting for the majority of abnormal HPRT genes. Nonsense mutations and exon deletions were only found in HPRT cDNA isolated from classic Lesch-Nyhan patients. Mutations associated with partial HPRT-deficiency were frequently located in the amino terminal part of the molecule. A CpG mutational hot spot was identified at the position for Arg-51 in the HPRT protein. Two hyperuricemic patients exhibited unusual splice site mutations: in one this led to the creation of an additional exon in the HPRT gene and in the other part of exon 6 was missing in a subpopulation of the transcripts, producing the effect of a dominant, negative mutation.
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PMID:Characterization of mutations in phenotypic variants of hypoxanthine phosphoribosyltransferase deficiency. 130 16

To determine the clonal nature of hematopoiesis and to assess lineage involvement in patients with myelodysplastic syndromes (MDS), we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Eleven female MDS patients heterozygous for at least one of these probes were studied: 3 with refractory anemia (RA), 2 with RA with ringed sideroblasts (RARS), 2 with chronic myelomonocytic leukemia (CMML), and 4 with RA with excess of blasts in transformation (RAEB-t). All exhibited clonal hematopoiesis as determined by Southern analysis of DNA prepared from peripheral blood (PB) and/or bone marrow (BM) cells. In three of the six patients heterozygous for the PGK1 gene, purified cell suspensions of polymorphonuclear cells (PMN), monocytes, lymphocytes, and/or T cells prepared from PB were tested. In addition, five of these patients were analyzed by a polymerase chain reaction (PCR)-based procedure as described recently. This method was slightly adapted to facilitate the analysis of cell lysates of fluorescence-activated cell sorted (FACS) monocytes, T and B lymphocytes, and natural killer (NK) cells. The outcome of Southern and PCR analysis was concordant, showing that PMN and monocytes were clonally derived, whereas circulating T and B lymphocytes and NK cells exhibited random X-chromosome inactivation compatible with a polyclonal pattern. To address the question of whether T cells are derived from unaffected progenitor cells or that their origin had antedated the onset of MDS, naive and memory T cells were analyzed separately. Both subsets showed a polyclonal pattern. However, in one patient analysis of constitutive DNA suggested a skewed methylation, and the presence of clonal lymphocytes against a background of polyclonal lymphoid cells cannot be ruled out in this patient. PCR analysis of PB and BM cells showed a nonrandom, unilateral pattern of X-inactivation, compatible with a mixture of clonally (myeloid) and polyclonally (lymphoid) derived cells. In conclusion, in some patients, MDS represents a disorder with clonal hematopoiesis restricted to cells of myeloid origin, whereas a random X-inactivation pattern is found in lymphoid cells.
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PMID:Clonal involvement of granulocytes and monocytes, but not of T and B lymphocytes and natural killer cells in patients with myelodysplasia: analysis by X-linked restriction fragment length polymorphisms and polymerase chain reaction of the phosphoglycerate kinase gene. 135 10

MEL cells, undergoing erythroid differentiation and parasynchronized by dimethyl sulfoxide (DMSO) induction, were irradiated with a 3-s pulse of UV light at sublethal dose. A large number of clones deficient in different gene functions are found in the progeny of the treated cells, if the pulse irradiation is performed 18-24 h from the start of DMSO induction. Kinetics of thymidine incorporation into DNA show that the period of sensitivity corresponds to the S phase. The results show that the activities of the tested genes are differently affected depending on the exact time of cell irradiation. Maximum percent inhibition of cells not expressing glucose-6-phosphate dehydrogenase (G-6-PD) (70%) is produced by irradiating at 20 h from the start of DMSO induction; 6-phosphogluconate dehydrogenase (6-PGD) (55%), and hypoxanthine (guanine) phosphoribosyltransferase (HPRT) (33%), at 21 h; hemoglobin (50%), at 22 h. The time difference in the sensitivity to UV light is highly reproducible and has been exploited to isolate, with high efficiency, cellular clones deficient in any one of the tested functions. Determinations of enzymatic activities on cell lysates show that the expression of tested genes is actually altered in cells that, on the basis of cytochemical tests, appear unaffected by UV irradiation. While the production of mutant clones is observed only during the S phase of the cell cycle, immediate statistical damage of the cellular DNA is produced at all times of irradiation. This finding excludes that the two types of phenotypic alterations, blocked or altered gene expression, both propagated in the progeny of the cells as clonal properties, may derive from a preferential alteration of those functions during the S phase.
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PMID:Selective gene mutation in MEL cells. 137 Jul 18

The molecular nature of 254 nm ultraviolet light (UV)-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in UV24 Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, was determined. Sequence analysis of 19 hprt mutants showed that single base substitutions (9 mutants) and tandem base changes (7 mutants) dominated the UV mutation spectrum in this cell line. Sixty-five percent of the base substitutions were GC greater than AT transitions, whereas the rest consisted of transitions and transversions at AT base pairs. Analysis of the distribution of dipyrimidine sites over the two DNA strands, where the photoproducts causing these mutations presumably were formed, showed that 12 out of 14 mutations were located in the transcribed strand of the hprt gene. A similar strand distribution of mutagenic photoproducts as in UV24 has previously been found in two other UV-sensitive Chinese hamster cell lines (V-H1 and UV5), indicating that under defective nucleotide excision repair conditions the induction of mutations is strongly biased towards lesions in the transcribed strand of the hprt gene. A plausible explanation for this phenomenon is that during DNA replication large differences exist in the error rate with which DNA polymerase(s) bypass lesions in the templates for the leading and lagging strand, respectively.
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PMID:UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand. 137 6

Reactivation of the hypoxanthine phosphoribosyltransferase (HPRT) gene on an inactive human X chromosome in a somatic cell hybrid was analyzed following exposure to 5-aza-2'-deoxycytidine. Hemimethylation and chromatin hypersensitivity in the 5' CpG island appeared by 6 h after exposure and continued to increase for 24 h in an exponentially growing cell culture. These results imply that the conformation of inactive chromatin requires a symmetrically methylated 5' G+C-rich promoter region. In addition, quantitative analysis of the time course patterns suggest that chromatin sensitivity changes may depend on strand-specific demethylation. Symmetrically demethylated DNA was first detected at 24 h and continued to increase until 48 h. HPRT mRNA was first detected at 24 h and increased in a biphasic pattern until 48 h. These results suggest that hemimethylation permits nuclease attack but not transcription factor binding, which requires symmetrically demethylated DNA. We also show that in G1-arrested cells, 5-aza-2'-deoxycytidine has no effect on methylation, chromatin conformation, or transcription. We conclude that reactivation of the HPRT gene present on the inactive X chromosome of a somatic cell hybrid involves the initial events of DNA hemimethylation and chromatin hypersensitivity at the 5' CpG island, followed by symmetrical demethylation and transcriptional reactivation.
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PMID:Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster x human somatic cell hybrid. 138 Jun 47

The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.
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PMID:V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans. 138 Jun 58

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.
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PMID:Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE). 138 70


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