RESUMEN
Deficiency of uridine monophosphate synthase (DUMPS) is a lethal genetic disorder associated with early embryonic mortality. Murrah and Mehsana male buffaloes (n = 594) were screened for DUMPS by PCR-RFLP technique. A few Murrah buffalo male calves were found to be carriers of DUMPS in RFLP, which has not been reported earlier. On the Sanger sequencing, a novel A to G substitution mutation was identified in AvaI restriction recognition site of UMPS gene in buffaloes. This mutation hinders digestion of DNA by AvaI which leds to false positive results for DUMPS carrier in RFLP. The results indicated that genome sequencing must be performed before confirming results of RFLP in any new species. All the buffaloes that were tested had only wild-type genotype in exon 5 for DUMPS specific allele.
Asunto(s)
Búfalos/genética , Enfermedades de los Bovinos/genética , Enfermedades Genéticas Congénitas/veterinaria , Orotato Fosforribosiltransferasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Polimorfismo de Longitud del Fragmento de Restricción , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Alelos , Animales , Bovinos , Mapeo Cromosómico , Exones , Reacciones Falso Positivas , Genotipo , Masculino , Mutación , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Reacción en Cadena de la Polimerasa/métodos , Secuenciación Completa del GenomaAsunto(s)
Calcio/metabolismo , Suplementos Dietéticos/efectos adversos , Ácido Orótico/metabolismo , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Niño , Discapacidades del Desarrollo/genética , Humanos , Masculino , Orotato Fosforribosiltransferasa/deficiencia , Orotato Fosforribosiltransferasa/metabolismo , Ácido Orótico/efectos adversos , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Errores Innatos del Metabolismo de la Purina-Pirimidina/metabolismoRESUMEN
Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/terapia , Terapia Genética , Hiperamonemia/terapia , Amoníaco/metabolismo , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/uso terapéutico , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/genética , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/metabolismo , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/patología , Carbamoil Fosfato/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Glutamina/metabolismo , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperamonemia/patología , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Mutación , Orotato Fosforribosiltransferasa/deficiencia , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/patologíaRESUMEN
BACKGROUND: Elevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue. METHODS AND RESULTS: We identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members. CONCLUSIONS: We therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.
Asunto(s)
Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Orotato Fosforribosiltransferasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Errores Innatos del Metabolismo de la Purina-Pirimidina/metabolismo , Anemia Megaloblástica/genética , Anemia Megaloblástica/metabolismo , Niño , Preescolar , Femenino , Heterocigoto , Humanos , Lactante , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Masculino , Mutación/genética , Orotato Fosforribosiltransferasa/genética , Orotato Fosforribosiltransferasa/metabolismo , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilasa/genética , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Pirimidinas/metabolismo , Trastornos Innatos del Ciclo de la Urea/genética , Trastornos Innatos del Ciclo de la Urea/metabolismo , Uridina/metabolismoRESUMEN
The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura+ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10-8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.
Asunto(s)
Candida tropicalis/genética , Marcación de Gen/métodos , Genética Microbiana/métodos , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Orotidina-5'-Fosfato Descarboxilasa/deficienciaRESUMEN
Objective Orotic aciduria and deficiency of uridine monophosphate synthetase have been observed in a patient, studied over 10 years, who had no megaloblastic anemia. Excretion of orotic acid and orotidine were 8.24 and 0.52 mmol/mol of creatinine. The ratio of 15.85 differed appreciably from that of 6 patients reported with no megaloblastic anemia. Methods The analysis of orotidine by gas chromotography mass spectrometry was conducted. Conclusion Patients with orotic aciduria with and without megaloblastic anemia cannot be distinguished by ratio of orotic acid to orotidine.
Asunto(s)
Orotato Fosforribosiltransferasa/deficiencia , Ácido Orótico/orina , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Errores Innatos del Metabolismo de la Purina-Pirimidina/orina , Uridina/análogos & derivados , Niño , Femenino , Humanos , Orotato Fosforribosiltransferasa/efectos de los fármacos , Orotato Fosforribosiltransferasa/orina , Orotidina-5'-Fosfato Descarboxilasa/efectos de los fármacos , Orotidina-5'-Fosfato Descarboxilasa/orina , Uridina/uso terapéutico , Uridina/orina , Adulto JovenAsunto(s)
Acetatos/uso terapéutico , Orotato Fosforribosiltransferasa/deficiencia , Ácido Orótico/orina , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Errores Innatos del Metabolismo de la Purina-Pirimidina/tratamiento farmacológico , Uridina/uso terapéutico , Acetatos/administración & dosificación , Acetatos/efectos adversos , Acetatos/economía , Administración Oral , Biomarcadores/orina , Costos de los Medicamentos , Humanos , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Errores Innatos del Metabolismo de la Purina-Pirimidina/enzimología , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/orina , Resultado del Tratamiento , Uridina/administración & dosificación , Uridina/efectos adversos , Uridina/análogos & derivados , Uridina/economíaAsunto(s)
Anemia Megaloblástica/complicaciones , Epilepsia/complicaciones , Enfermedades Metabólicas/complicaciones , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Humanos , MasculinoRESUMEN
Hereditary orotic aciduria is a rare metabolic disease that results from a defect of uridine-5-monophosphate synthase (UMPS). In affected patients, main clinical symptoms are a markedly increased urinary excretion of orotic acid combined with megaloblastic anemia. This report describes a new case of UMPS deficiency without megaloblastic anemia but with epilepsy.
Asunto(s)
Anemia Megaloblástica/complicaciones , Epilepsia/complicaciones , Enfermedades Metabólicas/complicaciones , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Anemia Megaloblástica/metabolismo , Preescolar , Epilepsia/metabolismo , Humanos , Masculino , Enfermedades Metabólicas/genéticaRESUMEN
Among the various hereditary diseases that have been widely studied in dairy cattle, bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine-5-monophosphate synthase (DUMPS), and complex vertebral malformation (CVM) are noteworthy because of their high impact on overall herd productivity as a consequence of increased calf mortality. The aim of this study was to verify the frequency of carriers of BLAD, CVM, and DUMPS mutant alleles in cows and bulls from the National Girolando Progeny Test carried out in Brazil by using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR assays. A total of 777 animals were genotyped for BLAD, 783 for CVM, and 122 for DUMPS. The frequencies of carriers for BLAD and CVM were 0.77 and 1.53%, respectively, whereas no carriers of DUMPS were observed.
Asunto(s)
Enfermedades de los Bovinos/genética , Frecuencia de los Genes/genética , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Animales , Brasil , Bovinos , Enfermedades de los Bovinos/patología , Femenino , Genotipo , Síndrome de Deficiencia de Adhesión del Leucocito/veterinaria , Masculino , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Polimorfismo de Longitud del Fragmento de Restricción , Columna Vertebral/patologíaRESUMEN
A 2- year- old boy presented with non responsive megaloblastic anemia, growth failure and developmental delay. Blood levels of B(12), folic acid and iron were normal. Tandem mass spectroscopy for common inborn errors of metabolism did not reveal any abnormality. There was an increased excretion of orotic acid in urine. The authors report this as a rare cause of megaloblastic anemia.
Asunto(s)
Anemia Megaloblástica/etiología , Errores Innatos del Metabolismo de la Purina-Pirimidina/diagnóstico , Preescolar , Humanos , Masculino , Orotato Fosforribosiltransferasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/deficienciaRESUMEN
BACKGROUND: Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins. METHODS: Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not. RESULTS: Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID). CONCLUSION: This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.
Asunto(s)
Enfermedades de los Bovinos/genética , Citrulinemia/veterinaria , Deficiencia del Factor XI/veterinaria , Síndrome de Deficiencia de Adhesión del Leucocito/veterinaria , Columna Vertebral/anomalías , Secuencia de Aminoácidos , Animales , Bovinos , Enfermedades de los Bovinos/epidemiología , Citrulinemia/epidemiología , Citrulinemia/genética , ADN/química , ADN/genética , Deficiencia del Factor XI/epidemiología , Deficiencia del Factor XI/genética , Femenino , Genotipo , Heterocigoto , Síndrome de Deficiencia de Adhesión del Leucocito/epidemiología , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Datos de Secuencia Molecular , Orotato Fosforribosiltransferasa/deficiencia , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/genética , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Errores Innatos del Metabolismo de la Purina-Pirimidina/epidemiología , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/veterinaria , Alineación de Secuencia , Turquía/epidemiologíaRESUMEN
Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5'-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.
Asunto(s)
Proteínas Fúngicas/metabolismo , Ingeniería Genética/métodos , Vectores Genéticos , Genética Microbiana/métodos , Monascus/enzimología , Monascus/genética , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Clonación Molecular , Cartilla de ADN/genética , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Monascus/aislamiento & purificación , Monascus/efectos de la radiación , Mutagénesis , Ácido Orótico/análogos & derivados , Ácido Orótico/toxicidad , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/genética , Plásmidos , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Selección Genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Rayos UltravioletaRESUMEN
Three subtypes of hereditary orotic aciduria are described in the literature, all related to deficiencies in uridine monophosphate synthase, the multifunctional enzyme that contains both orotate: pyrophosphoryl transferase and orotidine monophosphate decarboxylase activities. The type of enzyme defect present in the subtypes has been re-examined by steady-state modelling of the relative outputs of the three enzymic products, uridine monophosphate, urinary orotic acid and urinary orotidine. It is shown that the ratio of urinary outputs of orotidine to orotate provides a means of testing for particular forms of enzyme defect. It is confirmed that the type I defect is caused by loss of uridine monophosphate synthase activity. Cells and tissue of type I cases have a residual amount of activity that is qualitatively unchanged: the relative rates of the transferase and decarboxylase do not differ from those of wild-type enzyme. The single claimed case of type II, thought to be due to specific inactivation of orotidine monophosphate decarboxylase, is shown to have a product spectrum inconsistent with that claim. It is proposed that this type II form does not differ sufficiently to be accepted as separate from type I. The third subtype, hereditary orotic aciduria without megaloblastic anaemia, occurs in two cases. It has the product spectrum expected of a defect in orotidine monophosphate decarboxylase. This form is the only one that appears to have a qualitatively different uridine monophosphate synthase. The possibility that orotidine monophosphate may control flux through the pyrimidine biosynthesis pathway in hereditary orotic aciduria is discussed.
Asunto(s)
Complejos Multienzimáticos/deficiencia , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/deficiencia , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/genética , Errores Innatos del Metabolismo de la Purina-Pirimidina/enzimología , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , Adulto , Anemia Megaloblástica/complicaciones , Niño , Preescolar , Humanos , Lactante , Cinética , Redes y Vías Metabólicas , Modelos Biológicos , Orotato Fosforribosiltransferasa/metabolismo , Ácido Orótico/orina , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Errores Innatos del Metabolismo de la Purina-Pirimidina/complicaciones , Errores Innatos del Metabolismo de la Purina-Pirimidina/metabolismo , Uridina/análogos & derivados , Uridina/orina , Uridina Monofosfato/metabolismoRESUMEN
A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.
Asunto(s)
Enfermedades de los Bovinos/orina , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Ácido Orótico/orina , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Animales , Bovinos , Análisis Mutacional de ADN/veterinaria , Enfermedades Carenciales/orina , Enfermedades Carenciales/veterinaria , Resultado Fatal , Cromatografía de Gases y Espectrometría de Masas/veterinaria , Masculino , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/genética , Ácido Orótico/sangre , Orotidina-5'-Fosfato Descarboxilasa/genética , LinajeRESUMEN
The present study investigated the occurrence of 2 autosomal recessive genetic diseases, bovine citrullinaemia and deficiency of uridine monophosphate synthase (DUMPS), in Indian Holstein cattle. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed on a group of 642 animals, mainly HF and HF crossbred cattle, to identify carriers of these diseases. None of the animals were carriers of citrullinaemia or DUMPS. It is possible that with the mounting selection pressure, the international gene pool may diminish, and consequently the risk of dissemination of inherited defects will increase. It is therefore recommended to screen breeding bulls for their breed-specific genetic diseases before they are inducted in artificial insemination programmes, to minimize the risk.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/genética , Citrulinemia/veterinaria , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Animales , Cruzamiento/métodos , Bovinos , Citrulinemia/epidemiología , Citrulinemia/genética , Tamización de Portadores Genéticos , India/epidemiología , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan.
Asunto(s)
Enfermedades de los Bovinos/enzimología , Enfermedades de los Bovinos/genética , Deleción Cromosómica , Feto/enzimología , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Cromosoma X , Animales , Southern Blotting , Bovinos , ADN/análisis , Femenino , Amplificación de Genes , Complejos Multienzimáticos/genética , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Embarazo , Procesos de Determinación del Sexo , Cromosoma X/genética , Cromosoma Y/genéticaRESUMEN
DUMPS (Deficiency of Uridine Monophosphate Synthase) is a hereditary recessive disorder in Holstein cattle causing early embryo mortality during its implantation in the uterus. The only way to avoid the economic losses is early detection of DUMPS carriers. Because American Holstein semen has been intensively imported to Poland since 1970, there was a risk that DUMPS could have spread in Polish dairy cattle. In our study, 2209 dairy cattle of the Polish Holstein breed have been screened by the DNA test. The dominant group was young bulls entering the testing program (1171) and proven bulls (781). They represented all sires entering Polish breeding programs between 1999 and 2003. Also, 257 sire dams were included in the screening program. No DUMPS carrier has been found. Our results then indicate that the population of dairy cattle reared in Poland is free from DUMPS. Because of the economical significance of the DUMPS mutation and its recessive mode of inheritance, attention has to be paid to any case of a bull having in his origin any known DUMPS carrier. Such a bull should be tested and if positive eliminated from the active population. Also, young bulls (testing bulls) should be screened for DUMPS if in their progeny a high incidence of embryo mortality is observed and their genealogy cannot exclude their relatedness to any DUMPS carriers.
Asunto(s)
Cruzamiento/métodos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/genética , Tamización de Portadores Genéticos , Tamizaje Masivo/veterinaria , Errores Innatos del Metabolismo/veterinaria , Complejos Multienzimáticos/deficiencia , Orotato Fosforribosiltransferasa/deficiencia , Orotidina-5'-Fosfato Descarboxilasa/deficiencia , Animales , Bovinos , Cartilla de ADN , Tamizaje Masivo/métodos , Errores Innatos del Metabolismo/epidemiología , Errores Innatos del Metabolismo/genética , Polonia/epidemiología , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
Although the nuclear genome sequence of Cyanidioschyzon merolae 10D, a unicellular red alga, was recently determined, DNA transformation technology that is important as a model plant system has never been available thus far. In this study, improved culture conditions resulted in a faster growth rate of C. merolae in liquid medium (doubling time = 9.2 h), and colony formation on gellan gum plates. Using these conditions, spontaneous mutants (5-fluoroortic acid resistant) deficient in the UMP synthase gene were isolated. The lesions were then restored by introducing the wild-type UMP synthase gene into the cells suggesting DNA transformation by homologous recombination.