Functional Genomics Projects Research Articles

TOWARDS IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF DISEASE RESISTANT GENES FROM THE NATIVE NORTH AMERICAN GRAPE SPECIES VITIS SHUTTLEWORTHII

Diseases such as Pierce's disease, anthracnose, and downy mildew are the limited factors of growing the European grapes (Vitis vinifera) in the southeast United States. On the other hand, many native North American grape species are tolerant/ resistant to these diseases. However, little has been know about the mechanism and genetic basis of the host resistance, which has been greatly hampering our ability to genetically manipulate the host resistant genes for cultivar improvement. To improve disease resistance of the European grape varieties by using the host resistance from the native American species, a functional genomic project with emphasis on identifying, isolating, and characterizing disease resistant genes was initiated at the Center for Viticulture and Small Fruit Research, Florida A&M University, in collaboration with the USDA-ARS Horticultural Research Laboratory at Fort Pierce, Florida. Vitis shuttleworthii, one of the most disease/pest resistant grape species originated from the southeastern United States, was chosen for the EST sequencing. We have sequenced 22,500 ESTs, from which about 9,000 unigenes were generated. Functional annotation revealed that more than 7% of them were correlated to disease resistance. Comparative genomic analysis indicated that about 20% of the genes found in the native grapes did not have homology in the European grapes (screening approximately 170,000 ESTs and 23,000 unigenes). The potential usefulness of these unique genes as genetic markers linked to disease resistance, and their role in disease resistance pathways are being further examined using microarray analysis. Among the homologous gene sequences between V. shualwworthii and V. vinifera, a high frequency of SNPs was detected between the two genomes. Functional analysis revealed that high percentage of these SNP variations may be correlated to disease resistance.

Assessing the efficiency of RNA interference for maize functional genomics.

A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.

JUICE: a data management system that facilitates the analysis of large volumes of information in an EST project workflow

BackgroundExpressed sequence tag (EST) analyses provide a rapid and economical means to identify candidate genes that may be involved in a particular biological process. These ESTs are useful in many Functional Genomics studies. However, the large quantity and complexity of the data generated during an EST sequencing project can make the analysis of this information a daunting task.ResultsIn an attempt to make this task friendlier, we have developed JUICE, an open source data management system (Apache + PHP + MySQL on Linux), which enables the user to easily upload, organize, visualize and search the different types of data generated in an EST project pipeline. In contrast to other systems, the JUICE data management system allows a branched pipeline to be established, modified and expanded, during the course of an EST project.The web interfaces and tools in JUICE enable the users to visualize the information in a graphical, user-friendly manner. The user may browse or search for sequences and/or sequence information within all the branches of the pipeline. The user can search using terms associated with the sequence name, annotation or other characteristics stored in JUICE and associated with sequences or sequence groups. Groups of sequences can be created by the user, stored in a clipboard and/or downloaded for further analyses.Different user profiles restrict the access of each user depending upon their role in the project. The user may have access exclusively to visualize sequence information, access to annotate sequences and sequence information, or administrative access.ConclusionJUICE is an open source data management system that has been developed to aid users in organizing and analyzing the large amount of data generated in an EST Project workflow. JUICE has been used in one of the first functional genomics projects in Chile, entitled "Functional Genomics in nectarines: Platform to potentiate the competitiveness of Chile in fruit exportation". However, due to its ability to organize and visualize data from external pipelines, JUICE is a flexible data management system that should be useful for other EST/Genome projects. The JUICE data management system is released under the Open Source GNU Lesser General Public License (LGPL). JUICE may be downloaded from or .

Thematic review series: Systems Biology Approaches to Metabolic and Cardiovascular Disorders Approaches to lipid metabolism gene identification and characterization in the postgenomic era

The availability of genomic resources has already had a tremendous impact on biomedical research. In this review, we describe how whole genome sequence and high-throughput functional genomics projects have facilitated the identification and characterization of important genes in lipid metabolism and disease. We review key approaches and lipid genes identified in the first years of this century and discuss how genomic resources are likely to streamline gene identification and functional characterization in the future.

SgTarget: a target selection resource for structural genomics

sgTarget () is a web-based resource to aid the selection and prioritization of candidate proteins for structure determination. The system annotates user submitted gene or protein sequences, identifying sequence families with no homologues of known structure, and characterizing each protein according to a range of physicochemical properties that may affect its expression, solubility and likelihood to crystallize. Summaries of these analyses are available for individual sequences, as well as whole datasets. This type of analysis enables structural biologists to iteratively select targets from their genomic sequences of interest and according to their research needs. All sequence datasets submitted to sgTarget are available for users to select and rank using their choice of criteria. sgTarget was developed to support individual laboratories collaborating in structural and functional genomics projects and should be valuable to structural biologists wishing to employ the wealth of available genome sequences in their structural quests.

The Work of the Human Proteome Organisation's Proteomics Standards Initiative (HUPO PSI)

This article describes the origins, working practices and various development projects of the HUman Proteome Organisation's Proteomics Standards Initiative (HUPO PSI), specifically, our work on reporting requirements, data exchange formats and controlled vocabulary terms. We also offer our view of the two functional genomics projects in which the PSI plays a role (FuGE and FuGO), discussing their impact on our process and laying out the benefits we see as accruing, both to the PSI and to biomedical science as a whole as a result of their widespread acceptance.

EHPnet: Wellcome Trust Sanger Institute

Vol. 114, No. 3 EnvironewsOpen AccessEHPnet: Wellcome Trust Sanger Institute Erin E. Dooley Erin E. Dooley Search for more papers by this author Published:1 March 2006https://doi.org/10.1289/ehp.114-a155AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit The Cambridge, United Kingdom–based Wellcome Trust Sanger Institute (WTSI) was founded in 1993 to provide a center for research on the genomes of humans and other organisms. The institute has been a leading contributor to the Human Genome Project and now focuses much of its work on studying human DNA sequence variations and how these correlate with genetic diseases. The institute has developed a website located at http://www.sanger.ac.uk/ to serve as a central repository for information on its varied areas of study.The densely packed website offers a wealth of scientific resources related to its research on human genetics, model organisms, pathogens, bioinformatics, sequencing, and proteomics. Visitors also have several points of access to databases including Ensembl, COSMIC, Pfam, GeneDB, Wormbase, Vega, MEROPS, and DECIPHER.Clicking on any of the topic headers in the center of the homepage leads to an assemblage of resources, project information, and laboratory homepage links related to the WTSI’s six main areas of study. Clicking on Human Genetics, for example, takes visitors to a lengthy menu of team links, each of which leads to information on what that team is working on, along with relevant references and illustrations. The Model Organisms link yields resources categorized by organism: mouse, zebrafish, Caenorhabditis elegans, Schizosaccharomyces pombe, and Xenopus tropicalis. The Pathogens link leads to organism-specific sequencing information for more than 100 bacteria, fungi, protozoa, helminths, vectors, and plasmids, as well as a variety of computational tools. The Bioinformatics header leads to links for downloading a number of programs, including packages for production sequencing, physical mapping, informatics analysis, and special data descriptions and specifications used at the WTSI. The Sequencing link pulls together in one area those resources specific to sequencing of various organisms. And the Proteomics link offers information on three groups at the institute that are working in this field of study.Visitors can also navigate through a bar down the left-hand side of the homepage, which offers quick links to biological resources, the database resources mentioned above, and news releases from the institute. Biological resources listed here include the Mutagenic Insertion and Chromosome Engineering Resource and the Sanger Institute Gene Trap Resource (both of which aid in developing genetically modified animal models), the WTSI Microarray Facility (which produces arrays for a range of organisms largely for use by Sanger researchers), and Clone Ordering (which allows external scientists to order mouse and human clone cells free of charge).Delving deeper into the site through the Site Map, visitors will find a page on Functional Genomics projects including the Human Epigenome Project (HEP), a public–private consortium of the WTSI, Epigenomics AG, and France’s Centre National de Génotypage. The goal of the HEP is to locate, identify, and catalog methylation variable positions within the human genome. The partners have conducted a pilot study of the methylation patterns within the major histocompatibility complex, a region of chromosome 6 that is associated with more diseases than any other region of the human genome. An overview of this pilot study is available within the HEP section, along with a look at the data analysis process employed by this study, information on epigenotyping, and links to the HEP partners.FiguresReferencesRelatedDetails Vol. 114, No. 3 March 2006Metrics About Article Metrics Publication History Originally published1 March 2006Published in print1 March 2006 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Computational models with thermodynamic and composition features improve siRNA design

BackgroundSmall interfering RNAs (siRNAs) have become an important tool in cell and molecular biology. Reliable design of siRNA molecules is essential for the needs of large functional genomics projects.ResultsTo improve the design of efficient siRNA molecules, we performed a comparative, thermodynamic and correlation analysis on a heterogeneous set of 653 siRNAs collected from the literature. We used this training set to select siRNA features and optimize computational models. We identified 18 parameters that correlate significantly with silencing efficiency. Some of these parameters characterize only the siRNA sequence, while others involve the whole mRNA. Most importantly, we derived an siRNA position-dependent consensus, and optimized the free-energy difference of the 5' and 3' terminal dinucleotides of the siRNA antisense strand. The position-dependent consensus is based on correlation and t-test analyses of the training set, and accounts for both significantly preferred and avoided nucleotides in all sequence positions. On the training set, the two parameters' correlation with silencing efficiency was 0.5 and 0.36, respectively. Among other features, a dinucleotide content index and the frequency of potential targets for siRNA in the mRNA added predictive power to our model (R = 0.55). We showed that our model is effective for predicting the efficiency of siRNAs at different concentrations.We optimized a neural network model on our training set using three parameters characterizing the siRNA sequence, and predicted efficiencies for the test siRNA dataset recently published by Novartis. On this validation set, the correlation coefficient between predicted and observed efficiency was 0.75. Using the same model, we performed a transcriptome-wide analysis of optimal siRNA targets for 22,600 human mRNAs.ConclusionWe demonstrated that the properties of the siRNAs themselves are essential for efficient RNA interference. The 5' ends of antisense strands of efficient siRNAs are U-rich and possess a content similarity to the pyrimidine-rich oligonucleotides interacting with the polypurine RNA tracks that are recognized by RNase H. The advantage of our method over similar methods is the small number of parameters. As a result, our method requires a much smaller training set to produce consistent results. Other mRNA features, though expensive to compute, can slightly improve our model.

Simultaneous high‐throughput recombinational cloning of open reading frames in closed and open configurations

Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.

IN SILICO PHENOTYPIC SCREENING METHOD OF MUTANTS BASED ON STATISTICAL MODELING OF GENETICALLY MIXED SAMPLES

In comprehensive functional genomics projects, systematic analysis of phenotypes is vital. However, conventional phenotypic screening is done mainly by imprecise visual observation of qualitative traits, and, therefore, in silico screening techniques for quantitative traits are required. In this report, we propose in silico phenotypic screening method that utilizes a Gaussian mixture model for the trait distribution in the offspring of a mutagenized line and the likelihood ratio test between the estimated Gaussian mixture model and the wild-type single Gaussian model. In order to evaluate the proposed method, we performed a screening experiment using real trait data of Arabidopsis. In this experiment, the proposed screening method properly distinguished the mutant line from the wild-type line. Furthermore, we conducted power analysis of the proposed method and two conventional methods under various simulated conditions of sample size and distribution of trait frequency. The result of the power analysis confirmed the effectiveness of the proposed method compared to the conventional methods.

GENOMIC APPROACHES TO POSTHARVEST BIOTIC AND ABIOTIC STRESSES OF CITRUS FRUIT

Citrus fruit may be subjected to biotic and abiotic stresses during postharvest handling and storage that influence their marketability. The major biotic stress is caused by phytopathogenic fungi of the genus Penicillium, being P. digitatum the most important pathogen. Among the abiotic stresses, we are particularly interested in chilling injury and in the heat-induced chilling tolerance of chilling-sensitive citrus cultivars. To gain a deeper knowledge on the mechanisms underlying the defence response of Citrus fruit to pathogen attack and on the heat-induced chilling tolerance, we have undertaken two complementary approaches. In the framework of the Spanish Citrus Functional Genomic Project, CFGP we have generated one cDNA library for each condition that will allow us to get a general picture of the genes being transcribed under these conditions.[...]

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

New genes with roles in theC. elegansembryo revealed using RNAi of ovary-enriched ORFeome clones

Several RNA interference (RNAi)-based functional genomic projects have been performed in Caenorhabditis elegans to identify genes required during embryogenesis. These studies have demonstrated that the ovary is enriched for transcripts essential for the first cell divisions. However, comparing RNAi results suggests that many genes involved in embryogenesis have yet to be identified, especially those eliciting partially penetrant phenotypes. To discover additional genes required for C. elegans embryonic development, we tested by RNAi 1123 ORFeome clones selected to represent ovary-enriched genes not associated with an embryonic phenotype. We discovered 155 new ovary-enriched genes with roles during embryogenesis, of which 69% show partial penetrance lethality. Time-lapse microscopy revealed specific phenotypes during early embryogenesis for genes giving rise to high penetrance lethality. Together with previous studies, we now have evidence that 1843 C. elegans genes have roles in embryogenesis, and that many more remain to be found. Using all available RNAi phenotypic data for the ovary-enriched genes, we re-examined the distribution of genes by chromosomal location, functional class, ovary enrichment, and conservation and found that trends are driven almost exclusively by genes eliciting high-penetrance phenotypes. Furthermore, we discovered a striking direct relationship between phylogenetic distribution and the penetrance level of embryonic lethality elicited by RNAi.

Bioinformatic approaches to assigning protein function from novel sequence data.

The current pace of functional genomic initiatives and genome sequencing projects has provided researchers with a bewildering array of sequence and biological data to analyze. The disease system-driven approach to identifying key genes frequently identifies nucleotide and protein sequences for which the gene and protein function are not known in sufficient detail to allow informed follow-up. Using a range of bioinformatic tools and sequence-based clues, most of unassigned sequences can now be annotated. This chapter takes as an example an unannotated expressed sequence tag, describing how to identify its related gene, and how to annotate the encoded protein using sequence, profile, and structure-based annotation methodologies.

Using siRNA Technique to Generate Transgenic Animals with Spatiotemporal and Conditional Gene Knockdown

Based on the RNAi technique, we have developed a new approach that generates transgenic animals capable of mimicking human genetic diseases. The new system is a combination of siRNA with Cre-loxP and tetracycline-on. It has the characteristics of being stable, inheritable, and inducible, with the siRNA able to be transcribed tissue specifically. To support the ability of this new method to generate a model for a disease, we created an ABCA1-deficient mouse line that mimics Tangier disease under controlled conditions. Thus, it should now be possible to rapidly establish human genetic diseases as a whole animal model without the use of embryonic stem cell and gene targeting. This system also provides a tool for pathological and pharmacological studies of aspects peculiar to particular human genetic diseases.

Schistosome transcriptome: insights and perspectives for functional genomics.

Progress on schistosome research has, until recently, suffered from limited genomic information. This situation changed dramatically in October 2003 with the simultaneous publication of the Schistosoma mansoni and Schistosome japonicum transcriptomes. A total of 45 000 expressed-sequence tags was obtained for S. japonicum, derived from adult worms and eggs. For S. mansoni, a total of ∼125 000 ESTs was analyzed, derived from six different life cycle stages, and estimated to represent 92% of the complement of >14 000 genes. The wealth of information generated is essential for annotation of the now-sequenced S. mansoni genome. It has permitted a list of potential drug targets and vaccine candidates to be drawn up, and has set in train proteomic and functional genomic projects to exploit the data, and ultimately lead to improved control strategies.

Functional genomics in chickens: development of integrated‐systems microarrays for transcriptional profiling and discovery of regulatory pathways

The genetic networks that govern the differentiation and growth of major tissues of economic importance in the chicken are largely unknown. Under a functional genomics project, our consortium has generated 30 609 expressed sequence tags (ESTs) and developed several chicken DNA microarrays, which represent the Chicken Metabolic/Somatic (10 K) and Neuroendocrine/Reproductive (8 K) Systems (http://udgenome.ags.udel.edu/cogburn/). One of the major challenges facing functional genomics is the development of mathematical models to reconstruct functional gene networks and regulatory pathways from vast volumes of microarray data. In initial studies with liver-specific microarrays (3.1 K), we have examined gene expression profiles in liver during the peri-hatch transition and during a strong metabolic perturbation—fasting and re-feeding—in divergently selected broiler chickens (fast vs. slow-growth lines). The expression of many genes controlling metabolic pathways is dramatically altered by these perturbations. Our analysis has revealed a large number of clusters of functionally related genes (mainly metabolic enzymes and transcription factors) that control major metabolic pathways. Currently, we are conducting transcriptional profiling studies of multiple tissues during development of two sets of divergently selected broiler chickens (fast vs. slow growing and fat vs. lean lines). Transcriptional profiling across multiple tissues should permit construction of a detailed genetic blueprint that illustrates the developmental events and hierarchy of genes that govern growth and development of chickens. This review will briefly describe the recent acquisition of chicken genomic resources (ESTs and microarrays) and our consortium's efforts to help launch the new era of functional genomics in the chicken.

AGRIS: Arabidopsis Gene Regulatory Information Server, an information resource of Arabidopsis cis-regulatory elements and transcription factors

BackgroundThe gene regulatory information is hardwired in the promoter regions formed by cis-regulatory elements that bind specific transcription factors (TFs). Hence, establishing the architecture of plant promoters is fundamental to understanding gene expression. The determination of the regulatory circuits controlled by each TF and the identification of the cis-regulatory sequences for all genes have been identified as two of the goals of the Multinational Coordinated Arabidopsis thaliana Functional Genomics Project by the Multinational Arabidopsis Steering Committee (June 2002).ResultsAGRIS is an information resource of Arabidopsis promoter sequences, transcription factors and their target genes. AGRIS currently contains two databases, AtTFDB (Arabidopsis thaliana transcription factor database) and AtcisDB (Arabidopsis thaliana cis-regulatory database). AtTFDB contains information on approximately 1,400 transcription factors identified through motif searches and grouped into 34 families. AtTFDB links the sequence of the transcription factors with available mutants and, when known, with the possible genes they may regulate. AtcisDB consists of the 5' regulatory sequences of all 29,388 annotated genes with a description of the corresponding cis-regulatory elements. Users can search the databases for (i) promoter sequences, (ii) a transcription factor, (iii) a direct target genes for a specific transcription factor, or (vi) a regulatory network that consists of transcription factors and their target genes.ConclusionAGRIS provides the necessary software tools on Arabidopsis transcription factors and their putative binding sites on all genes to initiate the identification of transcriptional regulatory networks in the model dicotyledoneous plant Arabidopsis thaliana. AGRIS can be accessed from .

Fly developments: an introspective.

The EMBO Workshop on the Molecular and Developmental Biology of Drosophila took place on June 23–29, 2002 at the Orthodox Academy of Crete, Greece, for the 13th time since its inception in 1978. As has been customary in these meetings, cutting-edge and mostly unpublished research was presented inabroad rangeof topics.With the completionof the genome sequence, new insight is beginning to emerge from the use of global approaches to examine the regulatory pathwaysgoverningmulticellular processes, and suchapproaches were evident in a number of talks. However, from the majority of the presentations, it became clear that fresh insight and exciting advances are also being gained by looking inward, to the innerworkings of cells. Thepresentationswerepunctuated by the addition of new components to established pathways, as well as the joining of previously unlinked pathways and processes. The most-prominent topics were developmental decisions and patterning, although studies on gene expression, cell biology, neurobiology and immunology were also included. Finally, progress on the annotation of the genome and on a number of functional genomic projects was reported. The large quantity and high quality of research presented during this exciting week prohibits reporting on a fair crosssection of the talks in a limited space, so we will (somewhat arbitrarily) limit ourselves to a selected number of topics.

Improving the efficiency of gene replacements in Neurospora crassa: a first step towards a large-scale functional genomics project

Here, we report the use of the mating type heterokaryon incompatibility system as a counterselection to increase the probability of identifying gene replacements in Neurospora crassa. We compared the frequencies of gene replacements observed among transformants obtained by using plasmids with or without the mat a-1 + gene (hereby called “Toxic Gene”) placed adjacent to disruption cassettes. On an average, we were 20× more likely to identify a correct gene replacement by incorporating the toxic gene in our constructs. Using this strategy, we constructed strains containing a deletion of the inl ( 1 l -myo-inositol-1-phosphate synthase) gene. Finally, we demonstrated that we were able to remove the transformation marker (the hygromycin B phosphotransferase- thymidine kinase gene fusion [ hph +:: tk +]) from the genome by using a strategy similar to the “URA-blaster” strategy used in yeast, which we call “tk-blaster.”