Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide

The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by β1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdo

The social and scientific values that shape national climate scenarios: a comparison of the Netherlands, Switzerland and the UK

This paper seeks to understand why climate information is produced differently from country to country. To do this, we critically examined and compared the social and scientific values that shaped the production of three national climate scenarios in the Netherlands, Switzerland and the UK. A comparative analysis of documentary materials and expert interviews linked to the climate scenarios was performed. Our findings reveal a new typology of use-inspired research in climate science for decision-making: (i) innovators, where the advancement of science is the main objective; (ii) consolidators, where knowledge exchanges and networks are prioritised; and (iii) collaborators, where the needs of users are put first and foremost. These different values over what constitutes ‘good’ science for decision-making are mirrored in the way users were involved in the production process: (i) elicitation, where scientists have privileged decision-making power; (ii) representation, where multiple organisations mediate on behalf of individual users; and (iii) participation, where a multitude of users interact with scientists in an equal partnership. These differences help explain why climate knowledge gains its credibility and legitimacy differently even when the information itself might not be judged as salient and usable. If the push to deliberately co-produce climate knowledge is not sensitive to the national civic epistemology at play in each country, scientist–user interactions may fail to deliver more ‘usable’ climate information

A smoothing algorithm for estimating stochastic, continuous time model parameters and its application to a simple climate model

Even after careful calibration, the output of deterministic models of environmental systems usually still show systematic deviations from measured data. To analyse possible causes of these discrepancies, we make selected model parameters time variable by treating them as continuous time stochastic processes. This extends an approach that was proposed earlier using discrete time stochastic processes. We present a Markov chain Monte Carlo algorithm for Bayesian estimation of such parameters jointly with the other, constant, parameters of the model. The algorithm consists of Gibbs sampling between constant and time varying parameters by using a Metropolis-Hastings algorithm for each parameter type. For the time varying parameter, we split the overall time period into consecutive intervals of random length, over each of which we use a conditional Ornstein-Uhlenbeck process with fixed end points as the proposal distribution in a Metropolis-Hastings algorithm. The hyperparameters of the stochastic process are selected by using a cross-validation criterion which maximizes a pseudolikelihood value, for which we have derived a computationally efficient estimator. We tested our algorithm by using a simple climate model. The results show that the algorithm behaves well, is computationally tractable and improves the fit of the model to the data when applied to an additional time-dependent forcing component. However, this additional forcing term is too large to be a reasonable correction of estimated forcing and it alters the posterior distribution of the other, time constant parameters to unrealistic values. This difficulty, and the impossibility of achieving a good simulation when making other parameters time dependent, indicates a more fundamental, structural deficit of the climate model. This is probably related to the poor resolution of the ocean in the model. Our study demonstrates the technical feasibility of the smoothing technique but also the need for a careful interpretation of the results. Copyright (c) 2009 Royal Statistical Society.

Targeted Gene Silencing in the Model Mushroom Coprinopsis cinerea (Coprinus cinereus) by Expression of Homologous Hairpin RNAs

The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes. Mutant screens and expression studies have implicated a number of genes in this developmental process. Functional analysis of these genes, however, is hampered by the lack of reliable reverse genetics tools for C. cinerea. Here, we report the applicability of gene targeting by RNA silencing for this organism. Efficient silencing of both an introduced GFP expression cassette and the endogenous cgl1 and cgl2 isogenes was achieved by expression of homologous hairpin RNAs. In latter case, silencing was the result of a hairpin construct containing solely cgl2 sequences, demonstrating the possibility of simultaneous silencing of whole gene families by a single construct. Expression of the hairpin RNAs reduced the mRNA levels of the target genes by at least 90%, as determined by quantitative real-time PCR. The reduced mRNA levels were accompanied by cytosine methylation of transcribed and nontranscribed DNA at both silencing and target loci in the case of constitutive high-level expression of the hairpin RNA but not in the case of transient expression. These results suggest the presence of both posttranscriptional and transcriptional gene silencing mechanisms in C. cinerea and demonstrate the applicability of targeted gene silencing as a powerful reverse genetics approach in this organism

Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide

ISSN:0959-665