Plant Biol (Stuttg), 2004 Mar-Apr, 6(2), 119 - 27 High throughput cryopreservation of 140,000 Physcomitrella patens mutants; Schulte J et al.; A high throughput protocol was established to preserve 140,000 mutants of a moss, Physcomitrella patens, a model plant for functional genomics studies, over liquid nitrogen . Regarding the reliable long-term storage of diverse mutant phenotypes, as well as time and cost effectiveness, each working step was optimized: 1) plant preparation, 2) freezing regime, cryogenic conditions, 3) regrowth after thawing . A prerequisite for maximum regrowth was a 1-week preculture of chopped plant material on a supplemented medium prior to freezing . Cryo vials as preculture vessels resulted in identical regrowth rates, compared to petri dishes . The cryo vial type had a significant influence on regrowth rates . A cooling rate of - 1 degrees C/min down to - 35 degrees C with a 10 min holding time before transferring plants to - 152 degrees C was the most suitable freezing regime . This protocol allows a cryopreservation of 1100 plants during a 5-day working week, practicable by one person . For more than 650 cryopreserved mutants a maximum regrowth rate of 100 % was obtained, independently of mutant phenotypes. Prostaglandins Leukot Essent Fatty Acids, 2004 Apr, 70(4), 345 - 7 Can fat explain the human brain's big bang evolution?-Horrobin's leads for comparative and functional genomics; Erren TC et al.; When David Horrobin suggested that phospholipid and fatty acid metabolism played a major role in human evolution, his 'fat utilization hypothesis' unified intriguing work from paleoanthropology, evolutionary biology, genetic and nervous system research in a novel and coherent lipid-related context . Interestingly, unlike most other evolutionary concepts, the hypothesis allows specific predictions which can be empirically tested in the near future . This paper summarizes some of Horrobin's intriguing propositions and suggests as to how approaches of comparative genomics published in Cell, Nature, Science and elsewhere since 1997 may be used to examine his evolutionary hypothesis . Indeed, systematic investigations of the genomic clock in the species' mitochondrial DNA, the Y and autosomal chromosomes as evidence of evolutionary relationships and distinctions can help to scrutinize associated predictions for their validity, namely that key mutations which differentiate us from Neanderthals and from great apes are in the genes coding for proteins which regulate fat metabolism, and particularly the phospholipid metabolism of the synapses of the brain . It is concluded that beyond clues to humans' relationships with living primates and to the Neanderthals' cognitive performance and their disappearance, the suggested molecular clock analyses may provide crucial insights into the biochemical evolution-and means of possible manipulation-of our brain. Appetite, 2004 Feb, 42(1), 11 - 4 The future of genetic research on appetitive behavior; Kowalski TJ; The risk for developing obesity has a significant genetic component . Several quantitative trait loci and candidate genes have been identified using current methodological approaches however the information gained thus far is insufficient to adequately explain the genetics underlying human obesity . The completion of a draft of the human genome sequence, the potential benefit of single nucleotide polymorphisms association studies for identifying risk conferring alleles, and developing functional genomics technologies promise to accelerate obesity gene discovery . These advances, used with current evaluative tools (murine molecular genetic techniques), may increase our understanding of human obesity, and ultimately provide better approaches to diagnosis and treatment. Curr Opin Microbiol, 2004 Feb, 7(1), 33 - 8 Proteomic and gene profiling approaches to study host responses to bacterial infection; Walduck A et al.; Infectious disease is the result of an intimate relationship between the pathogen and host, which involves cross-talk . After an initial flood of mainly descriptive reports on the influence of acute bacterial infection on cells, transcriptome and proteome studies are now becoming more refined in their approach, and are shedding light on the role of pathogen-specific mechanisms/structures in pathogenesis . In addition, studies of gene expression in vivo have shed new light on how the host influences the niche occupied by bacteria . Elegant refinements to proteomics using beads coated with bacterial invasins, or purifying subcellular fractions are producing a picture of invasion specific processes . Such approaches combined with modern functional genomics technologies such as RNAi represent the next phase in understanding host-bacteria interactions. Anal Biochem, 2004 Apr 1, 327(1), 23 - 34 High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae; Weiss A et al.; Cell-based assays are widely used in high-throughput screening to determine the effects of toxicants and drugs on their biological targets . To enable a functional genomics modeling of gene-environment interactions, quantitative assays are required both for gene expression and for the phenotypic responses to environmental challenge . To address this need, we describe an automated high-throughput methodology that provides phenotypic profiling of the cellular responses to environmental stress in Saccharomyces cerevisiae . Standardized assay conditions enable the use of a single metric value to quantify yeast microculture growth curves . This assay format allows precise control of both genetic and environmental determinants of the cellular responses to oxidative stress, a common mechanism of environmental insult . These yeast-cell-based assays are validated with hydrogen peroxide, a simple direct-acting oxidant . Phenotypic profiling of the oxidative stress response of a yap1 mutant strain demonstrates the mechanistic analysis of genetic susceptibility to oxidative stress . As a proof of concept for analysis of more complex gene-environment interactions, we describe a combinatorial assay design for phenotypic profiling of the cellular responses to tert-butyl hydroperoxide, a complex oxidant that is actively metabolized by its target cells . Thus, the yeast microculture assay format supports comprehensive applications in toxicogenomics. Curr Cancer Drug Targets, 2004 Mar, 4(2), 111 - 24 The role of emerging genomics and proteomics technologies in cancer drug target discovery; Onyango P; Cancer drugs have traditionally been identified in screens designed to produce broad biological end points such as cell death . A serious undesired outcome of drugs discovered in these screens is that the mechanism of drug action is unknown and such drugs often have adverse side effects . Designing cancer drugs that act on specific targets offer the advantage that the mechanism of drug action can be understood and accurately monitored in clinical trials leading to development of better drugs . The pharmacological industry has recently shifted to a target directed drug discovery model . However, until recently potential cancer drug targets comprised of only a small fraction of the human genome . The human genome project and high-throughput structural and functional genomics have dramatically increased the number of cancer drug targets . Deciphering cancer drug targets requires the understanding of biochemical pathways that are affected in the cancer genome . It has been suggested that utilization of Single-nucleotide polymorphisms (SNPs) will aid in identifying individuals at high risk of developing certain cancers, and will also help in development of tailored medication or identify genetic profiles of specific drug action and toxicity . Achieving successful new cancer drug development schemes will require a merger of research disciplines that include pharmacology, genomics, comparative genomics, functional genomics, proteomics and bioinformatics . In this review the significance and challenges of these rapidly evolving technologies in cancer drug target discovery are discussed. J Agric Food Chem, 2004 Mar 24, 52(6), 1427 - 34 Metabolic profiling: a new tool in the study of wood formation; Morris CR et al.; In the realm of plant genomics, metabolic profiling has become a valuable tool with which to assess the effect of genetic and/or environmental factors on plant development . This paper reports the first application of metabolic profiling on differentiating xylem tissue of loblolly pine . A protocol is presented for the analysis of loblolly pine xylem tissue . The effects of sample preparation, extraction, and derivatization on the corresponding metabolite profiles and yields have been investigated and are reported . Gas chromatography-mass spectroscopy has been used to quantify >60 polar and lipophilic metabolites from wood-forming tissue . It was possible to assign chemical structures to approximately half of these compounds . Comparison of six loblolly pine genotypes, three high cellulose (50-52%) and three medium (45-48%) cellulose, showed distinct metabolic profiles . Principal component analysis enabled the assignment of metabolic phenotypes using these large data sets . Metabolic phenotype clustering occurred in which the three high-cellulose genotypes were segregated from the medium-cellulose genotypes . These results demonstrate the use of metabolic profiling for the study of wood-forming tissue and as a tool in functional genomics. J Neurol Neurosurg Psychiatry, 2004 Apr, 75(4), 529 - 38 Functional genomics and proteomics: application in neurosciences; Wilson KE et al.; The sequencing of the complete genome for many organisms, including man, has opened the door to the systematic understanding of how complex structures such as the brain integrate and function, not only in health but also in disease . This blueprint, however, means that the piecemeal analysis regimes of the past are being rapidly superseded by new methods that analyse not just tens of genes or proteins at any one time, but thousands, if not the entire repertoire of a cell population or tissue under investigation . Using the most appropriate method of analysis to maximise the available data therefore becomes vital if a complete picture is to be obtained of how a system or individual cell is affected by a treatment or disease . This review examines what methods are currently available for the large scale analysis of gene and protein expression, and what are their limitations. Mol Cell Endocrinol, 2004 Feb 27, 215(1-2), 19 - 30 Identification and characterization of 17 beta-hydroxysteroid dehydrogenases in the zebrafish, Danio rerio; Mindnich R et al.; The 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs) are key enzymes in the final steps of steroid hormone synthesis . 17beta-HSD type 1 (HSD17B1) catalyzes the reduction of estrone to estradiol, while type 3 (HSD17B3) performs the conversion of androstenedione to testosterone . Here we present a functional genomics study of putative candidates of these enzymes in the zebrafish . By an in silico screen of zebrafish EST databases we identified three candidate homologs for both HSD17B1 and HSD17B3 . Phylogenetic analysis, unique expression patterns (RT-PCR) during embryogenesis and adulthood, as well as activity measurements revealed that one of the HSD17B1 candidates is the zebrafish homolog, while the other two are paralogous photoreceptor-associated retinol dehydrogenases . All three HSD17B3 candidate genes showed nearly identical, ubiquitous expressions in embryogenesis and adult tissues and were identified to be paralogs of HSD17B12 and a yet uncharacterized putative steroid dehydrogenase . Phylogenetic analysis shows that HSD17B3 and HSD17B12 are descendants from a common ancestor. Methods Mol Biol, 2004, 260, 255 - 76 Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies; Ivics Z et al.; Functional genomic analyses in vertebrate model systems, including fish, frogs, and mice, have greatly contributed to our understanding of embryonic development and human disease . However, new molecular tools and strategies are needed to meet the increasing demands of linking sequence information to gene function . Transposable elements (TEs) are very efficient at integrating into DNA, and are therefore useful vectors for transferring new genetic material into genomes . In particular, members of the Tc1/mariner superfamily of elements are able to transpose in species other than their hosts, and are therefore emerging tools for functional genomics in several organisms . This chapter describes strategies of using retrovirus vectors and DNA-based TEs for transgenesis and insertional mutagenesis in vertebrates, with special emphasis on the Sleeping Beauty (SB) element, a reconstructed Tc1/mariner-like transposon from fish . SB jumps efficiently in cells of diverse vertebrate species in culture, as well as in somatic and germline tissues of the mouse in vivo . Simple structure and easy laboratory handling of transposon vectors are coupled with efficient and stable transgene integration and persistent, long-term transgene expression by transposon-mediated gene transfer . These features all contribute to the usefulness of TEs as tools for vertebrate functional genomics, as well as for animal biotechnology and human gene therapy. Curr Opin Pharmacol, 2004 Feb, 4(1), 72 - 8 Pharmacogenetics and psychopharmacology; Bondy B et al.; Genetic factors play a major role in the differential responses to treatment or the incidence of adverse drug effects in psychopharmacotherapy . The aim of pharmacogenetics is to elucidate this variability caused by hereditary differences . From hypotheses on the mechanisms of drug action, several mutations in genes coding for neurotransmitter receptors, neurotransmitter-degrading enzymes, transport proteins or enzymes of the drug metabolizing system have been identified and investigated . Although there exists some controversy among the results, many studies are supportive of the hypothesis that psychopharmacogenetics will be helpful in predicting an individual's drug response while minimizing the rate of side effects . The inclusion of functional genomics, which investigates the complex gene and/or protein expression in response to a given drug, could help lead to the development of novel and safer drugs. Methods Mol Biol, 2004, 252, 523 - 32 Adenovirus-delivered siRNA; Shen C et al.; RNA interference is the process that double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA mediated by 21- to 23-nt small interfering RNA (siRNA) . Successful application of RNAi in functional genomics and proteomics, cancer gene therapy, and virus protection depends on the efficient delivery of siRNA into mammalian cells . The availability of high virus titer, infection of a broad spectrum of cell types, and independence on active cell division makes adenovirus the vector of choice for siRNA delivery . To this end, we developed a new adenovirus shuttle vector designated as pShuttle-H1 to host H1-RNA promoter and unique BglII and HindIII sites for insertion of oligos for expression of siRNA . In this chapter, we describe an adenovirus system that uses a commercially available adenovirus system and pShuttle-H1 to deliver siRNA-expressing cassette into cells to silence a specific gene in mammalian cells. Expert Opin Biol Ther, 2004 Mar, 4(3), 319 - 27 Therapeutic potential of retroviral RNAi vectors; Devroe E et al.; The ability of small interfering RNA (siRNA) to mediate gene-specific post-transcriptional silencing in mammalian cells will undoubtedly revolutionise functional genomics, as well as drug target identification and validation . Furthermore, there is widespread excitement that siRNA itself might prove useful in the clinical setting . For those wishing to develop siRNA as a therapeutic agent, the most difficult obstacle to overcome will be delivery . Recently, several breakthroughs have highlighted viruses as excellent vehicles for siRNA delivery . Retroviruses, the transgene-delivery vector of choice for many experimental gene therapy studies, have been engineered to deliver and stably express therapeutic siRNA within cells, both in vitro and in vivo . These findings are important milestones for the development of siRNA as a gene therapy for treatment of viral infections, cancer, autoimmune syndromes and numerous genetic disorders . This review describes the development of retroviral siRNA vectors, highlights proof-of-concept experiments demonstrating their therapeutic efficacy and explores therapeutic targets particularly suitable for retroviral-mediated gene silencing. Virology, 2004 Mar 1, 320(1), 135 - 43 Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus; Zhang J et al.; Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases . Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses . However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV) . To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33) . Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells . Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects. Methods, 2004 Apr, 32(4), 437 - 44 Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro; Keppler A et al.; The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics . We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives . Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro. Curr Opin Plant Biol, 2004 Apr, 7(2), 226 - 30 Genomics applications to biotech traits: a revolution in progress? Gutterson N, Zhang JZ. Twenty years since the inception of the agricultural biotechnology era, only two products have had a significant impact in the market place: herbicide-resistant and insect-resistant crops . Additional products have been pursued but little success has been achieved, principally because of limited understanding of key genetic intervention points . Genomics tools have fueled a new strategy for identifying candidate genes . Primarily thanks to the application of functional genomics in Arabidopsis and other plants, the industry is now overwhelmed with candidate genes for transgenic intervention points . This success necessitates the application of genomics to the rapid validation of gene function and mode of action . As one example, the development of C-box binding factors (CBFs) for enhanced freezing and drought tolerance has been rapidly advanced because of the improved understanding generated by genomics technologies. Curr Opin Plant Biol, 2004 Apr, 7(2), 219 - 25 Target-based discovery of novel herbicides; Lein W et al.; In the past 10 years, strategies for the first steps of herbicide discovery have switched from the testing of chemicals for efficacy on whole plants towards the use of in-vitro assays against molecular targets . Many different approaches have been developed to identify bona fide targets for in-vitro screening . Developments in functional genomics and in pharmaceutical research could aid the development of assay systems for the evaluation of chemicals for their suitability as lead structures in herbicide discovery. Oligonucleotides, 2003, 13(5), 411 - 9 New tools for gene manipulation in chicken embryos; Bourikas D et al.; Genomics has changed the pace by which genes are analyzed . Rather than looking at genes one by one, gene expression today is studied at the genome level . Unfortunately, the data we get from microarray analysis do not give us any clues about the function of these genes . Functional analyses are still refractory to large-scale, high-throughput studies, particularly in vertebrates . With the development of in ovo RNAi as a tool for specific gene silencing, the chicken embryo has become an efficient in vivo system to study gene function during development . A major advantage of in ovo RNAi is the fact that the knowledge of a cDNA fragment of the gene of interest is sufficient to get loss-of-function phenotypes . Thus, this new approach is a valuable tool for functional genomics. Plant J, 2004 Mar, 37(6), 897 - 905 A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis; Kuromori T et al.; More than 10 000 transposon-tagged lines were constructed by using the Activator (Ac)/Dissociation (Ds) system in order to collect insertional mutants as a useful resource for functional genomics of Arabidopsis . The flanking sequences of the Ds element in the 11 800 independent lines were determined by high-throughput analysis using a semi-automated method . The sequence data allowed us to map the unique insertion site on the Arabidopsis genome in each line . The Ds element of 7566 lines is inserted in or close to coding regions, potentially affecting the function of 5031 of 25 000 Arabidopsis genes . Half of the lines have Ds insertions on chromosome 1 (Chr . 1), in which donor lines have a donor site . In the other half, the Ds insertions are distributed throughout the other four chromosomes . The intrachromosomal distribution of Ds insertions varies with the donor lines . We found that there are hot spots for Ds transposition near the ends of every chromosome, and we found some statistical preference for Ds insertion targets at the nucleotide level . On the basis of systematic analysis of the Ds insertion sites in the 11 800 lines, we propose the use of Ds-tagged lines with a single insertion in annotated genes for systematic analysis of phenotypes (phenome analysis) in functional genomics . We have opened a searchable database of the insertion-site sequences and mutated genes and are depositing these lines in the RIKEN BioResource Center as available resources . Genome Res, 2004 Mar, 14(3), 391 - 7 Quantifying modularity in the evolution of biomolecular systems; Snel B et al.; Functional modules are considered the primary building blocks of biomolecular systems . Here we study to what extent functional modules behave cohesively across genomes:That is, are functional modules also evolutionary modules? We probe this question by analyzing for a large collection of functional modules the phyletic patterns of their genes across 110 genomes . The majority of functional modules display limited evolutionary modularity . This result confirms certain comparative genome analyses, but is in contrast to implicit assumptions in the systems analysis of functional genomics data . We show that this apparent interspecies flexibility in the organization of functional modules depends more on functional differentiation within orthologous groups of genes, than on noise in the functional module definitions . When filtering out these sources of nonmodularity, even though very few functional modules behave perfectly modular in evolution, about half behave at least significantly more modular than a random set of genes . There are substantial differences in the evolutionary modularity between individual functional modules as well as between collections of functional modules, partly corresponding to conceptual differences in the functional module definition, which make comparisons between functional module collections biologically difficult to interpret . Analysis within one collection does not suffer from such differences, and we show that within the EcoCyc metabolic pathway database, biosynthetic pathways are evolutionarily more modular than catabolic pathways. Am J Pharmacogenomics, 2004, 4(1), 29 - 43 Functional genomics approaches in arthritis; Attur MG et al.; The post-genomic era of functional genomics and target validation will allow us to narrow the bridge between clinically correlative data and causative data for complex diseases, such as arthritis, for which the etiological agent remains elusive . The availability of human and other annotated genome sequences, and parallel developments of new technologies that allow analysis of minute amounts of human and animal cells (peripheral blood cells and infiltrating cells) and tissues (synovium and cartilage) under different pathophysiological conditions, has facilitated high-throughput gene mining approaches that can generate vast amounts of clinically correlative data . Characterizing some of the correlative/causative genes will require reverting to the hypothesis-driven, low throughput method of complementary experimental biology using genomic approaches as a tool . This will include in silico gene expression arrays, genome-wide scans, comparative genomics using various animal models (such as rodents and zebrafish), bioinformatics and a team of well trained translational scientists and physicians . For the first time, the "genomic tools" will allow us to analyze small amounts of surgical samples (such as needle biopsies) and clinical samples in the context of the whole genome . Preliminary genomic analysis in osteoarthritis has already resurrected the debate on the semantic issues in the definition of inflammation . Further analyses will not only facilitate the development of unbiased hypotheses at the molecular level, but also assist us in the identification and characterization of novel targets and disease markers for pharmacological intervention, gene therapy, and diagnosis. Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2888 - 93 Epub 2004 Feb 23. Whole-genome annotation by using evidence integration in functional-linkage networks; Karaoz U et al.; The advent of high-throughput biology has catalyzed a remarkable improvement in our ability to identify new genes . A large fraction of newly discovered genes have an unknown functional role, particularly when they are specific to a particular lineage or organism . These genes, currently labeled "hypothetical," might support important biological cell functions and could potentially serve as targets for medical, diagnostic, or pharmacogenomic studies . An important challenge to the scientific community is to associate these newly predicted genes with a biological function that can be validated by experimental screens . In the absence of sequence or structural homology to known genes, we must rely on advanced biotechnological methods, such as DNA chips and protein-protein interaction screens as well as computational techniques to assign putative functions to these genes . In this article, we propose an effective methodology for combining biological evidence obtained in several high-throughput experimental screens and integrating this evidence in a way that provides consistent functional assignments to hypothetical genes . We use the visualization method of propagation diagrams to illustrate the flow of functional evidence that supports the functional assignments produced by the algorithm . Our results contain a number of predictions and furnish strong evidence that integration of functional information is indeed a promising direction for improving the accuracy and robustness of functional genomics. Infect Immun, 2004 Mar, 72(3), 1265 - 74 A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp . paratuberculosis encodes cell surface proteins expressed in the host; Stratmann J et al.; We have used representational difference analysis to identify a novel Mycobacterium avium subsp . paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes . Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases . Together these genes form a 38-kb M . avium subsp . paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110 . Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M . avium subsp . paratuberculosis . The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants . The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host . Together, these data suggest that the 38-kb locus constitutes an M . avium subsp . paratuberculosis pathogenicity island. Reprod Fertil Dev, 2004, 16(2), 87 - 92 Serial analysis of gene expression (SAGE) during porcine embryo development; Blomberg le A et al.; Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos . High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig . The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications . Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos . However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis . The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12 . First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development . Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived. Biochemistry, 2004 Feb 24, 43(7), 1921 - 7 Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids; Liu Y et al.; Peptide nucleic acids (PNAs) are a potentially powerful approach for the recognition of cellular mRNA and the inhibition of gene expression . Despite their promise, the rules for using antisense PNAs have remained obscure, and antisense PNAs have been used sparingly in research . Here we investigate the ability of PNAs to be effective antisense agents inside mammalian cells, to inhibit expression of human caveolin-1 (hCav-1), and to discriminate between its alpha and beta isoforms . Many human genes are expressed as isoforms . Isoforms may play different roles within a cell or within different tissues, and defining these roles is a challenge for functional genomics and drug discovery . PNAs targeted to the translation start codons for the alpha and beta isoforms inhibit expression of hCav-1 . Inhibition is dependent on PNA length . The potency and duration of inhibition by PNAs are similar to inhibition of gene expression by short interferring RNA (siRNA) . Expression of the alpha isoform can be blocked selectively by a PNA . Cell proliferation is halted by inhibition of expression of both hCav-1 isoforms, but not by inhibition of the alpha hCav-1 isoform alone . Efficient antisense inhibition and selective modulation of isoform expression suggest that PNAs are versatile tools for controlling gene expression and dissecting the roles of closely related protein variants . Potent inhibition by PNAs may supply a "knock down" technology that can complement and "cross-check" siRNA and other approaches to antisense gene inhibition that rely on oligomers with phosphate or phosphorothioate backbone linkages. Plant Physiol, 2004 Feb, 134(2), 548 - 59 Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry; von Roepenack-Lahaye E et al.; Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology . The metabolome of a cell or an organism is chemically highly complex . Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques . Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry . About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves . Many of these originate from Arabidopsis secondary metabolites . Detection based on retention times and exact masses is robust and reproducible . The dynamic range is sufficient for the quantification of metabolites . Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability . Tools were optimized or established for the automatic data deconvolution and data processing . Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant . The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites . In-source fragmentation and tandem mass spectrometry can be used to gain structural information . This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems . The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling. Mol Plant Microbe Interact, 2004 Feb, 17(2), 175 - 83 Establishment of a functional genomics platform for Leifsonia xyli subsp . xyli; Brumbley SM et al.; Leifsonia xyli subsp . xyli, the causal agent of ratoon stunting disease in sugarcane, is a xylem-limited, nutritionally fastidious, slow growing, gram-positive coryneform bacterium . Because of the difficulties in growing this bacterium in pure culture, little is known about the molecular mechanisms of pathogenesis . Currently, the genome sequence of L . xyli subsp . xyli is being completed by the Agronomical and Environmental Genomes group from the Organization for Nucleotide Sequencing and Analysis in Brazil . To complement this work, we produced 712 Lxx::Tn4431 transposon mutants and sequenced flanking regions from 383 of these, using a rapid polymerase chain reaction-based approach . Tn4431 insertions appeared to be widespread throughout the L . xyli subsp . xyli genome; however, there were regions that had significantly higher concentrations of insertions . The Tn4431 mutant library was screened for individuals unable to colonize sugarcane, and one noncolonizing mutant was found . The mutant contained a transposon insertion disrupting two open reading frames (ORF), one of which had homology to an integral membrane protein from Mycobacterium leprae . Sequencing of the surrounding regions revealed two operons, pro and cyd, both of which are believed to play roles in disease . Complementation studies were carried out using the noncolonizing Lxx::Tn4431 mutant . The noncolonizing mutant was transformed with a cosmid containing 40 kbp of wild-type sequence, which included the two ORF disrupted in the mutant, and several transformants were subsequently able to colonize sugarcane . However, analysis of each of these transformants, before and after colonization, suggests that they have all undergone various recombinant events, obscuring the roles of these ORF in L . xyli subsp . xyli pathogenesis. Mar Biotechnol (NY), 2002 Dec, 4(6), 526 - 36 Expression profile of the channel catfish spleen: analysis of genes involved in immune functions; Kocabas AM et al.; Both qualitative and quantitative patterns of tissue-specific gene expression can be determined using gene profiling . Expressed sequence tag (EST) analysis is an efficient approach not only for gene discovery and examining gene expression, but also for development of molecular resources useful for functional genomics . As part of an ongoing transcriptome analysis of channel catfish (Ictalurus punctatus), EST analysis was conducted for gene annotations and profiling using a complementary DNA library developed from messenger RNA of the spleen . A total of 1204 spleen cDNA clones were analyzed . Of the 1204 clones, 665 clones (55.2%) were identified as orthologs of known genes from other organisms by BLAST searches and 539 clones (44.8%) as unknown gene clones . In total 147 novel genes were identified, and annotations were made to 118 of them . In addition, 389 novel EST clusters were identified . Expression profile was analyzed in relation to metabolic functional groups . A total of 28 known genes were involved in immune functions, of which 10 were identified for the first time in channel catfish . Microsatellite-containing clones were also identified that may be potentially useful for genome mapping . This work contributed to the Catfish Gene Index, and toward a Unigene set useful for functional genomics research concerning spleen gene functions in relation to disease defenses. Bioinformatics, 2004 Feb 12, 20(3), 430 - 2 Epub 2004 Jan 22. Gene specific siRNA selector; Levenkova N et al.; Small interfering RNA (siRNA) is used in functional genomics applications to decrease the expression of a target gene, which may yield a biological effect that suggests a function for the target gene . The siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules . Selected candidates are then screened to identify those siRNA sequences that are specific to the gene of interest. Nucleic Acids Res, 2004 Feb 09, 32(3), 936 - 48 Print 2004. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference; Ui-Tei K et al.; In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells . We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed . These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length . siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells . Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos . In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi. Prog Biophys Mol Biol, 2004 Feb-Apr, 84(2-3), 107 - 23 Functional genomics as an emerging strategy for the investigation of central mechanisms in experimental hypertension; Veerasingham SJ et al.; 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. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2004 Feb, 21(1), 47 - 51 {Mouse whole mount RNA in situ hybridization: an effective technique for analyzing gene expression}; Xiao CY et al.; OBJECTIVE: To set up a method of analyzing gene expression profile from mouse whole embryos . METHODS: Mouse whole mount RNA in situ hybridization(WM-ISH) of E10.5-E14 embryos was carried out by using digoxigenin-labeled Runx1 and Runx3 RNA probes and their expression profile was observed by detecting the existence and status of corresponding mRNAs in the embryonic tissues.RESULTS: Clear hybridization signals were observed in different tissues and organs hybridized by Runx1 or Runx3 RNA probe . Different probes and ages of embryos had need of their own optimal proteinase K treatment conditions . CONCLUSION: Mouse whole mount RNA in situ hybridization is an effective method of analyzing gene expression . It is useful for revealing whole gene expression profile and has a great potentiality in the era of functional genomics . It provides an alternative method of studies on gene expression which is at least as good as LacZ staining and immunohistochemistry . The key factor of the success to mouse whole mount RNA in situ hybridization is whether the proteinase K treatment conditions are optimal or not. Nucleic Acids Res, 2004 Feb 03, 32(2), 742 - 8 Print 2004. High-throughput protein analysis integrating bioinformatics and experimental assays; del Val C et al.; The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis . Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated . We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium . The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity . Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g . PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT) . Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis . The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins. J Biochem Mol Biol, 2004 Jan 31, 37(1), 133 - 8 Proteomic studies in plants; Park OK; Proteomics is a leading technology for the high-throughput analysis of proteins on a genome-wide scale . With the completion of genome sequencing projects and the development of analytical methods for protein characterization, proteomics has become a major field of functional genomics . The initial objective of proteomics was the large-scale identification of all protein species in a cell or tissue . The applications are currently being extended to analyze various functional aspects of proteins such as post-translational modifications, protein-protein interactions, activities and structures . Whereas the proteomics research is quite advanced in animals and yeast as well as Escherichia coli, plant proteomics is only at the initial phase . Major studies of plant proteomics have been reported on subcellular proteomes and protein complexes (e.g . proteins in the plasma membranes, chloroplasts, mitochondria and nuclei) . Here several plant proteomics studies will be presented, followed by a recent work using multidimensional protein identification technology (MudPIT). J Biochem Mol Biol, 2004 Jan 31, 37(1), 122 - 32 Functional genomics approach using mice; Sung YH et al.; The rapid development and characterization of the mouse genome sequence, coupled with comparative sequence analysis of human, has been paralleled by a reinforced enthusiasm for mouse functional genomics . The way to uncover the in vivo function of genes is to analyze the phenotypes of the mutant animals . From this standpoint, the mouse is a suitable and valuable model organism in the studies of functional genomics . Therefore, there have been enormous efforts to enrich the list of the mutant mice . Such a trend emphasizes the random mutagenesis, including ENU mutagenesis and gene-trap mutagenesis, to obtain a large stock of mutant mice . However, since various mutant alleles are needed to precisely characterize the role of a gene in vivo, mutations should be designed . The simplicity and utility of transgenic technology can satisfy this demand . The combination of RNA interference with transgenic technology will provide more opportunities for researchers . Nevertheless, gene targeting can solely define the in vivo function of a gene without a doubt . Thus, transgenesis and gene targeting will be the major strategies in the field of functional genomics. J Biochem Mol Biol, 2004 Jan 31, 37(1), 93 - 106 Yeast as a touchstone in post-genomic research: strategies for integrative analysis in functional genomics; Castrillo JI et al.; The new complexity arising from the genome sequencing projects requires new comprehensive post-genomic strategies: advanced studies in regulatory mechanisms, application of new high-throughput technologies at a genome-wide scale, at the different levels of cellular complexity (genome, transcriptome, proteome and metabolome), efficient analysis of the results, and application of new bioinformatic methods in an integrative or systems biology perspective . This can be accomplished in studies with model organisms under controlled conditions . In this review a perspective of the favourable characteristics of yeast as a touchstone model in post-genomic research is presented . The state-of-the art, latest advances in the field and bottlenecks, new strategies, new regulatory mechanisms, applications (patents) and high-throughput technologies, most of them being developed and validated in yeast, are presented . The optimal characteristics of yeast as a well-defined system for comprehensive studies under controlled conditions makes it a perfect model to be used in integrative, "systems biology" studies to get new insights into the mechanisms of regulation (regulatory networks) responsible of specific phenotypes under particular environmental conditions, to be applied to more complex organisms (e.g . plants, human). J Biochem Mol Biol, 2004 Jan 31, 37(1), 83 - 92 From the sequence to cell modeling: comprehensive functional genomics in Escherichia coli; Mori H; As a result of the enormous amount of information that has been collected with E . coli over the past half century (e.g . genome sequence, mutant phenotypes, metabolic and regulatory networks, etc.), we now have detailed knowledge about gene regulation, protein activity, several hundred enzyme reactions, metabolic pathways, macromolecular machines, and regulatory interactions for this model organism . However, understanding how all these processes interact to form a living cell will require further characterization, quantification, data integration, and mathematical modeling, systems biology . No organism can rival E . coli with respect to the amount of available basic information and experimental tractability for the technologies needed for this undertaking . A focused, systematic effort to understand the E . coli cell will accelerate the development of new post-genomic technologies, including both experimental and computational tools . It will also lead to new technologies that will be applicable to other organisms, from microbes to plants, animals, and humans . E . coli is not only the best studied free-living model organism, but is also an extensively used microbe for industrial applications, especially for the production of small molecules of interest . It is an excellent representative of Gram-negative commensal bacteria . E . coli may represent a perfect model organism for systems biology that is aimed at elucidating both its free-living and commensal life-styles, which should open the door to whole-cell modeling and simulation. Ann N Y Acad Sci, 2003 Dec, 1002, 56 - 62 Short interfering RNA (siRNA)-mediated RNA interference (RNAi) in human cells; Caplen NJ et al.; Transient gene silencing in mammalian cells can be mediated by double stranded RNA (dsRNAs) molecules of approximately 20-25 nucleotides termed short interfering (siRNAs) . Naturally occurring siRNAs in lower eukaryotes have characteristic structural elements; however, little is known about what features are critical for an exogenous siRNA to mediate RNAi in mammals . We have recently determined some of the critical parameters that influence the efficiency of siRNA-mediated RNAi in mammalian cells and have been considering the use of RNAi as a functional genomics tool, particularly for high throughput analysis, and the potential use of RNAi as a therapeutic tool. Diabetes, 2004 Feb, 53 Suppl 1, S159 - 65 Chronic effects of fatty acids on pancreatic beta-cell function: new insights from functional genomics; Biden TJ et al.; Type 2 diabetes can be viewed as a failure of the pancreatic beta-cell to compensate for peripheral insulin resistance with enhanced insulin secretion . This failure is explained by both a relative loss of beta-cell mass as well as secretory defects that include enhanced basal secretion and a selective loss of sensitivity to glucose . These features are reproduced by chronic exposure of beta-cells to fatty acids (FAs), suggesting that hyperlipidemia might contribute to decompensation . Using MIN6 cells pretreated for 48 h with oleate or palmitate, we have previously defined alterations in global gene expression by transcript profiling and described additional secretory changes to those already established (Busch A-K, Cordery D, Denyer G, Biden TJ: Diabetes 51:977-987, 2002) . In contrast to a modest decoupling of glucose-stimulated insulin secretion, FA pretreatment markedly enhanced the secretory response to an acute subsequent challenge with FAs . We propose that this apparent switch in sensitivity from glucose to FAs would be an appropriate response to hyperlipidemia in vivo and thus plays a positive role in beta-cell compensation for insulin resistance . Altered expression of dozens of genes could contribute to this switch, and allelic variations in any of these genes could (to varying degrees) impair beta-cell compensation and thus contribute to conditions ranging from impaired glucose tolerance to frank diabetes. Physiol Genomics, 2004 Apr 13, 17(2), 140 - 9 An integrated reverse functional genomic and metabolic approach to understanding orotic acid-induced fatty liver; Griffin JL et al.; In functional genomics, DNA microarrays for gene expression profiling are increasingly being used to provide insights into biological function or pathology . To better understand the significance of the multiple transcriptional changes across a time period, the temporal changes in phenotype must be described . Orotic acid-induced fatty liver disease was investigated at the transcriptional and metabolic levels using microarrays and metabolic profiling in two strains of rats . High-resolution 1H-NMR spectroscopic analysis of liver tissue indicated that Kyoto rats compared with Wistar rats are predisposed to the insult . Metabolite analysis and gene expression profiling following orotic acid treatment identified perturbed metabolic pathways, including those involved in fatty acid, triglyceride, and phospholipid synthesis, beta-oxidation, altered nucleotide, methyl donor, and carbohydrate metabolism, and stress responses . Multivariate analysis and statistical bootstrapping were used to investigate co-responses with transcripts involved in metabolism and stress responses . This reverse functional genomic strategy highlighted the relationship between changes in the transcription of stearoyl-CoA desaturase 1 and those of other lipid-related transcripts with changes in NMR-derived lipid profiles . The results suggest that the integration of 1H-NMR and gene expression data sets represents a robust method for identifying a focused line of research in a complex system. C R Biol, 2003 Oct-Nov, 326(10-11), 1083 - 7 Microarray analysis using bioinformatics analysis audit trails (BAATs); Bellgard M et al.; Bioinformatics analysis plays an integrative role in genomics and functional genomics . The ability to conduct quality managed, hypothesis-driven bioinformatics analysis with the plethora of data available is mandatory . Biological interpretation of this data is dependent on versions of databases, programs and the parameters used . Thus, tracking and auditing the analyses process is important . This paper outlines what we term Bioinformatics Analysis Audit Trails (BAATs) and describes YABI, a bioinformatics environment that implements BAATs . YABI can incorporate most bioinformatics tools within the same environment, making it a valuable resource. C R Biol, 2003 Oct-Nov, 326(10-11), 1003 - 9 CDNAs for functional genomics and proteomics: the German Consortium; Wiemann S et al.; To functionally characterize numerous novel proteins encoded by cDNAs sequenced by the German Consortium, 800 were tagged with green fluorescent protein . The subcellular localizations of the fusion proteins were examined in living cells, enabling their classification in subcellular groups . Their activity in cell growth, cell death, and protein transport was screened in high throughput using robotic liquid handling and reading stations . The resulting information is integrated with functional genomics and proteomics data for further understanding of protein functions in the cellular context. C R Biol, 2003 Oct-Nov, 326(10-11), 879 - 92 From functional genomics to systems biology: concepts and practices; Auffray C et al.; Systems biology is the iterative and integrative study of biological systems as systems in response to perturbations . It is founded on hypotheses formalized in models built from the results of global functional genomics analyses of the complexity of the genome, transcriptome, proteome, metabolome, etc . Its implementation by cross-disciplinary teams in a standardized mode under quality assurance should allow accessing the small variations of the large number of elements determining functioning of biological systems . Galactose utilization in yeast, and sea urchin development are two examples of emerging systems biology. C R Biol, 2003 Oct-Nov, 326(10-11), 867 - 75 Foreword: from the TRANSCRIPTOME conferences to the SYSTEMOSCOPE international consortium; Auffray C et al.; This thematic issue issue of the Comptes rendus Biologies contains review articles, original papers and conference reports presented at the first two TRANSCRIPTOME conferences From Functional Genomics to Systems Biology and IMAGE Consortium Invitational workshops (Paris, November 2000 and Seattle, March 2002), and discussed during the inaugural meetings of the SYSTEMOSCOPE International Consortium (Paris, June 2003) . We describe the founding principles, missions, working plan and policy for partnership and industrial development of SYSTEMOSCOPE to promote the study of the complexity of biological systems by integrating scientific, medical, ethical and economic issues in implementation of interdisciplinary projects for human health. Nucleic Acids Res . 2004 Jan 22;32(2):e17. A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells; Wu W et al.; An important aim in the post-sequencing age of functional genomics is to translate gene sequences into protein functions . This shift of focus is particularly necessary for a very large number of human genes, referred to as novel genes, where we have no or very rudimentary information about their biochemical functions . Recently, a new method for investigating human gene functions using small interfering RNA molecules (siRNAs) has become available . siRNAs are powerful reagents for post-transcriptional silencing, where mRNA targeted by the siRNAs is degraded in vivo and the level of the encoded protein is reduced . However, the lack of antibodies against proteins encoded by novel genes restricts the general value of siRNAs as functional tools, as only the mRNA levels can be measured for these genes . We report a method that combines measurements of protein levels in cell culture for novel, exogenously expressed genes, with parallel measurements of the endogenous mRNA levels of the same genes . We find that this combinatorial approach correctly predicts siRNAs that efficiently reduce mRNA and protein levels in cultured cells . Furthermore, this method identifies proteins that have a slow turnover, which weakens the value of the RNA interference method as a tool for functional studies of such genes . The described method should prove to be valuable for large-scale functional studies of novel human genes. Semin Nucl Med, 2004 Jan, 34(1), 4 - 22 Impact of functional genomics and proteomics on radionuclide imaging; Haberkorn U et al.; The assessment of gene function following the completion of human genome sequencing may be performed using radionuclide imaging procedures . These procedures are needed for the evaluation of genetically manipulated animals or newly designed biomolecules, which requires a thorough understanding of physiology, biochemistry, and pharmacology . The experimental approaches will involve many new technologies, including in vivo imaging with single photon emission computed tomography and positron emission tomography . Nuclear medicine procedures may be applied for the determination of gene function and regulation using established and new tracers, or using in vivo reporter genes, such as genes encoding enzymes, receptors, antigens, or transporters . Visualization of in vivo reporter gene expression can be performed using radiolabeled substrates, antibodies, or ligands . Combinations of specific promoters and in vivo reporter genes may deliver information about the regulation of the corresponding genes . Furthermore, protein-protein interactions and activation of signal transduction pathways may be visualized noninvasively . The role of radiolabeled antisense molecules for the analysis of messenger ribonucleic acid (RNA) content has to be investigated . However, possible applications are therapeutic intervention using triplex oligonucleotides with therapeutic isotopes, which can be brought near to specific deoxyribonucleic acid sequences to induce deoxyribonucleic acid strand breaks at selected loci . Imaging of labeled siRNA makes sense if these are used for therapeutic purposes to assess the delivery of these new drugs to their target tissue . Pharmacogenomics will identify new surrogate markers for therapy monitoring, which may represent potential new tracers for imaging . Drug distribution studies for new therapeutic biomolecules are needed at least during preclinical stages of drug development . New treatment modalities, such as gene therapy with suicide genes, will need procedures for therapy planning and monitoring . Finally, new biomolecules will be developed by bioengineering methods, which may be used for the isotope-based diagnosis and treatment of disease. Bioinformatics, 2004 Jan 22, 20(2), 282 - 3 FGDP: functional genomics data pipeline for automated, multiple microarray data analyses; Grant JD et al.; Gene expression microarrays and oligonucleotide GeneChips have provided biologists with a means of measuring, in a single experiment, the expression levels of entire genomes under a variety of conditions . As with any nascent field, there is no single accepted method for analyzing the new data types, with new methods appearing monthly . Investigators using the new technology must constantly seek access to the latest tools and explore their data in multiple ways . The functional genomics data pipeline provides an integrated, extendable analysis environment permitting multiple, simultaneous analyses to be automatically performed and provides a web server and interface for presenting results . AVAILABILITY: Source code and executables are available under the GNU public license at http://bioinformatics.fccc.edu/ Bioinformatics, 2004 Jan 22, 20(2), 161 - 9 Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome; Zhu Q et al.; MOTIVATION: Multiple chitinases as well as lectins closely related to them have been characterized previously from many insect species and the corresponding genes/cDNAs have been cloned . However, the identification of the entire assortment of genes for chitinase family proteins and their differences in biochemical properties have not been carried out in any individual insect species . The completion of the entire DNA sequence of Drosophila melanogaster (fruit fly) genome and identification of open reading frames presents an opportunity to study the structures and functions of chitinase-like proteins, and also to identify new members of this family in DROSOPHILA: We are, therefore, interested in studying the functional genomics of chitinase-like gene families in insects . METHODS: We searched the Drosophila protein sequences database using fully characterized insect chitinase sequences and BLASTP software, identified all the putative chitinase-like proteins encoded in Drosophila genome, and predicted their structures using domain analysis tools . A phylogenetic analysis of the chitinase-like proteins from Drosophila and several other insect species was carried out . The structures of these chitinases were modeled using homology modeling software . RESULTS: Our analysis revealed the presence of 18 chitinase-like proteins in the Drosophila protein database . Among these are seven novel chitinase-like proteins that contain four signature amino acid sequences of chitinases belonging to family 18 glycosylhydrolases, including both acidic and hydrophobic amino acid residues critical for enzyme activity . All the proteins contain at least one catalytic domain with one having four catalytic domains . Phylogenetic analysis of chitinase-like proteins from Drosophila and other insects revealed an evolutionary relationship among all these proteins, which indicated gene duplication and domain shuffling to generate the observed diversity in the encoded proteins . Homology modeling showed that all the Drosophila chitinase-like proteins contain one or more catalytic domains with a (alpha/beta)8 barrel-like structure . Our results suggest that insects utilize multiple family 18 chitinolytic enzymes and also non-enzymatic chitinase-like proteins for degrading/remodeling/binding to chitin in different insect anatomical extracellular structures, such as the cuticle, peritrophic membrane, trachea and mouth parts during insect development, and possibly for other roles including chitin synthesis . AVAILABILITY: Perl program and supplementary material are available at http://www.ksu.edu/bioinformatics/supplementary.htm Metab Eng, 2004 Jan, 6(1), 85 - 100 New stable isotope-mass spectrometric techniques for measuring fluxes through intact metabolic pathways in mammalian systems: introduction of moving pictures into functional genomics and biochemical phenotyping; Hellerstein MK; The thesis of this review is that fully assembled metabolic pathways in living systems, rather than genes or proteins, are the true units of function in biology and biochemistry . A corollary is that measurement of metabolic fluxes (biochemical kinetics) is thereby required to understand biochemical control and gene function . Recent methodologic advances for improving observability of metabolic pathway fluxes in vivo are reviewed . Stable isotope-mass spectrometric techniques discussed here include mass isotopomer distribution analysis (combinatorial analysis), for measurement of polymerization biosynthesis; 2H(2)O administration, for measuring synthesis of DNA (i.e., cell proliferation), RNA, proteins, lipids, glycolipids and other classes of molecules; non-invasive probes of intracellular metabolism, by sampling secreted metabolites in accessible body fluids, after isotopic labeling of the intracellular pathway; and measurement of multiple molecular fluxes concurrently, particularly through use of 2H(2)O . Examples are given of pathway fluxes measured by each of these techniques, noting the often-surprising results . It is concluded that the introduction of "moving pictures" as tools for biochemical phenotyping could radically alter many signature areas of contemporary biology, including functional genomics, drug discovery and development, and disease research. Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2674 - 7 A transient RNA interference assay system using Arabidopsis protoplasts; An CI et al.; Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi) . RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants . We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87) . Introduction of dsRNA into protoplasts led to marked silencing of target transgenes . Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana. Plant Physiol, 2004 Jan, 134(1), 67 - 80 The Arabidopsis root transcriptome by serial analysis of gene expression . Gene identification using the genome sequence; Fizames C et al.; Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs . For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome . The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis . This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases . Moreover, 85% of the tags were specific for one gene . To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families . These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies . However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes . The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system . As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3- or NH4NO3 as N source. Am J Cardiovasc Drugs, 2002, 2(5), 287 - 96 Pharmacogenetics, pharmacogenomics, and cardiovascular therapeutics: the way forward; Terra SG et al.; The completion of sequencing of the human genome will be the vanguard for numerous advances in medicine . The first discernible application is likely to occur in pharmacogenomics, a field focused on the influence of genetic differences on the variability in patients' response to medications . While an inherited basis for drug response has been recognized for some time, it is the confluence of molecular biology, high-throughput genotyping, and bioinformatics that has made it practical to study the genetic basis of variability to medications on a large scale . Pharmacogenomics may enable clinicians to prospectively identify patients most likely to derive benefit from a drug, with minimal likelihood of adverse events . This DNA-based approach to predicting clinical drug efficacy and toxicity would shift the current prescribing paradigm from its empirical nature to a more patient-specific model, ushering in a new era of personalized medicine . Polymorphisms in drug metabolizing enzymes, drug targets, and disease pathogenesis genes are associated with therapeutic effect to cardiovascular pharmacotherapy . Moreover, pharmacogenomics and functional genomics are expected to have a profound impact on the process of drug discovery and development . Finally, pharmacogenomics is likely to transform the way clinical trials are conducted by allowing for the selection of a more homogeneous study population, thereby reducing the size and cost of clinical investigation. J Pers Soc Psychol, 2004 Jan, 86(1), 112 - 29 Intelligence: genetics, genes, and genomics; Plomin R et al.; More is known about the genetics of intelligence than about any other trait, behavioral or biological, which is selectively reviewed in this article . Two of the most interesting genetic findings are that heritability of intelligence increases throughout the life span and that the same genes affect diverse cognitive abilities . The most exciting direction for genetic research on intelligence is to harness the power of the Human Genome Project to identify some of the specific genes responsible for the heritability of intelligence . The next research direction will be functional genomics--for example, understanding the brain pathways between genes and intelligence . Deoxyribonucleic acid (DNA) will integrate life sciences research on intelligence; bottom-up molecular biological research will meet top-down psychological research in the brain. J Appl Physiol, 2004 Feb, 96(2), 765 - 73 Functional genomics approach to hypoxia signaling; Seta KA et al.; Mammalian cells require a constant supply of oxygen to maintain energy balance, and sustained hypoxia can result in cell death . It is therefore not surprising that sophisticated adaptive mechanisms have evolved that enhance cell survival during hypoxia . During the past few years, there have been a growing number of reports on hypoxia-induced transcription of specific genes . In this review, we describe a unique experimental approach that utilizes focused cDNA libraries coupled to microarray analyses to identify hypoxia-responsive signal transduction pathways and genes that confer the hypoxia-tolerant phenotype . We have used the subtractive suppression hybridization (SSH) method to create a cDNA library enriched in hypoxia-regulated genes in oxygen-sensing pheochromocytoma cells and have used this library to create microarrays that allow us to examine hundreds of genes at a time . This library contains over 300 genes and expressed sequence tags upregulated by hypoxia, including tyrosine hydroxylase, vascular endothelial growth factor, and junB . Hypoxic regulation of these and other genes in the library has been confirmed by microarray, Northern blot, and real-time PCR analyses . Coupling focused SSH libraries with microarray analyses allows one to specifically study genes relevant to a phenotype of interest while reducing much of the biological noise associated with these types of studies . When used in conjunction with high-throughput, dye-based assays for cell survival and apoptosis, this approach offers a rapid method for discovering validated therapeutic targets for the treatment of cardiovascular disease, stroke, and tumors. Adv Parasitol, 2003, 54, 101 - 95 Nematoda: genes, genomes and the evolution of parasitism; Blaxter ML; Nematodes are remarkably successful, both as free-living organisms and as parasites . The diversity of parasitic lifestyles displayed by nematodes, and the diversity of hosts used, reflects both a propensity towards parasitism in the phylum, and an adaptability to new and challenging environments . Parasitism of plants and animals has evolved many times independently within the Nematoda . Analysis of these origins of parasitism using a molecular phylogeny highlights the diversity underlying the parasitic mode of life . Many vertebrate parasites have arthropod-associated sister taxa, and most invade their hosts as third stage larvae: these features co-occur across the tree and thus suggest that this may have been a shared route to parasitism . Analysis of nematode genes and genomes has been greatly facilitated by the Caenorhabditis elegans project . However, the availability of the whole genome sequence from this free-living rhabditid does not simply permit definition of 'parasitism' genes; each nematode genome is a mosaic of conserved features and evolutionary novelties . The rapid progress of parasitic nematode genome projects focussing on species from across the diversity of the phylum has defined sets of genes that have patterns of evolution that suggest their involvement with various facets of parasitism, in particular the problems of acquisition of nutrients in new hosts and the evasion of host immune defences . With the advent of functional genomics techniques in parasites, and in particular the possibility of gene knockout using RNA interference, the roles of many putative parasitism genes call now be tested. Mol Psychiatry, 2004 Feb, 9(2), 128 - 43 Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE); Swatton JE et al.; Two-dimensional gel electrophoresis (2-D GE) is a key tool for comparative proteomics research . With its ability to separate complex protein mixtures with high resolution, 2-D GE is a technique commonly employed for protein profiling studies . Significant improvements have been made in 2-D GE technology with the development of two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), where proteins are first labelled with one of three spectrally resolvable fluorescent cyanine dyes before being separated over first and second dimensions according to their charge and size, respectively . When used in conjunction with automated analysis packages, this multiplexing approach can accurately and reproducibly quantify protein expression for control and experimental groups . Differentially expressed proteins can be subsequently identified by mass spectrometric methods . Here, we describe the successful application and optimisation of 2-D DIGE technology for human postmortem brain studies . This technology, especially when coupled with other functional genomics approaches, such as transcriptomics and metabolomics studies, will enhance our current understanding of human disease and lead to new therapeutic and diagnostic possibilities. Proteins, 2004 Jan 1, 54(1), 49 - 57 Detection of functional modules from protein interaction networks; Pereira-Leal JB et al.; Complex cellular processes are modular and are accomplished by the concerted action of functional modules (Ravasz et al., Science 2002;297:1551-1555; Hartwell et al., Nature 1999;402:C47-52) . These modules encompass groups of genes or proteins involved in common elementary biological functions . One important and largely unsolved goal of functional genomics is the identification of functional modules from genomewide information, such as transcription profiles or protein interactions . To cope with the ever-increasing volume and complexity of protein interaction data (Bader et al., Nucleic Acids Res 2001;29:242-245; Xenarios et al., Nucleic Acids Res 2002;30:303-305), new automated approaches for pattern discovery in these densely connected interaction networks are required (Ravasz et al., Science 2002;297:1551-1555; Bader and Hogue, Nat Biotechnol 2002;20:991-997; Snel et al., Proc Natl Acad Sci USA 2002;99:5890-5895) . In this study, we successfully isolate 1046 functional modules from the known protein interaction network of Saccharomyces cerevisiae involving 8046 individual pair-wise interactions by using an entirely automated and unsupervised graph clustering algorithm . This systems biology approach is able to detect many well-known protein complexes or biological processes, without reference to any additional information . We use an extensive statistical validation procedure to establish the biological significance of the detected modules and explore this complex, hierarchical network of modular interactions from which pathways can be inferred . Nat Genet, 2004 Jan, 36(1), 40 - 5 Epub 2003 Dec 21. Complete sequencing and characterization of 21,243 full-length human cDNAs; Ota T et al.; As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs . We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection . About half of them (5,416) seemed to be protein-coding . Of those, 1,999 clusters had not been predicted by computational methods . The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs . Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures . The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs . About one-fourth of them (1,378) showed a clear pattern of splicing . The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs. Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 716 - 20 Retrovirus vector-mediated stable gene silencing in human cell; Liu CM et al.; RNA interference (RNAi) is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by short interfering RNAs (siRNAs) homologous to the gene being suppressed . siRNAs, which mediate sequence-specific mRNA degradation, are duplexes of about 21-23 nucleotides with 3'-overhangs synthesized in vitro or expressed by DNA-based vector . However, these systems rely on transfection for delivery and cannot generate long-term gene silencing in vivo . This obstacle may be circumvented by recently developed retrovirus- and lentivirus-delivered RNAi . Here, we describe a retroviral system for delivery of siRNA into cells, which can substantially down-regulate the expression of human p53 gene in human HepG2 cells . What's more, the G1 and S phases of cell cycle change dramatically in p53-down-regulated cells . These results indicate that retrovirus vector-delivered RNAi may be used in functional genomics and in gene therapy. Bioessays, 2004 Jan, 26(1), 39 - 49 Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans; Jones AK et al.; Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that bring about a diversity of fast synaptic actions . Analysis of the Caenorhabditis elegans genome has revealed one of the most-extensive and diverse nAChR gene families known, consisting of at least 27 subunits . Striking variation with possible functional implications has been observed in normally conserved motifs at the acetylcholine-binding site and in the channel-lining region . Some nAChR subunits are particular to neurons whilst others are present in both neurons and muscles . The localization of subunits in non-synaptic regions suggests novel roles for nAChRs . Genetic and heterologous expression studies have identified a subset of nAChR subunits that are important drug targets while the study of mutants has identified genes functionally-linked to nAChRs . Future studies using C . elegans offer the prospect of increasing our understanding of the functional diversity of a complex nAChR gene family as well as addressing the role of nAChRs and associated proteins in human disorders . Bioinformatics, 2004 Jan 1, 20(1), 45 - 50 ClusPro: an automated docking and discrimination method for the prediction of protein complexes; Comeau SR et al.; MOTIVATION: Predicting protein interactions is one of the most challenging problems in functional genomics . Given two proteins known to interact, current docking methods evaluate billions of docked conformations by simple scoring functions, and in addition to near-native structures yield many false positives, i.e . structures with good surface complementarity but far from the native . RESULTS: We have developed a fast algorithm for filtering docked conformations with good surface complementarity, and ranking them based on their clustering properties . The free energy filters select complexes with lowest desolvation and electrostatic energies . Clustering is then used to smooth the local minima and to select the ones with the broadest energy wells-a property associated with the free energy at the binding site . The robustness of the method was tested on sets of 2000 docked conformations generated for 48 pairs of interacting proteins . In 31 of these cases, the top 10 predictions include at least one near-native complex, with an average RMSD of 5 A from the native structure . The docking and discrimination method also provides good results for a number of complexes that were used as targets in the Critical Assessment of PRedictions of Interactions experiment . AVAILABILITY: The fully automated docking and discrimination server ClusPro can be found at http://structure.bu.edu Mol Cell, 2003 Dec, 12(6), 1343 - 4 Functional Genomics: all the king's horses and all the king's men can put Humpty together again; Oliver S; The integration of data from different kinds of high-throughput functional genomic analyses not only permits the accurate determination of the functions of novel genes, but also presents the prospect of a "systems" approach to biology in the future. Curr Cancer Drug Targets, 2003 Dec, 3(6), 445 - 53 Metabolic biomarker and kinase drug target discovery in cancer using stable isotope-based dynamic metabolic profiling (SIDMAP); Boros LG et al.; Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and re-distribution among biosynthetic pathways . This results in a proliferating phenotype with altered cellular function . These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty acid synthesis and TCA cycle glucose oxidation . This primarily non-oxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment . Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways . Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation . This can be demonstrated by the broad labeling and unique specificity of {1,2-(13)C(2)}glucose to trace a large number of metabolites in the metabolome . Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs . It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing. Curr Pharm Biotechnol, 2003 Dec, 4(6), 417 - 37 Software packages for quantitative microarray-based gene expression analysis; Dresen IM et al.; Microarray technology enables researchers to investigate the expression of several thousand genes simultaneously . The whole transcriptional response of these genes in normal cells or tissue, in disease condition, as an response to biological, genetical or chemical stimuli or during normal biological processes such as cell cycle or embryonic development can be investigated . This leads to a huge amount of data, from which the relevant information has to be extracted by statistical and computational methods . Several software packages for the analysis of gene expression data are available, both commercially and freely . They differ particularly with regard to the implemented analytical methods, the graphical display and the manageability . In this paper the commercial software packages arraySCOUT, GeneSpring and Spotfire DecisionSite for Functional Genomics are compared and their applicability for analysis of gene expression data is studied . Small artificial and application test datasets are used to compare the computational results of the software packages . As far as possible results are verified with standard statistical software package SAS. Curr Pharm Biotechnol, 2003 Dec, 4(6), 351 - 78 Human inter-individual DNA sequence variation in candidate genes, drug targets, the importance of haplotypes and pharmacogenomics; Hoehe MR et al.; The identification of genes predisposing to human diseases is of paramount importance for understanding the molecular basis of the disease and individually different drug response, and will establish new routes to diagnosis and therapeutic advances of immense medical benefit . A key step common to all strategies for disease gene identification is the systematic analysis of candidate gene sequences to identify specific sequence variations associated with disease or any other phenotype of pharmaceutical relevance . In this article, current concepts and approaches to haplotype-based candidate gene analysis are reviewed . Moreover, a comprehensive summary of recent studies and data on the amount, nature, pattern and structure of genetic variation in candidate genes is given . These data demonstrate altogether remarkable gene sequence and haplotype diversity . Numerous individually different forms of a gene may exist . This presents challenges to the traditional views of the concept of "a" gene with far-reaching implications on the functional analysis of candidate gene variation, on the establishment of "sequence"-"structure"-"function" and complex haplotype/genotype-phenotype relationships, on the identification, evaluation and prioritization of drug targets and the concept of a "personalized medicine" in general . Moreover, present and future approaches to the identification of candidate and disease genes will be addressed . These include whole genome-based approaches such as integrative genomics as well as functional genomics-based approaches to analyze and model complex biological and medical processes . The analysis of whole complex systems in particular will provide the basis to make "maximally informed" guesses on candidate genes and address complex variability patterns in genes as well as complex genotype-phenotype relationships comprehensively at an advanced level. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D505 - 8 LIFEdb: a database for functional genomics experiments integrating information from external sources, and serving as a sample tracking system; Bannasch D et al.; We have implemented LIFEdb to link information regarding novel human full-length cDNAs generated and sequenced by the German cDNA Consortium with functional information on the encoded proteins produced in functional genomics and proteomics approaches . The database also serves as a sample-tracking system to manage the process from cDNA to experimental read-out and data interpretation . A web interface enables the scientific community to explore and visualize features of the annotated cDNAs and ORFs combined with experimental results, and thus helps to unravel new features of proteins with as yet unknown functions. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D383 - 7 The Rice PIPELINE: a unification tool for plant functional genomics; Yazaki J et al.; The Rice Genome Research Project in Japan performs genome sequencing and comprehensive expression profiling, constructs genetic and physical maps, collects full-length cDNAs and generates mutant lines, all aimed at improving the breeding of the rice plant as a food source . The National Institute of Agrobiological Sciences in Tsukuba, Japan, has accumulated numerous rice biological resources and has already successfully produced a high-quality genome sequence, a high-density genetic map with 3000 markers, 30,000 full-length cDNAs, over 700 expression profiles with a 9000 cDNA microarray and 15,000 flanking sequences with Tos17 insertions in about 3765 mutant lines from about 50,000 transposon insertion lines . These resources are available in the public domain . A new unification tool for functional genomics, called Rice PIPELINE, has also been developed for the dynamic collection and compilation of genomics data (genome sequences, full-length cDNAs, gene expression profiles, mutant lines, cis elements) from various databases . The mission of Rice PIPELINE is to provide a unique scientific resource that pools publicly available rice genomic data for search by clone sequence, clone name, GenBank accession number, or keyword . The web-based form of Rice PIPELINE is available at http://cdna01.dna.affrc.go.jp/PIPE/. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D373 - 6 MIPS Arabidopsis thaliana Database (MAtDB): an integrated biological knowledge resource for plant genomics; Schoof H et al.; Arabidopsis thaliana is the most widely studied model plant . Functional genomics is intensively underway in many laboratories worldwide . Beyond the basic annotation of the primary sequence data, the annotated genetic elements of Arabidopsis must be linked to diverse biological data and higher order information such as metabolic or regulatory pathways . The MIPS Arabidopsis thaliana database MAtDB aims to provide a comprehensive resource for Arabidopsis as a genome model that serves as a primary reference for research in plants and is suitable for transfer of knowledge to other plants, especially crops . The genome sequence as a common backbone serves as a scaffold for the integration of data, while, in a complementary effort, these data are enhanced through the application of state-of-the-art bioinformatics tools . This information is visualized on a genome-wide and a gene-by-gene basis with access both for web users and applications . This report updates the information given in a previous report and provides an outlook on further developments . The MAtDB web interface can be accessed at http://mips.gsf.de/proj/thal/db. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D332 - 3 dictyBase: a new Dictyostelium discoideum genome database; Kreppel L et al.; Dictyostelium discoideum is a powerful and genetically tractable model system used for the study of numerous cellular molecular mechanisms including chemotaxis, phagocytosis and signal transduction . The past 2 years have seen a significant expansion in the scope and accessibility of online resources for Dictyostelium . Recent advances have focused on the development of a new comprehensive online resource called dictyBase . This database not only provides access to genomic data including functional annotation of genes, gene products and chromosomal mapping, but also to extensive biological information such as mutant phenotypes and corresponding reference material . In conjunction with additional sites from the genome sequencing and assembly centers, these improvements have expanded the scope of the Dictyostelium databases making them accessible and useful to any researcher interested in comparative and functional genomics in metazoan organisms. Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D165 - 7 Update of NUREBASE: nuclear hormone receptor functional genomics; Ruau D et al.; Nuclear hormone receptors are an abundant class of ligand-activated transcriptional regulators, found in varying numbers in all animals . Based on our experience of managing the official nomenclature of nuclear receptors, we have developed NUREBASE, a database containing protein and DNA sequences, reviewed protein alignments and phylogenies, taxonomy and annotations for all nuclear receptors . New developments in NUREBASE include explicit declaration of alternative transcripts of each gene, and expression data for human and mouse nuclear receptors . The core of NUREBASE is reviewed, and it is completed by NUREBASE_DAILY, automatically updated every 24 h . All information on accessing and installing NUREBASE may be found at ens-lyon.fr/LBMC/laudet/nurebase/nurebase.html. Expert Opin Investig Drugs, 2004 Jan, 13(1), 1 - 19 Cancer metastasis therapeutic targets and drug discovery: emerging small-molecule protein kinase inhibitors; Sawyer TK; Cancer metastasis is a significant problem and a tremendous challenge to drug discovery relative to identifying key therapeutic targets as well as developing breakthrough medicines . Recent progress in unravelling the complex molecular circuitry of cancer metastasis, including receptors, intracellular proteins and genes, is highlighted . Furthermore, recent advances in drug discovery to provide novel proof-of-concept ligands, in vivo effective lead compounds and promising clinical candidates, are summarised . Such drug discovery efforts illustrate the integration of functional genomics, cell biology, structural biology, drug design, molecular/cellular screening and chemical diversity (e.g., small molecules, peptides/peptidomimetics, natural products, antisense, vaccines and antibodies) . Promising therapeutic targets for cancer metastasis have been identified, including Src, focal adhesion kinase, the integrin receptor, the vascular endothelial growth factor receptor, the epidermal growth factor receptor, Her-2/neu, c-Met, Ras/Rac GTPases, Raf kinase, farnesyl diphosphate synthase (i.e., amino-bisphosphonate therapeutic target) and matrix metalloproteases within the context of their implicated functional roles in cancer growth, invasion, angiogenesis and survival at secondary sites . Clinical and preclinical drug discovery is described and emerging small-molecule inhibitors of protein kinases are highlighted. Sci STKE . 2003 Dec 16;2003(213):re17. EH and UIM: endocytosis and more; Polo S et al.; Exogenously and endogenously originated signals are propagated within the cell by functional and physical networks of proteins, leading to numerous biological outcomes . Many protein-protein interactions take place between binding domains and short peptide motifs . Frequently, these interactions are inducible by upstream signaling events, in which case one of the two binding surfaces may be created by a posttranslational modification . Here, we discuss two protein networks . One, the EH-network, is based on the Eps15 homology (EH) domain, which binds to peptides containing the sequence Asp-Pro-Phe (NPF) . The other, which we define as the monoubiquitin (mUb) network, relies on monoubiquitination, which is emerging as an important posttranslational modification that regulates protein function . Both networks were initially implicated in the control of plasma membrane receptor endocytosis and in the regulation of intracellular trafficking routes . The ramifications of these two networks, however, appear to extend into many other aspects of cell physiology as well, such as transcriptional regulation, actin cytoskeleton remodeling, and DNA repair . The focus of this review is to integrate available knowledge of the EH- and mUb networks with predictions of genetic and physical interactions stemming from functional genomics approaches. J Physiol, 2004 Jan 1, 554(Pt 1), 56 - 63 Functional genomics in rodent models of hypertension; McBride MW et al.; Inbred strains of rodents have been used to study mammalian physiology and pathophysiology in an attempt to understand the contribution of genes in the pathogenesis of the disease process . In this review we focus on experimental animal models to identify quantitative trait loci (QTL) and possible strategies for identifying underlying genetic determinants responsible for hypertension . Confirmation of the existence of the QTL and dissection of the implicated region can be undertaken by production of either recombinant inbred, consomic or congenic strains . Despite complex interactions and the relatively few confirmed causative genes underlying QTL, recent developments in rat genome resources and advancement in statistical and bioinformatic methods will facilitate the identification of major gene(s) responsible for complex, polygenic traits. Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15764 - 9 Epub 2003 Dec 11. Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus; Kushner DB et al.; Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS) . Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae . By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps . To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants . Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA replication template with the capsid gene replaced by a luciferase reporter . Luciferase expression, which is dependent on viral RNA replication and RNA-dependent mRNA synthesis, was measured in intact yeast cells . Approximately 4500 yeast deletion strains ( approximately 80% of yeast genes) were screened in duplicate and selected strains analyzed further . This functional genomics approach revealed nearly 100 genes whose absence inhibited or stimulated BMV RNA replication and/or gene expression by 3- to >25-fold . Several of these genes were shown previously to function in BMV replication, validating the approach . Newly identified genes include some in RNA, protein, or membrane modification pathways and genes of unknown function . The results further illuminate virus and cell pathways . Further refinement of virus screening likely will reveal contributions from additional host genes. Hum Gene Ther, 2003 Dec 10, 14(18), 1799 - 807 Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain; Van den Haute C et al.; RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene-specific silencing . For in vivo applications, RNAi has been hampered until recently by inefficient delivery methods and by the transient nature of the gene suppression . Lentiviral vectors (LVs) hold great promise for gene therapeutic applications, pharmaceutical target validation, and functional genomics because stable gene transfer is mediated both in dividing and nondividing cells . We have used a lentiviral vector-based system for RNAi . We produced human immunodeficiency virus type 1-derived LVs encoding a short hairpin RNA specific for enhanced green fluorescent protein (EGFP) mRNA that were capable of inhibiting EGFP expression in mammalian cells . EGFP knockdown persisted after multiple passages of the cells . Of particular interest, our RNAi LVs were equally effective in suppression and prevention of EGFP expression after stereotactic injection in adult mouse brain . Therefore, we believe that the use of LVs for stable RNAi in brain will become a powerful aid to probe gene function in vivo and for gene therapy of diseases of the central nervous system. Yi Chuan Xue Bao, 2003 Oct, 30(10), 983 - 8 {Recombineering and its application}; Zhou JG et al.; Driven by the need of functional genomics, a homologous recombination-based, highly efficient genetic engineering system that termed "recombineering" has recently been developed . Recombineering has been defined as a genetic engineering with phage-encoded recombination function that utilizes short homologies, a convenient term to describe homologous-dependent, recombination-mediated, genetic engineering . The bacteriophage lambda Red recombination system has critical differences from standard E . coli RecA-dependent recombination pathway . The phage systems have unique advantage in that they can catalyze efficient recombination with very short regions of sequence homology (< 50 bp) . Recombineering does not require construction of plasmid or phage DNA intermediates containing the appropriately pre-engineered homology segment . All that is required in vitro is the synthesis of standard oligonucleotides or construction of PCR products that provide the homology . Importantly, they function even in the absence of RecA . These approaches do not rely on the presence of suitable restriction site, and can be used to insert, delete, clone or substitute genomic DNA sequences at any desired position on a target molecule in Escherichia Coli . Recombineering also facilitates many kinds of genomic experiments difficult to be carried out . In this article, the bacteriophage lambda Red recombinase system, the progression and applications of this powerful new technique are reviewed according to the data published recently. Int Rev Cytol, 2003, 228, 85 - 139 Homologous recombination and gene targeting in plant cells; Bernd R; Gene targeting has become an indispensable tool for functional genomics in yeast and mouse; however, this tool is still missing in plants . This review discusses the gene targeting problem in plants in the context of general knowledge on recombination and gene targeting . An overview on the history of gene targeting is followed by a general introduction to genetic recombination of bacteria, yeast, and vertebrates . This abridged discussion serves as a guide to the following sections, which cover plant-specific aspects of recombination assay systems, the mechanism of recombination, plant recombination genes, the relationship of recombination to the environment, approaches to stimulate homologous recombination and gene targeting, and a description of two plant systems, the moss Physcomitrella patens and the chloroplast, that naturally have high efficiencies of gene targeting . The review concludes with a discussion of alternatives to gene targeting. Microbiol Mol Biol Rev, 2003 Dec, 67(4), 657 - 85 RNA interference: biology, mechanism, and applications; Agrawal N et al.; Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms . The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA . The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants . Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification . Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing . The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity . In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it . Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi . Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs . The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent . Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination . As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible . Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes. Trends Plant Sci, 2003 Dec, 8(12), 576 - 81 Lignins and lignocellulosics: a better control of synthesis for new and improved uses; Boudet AM et al.; The composition and structure of lignified walls has a dramatic impact on the technological value of raw materials . The chemical flexibility of the secondary cell wall has been demonstrated and it is now possible to develop strategies to optimize its composition through genetic engineering . Thanks to functional genomics, new target genes of both plant and microbial origin are rapidly becoming available for this purpose and their use will open new avenues for producing tailor-made plant products with improved properties . Moreover, the major proportion of terrestrial plant biomass comprises lignified cell walls and this reservoir of carbon should be increasingly exploited for the production of chemicals and energy within the context of sustainable development . For example, the design of plants suitable for downstream conversion processes, such as the production of bioethanol, and the exploitation of microorganisms and microbial enzymes for biomass pretreatments or for the production of novel chemicals. Genome Biol . 2003;4(12):352 . Epub 2003 Nov 24. Yeast functional genomics and cell biology: no longer in the dark; Friesen H et al.; A report on the Cold Spring Harbor Laboratory meeting 'Yeast Cell Biology', Cold Spring Harbor, USA, 12-17 August 2003. Genome Biol . 2003;4(12):241 . Epub 2003 Nov 24. Genetics and functional genomics of type 2 diabetes mellitus; Toye A et al.; Genome-wide studies of transcription in the skeletal muscle of type 2 diabetic patients have identified coordinated changes in the expression of genes involved in oxidative phosphorylation, and have underlined the central role of the oxidative-phosphorylation regulator, PCG1alpha . These findings help unravel the complex pathogenesis and inheritance of polygenic type 2 diabetes mellitus. Neuroendocrinology, 2003 Nov, 78(5), 253 - 9 Brain renin-angiotensin system . Lessons from functional genomics; Baltatu O et al.; The existence of a brain renin-angiotensin system (RAS) was postulated 30 years ago . Since then our knowledge on the biology of the brain RAS has advanced considerably . The brain RAS has been found to be involved in the modulation of cardiovascular and fluid-electrolyte homeostasis, generally complementing the classical roles of the endocrine RAS . The RAS has additionally been implicated in other brain-specific functions, such as memory, cognition and stress . During the last years, the development of transgenic technologies allowed to get further insight into the functionality and relevance of the brain RAS . This paper is attempting to summarize our recent experience from transgenic animals . FEMS Yeast Res, 2003 Dec, 4(3), 259 - 69 Comparative genotyping of the Saccharomyces cerevisiae laboratory strains S288C and CEN.PK113-7D using oligonucleotide microarrays; Daran-Lapujade P et al.; To analyse the reliability and accuracy of genotype analysis with high-density oligonucleotide microarrays, this method and other experimental approaches were used to analyse genomic DNA of two popular Saccharomyces cerevisiae laboratory strains . S288C was used for systematic sequencing of 'the' S . cerevisiae genome; CEN.PK113-7D is a popular strain for physiological studies and functional genomics . Random amplified polymorphic DNA, electrophoretic karyotyping and microarray analysis all indicated a high level of sequence similarity between the two strains . In the microarray analysis, as few as 288 (4.5%) of the ca . 6300 represented yeast genes were identified that yielded significantly different hybridisation intensities between the two strains . These could be classified as amplified, absent, or with sequence polymorphism in CEN.PK113-7D compared to S288C . A detailed analysis focused on the subset of 25 genes called absent in CEN.PK113-7D . Among these absent genes, 17 were clustered together on five chromosomes, mainly in subtelomeric regions . Thorough analysis of these regions by polymerase chain reaction (PCR) and restriction fragment length polymorphism confirmed the absence of these genes in CEN.PK113-7D . Surprisingly, three of these regions were not smaller in CEN.PK113-7D chromosomes, indicating that they may harbour unidentified and potentially new sequences . In addition, eight genes called absent by the microarrays were scattered over the chromosomes