Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum

Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 ¹, and pvr1 ². These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.     

Buccal DNA samples for DNA typing: new collection and processing methods

With the recent expansion of DNA database laws in many states, there is a critical need for the rapid and simple collection of DNA samples and streamlined processing for downstream applications. The Buccal DNA Collector was developed to address the need for a reliable, practical alternative to blood collection that is compatible with high-throughput operations. The collection area consists of filter paper that is placed against the inside of the cheek, and the sample is taken by swiping the cheek several times while pulling the device out of the mouth. Using this method, DNA profiles have been obtained from samples stored for 2 years at room temperature. Cells are collected on all regions of the filter paper with the maximum DNA recovery from the tip. The processing of DNA for DNA typing is accomplished with BodeElute, a new product that prepares DNA for amplification in a single 30-min heating step. Extracted DNA samples were successfully amplified with four commonly used multiplex short tandem repeat (STR) amplification kits. These products provide simplified approaches for collecting and processing buccal cell samples.     

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.     

Inactivation of NF1 in CNS causes increased glial progenitor proliferation and optic glioma formation

The gene responsible for neurofibromatosis type 1 (NF1) encodes a tumor suppressor that functions as a negative regulator of the Ras proto-oncogene. Individuals with germline mutations in NF1 are predisposed to the development of benign and malignant tumors of the peripheral and central nervous system (CNS). Children with this disease suffer a high incidence of optic gliomas, a benign but potentially debilitating tumor of the optic nerve; and an increased incidence of malignant astrocytoma, reactive astrogliosis and intellectual deficits. In the present study, we have sought insight into the molecular and cellular basis of NF1-associated CNS pathologies. We show that mice genetically engineered to lack NF1 in CNS exhibit a variety of defects in glial cells. Primary among these is a developmental defect resulting in global reactive astrogliosis in the adult brain and increased proliferation of glial progenitor cells leading to enlarged optic nerves. As a consequence, all of the mutant optic nerves develop hyperplastic lesions, some of which progress to optic pathway gliomas. These data point to hyperproliferative glial progenitors as the source of the optic tumors and provide a genetic model for NF1-associated astrogliosis and optic glioma.     

Crystal structures of interleukin-2 tyrosine kinase and their implications for the design of selective inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.     

Effect of Volumetric Water Content and Clover (Trifolium incarnatum) on the Survival of Escherichia coli O157:H7 in a Soil Matrix

Studies aimed at understanding Escherichia coli O157:H7 soil survival dynamics are paramount due to their inevitable introduction into the organic vegetable production systems via animal manure-based fertilizer. Therefore, a greenhouse study was conducted to determine the survival of E. coli O157:H7 in highly controlled soil matrices subjected to two variable environmental stressors: (1) soil volumetric water content (25 or 45 % VWC), and (2) the growth of clover (planted or unplanted). During the 7-week study, molecular-based qPCR analyses revealed that E. coli O157:H7 survival was significantly lower in soils maintained at either near water-holding capacity (45 % VWC) or under clover growth. The significant reduction under clover growth was only observed when E. coli populations were determined relative to all bacteria, indicating the need to further study the competition between E. coli O157:H7 and the total bacterial community in organic soils. Given the significant effect of clover on E. coli O157:H7 survival under different moisture conditions in this greenhouse-based study, this work highlights the antimicrobial potential of clover exudates in arable soils, and future work should concentrate on their specific mechanisms of inhibition; ultimately leading to the development of crop rotations/production systems to improve pre-harvest food safety and security in minimally processed, ready-to-eat and organic production systems.     

Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.     

Elevated CO2 affects plant responses to variation in boron availability

Aim

Effects of elevated CO₂ on N relations are well studied, but effects on other nutrients, especially micronutrients, are not. We investigated effects of elevated CO₂ on response to variation in boron (B) availability in three unrelated species: seed geranium (Pelargonium x hortorum), barley (Hordeum vulgare), and water fern (Azolla caroliniana).

Methods

Plants were grown at two levels of CO₂ (370, 700?ppm) and low, medium, and high B. Treatment effects were measured on biomass, net photosynthesis (P_n) and related variables, tissue nutrient concentrations, and B transporter protein BOR1.

Results

In geranium, there were interactive effects (P?<?0.05) of B and CO₂ on leaf, stem, and total plant mass, root:shoot ratio, leaf [B], B uptake rate, root [Zn], and P_n. Elevated CO₂ stimulated growth at 45?μM B, but decreased it at 450?μM B and did not affect it at 4.5?μM B. P_n was stimulated by elevated CO₂ only at 45?μM B and chlorophyll was enhanced only at 450?μM B. Soluble sugars increased with high CO₂ only at 4.5 and 45?μM B. High CO₂ decreased leaf [B] and B uptake rate, especially at 450?μM B. Though CO₂ and B individually affected the concentration of several other nutrients, B x CO₂ interactions were evident only for Zn in roots, wherein [Zn] decreased under elevated CO₂. Interactive effects of B and CO₂ on growth were confirmed in (1) barley grown at 0, 30, or 1,000?μM B, wherein growth at high CO₂ was stimulated more at 30?μM B, and (2) Azolla grown at 0, 10, and 1,000?μM B, wherein growth at high CO₂ was stimulated at 0 and 10?μM B.

Conclusion

Thus, low and high B both may limit growth stimulation under elevated vs. current [CO₂], and B deficiency and toxicity, already common, may increase in the future.     

GM-CSF priming drives bone marrow-derived macrophages to a pro-inflammatory pattern and downmodulates PGE2 in response to TLR2 ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB(4). However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-α and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-γ. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NFκB but was not dependent on peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-IκBα formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.