RNAi分子机制及在植物功能基因组研究中的应用

RNA干涉现象自20世纪90年代被发现以来,现在已逐渐成为分子生物学和细胞生物学研究的有用工具之一,已被广泛应用到植物功能基因组研究和植物品质营养改良中。RNA干涉机制的深入研究以及该技术在植物基因功能分析中的应用,建立了新的功能基因组学研究平台。阐述了RNAi的分子作用机制、基因沉默的主要类型以及该技术在植物功能基因组研究和品质营养改良上的应用。     

Structural proteomics: a tool for genome annotation

In any newly sequenced genome, 30% to 50% of genes encode proteins with unknown molecular or cellular function. Fortunately, structural genomics is emerging as a powerful approach of functional annotation. Because of recent developments in high-throughput technologies, ongoing structural genomics projects are generating new structures at an unprecedented rate. In the past year, structural studies have identified many new structural motifs involved in enzymatic catalysis or in binding ligands or other macromolecules (DNA, RNA, protein). The efficiency by which function is deduced from structure can be further improved by the integration of structure with bioinformatics and other experimental approaches, such as screening for enzymatic activity or ligand binding.     

Residual dipolar couplings: Synergy between NMR and structural genomics

Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However, comprehensive association of gene products with functions also requires systematic determination of more complex protein structures and other biomolecules participating in cellular processes such as nucleic acids, and characterization of biomolecular interactions and dynamics relevant to function. Such NMR investigations are becoming more feasible, not only due to recent advances in NMR methodology, but also because structural genomics is providing valuable structural information and new experimental and computational tools. The measurement of residual dipolar couplings in partially oriented systems and other new NMR methods will play an important role in this synergistic relationship between NMR and structural genomics. Both an expansion in the domain of NMR application, and important contributions to future structural genomics efforts can be anticipated.     

Mapping the gene expression universe

Methods to globally survey gene expression provide valuable insights into gene function during development. In particular, comprehensive in situ hybridization studies have demonstrated that gene expression patterns are extraordinarily diverse and new imaging techniques have been introduced to capture these patterns with higher resolution at the tissue, cellular, and subcellular levels. The analysis of massive image databases can be greatly facilitated by computer vision techniques once annotated image sets reach the crucial mass sufficient to train the computer in pattern recognition. Ultimately, genome-wide atlases of gene expression during development will record gene activity in living animals with at least cellular resolution and in the context of morphogenetic events. These emerging datasets will lead to great advances in the field of comparative genomics and revolutionize our ability to decipher and model developmental processes for a variety of organisms.     

基于mRNA水平的差异表达基因筛选技术

随着分子生物学技术的深入发展,基因组研究重点已经由基因组测序转向基因功能鉴定,即由结构基因组学向功能基因组学转变。研究获得不同处理下基因的差异表达谱是功能基因组学的重要一环,目前已经有多种检测基因差异表达的技术可供选用。在减法杂交技术和PCR基础上发展起来的DDRT—PCR、cDNA—RDA、cDNA—AFLP和SSH技术因其实用性而得到广泛的应用,并取得了令人满意的结果。     

Functional genomics as an emerging strategy for the investigation of central mechanisms in experimental hypertension

Centrally mediated increases in sympathetic nerve activity and attenuated arterial baroreflexes contribute to the pathogenesis of hypertension. Despite the characterization of cellular and physiological mechanisms that regulate blood pressure and alterations that contribute to hypertension, the genetic and molecular basis of this pathophysiology remains poorly understood. Strategies to identify genes that contribute to central pathophysiologic mechanisms in hypertension include integrative biochemistry and physiology as well as functional genomics. This article summarizes recent progress in applying functional genomics to elucidate the genetic basis of altered central blood pressure regulatory mechanisms in hypertension. We describe approaches others and we have undertaken to investigate gene expression profiles in hypertensive models in order to identify genes that contribute to the pathogenesis of hypertension. Finally, we provide the readers a roadmap for negotiating the route from experimental findings of gene expression profiling to translating their therapeutic potential. The combination of gene expression profiling and the phenotypic characterization of in vitro and in vivo loss or gain of function experiments for candidate genes have the potential to identify genes involved in the pathogenesis of hypertension and may present novel targets for therapy.     

从基因组学到功能蛋白质组学的研究

人类基因组草图绘制的完成,标志着生命科学已实质性地跨入了后基因组时代,研究重心已从揭示生命的所有遗传信息转移到在分子整体水平对功能的研究[1]。这种转向表明目前已进入功能基因组学(functional genom ics)以及随之产生的功能蛋白质组学(functional proteomics)等新学科领域的研究。     

An application of gene comparative image for predicting the effect on replication ratio by HBV virus gene missense mutation

Hepatitis B viruses (HBVs) show instantaneous and high-ratio mutations when they are replicated, some sorts of which significantly affect the efficiency of virus replication through enhancing or depressing the viral replication, while others have no influence at all. The mechanism of gene expression is closely correlated with its gene sequence. With the rapid increase in the number of newly found sequences entering into data banks, it is highly desirable to develop an automated method for simulating the gene regulating function. The establishment of such a predictor will no doubt expedite the process of prioritizing genes and proteins identified by genomics efforts as potential molecular targets for drug design. Based on the power of cellular automata (CA) in treating complex systems with simple rules, a novel method to present HBV gene image has been introduced. The results show that the images thus obtained can very efficiently simulate the effects of the gene missense mutation on the virus replication. It is anticipated that CA may also serve as a useful vehicle for many other studies on complicated biological systems.     

Shared mechanisms among neurodegenerative diseases: from genetic factors to gene networks

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis are pressing health concerns in modern societies for which effective therapies are still lacking. Recent high-throughput genomic technologies have enabled genome-scale, multidimensional investigations to facilitate a better understanding of the underlying mechanisms and the identification of novel targets. Here we review the molecular insights gained through such studies, and compare the similarities and differences between neurodegenerative diseases revealed by systems genomics and gene network modelling approaches. We focus specifically on the shared mechanisms at multiple molecular scales ranging from genetic factors to gene expression to network-level features of gene regulation, and whenever possible also point out mechanisms that distinguish one disease from another. Our review sets the stage for similar genomewide inspection in the future on shared/distinct features of neurodegenerative diseases at the levels of cellular, proteomic or epigenomic signatures, and how these features may interact to determine the progression and treatment response of different diseases afflicting the same individual.     

The intriguing complexities of mammalian gene regulation: How to link enhancers to regulated genes. Are we there yet?

The information encoded in genomes supports the differentiation and function of the more than 200 unique cell types, which exist in various mammalian species. The major mechanism driving cellular differentiation and specification is differential gene expression regulation. Cis-acting enhancers and silencers appear to have key roles in regulating the expression of mammalian genes. However, these cis-acting elements are often located very far away from the regulated gene. Therefore, it is hard to find all of them and link them to the regulated gene. An intriguing and unresolved issue of the field is to identify all of the enhancers of a particular gene and link these short regulatory sequences to the genes they regulate and thus, reliably identify gene regulatory enhancer networks. Recent advances in molecular biological methods coupled with Next-Generation Sequencing (NGS) technologies have opened up new possibilities in this area of genomics. In this review we summarize the technological advances, bioinformatics challenges and the potential molecular mechanisms allowing the construction of enhancer networks operating in specific cell types and/or activated by various signals.     

Nuclear microenvironments and cancer

Nucleic acids and regulatory proteins are architecturally organized in nuclear microenvironments. The compartmentalization of regulatory machinery for gene expression, replication and repair, is obligatory for fidelity of biological control. Perturbations in the organization, assembly and integration of regulatory machinery have been functionally linked to the onset and progression of tumorigenesis. The combined application of cellular, molecular, biochemical and in vivo genetic approaches, together with structural biology, genomics, proteomics and bioinformatics, will likely lead to new approaches in cancer diagnostics and therapy.     

Molecular approaches in pig breeding to improve meat quality.

This article reviews the advances in molecular genetics that have led to the identification of genes and markers associated with meat quality in pig. The development of a considerable number of annotated livestock genome sequences represents an incredibly rich source of information that can be used to identify candidate genes responsible for complex traits and quantitative trait loci effects. In pig, the huge amount of information emerging from the study of the genome has helped in the acquisition of new knowledge concerning biological systems and it is opening new opportunities for the genetic selection of this specie. Among the new fields of genomics recently developed, functional genomics and proteomics that allow considering many genes and proteins at the same time are very useful tools for a better understanding of the function and regulation of genes, and how these participate in complex networks controlling the phenotypic characteristics of a trait. In particular, global gene expression profiling at the mRNA and protein level can provide a better understanding of gene regulation that underlies biological functions and physiology related to the delivery of a better pig meat quality. Moreover, the possibility to realize an integrated approach of genomics and proteomics with bioinformatics tools is essential to obtain a complete exploitation of the available molecular genetics information. The development of this knowledge will benefit scientists, industry and breeders considering that the efficiency and accuracy of the traditional pig selection schemes will be improved by the implementation of molecular data into breeding programs.     

Focusing in on structural genomics: the University of Queensland structural biology pipeline

The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis.     

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.