RESUMEN
In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.
Asunto(s)
Bacterias/genética , Etiquetas de Secuencia Expresada , Expresión Génica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Algoritmos , Análisis de Varianza , Bacterias/citología , Bacterias/crecimiento & desarrollo , Recuento de Colonia Microbiana/métodos , ADN Complementario/genética , Cinética , Viabilidad Microbiana , Plásmidos/genéticaRESUMEN
Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80 similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme
Asunto(s)
Fosfotransferasas/genética , Pirofosfatasas/metabolismo , Saccharum/enzimología , Secuencia de Aminoácidos , ADN Complementario/análisis , Fosfotransferasas/metabolismo , Datos de Secuencia Molecular , Saccharum/genéticaRESUMEN
Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme.
Asunto(s)
Fosfotransferasas/genética , Pirofosfatasas/metabolismo , Saccharum/enzimología , Secuencia de Aminoácidos , ADN Complementario/análisis , Datos de Secuencia Molecular , Fosfotransferasas/metabolismo , Saccharum/genéticaRESUMEN
The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 x 2]-factorial arrangement, using two levels of rbST (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A cDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus cDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbST treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone mRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone mRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9 higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds
Asunto(s)
Humanos , Masculino , Bovinos/crecimiento & desarrollo , Expresión Génica/efectos de los fármacos , Hipófisis/efectos de los fármacos , Hormona del Crecimiento/farmacología , Bovinos/genética , ADN Complementario/análisis , ADN Complementario/genética , Expresión Génica/genética , Hipófisis , Hormona del Crecimiento/genética , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Análisis de Secuencia de ADNRESUMEN
The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 x 2]-factorial arrangement, using two levels of rbST (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A cDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus cDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbST treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone mRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone mRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9% higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds.
Asunto(s)
Bovinos/crecimiento & desarrollo , Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/farmacología , Hipófisis/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Bovinos/genética , ADN Complementario/análisis , ADN Complementario/genética , Expresión Génica/genética , Hormona del Crecimiento/genética , Masculino , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/genética , Hipófisis/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Análisis de Secuencia de ADNRESUMEN
We describe 8 patients with muscle carnitine deficiency, 7 males and 1 female, varying in age from 5 days to 64 years. Seven had decreased muscle strength and all had increased lipids droplets in the muscle biopsy. The symptoms began in the first days of life in three cases, in childhood in two, in adult life in two, while one case was free of symptoms at age 64 (heterozygote?). Some patients had difficulty chewing, dysphagia, hypotonia and splenomegaly; one patient had a fluctuating clinical course. All had elevated serum enzymes, mainly creatine-kinase. The electromyogram showed primary muscle involvement in one case, denervation in two, "mixed" features in two and was not done in three. The muscle biopsy, beside lipid storage, showed denervation in four, chronic myopathy in four and type II fiber atrophy in one. In two cases, histological findings suggested infantile spinal muscle atrophy. One patient appeared to have a systemic form of carnitine deficiency, with severe myocardial involvement and died of heart failure before treatment was initiated. A discussion about clinical findings, metabolism and therapeutic aspects of muscle carnitine deficiency is made.
Asunto(s)
Carnitina/deficiencia , Enfermedades Musculares/etiología , Adulto , Preescolar , Electromiografía , Femenino , Humanos , Lactante , Recién Nacido , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Hipotonía Muscular/etiología , Músculos/metabolismo , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patologíaRESUMEN
We describe two brothers, 25 and 19 years-old, with muscle pain and decreased strength after prolonged exercise; these symptoms are worsened by cold whether of fasting. One of the patients developed recurrent myoglobinuria and had one episode of renal failure. Laboratory investigations were normal between the crises, but during myoglobinuria, serum creatine kinase activity increased 100 times. Electromyography was suggestive of denervation. Muscle biopsy showed increased lipid droplets by the "oil red O" stain and increased activity of succinic dehydrogenase histochemical reaction. Lactate production during ischemia was normal. Biochemical analysis showed decreased carnitine-palmityl-transferase activity in muscle (7.23 and 10.58 nmoles/min/gr; normal range 66.7 +/- 17.3), with normal values for carnitine-octanoyl-transferase and carnitine-acetyl-transferase. The metabolic pathway of fatty acid utilization as an energy source for muscle during exercise in normal and in pathological conditions is discussed.