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PubMed2005

Flux an important, but neglected, component of functional genomics.

Genomics approaches aimed at understanding metabolism currently tend to involve mainly expression profiling, although proteomics and steady-state metabolite profiling are increasingly being carried out as alternative strategies. These approaches provide rich information on the inventory of the cell. It is, however, of growing importance that such approaches are augmented by sophisticated integrative analyses and a higher-level understanding of cellular dynamics to provide insights into mechanisms that underlie biological processes. We argue the need for, and discuss theoretical and practical aspects of, the determination of metabolic flux as a component of functional genomics.

Genome, Plant↗
PubMed2002

Functional genomics and sexual differentiation in amphibians.

In Xenopus laevis the basic mechanisms underlying sexual differentiation were investigated by determining time courses of sexual steroids and their corresponding receptors during complete larval development from egg to juveniles. Androgens as well as estradiol (E2) are derived from maternal origin and accumulate in hatching tadpoles. Sexual steroid contents decreased rapidly after hatching and rose again at the end of metamorphosis indicating endogenous production. In parallel the mRNA expression for corresponding androgen (AR) and estrogen receptors (ER) was measured by means of semiquantitative RT-PCR. Both receptor mRNAs increased dramatically just after hatching and decreased only moderately until end of metamorphosis. In female juveniles E2 and ER-mRNA levels were higher compared with males. Treatment by exogenous E2 elevated both, ER- and AR-mRNA, indicating stimulatory functions of E2 for gene expression of both receptors. Effects on sexual differentiation during larval development were achieved by treatment with E2 and the antiandrogen cyproterone acetate both causing feminization, the antiestrogen tamoxifen resulting in neutralization, and the androgens, methyltestosterone and dihydrotestosterone, but not testosterone, leading to masculinization. The data presented are in accordance with further recent findings and suggest a new hypothesis for functional genomics in sexual differentiation of amphibians.

Androgens↗
PubMed2003

Transposon-based strategies for microbial functional genomics and proteomics.

Transposons are mobile genetic elements that can relocate from one genomic location to another. As well as modulating gene expression and contributing to genome plasticity and evolution, transposons are remarkably diverse molecular tools for both whole-genome and single-gene studies in bacteria, yeast, and other microorganisms. Efficient but simple in vitro transposition reactions now allow the mutational analysis of previously recalcitrant microorganisms. Transposon-based signature-tagged mutagenesis and genetic footprinting strategies have pinpointed essential genes and genes that are crucial for the infectivity of a variety of human and other pathogens. Individual proteins and protein complexes can be dissected by transposon-mediated scanning linker mutagenesis. These and other transposon-based approaches have reaffirmed the usefulness of these elements as simple yet highly effective mutagens for both functional genomic and proteomic studies of microorganisms.

Artificial Gene Fusion↗
PubMed2006

Genetics and functional genomics of legume nodulation.

Gram-negative soil bacteria (rhizobia) within the Rhizobiaceae phylogenetic family (alpha-proteobacteria) have the unique ability to infect and establish a nitrogen-fixing symbiosis on the roots of leguminous plants. This symbiosis is of agronomic importance, reducing the need for nitrogen fertilizer for agriculturally important plants (e.g. soybean and alfalfa). The establishment of the symbiosis involves a complex interplay between host and symbiont, resulting in the formation of a novel organ, the nodule, which the bacteria colonize as intracellular symbionts. This review focuses on the most recent discoveries relating to how this symbiosis is established. Two general developments have contributed to the recent explosion of research progress in this area: first, the adoption of two genetic model legumes, Medicago truncatula and Lotus japonicus, and second, the application of modern methods in functional genomics (e.g. transcriptomic, proteomic and metabolomic analyses).

Amino Acid Sequence↗
PubMed2005

Functional Genomics meets neurodegenerative disorders Part I: transcriptomic and proteomic technology.

Transcriptomics and proteomics are increasingly applied to gain a mechanistic insight into neurodegenerative disorders. These techniques not only identify distinct, differentially expressed mRNAs and proteins but are also employed to dissect signaling pathways and reveal networks by using an integrated approach. In part I of this back-to-back review, technical aspects are discussed: in the transcriptomics section, which includes enrichment by laser microcapture dissection, we comment on qRT-PCR, SAGE, subtractive hybridization, differential display and microarrays, including software packages. In the proteomics section we discuss two-dimensional (2D) gel electrophoresis, liquid chromatography, methods to label and enrich specific proteins or peptides, and different types of mass spectrometers. These tools have been applied to a range of neurodegenerative disorders and are discussed and integrated in part II (Functional Genomics meets neurodegenerative disorders. Part II: application and data integration).

Animals↗
PubMed2002

Integrated functional genomics approach for the design of patient-individual antitumor vaccines.

Our aim is to identify as many candidates as possible for tumor-associated T-cell epitopes in individual patients. First, we performed expression profiling of tumor and normal tissue to identify genes exclusively expressed or overexpressed in the tumor sample. Then, using mass spectrometry, we characterized up to 77 different MHC ligands from the same tumor sample. Several of the MHC ligands were derived from overexpressed gene products, one was derived from a proto-oncogene, and another was derived from a frameshift mutation. At least one was identified as an actual T-cell epitope. Thus, we could show that by combining these two analytic tools, it is possible to propose several candidates for peptide-based immunotherapy. We envision the use of this novel integrated functional genomics approach for the design of antitumor vaccines tailored to suit the needs of each patient.

Antigens, Neoplasm↗
PubMed2004

Mouse functional genomics requires standardization of mouse handling and housing conditions.

The study of mouse models is crucial for the functional annotation of the human genome. The recent improvements in mouse genetics now moved the bottleneck in mouse functional genomics from the generation of mutant mice lines to the phenotypic analysis of these mice lines. Simple, validated, and reproducible phenotyping tests are a prerequisite to improving this phenotyping bottleneck. We analyzed here the impact of simple variations in animal handling and housing procedures, such as cage density, diet, gender, length of fasting, as well as site (retro-orbital vs. tail), timing, and anesthesia used during venipuncture, on biochemical, hematological, and metabolic/endocrine parameters in adult C57BL/6J mice. Our results, which show that minor changes in procedures can profoundly affect biological variables, underscore the importance of establishing uniform and validated animal procedures to improve reproducibility of mouse phenotypic data.

Animals↗
PubMed2005

Comparative functional genomics for identifying models of human cancer.

Genetically modified mice with overexpressed and/or deleted genes have been used extensively to model human cancer. However, it is uncertain as to what extent the mouse models reproduce the corresponding cancers in humans. We have compared the global gene expression patterns in human and mouse hepatocellular carcinomas (HCCs) in an attempt to identify the mouse models that most extensively reproduce the molecular pathways in the human tumors. The comparative analysis of the gene expression patterns in murine and human HCC indicates that certain genetic mouse models closely reproduce the gene expression patterns of HCC in humans, while others do not. Identification of mouse models that reproduce the molecular features of specific human cancers (or subclasses of specific human cancers) promises to accelerate both the understanding of the molecular pathogenesis of cancer and the discovery of therapeutic targets. We propose that this method, comparative functional genomics, could be effectively applied to the analysis of mouse models for other human cancers.

Animals↗
PubMed2006

Functional genomics of abiotic stress tolerance in cereals.

Abiotic stresses such as extreme temperatures, low water availability, high salt and mineral deficiencies or toxicities severely diminish productivity of cereal crops. These stresses are becoming increasingly important because of the declining availability of good quality water, land degradation and community pressures to move away from chemical intervention in agriculture. Of the major cereals, wheat and barley are grown in the most hostile and consequently lowest yielding environments. Extensive genetic studies and surveys of landrace and wild germplasm have indicated extensive variation for abiotic stress tolerance but this has been difficult to exploit due to the relatively poor background knowledge of the molecular basis for stress in these species. Interconnected signal transduction pathways that lead to multiple responses to abiotic stresses have been difficult to study using traditional approaches because of their complexity and the large number of genes and gene products involved in the various defensive and developmental responses of the plant. Functional genomics is now widely seen as providing tools for dissecting abiotic stress responses in wheat and barley, through which networks of stress perception, signal transduction and defensive responses can be examined from gene transcription, through protein complements of cells, to the metabolite profiles of stressed tissues.

Adaptation, Physiological↗
PubMed2004

Exploring tick saliva: from biochemistry to 'sialomes' and functional genomics.

Tick saliva, a fluid once believed to be only relevant for lubrication of mouthparts and water balance, is now well known to be a cocktail of potent anti-haemostatic, anti-inflammatory and immunomodulatory molecules that helps these arthropods obtain a blood meal from their vertebrate hosts. The repertoire of pharmacologically active components in this cocktail is impressive as well as the number of targets they specifically affect. These salivary components change the physiology of the host at the bite site and, consequently, some pathogens transmitted by ticks take advantage of this change and become more infective. Tick salivary proteins have therefore become an attractive target to control tick-borne diseases. Recent advances in molecular biology, protein chemistry and computational biology are accelerating the isolation, sequencing and analysis of a large number of transcripts and proteins from the saliva of different ticks. Many of these newly isolated genes code for proteins with homologies to known proteins allowing identification or prediction of their function. However, most of these genes code for proteins with unknown functions therefore opening the road to functional genomic approaches to identify their biological activities and roles in blood feeding and hence, vaccine development to control tick-borne diseases.

Animals↗
PubMed2002

Microarray data quality analysis: lessons from the AFGC project. Arabidopsis Functional Genomics Consortium.

Genome-wide expression profiling with DNA microarrays has and will provide a great deal of data to the plant scientific community. However, reliability concerns have required the development data quality tests for common systematic biases. Fortunately, most large-scale systematic biases are detectable and some are correctable by normalization. Technical replication experiments and statistical surveys indicate that these biases vary widely in severity and appearance. As a result, no single normalization or correction method currently available is able to address all the issues. However, careful sequence selection, array design, experimental design and experimental annotation can substantially improve the quality and biological of microarray data. In this review, we discuss these issues with reference to examples from the Arabidopsis Functional Genomics Consortium (AFGC) microarray project.

Arabidopsis↗
PubMed2006

High-throughput functional genomic methods to analyze the effects of dietary lipids.

The applications of 'omics' (genomics, transcriptomics, proteomics and metabolomics) technologies in nutritional studies have opened new possibilities to understand the effects and the action of different diets both in healthy and diseased states and help to define personalized diets and to develop new drugs that revert or prevent the negative dietary effects. Several single nucleotide polymorphisms have already been investigated for potential gene-diet interactions in the response to different lipid diets. It is also well-known that besides the known cellular effects of lipid nutrition, dietary lipids influence gene expression in a tissue, concentration and age-dependent manner. Protein expression and post-translational changes due to different diets have been reported as well. To understand the molecular basis of the effects and roles of dietary lipids high-throughput functional genomic methods such as DNA- or protein microarrays, high-throughput NMR and mass spectrometry are needed to assess the changes in a global way at the genome, at the transcriptome, at the proteome and at the metabolome level. The present review will focus on different high-throughput technologies from the aspects of assessing the effects of dietary fatty acids including cholesterol and polyunsaturated fatty acids. Several genes were identified that exhibited altered expression in response to fish-oil treatment of human lung cancer cells, including protein kinase C, natriuretic peptide receptor-A, PKNbeta, interleukin-1 receptor associated kinase-1 (IRAK-1) and diacylglycerol kinase genes by using high-throughput quantitative real-time PCR. Other results will also be mentioned obtained from cholesterol and polyunsaturated fatty acid fed animals by using DNA- and protein microarrays.

Animals↗
PubMed2004

Root growth maintenance during water deficits: physiology to functional genomics.

Progress in understanding the network of mechanisms involved in maize primary root growth maintenance under water deficits is reviewed. These include the adjustment of growth zone dimensions, turgor maintenance by osmotic adjustment, and enhanced cell wall loosening. The role of the hormone abscisic acid (ABA) in maintaining root growth under water deficits is also addressed. The research has taken advantage of kinematic analysis, i.e. characterization of spatial and temporal patterns of cell expansion within the root growth zone. This approach revealed different growth responses to water deficits and ABA deficiency in distinct regions of the root tip. In the apical 3 mm region, elongation is maintained at well-watered rates under severe water deficit, although only in ABA-sufficient roots, whereas the region from 3-7 mm from the apex exhibits maximum elongation in well-watered roots, but progressive inhibition of elongation in roots under water deficit. This knowledge has greatly facilitated discovery of the mechanisms involved in regulating the responses. The spatial resolution with which this system has been characterized and the physiological knowledge gained to date provide a unique and powerful underpinning for functional genomics studies. Characterization of water deficit-induced changes in transcript populations and cell wall protein profiles within the growth zone of the maize primary root is in progress. Initial results from EST and unigene analyses in the tips of well-watered and water-stressed roots highlight the strength of the kinematic approach to transcript profiling.

Adaptation, Physiological↗
PubMed2001

Generation of RCAS vectors useful for functional genomic analyses.

Avian leukosis type A virus-derived retroviral vectors have been used to introduce genes into cells expressing the corresponding avian receptor tv-a. This includes the use of Replication-Competent Avian sarcoma-leukosis virus (ASLV) long terminal repeat (LTR) with Splice acceptor (RCAS) vectors in the analysis of avian development, human and murine cell cultures, murine cell lineage studies and cancer biology. Previously, cloning of genes into this virus was difficult due to the large size of the vector and sparse cloning sites. To overcome some of the disadvantages of traditional cloning using the RCASBP-Y vector, we have modified the RCASBP-Y to incorporate "Gateway" site-specific recombination cloning of genes into the construct, either with or without HA epitope tags. We have found the repetitive "att" sequences, which are the targets for site-specific recombination, do not impair the production of infectious viral particles or the expression of the gene of interest. This is the first instance of site-specific recombination being used to generate retroviral gene constructs. These viral constructs will allow for the efficient transfer and expression of cDNAs needed for functional genomic analyses.

Amino Acid Sequence↗
PubMed2003

Functional genomic analysis reveals distinct neoplastic phenotypes associated with c-myb mutation in the bursa of Fabricius.

Avian retroviral integration into the c-myb locus is casually associated with the development of lymphomas in the bursa of Farbricius of chickens; these arise with a shorter latency than bursal lymphomas caused by deregulation of c-myc. This study indicates that c-myb mutation in embryonic bursal precursors leads to an oligoclonal population of developing bursal follicles, showing a variable propensity to form a novel lesion, the neoplastic follicle (NF). About half of such bursas rapidly developed lymphomas. Detection of changes in gene expression, during the development of neoplasms, was carried out by cDNA microarray analysis. The transcriptional signature of lymphomas with mutant c-myb was more limited than, and only partially shared with, those of bursal lymphomas caused by Myc or Rel oncogenes. The c-myb-associated lymphomas frequently showed overexpression of c-myc and altered expression of other genes involved in cell cycle control and proliferation-related signal transduction. Oligoclonal, NF-containing bursas lacked detectable c-myc overexpression and demonstrated a pattern of gene expression distinct from that of normal bursa and partially shared with the short-latency lymphomas. This functional genomic analysis uncovered several different pathways of lymphomagenesis by oncogenic transcription factors acting in a B-cell lineage.

Animals↗
PubMed2005

Functional genomics identifies a mechanism for estrogen activation of the retinoic acid receptor alpha1 gene in breast cancer cells.

The identification of estrogen receptor (ERalpha) target genes is crucial to our understanding of its predominant role in breast cancer. In this study, we used a chromatin immunoprecipitation (ChIP)-cloning strategy to identify ERalpha-regulatory modules and associated target genes in the human breast cancer cell line MCF-7. We isolated 12 transcriptionally active genomic modules that recruit ERalpha and the coactivator steroid receptor coactivator (SRC)-3 to different intensities in vivo. One of the ERalpha-regulatory modules identified is located 3.7 kb downstream of the first transcriptional start site of the RARA locus, which encodes retinoic acid receptor alpha1 (RARalpha1). This module, which includes an estrogen response element (ERE), is conserved between the human and mouse genomes. Direct binding of ERalpha to the ERE was shown using EMSAs, and transient transfections in MCF-7 cells demonstrated that endogenous ERalpha can induce estrogen-dependent transcriptional activation from the module or the ERE linked to a heterologous promoter. Furthermore, ChIP assays showed that the coregulators SRC-1, SRC-3, and receptor-interacting protein 140 are recruited to this intronic module in an estrogen-dependent manner. As expected from previous studies, the transcription factor Sp1 can be detected at the RARA alpha1 promoter by ChIP. However, treatment with estradiol did not influence Sp1 recruitment nor help recruit ERalpha to the promoter. Finally, ablation of the intronic ERE was sufficient to abrogate the up-regulation of RARA alpha1 promoter activity by estradiol. Thus, this study uncovered a mechanism by which ERalpha significantly activates RARalpha1 expression in breast cancer cells and exemplifies the utility of functional genomics strategies in identifying long-distance regulatory modules for nuclear receptors.

Acetyltransferases↗
PubMed2002

Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.

The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.

Actins↗
PubMed2005

In vivo application of RNA interference: from functional genomics to therapeutics.

RNAi has rapidly become a powerful tool for drug target discovery and validation in cell culture, and now has largely displaced efforts with antisense and ribozymes. Consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Studies on RNAi have resulted in two basic methods for its use for gene selective inhibition: 1) cytoplasmic delivery of short dsRNA oligonucleotides (siRNA), which mimics an active intermediate of an endogenous RNAi mechanism and 2) nuclear delivery of gene expression cassettes that express a short hairpin RNA (shRNA), which mimics the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism. Non-viral gene delivery systems are a diverse collection of technologies that are applicable to both of these forms of RNAi. Importantly, unlike antisense and ribozyme systems, a remarkable trait of siRNA is a lack of dependence on chemical modifications blocking enzymatic degradation, although chemical protection methods developed for the earlier systems are being incorporated into siRNA and are generally compatible with non-viral delivery systems. The use of siRNA is emerging more rapidly than for shRNA, in part due to the increased effort required to construct shRNA expression systems before selection of active sequences and verification of biological activity are obtained. In contrast, screens of many siRNA sequences can be accomplished rapidly using synthetic oligos. It is not surprising that the use of siRNA in vivo is also emerging first. Initial in vivo studies have been reported for both viral and non-viral delivery but viral delivery is limited to shRNA. This review describes the emerging in vivo application of non-viral delivery systems for RNAi for functional genomics, which will provide a foundation for further development of RNAi therapeutics. Of interest is the rapid adaptation of ligand-targeted plasmid-based nanoparticles for RNAi agents. These systems are growing in capabilities and beginning to pose a serious rival to viral vector based gene delivery. The activity of siRNA in the cytoplasm may lower the hurdle and thereby accelerate the successful development of therapeutics based on targeted non-viral delivery systems.

Animals↗