Amphibian oocytes and sphere organelles: are the U snRNA genes amplified?

The sphere organelles (spheres) ofXenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in theXenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified inXenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification inXenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.by A. Spradling     

Real-Time, label-free monitoring of tumor antigen and serum antibody interactions

Conventional techniques for the detection of biomolecular interactions can be limited by the need for exogenous labels, time- and labor-intensive protocols, as well as by poor sensitivity levels. A refractometer instrument has been reconfigured to detect biomolecular interactions through changes in surface plasmon resonance (SPR). The binding kinetics and affinity values of anti-NY-ESO-1 monoclonal antibody, ES121, to the cancer-testis antigen NY-ESO-1 were determined according to the surface heterogeneity model and resulted in K(D) values of 1.3x10(-9) and 2.1x10(-10) M. The reconfigured instrument was then used to measure the interaction between tumor antigens and serum antibodies against these antigens in preselected cancer patient sera samples. The tumor antigens assayed included NY-ESO-1, SSX2 and p53, all used as recombinant proteins containing polyhistidine tags. These results demonstrated that the instrument is capable of detecting the binding of serum antibodies from cancer patient sera to immobilized tumor antigens, consistent with those observed previously in ELISA-based experiments. These results demonstrate the potential of SPR technology for the rapid diagnosis and monitoring immune responses.     

Increased signal complexity is associated with increased mating success

The evolution of complex signals has often been explored by testing multiple functional hypotheses regarding how independent signal components provide selective benefits to offset the costs of their production. In the present study, we take a different approach by exploring the function of complexity per se. We test the hypothesis that increased vibratory signal complexity—based on both proportional and temporal patterning—provides selective benefits to courting male Schizocosa stridulans wolf spiders. In support of this hypothesis, all of our quantified metrics of vibratory signal complexity predicted the mating success of male S. stridulans. The rate of visual signalling, which is mechanistically tied to vibratory signal production, was also associated with mating success. We additionally found evidence that males can dynamically adjust the complexity of their vibratory signalling. Together, our results suggest that complexity per se may be a target of female choice.     

Trade-offs in the genetic control of functional and nutritional quality traits in UK winter wheat

A complex network of trade-offs exists between wheat quality and nutritional traits. We investigated the correlated relationships among several milling and baking traits as well as mineral density in refined white and whole grain flour. Our aim was to determine their pleiotropic genetic control in a multi-parent population over two trial years with direct application to practical breeding. Co-location of major quantitative trait loci (QTL) and principal component based multi-trait QTL mapping increased the power to detect QTL and revealed pleiotropic effects explaining many complementary and antagonistic trait relationships. High molecular weight glutenin subunit genes explained much of the heritable variation in important dough rheology traits, although additional QTL were detected. Several QTL, including one linked to the TaGW2 gene, controlled grain size and increased flour extraction rate. The semi-dwarf Rht-D1b allele had a positive effect on Hagberg falling number, but reduced grain size, specific weight, grain protein content and flour water absorption. Mineral nutrient concentrations were lower in Rht-D1b lines for many elements, in wholemeal and white flour, but potassium concentration was higher in Rht-D1b lines. The presence of awns increased calcium content without decreasing extraction rate, despite the negative correlation between these traits. QTL were also found that affect the relative concentrations of key mineral nutrients compared to phosphorus which may help increase bioavailability without associated anti-nutritional effects of phytic acid. Taken together these results demonstrate the potential for marker-based selection to optimise trait trade-offs and enhance wheat nutritional value by considering pleiotropic genetic effects across multiple traits.Subject terms: Plant breeding, Quantitative trait, Genetic variation     

Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.     

Enhancer trap expression patterns provide a novel teaching resource

A collection of Arabidopsis enhancer trap transposants has been identified for use as a teaching tool. This collection serves to assist students in understanding the patterning and organization of plant tissues and cells, and will be useful in plant anatomy, morphology, and developmental biology courses. Each transposant exhibits reporter gene expression in a specific tissue, cell type, or domain, and these lines collectively offer a glimpse of compartments of gene expression. Some compartments correspond to classical definitions of botanical anatomy and can assist in anatomical identification. Other patterns of reporter gene expression are more complex and do not necessarily correspond to known anatomical features. The sensitivity of the beta-glucuronidase histochemical stain provides the student with a colorful and direct way to visualize difficult aspects of plant development and anatomy, and provides the teacher with an invaluable tool for a practical laboratory session.     

Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts

Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome p450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate beta-lyase (beta-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of beta-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of beta-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome p450-mediated pathway. Western blot analysis detected the presence of cytochrome p450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome p450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.     

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.