J Biol Chem, 2002 Apr 26, 277(17), 14363 - 6 Epub 2002 Mar 06. Transcript abundance in yeast varies over six orders of magnitude; Holland MJ; In the current era of functional genomics, it is remarkable that the intracellular range of transcript abundance is largely unknown . For the yeast Saccharomyces cerevisiae, hybridization-based complexity analysis and SAGE analysis showed that the majority of yeast mRNAs are present at one or fewer copies per cell; however, neither method provides an accurate estimate of the full range of low abundance transcripts . Here we examine the range of intracellular transcript abundance in yeast using kinetically monitored, reverse transcriptase-initiated PCR (kRT-PCR) . Steady-state transcript levels encoded by all 65 genes on the left arm of chromosome III and 185 transcription factor genes are quantitated . Abundant transcripts encoded by glycolytic genes, previously quantitated by kRT-PCR, are present at a few hundred copies per cell whereas genes encoding physiologically important transcription factors are expressed at levels as low as one-thousandth transcript per cell . Of the genes assessed, only the silent mating type loci, HML and HMR, are transcriptionally silent . The results show that transcript abundance in yeast varies over six orders of magnitude . Finally, kRT-PCR, cDNA microarray, and high density oligonucleotide array assays are compared for their ability to detect and quantitate the complete yeast transcriptome. Trends Neurosci, 2001 Nov, 24(11 Suppl), S56 - 62 Antisense as a neuroscience tool and therapeutic agent; Estibeiro P et al.; Gene expression in the mammalian brain is highly complex and requires an immensely powerful functional genomics tool to unravel it . Antisense has the potential to meet this requirement, but has always been plagued by biological and technological hurdles that have made the technology unreliable . With recent progress in developing potent, low-toxicity nucleic acid chemistries and novel drug delivery methods to cross the blood-brain barrier, the use of antisense is gathering momentum. Nat Biotechnol, 2002 Mar, 20(3), 243 - 9 Metabolic control analysis in drug discovery and disease; Cascante M et al.; Metabolic control analysis (MCA) provides a quantitative description of substrate flux in response to changes in system parameters of complex enzyme systems . Medical applications of the approach include the following: understanding the threshold effect in the manifestation of metabolic diseases; investigating the gene dose effect of aneuploidy in inducing phenotypic transformation in cancer; correlating the contributions of individual genes and phenotypic characteristics in metabolic disease (e.g., diabetes); identifying candidate enzymes in pathways suitable as targets for cancer therapy; and elucidating the function of "silent" genes by identifying metabolic features shared with genes of known pathways . MCA complements current studies of genomics and proteomics, providing a link between biochemistry and functional genomics that relates the expression of genes and gene products to cellular biochemical and physiological events . Thus, it is an important tool for the study of genotype-phenotype correlations . It allows genes to be ranked according to their importance in controlling and regulating cellular metabolic networks . We can expect that MCA will have an increasing impact on the choice of targets for intervention in drug discovery. Genome Res, 2002 Mar, 12(3), 487 - 92 Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions; Doi N et al.; We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS) . By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation . We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments . These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics. Sleep Breath, 2001, 5(1), 47 - 51 Incorporating inheritance into models for understanding ventilatory behavior; Strohl KP et al.; Ventilation and its components (frequency and tidal volume) appear to be determined to a significant extent by inheritance . Gene manipulation, gene identification, and functional genomics now offer powerful tools to identify the strength and mode of inheritance for ventilatory behavior under steady-state and non-steady-state conditions, in health and in disease . Conscious integration of genetic principles into existing explanatory models may increase the likelihood of detecting traits that correlate with protein systems responsible for the structures and the functional components of respiration. Plant Mol Biol, 2002 Jan, 48(1-2), 99 - 118 DNA microarrays for functional plant genomics; Aharoni A et al.; DNA microarray technology is a key element in today's functional genomics toolbox . The power of the method lies in miniaturization, automation and parallelism permitting large-scale and genome-wide acquisition of quantitative biological information from multiple samples . DNA microarrays are currently fabricated and assayed by two main approaches involving either in situ synthesis of oligonucleotides ('oligonucleotide microarrays') or deposition of pre-synthesized DNA fragments ('cDNA microarrays') on solid surfaces . To date, the main applications of microarrays are in comprehensive, simultaneous gene expression monitoring and in DNA variation analyses for the identification and genotyping of mutations and polymorphisms . Already at a relatively early stage of its application in plant science, microarrays are being utilized to examine a range of biological issues including the circadian clock, plant defence, environmental stress responses, fruit ripening, phytochrome A signalling, seed development and nitrate assimilation . Novel insights are obtained into the molecular mechanisms co-ordinating metabolic pathways, regulatory and signalling networks . Exciting new information will be gained in the years to come not only from genome-wide expression analyses on a few model plant species, but also from extensive studies of less thoroughly studied species on a more limited scale . The value of microarray technology to our understanding of living processes will depend both on the amount of data to be generated and on its clever exploration and integration with other biological knowledge arising from complementary functional genomics tools for 'profiling' the genome, proteome, metabolome and phenome. Plant Mol Biol, 2002 Jan, 48(1-2), 155 - 71 Metabolomics--the link between genotypes and phenotypes; Fiehn O; Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes . In parallel to the terms 'transcriptome' and proteome', the set of metabolites synthesized by a biological system constitute its 'metabolome' . Yet, unlike other functional genomics approaches, the unbiased simultaneous identification and quantification of plant metabolomes has been largely neglected . Until recently, most analyses were restricted to profiling selected classes of compounds, or to fingerprinting metabolic changes without sufficient analytical resolution to determine metabolite levels and identities individually . As a prerequisite for metabolomic analysis, careful consideration of the methods employed for tissue extraction, sample preparation, data acquisition, and data mining must be taken . In this review, the differences among metabolite target analysis, metabolite profiling, and metabolic fingerprinting are clarified, and terms are defined . Current approaches are examined, and potential applications are summarized with a special emphasis on data mining and mathematical modelling of metabolism. Plant Mol Biol, 2002 Jan, 48(1-2), 119 - 31 Microarray data quality analysis: lessons from the AFGC project . Arabidopsis Functional Genomics Consortium; Finkelstein D et al.; Genome-wide expression profiling with DNA microarrays has and will provide a great deal of data to the plant scientific community . However, reliability concerns have required the development data quality tests for common systematic biases . Fortunately, most large-scale systematic biases are detectable and some are correctable by normalization . Technical replication experiments and statistical surveys indicate that these biases vary widely in severity and appearance . As a result, no single normalization or correction method currently available is able to address all the issues . However, careful sequence selection, array design, experimental design and experimental annotation can substantially improve the quality and biological of microarray data . In this review, we discuss these issues with reference to examples from the Arabidopsis Functional Genomics Consortium (AFGC) microarray project. J Exp Zool, 2002 Feb 15, 292(3), 217 - 20 Generating new marine cell lines and transgenic species--conference summary; Wise JP Sr et al.; Marine species offer a tremendous diversity of life histories, physiologies, genetics, behaviors, and biologies, reflecting myriad adaptations to the water environment . Historically, marine vertebrates, particularly fish, have played significant roles in a wide range of disciplines, including environmental toxicology, genetics, developmental biology, and physiology, among others . Much still remains to be learned from these animals, and there is a growing need for new marine models . Models for expression of marine animal genes have been limited to heterologous expression systems . While there is still a great deal to gain from heterologous expression systems, the interactions of genes with one another can best be determined in homologous expression systems where appropriate interactions are possible . This has become particularly important with the development of functional genomics in marine models . These homologous gene expression systems will be key to the use of functional genomics for marine animal molecular physiology and toxicology . Curr Opin Plant Biol, 2002 Apr, 5(2), 112 - 7 Tomato mutants as tools for functional genomics; Emmanuel E et al.; Tomato mutants have been used in genetic studies and breeding for decades, yet only a few tomato mutants have been characterized at the molecular level . Similarly, a wealth of sequence information for tomato is now available but the functions of only a few genes are known . New developments - such as the use of saturated mutant populations, new methods for the detection of mutants and new sequence data - are bridging the gap between tomato genes and their functions. Curr Opin Biotechnol, 2001 Dec, 12(6), 622 - 5 Functional genomics and target validation approaches using antisense oligonucleotide technology; Dean NM; The recent increase in the amount and rate of accumulation of genomic information has created new challenges for the pharmaceutical industry . These include how best to rapidly and efficiently identify key genes responsible for complex disease phenotypes and how to use this information to develop new and specific classes of drugs . Antisense technology offers a powerful approach to identify novel cellular networks and signaling "cassettes" and provides a method to validate genes in vivo as attractive drug targets. Curr Opin Biotechnol, 2001 Dec, 12(6), 613 - 21 The use of retroviruses as pharmaceutical tools for target discovery and validation in the field of functional genomics; Lorens JB et al.; Retrovirally mediated functional genomics enables identification of physiologically relevant cellular therapeutic targets . Unique properties of retroviruses make them ideal tools for the introduction of large and diverse libraries of potential genetic effectors to a variety of cell types . The identification and recovery of intracellular library elements responsible for altered disease responses establishes a direct basis for pharmaceutical development . Recent innovations in retroviral infection efficiency and expression control have broadened application of the methodology to include libraries of mutagenized cDNAs, peptides and ribozyme genetic effectors. J Cell Biochem Suppl, 2001, Suppl 37, 99 - 105 Object-oriented approach to drug design enabled by NMR SOLVE: first real-time structural tool for characterizing protein-ligand interactions; Sem DS et al.; As a result of genomics efforts, the number of protein drug targets is expected to increase by an order of magnitude . Functional genomics efforts are identifying these targets, while structural genomics efforts are determining structures for many of them . However, there is a significant gap in going from structural information for a protein target to a high affinity (K(d) < 100 nM) inhibitor, and the problem is multiplied by the sheer number of new targets now available . nature frequently designs proteins in classes that are related by the reuse, through gene duplication events, of cofactor binding domains . This reuse of functional domains is an efficient way to build related proteins in that it is object-oriented . There is a growing realization that the most efficient drug design strategies for attacking the mass of targets coming from genomics efforts will be systems-based approaches that attack groups of related proteins in parallel . We propose that the most effective drug design strategy will be one that parallels the object-oriented manner by which nature designed the gene families themselves . IOPE (Integrated Object-Oriented PharmacoEngineering) is such an approach . It is a three-step technology to build focused combinatorial libraries of potential inhibitors for major families and sub-families of enzymes, using cogent NMR data derived from representatives of these protein families . The NMR SOLVE (Structurally Oriented Library Valency Engineering) data used to design these libraries are gathered in days, and data can be obtained for large proteins (> 170 kDa) . Furthermore, the process is fully object-oriented in that once a given bi-ligand is identified for a target, potency is retained if different cofactor mimics are swapped . This gives the drug design process maximum flexibility, allowing for the more facile transition from in vitro potency to in vivo efficacy . Philos Trans R Soc Lond B Biol Sci, 2002 Jan 29, 357(1417), 47 - 53 Putting the Leishmania genome to work: functional genomics by transposon trapping and expression profiling; Beverley SM et al.; Leishmania are important protozoan pathogens of humans in temperate and tropical regions . The study of gene expression during the infectious cycle, in mutants or after environmental or chemical stimuli, is a powerful approach towards understanding parasite virulence and the development of control measures . Like other trypanosomatids, Leishmania gene expression is mediated by a polycistronic transcriptional process that places increased emphasis on post-transcriptional regulatory mechanisms including RNA processing and protein translation . With the impending completion of the Leishmania genome, global approaches surveying mRNA and protein expression are now feasible . Our laboratory has developed the Drosophila transposon mariner as a tool for trapping Leishmania genes and studying their regulation in the form of protein fusions; a classic approach in other microbes that can be termed 'proteogenomics' . Similarly, we have developed reagents and approaches for the creation of DNA microarrays, which permit the measurement of RNA abundance across the parasite genome . Progress in these areas promises to greatly increase our understanding of global mechanisms of gene regulation at both mRNA and protein levels, and to lead to the identification of many candidate genes involved in virulence. Philos Trans R Soc Lond B Biol Sci, 2002 Jan 29, 357(1417), 17 - 23 Functional genomics: lessons from yeast; Oliver SG; Functional genomics represents a systematic approach to elucidating the function of the novel genes revealed by complete genome sequences . Such an approach should adopt a hierarchical strategy since this will both limit the number of experiments to be performed and permit a closer and closer approximation to the function of any individual gene to be achieved . Moreover, hierarchical analyses have, in their early stages, tremendous integrative power and functional genomics aims at a comprehensive and integrative view of the workings of living cells . The first draft of the human genome sequence has just been produced, and the complete genome sequences of a number of eukaryotic human pathogens (including the parasitic protozoa Plasmodium, Leishmania, and Trypanosoma) will soon be available . However, the most rapid progress in the elucidation of gene function will initially be made using model organisms . Yeast is an excellent eukaryotic model and at least 40% of single-gene determinants of human heritable diseases find homologues in yeast . We have adopted a systematic approach to the functional analysis of the Saccharomyces cerevisiae genome . A number of the approaches for the functional analysis of novel yeast genes are discussed . The different approaches are grouped into four domains: genome, transcriptome, proteome, and metabolome . The utility of genetic, biochemical, and physico-chemical methods for the analysis of these domains is discussed, and the importance of framing precise biological questions, when using these comprehensive analytical methods, is emphasized . Finally, the prospects for elucidating the function of protozoan genes by using the methods pioneered with yeast, and even exploiting Saccharomyces itself, as a surrogate, are explored. Rev Med Chil, 2001 Nov, 129(11), 1328 - 32 {Medical applications of genome discovery}; Cruz-Coke R; The discovery of the complete base sequence of human genome unveils several perspectives to understand human diseases and develop new therapies . Human genome contains approximately 39,000 genes of which 26,000 code specific proteins that have been identified . There are approximately 1,500 diseases with identified molecular disturbances . Genes can modify signs and symptoms of common diseases . Thus, there are no pure monogenic diseases . Chronic diseases of adults are complex and dependent on multiple factors . Several genes that predispose to chronic degenerative diseases have been identified . This is revealing the complex nature and the interaction of these ailments with the environment . The discovery of bacterial and viral genomic sequences will allow the manufacturing of new vaccines and specific molecular antimicrobials . The new pharmacogenomics will devise treatments for each subject according to her specific genomic profile . The new applications of genomic technology is creating new paradigms in biomedical research such as functional genomics, proteonomics, epigenetic regulation . Gene diagnosis and therapy will considerably improve the future of medicine. Bioinformatics, 2002 Jan, 18(1), 160 - 6 Machine learning of functional class from phenotype data; Clare A et al.; MOTIVATION: Mutant phenotype growth experiments are an important novel source of functional genomics data which have received little attention in bioinformatics . We applied supervised machine learning to the problem of using phenotype data to predict the functional class of Open Reading Frames (ORFs) in Saccaromyces cerevisiae . Three sources of data were used: TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces (TRIPLES), European Functional Analysis Network (EUROFAN) and Munich Information Center for Protein Sequences (MIPS) . The analysis of the data presented a number of challenges to machine learning: multi-class labels, a large number of sparsely populated classes, the need to learn a set of accurate rules (not a complete classification), and a very large amount of missing values . We modified the algorithm C4.5 to deal with these problems . RESULTS: Rules were learnt which are accurate and biologically meaningful . The rules predict function of 83 ORFs of unknown function at an estimated accuracy of > or = 80%. Lepr Rev, 2001 Dec, 72(4), 462 - 9 The integrated genome map of Mycobacterium leprae; Eiglmeier K et al.; The integrated map of the Mycobacterium leprae genome unveiled for the first time the genomic organization of this obligate intracellular parasite . Selected cosmid clones, isolated from a genomic library created in the cosmid vector Lorist6, were identified as representing nearly the complete genome and were subsequently used in the M . leprae genome sequencing project . Now a new version of the integrated map of M . leprae can be presented, combining the mapping results from the Lorist6 cosmids with data obtained from a second genomic library constructed in an Escherichia coli-mycobacterium shuttle cosmid, pYUB18 . More than 98% of the M . leprae genome is now covered by overlapping large insert genomic clones representing a renewable source of well defined DNA segments and a powerful tool for functional genomics. Nat Rev Genet, 2002 Jan, 3(1), 43 - 52 Mapping and analysis of quantitative trait loci in experimental populations; Doerge RW; Simple statistical methods for the study of quantitative trait loci (QTL), such as analysis of variance, have given way to methods that involve several markers and high-resolution genetic maps . As a result, the mapping community has been provided with statistical and computational tools that have much greater power than ever before for studying and locating multiple and interacting QTL . Apart from their immediate practical applications, the lessons learnt from this evolution of QTL methodology might also be generally relevant to other types of functional genomics approach that are aimed at the dissection of complex phenotypes, such as microarray assessment of gene expression. News Physiol Sci, 2002 Feb, 17, 11 - 6 Of mice and worms: novel insights into ClC-2 anion channel physiology; Strange K; ClC anion channels are found in all major groups of organisms . Recent studies in nematodes and mice suggest that the function and regulation of ClC-2 have been conserved over vast evolutionary time spans . These studies illustrate the experimental advantages of using genomically defined nonmammalian model organisms for characterizing ClC channel functional genomics. Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 251 - 9 Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes; Baltz RH; The foundation for any strain improvement program is efficient random chemically-induced mutagenesis coupled with highly reproducible fermentation and product assays . The broad spectrum of spontaneous mutations can be leveraged in some cases by direct selection of mutants with desired traits . Transposons containing outward-reading promoter activity might be used to enhance yields by inducing promoter fusions, disrupting negative regulatory elements, or disrupting genes involved in competing pathways . Transposons might also be used to identify and clone positive regulatory genes . As knowledge of the key elements in the fermentation process and secondary metabolite biosynthesis grows, gene cloning and targeted gene duplication becomes an important tool . Duplication of genes involved in rate limiting steps can be achieved to improve product yields by inserting the desired gene(s) into neutral sites in the chromosome by homologous recombination or by site-specific integration . The probabilities and frequencies of success of the molecular genetic approaches should increase with an increasing knowledge of key factors influencing product yields . This knowledge can be broadened dramatically by a combination of structural and functional genomics, gene disruption analysis and metabolic modeling . Protoplast fusion can be used to recombine beneficial traits from any of the other approaches. Adv Biochem Eng Biotechnol, 2001, 73, 1 - 8 After a decade of progress, an expanded role for metabolic engineering; Stephanopoulos G et al.; Over the past decade, metabolic engineering has emerged as an active and distinct discipline characterized by its over-arching emphasis on integration . In practice, metabolic engineering is the directed improvement of cellular properties through the application of modern genetic methods . Although it was applied on an ad hoc basis for several years following the introduction of recombinant techniques {1,2}, metabolic engineering was formally defined as a new field approximately a decade ago {3} . Since that time, many creative applications, directed primarily to metabolite overproduction, have been reported {4} . In parallel, recent advances in the resolution and acquisition time of biological data, especially structural and functional genomics, has amplified interest in the systemic view of biology that metabolic engineering provides . To facilitate the burgeoning scientific exchange in this area on a more regular and convenient basis, a new conference series was launched in 1996 followed by a new journal in 1999. Trends Biotechnol, 2002 Feb, 20(2), 79 - 84 Structural proteomics: developments in structure-to-function predictions; Norin M et al.; The major challenge for post-genomic research is to functionally assign and validate a large number of novel target genes and their corresponding proteins . Functional genomics approaches have, therefore, gained considerable attention in the quest to convert this massive data set into useful information . One of the crucial components for the functional understanding of unassigned proteins is the analysis of their experimental or modeled 3D structures . Structural proteomics initiatives are generating protein structures at an unprecedented rate but our current knowledge of 3D-structural space is still limited . Estimates on the completeness of the 3D-structural coverage of proteins vary but it is generally accepted that only a minority of the structural proteome has a template structure from which reliable conclusions can be drawn . Thus, structural proteomics has set out to build a map of protein structures that will represent all protein folds included in the 'global proteome'. J Autism Dev Disord, 2001 Dec, 31(6), 551 - 5 Functional genomics approaches to a primate model of autistic symptomology; Hemby SE et al.; Several studies indicate a primary dysfunction of the temporal lobe in autism, specifically the hippocampal formation and entorhinal cortex (EC) . Assessment of gene expression in the EC and hippocampus will provide insight into the subtle alterations in neuronal function associated with autism . To this end, evaluations in a primate model of social attachment, which produces behaviors associated with autism, in addition to the use of human post-mortem tissue from individuals diagnosed with autism will provide heretofore unattainable information of how the complex neural circuitry of this region is altered in autism . Identification of altered expression of multiple genes should provide a molecular "fingerprint" of autism and may provide new targets for pharmacotherapeutic intervention. J Cardiovasc Pharmacol, 2001 Oct, 38 Suppl 1, S1 - 5 Functional genomic research of alpha 1-adrenoceptors; Tsujimoto G et al.; The Human Genome Project is now almost completed, and we are about to move into the post-genome sequence era of functional genomics . The advent of genome science has markedly changed the way life science research including pharmacological study is conducted; thus, systematic and integrated 'genome-wide' survey is feasible . The stream of 'Genome-->Transcriptome--> Proteomics' is logical and, in each aspect, approaches for functional genomics are now pursued at a high pace . We have recently developed a standardized technical platform (in various levels, such as transcription, cell and whole animal levels, etc.), and applied these techniques to the study of functional genomics of G-protein-coupled receptors, particularly alpha1-adrenoceptors as a model . Combining the genome information and technology, future pharmacological studies would become the genome-based search and research. Eur J Nucl Med Mol Imaging, 2002 Jan, 29(1), 115 - 32 Epub 2001 Nov 14. Functional genomics and proteomics--the role of nuclear medicine; Haberkorn U et al.; Now that the sequencing of the human genome has been completed, the basic challenges are finding the genes, locating their coding regions and predicting their functions . This will result in a new understanding of human biology as well as in the design of new molecular structures as potential novel diagnostic or drug discovery targets . The assessment of gene function may be performed using the tools of the genome program . These tools represent high-throughput methods used to evaluate changes in the expression of many or all genes of an organism at the same time in order to investigate genetic pathways for normal development and disease . This will lead to a shift in the scientific paradigm: In the pre-proteomics era, functional assignments were derived from hypothesis-driven experiments designed to understand specific cellular processes . The new tools describe proteins on a proteome-wide scale, thereby creating a new way of doing cell research which results in the determination of three-dimensional protein structures and the description of protein networks . These descriptions may then be used for the design of new hypotheses and experiments in the traditional physiological, biochemical and pharmacological sense . The evaluation of genetically manipulated animals or newly designed biomolecules will require a thorough understanding of physiology, biochemistry and pharmacology and the experimental approaches will involve many new technologies, including in vivo imaging with single-photon emission 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 enzymes, receptors, antigens or transporters . Pharmacogenomics will identify new surrogate markers for therapy monitoring which may represent potential new tracers for imaging . Also, drug distribution studies for new therapeutic biomolecules are needed, at least during preclinical stages of drug development . Finally, new biomolecules will be developed by bioengineering methods which may be used for isotope-based diagnosis and treatment of disease. Genome Biol . 2002;3(1):REVIEWS1002 . Epub 2001 Dec 21. Functional genomics in the study of seed germination; Bove J et al.; A recent proteomic analysis of germinating Arabidopsis thaliana seeds demonstrates the effectiveness of functional genomics for investigating the complexity of developmental regulatory networks, such as the development of the embryo into a young plant. C R Acad Sci III, 2001 Dec, 324(12), 1103 - 10 From Arabidopsis to rice genomics: a survey of French programmes; Delseny M et al.; During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases . The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases . This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation . French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing . In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants . This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement. Trends Neurosci, 2002 Jan, 25(1), 45 - 50 Hormonal and genetic influences on arousal--sexual and otherwise; Pfaff D et al.; Genetic influences on lordosis, a mammalian social behavior, are amenable for study because of the relative simplicity of both stimuli and response . The neural circuit for lordosis involves a supraspinal loop, which is controlled by an estrogen- and progesterone-dependent signal from the medial hypothalamus and results in heightened sexual motivation . In turn, this involves elevated states of arousal, defined by increased sensory alertness, motor activity and emotional reactivity . Mice in which the gene encoding the alpha form of the estrogen receptor (ERalpha) has been knocked out show that ERalpha is crucial for lordosis behavior . Comparing ERalpha-, ERbeta- and double knockouts reveals that different patterns of sexual behaviors in mice require different patterns of ER activity . Understanding how hormonal and genetic effects on deep motivational and arousal processes contribute to their effects on specific sexual and aggressive behaviors pose significant challenges for mouse functional genomics. Metab Eng, 2002 Jan, 4(1), 98 - 106 Functional genomics: high-throughput mRNA, protein, and metabolite analyses; Oliver DJ et al.; A tremendous amount of DNA sequence information is now available to scientists and engineers . These DNA sequences provide the foundation for studying how the genome of an organism is functioning and they are particularly useful for metabolic engineers interested in manipulating plants for the production of chemicals and enzymes . Functional genomics relies on high-throughput techniques for measuring the mRNA (the transcriptome), protein (the proteome), and metabolite (the metabolome) components of plants as well as their organs and tissues . Microarray technologies, recent advances in protein mass spectrometry, and high-throughput metabolite analyses are beginning to provide detailed information on the total mRNA, protein, and metabolite components of plants . This knowledge will allow scientists to monitor changes in proteins and metabolites in plants . Ultimately, it may allow them to discover new metabolic pathways and to model metabolic and regulatory networks in plants. J Allergy Clin Immunol, 2002 Jan, 109(1), 14 - 21 The functional genomics of CD14 and its role in IgE responses: an integrated view; Vercelli D; Several studies in recent years have suggested that there is a strong genetic component in the pathogenesis of IgE-mediated diseases . Epidemiologic studies have identified a number of genes that carry single base changes (single nucleotide polymorphisms) associated with parameters of allergy . What remain to be established are the mechanisms whereby genetic variation results in dysregulation of IgE-mediated responses . This is the task of functional genomics . In this article, some of the most powerful approaches that have been devised to provide a mechanistic explanation for the effects of genetic variation on the regulation of gene expression and function are discussed . Recent data on the impact of genetic variation on the regulation of CD14 are explored in the context of the potential role played by this gene in the pathogenesis of allergy . Also discussed is the notion that taken individually, each instance of variation might result in small effects . It is the combination of variations in the same gene and/or in genes arrayed along one functional pathway that might eventually lead to dysregulation strong enough to cause disease . In this scenario, the environment is likely to play an essential role in determining the functional outcome of genetic variation. Q Rev Biol, 2001 Dec, 76(4), 417 - 32 Evolution in health and disease: work in progress; Stearns SC et al.; This article surveys progress in Darwinian medicine since 1991 . Evolutionary thinking has been providing an increasing flow of fresh ideas into medical science, ideas that would not be suggested by other perspectives . Recent contributions have shed new light on the evolution of virulence, of antibiotic resistance, of oocytic atresia, of menopause, of the timing of the expression of genetic disease, of links between mate choice and disease resistance, and of genomic conflict between mother and fetus over resource provisioning . An important consequence of changes from the environment of evolutionary adaptedness concerns reproductive cancers; the incidence of reproductive cancers may be linked to changes in the frequency of menstruation in postindustrial societies . Other intriguing developments include some unanticipated and undesirable consequences of good hygiene, hope from an unexpected quarter for progress on nerve and muscle regeneration, evolutionary interpretations of mental disease, and insights from functional genomics into the nature of tradeoffs . The application of evolutionary thinking to problems in medical research and practice has thus yielded an abundant and growing harvest of insights . Some are well founded, others remain speculative . The field is moving from an initial phase dominated by speculation and hypothesis formation into a more rigorous phase of experimental testing of explicit alternatives . Currently the most promising areas, those in which experimental rigor can be applied efficiently, include experimental evolution and functional genomics . The pioneers can be proud of what they have set in motion. J Mol Biol, 2002 Jan 4, 315(1), 1 - 8 High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm; Venturi M et al.; The antigen-binding fragments (Fab) of antibodies are powerful tools in clinical therapy, molecular diagnostics and basic research . However, their principal applications require pure recombinant molecules in large amounts, which are challenging to obtain . Severe limitations in yield, folding and functionality are commonly encountered in bacterial production of Fab fragments . Secretion into the oxidizing periplasm generally results in low yield, whereas expression in the reducing cytoplasmic environment produces unfolded or non-functional protein . We hypothesized that an impaired reducing environment of the cytoplasm would permit correctly folded, functional cytoplasmic expression of Fabs with high yield . We used the Escherichia coli strain FA113, which has no activity of both thioredoxin and glutathione reductase, and thus has an oxidizing cytoplasmic environment . With the newly constructed vector pFAB1 we tested the cytoplasmic expression of two Fab fragments, which recognize the integral membrane protein NhaA, a bacterial Na(+)/H(+) antiporter . These antibodies differ in terms of DNA sequence and stability . Both antibody fragments were produced to very high yields (10-30 mg/l from bacterial cultures at an A(600 nm)=1.2-1.3) . This is a factor 50-250 times higher than any other reported over-expression strategy for Fab fragments and currently represents the highest production rate ever been reported for antibody Fab fragments in bacteria grown to similar cell densities . The fragments are fully functional and can be efficiently purified by His-tag chromatography . Expression of active Fab fragments in the bacterial cytoplasm unlocks the possibility of using antibody specific targeting in an intracellular environment . Such a capacity opens new perspectives for investigating metabolic and regulatory pathways in vivo and also provides a powerful selection system for functional genomics . J Neural Transm Suppl, 2001, (61), 131 - 48 Functional genomics of Down syndrome: a multidisciplinary approach; Dierssen M et al.; The availability of the DNA sequence of human chromosome 21 (HSA21) is a landmark contribution that will have an immediate impact on the study of the role of specific genes to Down syndrome (DS) . Trisomy 21, full or partial, is a major cause of mental retardation and other phenotypic abnormalities, collectively known as Down syndrome (DS), a disorder affecting 1 in 700 births . The identification of genes on HSA21 and the elucidation of the function of the proteins encoded by these genes have been a major challenge for the human genome project and for research in DS . Over 100 of the estimated 300-500 genes of HSA21 have been identified, but the function of most remains largely unknown . It is believed that the overexpression of an unknown number of HSA21 genes is directly or indirectly responsible for the mental retardation and the other clinical features of DS . For this reason, HSA21 genes that are expressed in tissues affected in DS patients are of special interest. Yakugaku Zasshi, 2001 Dec, 121(12), 845 - 73 Fruits of human genome project and private venture, and their impact on life science; Ikekawa A et al.; A small knowledge base was created by organizing the Human Genome Project (HGP) and its related issues in "Science" magazines between 1996 and 2000 . This base revealed the stunning achievement of HGP and a private venture and its impact on today's biology and life science . In the mid-1990, they encouraged the development of advanced high throughput automated DNA sequencers and the technologies that can analyse all genes at once in a systematic fashion . Using these technologies, they completed the genome sequence of human and various other organisms . These fruits opened the door to comparative genomics, functional genomics, the interdisprinary field between computer and biology, and proteomics . They have caused a shift in biological investigation from studying single genes or proteins to studying all genes or proteins at once, and causing revolutional changes in traditional biology, drug discovery and therapy . They have expanded the range of potential drug targets and have facilitated a shift in drug discovery programs toward rational target-based strategies . They have spawned pharmacogenomics that could give rise to a new generation of highly effective drugs that treat causes, not just symptoms . They should also cause a migration from the traditional medications that are safe and effective for every members of the population to personalized medicine and personalized therapy. Adv Exp Med Biol, 2001, 500, 479 - 87 Functional genomics of oxidant-induced lung injury; Leikauf GD et al.; In summary, acute lung injury is a severe (>40% mortality) respiratory disease associated with numerous precipitating factors . Despite extensive research since its initial description over 30 years ago, questions remain about the basic pathophysiological mechanisms and their relationship to therapeutic strategies . Histopathology reveals surfactant disruption, epithelial perturbation and sepsis, either as initiating factors or as secondary complications, which in turn increase the expression of cytokines that sequester and activate inflammatory cells, most notably, neutrophils . Concomitant release of reactive oxygen and nitrogen species subsequently modulates endothelial function . Together these events orchestrate the principal clinical manifestations of the syndrome, pulmonary edema and atelectasis . To better understand the gene-environmental interactions controlling this complex process, we examined the relative sensitivity of inbred mouse strains to acute lung injury induced by ozone, ultrafine PTFE, or fine particulate NiSO4 (0.2 microm MMAD, 15-150 microg/m3) . Measuring survival time, protein and neutrophils in bronchoalveolar lavage, lung wet: dry weight, and histology, we found that these responses varied between inbred mouse strains, and susceptibility is heritable . To assess the molecular progression of NiSO4-induced acute lung injury, temporal relationships of 8734 genes and expressed sequence tags were assessed by cDNA microarray analysis . Clustering of co-regulated genes (displaying similar temporal expression patterns) revealed the altered expression of relatively few genes . Enhanced expression occurred mainly in genes associated with oxidative stress, anti-proteolytic function, and repair of the extracellular matrix . Concomitantly, surfactant proteins and Clara cell secretory protein mRNA expression decreased . Genome wide analysis of 307 mice generated from the backcross of resistant B6xA F1 with susceptible A strain identified significant linkage to a region on chromosome 6 (proposed as Aliq4) and suggestive linkages on chromosomes 1, 8, and 12 . Combining of these QTLs with two additional possible modifying loci (chromosome 9 and 16) accounted for the difference in survival time noted in the A and B6 parental strains . Combining these findings with those of the microarray analysis has enabled prioritization of candidate genes . These candidates, in turn, can be directed to the lung epithelium in transgenic mice or abated in inducible and constitutive gene-targeted mice . Initial results are encouraging and suggest that several of these mice vary in their susceptibility to oxidant-induced lung injury . Thus, these combined approaches have led to new insights into functional genomics of lung injury and diseases. Eur Respir J, 2001 Nov, 18(5), 882 - 9 Identification of disease genes by expression profiling; Bals R et al.; The human genome has been completely sequenced . The development of innovative methodologies and tools to understand the functions of human genes in health and disease will allow the data of the human genome project to be utilized . This paper reviews methods that can be used to detect and isolate genes that are specifically expressed in certain diseases or that are specific to cell types . First, classical methods, such as differential screening of complementary deoxyribonucleic acid libraries and subtractive techniques, are described . Methods based on polymerase chain reaction (PCR), such as differential display PCR or serial analysis of gene expression, will then be discussed . Finally, recent developments in gene chip technology and basic principles of functional genomics will be illustrated . Future developments will link the results of genomic approaches to data obtained by other systematic methods, such as proteomics (i.e . the systematic, large scale analysis of proteins), and will allow the production of a detailed molecular characterization of diseases, disease stages, tissues, or cell types . Methods to detect disease or cell type-specific gene expression patterns will play an important role in the future of basic research, as well as the development of novel diagnostic procedures and identification of therapeutic targets. Nucleic Acids Res, 2002 Jan 1, 30(1), 385 - 6 FUGOID: functional genomics of organellar introns database; Li F et al.; FUGOID is a web-based, taxonomically broad organelle intron database that collects and integrates various functional and structural data on organellar (mitochondrial and chloroplast) introns . The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism . It is implemented in a relational database management system, allowing sophisticated, user-friendly searching, data entry and revision . Users can access the database by any common web browser using a variety of operating systems . The main page of the database is available at http://wnt.cc.utexas.edu/~ifmr530/introndata/main.htm. Nucleic Acids Res, 2002 Jan 1, 30(1), 351 - 3 HOX Pro DB: the functional genomics of hox ensembles; Spirov AV et al.; The HOX Pro database contains information about the organization, function and evolution of gene ensembles, notably the homeobox-containing genes . It is now clear that a subset of genes containing the homeobox motif play key roles in the orchestration of genes which control embryonic patterning, morphogenesis, cell differentiation and malignant transformation . The HOX Pro contains a broad spectrum of information including images, diagrams and animations . Currently this amounts to approximately 700 HTML pages together with 400 images which contain information on 200 groups of genes and 90 promoters, in turn linked to maps of 13 HOX clusters and nine genetic networks . There are about 700 sequences of individual hox-genes of animals classified in approximately 200 homologous or paralogous groups . Graphical representation of HOX clusters and Hox-based networks is accomplished by means of flow and 3D diagrams, JavaScript animations and Java applets . The HOX Pro now includes sections presenting data mining and data simulation issues . The DB is located at http://www.iephb.nw.ru/hoxpro. Bioinformatics, 2001 Dec, 17(12), 1183 - 97 How to reconstruct a large genetic network from n gene perturbations in fewer than n(2) easy steps; Wagner A; MOTIVATION: The reconstruction of genetic networks is the holy grail of functional genomics . Its core task is to identify the causal structure of a gene network, that is, to distinguish direct from indirect regulatory interactions among gene products . In other words, to reconstruct a genetic network is to identify, for each network gene, which other genes and their activity the gene influences directly . Crucial to this task are perturbations of gene activity . Genomic technology permits large-scale experiments perturbing the activity of many genes and assessing the effect of each perturbation on all other genes in a genome . However, such experiments cannot distinguish between direct and indirect effects of a genetic perturbation . RESULTS: I present an algorithm to reconstruct direct regulatory interactions in gene networks from the results of gene perturbation experiments . The algorithm is based on a graph representation of genetic networks and applies to networks of arbitrary size and complexity . Algorithmic complexity in both storage and time is low, less than O(n(2)) . In practice, the algorithm can reconstruct networks of several thousand genes in mere CPU seconds on a desktop workstation . AVAILABILITY: A perl implementation of the algorithm is given in the Appendix . CONTACT: wagnera@unm.edu Plant Physiol, 2001 Dec, 127(4), 1539 - 55 Brachypodium distachyon . A new model system for functional genomics in grasses; Draper J et al.; A new model for grass functional genomics is described based on Brachypodium distachyon, which in the evolution of the Pooideae diverged just prior to the clade of "core pooid" genera that contain the majority of important temperate cereals and forage grasses . Diploid ecotypes of B . distachyon (2n = 10) have five easily distinguishable chromosomes that display high levels of chiasma formation at meiosis . The B . distachyon nuclear genome was indistinguishable in size from that of Arabidopsis, making it the simplest genome described in grasses to date . B . distachyon is a self-fertile, inbreeding annual with a life cycle of less than 4 months . These features, coupled with its small size (approximately 20 cm at maturity), lack of seed-head shatter, and undemanding growth requirements should make it amenable to high-throughput genetics and mutant screens . Immature embryos exhibited a high capacity for plant regeneration via somatic embryogenesis . Regenerated plants display very low levels of albinism and have normal fertility . A simple transformation system has been developed based on microprojectile bombardment of embryogenic callus and hygromycin selection . Selected B . distachyon ecotypes were resistant to all tested cereal-adapted Blumeria graminis species and cereal brown rusts (Puccinia reconditia) . In contrast, different ecotypes displayed resistance or disease symptoms following challenge with the rice blast pathogen (Magnaporthe grisea) and wheat/barley yellow stripe rusts (Puccinia striformis) . Despite its small stature, B . distachyon has large seeds that should prove useful for studies on grain filling . Such biological characteristics represent important traits for study in temperate cereals. Mod Pathol, 2001 Dec, 14(12), 1294 - 9 Functional genomics, gene arrays, and the future of pathology; Gabrielson E et al.; The human genome project has attracted a great deal of attention in recent years among the general public as well as the scientific community . Although it is likely to be a number of years before many of the expected benefits of the genomics revolution are realized, the impact of these scientific breakthroughs on diagnostic pathology is likely to become apparent relatively quickly . In particular, gene array technology, which allows gene expression measurements of thousands of genes in parallel, provides a powerful tool for pathologists seeking new markers for diagnosis . Several recent studies demonstrate how the gene array approach can not only recognize markers for known categories of neoplasia but also lead to recognition of different categories not previously appreciated . Although this approach shows great potential, the successful application of gene arrays to diagnostic problems will require thoughtful interpretation, just as immunochemical technologies require careful planning and analysis. Mol Biochem Parasitol, 2001 Dec, 118(2), 155 - 65 FLEXGene repository: from sequenced genomes to gene repositories for high-throughput functional biology and proteomics; Brizuela L et al.; The vast amount of information generated by the human genome sequencing project and related projects has given rise to a new paradigm in experimental biology . This new paradigm invokes the experimentation and data analysis at genome-wide scales, as well as the generation of new technologies and resources that take full advantage of the available sequence information . The Institute of Proteomics at Harvard Medical School is building a comprehensive, characterized, arrayed and flexible gene repository that will allow full exploitation of the genomic information by enabling functional genomics as well as protein expression, purification and analysis at genome wide scale . The FLEXGene repository (Full Length EXpression-ready) will contain clones representing the complete set of open reading frames (ORFs) of different organisms including H . sapiens and several pathogens and model organisms . The clones are constructed using recombination-based cloning technology so that hundreds or thousands of coding regions can be transferred into any expression vector in a parallel and timely mode, allowing the broadest variety of experiments to be carried out. Mol Biochem Parasitol, 2001 Dec, 118(2), 139 - 45 Bioinformatics and the malaria genome: facilitating access and exploitation of sequence information; Coppel RL; The torrent of sequence information unleashed by the various genome sequencing projects, including that of Plasmodium falciparum, will lead to an unprecedented increase in the data available for research purposes . The scientific community is struggling to develop ways to assimilate this information and ensure that it is fully analysed in a way that enables rapid development of new therapeutic and diagnostic advances . This is particularly so for the field of tropical medicine where many of the scientists have had limited training in the area of Bioinformatics and may be further hampered by poor access to the sequence data . A number of collections of malaria genome sequence are available, each with their own advantages and disadvantages, however further improvements in these information resources are needed . In particular, there would be great benefit in integrating genomic sequence and functional genomics results with the large amount of pre-existing knowledge related to parasite biology and immunological interactions with the host . Attempts to achieve this include the PlasmoDB database, and the lessons learned in this effort could be of great utility to other organism-specific databases. Genome Biol . 2001;2(11):REVIEWS1031 . Epub 2001 Oct 22. Intraepithelial gamma delta T cells exposed by functional genomics; Boismenu R et al.; Epithelial tissues house gammadelta T cells, which are important for the mucosal immune system and may be involved in controlling malignancies, infections and inflammation . Whole-genome gene-expression analysis provides a new way to study the signals required for the activation of gammadelta T cells, their mode of action and relationships among cells of the mucosal immune system. Nat Biotechnol, 2001 Dec, 19(12), 1162 - 7 Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria; Karberg M et al.; Mobile group II introns can be retargeted to insert into virtually any desired DNA target . Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22% . Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations . Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria. FEBS Lett, 2001 Nov 23, 508(3), 407 - 12 Antigen-independent selection of stable intracellular single-chain antibodies; Auf der Maur A et al.; The intracellular expression of single-chain Fv antibody fragments (scFv) in eukaryotic cells has an enormous potential in functional genomics and therapeutics {Marasco (1997) Gene Ther . 4, 11-15; Richardson and Marasco (1995) Trends Biotechnol . 13, 306-310} . However, the application of these so-called intrabodies is currently limited by their unpredictable behavior under the reducing conditions encountered inside eukaryotic cells, which can affect their stability and solubility properties {Worn et al . (2000) J . Biol . Chem . 275, 2795-2803; Biocca et al . (1995) Bio/Technology 13, 1110-1115} . We present a novel system that enables selection of stable and soluble intrabody frameworks in vivo without the requirement or knowledge of antigens . This system is based on the expression of single-chain antibodies fused to a selectable marker that can control gene expression and cell growth . Our results show that the activity of a selectable marker fused to well characterized scFvs {Worn et al . (2000) J . Biol . Chem . 275, 2795-2803} correlates with the solubility and stability of the scFv moieties . This method provides a unique tool to identify stable and soluble scFv frameworks, which subsequently serve as acceptor backbones to construct intrabody complementarity determining region libraries by randomization of hypervariable loops. Pharmacogenomics, 2001 Nov, 2(4), 361 - 72 Recent advances in computational genomics; Claverie JM et al.; In the post-genomic era, the new discipline of functional genomics is now facing the challenge of associating a function (as well as estimating its relevance to industrial applications) to about 100,000 microbial, plant or animal genes of known sequence but unknown function . Besides the design of databases, computational methods are increasingly becoming intimately linked with the various experimental approaches . Consequently, bioinformatics is rapidly evolving into independent fields addressing the specific problems of interpreting i) genomic sequences, ii) protein sequences and 3D-structures, as well as iii) transcriptome and macromolecular interaction data . It is thus increasingly difficult for the biologist to choose the computational approaches that perform best in these various areas . This paper attempts to review the most useful developments of the last 2 years. Vet Parasitol, 2001 Nov 22, 101(3-4), 215 - 30 PCR as a diagnostic and quantitative technique in veterinary parasitology; Zarlenga DS et al.; Over the past 15 years, there has been a dramatic evolution in molecular approaches to study parasites and parasitic diseases . Many of these advancements have been brought about through the development of new applications of the polymerase chain reaction (PCR) . Enhancements in sensitivity that can be achieved using PCR now permit scientists to investigate changes at the level of a single cell, far below what is often needed for parasite-derived applications . PCR has had a substantial impact on advances made in the areas of parasite systematics and epidemiology, immunology and host-parasite interactions, recombinant DNA vaccine development and most recently, the analysis of whole genomes either through directly sequencing the DNA, the analysis of expressed sequence tags (ESTs) or through the rapidly growing field of functional genomics . This paper, however, focuses on the application of PCR methodology to parasite detection and differentiation, and the diagnosis of disease . Specific attention is given to advances provided by multiplex PCR, fluorescence-based "real-time" PCR, and the utilization of PCR as a quantitative technique. Genomics, 2001 Nov, 78(1-2), 3 - 6 Viral vectors as part of an integrated functional genomics program; Janson CG et al.; Over the past decade, viral vectors have slowly gained mainstream acceptance in the neuroscience and genetics communities for the in vivo study of gene function {1} . Using stereotactic techniques, it is possible to characterize neuroanatomical relationships through the delivery of neurotropic viral vectors to specific brain regions . More sophisticated studies combine viral vectors with other methods of genetic manipulation such as germline transgenic mice . As more is learned about the properties of different viral vectors, it has become possible to use viral vectors to test hypotheses about the function of genes, through targeted in vivo delivery to the central nervous system (CNS) . The effects of gene expression in the brain can be measured on the molecular, biochemical, electrophysiological, morphological, and behavioral levels . We propose that viral vectors should be considered as part of an integrated functional genomics platform in the CNS. Nat Biotechnol, 2001 Nov, 19(11), 1047 - 52 Alteration of Cre recombinase site specificity by substrate-linked protein evolution; Buchholz F et al.; Directed molecular evolution was applied to generate Cre recombinase variants that recognize a new DNA target sequence . Cre was adapted in a three-stage strategy to evolve recombinases to specifically recombine the new site . This complex multicycle task was made feasible by an improved directed-evolution procedure that relies on placing the recombination substrate next to the recombinase coding region . Consequently, those DNA molecules carrying the coding region for a successful recombinase are physically marked by the action of that recombinase on the linked substrate and are easily retrieved from a large background of unsuccessful candidates by PCR amplification . We term this procedure substrate-linked protein evolution (SLiPE) . The method should facilitate the development of new recombinases and other DNA-modifying enzymes for applications in genetic engineering, functional genomics, and gene therapy. Trends Mol Med, 2001 Nov, 7(11), 521 - 6 Genetics and genomics in infectious disease susceptibility; Blackwell JM; The past decade has witnessed a rapid transition from the first positional cloning of an infectious disease susceptibility gene (Slc11a1, also called Nramp1) in the mouse to genome-wide scans in human multicase families and the identification of potential disease-causing genes by simple inspection of the public human genome databases . Pathogen genome projects have facilitated multilocus sequence typing of pathogen isolates and studies of ecological fitness and virulence patterns in disease-causing isolates . Comparative sequence analysis of pathogen strains and functional genomics studies are now underway, hopefully providing new insight into infectious disease susceptibility. Trends Mol Med, 2001 Nov, 7(11), 494 - 501 Taking a functional genomics approach in molecular medicine; Yaspo ML; The elucidation of genetic components of human diseases at the molecular level provides crucial information for developing future causal therapeutic intervention . High-throughput genome sequencing and systematic experimental approaches are fuelling strategic programs designed to investigate gene function at the biochemical, cellular and organism levels . Bioinformatics is one important tool in functional genomics, although showing clear limitations in predicting ab initio gene structures, gene function and protein folds from raw sequence data . Systematic large-scale data-set generation, using the same type of experiments that are used to decipher the function of single genes, are being applied on entire genomes . Comparative genomics, establishment of gene catalogues, and investigation of cellular and tissue molecular profiles are providing essential tools for understanding gene function in complex biological networks. Oncogene, 2001 Oct 18, 20(47), 6871 - 80 Studies on human colon cancer gene APC by targeted expression in Drosophila; Bhandari P et al.; Mutations in human Adenomatous Polyposis Coli (APC) gene are associated with both familial and sporadic colorectal tumors . APC is known to down regulate beta-catenin levels, a transducer of Wnt signaling . The aim of this study is to provide transgenic Drosophila expressing either full-length or truncated forms of human APC (hAPC) protein and methods for using them in functional genomics and drug screening . Consistent with its biochemical properties, targeted expression of either full-length hAPC or its beta-catenin binding domain alone negatively regulated the function of the beta-catenin homologue, Armadillo (Arm) and thereby, inhibited Wnt/Wg signaling during fly development . hAPC inhibited Arm function even in the absence of GSK-3beta activity, although the latter was required to mediate the degradation of Arm . Consistent with this, hAPC suppressed the phenotypes induced by the over-expression of degradation-resistant forms of Arm . Subsequently, using hAPC-induced eye phenotypes as the assay in a suppressor-enhancer screen, we have identified two new loci in Drosophila, which modulate Wnt/Wg signaling . In addition, an anti-colon cancer drug, indomethacin, specifically enhanced hAPC-induced phenotypes. Biotechnol Annu Rev, 2001, 7, 195 - 238 Genomics and plant breeding; Aljanabi S; Much of our most basic understanding of genetics has its roots in plant genetics and crop breeding . The study of plants has led to important insights into highly conserved biological process and a wealth of knowledge about development . Agriculture is now well positioned to take its share benefit from genomics . The primary sequences of most plant genes will be determined over the next few years . Informatics and functional genomics will help identify those genes that can be best utilized to crop production and quality through genetic engineering and plant breeding . Recent developments in plant genomics are reviewed. Biotechnol Annu Rev, 2001, 7, 131 - 64 P53-responsive genes and the potential for cancer diagnostics and therapeutics development; Xu H et al.; P53 protein regulates cell responses to DNA damage to keep genomic stability by transactivation and trans-repression of its downstream target genes . P53 protein also has activators, inactivators, or co-factors via interaction with other proteins . Both the p53-regulated genes and interacted proteins form a huge network . As tumors usually escape from proliferating controls by means of accumulation of genetic alterations, p53 is one of the most important tumor suppressor genes that can be targeted for diagnosis, prognosis, and therapeutic intervention . Reviewing the p53-network is of great importance . In this review, we are focusing on cancer-related p53 downstream-regulated genes . Various methods dealing with the discovery of p53-regulated genes by the detection of gene expression have been applied . Recently high throughput functional genomics methods, such as DNA microarray, serial analysis of gene expression (SAGE), differential display, and protein two-dimensional gel electrophoresis, have provided a wealth of information on the dynamics of cell context responses . Hundreds of genes have been discovered whose transcriptions are regulated by p53 protein . They were grouped, based on their functions, into sub-classes including cell-cycle regulation, DNA repair, angiogenesis, metastasis, and multidrug resistance . P53 plays a pivotal role in keeping genomic stability and tumor suppression . The deeper we investigate the cell responses as mediated by p53, the more complex p53-network becomes . However, understanding p53-network, offers great opportunities to develop more sensitive and accurate diagnostic/prognostic tools, as well as more efficient therapies for cancer. Hum Mol Genet, 2001 Oct 1, 10(20), 2209 - 14 Sequence variation and disease in the wake of the draft human genome; Goodstadt L et al.; The sequencing phase of the human genome project will soon be over . In its wake, repertoires of sequence polymorphisms among the human population are being sampled and a battery of functional genomics projects, from gene and protein expression studies to whole proteome interaction experiments, are generating vast quantities of data . Now that the data, or the means to generate data, are available it is the application of this information in enhancing our understanding of biology that represents the next formidable challenge . Two prominent issues should be considered . First, existing data must be analysed using the best methods available . The prediction of enzymatic activity for bestrophin, whose gene is mutated in Best macular dystrophy, is described in this review . This is an example of the experimentally testable hypotheses that can result from such detailed and exhaustive analyses . Secondly, the torrents of data from high-throughput studies will need to be made more accessible to all using web-based resources that integrate and digest complementary data types . The internet sites that showcase the human genome sequence are blazing a new trail . Ultimately, the success of genome sequencing and functional genomics will be measured not by the quantity and accuracy of raw data generated, but how rapidly they can be harnessed to span the divide between genotype and phenotype. Swiss Med Wkly, 2001 Aug 11, 131(31-32), 459 - 70 Functional genomics in sarcoidosis--reduced or increased apoptosis? Rutherford RM, Kehren J, Staedtler F, Chibout SD, Egan JJ, Tamm M, Gilmartin JJ, Brutsche MH. BACKGROUND: A variety of studies have stressed the importance of the control of inflammatory cell longevity and the balance of pro-survival and pro-apoptotic signaling pathways . The aim of the study was to investigate the systemic activation of apoptosis pathways using cDNA array technology in patients with acute onset sarcoidosis . METHOD: We have performed a comprehensive genomic analysis, applying high-density human GeneChip probe arrays (HGU95A, Affymetrix) for RNA expression profiling from peripheral blood mononuclear cells from patients with acute pulmonary sarcoidosis and matched healthy controls . Twelve patients and 12 controls were assessed, mean age 36 +/- 12 and 33 +/- 10 years respectively . Results focus on apoptosis-related gene products . Group differences were assessed with the Mann-Whitney U-test . RESULTS: Seven patients had self-limited disease (all type I sarcoidosis) and 5 progressive disease requiring immunosuppression (all type II or III sarcoidosis) . We found 53 of 112 (47%) apoptosis-related gene products dysregulated in sarcoidosis compared to controls . Particular growth factors, especially heparin-binding EGF-like GF, EGF, PDEGF, SISPDGF2 and VEGF, were upregulated in patients consistent with a pro-survival profile . The Bcl-2 family of genes also showed a net pro-survival profile in sarcoidosis patients . In contrast, alterations in the TNF-pathway were compatible with increased apoptosis signals in both, type I and type II/III sarcoidosis patients . Other cell death receptors were equally expressed, as were caspases and p53-associated genes . In contrast to patients with type I-sarcoidosis, patients with progressive type II or III disease showed an upregulation of NFKB and a leak of downregulation of inhibitor of apoptosis 1 . CONCLUSION: Significant differences in the expression of apoptosis-related genes were found in peripheral blood of patients with acute onset sarcoidosis . Gene expression did not show a definite pattern that was suggestive of pro-survival or proapoptosis . However, the number of genes whose altered expression would be predicted to favour increased survival exceeded that of genes likely to reduce survival . Protein-based confirmation of the differences in the activity of apoptosis-pathways needs to be done in further studies. Radiat Res, 2001 Nov, 156(5 Pt 2), 657 - 61 Induction of gene expression as a monitor of exposure to ionizing radiation; Amundson SA et al.; The complex molecular responses to genotoxic stress are mediated by a variety of regulatory pathways . The transcription factor TP53 plays a central role in the cellular response to DNA-damaging agents such as ionizing radiation, but other pathways also play important roles . In addition, differences in radiation quality, such as the exposure to high-LET radiation that occurs during space travel, may influence the pattern of responses . The premise is developed that stress gene responses can be employed as molecular markers for radiation exposure using a combination of informatics and functional genomics approaches . Published studies from our laboratory have already demonstrated such transcriptional responses with doses of gamma rays as low as 2 cGy, and in peripheral blood lymphocytes (PBLs) irradiated ex vivo with doses as low as 20 cGy . We have also found several genes elevated in vivo 24 h after whole-body irradiation of mice with 20 cGy . Such studies should provide insight into the molecular responses to physiologically relevant doses, which cannot necessarily be extrapolated from high-dose studies . In addition, ongoing experiments are identifying large numbers of potential biomarkers using microarray hybridization and various irradiation protocols including expression at different times after exposure to low- and high-LET radiation . Computation-intensive informatics analysis methods are also being developed for management of the complex gene expression profiles resulting from these experiments . With further development of these approaches, it may be feasible to monitor changes in gene expression after low-dose radiation exposure and other physiological stresses that may be encountered during manned space flight, such as the planned mission to Mars. Genome Biol . 2001;2(10):REVIEWS1028 . Epub 2001 Sep 14. Functional genomics and metal metabolism; Eide DJ; Metal ions are essential nutrients, yet they can also be toxic if they over-accumulate . Homeostatic mechanisms and detoxification systems therefore precisely control their intracellular levels and distribution . The tools of functional genomics are rapidly accelerating understanding in this field, particularly in the yeast Saccharomyces cerevisiae. Angew Chem Int Ed Engl, 2001 Oct 1, 40(19), 3508 - 3533 Carbohydrate-Based Antibiotics: A New Approach to Tackling the Problem of Resistance; Ritter TK et al.; Recent interest in the problem of antibiotic resistance has led to the identification of new targets and strategies for antibiotic discovery . Among these efforts, the development of small molecules as antibiotics to target carbohydrate receptors or carbohydrate-modifying enzymes represents a new direction . This review covers recent work in this regard and discusses the impact of each strategy on the development of drug resistance . Particularly interesting targets include unique cell-surface carbohydrates, the transglycosylase involved in peptidoglycan biosynthesis, and bacterial RNA . With a greater understanding of the genome of different bacteria as well as advances in functional genomics and proteomics, we can expect the discovery of a variety of targets for the development of novel antibiotics. Rev Med Virol, 2001 Sep-Oct, 11(5), 313 - 29 Post-genomic virology: the impact of bioinformatics, microarrays and proteomics on investigating host and pathogen interactions; Kellam P; Post-genomic research encompasses many diverse aspects of modern science . These include the two broad subject areas of computational biology (bioinformatics) and functional genomics . Laboratory based functional genomics aims to measure and assess either the messenger RNA (mRNA) levels (transcriptome studies) or the protein content (proteome studies) of cells and tissues . All of these methods have been applied recently to the study of host and pathogen interactions for both bacteria and viruses . A basic overview of the technology is given in this review together with approaches to data analysis . The wealth of information produced from even these preliminary studies has shown the generalities, subtleties and specificities of host-pathogen interactions . Such research should ultimately result in new methods for diagnosing and treating infectious diseases . Chem Biol, 2001 Oct, 8(10), 931 - 9 Intramers as promising new tools in functional proteomics; Famulok M et al.; Aptamers are valuable tools for studying numerous aspects of biological processes, opening up new experimental opportunities to analyse the function of a wide range of cellular molecules . Functional RNA molecules can be rapidly selected in vitro from complex combinatorial mixtures of different sequences . Recently, it was shown that in vitro selection processes can be automated: the first generation selection robots will soon mean aptamers for several targets can be isolated in parallel within days rather than weeks . Aptamers not only exhibit highly specific molecular recognition properties but are also able to modulate the function of their cognate targets in a highly specific manner by agonistic or antagonistic mechanisms . These properties prompted the development of novel technologies to exploit the use of aptamers to modulate distinct functions of biological targets . Recent controlled expression of aptamers inside cells demonstrated their impressive potential as rapidly generated intracellular inhibitors of biomolecules . Intracellularly applied aptamers are also called 'intramers' . Here we discuss recent developments and strategies for intramer-based technologies that have the potential to greatly facilitate characterisation of unknown protein functions in the context of their natural expression status in vivo . Thus, intramer-based technologies offer many promising applications in functional genomics, proteomics and drug discovery. Nat Rev Genet, 2001 Oct, 2(10), 769 - 79 Recombineering: a powerful new tool for mouse functional genomics; Copeland NG et al.; Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases . This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means . Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome. Anal Biochem, 2001 Oct 1, 297(1), 79 - 85 Screening for soluble expression of recombinant proteins in a 96-well format; Knaust RK et al.; For future structural and functional genomics programs new tools will be required . The implementation of high-throughput (HTP) methods for protein production will be an essential element . Present HTP protein production developments in structural genomics are aimed at obtaining well-expressing and highly soluble proteins, which are preferred candidates for structure-function studies . Here, we describe a cheap and efficient procedure to identify well-expressing soluble proteins in Escherichia coli in a compact 96-well format . Reproducible lysis on filter plates, followed by a filtration step on 96-well filter plates, allows the efficient separation of inclusion bodies from the soluble fraction . In the following step a dot blot procedure using anti-RGS-His(4) antibody (Qiagen) to detect expression of recombinant His-tagged protein is applied allowing direct detection of the target protein in the soluble fraction . The method is well suited for automation and should be applicable to expression screening of most proteins and fusion domains to which specific antibodies are available . Comb Chem High Throughput Screen, 2001 Sep, 4(6), 497 - 509 Genotype-phenotype linkage for directed evolution and screening of combinatorial protein libraries; Doi N et al.; The technologies for screening peptide and protein libraries for studies in the fields of directed protein evolution and functional genomics have advanced with astonishing speed . For screening of functional proteins, three technologies are required: (i) the construction of a gene library (genotype), (ii) the establishment of a linkage between each protein (phenotype) and its encoding gene (genotype), and (iii) the selection of desired proteins (phenotype) from the library . This review highlights the genotype-phenotype linkage technologies, which can be classified into three types; that is, cell-type linkage, virus-type linkage, and array-type linkage methods . These methods are summarized, and their advantages and disadvantages are discussed. EMBO Rep, 2001 Sep, 2(9), 821 - 8 A protein-protein interaction map of the Caenorhabditis elegans 26S proteasome; Davy A et al.; The ubiquitin-proteasome proteolytic pathway is pivotal in most biological processes . Despite a great level of information available for the eukaryotic 26S proteasome-the protease responsible for the degradation of ubiquitylated proteins-several structural and functional questions remain unanswered . To gain more insight into the assembly and function of the metazoan 26S proteasome, a two-hybrid-based protein interaction map was generated using 30 Caenorhabditis elegans proteasome subunits . The results recapitulate interactions reported for other organisms and reveal new potential interactions both within the 19S regulatory complex and between the 19S and 20S subcomplexes . Moreover, novel potential proteasome interactors were identified, including an E3 ubiquitin ligase, transcription factors, chaperone proteins and other proteins not yet functionally annotated . By providing a wealth of novel biological hypotheses, this interaction map constitutes a framework for further analysis of the ubiquitin-proteasome pathway in a multicellular organism amenable to both classical genetics and functional genomics. Acta Biotheor, 2001, 49(3), 191 - 202 Concept of immunomics: a new frontier in the battle for gene function? Klysik J. At the beginning of the 21st century, biology will try to address the function of a large number of new genes . From the perspective of technologies applied today to functional genomics, this task appears to be more complex than the effort invested in the sequencing of the human genome . Conceptually, a high-throughput approach permitting correlation between newly discovered genes and functional properties of their protein products has yet to be developed . To address relationships between tens of thousands of genes and their cognate proteins, novel interdisciplinary technologies need to emerge . In this paper, a new idea of immunomics is presented and an experimental strategy is outlined to circumvent some of the restrictions associated with methodologies currently in use . It is proposed that cloned segments of genomic DNA are used for genetic immunization to obtain a large collection of antibodies, and to generate microarrays of these antibodies for tracing differentially expressed cellular proteins. Clin Invest Med, 2001 Aug, 24(4), 179 - 95 Henry Friesen Award Lecture . Work, the clinician-scientist and human biochemical genetics; Scriver CR; The pursuit of human biochemical genetics has allowed us to understand better how the person with the (genetic) disease differs from the disease the person has and to develop the concept that genetics belongs in all aspects of health care . It is a perspective that comes quite readily to the clinician-scientist, and the restoration of that "species" in the era of functional genomics is strongly recommended . Garrod, the initial founder of human "biochemical genetics" belonged to the clinician-scientist community . Archibald Edward Garrod introduced a paradigm, new for its day, in medicine: biochemistry is dynamic and different from the static nature of organic chemistry . It led him to think about metabolic pathways and to recognize that variation in Mendelian heredity could explain an "inborn error of metabolism." At the time, Garrod had no idea about the nature of a gene . Genes are now well understood; genomes are being described for one organism after another (including Homo sapiens) and it is understood that genomes "speak biochemistry (not phenotype)." Accordingly, in the era of genomics, biochemistry and physiology become the bases of functional genomics, and it is possible to appreciate why "nothing in biology makes sense without evolution" (and nothing in medicine will make sense without biology) . Mendelian, biochemical and molecular genetics together have revealed what lies behind the 4 canonical inborn errors described by Garrod (albinisn, alkaptonuria, cystinuria and pentosuria) . Both older and newer ideas in genetics, new tools for applying them (and renewed respect for the clinician-scientist) will enhance our understanding of the human biological variation that accounts for variant states of health and overt disease . A so-called monogenic phenotype (phenylketonuria) is used to illustrate, in some detail, that all disease phenotypes are, in one way or another, likely to be complex in nature . What can be known and what ought to be done, with knowledge about human genetics, to benefit individuals, families and communities (society), is both opportunity and challenge. Anal Biochem, 2001 Sep 15, 296(2), 179 - 87 Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Todd MJ, Gomez J. Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented . The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated . (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained . (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored . Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition . Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1) . This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates . Such a universal assay may have wide applicability in functional genomics . Plant Mol Biol, 2001 Sep, 47(1-2), 275 - 91 Functional genomics and cell wall biosynthesis in loblolly pine; Whetten R et al.; Loblolly pine (Pinus taeda L.) is the most widely planted tree species in the USA and an important tree in commercial forestry world-wide . The large genome size and long generation time of this species present obstacles to both breeding and molecular genetic analysis . Gene discovery by partial DNA sequence determination of cDNA clones is an effective means of building a knowledge base for molecular investigations of mechanisms governing aspects of pine growth and development, including the commercially relevant properties of secondary cell walls in wood . Microarray experiments utilizing pine cDNA clones can be used to gain additional information about the potential roles of expressed genes in wood formation . Different methods have been used to analyze data from first-generation pine microarrays, with differing degrees of success . Disparities in predictions of differential gene expression between cDNA sequencing experiments and microarray experiments arise from differences in the nature of the respective analyses, but both approaches provide lists of candidate genes which should be further investigated for potential roles in cell wall formation in differentiating pine secondary xylem . Some of these genes seem to be specific to pine, while others also occur in model plants such as Arabidopsis, where they could be more efficiently investigated. Plant Mol Biol, 2001 Sep, 47(1-2), 115 - 30 Golgi enzymes that synthesize plant cell wall polysaccharides: finding and evaluating candidates in the genomic era; Perrin R et al.; Although the synthesis of cell wall polysaccharides is a critical process during plant cell growth and differentiation, many of the wall biosynthetic genes have not yet been identified . This review focuses on the synthesis of noncellulosic matrix polysaccharides formed in the Golgi apparatus . Our consideration is limited to two types of plant cell wall biosynthetic enzymes: glycan synthases and glycosyltransferases . Classical means of identifying these enzymes and the genes that encode them rely on biochemical purification of enzyme activity to obtain amino acid sequence data that is then used to identify the corresponding gene . This type of approach is difficult, especially when acceptor substrates for activity assays are unavailable, as is the case for many enzymes . However, bioinformatics and functional genomics provide powerful alternative means of identifying and evaluating candidate genes . Database searches using various strategies and expression profiling can identify candidate genes . The involvement of these genes in wall biosynthesis can be evaluated using genetic, reverse genetic, biochemical, and heterologous expression methods . Recent advances using these methods are considered in this review. Methods Inf Med, 2001, 40(4), 346 - 58 What is bioinformatics? A proposed definition and overview of the field; Luscombe NM et al.; BACKGROUND: The recent flood of data from genome sequences and functional genomics has given rise to new field, bioinformatics, which combines elements of biology and computer science . OBJECTIVES: Here we propose a definition for this new field and review some of the research that is being pursued, particularly in relation to transcriptional regulatory systems . METHODS: Our definition is as follows: Bioinformatics is conceptualizing biology in terms of macromolecules (in the sense of physical-chemistry) and then applying "informatics" techniques (derived from disciplines such as applied maths, computer science, and statistics) to understand and organize the information associated with these molecules, on a large-scale . RESULTS AND CONCLUSIONS: Analyses in bioinformatics predominantly focus on three types of large datasets available in molecular biology: macromolecular structures, genome sequences, and the results of functional genomics experiments (e.g . expression data) . Additional information includes the text of scientific papers and "relationship data" from metabolic pathways, taxonomy trees, and protein-protein interaction networks . Bioinformatics employs a wide range of computational techniques including sequence and structural alignment, database design and data mining, macromolecular geometry, phylogenetic tree construction, prediction of protein structure and function, gene finding, and expression data clustering . The emphasis is on approaches integrating a variety of computational methods and heterogeneous data sources . Finally, bioinformatics is a practical discipline . We survey some representative applications, such as finding homologues, designing drugs, and performing large-scale censuses . Additional information pertinent to the review is available over the web at http://bioinfo.mbb.yale.edu/what-is-it. Curr Opin Biotechnol, 2001 Aug, 12(4), 348 - 54 High-throughput three-dimensional protein structure determination; Heinemann U et al.; In the wake of finished genomic sequencing projects, high-throughput analysis techniques are being developed in various fields of functional genomics . Of special interest in this regard is the three-dimensional structure analysis of proteins by X-ray crystallography and NMR spectroscopy, which has been characterized by distinctly low-throughput in the past . A number of recent advances in instrumentation and software are promising to radically change this situation, leaving the production of suitable protein samples as the sole rate-limiting step in structural analyses. J Struct Biol, 2001 May-Jun, 134(2-3), 269 - 81 Review: protein design--where we were, where we are, where we're going; Pokala N et al.; Protein design has become a powerful approach for understanding the relationship between amino acid sequence and 3-dimensional structure . In the past 5 years, there have been many breakthroughs in the development of computational methods that allow the selection of novel sequences given the structure of a protein backbone . Successful design of protein scaffolds has now paved the way for new endeavors to design function . The ability to design sequences compatible with a fold may also be useful in structural and functional genomics by expanding the range of proteins used for fold recognition and for the identification of functionally important domains from multiple sequence alignments . J Struct Biol, 2001 May-Jun, 134(2-3), 257 - 68 On the structure of hisH: protein structure prediction in the context of structural and functional genomics; O'Donoghue P et al.; We predict a structure of the glutamine amidotransferase subunit (hisH) of imidazole glycerol phosphate synthase (IGPS) which catalyzes the fifth step of the histidine biosynthesis in Escherichia coli . The model is constructed using an energy-based threading program augmented by a multiple sequence to structure profile analysis . In developing our model we identified a conserved core region within hisH and a variable domain which is the likely site of interaction with the synthase subunit (hisF) of IGPS . Information available from structural and functional genomics studies was used to improve the structure prediction, to discuss parallels between histidine biosynthesis and other amino acid and nucleotide metabolic pathways, and to better understand the protein-protein interactions between the hisH and hisF domains of IGPS . This work allows us to develop a preliminary model for the structure of the entire IGPS holoenzyme . Genome Res, 2001 Sep, 11(9), 1463 - 8 Interrelating different types of genomic data, from proteome to secretome: 'oming in on function; Greenbaum D et al.; With the completion of genome sequences, the current challenge for biology is to determine the functions of all gene products and to understand how they contribute in making an organism viable . For the first time, biological systems can be viewed as being finite, with a limited set of molecular parts . However, the full range of biological processes controlled by these parts is extremely complex . Thus, a key approach in genomic research is to divide the cellular contents into distinct sub-populations, which are often given an "-omic" term . For example, the proteome is the full complement of proteins encoded by the genome, and the secretome is the part of it secreted from the cell . Carrying this further, we suggest the term "translatome" to describe the members of the proteome weighted by their abundance, and the "functome" to describe all the functions carried out by these . Once the individual sub-populations are defined and analyzed, we can then try to reconstruct the full organism by interrelating them, eventually allowing for a full and dynamic view of the cell . All this is, of course, made possible because of the increasing amount of large-scale data resulting from functional genomics experiments . However, there are still many difficulties resulting from the noisiness and complexity of the information . To some degree, these can be overcome through averaging with broad proteomic categories such as those implicit in functional and structural classifications . For illustration, we discuss one example in detail, interrelating transcript and cellular protein populations (transcriptome and translatome) . Further information is available at http://bioinfo.mbb.yale.edu/what-is-it. Biosens Bioelectron, 2001 Sep, 16(7-8), 593 - 601 Identification of differential gene expression profiles in rat cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline; Andreadis JD et al.; Recent technological advancements in microfabrication combined with the rapid acquisition of full genome sequence data have led to the development of DNA arrays that have the capacity to monitor the expression levels of thousands of genes simultaneously . The development of this technology enables the use of functional genomics approaches to identify molecular markers associated with cellular responsiveness to cytotoxic exposures . Databases containing unique cell-response profiles associated with specific toxicants or classes of toxicants can then be used in conjunction with cell-based biosensor platforms for environmental surveillance and toxicological assessment . An important issue that must be addressed, however, is whether DNA arrays can be used to identify transient gene modulation events in a reproducible manner . To address this issue, we utilized a primary embryonic rat (day 18) cortical cell model system and examined the RNA of both chemically treated and untreated cells using radioisotope-labeled cDNA probes and commercially available nylon membrane arrays . Using this approach, we examined experimental variability, basal gene expression variability, the occurrence of false positives, and the reproducibility of gene expression profiles obtained after chemical exposure . Minimal differences in gene modulation were observed between RNA samples from independently cultured cortical cells when array experiments were conducted in parallel (Pearson correlation coefficient for gene intensities =0.98) . In contrast, significant differences in gene expression were observed between array experiments conducted at different times with an identical RNA source (Pearson correlation coefficient for gene intensities=0.91) . Our results suggest the effect of basal gene activity differences in independently isolated cell cultures is negligible and that experimental variability possibly associated with the handling of RNA samples, differences in reverse transcription efficiency, hybridization, and/or signal acquisition are the primary contributors to variability in measurements . Using cDNA array analysis of unexposed cells from three independent cell culture preparations, we calculated false positive gene modulation events as a function of the threshold absolute value of log(2) >1.0 . The number of false positives using this criteria was 1-10 gene/ESTs/5109 actively transcribed gene/ESTs represented on the array . Using three independent replicate experiments of untreated cortical cell cultures, we determined that a threshold criterion of absolute value of log(2) >0.63 for triplicate experiments would reduce the expected number of false positives in our experiments to less than one . Using this criterion, reproducible gene expression profiles were identified in cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline. J Comput Biol, 2001, 8(3), 221 - 34 Mathematical modeling for functional divergence after gene duplication; Gu X; In this paper, I present a statistical framework for modeling the functional divergence after gene duplication . A rate-component model to describe the rate covariation among homologous genes of a gene family is implemented when a phylogenetic tree is known . The Markov chain model is rigorous but may require a huge amount of computational time when the number of sequences is large . On the other hand, the Poisson-based model is mathematically analytical so that computation is very fast even for a large dataset . Moreover, under the posterior framework, we have developed a site-specific profile for predicting important amino acid residues responsible for these functional differences between member genes of a gene family . Our study may have great potential for functional genomics because it is cost-effective, and these predictions can be further tested by biological experimentation. Nat Rev Genet, 2001 Sep, 2(9), 681 - 9 Http://C . elegans: mining the functional genomic landscape; Kim SK; Caenorhabditis elegans is a powerful animal model for the study of functional genomics . The completed and well-annotated DNA sequence is available and a systematic study of gene function by RNA-interference-mediated knockdown of every gene is in progress . Full-genome DNA microarrays and DNA chips can be used to determine expression changes at different stages of development and in different mutant backgrounds, and a protein-interaction map based on the yeast two-hybrid approach is in progress . These high-capacity approaches to studying gene function will provide new insights into invertebrate and vertebrate biology. Nat Biotechnol, 2001 Sep, 19(9), 851 - 5 A high-throughput alphavirus-based expression cloning system for mammalian cells; Koller D et al.; We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors . The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand . Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus . Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS) . Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step . Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced . Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins. Anim Genet, 2001 Aug, 32(4), 169 - 88 Transcriptome of channel catfish (Ictalurus punctatus): initial analysis of genes and expression profiles of the head kidney; Cao D et al.; Analysis of expressed sequence tags (ESTs) is an efficient approach for gene discovery, expression profiling, and development of resources useful for functional genomics studies . As part of the transcriptome analysis in channel catfish (Ictalurus punctatus), we have conducted EST analysis using a cDNA library made from the head kidney . We analysed 2228 EST clones . Orthologues were established for 1495 (67.1%) clones representing 748 genes, of which 545 (36.5%) clones were singletons . The remaining 733 (32.9%) clones represent unknown gene clones, for which the number of genes has not yet been determined. Biol Chem, 2001 Jul, 382(7), 993 - 9 Functional genomics in HIV-1 virus replication: protein-protein interactions as a basis for recruiting the host cell machinery for viral propagation; Tasara T et al.; Identification and characterization of protein-protein interactions between the host cell and parasites both enhance our understanding of basic cell biology and provide insights into central processes of parasite life cycles . Research on HIV-1 has broadened our knowledge of the various molecular events involved . However, our understanding of how this virus interacts with the host cell at the level of protein-protein interaction is still limited . Through these interactions the virus is able to recruit certain cellular metabolic pathways for its replication . Here we summarize our current knowledge of protein-protein interactions between HIV-1 and host cell factors during viral replication. Proc R Soc Lond B Biol Sci, 2001 Sep 7, 268(1478), 1803 - 10 The small world inside large metabolic networks; Wagner A et al.; The metabolic network of the catabolic, energy and biosynthetic metabolism of Escherichia coli is a paradigmatic case for the large genetic and metabolic networks that functional genomics efforts are beginning to elucidate . To analyse the structure of previously unknown networks involving hundreds or thousands of components by simple visual inspection is impossible, and quantitative approaches are needed to analyse them . We have undertaken a graph theoretical analysis of the E . coli metabolic network and find that this network is a small-world graph, a type of graph distinct from both regular and random networks and observed in a variety of seemingly unrelated areas, such as friendship networks in sociology, the structure of electrical power grids, and the nervous system of Caenorhabditis elegans . Moreover, the connectivity of the metabolites follows a power law, another unusual but by no means rare statistical distribution . This provides an objective criterion for the centrality of the tricarboxylic acid cycle to metabolism . The small-world architecture may serve to minimize transition times between metabolic states, and contains evidence about the evolutionary history of metabolism. Nutr Rev, 2001 Aug, 59(8 Pt 1), 259 - 63 Functional genomics and proteomics applied to the study of nutritional metabolism; Guengerich FP; Functional genomics, commonly applied to the genes and enzymes involved in metabolism of chemicals, can also be applied to enzymes involved in the metabolism of nutrients . Although in its infancy, genomics can be used to determine relationships between nutrition and toxicology, drug metabolism, and cancer. J Gen Virol, 2001 Sep, 82(Pt 9), 2051 - 60 A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors; Sollerbrant K et al.; The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells . Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest . We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV) . AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method . We show here that co-infection of mammalian producer cells with three viruses - a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene - produces infectious rAAV particles . This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production. Mol Biotechnol, 2001 Jul, 18(3), 213 - 32 Pathophysiological mechanisms of asthma . Application of cell and molecular biology techniques; Chung KF et al.; Asthma is a common increasing and relapsing disease that is associated with genetic and environmental factors such as respiratory viruses and allergens . It causes significant morbidity and mortality . The changes occurring in the airways consist of a chronic eosinophilic and lymphocytic inflammation, together with epithelial and structural remodeling and proliferation, and altered matrix proteins, which underlie airway wall narrowing and bronchial hyperresponsiveness (BHR) . Several inflammatory mediators released from inflammatory cells such as histamine and cysteinyl-leukotrienes induce bronchoconstriction, mucus production, plasma exudation, and BHR . Increased expression of T-helper 2 (Th2)-derived cytokines such as interleukin-4 and 5 (IL-4, 5) have been observed in the airway mucosa, and these may cause IgE production and terminal differentiation of eosinophils . Chemoattractant cytokines (chemokines) such as eotaxin may be responsible for the chemoattraction of eosinophils to the airways . The initiating events are unclear but may be genetically determined and may be linked to the development of a Th2-skewed allergen-specific immunological memory . The use of molecular biology techniques on tissues obtained from asthmatics is increasing our understanding of the pathophysiology of asthma . With the application of functional genomics and the ability to transfer or delete genes, important pathways underlying the cause if asthma will be unraveled . The important outcome of this is that new preventive and curative treatments may ensue. Plant Physiol, 2001 Aug, 126(4), 1527 - 38 T-DNA-associated duplication/translocations in Arabidopsis . Implications for mutant analysis and functional genomics; Tax FE et al.; T-DNA insertion mutants have become a valuable resource for studies of gene function in Arabidopsis . In the course of both forward and reverse genetic projects, we have identified novel interchromosomal rearrangements in two Arabidopsis T-DNA insertion lines . Both rearrangements were unilateral translocations associated with the left borders of T-DNA inserts that exhibited normal Mendelian segregation . In one study, we characterized the embryo-defective88 mutation . Although emb88 had been mapped to chromosome I, molecular analysis of DNA adjacent to the T-DNA left border revealed sequence from chromosome V . Simple sequence length polymorphism mapping of the T-DNA insertion demonstrated that a >40-kbp region of chromosome V had inserted with the T-DNA into the emb88 locus on chromosome I . A similar scenario was observed with a prospective T-DNA knockout allele of the LIGHT-REGULATED RECEPTOR PROTEIN KINASE (LRRPK) gene . Whereas wild-type LRRPK is on lower chromosome IV, mapping of the T-DNA localized the disrupted LRRPK allele to chromosome V . In both these cases, the sequence of a single T-DNA-flanking region did not provide an accurate picture of DNA disruption because flanking sequences had duplicated and inserted, with the T-DNA, into other chromosomal locations . Our results indicate that T-DNA insertion lines--even those that exhibit straightforward genetic behavior--may contain an unexpectedly high frequency of rearrangements . Such duplication/translocations can interfere with reverse genetic analyses and provide misleading information about the molecular basis of mutant phenotypes . Simple mapping and polymerase chain reaction methods for detecting such rearrangements should be included as a standard step in T-DNA mutant analysis. Curr Opin Mol Ther, 2001 Jun, 3(3), 2