DNA cloning in Streptomyces: resistance genes from antibiotic-producing species

The biochemical and morphological differentiation of actinomycetes makes them academically and economically interesting. Their secondary metabolites provide the majority of medically and agriculturally important antibiotics (streptomycete genes may also be the primary source of clinically important antibiotic resistance); their complex morphological developmental cycle involves a series of changes from vegetative mycelial growth to spore formation. Recombinant DNA technology would add a powerful new dimension to the analysis of these various aspects of actinomycete biology and would also facilitate the development of industrial strains with increased antibiotic yield, or capable of making new antibiotics. For most of these purposes, cloning of genes within and between actinomycetes is required to study the expression of particular genes in genetic backgrounds defined by mutations of the characters under study. To achieve this, we have now developed a method for molecular cloning involving the transfer of genes between unrelated streptomycetes.     

Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host

The application of molecular cloning to antibiotic-producing microorganisms should lead to enhanced antibiotic productivity and to the biosynthesis of novel antibiotics by in vitro interspecific recombination. To allow such approaches, the genes for antibiotic synthesis must be isolated, analysed and perhaps modified. Certain Streptomyces species produce nearly two-thirds of the known natural antibiotics; the recent development of cloning systems in the genus makes it possible to isolate and analyse Streptomyces genes. However, antibiotics are metabolites which require sets of several enzymes for their synthesis and attempts to isolate the corresponding genes have so far yielded clones carrying either individual genes of the set, or only incomplete gene sets. We describe here the isolation of a large continuous segment of Streptomyces coelicolor DNA which apparently carries the complete genetic information required for synthesis of an antibiotic, actinorhodin , from simple primary metabolites. Not only can the cloned DNA 'complement' all available classes of actinorhodin non-producing mutants of S. coelicolor but, on introduction into a different host, Streptomyces parvulus , it directs the synthesis of the antibiotic. The tendency for the genes for antibiotic synthesis to be clustered together on the chromosomes of Streptomyces species and the availability of plasmid vectors which can carry stable inserts of DNA larger than 30 kilobase pairs (kb) and which can be introduced efficiently into Streptomyces protoplasts, suggest that the experiments described have general significance for this area of biotechnology.     

Expression pattern of zebrafish pax genes suggests a role in early brain regionalization.

In vertebrates the developing hindbrain is organized in segmental units. These units provide the primary grid for differentiation and axonal outgrowth. In the more anterior regions of the brain, however, the subdivisions remain more controversial. Cellular and molecular studies of the embryonic brain in lower vertebrates such as the zebrafish, Brachydanio rerio, may reveal remnants of such subdivisions. We have isolated complementary DNA clones for two zebrafish pax genes related to Drosophila and mouse paired-box-containing segmentation genes. The expression of these two genes is confined to specific regions in the embryonic forebrain and midbrain. Strikingly, the borders of expression of the two pax genes coincide with morphological landmarks corresponding to the primary axon tracts that are generated in the embryonic brain a few hours after the initiation of expression of these genes.     

SOS response promotes horizontal dissemination of antibiotic resistance genes

Mobile genetic elements have a crucial role in spreading antibiotic resistance genes among bacterial populations. Environmental and genetic factors that regulate conjugative transfer of antibiotic resistance genes in bacterial populations are largely unknown. Integrating conjugative elements (ICEs) are a diverse group of mobile elements that are transferred by means of cell-cell contact and integrate into the chromosome of the new host. SXT is a approximately 100-kilobase ICE derived from Vibrio cholerae that encodes genes that confer resistance to chloramphenicol, sulphamethoxazole, trimethoprim and streptomycin. SXT-related elements were not detected in V. cholerae before 1993 but are now present in almost all clinical V. cholerae isolates from Asia. ICEs related to SXT are also present in several other bacterial species and encode a variety of antibiotic and heavy metal resistance genes. Here we show that SetR, an SXT encoded repressor, represses the expression of activators of SXT transfer. The 'SOS response' to DNA damage alleviates this repression, increasing the expression of genes necessary for SXT transfer and hence the frequency of transfer. SOS is induced by a variety of environmental factors and antibiotics, for example ciprofloxacin, and we show that ciprofloxacin induces SXT transfer as well. Thus, we present a mechanism by which therapeutic agents can promote the spread of antibiotic resistance genes.     

笠贝幼虫变态相关基因筛选及分子网络构建

利用NCBI在线数字化差异显示工具（Digital Differential Display）对笠贝(Lottia gigantea)EST数据库进行筛选,获得笠贝幼虫时期显著差异表达基因203个,其中124个基因有Blast2Go注释结果,这124个基因可以归类成细胞组分、分子功能和生物学过程三大类;参与MAPK、Ras、PI3K-Akt、Rap1、cAMP等信号通路;涉及能量代谢、免疫应激、细胞分化、神经发育、细胞骨架、信号传递等生物学过程.将获得的转录子运用Cytoscape软件构建分子相互作用网络,最终获得7个核心转录子.涉及到神经发育、次生壳形成等相关基因调控关系.     

Gene expression in Pseudomonas aeruginosa biofilms.

Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms. Some of the regulated genes are known to affect antibiotic sensitivity of free-living P. aeruginosa. Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes. We propose that this response is critical for the development of biofilm resistance to tobramycin. Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences. Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics.     

硬骨鱼类性别决定与分化相关基因研究进展

鱼类是脊椎动物中最低等但分布最广、种类最多的一类生物.与高等脊椎动物不同,鱼类的性别决定除了受遗传因素的影响,外界环境(激素水平、温度、盐度、氧气等)和自身内分泌调节也发挥了重要作用,因而其性别决定与分化机制极其复杂.尽管如此,遗传因素仍然是鱼类性别决定与分化的关键因素.本文通过对影响硬骨鱼类性别决定及分化的遗传因素(包括sox,dmrt1,amh,gsdf,cyp19a1a,foxl2等性别决定及分化相关基因和Rspo1/Wnt/β-catenin信号通路)的研究动态与进展进行综述,为更深入的探索鱼类性别决定与分化机制提供参考.     

南京地区簸箕柳雌、雄花序建成动态观察

【目的】 柳树雌株性成熟后会产生严重的飞絮污染,阐明其性别分化的分子机制是控制飞絮污染的关键。研究柳树雌、雄花芽分化以及花序建成的关键时期,为性别分化分子调控机理研究奠定基础。【方法】 以木本植物簸箕柳(Salix suchowensis)雌株和雄株花芽及花序为材料,从当年6月至翌年2月分别在17个时间点采集样品,利用石蜡切片技术对其雌、雄花芽分化及花序建成过程进行观察,分析不同发育阶段的相关特征。【结果】 簸箕柳花序建成初期为6月中旬至7月初,小花原基分化期在7月中旬至8月下旬,雌、雄蕊分化期在9月上旬至11月初,11月中旬至翌年2月初雌雄花序进入休眠期,翌年2月中旬至3月为大小孢子发生及雌雄配子体发育期,随后雌雄花序开放。研究表明,花序外观形态变化与内部解剖结构变化之间具有一定的相关性。【结论】 簸箕柳雌、雄花均为单性花。花序生长趋势总体呈“S”形上升趋势,在整个发育过程中,雄花序比雌花序外形稍大。簸箕柳花序外观形态可以用于判断花发育的关键时期。研究结果为全面了解簸箕柳花芽分化以及花序建成的完整过程提供了详细信息,为进一步阐明不同关键发育阶段的分子调控机制奠定了基础。     

Novel gene expressed during early embryogenesis of zebrafish identified by mRNA differential display

Following the revelation of the molecular mechanism of morphogenesis in fruitfly, research on the molecular mechanism of morphogenesis in vertebrate becomes the focus of developmental biology. The isolation of genes controlling the embryogenesis of zebrafish, a vertebrate model animal, is considered as an initial step toward investigating this issue. There are several approaches that can be used to isolate developmental genes, each of which is suited to a particular situation. In this note, mRNA differential display was utilized to demonstrate the mRNA differences among zebrafish embryos at 4, 5 and 6 h post fertilization (28.5℃, corresponding to oblong, dome and shield stages, respectively, called blastula, gastrula and neurula in this note). One cDNA tag that was specific to embryos at neurula stage was cloned and sequenced. After sequence comparison in Genbank, we found that this cDNA tag represents a novel gene. The expression of this gene in the developing zebrafish embryos was examined by whole mount in situ hybridization. The hybridization results confirmed that this gene was specifically expressed in zebrafish neurula embryos.     

水环境中抗生素和抗性基因污染特征及控制措施

 抗生素作为一类抗菌类药物被广泛用于医疗、农业和畜牧业等领域，因其使用量大并能诱导产生耐药菌株，对人类健康和生态环境造成巨大威胁。在梳理近年来地表水环境中抗生素相关研究的基础上，阐述了水环境中抗生素和抗性基因的污染来源和污染特征，分析了环境浓度水平下抗生素污染对人群和生态环境的影响，讨论了水环境中抗生素污染的控制措施及目前研究的主要问题，并对今后的研究进行了展望。     

Emergence of vancomycin tolerance in Streptococcus pneumoniae.

Streptococcus pneumoniae, the pneumococcus, is the most common cause of sepsis and meningitis. Multiple-antibiotic-resistant strains are widespread, and vancomycin is the antibiotic of last resort. Emergence of vancomycin resistance in this community-acquired bacterium would be catastrophic. Antibiotic tolerance, the ability of bacteria to survive but not grow in the presence of antibiotics, is a precursor phenotype to resistance. Here we show that loss of function of the VncS histidine kinase of a two-component sensor-regulator system in S. pneumoniae produced tolerance to vancomycin and other classes of antibiotic. Bacterial two-component systems monitor environmental parameters through a sensor histidine-kinase/phosphatase, which phosphorylates/dephosphorylates a response regulator that in turn mediates changes in gene expression. These results indicate that signal transduction is critical for the bactericidal activity of antibiotics. Experimental meningitis caused by the vncS mutant failed to respond to vancomycin. Clinical isolates tolerant to vancomycin were identified and DNA sequencing revealed nucleotide alterations in vncS. We conclude that broad antibiotic tolerance of S. pneumoniae has emerged in the community by a molecular mechanism that eliminates sensitivity to the current cornerstone of therapy, vancomycin.     

Homology between Streptomyces genes coding for synthesis of different polyketides used to clone antibiotic biosynthetic genes

Many important antibiotics such as tetracyclines, erythromycin, adriamycin, monensin, rifamycin and avermectins are polyketides. In their biosynthesis, multifunctional synthases catalyse iterated condensation of thio-esters derived from acetate, propionate or butyrate to yield aliphatic chains of varying length and carrying different alkyl substituents. Subsequent modifications, including aromatic or macrolide ring closure or specific methylations or glycosylations, generate further chemical diversity. It has been suggested that, if different polyketide synthases had a common evolutionary origin, cloned DNA coding for one synthase might be used as a hybridization probe for the isolation of others. We show here that this is indeed possible. Study of a range of such synthase genes and their products should help to elucidate what determines the choice and order of condensation of different residues in polyketide assembly, and might yield, by in vitro recombination or mutagenesis, synthase genes capable of producing novel antibiotics. Moreover, because genes for entire antibiotic pathways are usually clustered in Streptomyces, cloned polyketide synthase genes are valuable in giving access to groups of linked biosynthetic genes.     

Gene expression divergence recapitulates the developmental hourglass model

The observation that animal morphology tends to be conserved during the embryonic phylotypic period (a period of maximal similarity between the species within each animal phylum) led to the proposition that embryogenesis diverges more extensively early and late than in the middle, known as the hourglass model. This pattern of conservation is thought to reflect a major constraint on the evolution of animal body plans. Despite a wealth of morphological data confirming that there is often remarkable divergence in the early and late embryos of species from the same phylum, it is not yet known to what extent gene expression evolution, which has a central role in the elaboration of different animal forms, underpins the morphological hourglass pattern. Here we address this question using species-specific microarrays designed from six sequenced Drosophila species separated by up to 40 million years. We quantify divergence at different times during embryogenesis, and show that expression is maximally conserved during the arthropod phylotypic period. By fitting different evolutionary models to each gene, we show that at each time point more than 80% of genes fit best to models incorporating stabilizing selection, and that for genes whose evolutionarily optimal expression level is the same across all species, selective constraint is maximized during the phylotypic period. The genes that conform most to the hourglass pattern are involved in key developmental processes. These results indicate that natural selection acts to conserve patterns of gene expression during mid-embryogenesis, and provide a genome-wide insight into the molecular basis of the hourglass pattern of developmental evolution.     

维管植物导管元素分化与细胞程序性死亡

通过遗传控制的细胞死亡是植物正常生长和发育的重要过程，是近年来国内外的研究热点之一。维管植物的导管和筛管由死亡的管导分子组成，本文着重讨论了维管植物的导分子形成过程中的细胞程序性死亡（ｐｒｏｇｒａｍｍｅｄ ｃｅｌｌｄｅａｔｈＰＣＤ）的形态和化特性以及参与找在因，并介绍了植物生长发育过程中ＰＣＤ存在的时空广泛性。虽然植物发育生殖过程中的ＰＣＤ与动物细胞通过遗控制的细胞死亡是植物正常生长和发育的重要过程，是近年来国内外的研究热点之一，维管植物的导管和筛管由天文馆的导管分子组成。本文着重讨论了维管植物的导管分子形成过程中的细程序性死亡（programmed cell death PCD)的形态和生化特征以及参与的基因，并介绍了植物生长发育过程中ＰＣＤ存在的时空广泛性。虽然植物发育和生殖过程中的ＰＣＤ与动物细胞凋亡存在许多相似的形太必生化特征，但细胞通过自溶或自自我吞噬来死亡，而并不形成动物细胞凋亡中典型的凋亡小体。说明这些ＰＣＤ类型本质上凋亡型ＰＣＤ。导管分子的死亡是一种典型的植物ＰＣＤ。近年来体外系统研究表明，导管元素分析中的细胞死亡程序与动物细胞凋亡不同。     

Homoeobox gene expression in mouse embryos varies with position by the primitive streak stage

Pattern formation in animal development requires that genes be expressed differentially according to position in the sheets of cells that make up the early embryo. The homoeobox-containing genes of Drosophila are control genes active both in the establishment of a segmentation pattern and in the specification of segment identity. In situ hybridization experiments confirm that these genes are expressed in a segmentally-restricted manner and that their expression presages morphological differentiation of segmental structures. Homoeobox genes have recently been isolated from the mouse and have been shown to be expressed during mouse development. Using in situ hybridization, we show here that expression of the mouse homoeobox gene Mo-10 (ref. 7) is spatially restricted in the developing embryo and that localization of expression is already evident within the germ layers before their morphological differentiation. These findings support the suggestion that the homoeobox genes of mammals, like those of Drosophila, may be important in pattern formation.     

Identification of auxin responsive genes in Arabidopsis by cDNA array

The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.     

488例围手术期预防性应用抗菌素的调查分析

目的分析手术患者围手术期应用抗菌素的现状及合理性。方法按照自行设计的调查表对该院外科2008年1月至12月488例手术患者的出院病历作回顾性分析。结果488例手术患者围手术期抗菌药物使用率100％，前六类抗菌药物为头孢类、喹诺酮类、β-内酰胺类、哨基咪唑类、母内酰胺类、氨基糖苷类。结论加强抗菌药物的合理使用，规范围手术期抗菌药物的使用原则。