RESUMEN
Mammalian cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) sequence informations. These fragments were subsequently used to screen a rat liver cDNA library, yielding a 1573-bp clone. Expression of DNA fragment containing either of two possible open reading frames (nucleotide numbers 97-1431 and 148-1431) of the isolated clone in yeast led to the accumulation of gamma-linolenic acid in the presence of exogenous linoleic acid. In this system, the addition of alpha-linolenic acid also resulted in the accumulation of its Delta-6 desaturated product whereas dihomo-gamma-linolenic acid failed to be a substrate. These results indicate that the protein encoded by the rat cDNA is Delta-6 fatty acid desaturase, and the first 17 amino acids corresponding to the coding region 97-147 of the clone are not required to function in yeast.
Asunto(s)
Ácido Graso Desaturasas/genética , Hígado/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía de Gases , Clonación Molecular , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/análisis , Ácido Linoleico/metabolismo , Ácidos Linolénicos/metabolismo , Linoleoil-CoA Desaturasa , Masculino , Ratones , Ratones Endogámicos BALB C , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
We analyzed DNA from six Japanese patients with adenine phosphoribosyltransferase (APRT) deficiency who developed 2,8-dihydroxyadenine (DHA) urolithiasis. These six patients were selected for DNA analysis since they were expected to possess allele(s) with mutations other than two known abnormalities, i.e. a missense mutation at codon 136 (APRT*J allele) and a nonsense mutation at codon 98. In three of the six patients an insert of four bases CCGA was detected in exon 3 by sequencing clones obtained from the genomic DNA. In two of the three patients, both of the two alleles had this mutation (homozygotes) while the other patient had the APRT*J allele in addition to the allele with the 4-base insertion. To search for mutations other than the above three defined germline mutations, we amplified a genomic DNA segment including all the 5 exons of the APRT gene by PCR and cloned it into a plasmid. After selecting recombinant plasmids containing neither of the three defined mutations, we sequenced the entire APRT exons and introns. Abnormalities were found in neither the coding regions nor the exon-intron junctions. Disease-related mutations in these mutant alleles may exist in either 5' or 3' flanking sequences and remain to be elucidated.
Asunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Mutación de Línea Germinal , Adenina Fosforribosiltransferasa/genética , Secuencia de Bases , Clonación Molecular , ADN/aislamiento & purificación , Análisis Mutacional de ADN , Exones , Genotipo , Humanos , Japón , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
Adenine phosphoribosyltransferase (APRT) deficiency causes 2,8-dihydroxyadenine(DHA) urolithiasis and renal failure. Recently, two different common mutations were identified; one was APRT* J with a substitution of ACG for ATG at codon 136, called "Japanese-type", another was APRT* Q0 with TGA for TGG at codon 98. Approximately 98% of all Japanese patients with this disorder have been estimated to have these mutations APRT* J (approximately 80%) and/or APRT*Q0 (approximately 20%). We developed a diagnostic method to detect these genotypes. After gene amplification by PCR, target DNA was hybridized with a biotinylated specific probe in the presence of the non-labelled competitive probe on a dot-blotted membrane. To detect the APRT* J (or APRT* Q0) mutation, the biotinylated APRT* J (or APRT* Q0) probe and non-labelled normal probe for the same region were used as specific and competitive probes, respectively. After incubation at 60 degrees C for 30 min, the temperature was gradually decreased from 60 degrees C to 40 degrees C during 120 min, and then incubation was continued at 40 degrees C for 30 min. By using method, we were able to omit the posthybridization process, and the detecting signal was clear and highly specific. This method is useful for detecting point mutations in other genes.
Asunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Pueblo Asiatico/genética , Secuencia de Bases , Sondas de ADN , Femenino , Humanos , Japón , Masculino , Datos de Secuencia Molecular , Mutación Puntual , Cálculos Urinarios/genéticaRESUMEN
About 79% of all the Japanese patients with adenine phosphoribosyltransferase (APRT) deficiency have been estimated to possess at least one APRT*J allele with a substitution of ACG for ATG at codon 136. We developed a non-radioactive method for diagnosing genotypes of this disease. Part of the genomic DNA including the mutation site of the APRT*J allele was amplified using polymerase chain reaction and the amplified product was dot-blotted onto nylon membranes and then hybridized with either APRT*J-specific or non-APRT*J-specific synthetic oligonucleotides labelled at the 5' termini with biotin in the presence of non-labelled competitive synthetic sequences. The temperature was gradually decreased during the hybridization. When competitive sequences were omitted, difference in the intensity of the hybridization between APRT*J-containing and non-containing samples was not sufficiently clear to differentiate the genotypes. When an excess amount of competitive sequences was added in addition to biotin-labelled oligonucleotides, this method effectively differentiated samples containing only APRT*J alleles from those containing only non-APRT*J alleles. The present method was also useful to differentiate samples with both APRT*J and non-APRT*J alleles from those having only either of the alleles. An equivalent procedure using competitive sequence for hybridization and gradually decreasing the temperature will be useful for detecting point mutations in other genes.