Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system

The mammalian zygote-mediated CRISPR/Cas system can efficiently generate targeted genome-modified animals. However, this system is limited by the risk of off-target mutations. Here we show that offset-nicking by Cas9 nickase and paired gRNAs allows us to generate region deleted mice and targeted knock-in mice without off-target mutations.     

The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

Earliest colobine skeletons from Nakali,Kenya

Old World monkeys represent one of the most successful adaptive radiations of modern primates, but a sparse fossil record has limited our knowledge about the early evolution of this clade. We report the discovery of two partial skeletons of an early colobine monkey (Microcolobus) from the Nakali Formation (9.8–9.9 Ma) in Kenya that share postcranial synapomorphies with extant colobines in relation to arboreality such as mediolaterally wide distal humeral joint, globular humeral capitulum, distinctly angled zona conoidea, reduced medial trochlear keel, long medial epicondyle with weak retroflexion, narrow and tall olecranon, posteriorly dislocated fovea on the radial head, low projection of the femoral greater trochanter, wide talar head with a greater rotation, and proximodistally short cuboid and ectocuneiform. Microcolobus in Nakali clearly differs from the stem cercopithecoid Victoriapithecus regarding these features, as Victoriapithecus is postcranially similar to extant small‐sized terrestrial cercopithecines. However, degeneration of the thumb, a hallmark of modern colobines, is not observed, suggesting that this was a late event in colobine evolution. This discovery contradicts the prevailing hypothesis that the forest invasion by cercopithecids first occurred in the Plio‐Pleistocene, and shows that this event occurred by the late Miocene at a time when ape diversity declined. Am J Phys Anthropol 143:365‐382, 2010. © 2010 Wiley‐Liss, Inc.     

Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4 + concentrations in hydroponic conditions

Root system development is an important target for improving yield in cereal crops. Active root systems that can take up nutrients more efficiently are essential for enhancing grain yield. In this study, we attempted to identify quantitative trait loci (QTL) involved in root system development by measuring root length of rice seedlings grown in hydroponic culture. Reliable growth conditions for estimating the root length were first established to renew nutrient solutions daily and supply NH₄ ⁺ as a single nitrogen source. Thirty-eight chromosome segment substitution lines derived from a cross between ‘Koshihikari’, a japonica variety, and ‘Kasalath’, an indica variety, were used to detect QTL for seminal root length of seedlings grown in 5 or 500 μM NH₄ ⁺. Eight chromosomal regions were found to be involved in root elongation. Among them, the most effective QTL was detected on a ‘Kasalath’ segment of SL-218, which was localized to the long-arm of chromosome 6. The ‘Kasalath’ allele at this QTL, qRL6.1, greatly promoted root elongation under all NH₄ ⁺ concentrations tested. The genetic effect of this QTL was confirmed by analysis of the near-isogenic line (NIL) qRL6.1. The seminal root length of the NIL was 13.5–21.1% longer than that of ‘Koshihikari’ under different NH₄ ⁺ concentrations. Toward our goal of applying qRL6.1 in a molecular breeding program to enhance rice yield, a candidate genomic region of qRL6.1 was delimited within a 337 kb region in the ‘Nipponbare’ genome by means of progeny testing of F₂ plants/F₃ lines derived from a cross between SL-218 and ‘Koshihikari’.     

Phenotypic Plasticity in Photosynthetic Temperature Acclimation among Crop Species with Different Cold Tolerances

While interspecific variation in the temperature response of photosynthesis is well documented, the underlying physiological mechanisms remain unknown. Moreover, mechanisms related to species-dependent differences in photosynthetic temperature acclimation are unclear. We compared photosynthetic temperature acclimation in 11 crop species differing in their cold tolerance, which were grown at 15°C or 30°C. Cold-tolerant species exhibited a large decrease in optimum temperature for the photosynthetic rate at 360 μL L⁻¹ CO₂ concentration [Opt (A₃₆₀)] when growth temperature decreased from 30°C to 15°C, whereas cold-sensitive species were less plastic in Opt (A₃₆₀). Analysis using the C₃ photosynthesis model shows that the limiting step of A₃₆₀ at the optimum temperature differed between cold-tolerant and cold-sensitive species; ribulose 1,5-bisphosphate carboxylation rate was limiting in cold-tolerant species, while ribulose 1,5-bisphosphate regeneration rate was limiting in cold-sensitive species. Alterations in parameters related to photosynthetic temperature acclimation, including the limiting step of A₃₆₀, leaf nitrogen, and Rubisco contents, were more plastic to growth temperature in cold-tolerant species than in cold-sensitive species. These plastic alterations contributed to the noted growth temperature-dependent changes in Opt (A₃₆₀) in cold-tolerant species. Consequently, cold-tolerant species were able to maintain high A₃₆₀ at 15°C or 30°C, whereas cold-sensitive species were not. We conclude that differences in the plasticity of photosynthetic parameters with respect to growth temperature were responsible for the noted interspecific differences in photosynthetic temperature acclimation between cold-tolerant and cold-sensitive species.The temperature dependence of leaf photosynthetic rate shows considerable variation between plant species and with growth temperature (Berry and Björkman, 1980; Cunningham and Read, 2002; Hikosaka et al., 2006). Plants native to low-temperature environments and those grown at low temperatures generally exhibit higher photosynthetic rates at low temperatures and lower optimum temperatures, compared with plants native to high-temperature environments and those grown at high temperatures (Mooney and Billings, 1961; Slatyer, 1977; Berry and Björkman, 1980; Sage, 2002; Salvucci and Crafts-Brandner, 2004b). For example, the optimum temperature for photosynthesis differs between temperate evergreen species and tropical evergreen species (Hill et al., 1988; Read, 1990; Cunningham and Read, 2002). Such differences have been observed even among ecotypes of the same species (Björkman et al., 1975; Pearcy, 1977; Slatyer, 1977).Temperature dependence of the photosynthetic rate has been analyzed using the biochemical model proposed by Farquhar et al. (1980). This model assumes that the photosynthetic rate (A) is limited by either ribulose 1,5-bisphosphate (RuBP) carboxylation (A_c) or RuBP regeneration (A_r). The optimum temperature for photosynthetic rate in C₃ plants is thus potentially determined by (1) the temperature dependence of A_c, (2) the temperature dependence of A_r, or (3) both, at the colimitation point of A_c and A_r (Fig. 1; Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006).Open in a separate windowFigure 1.A scheme illustrating the shift in the optimum temperature for photosynthesis depending on growth temperature. Based on the C₃ photosynthesis model, the A₃₆₀ (white and black circles) is limited by A_c (solid line) or A_r (broken line). The optimum temperature for the photosynthetic rate is potentially determined by temperature dependence of A_c (A), temperature dependence of A_r (B), or the intersection of the temperature dependences of A_c and A_r (C). When the optimum temperature for the photosynthetic rate shifts to a higher temperature, there are also three possibilities determining the optimum temperature: temperature dependence of A_c (D), temperature dependence of A_r (E), or the intersection of the temperature dependences of A_c and A_r (F). Especially in the case that the optimum temperature is determined by the intersection of the temperature dependences of A_c and A_r, the optimum temperature can shift by changes in the balance between A_c and A_r even when the optimum temperatures for these two partial reactions do not change.In many cases, the photosynthetic rate around the optimum temperature is limited by A_c, and thus the temperature dependence of A_c determines the optimum temperature for the photosynthetic rate (Hikosaka et al., 1999, 2006; Yamori et al., 2005, 2006a, 2006b, 2008; Sage and Kubien, 2007; Sage et al., 2008). As the temperature increases above the optimum, A_c is decreased by increases in photorespiration (Berry and Björkman, 1980; Jordan and Ogren, 1984; von Caemmerer, 2000). Furthermore, it has been suggested that the heat-induced deactivation of Rubisco is involved in the decrease in A_c at high temperature (Law and Crafts-Brandner, 1999; Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a; Yamori et al., 2006b). Numerous previous studies have shown changes in the temperature dependence of A_c with growth temperature (Hikosaka et al., 1999; Bunce, 2000; Yamori et al., 2005). Also, the temperature sensitivity of Rubisco deactivation may differ between plant species (Salvucci and Crafts-Brandner, 2004b) and with growth temperature (Yamori et al., 2006b), which may explain variation in the optimum temperature for photosynthesis (Fig. 1, A and D).A_r is more responsive to temperature than A_c and often limits photosynthesis at low temperatures (Hikosaka et al., 1999, 2006; Sage and Kubien, 2007; Sage et al., 2008). Recently, several researchers indicated that A_r limits the photosynthetic rate at high temperature (Schrader et al., 2004; Wise et al., 2004; Cen and Sage, 2005; Makino and Sage, 2007). They suggested that the deactivation of Rubisco at high temperatures is not the cause of decreased A_c but a result of limitation by A_r. However, it remains unclear whether limitation by A_r is involved in the variation in the optimum temperature for the photosynthetic rate (Fig. 1, B and E).A shift in the optimum temperature for photosynthesis can result from changes in the balance between A_r and A_c, even when the optimum temperatures for these two partial reactions do not change (Fig. 1, C and F; Farquhar and von Caemmerer, 1982). The balance between A_r and A_c has been shown to change depending on growth temperature (Hikosaka et al., 1999; Hikosaka, 2005; Onoda et al., 2005a; Yamori et al., 2005) and often brings about a shift in the colimitation temperature of A_r and A_c. Furthermore, recent studies have shown that plasticity in this balance differs among species or ecotypes (Onoda et al., 2005b; Atkin et al., 2006; Ishikawa et al., 2007). Plasticity in this balance could explain interspecific variation in the plasticity of photosynthetic temperature dependence (Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006), although there has been no evidence in the previous studies that the optimum temperature for photosynthesis occurs at the colimitation point of A_r and A_c.Temperature tolerance differs between species and, with growth temperature, even within species from the same functional group (Long and Woodward, 1989). Bunce (2000) indicated that the temperature dependences of A_r and A_c to growth temperature were different between species from cool and warm climates and that the balance between A_r and A_c was independent of growth temperature for a given plant species. However, it was not clarified what limited the photosynthetic rate or what parameters were important in temperature acclimation of photosynthesis. Recently, we reported that the extent of temperature homeostasis of leaf respiration and photosynthesis, which is assessed as a ratio of rates measured at their respective growth temperatures, differed depending on the extent of the cold tolerance of the species (Yamori et al., 2009b). Therefore, comparisons of several species with different cold tolerances would provide a new insight into interspecific variation of photosynthetic temperature acclimation and their underlying mechanisms. In this study, we selected 11 herbaceous crop species that differ in their cold tolerance (Yamori et al., 2009b) and grew them at two contrasting temperatures, conducting gas-exchange analyses based on the C₃ photosynthesis model (Farquhar et al., 1980). Based on these results, we addressed the following key questions. (1) Does the plasticity in photosynthetic temperature acclimation differ between cold-sensitive and cold-tolerant species? (2) Does the limiting step of photosynthesis at several leaf temperatures differ between plant species and with growth temperature? (3) What determines the optimum temperature for the photosynthetic rate among A_c, A_r, and the intersection of the temperature dependences of A_c and A_r?     

Efficient extraction of thioreodoxin from Saccharomyces cerevisiae by ethanol

Thioredoxin, an antioxidant protein, is a promising molecule for development of functional foods because it protects the gastric mucosa and reduces the allergenicity of allergens. To establish a method for obtaining an ample amount of yeast thioredoxin, we found here that thioredoxin is released from Saccharomyces cerevisiae by treatment with 20% ethanol. We also found that Japanese sake contains a considerable amount of thioredoxin.     

Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization

Plant cell wall degradation is a premier event when Bacillus subtilis, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in B. subtilis strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (yesOPQRSTUVWXYZ, ytePQRST, and ybcMOPST-ybdABDE) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium, B. subtilis strain natto, fermenting soybeans also express the gene cluster including the yes series during the assimilation of soybean used as a carbon source. Among proteins encoded in the yes cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus Aspergillus aculeatus. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation.     

Fine mapping and allelic dosage effect of <Emphasis Type="Italic">Hwc1</Emphasis>, a complementary hybrid weakness gene in rice

Hybrid weakness is a reproductive barrier that is found in many plant species. In rice, the hybrid weakness caused by two complementary genes, Hwc1 and Hwc2, has been surveyed intensively. However, their gene products and the molecular mechanism that causes hybrid weakness have remained unknown. We performed linkage analyses of Hwc1, narrowed down the area of interest to 60 kb, and identified eight candidate genes. In the F(2) population, in which both Hwc1 and Hwc2 genes were segregated, plants were separable into four classes according to their respective phenotypes: severe type, semi-severe type, F(1) type, and normal type. Severe type plants show such severe symptoms that they could produce only tiny shoot-like structures; they were unable to generate roots. Genetic analyses using closely linked DNA markers of the two genes showed that the symptoms of the F(2) plants were explainable by the genotypes of Hwc1 and Hwc2. Weakness was observed in plants that have both Hwc1 and Hwc2. In Hwc1 homozygote, the symptoms worsened and severe type or semi-severe type plants appeared. Consequently, Hwc1 should have a gene dosage effect and be a semi-dominant gene. The dosage effect of Hwc2 was recognizable, but it was not so severe as that in Hwc1. These results are useful to elucidate the mechanism that causes the hybrid weakness phenomenon and the role of each causal gene in hybrid weakness.     

The evolution of conformist transmission in social learning when the environment changes periodically

Conformity is often observed in human social learning. Social learners preferentially imitate the majority or most common behavior in many situations, though the strength of conformity varies with the situation. Why has such a psychological tendency evolved? I investigate this problem by extending a standard model of social learning evolution with infinite environmental states (Feldman, M.W., Aoki, K., Kumm, J., 1996. Individual versus social learning: evolutionary analysis in a fluctuating environment. Anthropol. Sci. 104, 209-231) to include conformity bias. I mainly focus on the relationship between the strength of conformity bias that evolves and environmental stability, which is one of the most important factors in the evolution of social learning. Using the evolutionarily stable strategy (ESS) approach, I show that conformity always evolves when environmental stability and the cost of adopting a wrong behavior are small, though environmental stability and the cost of individual learning both negatively affect the strength of conformity.