东亚正钳蝎毒素基因BmKbpp的原核表达及其抗菌功能初步鉴定

一级结构分析和二级结构预测表明无二硫键蝎毒液多肽(NDBPs)BmKBPP具两亲性α-Helix结构,含有大量带净正电荷的碱性残基(Arg和Lys),符合多价阳离子抗菌肽的典型特征。采用PCR法和质粒载体pGEX-5X-1构建BmKbpp、EGFP及BmKbpp/EGFP与GST的原核融合表达载体,3个表达载体与pGEX-5X-1分别在Rossetta(DE3)菌中进行表达,并测定4个Rossetta(DE3)表达工程菌诱导表达的生长曲线。结果表明,BmKBPP是一个具有很强抑菌功能的抗菌肽,GST、BmKBPP及EGFP3个蛋白一起融合表达可获得少量蛋白的表达,这将为后续的功能研究和应用研究奠定基础。     

Evolution of a bacteria/plasmid association

Associations between bacteria and their accessory elements (viruses, plasmids and transposons) range from antagonistic to mutualistic. A number of previous studies have demonstrated that plasmid carriage reduces bacterial fitness in the absence of selection for specific functions such as antibiotic resistance. Many studies have demonstrated increased fitness of evolving microbial populations in laboratory environments, but we are aware of only one study in which fitness gains were partitioned between a plasmid and its host. Here, we examine the evolution of an association between a plasmid and its bacterial host. Carriage of the non-conjugative plasmid pACYC184 initially reduced the fitness of Escherichia coli B in the absence of antibiotic. We then cultured plasmid-bearing bacteria for 500 generations in the presence of antibiotic. The fitness of each combination of host and plasmid, with and without the culture history, was determined by competing it against a baseline strain. The results indicate adaptation by the host genome, but no plasmid adaptation. We also competed the evolved host, transformed with the baseline plasmid, against its isogenic plasmid-free counterpart. The plasmid now increased the fitness of its host.     

Chance and necessity in the evolution of minimal metabolic networks

It is possible to infer aspects of an organism's lifestyle from its gene content. Can the reverse also be done? Here we consider this issue by modelling evolution of the reduced genomes of endosymbiotic bacteria. The diversity of gene content in these bacteria may reflect both variation in selective forces and contingency-dependent loss of alternative pathways. Using an in silico representation of the metabolic network of Escherichia coli, we examine the role of contingency by repeatedly simulating the successive loss of genes while controlling for the environment. The minimal networks that result are variable in both gene content and number. Partially different metabolisms can thus evolve owing to contingency alone. The simulation outcomes do preserve a core metabolism, however, which is over-represented in strict intracellular bacteria. Moreover, differences between minimal networks based on lifestyle are predictable: by simulating their respective environmental conditions, we can model evolution of the gene content in Buchnera aphidicola and Wigglesworthia glossinidia with over 80% accuracy. We conclude that, at least for the particular cases considered here, gene content of an organism can be predicted with knowledge of its distant ancestors and its current lifestyle.     

中国白兔IL-6基因的分子克隆及其表达研究

运用RT-PCR技术对用ConA刺激的中国白兔外周血淋巴细胞(PMBCr)进行了扩增,将纯化后的PCR产物克隆入pMD18-T中进行核苷酸序列测定.结果中国白兔IL-6基因全长726 bp,编码242个氨基酸,其中前26个氨基酸残基构成信号肽序列.与不同物种IL-6基因相比,核苷酸和推导的氨基酸序列有一定的差异.在推导的中国白兔IL-6氨基酸序列中,在108-110位存在1个潜在的N-联糖基化位点,同时存在9个Cys残基.将pTIL-6双酶切,回收目的基因片段克隆到大肠杆菌表达载体pET28a中构建了重组质粒pETIL-6,转化大肠杆菌BL21(DE3),并用IPTG进行了诱导.结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子量为29.5kDa的重组目的蛋白.经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的16.2%.     

Using the Acropora digitifera genome to understand coral responses to environmental change

Despite the enormous ecological and economic importance of coral reefs, the keystone organisms in their establishment, the scleractinian corals, increasingly face a range of anthropogenic challenges including ocean acidification and seawater temperature rise. To understand better the molecular mechanisms underlying coral biology, here we decoded the approximately 420-megabase genome of Acropora digitifera using next-generation sequencing technology. This genome contains approximately 23,700 gene models. Molecular phylogenetics indicate that the coral and the sea anemone Nematostella vectensis diverged approximately 500 million years ago, considerably earlier than the time over which modern corals are represented in the fossil record (～240 million years ago). Despite the long evolutionary history of the endosymbiosis, no evidence was found for horizontal transfer of genes from symbiont to host. However, unlike several other corals, Acropora seems to lack an enzyme essential for cysteine biosynthesis, implying dependency of this coral on its symbionts for this amino acid. Corals inhabit environments where they are frequently exposed to high levels of solar radiation, and analysis of the Acropora genome data indicates that the coral host can independently carry out de novo synthesis of mycosporine-like amino acids, which are potent ultraviolet-protective compounds. In addition, the coral innate immunity repertoire is notably more complex than that of the sea anemone, indicating that some of these genes may have roles in symbiosis or coloniality. A number of genes with putative roles in calcification were identified, and several of these are restricted to corals. The coral genome provides a platform for understanding the molecular basis of symbiosis and responses to environmental changes.     

大肠杆菌和枯草杆菌的L-ter 到起始密码子的距离分析

基于第一ATG规则，分别对确定基因和不确定基因的L-ter到起始密码子(ATG、GTG或TTG)的距离和L-ter到起始密码子下游紧邻的ATG的距离进行统计，结果表明：L-ter到起始密码子的距离主要分布在20个氨基酸以内；在以第一ATG和第一GTG为起始密码子的基因中，L-ter到起始密码子下游紧邻的ATG是L-ter到起始密码子平均距离的4～5倍。这些可能是起始密码子的重要位置特征，也说明了少数基因以GTG起始的原因．对于不满足第一ATG、GTG规则的基因，分析了L-ter到起始密码子之间出现ATG、GTG和TTG的比率，发现大肠杆菌的不确定基因与确定基因之间有较大的差别，而枯草杆菌的差别不大．     

半胱氨酸的抑菌作用与类SOD活力

为了研究半胱氨酸的抑菌作用与类SOD活力关系，用不同浓度半胱氨酸溶液滤纸片对大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌做定性抑菌实验，并用连苯三酚自氧化法测定19种氨基酸的类SOD活力．结果表明：半胱氨酸的类SOD活力在19种氨基酸中最高，半胱氨酸饱和溶液滤纸片对大肠杆菌和金黄色葡萄球菌有很强的抑制作用．这说明半胱氨酸的抑菌作用和其类SOD活力间存在一定关系．     

Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution

Interactions between bacterial hosts and their viruses (phages) lead to reciprocal genome evolution through a dynamic co-evolutionary process. Phage-mediated transfer of host genes--often located in genome islands--has had a major impact on microbial evolution. Furthermore, phage genomes have clearly been shaped by the acquisition of genes from their hosts. Here we investigate whole-genome expression of a host and phage, the marine cyanobacterium Prochlorococcus MED4 and the T7-like cyanophage P-SSP7, during lytic infection, to gain insight into these co-evolutionary processes. Although most of the phage genome was linearly transcribed over the course of infection, four phage-encoded bacterial metabolism genes formed part of the same expression cluster, even though they are physically separated on the genome. These genes--encoding photosystem II D1 (psbA), high-light inducible protein (hli), transaldolase (talC) and ribonucleotide reductase (nrd)--are transcribed together with phage DNA replication genes and seem to make up a functional unit involved in energy and deoxynucleotide production for phage replication in resource-poor oceans. Also unique to this system was the upregulation of numerous genes in the host during infection. These may be host stress response genes and/or genes induced by the phage. Many of these host genes are located in genome islands and have homologues in cyanophage genomes. We hypothesize that phage have evolved to use upregulated host genes, leading to their stable incorporation into phage genomes and their subsequent transfer back to hosts in genome islands. Thus activation of host genes during infection may be directing the co-evolution of gene content in both host and phage genomes.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

Analysis of components of conserved “backbone sequences”among genomes of Shigella spp. strains

Difference in the genomic compositions of prokaryotes is the basis of the diversity in their biological characters. However, besides their flora-or strain-specific genes,those floras with closer relationship in the evolution also have conserved “backbone sequences”, which reveal the marks of their origin and evolution, and these “backbone sequences”are just the basis of their elementary living abilities and common biological properties. Shigella is very closely related to E. coli in the origin and evolution, and may turn out to belong to the same genus. In this study, a microarray containing E. coli K-12 whole genome and Sf301 specific ORFs is used to investigate the genomic components of four Shigella strains. The results indicate that 16%-22% K-12 ORFs sequences are not detected in the genome of Shigella strains while the genome of Shigella contains at least 2800 conserved ORFs, which compose the common “backbone sequences”.Advanced analysis indicated that the “backbone sequences”are the essential components in maintaining the normal physiological activities of intestinal bacteria. Furthermore,only 20% Sf301-specific ORFs exist in other strains simultaneously, which demonstrate the great genome heterogeneity and the genetic diversity among the strains.     

Evolution and metabolic significance of the urea cycle in photosynthetic diatoms

Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world's most productive marine food webs. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont), yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline and the precursors for long-chain polyamines required for silica precipitation during cell wall formation. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.     

用Markov链模型研究不同物各的氨基酸丰度

针对不同的和种,利用Markov链模型分别计算了E.coli和酿酒酵母（S-cerevisiae)的20种氨基酸的丰度和变异度,并与丰度的统计值进行比较,。二取得了较好的一致。     

The origin of red algae and the evolution of chloroplasts

Chloroplast structure and genome analyses support the hypothesis that three groups of organisms originated from the primary photosynthetic endosymbiosis between a cyanobacterium and a eukaryotic host: green plants (green algae + land plants), red algae and glaucophytes (for example, Cyanophora). Although phylogenies based on several mitochondrial genes support a specific green plants/red algae relationship, the phylogenetic analysis of nucleus-encoded genes yields inconclusive, sometimes contradictory results. To address this problem, we have analysed an alternative nuclear marker, elongation factor 2, and included new red algae and protist sequences. Here we provide significant support for a sisterhood of green plants and red algae. This sisterhood is also significantly supported by a multi-gene analysis of a fusion of 13 nuclear markers (5,171 amino acids). In addition, the analysis of an alternative fusion of 6 nuclear markers (1,938 amino acids) indicates that glaucophytes may be the closest relatives to the green plants/red algae group. Thus, our study provides evidence from nuclear markers for a single primary endosymbiosis at the origin of these groups, and supports a kingdom Plantae comprising green plants, red algae and glaucophytes.     

Direct role of the himA gene product in phage lambda integration

The integration of phage lambda into the Escherichia coli chromosome is accomplished by a site-specific recombination between two unique DNA sequences (attB on the bacterial genome and attP on the phage; reviewed in refs 2, 3) and requires proteins encoded by both the bacterium and the phage. Genetic and biochemical studies have shown that bacterial strains mutant in the himA gene, located at 38 min on the E. coli map, are defective in the activity of the host-encoded component. They are, moreover, defective for the growth of bacteriophage Mu, for precise excision of transposable antibiotic resistance determinants and for the synthesis of the lambda int gene product. We now show that the himA gene product (phimA) is not solely a regulator of genes involved in integration but is one of two host polypeptides required for integrative recombination.     

Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs

Two distinct but distantly related complementary DNAs encoding proteins sharing human interleukin-1 (IL-1) activity (termed IL-1 alpha and IL-1 beta), were isolated from a macrophage cDNA library. The primary translation products of the genes are 271 and 269 amino acids long, although expression in Escherichia coli of the carboxy-terminal 159 and 153 amino acids produces IL-1 biological activity.     

栖热菌噬菌体TSP4密码子偏嗜性及其对基因异源表达的影响

 为了探索噬菌体TSP4、栖热菌模式菌株Thermus thermophilus HB27中6种蛋白编码序列和Escherichia coli基因组遗传密码子使用偏嗜性,探索TSP4基因密码子偏嗜性对其基因异源表达的影响本研究利用生物学软件Editseq和RSCU算法统计噬菌体TSP4密码子使用频率并分析其偏嗜性,与栖热菌HB27的同源基因以及常温菌E.coli基因组的密码子使用偏嗜性进行对比分析.结果显示,噬菌体TSP4与栖热菌HB27优势密码子相似度高达85%,而与E.coli的相似性仅有65%.为了验证分析结果,选取TSP4基因组中一个潜在分子伴侣基因序列在E.coli BL21和E.coli Rosetta(补充了BL21菌株中缺失的6个稀有密码子)中进行异源表达.噬菌体TSP4与其宿主菌HB27之间密码子高相似性说明在长期的进化过程中TSP4在基因水平上形成对宿主的一种适应性机制.异源表达结果显示,分子伴侣基因在E.coli BL21中未见明显表达,但在Rosetta中高效表达,说明Rosetta中补充的 6个稀有密码子明显有助于分子伴侣基因的表达,该结果也进一步证明密码子偏嗜性是否一致在很大程度上影响基因的表达.     

HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product

Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).