RESUMEN
UDP-glucuronate decarboxylase (UDP-xylose synthase; UXS, EC 4.1.1.35) is an essential enzyme of the non-cellulosic polysaccharide biosynthetic pathway. In the present study, using transient expression of fluorescently labeled Gossypium hirsutum UXS (GhUXS3) protein in onion epidermal cells, we observed that this protein was distributed in the cytoplasm. The GhUXS3 cDNA of cotton was expressed in an antisense orientation in Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. Homozygous plants showing down-regulation of UXS were analyzed with northern blots. Compared to the untransformed control, transgenic plant showed shorter roots, earlier blossom formation, and delayed senescence. Biochemical analysis indicated that levels of rhamnose, mannose, galactose, glucose, xylose, and cellulose were reduced in some of the down-regulated antisense plants. These results suggest that GhUXS3 regulates the conversion of non-cellulosic polysaccharides and modulates their composition in plant cell walls. We also discuss a possible cellular function for GhUXS in determining the quality of cotton fibers.
Asunto(s)
Arabidopsis/genética , Metabolismo de los Hidratos de Carbono/genética , Carboxiliasas/genética , Pared Celular/metabolismo , ADN sin Sentido , Gossypium/enzimología , Envejecimiento , Arabidopsis/metabolismo , Arabidopsis/fisiología , Pared Celular/química , ADN de Plantas , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Gossypium/genética , Raíces de Plantas/anatomía & histología , Plantas Modificadas GenéticamenteRESUMEN
Previous studies have indicated that the protein tyrosine phosphatase nonreceptor type 22 gene (PTPN22) is associated with type 1 diabetes (T1DM) in the Caucasian population. In the present study, we investigated the relationship between PTPN22 genetic polymorphisms and T1DM in Chinese children. A total of 202 children and adolescents with T1DM and 240 healthy control subjects of Chinese Han origin were included in our analysis. Polymerase chain reaction-restriction fragment length polymorphism was used to determine the presence of the C1858T polymorphism in the PTPN22 gene. We found that the TT +TC genotype and the T allele of C1858T were more frequent in T1DM patients (19.40 and 10.0%, respectively) than in healthy subjects (7.51 and 4.0%, respectively), and the difference was significant (both P < 0.001). After adjusting for confounding variables such as gender, age, and family history of T1DM, the difference remained significant (P = 0.007, odds ratio = 2.88, 95% confidence interval 1.76-4.32). Our results indicate that genetic polymorphisms in the PTPN22 gene may increase the risk of T1DM in Chinese children and adolescents.
Asunto(s)
Pueblo Asiatico/genética , Diabetes Mellitus Tipo 1/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Adolescente , Niño , Preescolar , China , Femenino , Humanos , Modelos Logísticos , MasculinoRESUMEN
Keshan disease (KSD), a potentially fatal cardiomyopathy, has very high incidence in some selenium-poor regions of China. KSD may be accompanied with a variety of arrhythmia, which is associated with mutations in the gene coding for cardiac voltage-gated sodium channel (SCN5A). The molecular mechanism of KSD is still largely obscure. We aimed to determine the association between the H558R polymorphism of SCN5A and KSD. We recruited 71 patients with KSD and 80 geographical region-matched control subjects in our study. Vital sign and electrocardiographic (ECG) measurements were performed for heart rate, systolic pressure, diastolic pressure, PR interval, QT interval, QRS duration, ST-T changes and complete right bundle branch block (CRBBB), and H558R polymorphism was genotyped using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method and sequencing. A significant association was found between the H558R polymorphism of exon 12 and KSD. Allele C carriers had a decreased risk for KSD with an odds ratio of 0.332 [95% confidence interval (CI), 0.160-0.692] as well as for QRS prolongation in KSD patients with an odds ratio of 0.089 (95%CI, 0.022-0.361). Our results provide support to the association between H558R polymorphism and the decreased risk for KSD. H558R polymorphism might increase susceptibility to KSD, and SCN5A containing the polymorphism might be a predisposing gene for QRS prolongation.
Asunto(s)
Cardiomiopatías/genética , Infecciones por Enterovirus/genética , Predisposición Genética a la Enfermedad/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Polimorfismo Genético , Anciano , Presión Sanguínea , Cardiomiopatías/fisiopatología , China , Estudios de Cohortes , Análisis Mutacional de ADN/métodos , Electrocardiografía , Infecciones por Enterovirus/fisiopatología , Exones/genética , Femenino , Frecuencia de los Genes , Genotipo , Frecuencia Cardíaca , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Factores de RiesgoRESUMEN
Sea Island cotton (Gossypium barbadense) is highly valued for its superior fiber qualities, especially fiber strength. Based on a transcript-derived fragment originated from transcriptome QTL mapping, a fiber strength related candidate gene of phosphatidylinositol 4-kinase cDNA, designated as GbPI4K, was first cloned, and its expression was characterized in the secondary cell wall thickening stage of G. barbadense fibers. The ORF of GbPI4K was found to be 1926 bp in length and encoded a predicted protein of 641 amino acid residues. The putative protein contained a clear PI3/4K kinase catalytic domain and fell into the plant type II PI4K cluster in phylogenetic analysis. In this study, the expression of cotton PI4K protein was also induced in Escherichia coli BL21 (DE3) as a fused protein. Semi-quantitative RT-PCR analysis showed that the gene expressed in the root, hypocotyl and leaf of the cotton plants. Real-time RT-PCR indicated that this gene in Sea Island cotton fibers expressed 10 days longer than that in Upland cotton fibers, and the main expression difference of PI4K between Sea Island cotton and Upland cotton in fibers was located in the secondary cell wall thickening stage of the fiber. Further analysis indicated that PI4K is a crucial factor in the ability of Rac proteins to regulate phospholipid signaling pathways.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/genética , Mapeo Cromosómico , Fibra de Algodón , Gossypium/enzimología , Gossypium/genética , Sitios de Carácter Cuantitativo/genética , Transcriptoma/genética , 1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Filogenia , Células Procariotas/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
BACKGROUND: . Streptomyces venezuelae produces two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolide pikromycin. Methymycin and pikromycin are derived from the corresponding precursors, YC-17 and narbomycin, respectively, by hydroxylation of the tertiary carbon position (C-10 in YC-17 or C-12 in narbomycin) on the macrolactone ring. In contrast, neomethymycin is derived from YC-17 by hydroxylation of the secondary carbon (C-12) of the propionyl starter unit sidechain. RESULTS: . Using a genetic and biochemical approach we have characterized a single P450 hydroxylase (PikC) in the methymycin/pikromycin biosynthetic gene cluster (pik) from S. venezuelae. Inactivation of pikC abolished production of all hydroxylated macrolides, with corresponding accumulation of YC-17 and narbomycin in the culture medium. The enzyme was produced efficiently and purified as a His-tagged protein from recombinant Escherichia coli cells. Purified PikC effectively converts YC-17 into methymycin and neomethymycin and narbomycin into pikromycin in vitro. CONCLUSIONS: . These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S. venezuelae. This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system. The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics.
Asunto(s)
Antibacterianos/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Genes Bacterianos/genética , Macrólidos/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Medios de Cultivo , Cinética , Datos de Secuencia Molecular , Plásmidos/genéticaRESUMEN
In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.