Asunto(s)
Isoenzimas/genética , Mutación Puntual , Nucleótidos de Purina/farmacología , Ribosa-Fosfato Pirofosfoquinasa/antagonistas & inhibidores , Ribosa-Fosfato Pirofosfoquinasa/genética , Cromosoma X , Secuencia de Aminoácidos , Mapeo Cromosómico , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/antagonistas & inhibidoresRESUMEN
Superactivity of phosphoribosylpyrophosphate synthetase (PRS) is an X chromosome-linked disorder of purine metabolism, characterized by gout with uric acid overproduction and, in some families, neurodevelopmental impairment. Two highly homologous isoforms of PRS (PRS1 and PRS2), each encoded by a distinct X chromosome-linked locus, have been identified, and PRS1 and 2 cDNAs have been cloned. The entire 954-base pair translated regions of PRS1 and 2 cDNAs derived from cultured lymphoblasts and fibroblasts from two patients in whom purine nucleotide feedback resistance of PRS is associated with enzyme superactivity and neurodevelopmental defects were examined by direct sequencing after polymerase chain reaction amplification of PRS transcripts. Nucleotide sequences of PRS2 cDNAs from the patients and normal individuals were identical. In contrast, PRS1 cDNAs from the patients differ from normal PRS1 cDNA, each by a single base substitution. PRS1 cDNA from patient N. B. showed an A to G transition at nucleotide 341, corresponding to an asparagine to serine change at amino acid residue 113 of mature PRS1. A G to C transversion at nucleotide 547, indicating an aspartic acid to histidine change at amino acid 182, was found for PRS1 cDNA from patient S. M. Point mutations at the sites identified in the PRS1 cDNAs of the two patients were confirmed by the results of RNase mapping analysis. Normal, N. B., and S. M. PRS1 cDNAs were introduced into Escherichia coli BL21 (DE3)/pLyS, and recombinant N. B. and S. M. PRS1s showed the purine nucleotide feedback resistance phenotypes characteristic of PRS from patients' cells.
Asunto(s)
Ligamiento Genético , Isoenzimas/metabolismo , Mutación Puntual , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Cromosoma X , Secuencia de Bases , Línea Celular , ADN Complementario , Escherichia coli/genética , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Nucleótidos de Purina/antagonistas & inhibidores , Ribosa-Fosfato Pirofosfoquinasa/genética , Análisis de Secuencia de ADNRESUMEN
Naturally occurring mutations in hypoxanthine-guanine phosphoribosyltransferase (HPRT) have been identified by amino acid sequencing, cDNA cloning, and direct nucleotide sequencing of PCR-amplified transcripts. To determine the effect these mutations have on the catalytic properties of the molecule, knowledge of the three-dimensional structure of HPRT is required. A prerequisite for this, however, is the availability of a large amount of purified product for crystallization and x-ray diffraction analysis. For these reasons we have developed an effective means of producing high levels of human HPRT in Escherichia coli using the expression cassette PCR. By taking advantage of a T7 polymerase/promoter system, we have expressed both normal and variant human hprt sequences in E. coli. The proteins synthesized from these sequences are immunologically and enzymatically active, and are physically indistinguishable from the HPRT in B-lymphoblasts derived from normal and three HPRT-deficient subjects.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/biosíntesis , Secuencia de Bases , Escherichia coli , Vectores Genéticos , Humanos , Hipoxantina Fosforribosiltransferasa/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Factores de TiempoRESUMEN
Hypoxanthine--guanine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Previous studies of mutant HPRT proteins analyzed at the molecular level have shown a significant heterogeneity. This investigation further verifies this heterogeneity and identifies insertions, deletions, and point mutations. The direct sequencing of the polymerase chain reaction-amplified product of reverse-transcribed HPRT mRNA enabled the rapid identification of the mutations found in 17 previously uncharacterized cell lines derived from patients with the Lesch-Nyhan syndrome.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Síndrome de Lesch-Nyhan/genética , Mutación , Línea Celular , Análisis Mutacional de ADN , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Síndrome de Lesch-Nyhan/genética , Mutación/genética , Deleción Cromosómica , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Síndrome de Lesch-Nyhan/enzimología , Mutagénesis InsercionalRESUMEN
1. ECPR is a rapid and effective means for generating recombinant human HPRT. 2. The Bl21 (DE3) T7 polymerase/T7 promoter system provides high level expression of human HPRT constructs after induction of the T7 polymerase gene with IPTG. 3. Human HPRT constructs expressed in E. coli mimic the variant properties originally demonstrated in lymphoblast extracts from affected individuals. 4. Human HPRT expressed in E. coli can be rapidly purified to near homogeneity by a two step purification scheme.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Secuencia de Bases , Clonación Molecular , ADN/genética , Escherichia coli/genética , Expresión Génica , Variación Genética , Humanos , Hipoxantina Fosforribosiltransferasa/deficiencia , Hipoxantina Fosforribosiltransferasa/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
Adenine phosphoribosyltransferase (APRT) deficiency causing 2,8-dihydroxyadenine urolithiasis and renal failure is present at a high frequency among the Japanese but not other ethnic groups. A special type of mutant allele, designated APRT*J, with a nucleotide substitution at codon 136 from ATG (Met) to ACG (Thr) is carried by approximately 79% of all Japanese 2,8-dihydroxyadenine urolithiasis patients. We analyzed mutant alleles of 39 APRT deficient patients using a specific oligonucleotide hybridization method after in vitro amplification of a part of the genomic APRT sequence. We found that 24 had only APRT*J alleles. Determination of the haplotypes of 194 APRT alleles from control Japanese subjects and of the 48 different APRT*J alleles indicated that normal alleles occur in four major haplotypes, whereas all APRT*J alleles occur in only two. These results suggest that all APRT*J alleles have a single origin and that this mutant sequence has been maintained for a long period, as calculated from the frequency of the recombinant alleles.
Asunto(s)
Adenina Fosforribosiltransferasa/genética , Intercambio Genético , Southern Blotting , Ligamiento Genético , Humanos , Enfermedades Renales/genética , Mutación , Sondas de Oligonucleótidos , Mapeo Restrictivo , Cálculos Urinarios/genéticaRESUMEN
DNA methylation is one of the mechanisms involved in the regulation of developmentally relevant genes. Previous experiments demonstrated that T cells treated with DNA methylation inhibitors reacquire some of the phenotypic and functional characteristics of thymocytes, suggesting that DNA methylation may be involved in regulating some of the changes in gene expression during thymic maturation. To further examine whether changes in DNA methylation occur during T cell differentiation, total DNA deoxymethylcytosine content was compared in human thymocyte subsets and mature T cells. A significant increase in deoxymethylcytosine was found at the end of T cell differentiation which then decreased with age. These results suggest that increased DNA methylation may serve to silence genes following T cell differentiation. The results also raise the possibility that age-related decreases in T cell DNA methylation may contribute to changes in T cell function occurring in the elderly.
Asunto(s)
Envejecimiento/inmunología , ADN , Regulación de la Expresión Génica , Activación de Linfocitos/fisiología , Linfocitos T/química , Adolescente , Southern Blotting , Niño , Preescolar , Humanos , Lactante , Metilación , Timo/inmunología , Transcripción GenéticaRESUMEN
Chronic perturbations of intracellular deoxyribonucleoside triphosphate (dNTP) pools have been associated with a mutator phenotype and increased mutation rates at several genetic loci. We have examined the specific effects of transient pharmacological purine dNTP pool perturbations on mutations induced at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in a cultured human T-lymphoblast cell line. Incubation of CEM cells with 50 microM 2'-deoxyguanosine for 6 h increased intracellular dGTP levels 43-fold and induced a 40-fold increase in mutation frequency at the HPRT locus. Six-h incubations with 5, 10, and 20 microM 2'-deoxyadenosine increased dATP pools 4.8-, 8-, and 14.5-fold, respectively, with 59-, 34-, and 43-fold increases in HPRT mutant fractions. In contrast, 24-h incubations with hydroxyurea at concentrations which inhibited cell growth to similar extents did not induce HPRT mutations. Sequencing of HPRT complementary DNA derived from mutant cell lines revealed that the mutations induced by transient purine dNTP pool perturbations exhibited no significant misincorporation of the nucleotide in excess or next-nucleotide effect, and were similar in nature and location to spontaneous HPRT mutations. We conclude that mutations caused by transient purine dNTP pool elevations in these dividing cells are most likely induced by inhibition of DNA repair processes.
Asunto(s)
Desoxirribonucleótidos/metabolismo , Hipoxantina Fosforribosiltransferasa/genética , Mutación , Nucleótidos de Purina/metabolismo , Células Tumorales Cultivadas/metabolismo , Secuencia de Bases , Línea Celular , Desoxiadenosinas/farmacología , Desoxiguanosina/farmacología , Inducción Enzimática , Amplificación de Genes , Humanos , Hipoxantina Fosforribosiltransferasa/biosíntesis , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfocitos T , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, the Lesch-Nyhan syndrome. This disorder has been identified as a candidate for initial attempts at somatic cell gene therapy. We have previously reported the construction of a recombinant herpes simplex virus type 1 (HSV-1) vector containing human hprt cDNA sequences under the regulatory control of the viral thymidine kinase gene (tk) [Palella et al., Mol. Cell. Biol. 8 (1988) 457-460]. Infection of HPRT- cultured rat neuronal cells with these vectors resulted in transient expression of human hprt. In this paper, we report the expression of human hprt mRNA transcripts in the brains of mice infected in vivo with this vector by direct intracranial inoculation. Human hprt transcripts were distinguished from endogenous mouse transcripts by RNase A mapping using riboprobes transcribed from human hprt cDNA. These initial studies demonstrate the transfer and transcription of a human gene in brain cells by direct in vivo infection with recombinant HSV-1 vectors.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Simplexvirus/genética , Animales , Encéfalo/enzimología , Encefalopatías/enzimología , Encefalopatías/genética , ADN/genética , Vectores Genéticos , Herpes Simple/enzimología , Herpes Simple/genética , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Hígado/enzimología , Ratones , Ratones Endogámicos DBA , Mapeo Nucleótido , Sondas ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
Generally, if mutant and normal proteins have similar molecular weights and electric charges, they cannot easily be distinguished from one another. We have developed a unique method by which a mutant enzyme of adenine phosphoribosyltransferase (APRT) can easily be distinguished from normal enzyme with nearly identical molecular weight and electric charge. DNA sequencing data have suggested that in this special type of disease (Japanese-type APRT deficiency) there is an amino acid substitution from Met to Thr at position 136 of APRT. Since normal APRT has only one Met residue, the Japanese-type mutant APRT should be a methionine-free protein. Using both an amino acid sequence-specific antiserum against APRT, and specific cleavage of peptide at the methionine residue with BrCN, we could distinguish between normal and mutant proteins. Thus, normal but not mutant APRT was cleaved with BrCN, indicating that the mutant APRT is a methionine-free protein. All tested patients with the Japanese-type APRT deficiency were found to synthesize exclusively methionine-free APRT. Usefulness of this method is not restricted to a single family, as 79% of all the patients with this disease among Japanese, and more than half of all the patients with this disease reported in the world, are likely to have this unique mutation. Thus, not only sequence-specific cleavage of DNA with restriction endonucleases but also that of protein with a chemical agent has been shown to be sometimes useful for the diagnosis and analysis of a genetic disease by careful examination of normal and mutant amino acid sequences.
Asunto(s)
Adenina Fosforribosiltransferasa/genética , Mutación , Pentosiltransferasa/genética , Adenina Fosforribosiltransferasa/deficiencia , Secuencia de Aminoácidos , Linfocitos B/enzimología , Línea Celular , Bromuro de Cianógeno , Humanos , Metionina , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/análisis , Valores de Referencia , TreoninaRESUMEN
To evaluate the frequency and the pathogenesis of hyperuricemia and gout during cyclosporine therapy, we studied renal-transplant recipients who were treated with either cyclosporine and prednisone (n = 129) or azathioprine and prednisone (n = 168). Among the patients with stable allograft function and serum creatinine concentrations below 265 mumol per liter, hyperuricemia was more common in the cyclosporine group than in the azathioprine group (84 percent vs. 30 percent; P = 0.0001). Gout developed in nine patients (7 percent) in the cyclosporine group, but no episodes occurred in the azathioprine group. Serum urate levels became elevated in 90 percent of the patients in the cyclosporine group who were treated with diuretics, as compared with 60 percent of those not treated with diuretics (P = 0.001); in the azathioprine group, the corresponding values were 47 percent and 15 percent (P = 0.0001). Serum urate levels did not correlate with trough blood cyclosporine levels in a selected subgroup (n = 40) of patients from the cyclosporine group, who were studied from 4 to 96 weeks after transplantation. Detailed studies of urate metabolism in six cyclosporine-treated patients revealed normal turnover rates for urate and decreases in creatinine and urate clearance, as compared with seven control subjects. We conclude that hyperuricemia is a common complication of cyclosporine therapy and is caused by decreased renal urate clearance. Gouty arthritis is the cause of considerable morbidity among renal-transplant recipients who receive cyclosporine.
Asunto(s)
Ciclosporinas/efectos adversos , Gota/inducido químicamente , Ácido Úrico/sangre , Adulto , Artritis Gotosa/inducido químicamente , Azatioprina/administración & dosificación , Creatinina/sangre , Ciclosporinas/administración & dosificación , Quimioterapia Combinada , Femenino , Humanos , Trasplante de Riñón , Masculino , Complicaciones Posoperatorias , Prednisona/administración & dosificación , Ácido Úrico/farmacocinéticaRESUMEN
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an inborn error of purine metabolism. Mutant HPRT gene sequences from patients deficient in enzyme activity have previously been characterized by cDNA cloning or amino acid sequencing techniques. The presence of HPRT-specific mRNA in nearly all deficient subjects, as well as the small size of the HPRT mRNA (1,400 bp), make the polymerase chain reaction (PCR) an alternative for the identification of mutations at this locus. In this report we use the PCR to identify previously undetermined mutations in HPRT mRNA from B lymphoblasts derived from 10 deficient individuals. Six of these variants contain single point mutations, three contain deletions, and one contains a single nucleotide insertion. Several of these mutations map near previously identified HPRT variants, and are located in evolutionarily conserved regions of the molecule.
Asunto(s)
Amplificación de Genes , Hipoxantina Fosforribosiltransferasa/deficiencia , Mutación , Transcripción Genética , Secuencia de Aminoácidos , ADN/análisis , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Datos de Secuencia MolecularAsunto(s)
Artritis , National Institutes of Health (U.S.) , Investigación , Humanos , Estados UnidosAsunto(s)
Granulomatosis con Poliangitis/diagnóstico , Metildopa/efectos adversos , Vasculitis Leucocitoclástica Cutánea/diagnóstico , Diagnóstico Diferencial , Humanos , Hipertensión/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Vasculitis Leucocitoclástica Cutánea/inducido químicamenteRESUMEN
We have cloned and sequenced a full length cDNA for HPRT cDNA for HPRTYale isolated from Lesch-Nyhan subject and identified a single nucleotide substitution which results in amino acid substitution of glycine to arginine. Since most HPRT mutants have normal levels of specific HPRT messenger RNA, mutant cDNA analysis is the method of choice to define the mutation in HPRT deficient subjects.