The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1

Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.     

Factors influencing the sporulation and cyst formation of Aphanomyces invadans, etiological agent of ulcerative mycosis in Atlantic menhaden, Brevoortia tyrannus

Oomycete infections caused by Aphanomyces invadans occur in freshwater and estuarine fishes around the world. Along the east coast of the USA, skin ulcers caused by A. invadans are prevalent in Atlantic menhaden, Brevoortia tyrannus. From laboratory observations low salinities appear crucial to transmission of the pathogen. To better understand aspects of transmission, we characterized sporulation and cyst formation of secondary zoospores of two isolates of A. invadans at different salinities and temperatures. Sporulation occurred only at low salinities. At room temperature (ca. 20-22 C), using "pond water" augmented with artificial sea salts, the endemic strain WIC and the Thailand strain PA7 of A. invadans produced free-swimming secondary zoospores at salinities of 0, 1 and 2 psu (practical salinity unit = per thousand), but not at 4 psu or higher. Secondary zoospores of another species, ATCC-62427 (Aphanomyces sp.), were observed at 1, 2, 4 and 8 psu but not at 0 and 12 psu. Secondary zoospores of all three isolates, especially WIC, were abundant and motile 1-2 d postsporulation. Sporulation was temperature dependent and occurred over a relatively narrow range. No sporulation occurred at 4, 30 or 35 C for either WIC or PA7. For both strains zoospore production within 1-3 d after the initiation of sporulation was more prolific at 25 C than at 20 and 15 C. At 15 C production of zoospores was sustained over 11 d for WIC and 5 d for PA7. At room temperature single WIC secondary zoospores remained motile 12-18 h. Salinities exceeding 4 psu or vigorous shaking caused immediate cyst formation of WIC secondary zoospores. Exposure to menhaden tissue, but not tissues of other fishes to secondary zoospores (WIC), caused rapid (2 h) cyst formation. Cysts were capable of excysting when transferred to 1 psu water within 2-3 h of cyst formation. Cysts that had remained encysted in 6.5 psu for 24 h did not excyst when transferred to 1 psu water. Salinity and temperature requirements for sporulation indicate that juvenile menhaden must acquire infections during rain or in low salinity oligohaline waters.     

Seed weight increases with altitude in the Swiss Alps between related species but not among populations of individual species

Seed weight is a crucial plant life history trait, determining establishment success and dispersal ability. Especially in stressful environments, larger seeds may be selected at the expense of seed number, because larger seeds have a better chance of giving rise to an established offspring. We tested the hypotheses that between related species-pairs and among populations of single species a similar trend for increasing seed weight with increasing altitude should be present. Firstly, we measured seed weights from 29 species-pairs, with one species occurring in lowland areas and a congeneric species from high altitudes. Seeds of the alpine species were 28±8% larger than seeds from lowland species (P<0.01). Compared to the related lowland species, 55% of the alpine species had heavier seeds, 3% (one species) had lighter, and 41% had seeds of approximately equal weight. Secondly, we compared seed weights among populations of four species from different habitats and with different life histories. Seeds from between 11 and 34 populations per species were sampled along altitudinal gradients of 800–1,500 m (ca. 800 m in Scabiosa lucida, ca. 1,000 m in Saxifraga oppositifolia, ca. 1,000 m in Epilobium fleischeri, and ca. 1,500 m in Carex flacca). In all the four species, we found no indication for heavier seeds at higher altitudes. Our results indicate a selection pressure for species with heavier seeds at higher altitude, but the trend does not seem to operate across all cases. Phylogenetic constraints may limit the correlation among altitude and seed weight, operating particularly against selection for larger seed size, the closer populations and species are related to each other.     

Phyllosphere nitrogen relations: reciprocal transfer of nitrogen between epiphyllous liverworts and host plants in the understorey of a lowland tropical wet forest in Costa Rica

Epiphyllous bryophytes on tropical rainforest plants acquire nutrients from throughfall and free-living N2-fixing organisms, but may also depend directly on host leaf leachates. By contrast, after drying events bryophytes lose significant quantities of nutrients through leaching that can be taken up by host leaves. To assess a potential nutritional interdependency, nitrogen fluxes between epiphyllous liverworts and their host leaves (Carludovica drudei, Costus laevis, Dieffenbachia concinna, Pentagonia wendlandii) were quantified by in situ15N-labelling techniques in a lowland rainforest, Piedras Blancas National Park, Costa Rica. Depending on host species, epiphyllous bryophytes met between 1 and 57% of their N demand from host leaf leachates. Externally supplied 15N was taken up both by epiphylls and host leaves, but N from epiphyll leachates accounted for < 2.5% of host leaf N after 14 d. Long-term observations (180 d) demonstrated the highly dynamic nature of phyllosphere N of the investigated tropical rainforest understorey and an intermittent sink capacity of epiphyllous bryophytes.     

Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants

Tobacco plants grown in vitro were supplied with a mixture of [U-13C6]glucose and unlabelled sucrose via the root system. After 20 days, leaves were harvested and extracted with water. Glucose was isolated from the extract and was analysed by 13C NMR spectroscopy. All 13C signals appeared as complex multiplets due to 13C-13C coupling. The abundance of 21 isotopologous glucose species was determined from the 13C NMR signal integrals by numerical deconvolution using a genetic algorithm. The relative fractions of specific isotopologs in the overall excess of 13C-labelled specimens establish flux contributions via glycolysis/glucogenesis, pentose phosphate pathway, citric acid cycle and Calvin cycle including 13CO2 refixation. The fluxes were modelled and reconstructed in silico by a novel rule-based approach yielding the contributions of circular pathways and the degree of multiple cycling events. The data indicate that the vast majority of the proffered [U-13C6]glucose molecules had been modified by catabolism and subsequent glucogenesis from catabolic fragments, predominantly via passage through the citric acid cycle and the pentose phosphate pathway.     

Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize

Developing kernels of the inbred maize line W22 were grown in sterile culture and supplied with a mixture of [U-13C6]glucose and unlabeled glucose during three consecutive intervals (11-18, 18-25, or 25-32 days after pollination) within the linear phase of starch formation. At the end of each labeling period, glucose was prepared from starch and analyzed by 13C isotope ratio mass spectrometry and high-resolution (13)C NMR spectroscopy. The abundances of individual glucose isotopologs were calculated by computational deconvolution of the NMR data. [1,2-(13)C2]-, [5,6-(13)C2]-, [2,3-(13)C2]-, [4,5-(13)C2]-, [1,2,3-(13)C3]-, [4,5,6-(13)C3]-, [3,4,5,6-(13)C4]-, and [U-(13)C6]-isotopologs were detected as the major multiple-labeled glucose species, albeit at different normalized abundances in the three intervals. Relative flux contributions by five different pathways in the primary carbohydrate metabolism were determined by computational simulation of the isotopolog space of glucose. The relative fractions of some of these processes in the overall glucose cycling changed significantly during maize kernel development. The simulation showed that cycling via the non-oxidative pentose phosphate pathway was lowest during the middle interval of the experiment. The observed flux pattern could by explained by a low demand for amino acid precursors recruited from the pentose phosphate pathway during the middle interval of kernel development.     

Free-energy landscape of the villin headpiece in an all-atom force field

We investigate the landscape of the internal free-energy of the 36 amino acid villin headpiece with a modified basin hopping method in the all-atom force field PFF01, which was previously used to predictively fold several helical proteins with atomic resolution. We identify near native conformations of the protein as the global optimum of the force field. More than half of the twenty best simulations started from random initial conditions converge to the folding funnel of the native conformation, but several competing low-energy metastable conformations were observed. From 76,000 independently generated conformations we derived a decoy tree which illustrates the topological structure of the entire low-energy part of the free-energy landscape and characterizes the ensemble of metastable conformations. These emerge as similar in secondary content, but differ in tertiary arrangement.     

Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.