Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: Module evolution of the filamentous phage genome

Summary The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism. Offprint requests to: R.N.H. Konings     

基于SNP 互作识别潜在冠心病致病基因

目的:基于全基因组关联分析(Genomewideassociationstudy,GWAS)数据与生物信息学方法,识别冠心病潜在致病基因。方法:利用生物信息学方法和GWAS数据,对单核苷酸多态性(SingleNucleotidePolymorphisms,SNP)进行疾病风险打分,依据特定距离阈值内的SNP-SNP互作关系,筛选出疾病相关SNP显著风险模块,识别潜在致病基因。结果:设定阈值20kb,经筛选获得279个SNP显著风险模块,映射到79个基因,文献验证率为71.01%。结论:基于SNP互作识别的潜在致病基因,能更加准确的分析冠心病的发生发展过程。     

Shoot dynamics and architecture of saplings in Fagus crenata across its geographical range

 Relationships between leaf or shoot size, number, and arrangement in response to light were investigated to test the hypothesis that these characteristics are linked. In order to test this hypothesis, the divergence in allometry and shoot dynamics in saplings of Japanese beech (Fagus crenata) obtained from four populations and having different leaf sizes were examined in a nursery under both full sun and shade conditions. Trees with different leaf sizes also showed large differences in canopy structure, particularly when shade-grown saplings were compared. The final leaf mass distributions of the large-leaf populations were conical or “bottom - heavy”, while those of the small-leaf populations were planar or “top - heavy”. The slope of the allometric relations between leaf mass and shoot and branch mass in small-leaved populations were steeper than those in large-leaf populations. The four populations were classified into two growth types: populations producing a few large leaf and shoot modules corresponded to “stem growth type”, and those producing many small leaf and shoot modules corresponded to “leaf growth type”. These kinds of intra-specific variation in architecture and growth of F. crenata trees may influence the structure and dynamics of forests in accordance with differences in competitive ability or sensitivity to disturbances such as windstorm. Received: 18 March 1997 / Accepted: 21 October 1997     

基于人类信号网络和表达谱数据研究静脉血栓复发机制

目的:静脉血栓是一种高复发风险和高致死率的疾病,其形成和复发的分子机制尚不明确。基于人类信号网络和基因表达谱数据可针对静脉血栓经华法令抗凝治疗后的复发机制进行研究。方法:结合表达谱数据和人类信号网络,设计差异模块筛选策略,通过功能分析、差异表达分析和已知血栓相关基因及药物靶基因的互作关联研究,获得与静脉血栓复发相关的显著差异模块。结果:最终获得8个与静脉血栓复发密切相关的显著差异模块,评估了华法令治疗静脉血栓的效能,提出了联合用药的3种可能途径。结论:应用本文提出的整合筛选策略,能识别与静脉血栓复发相关的模块,探究静脉血栓复发的分子机制和评估华法令的治疗效能。还提供了潜在的联合用药途径,这对治愈血栓、防治血栓复发及复发机制的研究具有重要意义。     

Inference of signaling and gene regulatory networks by steady-state perturbation experiments: structure and accuracy

One of the fundamental problems of cell biology is the understanding of complex regulatory networks. Such networks are ubiquitous in cells and knowledge of their properties is essential for the understanding of cellular behavior. In earlier work (Kholodenko et al. (PNAS 99: 12841), it was shown how the structure of biological networks can be quantified from experimental measurements of steady-state concentrations of key intermediates as a result of perturbations using a simple algorithm called "unravelling". Here, we study the effect of experimental uncertainty on the accuracy of the inferred structure (i.e. whether interactions are excitatory or inhibitory) of the networks determined using the unravelling algorithm. We show that the accuracy of the network structure depends not only on the noise level but on the strength of the interactions within the network. In particular, both very small and very large values of the connection strengths lead to large uncertainty in the inferred network. We describe a powerful geometric tool for the intuitive understanding of the effect of experimental error on the qualitative accuracy of the inferred network. In addition, we show that the use of additional data beyond that needed to minimally constrain the network not only improves the accuracy of the inferred network, but also may allow the detection of situations in which the initial assumptions of unravelling with respect to the network and the perturbations have been violated. Our ideas are illustrated using the mitogen-activated protein kinase (MAPK) signaling network as an example.     

Modelling gene networks at different organisational levels

Approaches to modelling gene regulation networks can be categorized, according to increasing detail, as network parts lists, network topology models, network control logic models, or dynamic models. We discuss the current state of the art for each of these approaches. There is a gap between the parts list and topology models on one hand, and control logic and dynamic models on the other hand. The first two classes of models have reached a genome-wide scale, while for the other model classes high throughput technologies are yet to make a major impact.     

Protein β-Sheet Nucleation Is Driven by Local Modular Formation

Despite its central role in the protein folding process, the specific mechanism(s) behind β-sheet formation has yet to be determined. For example, whether the nucleation of β-sheets, often containing strands separated in sequence by many residues, is local or not remains hotly debated. Here, we investigate the initial nucleation step of β-sheet formation by performing an analysis of the smallest β-sheets in a non-redundant dataset on the grounds that the smallest sheets, having undergone little growth after nucleation, will be enriched for nucleating characteristics. We find that the residue propensities are similar for small and large β-sheets as are their interstrand pairing preferences, suggesting that nucleation is not primarily driven by specific residues or interacting pairs. Instead, an examination of the structural environments of the two-stranded sheets shows that virtually all of them are contained in single, compact structural modules, or when multiple modules are present, one or both of the chain termini are involved. We, therefore, find that β-nucleation is a local phenomenon resulting either from sequential or topological proximity. We propose that β-nucleation is a result of two opposite factors; that is, the relative rigidity of an associated folding module that holds two stretches of coil close together in topology coupled with sufficient chain flexibility that enables the stretches of coil to bring their backbones in close proximity. Our findings lend support to the hydrophobic zipper model of protein folding (Dill, K. A., Fiebig, K. M., and Chan, H. S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1942–1946). Implications for protein folding are discussed.     

Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43

Exo-1,5-α-l-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.     

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.     

The M2 Module of the Cys-His-rich Domain (CHRD) of PCSK9 Protein Is Needed for the Extracellular Low-density Lipoprotein Receptor (LDLR) Degradation Pathway

PCSK9 enhances the cellular degradation of the LDL receptor (LDLR), leading to increased plasma LDL cholesterol. This multidomain protein contains a prosegment, a catalytic domain, a hinge region, and a cysteine-histidine rich domain (CHRD) composed of three tightly packed modules named M1, M2, and M3. The CHRD is required for the activity of PCSK9, but the mechanism behind this remains obscure. To define the contribution of each module to the function of PCSK9, we dissected the CHRD structure. Six PCSK9 deletants were generated by mutagenesis, corresponding to the deletion of only one (ΔM1, ΔM2, ΔM3) or two (ΔM12, ΔM13, ΔM23) modules. Transfection of HEK293 cells showed that all deletants were well processed and expressed compared with the parent PCSK9 but that only those lacking the M2 module were secreted. HepG2 cells lacking endogenous PCSK9 (HepG2/shPCSK9) were used for the functional analysis of the extracellular or intracellular activity of PCSK9 and its deletants. To analyze the ability of the deletants to enhance the LDLR degradation by the intracellular pathway, cellular expressions revealed that only the ΔM2 deletant retains a comparable total LDLR-degrading activity to full-length PCSK9. To probe the extracellular pathway, HepG2/shPCSK9 cells were incubated with conditioned media from transfected HEK293 or HepG2/shPCSK9 cells, and cell surface LDLR levels were analyzed by FACS. The results showed no activity of any secreted deletant compared with PCSK9. Thus, although M2 is dispensable for secretion, its presence is required for the extracellular activity of PCSK9 on cell surface LDLR.