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941.
Underwood BA Vanderhaeghen R Whitford R Town CD Hilson P 《Plant biotechnology journal》2006,4(3):317-324
Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones. 相似文献
942.
Microbial community dynamics in a humic lake: differential persistence of common freshwater phylotypes 总被引:4,自引:0,他引:4
In an effort to better understand the factors contributing to patterns in freshwater bacterioplankton community composition and diversity, we coupled automated ribosomal intergenic spacer analysis (ARISA) to analysis of 16S ribosomal RNA (rRNA) gene sequences to follow the persistence patterns of 46 individual phylotypes over 3 years in Crystal Bog Lake. Additionally, we sought to identify linkages between the observed phylotype variations and known chemical and biological drivers. Sequencing of 16S rRNA genes obtained from the water column indicated the presence of phylotypes associated with the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, TM7 and Verrucomicrobia phyla, as well as phylotypes with unknown affiliation. Employment of the 16S rRNA gene/ARISA method revealed that specific phylotypes varied independently of the entire bacterial community dynamics. Actinobacteria, which were present on greater than 95% of sampling dates, did not share the large temporal variability of the other identified phyla. Examination of phylotype relative abundance patterns (inferred using ARISA fragment relative fluorescence) revealed a strong correlation between the dominant phytoplankton succession and the relative abundance patterns of the majority of individual phylotypes. Further analysis revealed covariation among unique phylotypes, which formed several distinct bacterial assemblages correlated with particular phytoplankton communities. These data indicate the existence of unique persistence patterns for different common freshwater phylotypes, which may be linked to the presence of dominant phytoplankton species. 相似文献
943.
Sivan Vadakkadath Meethal Miguel J Gallego Ryan J Haasl Stephen J Petras III Jean-Yves Sgro Craig S Atwood 《BMC evolutionary biology》2006,6(1):103-17
Background
The Caenorhabditis elegans genome is known to code for at least 1149 G protein-coupled receptors (GPCRs), but the GPCR(s) critical to the regulation of reproduction in this nematode are not yet known. This study examined whether GPCRs orthologous to human gonadotropin-releasing hormone receptor (GnRHR) exist in C. elegans. 相似文献944.
945.
Liebscher RS Richards RC Lewis JM Short CE Muise DM Driedzic WR Ewart KV 《Physiological and biochemical zoology : PBZ》2006,79(2):411-423
In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression. 相似文献
946.
The aluminium cation Al(3+) is toxic to many plants at micromolar concentrations. A range of plant species has evolved mechanisms that enable them to grow on acid soils where toxic concentrations of Al(3+) can limit plant growth. Organic acids play a central role in these aluminium tolerance mechanisms. Some plants detoxify aluminium in the rhizosphere by releasing organic acids that chelate aluminium. In at least two species, wheat and maize, the transport of organic acid anions out of the root cells is mediated by aluminium-activated anion channels in the plasma membrane. Other plants, including species that accumulate aluminium in their leaves, detoxify aluminium internally by forming complexes with organic acids. 相似文献
947.
G Dou PG Sreekumar C Spee S He SJ Ryan R Kannan DR Hinton 《Free radical biology & medicine》2012,53(5):1111-1122
Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (-/-) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis. 相似文献
948.
We have developed an ELISA to determine the value of anti-glucosylceramide antibody for the prediction of disseminated cryptococcosis in immunocompromised subjects and performed a clinical prospective study at the Medical University of South Carolina. The study enrolled a total of 53 patients who were free of active fungal diseases at the time of enrollment but at risk of developing one because they were all immunocompromised, e.g., (1) patients positive for HIV and (2) patients post- or awaiting solid organ transplantation. Among 53 patients enrolled, two patients developed invasive cryptococcosis, and in both patients, IgM anti-GlcCer was detected in sera using the ELISA at least 6 weeks prior to the clinical presentation of the brain disease. These results were corroborated by a cryptococcal antigen lateral flow assay, which was also positive in serum prior to the development of meningoencephalitis. However, a high number of positive results were also detected in patients with no evidence of cryptococcosis. This study highlights the potential utility of this new assay in early diagnostic testing algorithms for patients at risk for cryptococcosis, but further investigations are needed to validate the sensitivity and specificity of the glucosylceramide ELISA as a predictor of cryptococcosis. 相似文献
949.
950.
Saigusa T Reichert R Guare J Siroky BJ Gooz M Steele S Fenton RA Bell PD Kolb RJ 《American journal of physiology. Renal physiology》2012,302(7):F801-F808
Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined. 相似文献