The Yeast GRASP Grh1 Colocalizes with COPII and Is Dispensable for Organizing the Secretory Pathway

In mammalian cells, the ‘Golgi reassembly and stacking protein’ (GRASP) family has been implicated in Golgi stacking, but the broader functions of GRASP proteins are still unclear. The yeast Saccharomyces cerevisiae contains a single non‐essential GRASP homolog called Grh1. However, Golgi cisternae in S. cerevisiae are not organized into stacks, so a possible structural role for Grh1 has been difficult to test. Here, we examined the localization and function of Grh1 in S. cerevisiae and in the related yeast Pichia pastoris, which has stacked Golgi cisternae. In agreement with earlier studies indicating that Grh1 interacts with coat protein II (COPII) vesicle coat proteins, we find that Grh1 colocalizes with COPII at transitional endoplasmic reticulum (tER) sites in both yeasts. Deletion of P. pastoris Grh1 had no obvious effect on the structure of tER–Golgi units. To test the role of S. cerevisiae Grh1, we exploited the observation that inhibiting ER export in S. cerevisiae generates enlarged tER sites that are often associated with the cis Golgi. This tER–Golgi association was preserved in the absence of Grh1. The combined data suggest that Grh1 acts early in the secretory pathway, but is dispensable for the organization of secretory compartments.     

Improving species distribution models for climate change studies: variable selection and scale

Statistical species distribution models (SDMs) are widely used to predict the potential changes in species distributions under climate change scenarios. We suggest that we need to revisit the conceptual framework and ecological assumptions on which the relationship between species distributions and environment is based. We present a simple conceptual framework to examine the selection of environmental predictors and data resolution scales. These vary widely in recent papers, with light inconsistently included in the models. Focusing on light as a necessary component of plant SDMs, we briefly review its dependence on aspect and slope and existing knowledge of its influence on plant distribution. Differences in light regimes between north‐ and south‐facing aspects in temperate latitudes can produce differences in temperature equivalent to moves 200 km polewards. Local topography may create refugia that are not recognized in many climate change SDMs using coarse‐scale data. We argue that current assumptions about the selection of predictors and data resolution need further testing. Application of these ideas can clarify many issues of scale, extent and choice of predictors, and potentially improve the use of SDMs for climate change modelling of biodiversity.     

Identification of close contacts between the sigma N (sigma 54) protein and promoter DNA in closed promoter complexes.

The complexes forming between the alternative sigma factor protein sigma N (sigma 54), its holoenzyme and promoter DNA were analysed using the hydroxyl radical probe and by photochemical footprinting of bromouridine-substituted DNA. Close contacts between the promoter, sigma N and its holoenzyme appear to be restricted predominantly to one face of the DNA helix, extending from -31 to -5. They all appear attributable to sigma N and no extra close contacts from the core RNA polymerase subunits in the holoenzyme-promoter DNA complex were detected. We suggest that the apparent absence of close core RNA polymerase contacts in the region of the promoter DNA to be melted during open complex formation is important for maintaining the closed complex. Results of the hydroxyl radical footprinting imply that sigma N makes multiple DNA backbone contacts across and beyond the -12, -24 consensus promoter elements, and the photochemical footprints indicate that consensus thymidine residues contribute important major groove contacts to sigma N. Formation of the open complex is shown to involve a major structural transition in the DNA contacted by sigma N, establishing a direct role for sigma N in formation of the activated promoter complex.     

Isolation and Characterization of φ80 Transducing Bacteriophage for a Ribonucleic Acid Polymerase Gene

A new method for isolating specialized transducing phages is described. It was used to isolate a group of phi80 transducing phages which carry various bacterial markers from the metB region of the Escherichia coli chromosome. Some of the phages selected for transduction of the supA36 marker were also shown to carry rif, a locus known to specify the beta subunit of ribonucleic acid polymerase. Expression of the prophage rif(r) gene in lysogens was demonstrated by its ability to confer rifampin resistance on part of the cellular ribonucleic acid polymerase pool.     

SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of beta(H)-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 alpha-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of alpha-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.     

Partition functions of unit-copy plasmids can stabilize the maintenance of plasmid pBR322 at low copy number.

The maintenance of plasmid pBR322 is highly unstable in a polA12 strain of Escherichia coli at 29 degrees C due to severely reduced copy number. Under these conditions, introduction of the par (partition) locus of plasmid P1 or the par (sop) region of F into pBR322 stabilizes it. A region with similar activity was detected in the P7 plasmid. The activity of the P1 par locus was dependent on the P1 parA gene product and was sensitive to par-specified incompatibility.     

Comparative analysis of monocytic and granulocytic myeloid-derived suppressor cell subsets in patients with gastrointestinal malignancies

Myeloid-derived suppressor cells (MDSC) are a heterogenous population of cells comprising myeloid progenitor cells and immature myeloid cells, which have the ability to suppress the effector immune response. In humans, MDSC have not been well characterized owing to the lack of specific markers, although it is possible to broadly classify the MDSC phenotypes described in the literature as being predominantly granulocytic (expressing markers such as CD15, CD66, CD33) or monocytic (expressing CD14). In this study, we set out to perform a direct comparative analysis across both granulocytic and monocytic MDSC subsets in terms of their frequency, absolute number, and function in the peripheral blood of patients with advanced GI cancer. We also set out to determine the optimal method of sample processing given that this is an additional source of heterogeneity. Our findings demonstrate consistent changes across sample processing methods for monocytic MDSC, suggesting that reliance upon cryopreserved PBMC is acceptable. Although we did not see an increase in the population of granulocytic MDSC, these cells were found to be more suppressive than their monocytic counterparts.     

Molecular evolution of the NF-κB signaling system

The mechanisms of innate immunity in vertebrates show certain overall resemblances to immune mechanisms of insects. Two hypotheses have been proposed to explain these resemblances. (1) According to the evolutionary continuity hypothesis, innate immune mechanisms evolved in the common ancestor of vertebrates and insects and have been conserved since that time. (2) In the independent-evolution hypothesis, the mechanisms of innate immunity in vertebrates evolved independently from invertebrate immune mechanisms. Phylogenetic analysis of five gene families (Pelle, Rel, IkappaB, Toll, and TRAF) whose members are involved in NF-kappaB signaling in vertebrates and insects were used to decide between these hypotheses. The phylogenies of the Rel and TRAF families strongly supported independent evolution of immune functions in vertebrates and invertebrates, and, except for a possible case in the Pelle family, orthologous molecules having immune functions in both vertebrates and invertebrates were not found. The results suggest that NF-kappaB represents an ancient, generalized signaling system that has been co-opted for immune system roles independently in vertebrate and insect lineages.     

Brief pre- and post-irrigation sprinkling with fresh water reduces foliar salt uptake in maize and barley sprinkler irrigated with saline water

Brief pre- and post-irrigation sprinkling treatments using freshwater were tested to determine if these practices could reduce the uptake of salts through leaves when saline water is used to sprinkler irrigate crops. Maize and barley were sprinkler irrigated 2 to 3 times per week for 30 min with saline water (4.2 dS m^–1, 30 mmol L^–1 NaCl and 2.8 mmoles L^–1 CaCl₂ for maize and 9.6 dS m^–1, 47 mmoles L^–1 NaCl and 23.5 mmoles L^–1 CaCl₂ for barley) in separate experiments with plants grown in pots outdoors. The soil surface of all pots was covered to prevent salinization of the soil by the sprinkling water. One half of the sprinkled plants was grown in nonsaline soil to study the effects of pre-wetting and post-washing when ion uptake was primarily through leaves. The other half of the sprinkled plants was grown in soil salinized by drip irrigation, in order to evaluate the effects of pre-wetting and post-washing when Na⁺ and Cl^- uptake was through both leaves and roots.Post-washing with freshwater (5 min) reduced the leaf sap concentrations of Cl^- in saline-sprinkled plants from 56 to 43 mmol L^–1 in maize and from 358 to 225 mmol L^–1 in barley (averages for plants grown in nonsaline and saline soil). Na⁺ concentrations in leaf sap were reduced from 93 to 65 mmoles L^–1 (maize) and from 177 to 97 mmoles L^–1 (barley) by the post-washing. Pre-wetting had a small effect on ion uptake through leaves, the only significant reduction in seasonal means being in leaf Na⁺ concentrations for plants grown in nonsaline soil. Pre-wetting and post-washing, when combined, reduced leaf Cl^- concentrations to levels similar to those of nonsprinkled plants grown in saline soil; however, Na⁺ concentrations in leaves remained 3.5 times (maize) and 1.5 times (barley) higher than those of nonsprinkled plants. When pre-wetting and post-washing were not applied, sprinkled barley plants grown in saline soil had grain yields which were 58% lower than nonsprinkled plants grown in saline soil, but the reduction in grain yield was only 17% when the freshwater treatments were given. We conclude that a brief period of post-washing with freshwater is essential when saline water is employed in sprinkler irrigation. By comparison, the benefits from pre-wetting were small in these experiments. ei]T J Flowers