Gravitropic plant growth regulation and ethylene: an unsought cardinal coordinate for a disused model

According to the Cholodny-Went hypothesis, gravitropic differential growth is brought about by the redistribution of auxin (indolyl-3-acetic acid, IAA). We reinvestigated the relevance of different auxins and studied the role of ethylene in hypocotyls of sunflower and shoots and roots of rye and maize seedlings. Incubation of coleoptiles and of sunflower hypocotyls in solutions of IAA and dichlorophenoxyacetic acid as well as naphthylacetic acid resulted in a two- to threefold length increase compared to water controls. In spite of this pronounced general effect on elongation growth, gravi-curvature was similar to water controls. In contrast to this, inhibition of ethylene synthesis by aminoethoxyvinylglycine prevented differential growth of both hypocotyls and coleoptiles and of roots of maize. In horizontally stimulated maize roots growing on surfaces, inhibition of ethylene perception by methylcyclopropene inhibited roots to adapt growth to the surface, resulting in a lasting vertical orientation of the root tips. This effect is accompanied by up- and down-regulation of a number of proteins as detected by two-dimensional matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Together the data query the regulatory relevance of IAA redistribution for gravitropic differential growth. They corroborate the crucial regulatory role of ethylene for gravitropic differential growth, both in roots and coleoptiles of maize as well as in hypocotyls.     

MSH2/MSH6 Complex Promotes Error-Free Repair of AID-Induced dU:G Mispairs as well as Error-Prone Hypermutation of A:T Sites

Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable Msh2^−/−Msh6^−/− mouse has for the first time allowed us to examine the impact of the complete loss of MutSα on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice are indistinguishable from Msh2^−/− mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSα heterodimers that maintain genomic stability. The similar defects on switching in Msh2^−/−, Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice confirm that MutSα but not MutSβ plays an important role in CSR. Analysis of SHM in Msh2^−/−Msh6^−/− mice not only confirmed the error-prone role of MutSα in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSα when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSα at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM.     

Effect of inhibition of protein glycosylation on auxin-induced growth and the occurrence of osmiophilic particles in maize (Zea mays L.) coleoptiles

The dependence of auxin (IAA)-induced elongation growth on proteinglycosylation was investigated in abraded maize (Zea mays L.)coleoptile segments, employing 2-deoxy-D-glucose (DOG) and tunicamycin(TUM) as inhibitors of protein glycosylation. TUM had no detectableeffect on growth at 100µg ml^–1. DOG impaired growthat concentrations larger than 1 mM. Total inhibition of growthoccurred at a concentration of 20 mM. Similar effects were observedwith mannose and glucosamine. The effect on wall-synthetic processesin the growth-limiting epidermis was analysed by tracer incorporationstudies. Within 30 min hemicellulose and cellulose synthesis,measured as ³H-glucose incorporation, was not affected by DOG,indicating that inhibition of growth is not causally relatedto synthesis of both wall components. In contrast, protein synthesisand secretion into the walls, measured as incorporation of ³H-leucineinto the TCA-precipitable protoplasmic and wall-bound protein,was rapidly inhibited by DOG. Concomitant with the effect ongrowth, DOG as well as mannose inhibited the occurrence of osmiophilicparticles (OPs) which specifically occur at the growth-limitingepidermis during IAA-induced growth. The results provide evidencethat IAA-induced wall loosening underlying elongation growthis dependent on O-glycosylation of proteins and their subsequentsecretion into the epidermal walls. It appears that interferencewith these processes is responsible for inhibition of IAA inducedgrowth by hexoses acting as anti-glucose antimetabolites. Key words: Auxin-induced growth, cell-wall synthesis, 2-deoxy-D-glucose, mannose, osmiophilic particles, tunicamycin     

Diverse functions of reactive cysteines facilitate unique biosynthetic processes of aggregate-prone interleukin-31

Interleukin-31 (IL-31) is a member of the four helical-bundle gp130/IL-6 cytokine family. Despite its implicated roles in inflammatory diseases, the biosynthetic processes of IL-31 have been poorly investigated. A detailed understanding of IL-31 biosynthesis and the nature of ligand–receptor interactions can provide insights into effective strategies for the design of therapeutic approaches. By using various heterologous protein expression systems, we demonstrated that murine IL-31 was secreted as inter-molecularly disulfide-bonded covalent aggregates. Covalently aggregated IL-31 appeared while trafficking in the secretory pathway, but was not actively retained in the ER. The aggregate formation was not caused by a dysfunctional ER quality control mechanism or an intrinsic limitation in protein folding capacity. Furthermore, secreted IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of aggregates may imply that IL-31 was erroneously folded, but it was capable of signaling through cognate receptors. Mutagenesis revealed the promiscuity of all five cysteines in inter-molecular disulfide formation with components of the hetero-aggregates, but no cysteine was required for IL-31 secretion itself. Our present study not only illustrated various functions that cysteines perform during IL-31 biosynthesis and secretion, but also highlighted their potential roles in cytokine effector functions.     

SLiCE: a novel bacterial cell extract-based DNA cloning method

We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.     

Hierarchical modularity and the evolution of genetic interactomes across species

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.     

Stability of Emulsions Using a New Natural Emulsifier: Sugar Beet Extract <Emphasis Type="Italic">(Beta vulgaris L.)</Emphasis>

This study describes the influence of environmental stresses on the stability of emulsions prepared by a natural sugar beet extract (Beta vulgaris L.). The emulsion stabilizing performance was compared to that of Quillaja extract, which is widely used within the food and beverage industry as natural surfactant. We investigated the influence of pH, ionic strength, heating and freeze-thawing on the mean particle size, ζ-potential and microstructure of oil-in-water emulsions (10% w/w oil, 0.75% w/w emulsifier). The emulsions stabilized by the anionic sugar beet extract were stable at pH 5–8 and against thermal treatments up to 60 °C. However, the prepared emulsions were unstable at acidic (pH 2–4) and basic pH conditions (pH 9), at high temperature (>60 °C), and at salt additions (> 0.1 M NaCl / CaCl₂). Moreover, they also phase separated upon freeze-thawing. Our results show that sugar beet extract is capable of stabilizing emulsions and may therefore be suitable as natural emulsifier for selected applications in the food and beverage industry.