The German cDNA Network: cDNAs,functional genomics and proteomics

Among the greatest challenges facing biology today is the exploitation of huge amounts of genomic data, and their conversion into functional information about the proteins encoded. For example, the large-scale cDNA sequencing project of the German cDNA Consortium is providing vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterization we have tagged over 500 of these with the green fluorescent protein (GFP), and examined the subcellular localizations of these fusion proteins in living cells. These data have allowed us to classify the proteins into subcellular groups which determines the next step towards a detailed functional characterization. To make further use of these GFP-tagged constructs, a series of functional assays have been designed and implemented to assess the effect of these novel proteins on processes such as cell growth, cell death, and protein transport.Functional assays with such a large set of molecules is only possible by automation. Therefore, we have developed, and adapted, functional assays for use by robotic liquid handling stations and reading stations. A transport assay allows to identify proteins which localize to distinct organelles of the secretory pathway and have the potential to be new regulators in protein transport, a proliferation assay helps identifying proteins that stimulate or repress mitosis. Further assays to monitor the effects of the proteins in apoptosis and signal transduction pathways are in progress. Integrating the functional information that is generated in the assays with data from expression profiling and further functional genomics and proteomics approaches, will ultimately allow us to identify functional networks of proteins in a morphological context, and will greatly contribute to our understanding of cell function.     

Gateway-compatible vectors for plant functional genomics and proteomics

Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.     

Miniaturization in functional genomics and proteomics

Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future.     

SPR技术与功能基因组和蛋白质组研究

表面等离子体共振(surface plasmon resonance,SPR)依据光学—介质相互作用原理建立,属于实时和非标记的测试方法。SPR方法在研究分子间相互作用方面具有其独特的优势,其非标记和实时检测以及可以进行动力学分析的特点,给研究生物大分子的相互作用提供了诱人的解决方案。近来,随着SPR成像技术和SPR芯片制备技术的进展,将为功能基因组学和蛋白质组学研究提供重要的新的技术平台。     

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。     

Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond

Yeast-based functional genomics and proteomics technologies developed over the past decade have contributed greatly to our understanding of bacterial, yeast, fly, worm, and human gene functions. In this review, we highlight some of these yeast-based functional genomic and proteomic technologies that are advancing the utility of yeast as a model organism in molecular biology and speculate on their future uses. Such technologies include use of the yeast deletion strain collection, large-scale determination of protein localization in vivo, synthetic genetic array analysis, variations of the yeast two-hybrid system, protein microarrays, and tandem affinity purification (TAP)-tagging approaches. The integration of these advances with established technologies is invaluable in the drive toward a comprehensive understanding of protein structure and function in the cellular milieu.     

Fluorescent labels for proteomics and genomics

Fluorescent labeling reagents are an essential component of a huge industry built on sensitive fluorescence detection. This technology has grown over 30 years and is in some ways mature. Excellent labeling reagents with close to maximum theoretical brightness are available in many different colors. Large fluorescent proteins like phycobiliproteins are also widely used that are exceedingly bright. Other fluorescent proteins like the GFP family can be obtained for creating genetically encoded protein labels in living cells. A new 'solid state' quantum dot technology is being exploited for large-scale multiparameter labeling. This technology provides the 'ultimate' photostable labeling reagent. Still, there are advances to be made. Not available is the ultimate tool kit of low molecular weight, strongly light absorbing, photostable labels with narrow emission bands ranging from the UV to the IR.     

Single-molecule reader for proteomics and genomics

Recent developments in ultrasensitive fluorescence microscopy enabled the detection and detailed characterization of individual biomolecules in their native environment. New types of information can be obtained from studying individual molecules, which is not accessible from ensemble measurements. Moreover, this methodological advance matches the need of bioscience to downscale the sample amount required for screening devices. It is envisioned that concentrations as low as approximately 1000 molecules contained in a sample of 1 nl can be detected in a chip-based assay. In this review, we overview state-of-the-art single molecule microscopy with respect to its applicability to ultrasensitive screening. Quantitative estimations will be given, based on a novel apparatus designed for large area screening at single molecule sensitivity.     

Bioinformatics,functional genomics,and proteomics study of Bacillus sp

The ability of bioinformatics to characterize genomic and proteomic sequences from bacteria Bacillus sp. for prediction of genes and proteins has been evaluated. Genomics coupling with proteomics, which is relied on integration of the significant advances recently achieved in two-dimensional (2-D) electrophoretic separation of proteins and mass spectrometry (MS), are now important and high throughput techniques for qualifying and analyzing gene and protein expression, discovering new gene or protein products, and understanding of gene and protein functions including post-genomic study. In addition, the bioinformatics of Bacillus sp. is embraced into many databases that will facilitate to rapidly search the information of Bacillus sp. in both genomics and proteomics. It is also possible to highlight sites for post-translational modifications based on the specific protein sequence motifs that play important roles in the structure, activity and compartmentalization of proteins. Moreover, the secreted proteins from Bacillus sp. are interesting and widely used in many applications especially biomedical applications that are the highly advantages for their potential therapeutic values.     

Insights into environmental bioremediation by microorganisms through functional genomics and proteomics

Environmental pollutants in the soil are a major concern worldwide. Bioremediation mediated by microorganisms is a highly promising technology that is environmentally friendly, safe, and effective. However, incomplete biological information regarding the cellular responses in many microbial communities restricts progress in the site-specific mineralization process. The application of proteomics in environmental bioremediation research provides a global view of the protein compositions of the microbial cells and offers a promising approach to address the molecular mechanisms of bioremediation. With the combination of proteomics, functional genomics provide an insight into global metabolic and regulatory networks that can enhance the understanding of gene functions. This article deals with the applications of functional genomics and proteomics to dissect the cellular responses to environmental stimuli, such as stress response, induction and expressions of regulatory proteins/enzymes in response to aromatic hydrocarbons and heavy metals. An understanding of the growth conditions governing the expression of the proteome (for example, enzymes and regulatory proteins of aromatic hydrocarbon degradation, energy generation pathways, transport and stress-related proteins) in a specific environment is essential for developing rational strategies for successful bioremediation.     

In vivo-applied functional RNAs as tools in proteomics and genomics research

Non-natural, functional RNA molecules, such as short interfering (si) RNAs, aptazymes, maxizymes and intramers, allow modulation of gene function at the mRNA or protein level. This review discusses recent advances made in the expression and application of these functional RNAs and illustrates how engineered, intracellularly active RNAs can serve as promising tools for understanding the function of genes and their protein products or as potential therapeutic agents.     

Untangling multi-gene families in plants by integrating proteomics into functional genomics

The classification and study of gene families is emerging as a constructive tool for fast tracking the elucidation of gene function. A multitude of technologies can be employed to undertake this task including comparative genomics, gene expression studies, sub-cellular localisation studies and proteomic analysis. Here we focus on the growing role of proteomics in untangling gene families in model plant species. Proteomics can specifically identify the products of closely related genes, can determine their abundance, and coupled to affinity chromatography and sub-cellular fractionation studies, it can even provide location within cells and functional assessment of specific proteins. Furthermore global gene expression analysis can then be used to place a specific family member in the context of a cohort of co-expressed genes. In model plants with established reverse genetic resources, such as catalogued T-DNA insertion lines, this gene specific information can also be readily used for a wider assessment of specific protein function or its capacity for compensation through assessing whole plant phenotypes. In combination, these resources can explore partitioning of function between members and assess the level of redundancy within gene families.