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Forum (Genova), 1999 Jul-Dec, 9(3 Suppl 3), 37 - 46 Biotechnology and molecular diagnostics; Gulino A; Molecular diagnostic applications of biotechnology have allowed the exploitation of biological processes for the industrial production of bio-products that are used for diagnostic purposes . Advances in genetic-related technologies together with a multidisciplinary interplay of several fields led to the development of genomics which is focusing at the detection of pathogenetic events at the genome level . Both structural and functional genomic approaches are shaping the technological challenge of the discovery of disease-related genes and the identification of their structural alterations or elucidation of gene function . Some of the emerging technologies and diagnostic applications of both structural and functional genomics will be summarised, including the issues related to identification of disease-genes, detection of genetic alterations, mutation scanning and DNA chip technology or those related to expression genetics (hybridisation-, PCR- and sequence-based technologies), two-hybrid technology and bioinformatics and computational biology, respectively . Further advances of genetic engineering have also revolutionised immunoassay biotechnology via engineering of antibody-encoding genes and the phage display technology . A comment will also be given about some of the issues related to the commercialisation and widespread diffusion of genetic information derived from the exploitation of the biotechnology industry and the development and marketing of diagnostic services. Curr Biol, 1999 Dec 2, 9(23), R897 - 8 The genomics of adaptation in yeast; Sniegowski P; A recent study has combined methods of experimental evolution and DNA microarray technology to examine evolved changes in gene expression in yeast, providing intriguing insights into the genetics of adaptation and functional genomics, and pointing to future uses of microarray technology in evolutionary genetics. Mol Biol Evol, 1999 Dec, 16(12), 1664 - 74 Statistical methods for testing functional divergence after gene duplication; Gu X; Functional innovations after gene duplication may result in altered functional constraints between member gene clusters of a gene family . This type (type I) of functional divergence is measured by the coefficient of functional divergence (theta lambda), which can be interpreted as the decrease in rate correlation between gene clusters, or the probability that the evolutionary rate at a site is statistically independent between two gene clusters . A simple stochastic model has been developed for estimating theta lambda and testing its statistical significance . The current model includes the model of rate variation among sites as a special case when theta lambda = 0 . Moreover, we have developed a site-specific profile based on the hidden Markov model to identify critical amino acid residues that are responsible for these functional differences between two gene clusters, which may have great potential in functional genomics. Nat Biotechnol, 1999 Dec, 17(12), 1193 - 8 Mapping signal transduction pathways by phage display; Zozulya S et al.; Rapid identification of proteins that interact with a novel gene product is an important element of functional genomics . Here we describe a phage display-based technique for interaction screening of complex cDNA libraries using proteins or synthetic peptides as baits . Starting with the epidermal growth factor receptor (EGFR) cytoplasmic tail, we identified known protein interactions that link EGFR to the Ras/MAP kinase signal transduction cascade and several novel interactions . This approach can be used as a rapid and efficient tool for elucidating protein networks and mapping intracellular signal transduction pathways. Nat Biotechnol, 1999 Dec, 17(12), 1184 - 7 Oligonucleotide-based inhibition of embryonic gene expression; Driver SE et al.; We describe a technique to define gene function using antisense oligonucleotide (AS-ODN) inhibition of gene expression in mice . A single intravenous injection of an AS-ODN targeting vascular endothelial growth factor (VEGF) into pregnant mice between E7.5-8.5 resulted in a lack of primary angiogenesis . This enabled us to define the critical window required to inhibit VEGF expression and recapitulate the primary loss of function phenotype observed in VEGF (-/-) embryos . This phenotype was sequence-specific and time- and dose-dependent . Injection of an AS-ODN targeting a second gene, E-cadherin, into pregnant mice at E10 confirmed a hypothesized secondary phenotype . This is the first report of AS-ODN inhibition of gene expression in utero and provides a new strategy for target validation in functional genomics. Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 135 - 40 Functional genomic analysis reveals the utility of the I/LWEQ module as a predictor of protein:actin interaction; McCann RO et al.; The I/LWEQ module is a conserved sequence that we have identified as an actin-binding motif in the metazoan focal adhesion protein talin and the yeast protein Sla2p . Both of these proteins are associated with the actin cytoskeleton in cells . To better establish the value of the I/LWEQ module for prediction of actin-binding function, we have applied a functional genomics approach . Analysis of the 23 available I/LWEQ module sequences supports the division of I/LWEQ protein superfamily into four groups: (1) metazoan talin, (2) Dictyostelium discoideum talin homologs TalA/B, (3) metazoan Hip1p, and (4) yeast Sla2p . We show here that I/LWEQ modules from each major group bind to F-actin in vitro and that GFP-fusion proteins of the I/LWEQ modules of talin and Sla2p bind to F-actin in vivo . Therefore, the presence of an I/LWEQ module is strongly predictive of protein-actin interactions . The structural and functional conservation of the I/LWEQ module across the phylogenetic distance between cellular slime molds and mammals implies that the role of the I/LWEQ module is to connect diverse proteins involved in distinct cellular processes, including cell adhesion, cytoskeletal organization, and cell differentiation, to the actin cytoskeleton . Nutr Rev, 1999 Sep, 57(9 Pt 2), S13 - 8 Agricultural approaches to improving phytonutrient content in plants: an overview; Kochian LV et al.; Recent advances in plant molecular biology, functional genomics, and biochemistry have opened up a number of new avenues of research that will enable plant biologists to characterize, increase and modify plant content of a wide range of essential minerals and vitamins, as well as a number of secondary plant compounds that appear to play a role in improving human health and nutrition . In this review, several examples of exciting new research applying plant genomic and molecular genetic approaches to the improvement of phytonutrient content and composition in plants are presented . Research focusing on the elucidation of many of these complex biosynthetic and transport pathways in plants will require considerable resources in terms of funding, time, and personnel . As plant biologists move into interdisciplinary collaborations with nutritionists and food scientists, attention must be paid to a more complete identification and characterization of specific bioactive phytonutrients . Also, a more detailed assessment of the health-promoting properties of these compounds is needed, particularly for many of the secondary plant compounds for which clear epidemiologic and clinical data are still lacking . Finally, in order for significant progress to be made in modifying the nutrient composition of crops, a major investment must be made by funding agencies. Drug Discov Today, 1999 Dec, 4(12), 562 - 567 Antisense oligonucleotides: a systematic high-throughput approach to target validation and gene function determination; Taylor MF et al.; Antisense technology provides a high-throughput and systematic approach to drug target validation and gene function discovery . In combination with other emerging technologies (such as microarrays), this technology will enable efficient evaluation of the sequence data generated by the Human Genome Project . The authors review recent advances in the antisense field and discuss the potential use of antisense technology for functional genomics. Arch Surg, 1999 Nov, 134(11), 1209 - 15 Functional genomics: clinical effect and the evolving role of the surgeon; Hernandez A et al.; The genetic and molecular revolution that has occurred over the last 2 decades has dramatically increased our understanding of basic disease processes and will undoubtedly lead to improved detection methods and treatment . This will occur at an even more rapid rate after the completion of the Human Genome Project in the next 2 to 3 years . While these remarkable technological advances offer great hopes for novel therapeutic modalities, complicated medical, ethical, and legal issues will need to be addressed . This article briefly describes the advances that have occurred and their future ramifications for the field of surgery . Most assuredly, we will all be affected by these changes . Surgeons have the opportunity to be active participants and real leaders in the research and complex decisions regarding the optimal treatment of patients . However, formal training in these techniques and their potential applications will be required . Surgeons, as well as all physicians, must rise to the occasion or, otherwise, we will be relegated to a bystander status with clinical and moral decisions being made by nonclinicians. Novartis Found Symp, 1999, 223, 239 - 48; discussion 248-52 Genetics and evolution of insect resistance in Arabidopsis; Mitchell-Olds T; The genetic and molecular tools available in Arabidopsis allow identification of insect resistance genes . Many functional aspects of pest recognition and signal transduction are conserved in the defensive physiology of a broad range of plant species . Therefore, studies of insect resistance in Arabidopsis may be extended to functional genomics studies in many plant species of agricultural and ecological importance . Because of public concerns for field release of genetically modified organisms, naturally occurring genetic variation for resistance to insect herbivores will be valuable in plant breeding . Combined studies employing QTL mapping and candidate resistance genes are necessary to find and understand the genes responsible for variation in resistance . We review experiments showing that plant populations contain high levels of genetic variation for defensive physiology and disease and insect resistance, and that this variation can be manipulated to alter resistance and its components in a predictable fashion . In Arabidopsis, we can map the genes controlling physiological variation, and estimate the importance of regulatory or enzyme-encoding loci . Finally, we review functional genomics approaches for identification of insect resistance genes in Arabidopsis. Genome Res, 1999 Oct, 9(10), 950 - 9 Large-scale statistical analyses of rice ESTs reveal correlated patterns of gene expression; Ewing RM et al.; Large, publicly available collections of expressed sequence tags (ESTs) have been generated from Arabidopsis thaliana and rice (Oryza sativa) . A potential, but relatively unexplored application of this data is in the study of plant gene expression . Other EST data, mainly from human and mouse, have been successfully used to point out genes exhibiting tissue- or disease-specific expression, as well as for identification of alternative transcripts . In this report, we go a step further in showing that computer analyses of plant EST data can be used to generate evidence of correlated expression patterns of genes across various tissues . Furthermore, tissue types and organs can be classified with respect to one another on the basis of their global gene expression patterns . As in previous studies, expression profiles are first estimated from EST counts . By clustering gene expression profiles or whole cDNA library profiles, we show that genes with similar functions, or cDNA libraries expected to share patterns of gene expression, are grouped together . Promising uses of this technique include functional genomics, in which evidence of correlated expression might complement (or substitute for) those of sequence similarity in the annotation of anonymous genes and identification of surrogate markers . The analysis presented here combines the application of a correlation-based clustering method with a graphical color map allowing intuitive visualization of patterns within a large table of expression measurements. Science, 1999 Oct 15, 286(5439), 458 - 62, 479-81 The promise of comparative genomics in mammals; O'Brien SJ et al.; Dense genetic maps of human, mouse, and rat genomes that are based on coding genes and on microsatellite and single-nucleotide polymorphism markers have been complemented by precise gene homolog alignment with moderate-resolution maps of livestock, companion animals, and additional mammal species . Comparative genetic assessment expands the utility of these maps in gene discovery, in functional genomics, and in tracking the evolutionary forces that sculpted the genome organization of modern mammalian species. Plant Cell, 1999 Oct, 11(10), 1841 - 52 Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics; Tissier AF et al.; A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis . A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene . This construct was used to select for stable dSpm transpositions . Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale . To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events . DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest . By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences . This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations. Science, 1999 Oct 15, 286(5439), 487 - 91 Pharmacogenomics: translating functional genomics into rational therapeutics; Evans WE et al.; Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets have been linked to interindividual differences in the efficacy and toxicity of many medications . Pharmacogenomic studies are rapidly elucidating the inherited nature of these differences in drug disposition and effects, thereby enhancing drug discovery and providing a stronger scientific basis for optimizing drug therapy on the basis of each patient's genetic constitution. Science, 1999 Oct 15, 286(5439), 453 - 5 Biosequence exegesis; Boguski MS; Annotation of large-scale gene sequence data will benefit from comprehensive and consistent application of well-documented, standard analysis methods and from progressive and vigilant efforts to ensure quality and utility and to keep the annotation up to date . However, it is imperative to learn how to apply information derived from functional genomics and proteomics technologies to conceptualize and explain the behaviors of biological systems . Quantitative and dynamical models of systems behaviors will supersede the limited and static forms of single-gene annotation that are now the norm . Molecular biological epistemology will increasingly encompass both teleological and causal explanations. Proc Natl Acad Sci U S A, 1999 Oct 12, 96(21), 11723 - 8 Selection of antibodies for intracellular function using a two-hybrid in vivo system; Visintin M et al.; Expression of antibodies inside cells has been used successfully to ablate protein function . This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences . A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm . To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach . In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene . We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro . Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures . The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences. Bioessays, 1999 Nov, 21(11), 895 - 9 From global expression data to gene networks; Thieffry D; Allowing the parallel monitoring of the transcription of thousands of genes, microarrays constitute a powerful technique for functional genomics . In a recent paper, a clustering method and a local alignment software were combined to identify DNA motifs in sets of yeast genes endowed with similar transcription profiles throughout mitosis (1) . Identifying various known transcriptional binding sites together with new putative ones, the authors made a significant step towards a systematic characterization of the regulatory structure of genomic networks . BioEssays 1999;21:895-899 . J Bacteriol, 1999 Oct, 181(20), 6425 - 40 Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media; Tao H et al.; DNA arrays of the entire set of Escherichia coli genes were used to measure the genomic expression patterns of cells growing in late logarithmic phase on minimal glucose medium and on Luria broth containing glucose . Ratios of the transcript levels for all 4,290 E . coli protein-encoding genes (cds) were obtained, and analysis of the expression ratio data indicated that the physiological state of the cells under the two growth conditions could be ascertained . The cells in the rich medium grew faster, and expression of the majority of the translation apparatus genes was significantly elevated under this growth condition, consistent with known patterns of growth rate-dependent regulation and increased rate of protein synthesis in rapidly growing cells . The cells grown on minimal medium showed significantly elevated expression of many genes involved in biosynthesis of building blocks, most notably the amino acid biosynthetic pathways . Nearly half of the known RpoS-dependent genes were expressed at significantly higher levels in minimal medium than in rich medium, and rpoS expression was similarly elevated . The role of RpoS regulation in these logarithmic phase cells was suggested by the functions of the RpoS dependent genes that were induced . The hallmark features of E . coli cells growing on glucose minimal medium appeared to be the formation and excretion of acetate, metabolism of the acetate, and protection of the cells from acid stress . A hypothesis invoking RpoS and UspA (universal stress protein, also significantly elevated in minimal glucose medium) as playing a role in coordinating these various aspects and consequences of glucose and acetate metabolism was generated . This experiment demonstrates that genomic expression assays can be applied in a meaningful way to the study of whole-bacterial-cell physiology for the generation of hypotheses and as a guide for more detailed studies of particular genes of interest. Parasitol Today, 1999 Nov, 15(11), 430 - 1 Reports from the cutting edge of parasitic genome analysis; Blaxter M et al.; This new feature in Parasitology Today will host reports from the laboratories involved in genomics of parasites, be that sequencing, mapping or 'functional genomics' - the mining and analysis of the sequence datasets, and the development of postgenomics tools to examine gene expression, response to drugs and population variability . It will publicize new technology to wider audiences, let communities of researchers know about novel resources (particularly those available through the World Wide Web) and highlight significant advances in the understanding of parasitic genomes through functional genomics. Mol Microbiol, 1999 Sep, 33(6), 1103 - 17 Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens; Jungblut PR et al.; In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year . The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced . We compared the proteomes of two non-virulent vaccine strains of M . bovis BCG (Chicago and Copenhagen) with two virulent strains of M . tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics . The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE . Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database . Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified . Of these, 54 belong to the culture supernatant . Sixteen and 25 proteins differing in intensity or position between M . tuberculosis H37Rv and Erdman, and H37Rv and M . bovis BCG Chicago, respectively, were identified and categorized into protein classes . It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis. Curr Opin Biotechnol, 1999 Oct, 10(5), 511 - 6 Genomics for food biotechnology: prospects of the use of high-throughput technologies for the improvement of food microorganisms; Kuipers OP; Functional genomics is currently the most effective approach for increasing the knowledge at the molecular level of metabolic and adaptive processes in whole cells . High-throughput technologies, such as DNA microarrays, and improved two-dimensional electrophoresis methods combined with tandem mass-spectroscopy, supported by bioinformatics, are useful tools for food biotechnology, which depends on detailed knowledge of the properties of food microbes (and pathogens) in their industrial, food and consumer environments . Genomics of food microbes, based on rapidly emerging genome sequence information, generates valuable knowledge that can be used for metabolic engineering, improving cell factories and development of novel preservation methods . Furthermore, pre- and probiotic studies, characterization of stress responses, studies of microbial ecology and, last but not least, development of novel risk assessment procedures will be facilitated. Plant J, 1999 Sep, 19(5), 605 - 13 Ac as a tool for the functional genomics of rice; Enoki H et al.; To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations . All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions . In nine of the 12 independent transpositions analyzed, their germinal transmission was detected . Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences . These results indicate preferential Ac transposition into protein-coding rice genes . To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix . Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny . Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome. J Mol Med, 1999 Jun, 77(6), 469 - 80 Gene expression profiling, genetic networks, and cellular states: an integrating concept for tumorigenesis and drug discovery; Huang S; Genome-wide expression monitoring, a novel tool of functional genomics, is currently used mainly to identify groups of coregulated genes and to discover genes expressed differentially in distinct situations that could serve as drug targets . This descriptive approach . however, fails to extract "distributed" information embedded in the genomic regulatory network and manifested in distinct gene activation profiles . A model based on the formalism of boolean genetic networks in which cellular states are represented by attractors in a discrete dynamic system can serve as a conceptual framework for an integrative interpretation of gene expression profiles . Such a global (genome-wide) view of "gene function" in the regulation of the dynamic relationship between proliferation, differentiation, and apoptosis can provide new insights into cellular homeostasis and the origins of neoplasia . Implications for a rational approach to the identification of new drug targets for cancer treatment are discussed.
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