Coronalon: a powerful tool in plant stress physiology

Coronalon, a synthetic 6-ethyl indanoyl isoleucine conjugate, has been designed as a highly active mimic of octadecanoid phytohormones that are involved in insect and disease resistance. The spectrum of biological activities that is affected by coronalon was investigated in nine different plant systems specifically responding to jasmonates and/or 12-oxo-phytodienoic acid. In all bioassays analyzed, coronalon demonstrated a general strong activity at low micromolar concentrations. The results obtained showed the induction of (i) defense-related secondary metabolite accumulation in both cell cultures and plant tissues, (ii) specific abiotic and biotic stress-related gene expression, and (iii) root growth retardation. The general activity of coronalon in the induction of plant stress responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology. Moreover, coronalon might become employed in agriculture to elicit plant resistance against various aggressors.     

Proteomics: a powerful tool in the post-genomic era

Genomics is having a profound impact on biological research, including photosynthesis investigations. Genomes of many photosynthetic organisms have been sequenced. The information about ALL genes that govern and execute photoautotrophic metabolism provides many opportunities to understand genome function and details of known and uncharted pathways. Proteomics, analysis of the protein complement of the genome, is a powerful tool in understanding which proteins are present in a particular tissue under given conditions. Proteomics also allows us to estimate relative levels of proteins and to determine post-translational modifications of the gene products. In this minireview, we discuss the technology and its applications in plant sciences.     

Trans-kingdom rescue of Gln-tRNAGln synthesis in yeast cytoplasm and mitochondria

Aminoacylation of transfer RNA^Gln (tRNA^Gln) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNA^Gln is generated by direct glutaminylation of tRNA^Gln by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNA^Gln is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNA^Gln amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNA^Gln synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNA^Gln isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNA^Gln isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNA^Gln synthesis can be conferred by a single enzyme.     

Aptamer-guided gene targeting in yeast and human cells

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.     

嗜神经性疱疹病毒：神经科学研究的有力工具

病毒是研究现代神经科学的有力工具。对神经元的连接方式及功能研究大都是利用重组病毒完成的,嗜神经性疱疹病毒便是其中一种重要工具。随着基因工程学以及分子生物学技术的不断发展,多种嗜神经性疱疹病毒被改造为不同的重组病毒工具应用于神经科学研究。本文基于几种常见且应用较为广泛的嗜神经性疱疹病毒作为神经传导示踪工具、治疗神经性疾病的病毒载体和溶瘤病毒治疗神经肿瘤等应用进行阐述及讨论,为进一步开发嗜神经性疱疹病毒的功能提供参考。     

TREC-IN: gene knock-in genetic tool for genomes cloned in yeast

Background

With the development of several new technologies using synthetic biology, it is possible to engineer genetically intractable organisms including Mycoplasma mycoides subspecies capri (Mmc), by cloning the intact bacterial genome in yeast, using the host yeast’s genetic tools to modify the cloned genome, and subsequently transplanting the modified genome into a recipient cell to obtain mutant cells encoded by the modified genome. The recently described tandem repeat coupled with endonuclease cleavage (TREC) method has been successfully used to generate seamless deletions and point mutations in the mycoplasma genome using the yeast DNA repair machinery. But, attempts to knock-in genes in some cases have encountered a high background of transformation due to maintenance of unwanted circularization of the transforming DNA, which contains possible autonomously replicating sequence (ARS) activity. To overcome this issue, we incorporated a split marker system into the TREC method, enabling seamless gene knock-in with high efficiency. The modified method is called TREC-assisted gene knock-in (TREC-IN). Since a gene to be knocked-in is delivered by a truncated non-functional marker, the background caused by an incomplete integration is essentially eliminated.

Results

In this paper, we demonstrate applications of the TREC-IN method in gene complementation and genome minimization studies in Mmc. In the first example, the Mmc dnaA gene was seamlessly replaced by an orthologous gene, which shares a high degree of identity at the nucleotide level with the original Mmc gene, with high efficiency and low background. In the minimization example, we replaced an essential gene back into the genome that was present in the middle of a cluster of non-essential genes, while deleting the non-essential gene cluster, again with low backgrounds of transformation and high efficiency.

Conclusion

Although we have demonstrated the feasibility of TREC-IN in gene complementation and genome minimization studies in Mmc, the applicability of TREC-IN ranges widely. This method proves to be a valuable genetic tool that can be extended for genomic engineering in other genetically intractable organisms, where it may be implemented in elucidating specific metabolic pathways and in rationale vaccine design.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1180) contains supplementary material, which is available to authorized users.     

多肽配体技术在植物功能基因组学中的应用前景

人工智能配体或适配体(Aptamer)技术是近年来兴起的一项特异性极强的基因干扰技术。通过人工合成特异的智能配体结合靶基因的蛋白产物, 达到特异干扰靶基因的生物学功能, 这是人工智能配体技术的基本设想。文章综述了多肽配体(Peptide aptamer)技术在基因功能验证中的主要进展, 着重阐明它在植物基因功能验证和作物抗病毒育种中的应用前景, 并提出克服该技术主要风险对策。     

Phagosome proteomics: a powerful tool to assess bacteria-mediated immunomodulation

Bacteria-mediated immunomodulation has important implications in microbial infection and bacterial vaccines. Intraphagosomal bacteria negotiate survival niches with the intracellular environment by modulating the phagosome composition during invasion. The final phagosome composition determines the fate of the intraphagosomal bacterium or the efficacy of a bacterial vaccine. Therefore, the phagosome proteome is a valuable readout to assess the ability of a natural or genetically engineered bacterial strain to modulate the host immune response. Compared to a preparation of latex-bead-containing phagosomes, the preparation of bacterial phagosomes requires additional measures to ensure comparable purity due to their closer density to some other organelles. This bottleneck can be overcome with delicate preparation protocols, proper experimental designs to facilitate bioinformatics based discrimination against contaminating proteins, and the incorporation of stable-isotope labeled internal standards to correct for contaminating fractions of phagosomal proteins. The rapid progress in the proteomics and bioinformatics fields provides an array of techniques that promise to bring about an unprecedented coverage of both known and as yet undiscovered immunomodulation pathways within bacterial phagosome proteomes. A precise portrait of the bacteria-mediated immunomodulation pathways in phagosomes will likely aid in the intelligent design of bioengineered bacterial vaccine strains for important future biomedical applications.     

Intellectual Property: a powerful tool to develop biotech research

Today biotechnology is perhaps the most important technology field because of the strong health and food implications. However, due to the nature of said technology, there is the need of a huge amount of investments to sustain the experimentation costs. Consequently, investors aim to safeguard as much as possible their investments. Intellectual Property, and in particular patents, has been demonstrated to actually constitute a powerful tool to help them. Moreover, patents represent an extremely important means to disclose biotechnology inventions. Patentable biotechnology inventions involve products as nucleotide and amino acid sequences, microorganisms, processes or methods for modifying said products, uses for the manufacture of medicaments, etc. There are several ways to protect inventions, but all follow the three main patentability requirements: novelty, inventive step and industrial application.     

Second harmonic generation microscopy: a powerful tool for bio-imaging

Second harmonic generation (SHG) microscopy is an important optical imaging technique in a variety of applications. This article describes the history and physical principles of SHG microscopy and its more advanced variants, as well as their strengths and weaknesses in biomedical applications. It also provides an overview of SHG and advanced SHG imaging in neuroscience and microtubule imaging and how these methods can aid in understanding microtubule formation, structuration, and involvement in neuronal function. Finally, we offer a perspective on the future of these methods and how technological advancements can help make SHG microscopy a more widely adopted imaging technique.     

Recombineering: a powerful new tool for mouse functional genomics

Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.     

Cryo-EM as a powerful tool for drug discovery

The recent revolution in cryo-EM has produced an explosion of structures at near-atomic or better resolution. This has allowed cryo-EM structures to provide visualization of bound small-molecule ligands in the macromolecules, and these new structures have provided unprecedented insights into the molecular mechanisms of complex biochemical processes. They have also had a profound impact on drug discovery, defining the binding modes and mechanisms of action of well-known drugs as well as driving the design and development of new compounds. This review will summarize and highlight some of these structures. Most excitingly, the latest cryo-EM technology has produced structures at 1.2 Å resolution, further solidifying cryo-EM as a powerful tool for drug discovery. Therefore, cryo-EM will play an ever-increasing role in drug discovery in the coming years.     

Genetic transformation: a powerful tool for dissection of adaptive traits in trees

Plant transformation and regeneration systems have become indispensable parts of gene discovery and functional characterization over the last two decades. Adoption of transformation methods in studies of plant adaptation to natural environments has been slow. This is a result of poor genomic knowledge and inefficient transformation systems for species dominating terrestrial ecosystems, and logistical difficulties in conducting field tests of genetically engineered organisms. In trees, where long generation cycles, high background polymorphism, large sizes and outcrossing systems of mating make production of near-isogenic lines and large experiments difficult, transformation is an attractive alternative for establishing direct linkages between genes and adaptively significant phenotypes. Here, we outline the capabilities, challenges, and prospects for transformation to become a significant tool for studying the ecophysiological adaptation of trees to the environment. Focusing on poplars (genus Populus) as model system, we describe how transformation-based approaches can provide insights into the genes that control adaptive traits. The availability of the poplar genome sequence, along with its large expressed sequences tag (EST) databanks, facile transformation and rapid growth, enable reverse genetic approaches to be used to test virtually any hypothesis of gene function.