RESUMEN
The flowers of delphinium cultivars owe their coloration to anthocyanins such as delphinidin or pelargonidin derivatives. To date, no delphinium cultivars have been found with red flowers due to the presence of cyanidin derivatives. This suggests that delphiniums do not have cyanidin biosynthesis ability because of the loss of function of flavonoid 3' hydroxylase (F3'H). Here, we show that the wild delphinium species Delphinium zalil (synonym semibarbatum) can accumulate quercetin 3-glucosides in its sepals, presumably through F3'H activity. We isolated F3'H cDNA from D. zalil (DzF3'H) and produced a recombinant enzyme from a yeast transformant. The recombinant DzF3'H protein could convert naringenin, apigenin, dihydrokaempferol and kaempferol to eriodictyol, luteolin, dihydroquercetin and quercetin, respectively. An expression analysis confirmed that blue flowered D. grandiflorum does not express F3'H, and also showed that flavonoid 3',5'-hydroxylase and anthocyanidin synthase do not function in D. zalil sepals. DzF3'H can act as a flavonoid hydroxylase to produce cyanidin accumulation. The introduction of the DzF3'H gene into other delphinium species by conventional breeding may enable development of cultivars with novel flower colors.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Delphinium/enzimología , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Delphinium/genética , Flavonoles/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The recent advance in information technologies has bought about the borderlessness in every field of both science and business. The borderlessness has increasingly made activities in interdisciplinary field more important. This current situation produces a strong demand that people want to establish a virtual group, organization and society for their business and scientific purposes irrespective of the actual structure formed by organizations. Remarkably, bio sciences require a research platform that satisfies such demand for further development. In this paper, we present a research platform for bioinformatics in detail. The prominent feature of the research platform is the use of Grid and its location transparency, which means that bio scientists and researchers are able to utilize a large amount of computational power for their analysis and to access data of their interest without being aware of where data and computational resources are located. The usefulness and feasibility of the architecture of the research platform is shown as well as future issues to achieve toward the final goal of our research in this paper.
Asunto(s)
Investigación Biomédica , Biología Computacional/métodos , Interfaz Usuario-Computador , China , Simulación por Computador , Genómica , Humanos , Japón , ProteómicaRESUMEN
Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.
Asunto(s)
Chlorella/enzimología , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Chlorella/genética , Chlorella/crecimiento & desarrollo , Cromatografía/métodos , Ditiotreitol/química , Estabilidad de Enzimas , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Iones/química , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Metales/química , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/genética , Alineación de Secuencia , TemperaturaRESUMEN
In an attempt to clarify the involvement of fatty acid desaturases (FADs) in the freezing tolerance of Chlorella vulgaris IAM C-27, developed by hardening, we have isolated cDNA clones for two types of FADs from the Chlorella strain, based on the sequence information of genes for delta12 and omega-3 FADs, respectively desaturating oleic acid (18:1) to linoleic acid (18:2) and linoleic acid (18:2) to linolenic acid (18:3). The deduced amino acid sequence of the first clone, designated CvFad2, showed about 66% similarity to the microsomal delta12 FADs from several higher plants and this gene had delta12 FAD activity when expressed in Saccharomyces cerevisiae. The predicted protein encoded by a second gene, designated CvFad3, showed about 60% similarity to the microsomal and plastidial omega-3 FADs from several higher plants. The features of the amino acid sequences of the C- and N-terminal regions of CvFAD3 and fatty acid analysis of polar lipids in transgenic tobacco plant expressing the CvFad3 gene suggested that this gene encodes the microsomal omega-3 FAD. Southern blot analysis showed that both genes were single-copy genes in the genome of the Chlorella strain. Different transcriptional patterns were observed with the two genes during hardening in Northern blot analysis.
Asunto(s)
Chlorella/enzimología , Chlorella/genética , ADN Complementario/genética , Ácido Graso Desaturasas/genética , Nicotiana/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Cromatografía de Gases , Clonación Molecular , Ácido Graso Desaturasas/química , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/análisis , Expresión Génica , Datos de Secuencia Molecular , Filogenia , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/química , Temperatura , Nicotiana/químicaRESUMEN
The pbp3 gene encoding PBP3 of Bacillus cereus was cloned and sequenced. For this purpose, PBP3 was first purified from B. cereus ts-4, and N-terminal amino acid sequences of the peptides obtained from the protease digests of the protein were analyzed. The B. cereus ts-4 pbp3 gene consisted of an open reading frame of 1,986 bp encoding 662 amino acid residues with a calculated molecular mass of 73,044 Da. The active site-motifs SXXK, SXN, and KTG are present at the positions 393, 452, and 590, respectively, in the deduced amino acid sequence. The pbp3 structural gene was ligated into the pET17 x b expression vector and pET-pbp3 was constructed. A protein was produced by the cells of E. coli carrying pET-pbp3. The produced protein migrated at about 75 kDa in SDS-polyacrylamide gel and strongly reacted with biotinylated ampicillin.
Asunto(s)
Proteínas Portadoras/genética , Expresión Génica , Muramoilpentapéptido Carboxipeptidasa/genética , Secuencia de Aminoácidos , Bacillus cereus , Secuencia de Bases , Proteínas Portadoras/aislamiento & purificación , Clonación Molecular , ADN Complementario , Escherichia coli , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/aislamiento & purificación , Péptidos/análisis , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.
Asunto(s)
ADN Bacteriano/análisis , Escherichia coli O157/aislamiento & purificación , Carne/microbiología , Reacción en Cadena de la Polimerasa/métodos , Animales , Secuencia de Bases , Recuento de Colonia Microbiana , Cartilla de ADN , Microbiología de Alimentos , Peso Molecular , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non- Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non- Salmonella : Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella -positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.
RESUMEN
Monoclonal antibodies raised against dulcitol 1-phosphate dehydrogenase of Salmonella typhimurium IFO 12529 were screened against 20 serotypes of Salmonella and 13 non- Salmonella bacteria. A sandwich-capture, enzyme-linked immunosorbent assay (sandwich ELISA) was developed for detection of Salmonella in food. The assay utilizes two monoclonal antibodies (DUI2 and DU28) which show no cross-reactions with non- Salmonella bacteria. The limit of detection of the sandwich ELISA was about 1 × 107 CPU/ml. After cultivation in a medium containing dulcitol at 37°C for 18 h followed by the sandwich ELISA. 1 CPU of Salmonella was detected. Although a high inoculum level of E. coli interfered with the detection of Salmonella , the interference was minimized by using a selective dulcitol-magnesium chloride-pyridinesulfonic acid medium for enrichment. The novel ELISA procedure detected Salmonella in chicken filtrates inoculated with 1.4 CPU/50 m1 and 1.3 × 107 CPU/50 ml of E. coli within 25 h.