Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo

In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and is composed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase. It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway. To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins. These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains as compared to wild-type and G1 strains. Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae. Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S. cerevisiae.      

人肺腺癌细胞分化相关基因cDNAs的克隆

在用10^-5 mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆.     

Enhanced heterologous protein production in Pichia pastoris under increased air pressure

Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β‐galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1‐inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β‐galactosidase‐specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1040–1047, 2014     

Voting Behavior,Coalitions and Government Strength through a Complex Network Analysis

We analyze the network of relations between parliament members according to their voting behavior. In particular, we examine the emergent community structure with respect to political coalitions and government alliances. We rely on tools developed in the Complex Network literature to explore the core of these communities and use their topological features to develop new metrics for party polarization, internal coalition cohesiveness and government strength. As a case study, we focus on the Chamber of Deputies of the Italian Parliament, for which we are able to characterize the heterogeneity of the ruling coalition as well as parties specific contributions to the stability of the government over time. We find sharp contrast in the political debate which surprisingly does not imply a relevant structure based on established parties. We take a closer look to changes in the community structure after parties split up and their effect on the position of single deputies within communities. Finally, we introduce a way to track the stability of the government coalition over time that is able to discern the contribution of each member along with the impact of its possible defection. While our case study relies on the Italian parliament, whose relevance has come into the international spotlight in the present economic downturn, the methods developed here are entirely general and can therefore be applied to a multitude of other scenarios.     

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway

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Quantifying the climatic niche of symbiont partners in a lichen symbiosis indicates mutualist‐mediated niche expansions

The large distributional areas and ecological niches of many lichenized fungi may in part be due to the plasticity in interactions between the fungus (mycobiont) and its algal or cyanobacterial partners (photobionts). On the one hand, broad‐scale phylogenetic analyses show that partner compatibility in lichens is rather constrained and shaped by reciprocal selection pressures and codiversification independent of ecological drivers. On the other hand, sub‐species‐level associations among lichen symbionts appear to be environmentally structured rather than phylogenetically constrained. In particular, switching between photobiont ecotypes with distinct environmental preferences has been hypothesized as an adaptive strategy for lichen‐forming fungi to broaden their ecological niche. The extent and direction of photobiont‐mediated range expansions in lichens, however, have not been examined comprehensively at a broad geographic scale. Here we investigate the population genetic structure of Lasallia pustulata symbionts at sub‐species‐level resolution across the mycobiont's Europe‐wide range, using fungal MCM7 and algal ITS rDNA sequence markers. We show that variance in occurrence probabilities in the geographic distribution of genetic diversity in mycobiont‐photobiont interactions is closely related to changes in climatic niches. Quantification of niche extent and overlap based on species distribution modeling and construction of Hutchinsonian climatic hypervolumes revealed that combinations of fungal–algal interactions change at the sub‐species level along latitudinal temperature gradients and in Mediterranean climate zones. Our study provides evidence for symbiont‐mediated niche expansion in lichens. We discuss our results in the light of symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation and niche evolution in mutualisms.