Genetic Basis for Spontaneous Hybrid Genome Doubling during Allopolyploid Speciation of Common Wheat Shown by Natural Variation Analyses of the Paternal Species

The complex process of allopolyploid speciation includes various mechanisms ranging from species crosses and hybrid genome doubling to genome alterations and the establishment of new allopolyploids as persisting natural entities. Currently, little is known about the genetic mechanisms that underlie hybrid genome doubling, despite the fact that natural allopolyploid formation is highly dependent on this phenomenon. We examined the genetic basis for the spontaneous genome doubling of triploid F₁ hybrids between the direct ancestors of allohexaploid common wheat (Triticum aestivum L., AABBDD genome), namely Triticum turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome). An Ae. tauschii intraspecific lineage that is closely related to the D genome of common wheat was identified by population-based analysis. Two representative accessions, one that produces a high-genome-doubling-frequency hybrid when crossed with a T . turgidum cultivar and the other that produces a low-genome-doubling-frequency hybrid with the same cultivar, were chosen from that lineage for further analyses. A series of investigations including fertility analysis, immunostaining, and quantitative trait locus (QTL) analysis showed that (1) production of functional unreduced gametes through nonreductional meiosis is an early step key to successful hybrid genome doubling, (2) first division restitution is one of the cytological mechanisms that cause meiotic nonreduction during the production of functional male unreduced gametes, and (3) six QTLs in the Ae . tauschii genome, most of which likely regulate nonreductional meiosis and its subsequent gamete production processes, are involved in hybrid genome doubling. Interlineage comparisons of Ae . tauschii ’s ability to cause hybrid genome doubling suggested an evolutionary model for the natural variation pattern of the trait in which non-deleterious mutations in six QTLs may have important roles. The findings of this study demonstrated that the genetic mechanisms for hybrid genome doubling could be studied based on the intrinsic natural variation that exists in the parental species.     

Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.Cellulose, composed of β-1,4-linked glucosyl units, is the most abundant naturally produced biopolymer on earth and can be utilized as a sustainable and renewable energy resource in place of fossil fuel. Establishing conditions for the efficient degradation of cellulose will contribute to the enhanced use of bioethanol, a biobased alternative to gasoline, which will increase biomass recycling and reduce carbon dioxide emissions (21, 30). Hence, efficient degradation of cellulose is an issue of great importance today.Bacteria and fungi produce cellulases that catalyze the hydrolysis of β-1,4-glycosidic bonds and are involved in the degradation of cellulose. Cellulases are divided into three major types according to their substrate specificities and the mode of hydrolysis: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). The most efficient hydrolysis of cellulose is thought to result from the combined synergistic actions of cellulases, whereby the enzymatic activity of an enzyme mixture is substantially higher than the sum of the activities of the individual enzymes. Several types of synergy have been described as the cooperative actions of endo- and exo-acting enzymes (20, 25, 32, 33, 45). Such cellulose-degrading enzymes are routinely used in the manufacture of beverages and industrial products, e.g., beer and wine, animal feed, paper, textiles, laundry detergents, and food ingredients (5). Hence, reducing cellulase manufacturing costs by increasing the productivity of cellulases with high specific activities through biotechnological modification is a desired research goal.Fungal cellobiohydrolases belong to glycosyl hydrolase families 6 and 7 (GH-6 and -7) and act most efficiently on highly ordered crystalline cellulose, hydrolyzing from either the reducing or the nonreducing terminus to liberate predominantly cellobiose (C2) with a minor amount of cellotriose (C3) (6, 39, 40). Also, Trichoderma reesei Cel6A can hydrolyze 1,3-1,4-β-glucan (1, 18), but it is unclear whether in vivo it is hydrolysis of 1,3-1,4-β-glucan that occurs mainly or hydrolysis of cellulose derivatives. The resulting accumulation of cellobiose inhibits the activity of cellobiohydrolase (13, 15, 28, 31, 36, 37, 44). Some microorganisms possess cellulosomes, multienzyme complexes that contribute to the efficient degradation of cellulose. Cellobiohydrolase is a documented component of cellulosomes in Clostridium thermocellum (2, 31).The three-dimensional (3D) structures of two GH-6 family members have been elucidated, including the cellobiohydrolase of T. reesei and that of Humicola insolens in complex with glucose, cellooligosaccharide, and a nonhydrolyzable substrate analogue (35, 41-43). The proposed structures have identified the significant amino acids associated with the catalytic core domain, where the catalytic site is buried inside a tunnel-shaped cavity and an enzyme-cellooligosaccharide hydrogen bond network. The structure suggests that the mode of action proceeds in a processive manner as cellobiohydrolase progresses along the cellulose chain (7, 19, 34, 40).The ascomycete fungus Magnaporthe grisea is the pathogen that causes rice blast, the most devastating fungal disease of rice. Since the complete genome sequence of M. grisea has been published (10), mining the database for candidate genes involved in pathogen-plant interactions, cell wall degradation, etc., is quite feasible. The cell wall-degrading enzymes of the genus Magnaporthe that are involved in the infection process have been of particular interest (22-24). Based on the complete genome sequence, M. grisea has three putative GH-6 family cellobiohydrolases and four GH-7 family cellobiohydrolases. Using primers designed from the database of M. grisea cellobiohydrolases, we cloned putative GH-6 and GH-7 family cellobiohydrolases, designated MoCel6A and MoCel7A, respectively, from Magnaporthe oryzae by PCR. The cloned MoCel6A and MoCel7A from M. oryzae were completely identical to those of M. grisea. In this paper, we demonstrate the properties of MoCel6A prepared by homologous overexpression in M. oryzae and examine the effects of cellobiose on the hydrolytic activity of MoCel6A. Furthermore, the effects of cellobiose on the activities of both MoCel6A and a T. reesei GH family 6 cellobiohydrolase (TrCel6A, formerly referred to as CBH II), which were overexpressed in Aspergillus oryzae, were also examined.     

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5–ZNT6 heterodimers [ZNT5-6] and ZNT7–ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.     

RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells

Interleukin-12 (IL-12) is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB). Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR) 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA) of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells.     

Selenium-binding protein 1: Its physiological function,dependence on aryl hydrocarbon receptors,and role in wasting syndrome by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Background

Selenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1.

Methods

To address this issue, we generated Selenbp1-null [Selenbp1 (−/−)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse model.

Results

Selenbp1 (−/−) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (−/−) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (−/−) and (+/+) mice.

Conclusions

The above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity.

General significance

Selenbp1 (−/−) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.     

Unsaturated fatty acids induce cytotoxic aggregate formation of amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutants

Formation of misfolded protein aggregates is a remarkable hallmark of various neurodegenerative diseases including Alzheimer disease, Parkinson disease, Huntington disease, prion encephalopathies, and amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) immunoreactive inclusions have been found in the spinal cord of ALS animal models and patients, implicating the close involvement of SOD1 aggregates in ALS pathogenesis. Here we examined the molecular mechanism of aggregate formation of ALS-related SOD1 mutants in vitro. We found that long-chain unsaturated fatty acids (FAs) promoted aggregate formation of SOD1 mutants in both dose- and time-dependent manners. Metal-deficient SOD1s, wild-type, and mutants were highly oligomerized compared with holo-SOD1s by incubation in the presence of unsaturated FAs. Oligomerization of SOD1 is closely associated with its structural instability. Heat-treated holo-SOD1 mutants were readily oligomerized by the addition of unsaturated FAs, whereas wild-type SOD1 was not. The monounsaturated FA, oleic acid, directly bound to SOD1 and was characterized by a solid-phase FA binding assay using oleate-Sepharose. The FA binding characteristics were closely correlated with the oligomerization propensity of SOD1 proteins, which indicates that FA binding may change SOD1 conformation in a way that favors the formation of aggregates. High molecular mass aggregates of SOD1 induced by FAs have a granular morphology and show significant cytotoxicity. These findings suggest that SOD1 mutants gain FA binding abilities based on their structural instability and form cytotoxic granular aggregates.     

A model for oscillating chemical reactions.

A simple theoretical model for a class of oscillating chemical reactions is investigated, which is inspired by Degn's analysis of the mechanism of Belousov-Zhabotinsky reactions. Although the model involves only one auto-catalytic path, as distinct from models presented hitherto, a limit-cycle behaviour is found for the concentrations, corresponding to a definite region in the space of controllable parameters. Characteristics of the coherent oscillations are investigated together with the behaviour in the induction period. The critical slowing-down around the onset of instability is shown to behave very much like that found in the equilibrium phase transition. The existence of an "anti-Curie" point is found as well as the Curie point, corresponding to the fact that the domain for sustained oscillation is closed in certain directions. Possible extensions of the model and ways of improving the theory are discussed.     

Chaperonin Contributes to Cold Hardiness of the Onion Maggot Delia antiqua through Repression of Depolymerization of Actin at Low Temperatures

Winter-diapause and cold-acclimated non-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), show strong cold hardiness. To obtain insights into the mechanisms involved in the enhancement of cold hardiness, we investigated the expression patterns of genes encoding subunits of chaperonin (CCT) and the morphology of actin, a substrate of CCT, at low temperatures. Quantitative real-time PCR analyses showed the mRNA levels of CCT subunits in pupal tissues to be highly correlated with the cold hardiness of the pupae. While actin in the Malpighian tubules of non-cold-hardy pupae showed extensive depolymerization after a cold treatment, actin in the same tissue of cold-hardy pupae was not depolymerized. Damage to cell membranes became apparent after the depolymerization of actin. Moreover, administration of Latrunculin B, an inhibitor of actin polymerization, to the larvae markedly decreased the cold hardiness of the pupae obtained. These findings suggest that CCT contributes to the cold hardiness of D. antiqua through the repression of depolymerization of actin at low temperatures.