Functional genomics

Pharmacogenomics, 2004 Dec, 5(8), 1049 - 105
Genomic characterization of Alzheimer's disease and genotype-related phenotypic analysis of biological markers in dementia; Cacabelos R; More than 180 genes distributed across the human genome are potentially involved in the pathogenesis of Alzheimer's disease (AD) . The AD population shows a higher genetic variation rate than the control population . Significant differences in allelic distribution and frequency exist when AD-related polygenic clusters are compared with other forms of dementia, indicating that the genetic component in neurodegenerative dementia differs from that of other CNS disorders . The characterization of AD genotype-related phenotypic profiles reveals substantial differences in biological markers among AD clusters associated with different genes and/or allelic combinations . AD and dementia with vascular component (DVC) are the most prevalent forms of dementia . Both clinical entities share many similarities, but they differ in their major phenotypic and genotypic profiles, as revealed by structural and functional genomics studies . Comparative phenotypic studies have identified significant differences in 25% of more than 100 parametric variables, including anthropometric values, cardiovascular function, blood pressure, lipid metabolism, uric acid metabolism, peripheral calcium homeostasis, liver function, alkaline phosphatase, lactate dehydrogenase, red and white blood cells, regional brain atrophy, and brain blood flow velocity . Functional genomic studies incorporating apolipoprotein E (APOE)-related changes in biological markers extended the difference between AD and DVC by up to 57% . Structural genomic studies with AD-related genes, including APP, MAPT, APOE, PS1, PS2, A2M, ACE, AGT, cFOS, and PRNP, demonstrate different genetic profiles in AD and DVC, with an absolute genetic variation rate in the range of 30-80%, depending upon genes and genetic clusters . The relative polymorphic variation in genetic clusters integrated by two, three or four genes associated with AD ranges from 1 to 3% . The main phenotypic differences in AD are genotype dependent, indicating a powerful influence of polygenic factors on the AD phenotypic profile . All these genotypic and phenotypic variations bring about important consequences for the pharmacogenomics of AD.

Expert Opin Ther Targets, 2004 Dec, 8(6), 653 - 61
Chemical genetics for therapeutic target mining; Shim JS et al.; Chemical genetics is an emerging research field that utilises biologically active small molecules to study biological functions of genes and their products . The direct regulation of the protein function by the biologically active small molecules can alternate the gene mutagenesis studies utilised in conventional genetics . Like conventional genetics, chemical genetics can be divided into two concepts - 'forward' and 'reverse' chemical genetics . These approaches of chemical genetics have a tremendous impact on both functional genomics and drug development . This review focuses on the two ways in which chemical genetics can be used for therapeutic target mining and their practical application in drug development, particularly, in angiogenesis-related diseases.

Stem Cells, 2004, 22(7), 1330 - 7
Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord; Wang HS et al.; The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells . Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45) . Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3) . We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I . We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages . These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues.

Curr Med Chem, 2004 Dec, 11(23), 3119 - 45
Recent advances in chemical genomics; Darvas F et al.; Chemical genomics, which utilizes specially designed small chemical compounds early in the discovery phase of new drugs to explore the life science at various levels, can address biological questions that are not amenable to genetic manipulation or functional genomics/proteomics approaches . Following the development of HT phenotypic assays and DNA expression analysis, the integration of cell-based assays with activity / affinity-based approaches allows us to interrogate the cells by analyzing phenotypic alterations, changes of transcript signature or detecting the differences in protein expression levels . Furthermore, activity / affinity-based techniques directly provide a druggable subset of gene products, which interact with small molecules, greatly reducing the complexity of analyzing the proteome . In this paper, we give an account of the recent advances (approaches and strategies) in the field of chemical genomics, and discuss how these approaches enable the investigator to obtain a novel therapeutically relevant target as well as drug candidates acting on them in a target-specific manner . This novel post-genomic discovery strategy, where target identification/ validation is carried out by interactions with small molecules, could significantly reduce the time-scale for early drug discovery, and increase the success rate of finding novel, druggable targets, as well as more specific drug candidates.

Nucleic Acids Res . 2004 Dec;32(21):e171.
RNA silencing in plants by the expression of siRNA duplexes; Lu S et al.; In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants . Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown . Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters . Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics . The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts . Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants . Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality.

Science, 2004 Nov 26, 306(5701), 1555 - 8
A probabilistic functional network of yeast genes; Lee I et al.; A conceptual framework for integrating diverse functional genomics data was developed by reinterpreting experiments to provide numerical likelihoods that genes are functionally linked . This allows direct comparison and integration of different classes of data . The resulting probabilistic gene network estimates the functional coupling between genes . Within this framework, we reconstructed an extensive, high-quality functional gene network for Saccharomyces cerevisiae, consisting of 4681 (approximately 81%) of the known yeast genes linked by approximately 34,000 probabilistic linkages comparable in accuracy to small-scale interaction assays . The integrated linkages distinguish true from false-positive interactions in earlier data sets; new interactions emerge from genes' network contexts, as shown for genes in chromatin modification and ribosome biogenesis.

Br J Haematol, 2004 Dec, 127(5), 479 - 90
Genetic polymorphisms predicting the outcome of bone marrow transplants; Dickinson AM et al.; Analysis of non-histocompatibility leucocyte antigen (HLA) functional genomics, together with conventional risk factors in haematopoietic stem cell transplantation (HSCT) can lead to predicting outcome in HLA-matched sibling transplant recipients . Polymorphisms of cytokine genes including tumour necrosis factor alpha, interleukin-10, interferon gamma and interleukin (IL)-6, associate with more severe acute graft-versus-host disease (aGvHD) . Donor genotype for IL-1 receptor antagonist (IL-1Ra) has been associated with reduced aGvHD severity . Other genotypes (patient IL-1Ra, IL-6 and donor IL-1 alpha) have been associated with chronic GvHD, or overall survival (Vitamin D receptor and oestrogen receptor) . Polymorphisms within genes associated with host defence/inflammatory responses (mannose binding lectin genes, myeloperoxidase genes and the FC gamma receptors) have been associated with infections . Polymorphisms of pharmacogenes, such as methylenetetrahydrofolate-reductase, have been associated with aGvHD and other post-transplant complications . The NOD2 gene polymorphism, associated with Crohn's disease, has been shown to be associated with risk of gut GvHD . The majority of the studies have been carried out in single centre HLA-matched sibling cohorts and in relatively few matched unrelated donor transplants . This review gives an overall perspective of the current field of non-HLA genetics with regard to HSCT outcome, clinical relevance and potential application of the results to clinical management of HSCT.

Nat Genet, 2004 Dec, 36(12), 1306 - 11 Epub 2004 Dec.
Application of comparative functional genomics to identify best-fit mouse models to study human cancer; Lee JS et al.; Genetically modified mice have been extensively used for analyzing the molecular events that occur during tumor development . In many, if not all, cases, however, it is uncertain to what extent the mouse models reproduce features observed in the corresponding human conditions . This is due largely to lack of precise methods for direct and comprehensive comparison at the molecular level of the mouse and human tumors . Here we use global gene expression patterns of 68 hepatocellular carcinomas (HCCs) from seven different mouse models and 91 human HCCs from predefined subclasses to obtain direct comparison of the molecular features of mouse and human HCCs . Gene expression patterns in HCCs from Myc, E2f1 and Myc E2f1 transgenic mice were most similar to those of the better survival group of human HCCs, whereas the expression patterns in HCCs from Myc Tgfa transgenic mice and in diethylnitrosamine-induced mouse HCCs were most similar to those of the poorer survival group of human HCCs . Gene expression patterns in HCCs from Acox1(-/-) mice and in ciprofibrate-induced HCCs were least similar to those observed in human HCCs . We conclude that our approach can effectively identify appropriate mouse models to study human cancers.

Korean J Gastroenterol, 2004 Nov, 44(5), 239 - 45
{Proteomic approach in gastrointestinal and liver research}; Yeo M et al.; In the post-genomic era, the focus of research is now moving to functional genomics employing the information on predicted gene products provided by genome sequencing . Proteomics, the global analysis of structures, functions, and interactions of whole cellular proteins, draws the special attention as a tool for documenting the disease pathogenesis or progression . The high-throughput technology has become feasible by considerable improvement of two dimensional electrophoresis and mass fingerprinting . Thus proteome techniques can be used as tools to study the disease processes, develop new biomakers for diagnosis and early detection of diseases, and accelerate drug development . In this review, we discuss the background and techniques of proteomics, and potential applications to the research of gastrointestinal diseases.

Cell Biol Int, 2004, 28(11), 755 - 64
Cell biology, chemogenomics and chemoproteomics; Gagna CE et al.; The scientific techniques used in molecular biological research and drug discovery have changed dramatically over the past 10 years due to the influence of genomics, proteomics and bioinformatics . Furthermore, genomics and functional genomics are now merging into a new scientific approach called chemogenomics . Advancements in the study of molecular cell biology are dependent upon "omics" researchers realizing the importance of and using the experimental tools currently available to cell biologists . For example, novel microscopic techniques utilizing advanced computer imaging allow for the examination of live specimens in a fourth dimension, viz., time . Yet, molecular biologists have not taken full advantage of these and other traditional and novel cell biology techniques for the further advancement of genomic and proteomic-oriented research . The application of traditional and novel cellular biological techniques will enhance the science of genomics . The authors hypothesize that a stronger interdisciplinary approach must be taken between cell biology (and its closely related fields) and genomics, proteomics and bio-chemoinformatics . Since there is a lot of confusion regarding many of the "omics" definitions, this article also clarifies some of the basic terminology used in genomics, and related fields . It also reviews the current status and future potential of chemogenomics and its relationship to cell biology . The authors also discuss and expand upon the differences between chemogenomics and the relatively new term--chemoproteomics . We conclude that the advances in cell biology methods and approaches and their adoption by "omics" researchers will allow scientists to maximize our knowledge about life.

IDrugs, 2002 Mar, 5(3), 216 - 9
Proteolytic enzymes as therapeutic targets - keystone symposium; Creemers J; The Keystone Symposium 'Proteolytic Enzymes as Therapeutic Targets' was attended by approximately 150 scientists . Around two-thirds of the participants consisted of representatives from pharmaceutical companies, but representatives from academic institutes dominated the list of speakers . The meeting attracted scientists from many different fields, including biochemistry, molecular biology, structural biology, pharmacology, chemistry, and bioinformatics . The science ranged from the discovery and characterization of novel proteinases to the development and clinical trials of proteinase inhibitors and was presented as posters or in oral sessions . The discussions following the oral presentations were always very animated, but hardly ever heated . Although there were a few new drugs being presented, the real highlight was the enormous potential of recently discovered proteinases as new therapeutic targets . Both pharmaceutical companies and academic institutes are investing in programs to integrate the avalanche of new information coming from functional genomics, proteomics and structural information to create a platform for applied proteinase technology.

Semin Cell Dev Biol, 2004 Dec, 15(6), 721 - 31
Bioinformatics: harvesting information for plant and crop science; King GJ; Bioinformatics is an integral aspect of plant and crop science research . Developments in data management and analytical software are reviewed with an emphasis on applications in functional genomics . This includes information resources for Arabidopsis and crop species, and tools available for analysis and visualisation of comparative genomic data . Approaches used to explore relationships between plant genes and expressed sequences are compared, including use of ontologies . The impact of bioinformatics in forward and reverse genetics is described, together with the potential from data mining . The role of bioinformatics is explored in the wider context of plant and crop science.

Semin Cell Dev Biol, 2004 Dec, 15(6), 693 - 701
Integrative bioinformatics for functional genome annotation: trawling for G protein-coupled receptors; Flower DR et al.; G protein-coupled receptors (GPCR) are amongst the best studied and most functionally diverse types of cell-surface protein . The importance of GPCRs as mediates or cell function and organismal developmental underlies their involvement in key physiological roles and their prominence as targets for pharmacological therapeutics . In this review, we highlight the requirement for integrated protocols which underline the different perspectives offered by different sequence analysis methods . BLAST and FastA offer broad brush strokes . Motif-based search methods add the fine detail . Structural modelling offers another perspective which allows us to elucidate the physicochemical properties that underlie ligand binding . Together, these different views provide a more informative and a more detailed picture of GPCR structure and function . Many GPCRs remain orphan receptors with no identified ligand, yet as computer-driven functional genomics starts to elaborate their functions, a new understanding of their roles in cell and developmental biology will follow.

Anal Biochem, 2004 Dec 15, 335(2), 260 - 6
High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections; Eisenberger S et al.; Osteocytes, the most abundant bone cell type with important roles in tissue maintenance and pathological aberrations such as observed in bone metastases, are enclosed within a highly compact, calcified extracellular matrix . This location complicates analysis in native bone, with the consequence that despite their importance their in vivo molecular physiology is only poorly understood . We have examined the possibility of isolating osteocyte RNA for transcript profiling from native, frozen bone instead of employing the formalin-fixed, paraffin-embedded, decalcified version routinely used in histology, providing chemically modified and highly disintegrated RNAs . Bone tissue was tape-assisted cryosectioned and fixed to glass slides by support of UV-flash-triggered adhesive polymerization followed by quick hematoxylin-eosin staining to generate a guidance image for microdissection . Using an UVa-nitrogen laser, matrix-enclosed osteocytes were either excised and catapulted into RNA preparation vials or freed of accompanying nonosteocyte cellular material . The influences of bone sectioning, staining, and osteocyte capturing procedures on the prepared osteocyte RNAs were analyzed and the method was optimized accordingly . The obtained osteocyte RNAs showed the expected expression pattern of marker genes (reverse transcriptase-polymerase chain reaction), and, following conversion into fluorescent-labeled cDNAs, led to transcript profiles (cDNAchips; 2600 genes) with scatter-graph geometries indicating suitability for high-confidence evaluation . With the approach described here we introduce a methodological way for the characterization of the in vivo molecular physiology of osteocytes by functional genomics.

Curr Opin Chem Biol, 2004 Dec, 8(6), 570 - 9
RNA interference and chemically modified small interfering RNAs; Manoharan M; RNA interference (RNAi) is a powerful biological process for specific silencing of gene expression in diversified eukaryotic cells and has tremendous potential for functional genomics, drug discovery through in vivo target validation, and development of novel gene-specific medicine . The future success of this technology relies on identifying appropriate chemical modifications to improve stability, potency and in vivo cellular delivery . The present review summarizes the role of the chemist's toolbox in this emerging technology.

J Transl Med . 2004 Nov 22;2(1):39.
RNA interference: learning gene knock-down from cell physiology; Mocellin S et al.; SUMMARY: Over the past decade RNA interference (RNAi) has emerged as a natural mechanism for silencing gene expression . This ancient cellular antiviral response can be exploited to allow specific inhibition of the function of any chosen target gene . RNAi is proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes . More importantly, RNAi technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes.This review briefly describes the molecular principles underlying the biology of RNAi phenomenon and discuss the main technical issues regarding optimization of RNAi experimental design.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Oct, 21(5), 848 - 51
{RNA interference in functional genomics and medical research}; You Z et al.; RNA interference (RNAi) is a post-transcriptional gene silencing process by targeting mRNA for degradation in a sequence-specific manner . This powerful platform has enormous potential in functional genomics and medical research . As a tool to knock out expression of specific genes in a variety of organisms, RNAi was used to investigate gene function in a high throughput fashion . Highly conserved in evolution RNAi appears to have evolved as a cellular defense mechanism in plants and animals to suppress viral infection, transposon jumping and endogenous aberrant genes . Exploiting the natural mechanism, the researchers can shut down disease-causing genes and develop novel therapeutics against infection, tumor and other disease.

Physiol Genomics, 2004 Nov 17, 19(3), 233 - 46
Functional genomics of the dopaminergic system in hypertension; Zeng C et al.; Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension . Under normal conditions, D(1)-like receptors (D(1) and D(5)) inhibit sodium transport in the kidney and intestine . However, in the Dahl salt-sensitive and spontaneously hypertensive rats (SHRs) and in humans with essential hypertension, the D(1)-like receptor-mediated inhibition of epithelial sodium transport is impaired because of an uncoupling of the D(1)-like receptor from its G protein/effector complex . The uncoupling is receptor specific, organ selective, nephron-segment specific, precedes the onset of hypertension, and cosegregates with the hypertensive phenotype . The defective transduction of the renal dopaminergic signal is caused by activating variants of G protein-coupled receptor kinase type 4 (GRK4: R65L, A142V, A486V) . The GRK4 locus is linked to and GRK4 gene variants are associated with human essential hypertension, especially in salt-sensitive hypertensive subjects . Indeed, the presence of three or more GRK4 variants impairs the natriuretic response to dopaminergic stimulation in humans . In genetically hypertensive rats, renal inhibition of GRK4 expression ameliorates the hypertension . In mice, overexpression of GRK4 variants causes hypertension either with or without salt sensitivity according to the variant . GRK4 gene variants, by preventing the natriuretic function of the dopaminergic system and by allowing the antinatriuretic factors (e.g., angiotensin II type 1 receptor) to predominate, may be responsible for salt sensitivity . Subclasses of hypertension may occur because of additional perturbations caused by variants of other genes, the quantitative interaction of which may vary depending upon the genetic background.

Autoimmun Rev, 2004 Nov, 3(7-8), 541 - 9
Strategies using functional genomics in rheumatic diseases; Burmester GR et al.; For functional genomics of inflammatory disorders and infection, rheumatic diseases offer unique features to analyse the transition from infection to chronic inflammation, autoimmunity and immunopathology, both systemic and tissue specific . The diseases are frequent and of considerable socio-economic impact . Well-defined cohorts of patients are available . The tissues and cells involved are readily accessible for molecular analysis . Both genetic predisposition and infection are involved in the aetiopathogenesis of rheumatic diseases . The number of susceptibility and severity genes has been estimated to be at least 30, but only few of them have been identified so far . There is an urgent need for developing new therapies adapted to genetic risk and based on a functional genetic and molecular understanding of chronic inflammation . It is evident that gene analysis in inflammatory rheumatic diseases will not only be beneficial for the large number of patients involved, but will also lead to a better understanding of other inflammatory disorders, thereby possibly leading to novel diagnostic and therapeutic strategies in this important group of disorders.

Neurotox Res, 2004, 6(5), 363 - 72
Functional genomics strategies to identify susceptibility genes and treatment targets in alcohol dependence; Heilig M et al.; Genetic factors contribute to alcohol dependence through two main categories of mechanisms . The 50-60% heritability observed in this disorder is presumably conferred by polymorphic variants, encoding functionally altered proteins, or leading to differential transcriptional activity . Secondly, long term changes during the process of developing dependence are likely encoded by persistent changes in gene expression . Thus, genetic and environmental factors interact at the level of the transcriptome, making this an attractive level of analysis . For this purpose, we have applied differential display and more recently Affymetrix oligonucleotide gene arrays to models of genetic susceptibility and alcohol-induced neuroadaptation.

Mol Biotechnol, 2004 Nov, 28(3), 195 - 9
Knockdown of mouse adult beta-globin gene expression in MEL cells by retrovirus vector-mediated RNA interference; Zhao N et al.; RNA interference (RNAi) efficiently induces sequence-specific gene silencing in mammalian cells through short interfering RNA (siRNA) of 21-23 nucleotides synthesized in vitro or expressed by DNA-based vector . However, introduction of siRNA into mammalian cells by transfection limits the application of RNAi, especially when it is necessary to generate long-term gene silencing in vivo . Virus vector-mediated RNAi provides an alternative to transfection . In the present study, we investigated such transduction system and showed that retrovirus vector-mediated RNAi can substantially down-regulate expression of mouse adult beta-globin gene in MEL cells . The results suggest that retrovirus vector-delivered RNAi may find its use in functional genomics and in gene therapy.

Mol Cell Biol, 2004 Dec, 24(23), 10263 - 76
Multiple, distant Gata2 enhancers specify temporally and tissue-specific patterning in the developing urogenital system; Khandekar M et al.; Transcription factor GATA-2 is expressed in a complex temporally and tissue-specific pattern within the developing embryo . Loss-of-function studies in the mouse showed that GATA-2 activity is first required during very early hematopoiesis . We subsequently showed that a 271-kbp yeast artificial chromosome (YAC) transgene could fully complement the loss of Gata2 hematopoietic function but that these YAC-rescued Gata2 null mutant mice die perinatally due to defective urogenital development . The rescuing YAC did not display appropriate urogenital expression of Gata2, implying the existence of a urogenital-specific enhancer(s) lying outside the boundaries of this transgene . Here we outline a coupled general strategy for regulatory sequence discovery, linking bioinformatics to functional genomics based on the bacterial artificial chromosome (BAC) libraries used to generate the mouse genome sequence . Exploiting this strategy, we screened >1 Mbp of genomic DNA surrounding Gata2 for urogenital enhancer activity . We found that the spatially and tissue-specific functions for Gata2 in the developing urogenital system are conferred by at least three separate regionally and temporally specific urogenital enhancer elements, two of which reside far 3' to the Gata2 structural gene . Including the additional enhancers that were discovered using this strategy (called BAC trap) extends the functional realm of the Gata2 locus to greater than 1 Mbp.

Plant Physiol, 2004 Dec, 136(4), 4228 - 36 Epub 2004 Nov 12.
Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions; Prisic S et al.; Rice (Oryza sativa) produces ent-copalyl diphosphate for both gibberellin (GA) phytohormone and defensive phytoalexin biosynthesis, raising the question of how this initial biosynthetic step is carried out for these distinct metabolic processes . Here, a functional genomics approach has been utilized to identify two disparate ent-copalyl diphosphate synthases from rice (OsCPS1ent and OsCPS2ent) . Notably, it was very recently demonstrated that only one of these (OsCPS1ent) normally operates in GA biosynthesis as mutations in this gene result in severely impaired growth . Evidence is presented here strongly indicating that the other (OsCPS2ent) is involved in related secondary metabolism producing defensive phytochemicals . In particular, under appropriate conditions, OsCPS2ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins . Thus, transcriptional control of OsCPS2ent seems to be an important means of regulating defensive phytochemical biosynthesis . Finally, OsCPS1ent is significantly more similar to the likewise GA-specific gene An1/ZmCPS1ent in maize (Zea mays) than its class II terpene synthase paralogs involved in rice secondary metabolism . Hence, we speculate that this cross-species conservation by biosynthetic process reflects derivation of related secondary metabolism from the GA primary biosynthetic pathway prior to the early divergence between the separate lineages within the cereal/grass family (Poaceae) resulting in modern rice and maize.

Cancer Biol Ther . 2004 Nov 12;3(11) {Epub ahead of print}
siRNAs: Mechanism of RNA Interference, In Vivo and Potential Clinical Applications; Karagiannis TC et al.; Small interfering RNAs are currently the most widely used nucleic acid-based sequence-specific gene silencing molecules . These molecules mediate RNA interference-a natural post-transcriptional gene-silencing pathway . Given the high reliability and higher efficiency of small interfering RNA-mediated RNA interference, compared to earlier reverse genetic technologies, this is now the preferred technique in functional genomics . Furthermore, the exquisite specificity and exceptional gene-silencing potency of small interfering RNAs has resulted in intense research related to potential target-specific therapeutic applications of these molecules . This review will discuss the mechanism of RNA interference and applications of the pathway in molecular biology including functional genomics will be overviewed . The article will outline in vivo and potential clinical applications of small interfering RNA molecules.

Anal Chem, 2004 Nov 15, 76(22), 6555 - 9
Surface plasmon resonance-based sensors to identify cis-regulatory elements; Lin L et al.; In eukaryotes, transcription is regulated by multiprotein complexes binding to specific regions of genomic DNA, called cis-regulatory elements . Comprehensive identification of these elements is an important goal of functional genomics . Hence, it is of practical interest to develop a high-throughput assay to identify cis-regulatory elements . Toward that goal, we demonstrate that a surface plasmon resonance-based assay can identify whether a specific region of DNA binds to proteins present in raw nuclear lysate . Specifically, we immobilized a 16-basepair double-stranded DNA region of the SQSTM1 promoter to the Texas Instruments Spreeta, a surface plasmon resonance sensor . As a control, in a separate experiment, we immobilized a similar piece of DNA that differed by only a single base pair . We observed a significant difference in surface plasmon resonance signal when these two probes were exposed to raw nuclear lysate from NIH/3T3 cells . Using a luciferase-reporter vector transfected into live NIH/3T3 cells, we measured a significant difference in transcriptional activity between the two pieces of DNA . We conclude that a surface plasmon resonance-based sensor is capable of identifying physiologically significant cis-regulatory elements.

Microb Cell Fact . 2004 Nov 10;3(1):13.
The bag or the spindle: the cell factory at the time of systems' biology; Danchin A; Genome programs changed our view of bacteria as cell factories, by making them amenable to systematic rational improvement . As a first step, isolated genes (including those of the metagenome), or small gene clusters are improved and expressed in a variety of hosts . New techniques derived from functional genomics (transcriptome, proteome and metabolome studies) now allow users to shift from this single-gene approach to a more integrated view of the cell, where it is more and more considered as a factory . One can expect in the near future that bacteria will be entirely reprogrammed, and perhaps even created de novo from bits and pieces, to constitute man-made cell factories . This will require exploration of the landscape made of neighbourhoods of all the genes in the cell . Present work is already paving the way for that futuristic view of bacteria in industry.

Neuroendocrinology, 2004, 80(2), 92 - 9 Epub 2004.
Vasopressin stimulates ventromedial hypothalamic neurons via oxytocin receptors in oxytocin gene knockout male and female mice; Ragnauth AK et al.; A wealth of neuropharmacological data demonstrates that oxytocin (OT) actions in the mammalian forebrain support a wide variety of affiliative behaviors and repress aggressive behaviors . Based on that literature, it was expected that reproductive and affiliative behaviors would be vastly decreased and aggression markedly increased in OT gene knockout (OTKO) mice . The initial publications reporting the behaviors of these mice did not include such phenotypes . Here, we compared single-unit activities recorded from the ventromedial hypothalamus in tissue slices of male and female OTKO mice and their wild-type littermate to test two hypotheses about OT functional genomics . First, we proposed that in OTKO mice, a very similar 9-amino-acid neuropeptide, arginine vasopressin (a likely gene duplication product), can 'cross over' and compensate for the lack of OT . This hypothesis was confirmed in both males and females . Further, we proposed that because of the lifelong absence of OT in OTKO, OT receptors would be more sensitive to OT in the knockout animals . We tested this idea in males and found that it was correct . Thus, an answer to the 'OTKO paradox' is put forth, with implications for OT-sensitive behaviors in a variety of species.

J Am Acad Dermatol, 2004 Nov, 51(5), 681 - 92; quiz 693-6
DNA microarrays: from structural genomics to functional genomics . The applications of gene chips in dermatology and dermatopathology; Sellheyer K et al.; The human genome project was successful in sequencing the entire human genome and ended earlier than expected . The vast genetic information now available will have far-reaching consequences for medicine in the twenty-first century . The knowledge gained from the mapping and sequencing of human genes on a genome-wide scale--commonly referred to as structural genomics--is prerequisite for studies that focus on the functional aspects of genes . A recently invented technique, known as gene chip, or DNA microarray, technology, allows the study of the function of thousands of genes at once, thereby opening the door to the new field of functional genomics . At its core, the DNA microarray utilizes a unique feature of DNA known as complementary hybridization . As such, it is not different from Southern (DNA) blot or northern (RNA) blot hybridizations, or the polymerase chain reaction, with the exception that it allows expression profiling of the entire human genome in a single hybridization experiment . The article highlights the principles, technology, and applications of DNA microarrays as they pertain to the field of dermatology and dermatopathology . The most important applications are the gene expression profiling of skin cancer, especially of melanoma . Other potential applications include gene expression profiling of inflammatory skin diseases, the mutational analysis of genodermatoses, and polymorphism screening, as well as drug development and chemosensitivity prediction . cDNA microarrays will shape the diagnostic approach of the dermatology and the dermatopathology of the future and may lead to new therapeutic options.

J Gene Med . 2004 Nov 1; {Epub ahead of print}
Engineered Streptomyces quorum-sensing components enable inducible siRNA-mediated translation control in mammalian cells and adjustable transcription control in mice; Weber W et al.; BACKGROUND: Recent advances in functional genomics, gene therapy, tissue engineering, drug discovery and biopharmaceuticals production have been fostered by precise small-molecule-mediated fine-tuning of desired transgenes . METHODS: Capitalizing on well-evolved quorum-sensing regulatory networks in Streptomyces coelicolor we have designed a mammalian regulation system inducible by the non-toxic butyrolactone SCB1 . Fusion of the S . coelicolor SCB1 quorum-sensing receptor ScbR to the human Kox-1-derived transsilencing domain reconstituted a mammalian transsilencer (SCS) able to repress transcription from SCS-specific operator-containing promoters in a reverse SCB1-adjustable manner . RESULTS: This quorum-sensing-derived mammalian transgene control system (Q-ON) enabled precise SCB1-specific fine-tuning of (i) desired transgene transcription in a variety of mammalian/human cell lines and human primary cells, (ii) small interfering RNA-mediated posttranscriptional knockdown (siRNA) in mammalian cells, and (iii) dosing of a human glycoprotein in mice . CONCLUSIONS: As exemplified by Q-ON technology, bacterial quorum-sensing regulons may represent a near-infinite source for the design of mammalian gene control systems compatible with molecular interventions relevant to future gene therapy and tissue engineering scenarios . Copyright (c) 2004 John Wiley & Sons, Ltd.

Mamm Genome, 2004 Oct, 15(10), 768 - 83
Mouse functional genomics requires standardization of mouse handling and housing conditions; Champy MF et al.; The study of mouse models is crucial for the functional annotation of the human genome . The recent improvements in mouse genetics now moved the bottleneck in mouse functional genomics from the generation of mutant mice lines to the phenotypic analysis of these mice lines . Simple, validated, and reproducible phenotyping tests are a prerequisite to improving this phenotyping bottleneck . We analyzed here the impact of simple variations in animal handling and housing procedures, such as cage density, diet, gender, length of fasting, as well as site (retro-orbital vs . tail), timing, and anesthesia used during venipuncture, on biochemical, hematological, and metabolic/endocrine parameters in adult C57BL/6J mice . Our results, which show that minor changes in procedures can profoundly affect biological variables, underscore the importance of establishing uniform and validated animal procedures to improve reproducibility of mouse phenotypic data.

Nat Rev Drug Discov, 2004 Nov, 3(11), 965 - 72
Functional genomics to new drug targets; Kramer R et al.; The completion of the sequencing of the human genome, and those of other organisms, is expected to lead to many potential new drug targets in various diseases, and it is predicted that novel therapeutic agents will be developed against such targets . The role of functional genomics in modern drug discovery is to prioritize these targets and to translate that knowledge into rational and reliable drug discovery . Here, we describe the field of functional genomics and review approaches that have been applied to drug discovery, including RNA profiling, proteomics, antisense and RNA interference, model organisms and high-throughput, genome-wide overexpression or knockdowns, and outline the future directions that are likely to yield new drug targets from genomics.

J Exp Bot, 2005 Jan, 56(410), 255 - 65 Epub 2004 Nov 01.
Metabolite fingerprinting and profiling in plants using NMR; Krishnan P et al.; Although less sensitive than mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy provides a powerful complementary technique for the identification and quantitative analysis of plant metabolites either in vivo or in tissue extracts . In one approach, metabolite fingerprinting, multivariate analysis of unassigned (1)H NMR spectra is used to compare the overall metabolic composition of wild-type, mutant, and transgenic plant material, and to assess the impact of stress conditions on the plant metabolome . Metabolite fingerprinting by NMR is a fast, convenient, and effective tool for discriminating between groups of related samples and it identifies the most important regions of the spectrum for further analysis . In a second approach, metabolite profiling, the (1)H NMR spectra of tissue extracts are assigned, a process that typically identifies 20-40 metabolites in an unfractionated extract . These profiles may also be used to compare groups of samples, and significant differences in metabolite concentrations provide the basis for hypotheses on the underlying causes for the observed segregation of the groups . Both approaches generate a metabolic phenotype for a plant, based on a system-wide but incomplete analysis of the plant metabolome . However, a review of the literature suggests that the emphasis so far has been on the accumulation of analytical data and sample classification, and that the potential of (1)H NMR spectroscopy as a tool for probing the operation of metabolic networks, or as a functional genomics tool for identifying gene function, is largely untapped.

Oncogene, 2004 Nov 1, 23(51), 8401 - 9
RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age; Silva J et al.; Sequencing of complete genomes has provided researchers with a wealth of information to study genome organization, genetic instability, and polymorphisms, as well as a knowledge of all potentially expressed genes . The identification of all genes encoded in the human genome opens the door for large-scale systematic gene silencing using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) . With the recent development of siRNA and shRNA expression libraries, the application of RNAi technology to assign function to cancer genes and to delineate molecular pathways in which these genes affect in normal and transformed cells, will contribute significantly to the knowledge necessary to develop new and also improve existing cancer therapy.

Oncogene, 2004 Nov 1, 23(51), 8384 - 91
Oncology studies using siRNA libraries: the dawn of RNAi-based genomics; Sachse C et al.; High-throughput, human cell-based applications of RNA-mediated interference (RNAi) have emerged in recent years as perhaps the most powerful of a 'second wave' of functional genomics technologies . The available reagents and methodologies for RNAi screening studies now enable a wide range of different scopes and scales of investigation, from single-parameter assays applied to focused subsets of genes, to comprehensive genome-wide surveys based on rich, multiparameter readouts . As such, RNAi-based screens are offering important new avenues for the discovery and validation of novel therapeutic targets for several disease areas, including oncology . By enabling a 'clean' determination of gene function, that is the creation of direct causal links between gene and phenotype in human cells, RNAi investigations promise levels of pathophysiological relevance, efficiency, and range of applicability never before possible on this scale . The field of oncology, with its many assays using readily transfectable cell lines, has offered particularly fertile ground for showcasing the potential of RNAi-based genomics . However, like any other technology before it, RNAi is not without its own challenges, limitations, and caveats . Many of these issues stem directly from the choice of silencing reagent to be used in such studies, and the design of the overall screening strategy . Here, we discuss the basic design issues, potential advantages, and technical challenges of large-scale RNAi screens based on the use of chemically synthesized siRNA libraries.

Oncogene, 2004 Nov 1, 23(51), 8376 - 83
Use of RNA interference libraries to investigate oncogenic signalling in mammalian cells; Downward J; Over the past decade, 'RNA interference' has emerged as a natural mechanism of silencing of gene expression . This ancient cellular antiviral response can be manipulated to provide an effective research tool to knock down the level of expression of selected target genes, providing a very powerful new method for the analysis of cell signalling pathways . Systematic silencing of genes on a genome-wide scale using large rationally designed libraries targeting many thousands of genes provides a novel functional genomics approach to the investigation of many aspects of mammalian cell behaviour, including oncogenic transformation . Here, the different approaches taken to use RNA interference libraries to study the cancer phenotype will be considered, including both selective and high throughput screens and the use of both vector-based and synthetic oligonucleotide-based methods for inducing RNA interference . The advantages and drawbacks of the competing methodologies will be discussed . RNA interference library technology holds great promise for enabling somatic cell genetics in tissue culture systems . Whether it can provide significant new insights into cancer will be its greatest challenge.

Oncogene, 2004 Nov 1, 23(51), 8359 - 65
Using RNAi to catch Drosophila genes in a web of interactions: insights into cancer research; Dasgupta R et al.; The completion of whole-genome sequencing of various model organisms and the recent explosion of new technologies in the field of Functional Genomics and Proteomics is poised to revolutionize the way scientists identify and characterize gene function . One of the most significant advances in recent years has been the application of RNA interference (RNAi) as a means of assaying gene function . In the post-genomic era, advances in the field of cancer biology will rely upon the rapid identification and characterization of genes that regulate cell growth, proliferation, and apoptosis . Significant efforts are being directed towards cancer therapy and devising efficient means of selectively delivering drugs to cancerous cells . In this review, we discuss the promise of integrating genome-wide RNAi screens with proteomic approaches and small-molecule chemical genetic screens, towards improving our ability to understand and treat cancer.

Oncogene, 2004 Nov 1, 23(51), 8353 - 8
High-throughput gene silencing using cell arrays; Vanhecke D et al.; A recently established transfected cell array (TCA) technology has opened new experimental dimensions in the field of functional genomics . Cell arrays allow for transfection of several thousands different DNA molecules in microarray format . The effects of overexpression of hundreds of proteins on cellular physiology can be observed in a single experiment . The TCA technique has also found its application in RNA interference (RNAi) research . Small interfering RNAs (siRNA) as well as plasmid expressing short hairpin RNAs can be transferred into the cells through the process of reverse transfection . The silencing of numerous genes in spatially separated manner can be thus monitored . This review will provide an overview on current concepts concerning combination of cell array and RNAi for high-throughput loss-of-function studies.

J Nippon Med Sch, 2004 Oct, 71(5), 306 - 7
Thin is better!: ultrathin cryosection immunocytochemistry; Takizawa T et al.; In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images . One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope . The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm . Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections) . The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness . The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig . 1) . Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig . 2) . We show new methods of ultrathin cryosection immunocytochemistry(1-3) . Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen . Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM . Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles . For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol(1) . Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules(2,3).

Biol Reprod . 2004 Oct 27; {Epub ahead of print}
Human Myometrial Gene Expression Before and During Parturition; Havelock JC et al.; Identification of temporal and spatial changes in myometrial gene expression during parturition may further the understanding of the coordinated regulation of myometrial contractions during parturition . The objective of this study was to compare the gene expression profiles of human fundal myometrium from pregnant women before and after the onset of labor using a functional genomics approach, and to further characterize the spatial and temporal expression patterns of three genes thought to be important in parturition . Fundal myometrial mRNA was isolated from 5 women in labor and 5 women not in labor and analyzed using human UniGEM-V microarrays with 9182 cDNA elements . Real-time PCR using myometrial RNA from pregnant women in labor or not in labor was used to examine mRNA levels for three of the genes, namely prostaglandin-endoperoxide synthase 2 (PTGS2), calgranulin B (S100A9), and oxytocin receptor (OXTR) . The spatial expression pattern of these genes throughout the pregnant uterus before and after labor was also determined . Immunolocalization of COX-2 (also known as PTGS2) and S100A9 within the uterine cervix and myometrium were analyzed by immunohistochemistry . Few genes were differentially expressed in fundal myometrial tissues at term with the onset of labor . However, there appears to be a subset of genes important in the parturition cascade . The cellular properties of S100A9, its spatial localization, and dramatic increase in cervix and myometrium of laboring women suggest that this protein may be very important in the initiation or propagation of human labor.

Am J Obstet Gynecol, 2004 Oct, 191(4), 1331 - 8
Genome-wide expression profiling of fetal membranes reveals a deficient expression of proteinase inhibitor 3 in premature rupture of membranes; Tromp G et al.; OBJECTIVE: We used a genome-wide approach to identify differentially expressed genes in patients with preterm premature rupture of membranes to improve the understanding of underlying molecular mechanisms . STUDY DESIGN: RNA was isolated from the fetal membranes of patients with preterm labor with intact membranes and preterm premature rupture of membranes and was stratified according to the presence or absence of histologic chorioamnionitis . Microarray experiments were used to identify differentially expressed genes, and real-time quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry were used in follow-up experiments . RESULTS: Microarray experiments identified decreased expression of proteinase inhibitor 3 in the preterm premature rupture of membranes cases . Quantitative reverse transcriptase-polymerase chain reaction confirmed these results . Immunohistochemistry demonstrated decreased proteinase inhibitor 3 protein expression in preterm premature rupture of membranes . CONCLUSION: A genome-wide approach identified deficient expression of proteinase inhibitor 3 in preterm premature rupture of membranes, which demonstrated the usefulness of functional genomics for the dissection of mechanisms of disease and identification of differentially regulated genes that were not suspected previously to play a role in parturition.

J Virol, 2004 Nov, 78(22), 12576 - 90
Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis; Song WJ et al.; Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV) . Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp . One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified . Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins . Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database . An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified . Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation . The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands . Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins . Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products.

Plant Cell Rep . 2004 Oct 19; {Epub ahead of print}
A rapid and efficient transformation protocol for the grass Brachypodium distachyon; Christiansen P et al.; A fast and efficient microprojectile bombardment-mediated transformation protocol is reported for the grass species Brachypodium distachyon, a proposed alternative model plant to Oryza sativa for functional genomics in grasses . Embryogenic calli derived from immature embryos were transformed by a construct containing the uidA (coding for beta-glucuronidase) and bar (coding for phosphinothricin acetyl transferase) genes, and bialaphos, a non-selective herbicide, was used as the selection agent throughout all phases of the tissue culture . Average transformation efficiencies of 5.3% were achieved, and for single bombardments transformation efficiencies of up to 14% were observed . The time frame from the bombardment of embryogenic callus to the harvesting of transgenic T(1) seeds was 29 weeks and 25 weeks for the diploid and two tetraploid accessions used, respectively . Since the seed-to-seed life cycle is 19 weeks for the diploid and 15 weeks for the tetraploid accessions, our B . distachyon transformation system allows testing of both the T(0) and the T(1) generation as well as production of T(2) seeds within 1 year.

Plant J, 2004 Nov, 40(4), 611 - 21
Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas; Rohr J et al.; RNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies . Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans . However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene . This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes . Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii . We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3'UTR of a transgene conferring resistance to bleomycin . However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes . To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform) . We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens.

Wien Klin Wochenschr, 2002, 114 Suppl 4, 4 - 9
{Pathophysiology and genetics of obesity}; Oberkofler H et al.; Obesity has become the most prevalent nutritional disorder in post-industrialised societies and it is associated with the development of severe and costly complications such as type 2 diabetes mellitus and coronary heart disease or cancer . A large proportion of the risk of obesity is determined by the genetic susceptibility of an individual, but environmental factors conducive for the disorder play an important role in its phenotypic expression . Several candidate genes emerged from studies in animal models of obesity, but human pathophysiology is likely to be more complex . Thus, most cases of human obesity probably result from subtle interactions of susceptibility genes with environmental factors favouring deposition of excess calories as fat . The recent surge of obesity may relate to past evolutionary pressure which favoured selection of mechanisms defending body-weight against caloric restriction rather than against caloric excess . Rapidly developing new techniques in quantitative genetics and growing information from functional genomics will help to understand the interaction of environmental factors with signalling networks that regulate energy metabolism . The role of previously unknown pathways in the aetiology of obesity will be uncovered . The typing of numerous genetic variants will become possible and allow individual risk assessment for obesity and/or its associated disorders . Thus, rational and individually tailored therapies may be developed to combat obesity and its associated disorders.

Methods Mol Med, 2004, 105, 439 - 52
A functional genomics approach to hematopoietic stem cell regulation; Hackney JA et al.; Elucidation of the molecular mechanisms that are responsible for regulating the most basic properties of stem cells, self-renewal, and differentiation remains a major challenge in hematopoietic stem cell biology . We have taken a functional genomics approach towards revealing these mechanisms . Previous studies of the fetal liver genetic program led to the development of Stem Cell Database, a resource for the stem cell community . These studies have been expanded to include the microenvironmental component of hematopoiesis and are the focus herein . In our efforts to study the microenvironmental component we have identified a stromal cell line, AFT024, which serves as a surrogate stem cell niche . The line provides a milieu that facilitates the maintenance of transplantable mouse and human stem cells as well as the generation of large populations of committed progenitors . In a manner mirroring the work done with the SCDb, we provide an online resource, Stromal Cell Database, StroCDB, that is a compendium of information and data derived from biological and molecular studies of this surrogate niche . These include bioinformatic analyses of over 6000 clones derived from a subtracted library enriched for messages expressed in AFT024 as well as data derived from custom expression arrays developed from this library . Herein we describe these efforts and provide a guide for navigating the database and mining the information contained within.

Biochemistry, 2004 Oct 26, 43(42), 13348 - 56
Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA; Spagnou S et al.; RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells {Novina, C . D., and Sharp, P . A . (2004) The RNAi revolution, Nature 430, 161-164} . Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells . As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function . However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects . For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies . Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies . Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) . Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects . Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles . Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown . Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity . Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles . Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000 . Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection . By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.

Genome Res, 2004 Oct, 14(10B), 2176 - 89
Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications; Hilson P et al.; Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes . Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches . The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome . They were synthesized by PCR amplification from genomic DNA . Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments . The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols . These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics . We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells . We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines . These versatile GST resources provide novel and powerful tools for functional genomics.

Genome Res, 2004 Oct, 14(10B), 2136 - 44
From ORFeome to biology: a functional genomics pipeline; Wiemann S et al.; As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease . We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction . Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network . We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information . All information is presented in a Web database . It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

Genome Res, 2004 Oct, 14(10B), 2076 - 82
High-throughput generation of P . falciparum functional molecules by recombinational cloning; Aguiar JC et al.; Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression . We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes . Master clones and their ORFs were transferred en masse to multiple expression vectors . Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure . Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency . The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors . These transfers yielded a 100% success rate without any observed recombination based on single colony screening . The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite . From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages . We have overcome the potential limitation of producing large P . falciparum clone sets in multiple expression vectors . This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P . falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

Annu Rev Genomics Hum Genet, 2004, 5, 443 - 77
Plant genomics: the third wave; Borevitz JO et al.; Completing the primary genomic sequence of Arabidopsis thaliana was a major milestone, being the first plant genome and only the third high-quality finished eukaryotic genome sequence . Understanding how the genome sequence comprehensively encodes developmental programs and environmental responses is the next major challenge for all plant genome projects . This requires fully characterizing the genes, the regulatory sequences, and their functions . We discuss several functional genomics approaches to decode the linear sequence of the reference plant Arabidopsis thaliana, including full-length cDNA collections, microarrays, natural variation, knockout collections, and comparative sequence analysis . Genomics provides the essential tools to speed the work of the traditional molecular geneticist and is now a scientific discipline in its own right.

Annu Rev Genomics Hum Genet, 2004, 5, 267 - 93
Proteomics; de Hoog CL et al.; The genome sequences of important model systems are available and the focus is now shifting to large-scale experiments enabled by this data . Following in the footsteps of genomics, we have functional genomics, proteomics, and even metabolomics, roughly paralleling the biological hierarchy of the transcription, translation, and production of small molecules . Proteomics is initially concerned with determining the structure, expression, localization, biochemical activity, interactions, and cellular roles of as many proteins as possible . There has been great progress owing to novel instrumentation, experimental strategies, and bioinformatics methods . The area of protein-protein interactions has been especially fruitful . First pass interaction maps of some model organisms exist, and the proteins in many important organelles are about to be determined . Researchers are also beginning to integrate large-scale data sets from various "omics" disciplines in targeted investigations of specific biomedical areas and in pursuit of a general framework for systems biology.

Clin Orthop, 2004 Oct, (427 Suppl), S138 - 43
Functional genomics of osteoarthritis: on the way to evaluate disease hypotheses; Aigner T et al.; Functional genomics is a challenging new way to address complex diseases such as osteoarthritis on a molecular level . This complements previous research and will open up new areas of so far unrecognized molecular networks . In this respect, articular cartilage is a good target for functional genomics as it contains only one cell type to which all expression signals can be attributed to . Despite considerable limitations at present, such as a low sensitivity and insensitivity to alternative splicing, posttranscriptional regulation, and posttranslational modification, cDNA-array technology provides a powerful tool to obtain an overview on gene expression patterns hardly achievable with other techniques . This has been shown to be true for known genes as well as for the identification of new genes of interest . Therefore, gene expression analysis will help to identify single genes depending on the disease and experimental conditions investigated . However, the expression pattern of the plethora of expressed genes will paint a picture (network) of disease context, maybe even more pushing forward our understanding of complex diseases such as osteoarthritis.

FEMS Immunol Med Microbiol, 2004 Nov 1, 42(3), 271 - 9
Current concepts on Escherichia coli K1 translocation of the blood-brain barrier; Xie Y et al.; The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy . Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis . Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity . We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively . With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E . coli requires a high-degree of bacteremia, E . coli binding to and invasion of HBMEC, and E . coli traversal of the BBB as live bacteria . Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E . coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo . We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E . coli translocation of the BBB is the result of specific pathogen-host cell interactions . Recent studies using functional genomics techniques have identified additional E . coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis . In this review, we summarize the current knowledge on the mechanisms underlying the successful E . coli translocation of the BBB.

Sci Aging Knowledge Environ . 2004 Oct 06;2004(40):pe37.
Genes, longevity, and technology: meeting report from the 2nd conference on functional genomics of aging in Crete; Baumeister R et al.; Whole-genome sequences are now available, and methods have evolved for targeting, in parallel, each gene in a genome, offering for the first time the opportunity to study the entire dynamic network of genes involved in aging . At a recent conference in Hersonissos, Crete, around 200 internationally renowned experts gathered to discuss techniques and emerging results as the science of aging undergoes a shift toward systems biology.

Curr Opin Biotechnol, 2004 Oct, 15(5), 444 - 8
Using functional genomics to improve productivity in the manufacture of industrial biochemicals; Hermann T; Recent developments in the field of functional genomics have been used to increase productivity in the manufacture of industrial biochemicals . Technologies like transcriptomics and proteomics have profited from the increasing number of genome sequencing projects . Meanwhile functional genomics has evolved from several isolated technologies, such as DNA chip technology and proteomics, to combined approaches that can help us to understand why organisms produce a certain product . The combination of expression studies and kinetic studies, such as carbon flux determination or metabolite measurements, has significantly improved productivity in production processes.

Eur J Pharmacol, 2004 Oct 1, 500(1-3), 385 - 98
Proteomic approaches in brain research and neuropharmacology; Vercauteren FG et al.; Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions . Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents . Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time . In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed . The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed . It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.

Int J Cancer, 2005 Jan 20, 113(3), 434 - 9
High throughput functional genomics: identification of novel genes with tumor suppressor phenotypes; Koenig-Hoffmann K et al.; We have used a combination of high throughput functional genomics, computerized database mining and expression analyses to discover novel human tumor suppressor genes (TSGs) . A genome-wide high throughput cDNA phenotype screen was established to identify genes that induce apoptosis or reduce cell viability . TSGs are expressed in normal tissue and frequently act by reduction of growth of transformed cells or induce apoptosis . In agreement with that and thus serving as platform validation, our pro-apoptotic hits included genes for which tumor suppressing activities were known, such as kangai1 and CD81 antigen . Additional genes that so far have been claimed as putative TSGs or associated with tumor inhibitory activities (prostate differentiation factor, hRAS-like suppressor 3, DPH2L1-like and the metastasis inhibitor Kiss1) were confirmed in their proposed TSG-like phenotype by functionally defining their growth inhibitory or pro-apoptotic function towards cancer cells . Finally, novel genes were identified for which neither association with cell growth nor with apoptosis were previously described . A subset of these genes show characteristics of TSGs because they (i) reduce the growth or induce apoptosis in tumor cells; (ii) show reduced expression in tumor vs . normal tissue; and (iii) are located on chromosomal (LOH-) loci for which cancer-associated deletions are described . The pro-apoptotic phenotype and differential expression of these genes in normal and malignant tissue make them promising target candidates for the diagnosis and therapy of various tumors.

Biophys J, 2004 Oct, 87(4), 2647 - 55
Tertiary structure predictions on a comprehensive benchmark of medium to large size proteins; Zhang Y et al.; We evaluate tertiary structure predictions on medium to large size proteins by TASSER, a new algorithm that assembles protein structures through rearranging the rigid fragments from threading templates guided by a reduced Calpha and side-chain based potential consistent with threading based tertiary restraints . Predictions were generated for 745 proteins 201-300 residues in length that cover the Protein Data Bank (PDB) at the level of 35% sequence identity . With homologous proteins excluded, in 365 cases, the templates identified by our threading program, PROSPECTOR_3, have a root-mean-square deviation (RMSD) to native < 6.5 angstroms, with >70% alignment coverage . After TASSER assembly, in 408 cases the best of the top five full-length models has a RMSD < 6.5 angstroms . Among the 745 targets are 18 membrane proteins, with one-third having a predicted RMSD < 5.5 A . For all representative proteins less than or equal to 300 residues that have corresponding multiple NMR structures in the Protein Data Bank, approximately 20% of the models generated by TASSER are closer to the NMR structure centroid than the farthest individual NMR model . These results suggest that reasonable structure predictions for nonhomologous large size proteins can be automatically generated on a proteomic scale, and the application of this approach to structural as well as functional genomics represent promising applications of TASSER .

J Virol, 2004 Oct, 78(20), 10995 - 1006
Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response; Abate DA et al.; We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration . Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response . The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3) . In contrast, NF-kappaB is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus . pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells . pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state . Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response . By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response.

Curr Opin Microbiol, 2004 Oct, 7(5), 546 - 54
The promise of functional genomics: completing the encyclopedia of a cell; Hughes TR et al.; Genome sequencing provides complete parts lists of organisms . This presents the obvious challenge of determining how each gene contributes to the life of the organism . This task seems increasingly feasible; however, progress to date suggests that increased interaction between systematic efforts and individual investigators will be critical to completing the encyclopedia of the yeast cell.

Curr Opin Microbiol, 2004 Oct, 7(5), 535 - 45
Analyzing protein function on a genomic scale: the importance of gold-standard positives and negatives for network prediction; Jansen R et al.; The concept of 'protein function' is rather 'fuzzy' because it is often based on whimsical terms or contradictory nomenclature . This currently presents a challenge for functional genomics because precise definitions are essential for most computational approaches . Addressing this challenge, the notion of networks between biological entities (including molecular and genetic interaction networks as well as transcriptional regulatory relationships) potentially provides a unifying language suitable for the systematic description of protein function . Predicting the edges in protein networks requires reference sets of examples with known outcome (that is, 'gold standards') . Such reference sets should ideally include positive examples - as is now widely appreciated - but also, equally importantly, negative ones . Moreover, it is necessary to consider the expected relative occurrence of positives and negatives because this affects the misclassification rates of experiments and computational predictions . For instance, a reason why genome-wide, experimental protein-protein interaction networks have high inaccuracies is that the prior probability of finding interactions (positives) rather than non-interacting protein pairs (negatives) in unbiased screens is very small . These problems can be addressed by constructing well-defined sets of non-interacting proteins from subcellular localization data, which allows computing the probability of interactions based on evidence from multiple datasets.

Biol Chem, 2004 Aug, 385(8), 691 - 6
Functional genomics identifies novel and diverse molecular targets of nutrients in vivo; Gohil K; Genomic responses to nutrients are important determinants of physiological and pathological functions of living systems . Many of these responses are mediated by changes in mRNA concentrations that are primarily regulated by gene transcription . Transcriptional networks that regulate the expression and activities of transcription factors and structural genes in response to nutrients need to be defined . The tools of functional genomics and bioinformatics offer powerful means to address these needs . The application of global mRNA profiling tools to define genome-wide responses to nutrients and micronutrients with a primary focus on in vivo genomic responses of vital organs of laboratory mice is reviewed here . The studies show that major and minor nutrients affect the expression of mRNAs that are related to aging and inflammation, and chemically diverse micronutrients such as polyphenols and tocopherols may exert their effects through modulating the expression of functionally related genes.

Mycol Res, 2004 Aug, 108(Pt 8), 853 - 7
Combining transcriptome data with genomic and cDNA sequence alignments to make confident functional assignments for Aspergillus nidulans genes; Sims AH et al.; Whole genome sequencing of several filamentous ascomycetes is complete or in progress; these species, such as Aspergillus nidulans, are relatives of Saccharomyces cerevisiae . However, their genomes are much larger and their gene structure more complex, with genes often containing multiple introns . Automated annotation programs can quickly identify open reading frames for hypothetical genes, many of which will be conserved across large evolutionary distances, but further information is required to confirm functional assignments . We describe a comparative and functional genomics approach using sequence alignments and gene expression data to predict the function of Aspergillus nidulans genes . By highlighting examples of discrepancies between the automated genome annotation and cDNA or EST sequencing, we demonstrate that the greater complexity of gene structure in filamentous fungi demands independent data on gene expression and the gene sequence be used to make confident functional assignments.

Mol Genet Genomics, 2004 Sep, 272(2), 181 - 93 Epub 2004 Jul 30.
Mapping Ds insertions in barley using a sequence-based approach; Cooper LD et al.; A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley ( Hordeum vulgaresubsp . vulgare) . The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics . AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation . Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location . The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR . Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population . The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines . In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley . BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences . Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

J Exp Bot, 2004 Nov, 55(407), 2343 - 51 Epub 2004 Sep 24.
Root growth maintenance during water deficits: physiology to functional genomics; Sharp RE et al.; Progress in understanding the network of mechanisms involved in maize primary root growth maintenance under water deficits is reviewed . These include the adjustment of growth zone dimensions, turgor maintenance by osmotic adjustment, and enhanced cell wall loosening . The role of the hormone abscisic acid (ABA) in maintaining root growth under water deficits is also addressed . The research has taken advantage of kinematic analysis, i.e . characterization of spatial and temporal patterns of cell expansion within the root growth zone . This approach revealed different growth responses to water deficits and ABA deficiency in distinct regions of the root tip . In the apical 3 mm region, elongation is maintained at well-watered rates under severe water deficit, although only in ABA-sufficient roots, whereas the region from 3-7 mm from the apex exhibits maximum elongation in well-watered roots, but progressive inhibition of elongation in roots under water deficit . This knowledge has greatly facilitated discovery of the mechanisms involved in regulating the responses . The spatial resolution with which this system has been characterized and the physiological knowledge gained to date provide a unique and powerful underpinning for functional genomics studies . Characterization of water deficit-induced changes in transcript populations and cell wall protein profiles within the growth zone of the maize primary root is in progress . Initial results from EST and unigene analyses in the tips of well-watered and water-stressed roots highlight the strength of the kinematic approach to transcript profiling.

Mass Spectrom Rev . 2004 Aug 19; {Epub ahead of print}
Mass spectrometry in metabolome analysis; Villas-Boas SG et al.; I.Introduction000 II.Sample Preparation000A . Quenching000B . Extraction000C . Sample Concentration000 III.Analysis000A . GC-MS000 1 . Scope and Resolution of GC-MS000 2 . Analysis of Volatile Metabolites000 3 . Analysis of Semi-Volatile and Non-Volatile Metabolites000 4 . Sensitivity000 5 . Repeatability/Reproducibility of GC-MS for Metabolome Analysis000B . LC-MS000 1 . LC-MS for Target Analysis000 2 . LC-MS for Metabolite Profiling000C . CE-MS000 1 . CE-MS for Target Analysis000 2 . CE-MS for Metabolite Profiling000D . Direct-Infusion Mass Spectrometry000 1 . Direct-Infusion MS for Targeted Analysis000 2 . Direct-Infusion MS for Metabolite Profiling000E . Comparison of the Different Analytical Techniques000 IV.Application on Functional Genomics000 V.Future Outlook000Acknowledgments000References000In the post-genomic era, increasing efforts have been made to describe the relationship between the genome and the phenotype in cells and organisms . It has become clear that even a complete understanding of the state of the genes, messages, and proteins in a living system does not reveal its phenotype . Therefore, researchers have started to study the metabolome (or the metabolic complement of functional genomics) . Within this context, mass spectrometry (MS) has increasingly occupied a central position in the methodologies developed for determination of the metabolic state . This review is mainly focused on the status of MS in the metabolome field, trying to direct the reader to the main approaches for analysis of metabolites, reviewing basic methodologies in sample preparation, and the most recent MS techniques introduced . Apart from the description of the different methods, this review will try to state a general comparison between the several different techniques that involve MS and metabolite analysis, and will highlight their limitations and preferred applicability .

Exp Biol Med (Maywood), 2004 Oct, 229(9), 866 - 75
Genomics and clinical medicine: rationale for creating and effectively evaluating animal models; Swanson KS et al.; Because resolving human complex diseases is difficult, appropriate biomedical models must be developed and validated . In the past, researchers have studied diseases either by characterizing a human clinical disease and choosing the most appropriate animal model, or by characterizing a naturally occurring or induced mutant animal and identifying which human disease it best resembled . Although there has been a great deal of progress through the use of these methods, such models have intrinsic faults that limit their relevance to clinical medicine . The recent advent of techniques in molecular biology, genomics, transgenesis, and cloning furnishes investigators with the ability to study vertebrates (e.g., pigs, cows, chickens, dogs) with greater precision and utilize them as model organisms . Comparative and functional genomics and proteomics provide effective approaches for identifying the genetic and environmental factors responsible for complex diseases and in the development of prevention and treatment strategies and therapeutics . By identifying and studying homologous genes across species, researchers are able to accurately translate and apply experimental data from animal experiments to humans . This review supports the hypothesis that associated enabling technologies can be used to create, de novo, appropriate animal models that recapitulate the human clinical manifestation . Comparative and functional genomic and proteomic techniques can then be used to identify gene and protein functions and the interactions responsible for disease phenotypes, which aids in the development of prevention and treatment strategies.

Nucleic Acids Res, 2004 Sep 23, 32(17), 4955 - 61 Print 2004.
In silico identification of transcriptional regulators associated with c-Myc; Elkon R et al.; The development of powerful experimental strategies for functional genomics and accompanying computational tools has brought major advances in the delineation of transcriptional networks in organisms ranging from yeast to human . Regulation of transcription of eukaryotic genes is to a large extent combinatorial . Here, we used an in silico approach to identify transcription factors (TFs) that form recurring regulatory modules with c-Myc, a protein encoded by an oncogene that is frequently disregulated in human malignancies . A recent study identified, on a genomic scale, human genes whose promoters are bound by c-Myc and its heterodimer partner Max in Burkitt's lymphoma cells . Using computational methods, we identified nine TFs whose binding-site signatures are highly overrepresented in this promoter set of c-Myc targets, pointing to possible functional links between these TFs and c-Myc . Binding sites of most of these TFs are also enriched on the set of mouse homolog promoters, suggesting functional conservation . Among the enriched TFs, there are several regulators known to control cell cycle progression . Another TF in this set, EGR-1, is rapidly activated by numerous stress challenges and plays a central role in angiogenesis . Experimental investigation confirmed that c-Myc and EGR-1 bind together on several target promoters . The approach applied here is general and demonstrates how computational analysis of functional genomics experiments can identify novel modules in complex networks of transcriptional regulation.

Rev Med Univ Navarra, 2004 Apr-Jun, 48(2), 36 - 48
{Nutrigenomics and obesity}; Palou A et al.; Obesity is a multifactorial disorder affected by multiple genetic and environmental factors, in particular nutrients, and their interrelationships . Increasing knowledge of the genes and molecules involved in the development of obesity is paving the way for new methods of obesity control . In this sense, Nutrigenomics--which represents a new approach in nutrition research that joints the application of powerful functional genomics technologies, bioinformatics and molecular biology with more traditional methodologies--may orientate the design and development of new functional foods for obesity, based on the scientific knowledge of the impact of specific nutrients on the mammalian body weight control system and their mechanisms of action.

Epilepsy Res, 2004 Jul-Aug, 60(2-3), 173 - 8
Molecular profiling of temporal lobe epilepsy: comparison of data from human tissue samples and animal models; Majores M et al.; The advent of gene chip technology and the era of functional genomics have initially been accompanied by huge anticipations to quickly unravel the molecular pathogenesis of multifactorial diseases . Expectations have, today, given way to some concerns about this non-hypothesis driven approach . However, the careful and controlled application of expression microarrays in concert with refined bioinformatic tools may provide novel insights in major disorders particularly of highly complex organs such as the central nervous system (CNS) . Epilepsies are among the most frequent CNS disorders affecting approximately 1.5% of the population worldwide . In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation, a structure located in the mesial temporal lobe . Many TLE patients develop pharmacoresistance, i.e . seizures can no more be controlled by antiepileptic drugs . In order to achieve seizure control, surgical removal of the epileptogenic focus has been established as successful therapeutic strategy . Hippocampal biopsy tissue of pharmacoresistant TLE patients represents an excellent substrate to analyze molecular mechanisms related to structural and cellular reorganization in epilepsy . The complexity of alterations in TLE hippocampi suggests numerous genes and signaling cascades to be involved in the pathogenesis . By microarrays, genome wide expression profiles can be constituted from TLE tissues . However, hippocampi of pharmacoresistant TLE patients represent an advanced stage of the disease . Early stages of epilepsy development are not available for functional genome analysis in humans . Animal models of TLE appear particularly helpful to study molecular mechanisms of highly dynamic processes such as the development of hyperexcitability and pharmacoresistance . In this review, we summarize recent data of gene expression profiles in human and experimental TLE and discuss the relevance of novel tools for bioinformatic analysis and data mining.

Curr Opin Genet Dev, 2004 Oct, 14(5), 470 - 6
Genome-wide high-throughput screens in functional genomics; Friedman A et al.; The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function . Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals . Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function . Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells . Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms . Integration of such data with other genomic approaches will extend our understanding of cellular networks.

Mol Genet Genomics, 2004 Oct, 272(3), 344 - 52 Epub 2004 Sep 16.
Efficient gene disruption in the koji-mold Aspergillus sojae using a novel variation of the positive-negative method; Takahashi T et al.; When no phenotypic screen is available, gene disruption in the koji-mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination . To achieve efficient gene disruption in the koji-mold, we developed a novel positive-negative selection method to enrich for homologous recombinants . The pyrG gene from A . sojae was used as a positive selection marker for transformants, and the oliC31 gene of A . nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants . The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated . In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A . sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method . Despite the fact that this gene is not transcribed in A . sojae, aflR disruptants were efficiently obtained, suggesting that the method is indeed capable of targeting any locus, without additional ectopic integration, and is thus applicable for functional genomics studies in filamentous fungi, including A . sojae.

Mol Genet Genomics, 2004 Oct, 272(3), 308 - 27 Epub 2004 Sep 11.
Genetic mapping of EST-derived microsatellites from the diploid Gossypium arboreum in allotetraploid cotton; Han ZG et al.; To increase the numbers of microsatellites available for use in constructing a genetic map, and facilitate the use of functional genomics to elucidate fiber development and breeding in cotton, we sampled microsatellite sequences from expressed sequence tags (ESTs) transcribed during fiber elongation in the A-genome species Gossypium arboreum to evaluate their frequency of occurrence, level of polymorphism and distribution in the At and Dt subgenomes of tetraploid cotton . From among ESTs derived from G . arboreum fibers at 7-10 days post anthesis (dpa), 931 ESTs were found to contain simple sequence repeats (SSRs); 544 (58.4%) EST-SSR primer pairs were developed, and 468 (86%) amplified PCR products from allotetraploid cotton (G . hirsutumcv . TM-1 and G . barbadense cv . Hai7124) . However, only 99 (18.2%) of these were found to be polymorphic and segregating in our interspecific BC1 mapping population {(TM-1xHai7124)xTM-1} . In these amplified and informative EST-SSRs, hexa- and tri-nucleotide repeat motifs were the most frequent, representing 40.1 and 30%, respectively, of the total . A total of 111 loci detected with these 99 EST-SSRs were integrated into our backbone map including 511 SSR loci . The distribution of the EST-SSRs appeared to be non-random, since 72 loci were anchored to the At and 37 to the Dt subgenome of allotetraploid cotton based on linkage tests . Interestingly, out of the 10 pairs of duplicate loci amplified, seven were mapped to the corresponding homologous linkage groups and/or chromosomes . BLASTX analysis revealed that 69 of the 99 ESTs showed significant similarities to known genes . Some genes important for fiber development, such as sucrose synthase, were mapped to corresponding chromosomes . These EST-SSRs provide structural and functional genomic information that will be useful for understanding cotton fiber development.

Med Sci (Paris), 2004 Aug-Sep, 20(8-9), 815 - 9
{RNA interference: towards a functional genomics in mammalian cells?}; Dautry F et al.; The discovery of the induction of RNA degradation by double stranded RNA in C . elegans, "RNA interference", makes it possible to envision systematic studies of gene function in mammalian cells . Indeed, in spite of the existence in mammals of the interferon response to double stranded RNA, the introduction of small interfering RNA can induce a sequence specific inhibition of gene expression either through RNA degradation or by blocking translation . Although the inhibition is transient and usually not complete, strategies have been developed to achieve long term gene silencing . The issue of target specificity is still not completely clear and will probably constitute a limitation of this approach . However, because of the unprecedented ease with which large scale screens can be performed, RNA interference has already established itself as the tool of choice to initiate functional genomics in mammalian cells.

Bioinformatics . 2004 Sep 9; {Epub ahead of print}
VizRank: finding informative data projections in functional genomics by machine learning; Leban G et al.; SUMMARY: VizRank is a tool that finds interesting two-dimensional projections of class-labeled data . When applied to multi-dimensional functional genomics data sets, VizRank can systematically find relevant biological patterns . AVAILABILITY: SUPPLEMENTARY INFORMATION: http://www.ailab.si/supp/bi-vizrank.

Trends Endocrinol Metab, 2004 Aug, 15(6), 243 - 7
Flying through the genome: a comprehensive study of functional genomics using RNAi in Drosophila; Andres AJ; Sequencing the DNA of an entire mammalian genome now seems routine . The human sequence along with the mouse--the model for mammalian genetics--and the rat--the model for mammalian physiology--are now part of the data archive . However, the real challenges for the 21st century are what to do with this information and how to test the function of so many different genes in so many different cellular contexts . The potential payoffs are enormous . Examples include a better understanding of disease pathologies with effective strategies for therapeutic interventions that cause few, if any, side effects.

Trends Plant Sci, 2004 Aug, 9(8), 391 - 8
Weed genomics: new tools to understand weed biology; Basu C et al.; In spite of the large yield losses that weeds inflict on crops, we know little about the genomics of economically important weed species . Comparative genomics between plant model species and weeds, map-based approaches, genomic sequencing and functional genomics can play vital roles in understanding and dissecting weedy traits of agronomically important weed species that damage crops . Weed genomics research should increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make weeds a successful group of plants . Here, we propose specific weed candidates as genomic models, including economically important plants that have evolved herbicide resistance on several occasions and weeds with good comparative genomic qualities that can be anchored to the genomics of Arabidopsis and Oryza sativa.

BMC Genomics . 2004 Sep 09;5(1):64.
A method for detecting and correcting feature misidentification on expression microarrays; Tu IP et al.; BACKGROUND: Much of the microarray data published at Stanford is based on mouse and human arrays produced under controlled and monitored conditions at the Brown and Botstein laboratories and at the Stanford Functional Genomics Facility (SFGF) . Nevertheless, as large datasets based on the Stanford Human array began to accumulate, a small but significant number of discrepancies were detected that required a serious attempt to track down the original source of error . Due to a controlled process environment, sufficient data was available to accurately track the entire process leading to up to the final expression data . In this paper, we describe our statistical methods to detect the inconsistencies in microarray data that arise from process errors, and discuss our technique to locate and fix these errors . RESULTS: To date, the Brown and Botstein laboratories and the Stanford Functional Genomics Facility have together produced 40,000 large-scale (10-50,000 feature) cDNA microarrays . By applying the heuristic described here, we have been able to check most of these arrays for misidentified features, and have been able to confidently apply fixes to the data where needed . Out of the 265 million features checked in our database, problems were detected and corrected on 1.3 million of them . CONCLUSION: Process errors in any genome scale high throughput production regime can lead to subsequent errors in data analysis . We show the value of tracking multi-step high throughput operations by using this knowledge to detect and correct misidentified data on gene expression microarrays.

Pigment Cell Res, 2004 Oct, 17(5), 461 - 70
Functional genomics tools for the analysis of zebrafish pigment; Pickart MA et al.; Genetic model organisms are increasingly valuable in the post-genomics era to provide a basis for comparative analysis of the human genome . For higher order processes of vertebrate pigment cell biology and development, the mouse has historically been the model of choice . A complementary organism, the zebrafish (Danio rerio), shares many of the signaling and biological processes of vertebrates, e.g . neural crest development . The zebrafish has a number of characteristics that make it an especially valuable model for the study of pigment cell biology and disease . Large-scale genetic screens have identified a collection of pigmentation mutants that have already made valuable contributions to pigment research . An increasing repertoire of genomic resources such as an expressed sequence tag-based Gene Index (The Institute for Genomic Research) and improving methods of mutagenesis, transgenesis, and gene targeting make zebrafish a particularly attractive model . Morpholino phosphorodiamidate oligonucleotide (MO) 'knockdown' of pigment gene expression provides a non-conventional antisense tool for the analysis of genes involved in pigment cell biology and disease . In addition, an ongoing, reverse-genetic, MO-based screen for the rapid identification of gene function promises to be a valuable complement to other high-throughput microarray and proteomic approaches for understanding pigment cell biology . Novel reagents for zebrafish transgenesis, such as the Sleeping Beauty transposon system, continue to improve the capacity for genetic analysis in this system and ensure that the zebrafish will be a valuable genetic model for understanding a variety of biological processes and human diseases for years to come.

Proc Natl Acad Sci U S A, 2004 Sep 21, 101(38), 13951 - 6 Epub 2004 Sep 07.
A Populus EST resource for plant functional genomics; Sterky F et al.; Trees present a life form of paramount importance for terrestrial ecosystems and human societies because of their ecological structure and physiological function and provision of energy and industrial materials . The genus Populus is the internationally accepted model for molecular tree biology . We have analyzed 102,019 Populus ESTs that clustered into 11,885 clusters and 12,759 singletons . We also provide >4,000 assembled full clone sequences to serve as a basis for the upcoming annotation of the Populus genome sequence . A public web-based EST database (POPULUSDB) provides digital expression profiles for 18 tissues that comprise the majority of differentiated organs . The coding content of Populus and Arabidopsis genomes shows very high similarity, indicating that differences between these annual and perennial angiosperm life forms result primarily from differences in gene regulation . The high similarity between Populus and Arabidopsis will allow studies of Populus to directly benefit from the detailed functional genomic information generated for Arabidopsis, enabling detailed insights into tree development and adaptation . These data will also valuable for functional genomic efforts in Arabidopsis.

Proteomics, 2004 Sep, 4(9), 2672 - 84
Wheat leaf proteome analysis using sequence data of proteins separated by two-dimensional electrophoresis; Bahrman N et al.; Identifying wheat leaf protein expression is a major challenge of functional genomics . Using two-dimensional gel electrophoresis 541 wheat leaf proteins were separated and 55 of them were sequenced by nano liquid chromatography-tandem mass spectrometry . Peptide sequence data were screened against protein banks and expressed sequence tag public banks . Among these 55 spots, 20 proteins were found in wheat and 21 in other grass families . Twelve proteins showed similarities with other eukaryotic plant species . One protein showed homology to a bacterial sequence and another protein remained unknown . In 18 cases a significant score was found for the wheat TUC (Tentative Unique Contigs) of the PlantGDB data . In several cases, different spots were identified as corresponding to the same protein that can probably be attributed to the hexaploid structure of wheat . The identified proteins were classified in six groups and their role is discussed . Most of them (31/55) are involved in carbohydrate metabolism.

J Immunol Methods, 2004 Sep, 292(1-2), 59 - 71
Identification of compounds that enhance the anti-lymphoma activity of rituximab using flow cytometric high-content screening; Gasparetto M et al.; In this report, we describe a new flow cytometry technique termed flow cytometric high-content screening (FC-HCS) which involves semi-automated processing and analysis of multiparameter flow cytometry samples . As a first test of the FC-HCS technique, we used it to screen a 2000-compound library, called the National Cancer Institute (NCI) Diversity Set, to identify agents that would enhance the anti-lymphoma activity of the therapeutic monoclonal antibody rituximab . FC-HCS identified 15 compounds from the Diversity Set that significantly enhanced the ability of rituximab to inhibit cell cycle progression and induce apoptosis in lymphoma cells . The validity of the screening results was confirmed for several compounds using additional assays of cell proliferation, apoptosis and cell growth . The FC-HCS technique was relatively simple and reliable and could process up to 1000 samples/day on a single flow cytometer . The FC-HCS technique may be useful for a variety of applications including drug discovery, immunologic monitoring of patients, functional genomics studies and tissue engineering efforts.

Methods Find Exp Clin Pharmacol, 2004 Jul-Aug, 26(6), 421 - 44
Genomics and phenotypic profiles in dementia: implications for pharmacological treatment; Cacabelos R et al.; Constitutive genomics are probably determinant for the onset of dementia in conjunction with cerebrovascular and environmental factors . Furthermore, pharmacogenomic studies predict that the therapeutic response in Alzheimer's disease (AD) is genotype-specific, and that the expression of genes involved in the regulation of drug metabolism can influence efficacy and safety issues in pharmacotherapy . AD and dementia with a vascular component (DVC = VD + MXD) are the most prevalent forms of dementia . These clinical entities share many similarities, but they differ in major phenotypic and genotypic profiles, as revealed by structural and functional genomics studies . Comparative phenotypic studies have identified significant differences in 25% of more than 100 parametric variables, including anthropometry, cardiovascular function, aortic atherosclerosis, brain atrophy, blood pressure, blood biochemistry, hematology, thyroid function, folic acid and vitamin B(12) levels, brain hemodynamics and lymphocyte markers . The phenotypic profile of patients with DVC differs from that of AD patients in the following: (a) anthropometric values, (b) cardiovascular function, (c) blood pressure, (d) lipid metabolism, (e) uric acid levels, (f) peripheral calcium levels, (g) liver function (GOT, GPT, GGT), (h) alkaline phosphatase, (i) lactate dehydrogenase, (j) red and white blood cells, (k) regional brain atrophy (left temporal region, inter-hippocampal distance) and (l) brain blood flow velocity . Functional genomics studies incorporating APOE-related changes in biological markers extended the difference between AD and DVC up to 57% . Structural genomics studies with AD-related genes, including APP, MAPT, APOE, PS1, PS2, A2M, ACE, AGT, cFOS and PRNP genes, demonstrate different genetic profiles in AD and DVC, with an absolute genetic variation rate ranging from 30 to 80%, depending upon genes and genetic clusters . Single gene analysis identifies relative genetic variations ranging from 0 to 5% . The relative polymorphic variation in genetic clusters integrated by 2, 3 or 4 genes associated with AD ranges from 1 to 3% . The main phenotypic differences between AD and DVC are genotype-dependent, especially in AD, probably indicating that different genomic factors are essential for the expression of dementia symptoms that might be accelerated or induced by environmental and/or cerebrovascular factors.

BMC Genomics . 2004 Sep 03;5(1):62.
Enhancer trapping in zebrafish using the Sleeping Beauty transposon; Balciunas D et al.; BACKGROUND: Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate . Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem . RESULTS: We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio . We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny . We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event . Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly . We have identified both previously characterized as well as novel expression patterns from this screen . For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain . The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus . This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity . Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline . CONCLUSIONS: This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.

Genetics, 2004 Aug, 167(4), 1801 - 11
A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila; Bonin CP et al.; P-element-based gene and enhancer trap strategies have provided a wealth of information on the expression and function of genes in Drosophila melanogaster . Here we present a new vector that utilizes the simple insertion requirements of the piggyBac transposon, coupled to a splice acceptor (SA) site fused to the sequence encoding enhanced green fluorescent protein (EGFP) and a transcriptional terminator . Mobilization of the piggyBac splice site gene trap vector (PBss) was accomplished by heat-shock-induced expression of piggyBac transposase (PBase) . We show that insertion of PBss into genes leads to fusions between the gene's mRNA and the PBss-encoded EGFP transcripts . As heterozygotes, these fusions report the normal pattern of expression of the trapped gene . As homozygotes, these fusions can inactivate the gene and lead to lethality . Molecular characterization of PBss insertion events shows that they are single copy, that they always occur at TTAA sequences, and that splicing utilizes the engineered splice site in PBss . In those instances where protein-EGFP fusions are predicted to occur, the subcellular localization of the wild-type protein can be inferred from the localization of the EGFP fusion protein . These experiments highlight the utility of the PBss system for expanding the functional genomics tools that are available in Drosophila .

Mol Pharmacol, 2004 Dec, 66(6), 1361 - 71 Epub 2004 Dec.
Designing transcription factor architectures for drug discovery; Blancafort P et al.; Recent advances in the design, selection, and engineering of DNA binding proteins have led to the emerging field of designer transcription factors (TFs) . Modular DNA-binding protein domains can be assembled to recognize a given sequence of a DNA in a regulatory region of a targeted gene . TFs can be readily prepared by linking the DNA-binding protein to a variety of effector domains that mediate transcriptional activation or repression . Furthermore, the interaction between the TF and the genomic DNA can be regulated by several approaches, including chemical regulation by a variety of small molecules . Genome-wide single target specificity has been demonstrated using arrays of sequence-specific zinc finger (ZF) domains, polydactyl proteins . Any laboratory today can easily construct polydactyl ZF proteins by linkage of predefined ZF units that recognize specific triplets of DNA . The potential of this technology to alter the transcription of specific genes, to discover new genes, and to induce phenotypes in cells and organisms is now being applied in the areas of molecular therapeutics, pharmacology, biotechnology, and functional genomics.

Biometrics, 2004 Sep, 60(3), 812 - 9
Bayesian variable selection in multinomial probit models to identify molecular signatures of disease stage; Sha N et al.; Here we focus on discrimination problems where the number of predictors substantially exceeds the sample size and we propose a Bayesian variable selection approach to multinomial probit models . Our method makes use of mixture priors and Markov chain Monte Carlo techniques to select sets of variables that differ among the classes . We apply our methodology to a problem in functional genomics using gene expression profiling data . The aim of the analysis is to identify molecular signatures that characterize two different stages of rheumatoid arthritis.

Biometrics, 2004 Sep, 60(3), 774 - 82
Multiple-testing strategy for analyzing cDNA array data on gene expression; Delongchamp RR et al.; An objective of many functional genomics studies is to estimate treatment-induced changes in gene expression . cDNA arrays interrogate each tissue sample for the levels of mRNA for hundreds to tens of thousands of genes, and the use of this technology leads to a multitude of treatment contrasts . By-gene hypotheses tests evaluate the evidence supporting no effect, but selecting a significance level requires dealing with the multitude of comparisons . The p-values from these tests order the genes such that a p-value cutoff divides the genes into two sets . Ideally one set would contain the affected genes and the other would contain the unaffected genes . However, the set of genes selected as affected will have false positives, i.e., genes that are not affected by treatment . Likewise, the other set of genes, selected as unaffected, will contain false negatives, i.e., genes that are affected . A plot of the observed p-values (1 - p) versus their expectation under a uniform {0, 1} distribution allows one to estimate the number of true null hypotheses . With this estimate, the false positive rates and false negative rates associated with any p-value cutoff can be estimated . When computed for a range of cutoffs, these rates summarize the ability of the study to resolve effects . In our work, we are more interested in selecting most of the affected genes rather than protecting against a few false positives . An optimum cutoff, i.e., the best set given the data, depends upon the relative cost of falsely classifying a gene as affected versus the cost of falsely classifying a gene as unaffected . We select the cutoff by a decision-theoretic method analogous to methods developed for receiver operating characteristic curves . In addition, we estimate the false discovery rate and the false nondiscovery rate associated with any cutoff value . Two functional genomics studies that were designed to assess a treatment effect are used to illustrate how the methods allowed the investigators to determine a cutoff to suit their research goals.

Trends Biotechnol, 2004 Sep, 22(9), 440 - 5
Intelligent software for laboratory automation; Whelan KE et al.; The automation of laboratory techniques has greatly increased the number of experiments that can be carried out in the chemical and biological sciences . Until recently, this automation has focused primarily on improving hardware . Here we argue that future advances will concentrate on intelligent software to integrate physical experimentation and results analysis with hypothesis formulation and experiment planning . To illustrate our thesis, we describe the 'Robot Scientist' - the first physically implemented example of such a closed loop system . In the Robot Scientist, experimentation is performed by a laboratory robot, hypotheses concerning the results are generated by machine learning and experiments are allocated and selected by a combination of techniques derived from artificial intelligence research . The performance of the Robot Scientist has been evaluated by a rediscovery task based on yeast functional genomics . The Robot Scientist is proof that the integration of programmable laboratory hardware and intelligent software can be used to develop increasingly automated laboratories.

Mol Cancer Res, 2004 Aug, 2(8), 453 - 63
Molecular characterization of the transition to malignancy in a genetically engineered mouse-based model of ductal carcinoma in situ; Namba R et al.; A transplantable model of human ductal carcinoma in situ that progresses to invasive carcinoma was developed from a genetically engineered mouse (GEM) . Additional lines were established using early mammary premalignant lesions from transgenic MMTV-PyV-mT mice . These lines were verified to be premalignant and transplanted repeatedly to establish stable and predictable properties . Here, we report the first in-depth molecular analysis of neoplastic progression occurring in one premalignant transplantable GEM-derived line . Oligonucleotide microarrays showed that many genes are differentially expressed between the quiescent and prelactating mammary gland and the premalignant GEM outgrowth . In contrast, a small but consistent group of genes was associated with the transformation from premalignancy to tumor . This suggests that the majority of gene expression changes occur during the premalignant transition from normal to premalignancy, whereas many fewer changes occur during the malignant transition from premalignancy to invasive carcinoma . The premalignant transition is associated with several cell cycle-related genes and the up-regulation of oncogenes is associated with various cancers (Ccnd11, Cdk4, Myb, and Ect2) . The changes identified in the malignant transition included genes previously associated with human breast cancer progression . Misregulation of the insulin-like growth factor and transforming growth factor-beta signaling pathways and the stromal-epithelial interaction were implicated . Our results suggest that this transplantable GEM-based model recapitulates human ductal carcinoma in situ at both histologic and molecular levels . With consistent tumor latency and molecular profiles, this model provides an experimental platform that can be used to assess functional genomics and molecular pharmacology and to test promising chemoprevention strategies.

Cancer Cell, 2004 Aug, 6(2), 107 - 8
Toward a functional taxonomy of cancer; Golub TR; Interrogating the genomes of tumor cells with genomic and proteomic methods is becoming a mainstay of modern cancer classification efforts . This notion is brought to a new level by a paper in the July 23 issue of Cell, in which the dynamic responses of leukemia cells to perturbation are cataloged by flow cytometry, and the leukemias classified in terms of their functional responses . This study paves the way for more systematic attempts to bring functional genomics to the study of human cancer.

Plant J, 2004 Sep, 39(5), 682 - 96
Establishing gene function by mutagenesis in Arabidopsis thaliana; Ostergaard L et al.; The nuclear genome of Arabidopsis thaliana was sequenced to near completion a few years ago, and ahead lies the challenge of understanding its meaning and discerning its potential . How many genes are there? What are they? What do they do? Computer algorithms combined with genome array technologies have proven efficient in addressing the first two questions as shown in a recent report (Yamada et al., 2003) . However, assessing the function of every gene in every cell will require years of careful analyses of the phenotypes caused by mutations in each gene . Current progress in generating large numbers of molecular markers and near-saturation insertion mutant collections has immensely facilitated functional genomics studies in Arabidopsis . In this review, we focus on how gene function can be revealed through the analysis of mutants by either forward or reverse genetics . These mutants generally fall into two distinct classes . The first class typically includes point mutations or small deletions derived from chemical or fast neutron mutagenesis whereas the second class includes insertions of transferred-DNA or transposon elements . We describe the current methods that are used to identify the gene corresponding to these mutations, which can then be used as a probe to further dissect its function.

Mol Psychiatry, 2004 Nov, 9(11), 1007 - 29
Candidate genes, pathways and mechanisms for bipolar (manic-depressive) and related disorders: an expanded convergent functional genomics approach; Ogden CA et al.; Identifying genes for bipolar mood disorders through classic genetics has proven difficult . Here, we present a comprehensive convergent approach that translationally integrates brain gene expression data from a relevant pharmacogenomic mouse model (involving treatments with a stimulant--methamphetamine, and a mood stabilizer--valproate), with human data (linkage loci from human genetic studies, changes in postmortem brains from patients), as a bayesian strategy of crossvalidating findings . Topping the list of candidate genes, we have DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) located at 17q12, PENK (preproenkephalin) located at 8q12.1, and TAC1 (tachykinin 1, substance P) located at 7q21.3 . These data suggest that more primitive molecular mechanisms involved in pleasure and pain may have been recruited by evolution to play a role in higher mental functions such as mood . The analysis also revealed other high-probability candidates genes (neurogenesis, neurotrophic, neurotransmitter, signal transduction, circadian, synaptic, and myelin related), pathways and mechanisms of likely importance in pathophysiology.

Methods, 2004 Oct, 34(2), 215 - 24
Antigen-independent selection of intracellular stable antibody frameworks; Auf der Maur A et al.; The intracellular expression of highly specific antibody fragments ("intrabodies") in eukaryotes has a great potential in functional genomics and therapeutics . However, since the intracellular reducing environment prevents formation of the conserved intrachain disulfide bonds, most antibodies do not fold properly and are therefore inactive inside cells . The few antibodies that have been found to function in an intracellular environment and that have been characterized for their biophysical properties have generally shown a high degree of stability and solubility . Thus, for intracellular expression and application, very stable antibody frameworks are needed that can correctly fold even in the absence of disulfide bonds and that do not aggregate . Here, we present and discuss a novel method, named "Quality Control," which allows selection of stable and soluble antibody frameworks in vivo without the requirement or knowledge of antigens . This system is based on the expression of single-chain antibody fragments (scFvs) fused to a selectable marker that can control gene expression and cell growth . The activity of such a selectable marker fused to various scFvs that have been biophysically characterized correlated with the solubility and stability of the scFv moieties . This antigen-independent intrabody selection system was applied to screen scFv libraries for identifying stable and soluble frameworks, which subsequently served as acceptor backbones to construct intrabody libraries by randomization of hypervariable loops.

Neuron, 2004 Aug 19, 43(4), 527 - 37
A functional genomics strategy reveals Rora as a component of the mammalian circadian clock; Sato TK et al.; The mammalian circadian clock plays an integral role in timing rhythmic physiology and behavior, such as locomotor activity, with anticipated daily environmental changes . The master oscillator resides within the suprachiasmatic nucleus (SCN), which can maintain circadian rhythms in the absence of synchronizing light input . Here, we describe a genomics-based approach to identify circadian activators of Bmal1, itself a key transcriptional activator that is necessary for core oscillator function . Using cell-based functional assays, as well as behavioral and molecular analyses, we identified Rora as an activator of Bmal1 transcription within the SCN . Rora is required for normal Bmal1 expression and consolidation of daily locomotor activity and is regulated by the core clock in the SCN . These results suggest that opposing activities of the orphan nuclear receptors Rora and Rev-erb alpha, which represses Bmal1 expression, are important in the maintenance of circadian clock function.

Plant Physiol, 2004 Aug, 135(4), 2098 - 105 Epub 2004 Aug 06.
Identification of syn-pimara-7,15-diene synthase reveals functional clustering of terpene synthases involved in rice phytoalexin/allelochemical biosynthesis; Wilderman PR et al.; Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals . Accordingly, the committed step in biosynthesis of these natural products is catalyzed by the class I terpene synthase that converts syn-copalyl diphosphate to the corresponding polycyclic hydrocarbon intermediate syn-pimara-7,15-diene . Here, a functional genomics approach was utilized to identify a syn-copalyl diphosphate specific 9beta-pimara-7,15-diene synthase (OsDTS2) . To our knowledge, this is the first identified terpene synthase with this particular substrate stereoselectivity and, by comparison with the previously described and closely related ent-copalyl diphosphate specific cassa-12,15-diene synthase (OsDTC1), provides a model system for investigating the enzymatic determinants underlying the observed difference in substrate specificity . Further, OsDTS2 mRNA in leaves is up-regulated by conditions that stimulate phytoalexin biosynthesis but is constitutively expressed in roots, where momilactones are constantly synthesized as allelochemicals . Therefore, transcription of OsDTS2 seems to be an important regulatory point for controlling production of these defensive compounds . Finally, the gene identified here as OsDTS2 has previously been mapped at 14.3 cM on chromosome 4 . The class II terpene synthase producing syn-copalyl diphosphate from the universal diterpenoid precursor geranylgeranyl diphosphate was also mapped to this same region . These genes catalyze sequential cyclization steps in momilactone biosynthesis and seem to have been evolutionarily coupled by physical linkage and resulting cosegregation . Further, the observed correlation between physical proximity and common metabolic function indicates that other such class I and class II terpene synthase gene clusters may similarly catalyze consecutive reactions in shared biosynthetic pathways.

Exp Anim, 2004 Jul, 53(4), 311 - 20
Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus; Abe K et al.; Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics . Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods . This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection . More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes . In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained . A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.

Asia Pac J Clin Nutr . 2004;13(Suppl):S12.
Protein and amino acids: from building blocks to food and health; Bryden WL; Nutrition is an integrative science that brings together many aspects of agriculture, biochemistry, physiology and medicine . In the latter half of the 20th century there has been significant advances in our understanding of the nutrition of all domestic animal species and man; often through comparative studies . This has been particularly true of farm animals, namely poultry, pigs and ruminants, both cattle and sheep . Space and time have necessarily restricted the following review to amino acid and protein nutrition: it seems appropriate to discuss protein at this meeting as this macro-nutrient has been largely forgotten in the fierce debates that have raged over fats and carbohydrates during the last 20 years . Feed accounts for about 60% of the costs of animal production and this fact has justified the quest for efficiency in the use of feed resources . Protein and energy supply contribute 90% of feed costs and much research effort has been aimed at defining the requirements of animals for these nutrients and the corresponding nutrient attributes of feedstuffs . This has largely been achieved empirically in monogastric species but in ruminants, microbial fermentation in the rumen effectively transforms the nutrition of this species . The extensive fermentation of carbohydrate and protein in the rumen clearly established that the rational assessment of the nutritional needs of ruminants can only be made if the quantity of individual nutrients that are available to the animal post ruminally can be quantified . Isotope dilution techniques linked to arteriovenous difference measurements and blood flow data, have made important contributions to knowledge of cellular metabolism and quantitative nutrition of ruminants . Protein and amino acid requirements of monogastrics have been defined for different physiological states and attempts to balance nutrient intake with nutrient requirements have centred around detailed studies of the nutritional attributes of feed ingredients . There has been considerable effort recently to develop systems that allow compilation of the available nutrients, especially available amino acids within feedstuffs . Digestibility values of amino acids in the ileum are used widely as an estimate of availability . Various techniques to quantify the endogenous loss of amino acids during digestion and absorption have been developed and evaluated in an attempt to quantify this important aspect of protein metabolism . Strategies to reduce endogenous losses have been developed . Peptide metabolism in, and uptake from the gut is a significant aspect of intestinal amino acid utilisation . Studies in ruminants have quantified amino acid flows to defined tissues, especially muscle and the mammary gland and the uptake and utilisation of these nutrients by these organs . In addition, studies within the rumen have clearly elucidated the interactions of microbes with dietary feed sources and the production of microbial protein . Delineation of the production, absorption and utilisation of volatile fatty acids has largely come from studies in ruminant animals . The concept of a nutrient requirement which is pertinent only to a unique situation and is essentially a single point on a dose response curve is largely outdated . It is of far greater value to define the entire curve and thus have nutrient responses to different intakes and circumstances . The partitioning of amino acids between different tissues and organs, the effects of different physiological states, stress, disease and toxicological (mycotoxin) insults requires better definition in relation to the efficient use of dietary protein . Greater understanding will increase rates of protein deposition (growth and egg production) and secretion (lactation) in animal products and facilitate the development of functional foods of animal origin . In parallel with the rapid progress of nutrition over the last 50 years, there has been great developments in the area of genetics and molecular biology . The advancement of nutritional science and its application to human and animal nutritionimal nutrition will rely increasingly on molecular technologies . The application of genomic technology to nutrition (nutrigenomics) will allow the identification of modified gene expression in response to nutrients to be established for thousands of mammalian genes . This will enable the development of a much stronger theoretical and molecular bases for nutrient responses . However, in the post-genomic era, functional genomics will need to be coupled with techniques that allow integration with whole-body metabolism will enable the prediction of phenotypic outputs of metabolic pathways and the implications of amino acid fluxes on metabolism and nutrition . Molecular biology is likely to fuel major advances in our understanding of nutritional science . This knowledge and the development of efficient technologies for producing food conveys an optimism that a quality food supply will be sustained for an ever increasing human population in the 21st century.

Gene Ther, 2004 Aug, 11(16), 1241 - 8
Gene therapy progress and prospects . Downregulating gene expression: the impact of RNA interference; Caplen NJ; The control and maintenance of gene expression is critical for cell development and differentiation . Over the last 2 years, our understanding of the role of RNA as a regulator of gene expression has significantly increased . Small RNA molecules are key elements of a machinery that trigger chromosomal modifications, post-transcriptional gene silencing and protein translational blockade depending on the source, the RNA and the nature of the interaction with the target nucleic acid . Currently, the best characterized of this group of RNA-mediated gene regulation pathways is the post-transcriptional gene silencing mechanism known as RNA interference . RNAi is triggered by double-stranded RNA (dsRNA), which induces the formation of a ribonucleoprotein complex that mediates sequence-specific cleavage of the transcript cognate with the input dsRNA . RNAi has been adapted as a functional genomics tool and it has potential as a therapeutic approach . This review will summarize our current understanding of the RNAi mechanism and the various applications of RNAi-based technologies.

Mol Genet Genomics . 2004 Jul 30; {Epub ahead of print}
Mapping Ds insertions in barley using a sequence-based approach; Cooper LD et al.; A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley ( Hordeum vulgaresubsp . vulgare) . The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics . AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation . Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location . The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR . Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population . The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines . In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley . BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences . Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

Yeast, 2004 Jul 15, 21(9), 769 - 79
Phenotypic characterization of glucose repression mutants of Saccharomyces cerevisiae using experiments with 13C-labelled glucose; Raghevendran V et al.; In the field of metabolic engineering and functional genomics, methods for analysis of metabolic fluxes in the cell are attractive as they give an overview of the phenotypic response of the cells at the level of the active metabolic network . This is unlike several other high-throughput experimental techniques, which do not provide information about the integrated response a specific genetic modification has on the cellular function . In this study we have performed phenotypic characterization of several mutants of the yeast Saccharomyces cerevisiae through the use of experiments with (13)C-labelled glucose . Through GC-MS analysis of the (13)C incorporated into the amino acids of cellular proteins, it was possible to obtain quantitative information on the function of the central carbon metabolism in the different mutants . Traditionally, such labelling data have been used to quantify metabolic fluxes through the use of a suitable mathematical model, but here we show that the raw labelling data may also be used directly for phenotypic characterization of different mutant strains . Different glucose derepressed strains investigated employed are the disruption mutants reg1, hxk2, grr1, mig1 and mig1mig2 and the reference strain CEN.PK113-7D . Principal components analysis of the summed fractional labelling data show that deleting the genes HXK2 and GRR1 results in similar phenotype at the fluxome level, with a partial alleviation of glucose repression on the respiratory metabolism . Furthermore, deletion of the genes MIG1, MIG1/MIG2 and REG1 did not result in a significant change in the phenotype at the fluxome level .

Phytochemistry, 2004 Jun, 65(11), 1589 - 607
Mapping of the Physcomitrella patens proteome; Sarnighausen E et al.; The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA . The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics . To further extend the potentialities of this seed-less plant we aimed at exploring the P . patens proteome . Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss . Here we describe the identification of 306 proteins from the protonema of Physcomitrella . Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry . This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.

Phytochemistry, 2004 Jun, 65(11), 1517 - 30
Untangling multi-gene families in plants by integrating proteomics into functional genomics; Sappl PG et al.; The classification and study of gene families is emerging as a constructive tool for fast tracking the elucidation of gene function . A multitude of technologies can be employed to undertake this task including comparative genomics, gene expression studies, sub-cellular localisation studies and proteomic analysis . Here we focus on the growing role of proteomics in untangling gene families in model plant species . Proteomics can specifically identify the products of closely related genes, can determine their abundance, and coupled to affinity chromatography and sub-cellular fractionation studies, it can even provide location within cells and functional assessment of specific proteins . Furthermore global gene expression analysis can then be used to place a specific family member in the context of a cohort of co-expressed genes . In model plants with established reverse genetic resources, such as catalogued T-DNA insertion lines, this gene specific information can also be readily used for a wider assessment of specific protein function or its capacity for compensation through assessing whole plant phenotypes . In combination, these resources can explore partitioning of function between members and assess the level of redundancy within gene families.

Bioessays, 2004 Aug, 26(8), 901 - 15
Functional genomics studied by proteomics; Honore B et al.; The human genome contains about 30,000 genes, each creating several transcripts per gene . Transcript structures and expression are studied by high-throughput transcriptomic techniques using microarrays . Generally, transcripts are not directly operating molecules, but are translated into functional proteins, post-translationally modified by proteolysis, glycosylation, phosphorylation, etc., sometimes with great functional impact . Proteins need to be analyzed by proteomic techniques, less suited for high-throughput . Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), separating thousands of proteins has developed slowly over the past quarter of a century . This technique is now quite reproducible and suitable for differential proteomics, comparing normal and diseased cells/tissues revealing differentially regulated proteins . 2D-PAGE is combined with protein-identification methods, currently mass spectrometry (MS), which has been significantly improved over the last decade . Other proteomic techniques studying protein-protein interactions are now either established or still being developed, such as peptide or protein arrays, phage display, and the yeast two-hybrid system . The strengths and weaknesses of these techniques are discussed.

Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11227 - 32 Epub 2004 Jul 23.
Molecular-timetable methods for detection of body time and rhythm disorders from single-time-point genome-wide expression profiles; Ueda HR et al.; Detection of individual body time (BT) via a single-time-point assay has been a longstanding unfulfilled dream in medicine, because BT information can be exploited to maximize potency and minimize toxicity during drug administration and thus will enable highly optimized medication . To achieve this dream, we created a "molecular timetable" composed of >100 "time-indicating genes," whose gene expression levels can represent internal BT . Here we describe a robust method called the "molecular-timetable method" for BT detection from a single-time-point expression profile . The power of this method is demonstrated by the sensitive and accurate detection of BT and the sensitive diagnosis of rhythm disorders . These results demonstrate the feasibility of BT detection based on single-time-point sampling, suggest the potential for expression-based diagnosis of rhythm disorders, and may translate functional genomics into chronotherapy and personalized medicine.

Mol Ther, 2004 Jul, 10(1), 139 - 49
A single-LTR HIV-1 vector optimized for functional genomics applications; Ma H et al.; The development of high-throughput methods of converting simple expression cassettes into lentiviral vectors and expediting the process of retrieving vector genomes that carry candidate genes from host DNA will facilitate the use of lentiviral vectors as an efficient means of screening novel gene function . To optimize lentiviral vectors for functional genomic applications we have developed a shuttle HIV-1 vector containing a single LTR . Incorporation of a LoxP site and the Sbfl restriction enzyme site into the vector LTR allowed for the rescue of integrated vector genomes into individual bacterial clones . Vector DNA isolated from bacteria was used for a second round of functional screening . Furthermore, we identified a continuous DNA sequence containing all the cis elements required for vector production . Incorporating the isolated sequence into expression cassettes resulted in the generation of HIV-1 vectors in a single cloning step, which imparts a simplified procedure for converting cDNA expression cassettes into single-LTR lentiviral vectors.

Trends Plant Sci, 2004 Apr, 9(4), 159 - 61
Ethanol-inducible gene expression: first step towards a new green revolution?
Tomsett B, Tregova A, Garoosi A, Caddick M.
The introduction of dwarf varieties of cereals was fundamental to the green revolution, but in the post-genomic era, the manipulation of plant morphology could be more sophisticated . A recent publication by Tahar Ait-ali et al . describes the use of the ethanol-inducible transgene expression system to re-examine plant architecture, and the genes that determine it . Their findings have implications for the manipulation of plant height and yield, and demonstrate the efficacy of regulated transgene expression for functional genomics.

Nucleic Acids Res, 2004 Jul 22, 32(13), 3836 - 45 Print 2004.
siRNA-mediated gene silencing: a global genome view; Semizarov D et al.; The task of specific gene knockdown in vitro has been facilitated through the use of short interfering RNA (siRNA), which is now widely used for studying gene function, as well as for identifying and validating new drug targets . We explored the possibility of using siRNA for dissecting cellular pathways by siRNA-mediated gene silencing followed by gene expression profiling and systematic pathway analysis . We used siRNA to eliminate the Rb1 gene in human cells and determined the effects of Rb1 knockdown on the cell by using microarray-based gene expression profiling coupled with quantitative pathway analysis using the GenMapp and MappFinder software . Retinoblastoma protein is one of the key cell cycle regulators, which exerts its function through its interactions with E2F transcription factors . Rb1 knockdown affected G1/S and G2/M transitions of the cell cycle, DNA replication and repair, mitosis, and apoptosis, indicating that siRNA-mediated transient elimination of Rb1 mimics the control of cell cycle through Rb1 dissociation from E2F . Additionally, we observed significant effects on the processes of DNA damage response and epigenetic regulation of gene expression . Analysis of transcription factor binding sites was utilized to distinguish between putative direct targets and genes induced through other mechanisms . Our approach, which combines the use of siRNA-mediated gene silencing, mediated microarray screening and quantitative pathway analysis, can be used in functional genomics to elucidate the role of the target gene in intracellular pathways . The approach also holds significant promise for compound selection in drug discovery.

Methods Mol Biol, 2004, 267, 397 - 414
Enhancer detection and gene trapping as tools for functional genomics in plants; Acosta-Garcia G et al.; Although more than 25,000 genes of Arabidopsis thaliana have been sequenced and mapped, adequate expression or functional information is available for less than 15% of them . In the case of Oryza sativa (rice), about half of more than 55,000 predicted genes have been assigned to a vague functional category on the basis of their sequence, but fewer than 100 have been ascribed a precise, verified function after the identification of a mutant phenotype caused by the molecular disruption of the corresponding gene . Enhancer detection and gene trapping represent insertional mutagenesis strategies that report random expression of many genes and often generate loss-of-function mutations . Several trapping vectors have been designed in a limited number of species, and large-scale enhancer detection and gene trap screens that aim to generate a wide range of spatially and temporally restricted expression patterns have been initiated in both Arabidopsis and rice . These strategies are proving to be essential to the functional annotation of completely sequenced genomes, enabling the analysis of gene function in the context of the entire plant life cycle and substantially expanding our understanding of plant growth and development.

Curr Mol Med, 2004 Aug, 4(5), 519 - 28
Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules; Lobato MN et al.; The use of antibodies in medicine and research depends on their specificity and affinity in the recogniton and binding of individual molecules . However, these applications are limited to the extracellular targets . Advances in antibody engineering has allowed the manipulation of the antibody segments containing the antigen-binding regions and generation of small fragments that can be stably expressed in cells . These entities are called intracellular antibodies or intrabodies and have being successfully applied, mainly in the scFv format, to inhibit the function of intracellular target proteins in specific cellular compartments . As new techniques to select and isolate intrabody fragments have been developed, intrabodies are beginning to be used to interfere with the function of a greater number of relevant disease targets . Just as monoclonal antibodies are opening a new era in human therapeutics, intrabodies promise a new prospective for antibody tools for therapy and research . Their varied mode of action gives intrabodies great potential in different approaches in the treatment of human diseases, as well as in the area of functional genomics for characterisation of novel gene products and subsequent validation as potential drug targets . While techniques for identifying functional intrabodies have improved, there are still many significant problems to be overcome before intrabodies can actually be used in treatment of diseases such as cancer, AIDS or neuro-degenerative disorders.

Neurol Res, 2004 Jul, 26(5), 459 - 80
Phenotypic profiles and functional genomics in Alzheimer's disease and in dementia with a vascular component; Cacabelos R et al.; Alzheimer's disease (AD) and dementia with vascular component (DVC) are the most prevalent forms of dementia . Both clinical entities share many similarities, but they differ in major phenotypic and genotypic profiles as revealed by structural and functional genomics studies . Comparative phenotypic studies have identified significant differences in 25% of more than 100 parametric variables, including anthropometry, cardiovascular function, aortic atherosclerosis, brain atrophy, blood pressure, blood biochemistry, hematology, thyroid function, folate and vitamin B12 levels, brain hemodynamics and lymphocyte markers . The phenotypic profile of patients with DVC differs from that of AD patients in the following: anthropometric values (weight, height); cardiovascular function (ECG, heart rate); blood pressure; lipid metabolism (HDL-CHO, TGs); uric acid metabolism; peripheral calcium homeostasis; liver function (GOT, GPT, GGT); alkaline phosphatase; lactate dehydrogenase; red and white blood cells; regional brain atrophy (left temporal region, inter-hippocampal distance); and left anterior blood flow velocity . Functional genomics studies incorporating APOE-related changes in biological markers extended the difference between AD and DVC up to 57% . Brain perfusion studies show a severe brain hypoperfusion in dementia associated with enlarged age-dependent arterial perfusion times . Structural genomics studies with AD-related genes, including APP, MAPT, APOE, PS1, PS2, A2M, ACE, AGT, cFOS and PRNP genes, demonstrate different genetic profiles in AD and DVC, with an absolute genetic variation rate ranging from 30% to 80%, depending upon genes and genetic clusters . Single gene analysis identifies relative genetic variations ranging from 0% to 5% . The relative polymorphic variation in genetic clusters integrated by two, three or four genes associated with AD ranges from 1% to 3% . The main phenotypic differences between AD and DVC are genotype-dependent, especially in AD, probably indicating that different genomic factors are determinant for the expression of dementia symptoms which might be accelerated or induced by environmental and/or cerebrovascular factors.

J Struct Funct Genomics, 2004, 5(1-2), 69 - 74
Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins; Busso D et al.; For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential . A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system . Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E . coli cell-free extract . The results presented cover 24 open reading frames of unknown function from different micro-organisms . In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25 degrees C and 30 degrees C) . The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody . We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations.

Curr Opin Genet Dev, 2004 Aug, 14(4), 336 - 42
Development through the eyes of functional genomics; Fraser AG et al.; In many of the model organisms used to study development, it is becoming relatively routine to carry out global analyses of gene function . These analyses take many forms, from microarray analyses to the construction of physical interaction maps to the systematic analyses of loss-of-function phenotypes . Such large-scale datasets can be integrated to generate complex gene networks, and we explore how these gene networks can contribute to an understanding of developmental pathways . In particular, we examine how combining large-scale expression experiments and gene networks may move us towards a molecular description of the events of development, embodied in a succession of stage-specific subnetworks sampled from an organism's overall gene network .

Biol Pharm Bull, 2004 Jul, 27(7), 939 - 48
Pharmacogenomics of drug transporters: a new approach to functional analysis of the genetic polymorphisms of ABCB1 (P-glycoprotein/MDR1); Ishikawa T et al.; In the 21st century, emerging genomic technologies (i.e., bioinformatics, functional genomics, and pharmacogenomics) are shifting the paradigm of drug discovery research and improving the strategy of medical care for patients . In order to realize the personalized medicine, it is critically important to understand molecular mechanisms underlying inter-individual differences in the drug response, namely, pharmacological effect vs . side effect . Evidence is now accumulating to strongly suggest that drug transporters are one of the determinant factors governing the pharmacokinetic profile of drugs . Effort has been made to identify genetic variation in drug transporter genes . In particular, genetic variations of the human ABCB1 (P-glycoprotein/MDR1) gene have been most extensively studied . Hitherto more than fifty single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms in the ABCB1 gene have been reported . However, at the present time, information is still limited with respect to the actual effect of those genetic polymorphisms on the function of ABCB1 . In this context, we have undertaken functional analyses of ABCB1 polymorphisms . To quantify the impact of genetic polymorphisms on the substrate specificity of ABCB1, we have developed a high-speed screening system and a new structure-activity relationship (SAR) analysis method . This review addresses functional aspects of the genetic polymorphism of ABCB1 and provides the standard method to evaluate the effect of polymorphisms on the function.

Bioinformatics, 2004 Dec 12, 20(18), 3353 - 62 Epub 2004 Jul 15.
A graph-theoretic approach to testing associations between disparate sources of functional genomics data; Balasubramanian R et al.; MOTIVATION: The last few years have seen the advent of high-throughput technologies to analyze various properties of the transcriptome and proteome of several organisms . The congruency of these different data sources, or lack thereof, can shed light on the mechanisms that govern cellular function . A central challenge for bioinformatics research is to develop a unified framework for combining the multiple sources of functional genomics information and testing associations between them, thus obtaining a robust and integrated view of the underlying biology . RESULTS: We present a graph-theoretic approach to test the significance of the association between multiple disparate sources of functional genomics data by proposing two statistical tests, namely edge permutation and node label permutation tests . We demonstrate the use of the proposed tests by finding significant association between a Gene Ontology-derived predictome and data obtained from mRNA expression and phenotypic experiments for Saccharomyces cerevisiae . Moreover, we employ the graph-theoretic framework to recast a surprising discrepancy presented elsewhere between gene expression and knockout phenotype, using expression data from a different set of experiments . AVAILABILITY: An R software package, GraphAT, containing the data and statistical procedures is available from Bioconductor: http://www.bioconductor.org.

Metab Eng, 2004 Jul, 6(3), 212 - 9
Assaying gene function by growth competition experiment; Merritt J et al.; High-throughput screening and analysis is one of the emerging paradigms in biotechnology . In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts . In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition . Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

Metab Eng, 2004 Jul, 6(3), 204 - 11
Impact of 'ome' analyses on inverse metabolic engineering; Bro C et al.; Genome-wide or large-scale methodologies employed in functional genomics such as DNA sequencing, transcription profiling, proteomics, and metabolite profiling have become important tools in many metabolic engineering strategies . These techniques allow the identification of genetic differences and insight into their cellular effects . In the field of inverse metabolic engineering mapping of differences between strains with different degree of a certain desired phenotype and subsequent identification of factors conferring that phenotype are an essential part . Therefore, the tools of functional genomics in particular have the potential to promote and expand inverse metabolic engineering . Here, we review the use of functional genomics methods in inverse metabolic engineering, examples are presented, and we discuss the identification of targets for metabolic engineering with low fold changes using these techniques.

Nucleic Acids Res . 2004 Jul 12;32(12):e101.
Measuring specific interaction of transcription factor ZmDREB1A with its DNA responsive element at the molecular level; Jiang Y et al.; Specific interactions between transcription factors and DNA responsive elements are of fundamental importance in understanding how genetic regulatory proteins control gene transcription . Here we have developed a new method of using atomic force microscopy (AFM) to quantitatively study the single molecular specific interaction between ZmDREB1A, a transcription factor from maize, and its DNA responsive element, dehydration-responsive element (DRE) with core sequence A/GCCGAC . It was found that ZmDREB1A bound to both DRE ACCGAC and GCCGAC efficiently . The single molecular interaction forces of ZmDREB1A with DRE A/GCCGAC were determined to be 101 +/- 5 and 108 +/- 3 pN, respectively . The point mutation of ZmDREB1A in its DNA-binding domain or single base substitution of the DRE core sequence greatly reduced the binding affinity, demonstrating the high sensitivity of the AFM measurements . AFM is expected to be a simple, quick, sensitive and reliable method that offers valuable information for the characterization of transcription factors and the identification of their potential DNA responsive elements in functional genomics research.

Brief Funct Genomic Proteomic, 2003 Jul, 2(2), 121 - 7
The Drosophila phenotype gap - and how to close it; Dow JA; Functional genomics (the elucidation of gene function in the context of a sequenced genome) depends critically on functional biology . Genetic model organisms have hitherto not attracted much physiological input, however . This skills mismatch, termed the phenotype gap, can be quantified by analysis of the annotations of sequenced genomes . This is illustrated in the context of Drosophila . In this case, it seems as if a shift from developmental biology to transport physiology and metabolism will be required to provide a more balanced skills base for post-genomics.

Brief Funct Genomic Proteomic, 2003 Oct, 2(3), 244 - 53
Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery; Oker-Blom C et al.; Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures . The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell . Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions . This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a versatile system in molecular biology . Surface display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells . In addition, baculovirus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo . Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties . Finally, the viruses can accommodate large insertions of foreign DNA and replicate only in insect cells . Together, these are attributes that are very likely to make them important tools in functional genomics and proteomics.

Brief Funct Genomic Proteomic, 2003 Oct, 2(3), 175 - 84
Functional genomics and proteomics as a foundation for systems biology; Aggarwal K et al.; Developments in high-throughput measurement technologies for biological molecules have created a paradigm shift in modern life science research . The field of systems biology attempts to provide a systems-level understanding by systematically organising the genomic, functional genomic and proteomic data obtained from genetic and environmental perturbations of interest and using the data to build a descriptive and mechanistic model of the biological phenomena . The goal is to build a mathematical framework with some predictive abilities . This review highlights the need for system-level understanding, lists some of the high-throughput measurement tools of importance in systems biology, reviews various types of experimental and computational approaches being used in systems biology research and attempts to address some of the challenges facing this research community.

Brief Funct Genomic Proteomic, 2002 Oct, 1(3), 266 - 77
Regulation of alternative pre-mRNA splicing; Woodley L et al.; The first wave of bioinformatic studies that followed genome and complementary DNA sequencing projects revealed that alternative splicing of messenger RNA precursors (pre-mRNAs) contributes substantially to transcriptome complexity in higher eukaryotes . Together with the realisation of the impact of the process on cell differentiation, development and disease, these studies portray alternative splicing as a fundamental component of gene regulation . Both detailed mechanistic studies and genome-wide analyses will be necessary to unravel the molecular basis for cell type-specific splice site selection . This paper will highlight some recent progress and future challenges for functional genomics and bioinformatics in this rapidly developing area.

Brief Funct Genomic Proteomic, 2002 Oct, 1(3), 239 - 56
Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics?
Sumanas S, Larson JD.
Morpholino phosphorodiamidate anti-sense oligonucleotides (MPOs) have recently emerged as a tool for gene-specific knockdown . MPOs have a great potential for both therapeutic applications and functional genomics . In particular, zebrafish are well suited for gene function studies using MPOs owing to their rapid external development, transparent embryos and the ease of delivery of the intervening MPOs . This paper describes principles of MPO action and the application of MPOs for gene function studies and therapeutics . In the field of functional genomics, the MPO strategy has been most successfully used to study gene function in zebrafish . Over 35 mutations have been successfully phenocopied and over 30 novel gene functions have been analysed in zebrafish using MPOs during the last two years . The essential controls that are required to avoid misinterpretation of experimental data when using MPOs for gene function analysis will also be described.

Brief Funct Genomic Proteomic, 2003 Jan, 1(4), 342 - 55
Recent developments in the engineering of zinc finger proteins; Moore M et al.; Within the last 20 years, the understanding of the biology of the 'classical' or Cys(2)His(2) zinc finger domain has progressed rapidly from the initial identification of the zinc finger as a repetitive zinc-binding motif in transcription factors to its use in biotechnology . The domain is the most abundant DNA-binding motif in the human genome and is a component of many key eukaryotic transcription factors involved in growth and development . Numerous structures now exist for this domain and its mode of action is known in a variety of zinc finger-DNA complexes . Application of this knowledge has led to the development of 'designer' transcription factors where zinc fingers have been engineered to bind desired DNA sequences . Recently, advances have been made in this field that potentially allow the targeting of any DNA site . Consideration of chromatin structure and the use of effector domains in these 'designer' transcription factors have made possible the regulation of a number of endogenous genes . These advances in the customised regulation of genes will be discussed in detail, as well as the potential to use these proteins in functional genomics and gene therapy applications.

BMC Genomics . 2004 Jul 06;5(1):42.
Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray; Renn SC et al.; BACKGROUND: Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology . Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems . As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits . RESULTS: Here we present a microarray constructed with approximately 4500 features, derived from a brain-specific cDNA library for the African cichlid fish Astatotilapia burtoni (Perciformes) . Heterologous hybridization, targeting RNA to an array constructed for a different species, is used for eight different fish species . We quantified the concordance in gene expression profiles across these species (number of genes and fold-changes) . Although most robust when target RNA is derived from closely related species (<10 MA divergence time), our results showed consistent profiles for other closely related taxa (approximately 65 MA divergence time) and, to a lesser extent, even very distantly related species (>200 MA divergence time) . CONCLUSION: This strategy overcomes some of the restrictions imposed on model systems that are of importance for evolutionary and ecological studies, but for which only limited sequence information is available . Our work validates the use of expression profiling for functional genomics within a comparative framework and provides a foundation for the molecular and cellular analysis of complex traits in a wide range of organisms.

Cytogenet Genome Res, 2004, 105(2-4), 375 - 84
Chromosomes and speciation in Mus musculus domesticus; Capanna E et al.; Thirty years after its identification, the model of chromosomal speciation in Mus musculus domesticus is reevaluated using the methods of population biology, molecular cytogenetics and functional genomics . Three main points are considered: (1) the structural predisposition of M . m . domesticus chromosomes to Robertsonian fusion; (2) the impediment of structural heterozygosity to gene flow between populations of mice with karyotypes rearranged by Robertsonian fusion and between them and populations with the standard all-acrocentric 40-chromosome karyotype; (3) the selective advantage of chromosomal novelty, essential for the attainment of homozygosis and the rapid fixation of the new karyotype in the population .

Trends Plant Sci, 2004 Jul, 9(7), 358 - 64
Maize selection passes the century mark: a unique resource for 21st century genomics; Moose SP et al.; The Illinois Long-Term Selection Experiment for grain protein and oil concentration in maize (Zea mays) is the longest continuous genetics experiment in higher plants . A total of 103 cycles of selection have produced nine related populations that exhibit phenotypic extremes for grain composition and a host of correlated traits . The use of functional genomics tools in this unique genetic resource provides exciting opportunities not only to discover the genes that contribute to phenotypic differences but also to investigate issues such as the response of plant genomes to artificial selection, the genetic architecture of quantitative traits and the source of continued genetic variation within domesticated crop genomes.

Med Clin (Barc), 2004 Jun 19, 123(3), 104 - 17
{Genomics of body weight regulation: unraveling the molecular mechanisms predisposing to obesity}; Bastarrachea RA et al.; Obesity has become a worldwide public health problem which affects millions of people . Substantial progress has been made in elucidating the pathogenesis of energy homeostasis over the past few years . The fact that obesity is under strong genetic control has been well established . Twin, adoption and family studies have shown that genetic factors play a significant role in the pathogenesis of obesity . Human monogenic obesity is rare in large populations . The most common form of obesity is considered to be a polygenic disorder . New treatments are currently required for this common metabolic disease and type 2 diabetes . The identification of physiological and biochemical factors that underlie the metabolic disturbances observed in obesity is a key step in developing better therapeutic outcomes . The discovery of new genes and pathways involved in the pathogenesis of such a disease is critical to this process . However, identification of genes that contribute to the risk of developing the disease represents a significant challenge since obesity is a complex disease with many genetic and environmental causes . A number of diverse approaches have been used to discover and validate potential new genes for obesity . To date, DNA-based approaches using candidate genes and genome-wide linkage analysis have not had a great success in identifying genomic regions or genes involved in the development of these diseases . Recent advances in the ability to evaluate linkage analysis data from large family pedigrees (using variance components-based linkage analysis) show great promise in robustly identifying genomic regions associated with the development of obesity . Studying rare mutations in humans and animal models has provided fundamental insight into a complex physiological process, and has complemented population-based studies that seek to reveal primary causes . Remarkable progress has been made in both fronts and the pace of advance is likely to accelerate as functional genomics and the human genome project expand and mature . Approaches based on Mendelian and quantitative genetics may well converge, and ultimately lead to more rational and selective therapies.

Dev Biol, 2004 Jul 15, 271(2), 498 - 516
Defining a large set of full-length clones from a Xenopus tropicalis EST project; Gilchrist MJ et al.; Amphibian embryos from the genus Xenopus are among the best species for understanding early vertebrate development and for studying basic cell biological processes . Xenopus, and in particular the diploid Xenopus tropicalis, is also ideal for functional genomics . Understanding the behavior of genes in this accessible model system will have a significant and beneficial impact on the understanding of similar genes in other vertebrate systems . Here we describe the analysis of 219,270 X . tropicalis expressed sequence tags (ESTs) from four early developmental stages . From these, we have deduced a set of unique expressed sequences comprising approximately 20,000 clusters and 16,000 singletons . Furthermore, we developed a computational method to identify clones that contain the complete coding sequence and describe the creation for the first time of a set of approximately 7000 such clones, the full-length (FL) clone set . The entire EST set is cloned in a eukaryotic expression vector and is flanked by bacteriophage promoters for in vitro transcription, allowing functional experiments to be carried out without further subcloning . We have created a publicly available database containing the FL clone set and related clustering data and we make the FL clone set publicly available as a resource to accelerate the process of gene discovery and function in this model organism . The creation of the unique set of expressed sequences and the FL clone set pave the way toward a large-scale systematic analysis of gene sequence, gene expression, and gene function in this vertebrate species.

Mol Genet Genomics, 2004 Jul, 271(6), 639 - 50 Epub 2004 Jun 18.
Establishment of an enhancer trap system with Ds and GUS for functional genomics in rice; Ito Y et al.; To develop an efficient means of enhancer trapping, a two-element system employing Ds and an Ac transposase (AcTPase) gene was tested in rice . We generated 263 transgenic rice plants, each of which harboured the maize transposable element Ds together with a GUS coding sequence under the control of a minimal promoter (Ds-GUS), and a gene that confers resistance to the herbicide chlorsulfuron . Among the 263 lines generated, 42 were shown to have a single copy of the Ds-GUS element . Four single-copy lines were crossed with each of six transgenic plants that carried the AcTPase gene . Excision of the Ds-GUS in leaves of F1 plants was detected in eight combinations out of seventeen examined . The frequency of transposition of Ds-GUS in germ cells in the F1 plants was examined using 10,524 F2 plants, and 675 (6%) were judged to be transposants . Their frequencies differed among F1 plants depending on the AcTPase x Ds-GUS cross considered, and also among panicles on the same F1 plant . This suggests that Ds-GUS tends to transpose during panicle development . Southern analysis with a GUS probe showed different band patterns among transposants derived from different panicles . Therefore, the transposants derived from different panicles must have arisen independently . Transposants showing tissue-specific GUS activities were obtained, and enhancers thus trapped by the Ds-GUS element were identified . These results demonstrate that the system is suitable for the isolation of large numbers of independent Ds-GUS transposants, and for the identification of various tissue-specific enhancers . The Ds-GUS lines generated in this study offer a potentially powerful tool for studies on the functional genomics of rice.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W465 - 70
Expression Profiler: next generation--an online platform for analysis of microarray data; Kapushesky M et al.; Expression Profiler (EP, is a web-based platform for microarray gene expression and other functional genomics-related data analysis . The new architecture, Expression Profiler: next generation (EP:NG), modularizes the original design and allows individual analysis-task-related components to be developed by different groups and yet still seamlessly to work together and share the same user interface look and feel . Data analysis components for gene expression data preprocessing, missing value imputation, filtering, clustering methods, visualization, significant gene finding, between group analysis and other statistical components are available from the EBI (European Bioinformatics Institute) web site . The web-based design of Expression Profiler supports data sharing and collaborative analysis in a secure environment . Developed tools are integrated with the microarray gene expression database ArrayExpress and form the exploratory analytical front-end to those data . EP:NG is an open-source project, encouraging broad distribution and further extensions from the scientific community.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W124 - 9
siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference; Naito Y et al.; siDirect is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi) . Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity . Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook . The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing.

Biotechniques, 2004 Jun, 36(6), 1018 - 22, 1024, 1026-9
Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection; Faix PH et al.; Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature . However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires . Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage . The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs . The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display . The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection . Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently . The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.

CNS Spectr, 2002 Mar, 7(3), 215 - 26
Finding genes for bipolar disorder in the functional genomics era: from convergent functional genomics to phenomics and back; Kelsoe JR et al.; Psychiatric genetics, while promising to unravel the mechanisms of psychiatric disorders, has proven to be a challenging field . Psychiatric disorders, like other common genetic traits, are complex and heterogeneous . Psychiatric genetics has also suffered from a lack of quantifiable, biology-based phenotypes . However, the field is currently at an opportune moment . The work of various investigators is on the verge of paying rich dividends . Efforts at positional cloning are being greatly accelerated by the fruits of the Human Genome Project . New tools of functional genomics, such as expression profiling and proteomics, are being applied to animal models . These two methods can complement each other in an approach we have termed convergent functional genomics . Lastly, improvements in the measurement of biologically distinct endophenotypes--or phenomic--will lead to a better understanding of the mapping of genes to phenotypes in both animal and human systems.

Mech Dev, 2004 Jul, 121(7-8), 971 - 6
Large-scale expression screening by automated whole-mount in situ hybridization; Quiring R et al.; Gene expression profiling is an important component of functional genomics . We present a time and cost efficient high-throughput whole-mount in situ technique to perform a large-scale gene expression analysis in medaka fish (Oryzias latipes) embryos . Medaka is a model system ideally suited for the study of molecular genetics of vertebrate development . Random cDNA clones from an arrayed stage 20 medaka plasmid library were analyzed by whole-mount in situ hybridization on embryos of three representative stages of medaka development . cDNA inserts were colony PCR amplified in a 384-format . The PCR products were used to generate over 2000 antisense RNA digoxigenin probes in a high-throughput process . Whole-mount in situ hybridization was carried out in a robot and a broad range of expression patterns was observed . Partial cDNA sequences and expression patterns were documented with BLAST results, cluster analysis, images and descriptions, respectively; collectively this information was entered into a web-based database, "MEPD" , that is publicly accessible.

RNA, 2004 Jul, 10(7), 1019 - 25
Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo; Galy B et al.; We report the targeted mutagenesis of the murine iron regulatory protein (IRP)-1 and IRP2 genes, respectively, with a classical gene trap construct . Insertion of the targeting cassette into the second intron of either gene by homologous recombination interrupts their open reading frames near the N termini . Mice that are homozygous for the correctly modified IRP1 or IRP2 alleles, respectively, display a strong reduction (90%, IRP1(-/-)) or nondetectable levels (IRP2(-/-)) of the targeted proteins . Interestingly, the pre-mRNAs transcribed from the identical targeting cassettes are processed differently within the two different contexts . Detailed analysis of the respective products identifies the choice of alternative splice and 3' end processing sites in the same tissues in vivo . We discuss the implications for the understanding of RNA processing and for targeting strategies for functional genomics in the mouse.

Plant Physiol, 2004 Jun, 135(2), 622 - 9
Arabidopsis to rice . Applying knowledge from a weed to enhance our understanding of a crop species; Rensink WA et al.; Although Arabidopsis is well established as the premiere model species in plant biology, rice (Oryza sativa) is moving up fast as the second-best model organism . In addition to the availability of large sets of genetic, molecular, and genomic resources, two features make rice attractive as a model species: it represents the taxonomically distinct monocots and is a crop species . Plant structural genomics was pioneered on a genome-scale in Arabidopsis and the lessons learned from these efforts were not lost on rice . Indeed, the sequence and annotation of the rice genome has been greatly accelerated by method improvements made in Arabidopsis . For example, the value of full-length cDNA clones and deep expressed sequence tag resources, obtained in Arabidopsis primarily after release of the complete genome, has been recognized by the rice genomics community . For rice >250,000 expressed sequence tags and 28,000 full-length cDNA sequences are available prior to the completion of the genome sequence . With respect to tools for Arabidopsis functional genomics, deep sequence-tagged lines, inexpensive spotted oligonucleotide arrays, and a near-complete whole genome Affymetrix array are publicly available . The development of similar functional genomics resources for rice is in progress that for the most part has been more streamlined based on lessons learned from Arabidopsis . Genomic resource development has been essential to set the stage for hypothesis-driven research, and Arabidopsis continues to provide paradigms for testing in rice to assess function across taxonomic divisions and in a crop species.

Plant Cell, 2004 Jul, 16(7), 1679 - 91 Epub 2004 Jun 18.
Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution; Blanc G et al.; To study the evolutionary effects of polyploidy on plant gene functions, we analyzed functional genomics data for a large number of duplicated gene pairs formed by ancient polyploidy events in Arabidopsis thaliana . Genes retained in duplicate are not distributed evenly among Gene Ontology or Munich Information Center for Protein Sequences functional categories, which indicates a nonrandom process of gene loss . Genes involved in signal transduction and transcription have been preferentially retained, and those involved in DNA repair have been preferentially lost . Although the two members of each gene pair must originally have had identical transcription profiles, less than half of the pairs formed by the most recent polyploidy event still retain significantly correlated profiles . We identified several cases where groups of duplicated gene pairs have diverged in concert, forming two parallel networks, each containing one member of each gene pair . In these cases, the expression of each gene is strongly correlated with the other nonhomologous genes in its network but poorly correlated with its paralog in the other network . We also find that the rate of protein sequence evolution has been significantly asymmetric in >20% of duplicate pairs . Together, these results suggest that functional diversification of the surviving duplicated genes is a major feature of the long-term evolution of polyploids.

Tuberculosis (Edinb), 2004, 84(3-4), 131 - 7
Microarrays for Mycobacterium tuberculosis; Butcher PD; This special microarray issue of Tuberculosis recognises the important contributions of M . tuberculosis whole genome DNA microarrays to tuberculosis research by bringing together a range of papers that address M . tuberculosis physiology, host-pathogen interactions, mechanisms of drug action, in vitro and in vivo gene expression, host responses, comparative genomics and functional analysis of particular genes . A number of complete datasets of M . tuberculosis mRNA expression levels are provided to facilitate multiple cross-condition comparison . Microarrays represent one of the new functional genomics technologies exploiting genome sequence information that will bring us closer to realising the scientific and moral imperatives of better vaccines, diagnostics and new drugs for the control of tuberculosis throughout the world.

J Exp Biol, 2004 Jul, 207(Pt 15), 2551 - 63
Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics; Osta MA et al.; The resurgence of malaria is at least partly attributed to the absence of an effective vaccine, parasite resistance to antimalarial drugs and resistance to insecticides of the anopheline mosquito vectors . Novel strategies are needed to combat the disease on three fronts: protection (vaccines), prophylaxis/treatment (antimalarial drugs) and transmission blocking . The latter entails either killing the mosquitoes (insecticides), preventing mosquito biting (bednets and repellents), blocking parasite development in the vector (transmission blocking vaccines), genetic manipulation or chemical incapacitation of the vector . During the past decade, mosquito research has been energized by several breakthroughs, including the successful transformation of anopheline vectors, analysis of gene function by RNAi, genome-wide expression profiling using DNA microarrays and, most importantly, sequencing of the Anopheles gambiae genome . These breakthroughs helped unravel some of the mechanisms underlying the dynamic interactions between the parasite and the vector and shed light on the mosquito innate immune system as a set of potential targets to block parasite development . In this context, putative pattern recognition receptors of the mosquito that act as positive and negative regulators of parasite development have been identified recently . Characterizing these molecules and others of similar function, and identifying their ligands on the parasite surface, will provide clues on the nature of the interactions that define an efficient parasite-vector system and open up unprecedented opportunities to control the vectorial capacity of anopheline mosquitoes.

Plant J, 2004 Jul, 39(1), 98 - 112
Characterization of C-terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class A Hsfs with AHA and NES motifs essential for activator function and intracellular localization; Kotak S et al.; Heat stress transcription factors (Hsfs) are the major regulators of the plant heat stress (hs) response . Sequencing of the Arabidopsis genome revealed the existence of 21 open-reading frames (ORFs) encoding putative Hsfs assigned to classes A-C . Here we present results of a functional genomics approach to the Arabidopsis Hsf family focused on the analysis of their C-terminal domains (CTDs) harboring conserved modules for their function as transcription factors and their intracellular localization . Using reporter assays in tobacco protoplasts and yeast as well as glutathione-S-transferase (GST) pull-down assays, we demonstrate that short peptide motifs enriched with aromatic and large hydrophobic amino acid (aa) residues embedded in an acidic surrounding (AHA motifs) are essential for transcriptional activity of class A Hsfs . In contrast to this, class B and C Hsfs lack AHA motifs and have no activator function on their own . We also provide evidence for the function of a leucine (Leu)-rich region centered around a conserved QMGPhiL motif at the very C-terminus as a nuclear export signal (NES) of class A Hsfs . Sequence comparison indicates that the combination of a C-terminal AHA motif with the consensus sequence FWxxF/L,F/I/L as well as the adjacent NES represents a signature domain for plant class A Hsfs, which allowed to identify more than 60 new Hsfs from the expressed sequence tag (EST) database.

Immunol Rev, 2004 Apr, 198, 127 - 48
Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae; Christophides GK et al.; In much of Africa, the mosquito Anopheles gambiae is the major vector of human malaria, a devastating infectious disease caused by Plasmodium parasites . Vector and parasite interact at multiple stages and locations, and the nature and effectiveness of this reciprocal interaction determines the success of transmission . Many of the interactions engage the mosquito's innate immunity, a primitive but very effective defense system . In some cases, the mosquito kills the parasite, thus blocking the transmission cycle . However, not all interactions are antagonistic; some represent immune evasion . The sequence of the A . gambiae genome revealed numerous potential components of the innate immune system, and it established that they evolve rapidly, as summarized in the present review . Their rapid evolution by gene family expansion diversification as well as the prevalence of haplotype alleles in the best-studied families may reflect selective adaptation of the immune system to the exigencies of multiple immune challenges in a variety of ecologic niches . As a follow-up to the comparative genomic analysis, the development of functional genomic methodologies has provided novel opportunities for understanding the immune system and the nature of its interactions with the parasite . In this context, identification of both Plasmodium antagonists and protectors in the mosquito represents a significant conceptual advance . In addition to providing fundamental understanding of primitive immune systems, studies of mosquito interactions with the parasite open unprecedented opportunities for novel interventions against malaria transmission . The generation of transgenic mosquitoes that resist malaria infection in the wild and the development of antimalarial 'smart sprays' capable of disrupting interactions that are protective of the parasite, or reinforcing others that are antagonistic, represent technical challenges but also immense opportunities for improvement of global health.

Curr Opin Mol Ther, 2004 Apr, 6(2), 129 - 35
RNAi in functional genomics; Scherr M et al.; There has been a lack of powerful tools for systematic analysis of mammalian gene function, but RNA interference (RNAi) may now provide such a strategy . Stable transcription of RNAi triggers from suitable expression cassettes integrated into the host cell genome by viral gene transfer can induce long-term and heritable gene silencing in mammalian cells . However, the use of RNAi as a genetic tool is limited by difficulties in identifying efficient RNAi triggers, the problem of effective delivery and off-target effects, as well as potential genotoxic side effects of viral gene transfer strategies . Recent insights into the molecular mechanisms of silencing processes mediated by either siRNA or miRNA will allow further optimization of RNAi triggers as genetic tools.

Pol Merkuriusz Lek, 2004 Mar, 16(93), 205 - 7
{Functional genomics and its impact on the future medicine}; Klysik J; The sequencing of the human genome was completed in 2003 . These data will affect all aspects of biological and medical sciences . Since almost all the genes of the genome are known, the diagnosis and therapy of the future is expected to evolve towards more personalized and efficient methods . Before these medical advancements become applicable, functional genomics must successfully determine the cellular role all the genes in the genome . Comprehensive functional studies will require suitable animal models . Although many mammalian species are used in biological and medical experiments, the mouse emerges as a key model organism in studies of human genes . The power of the mouse model system comes from its extensive physiological and pathological similarities to humans and from technological advancements offered by mouse genetics . Recently, a project called ENCODE was launched to accelerate this monumental effort of gene analysis . Once gene analysis is completed, the face of medicine will change markedly . The greatest impact is expected on the front of individual diagnosis of disease genes and personalized treatments of patients.

Phytochemistry, 2004 May, 65(9), 1223 - 9
Identification of Vitis vinifera (-)-alpha-terpineol synthase by in silico screening of full-length cDNA ESTs and functional characterization of recombinant terpene synthase; Martin DM et al.; The flavour and aroma of certain Vitis vinifera grape varieties is dominated by volatile terpenes and small volatile aldehydes . Monoterpenes contribute to the final grape and wine aroma and flavour in form of free volatiles and as glycoside conjugates of monoterpene alcohols . Typical monoterpenol components of the cultivar Gewurztraminer and other aroma-rich grape varieties are linalool, geraniol, nerol, citronellol, and alpha-terpineol . In a functional genomics effort to identify genes for the formation of monoterpene alcohols in V . vinifera, a database of full-length cDNA sequences was screened in silico and yielded two clones for putative monoterpene synthases . The gene products were functionally characterized by expression in Escherichia coli, in vitro enzyme assay and gas chromatography-mass spectrometry (GC-MS) product identification as multi-product (-)-alpha-terpineol synthases.

Med Princ Pract, 2004 Jul-Aug, 13(4), 177 - 84
Defining the mandate of tuberculosis research in a postgenomic era; Chakhaiyar P et al.; The identification of Mycobacterium tuberculosis by Robert Koch in 1882 as the causative agent of tuberculosis, the release of the drug rifampicin in 1970 and the sequencing of the M . tuberculosis genome in 1998 are three major events that have revolutionized tuberculosis research . In spite of these breakthroughs, the continued status of tuberculosis as the largest killer amongst infectious diseases is an issue of major concern . Although directly observed short course chemotherapy exists to treat the disease, the emergence of drug-resistant strains has severely threatened the efficacy of the treatment . The recent sequencing of the M . tuberculosis genome holds promise for the development of new vaccines and the design of new drugs . This is all the more possible when the information from the genome sequence is combined with proteomics and structural and functional genomics . Such an integrated approach has led to the birth of a new field of research christened 'postgenomics' that holds substantial promise for the identification of novel drug targets and the potential to aid the development of new chemotherapeutic compounds to treat tuberculosis . The challenge before the scientific community therefore lies in elucidation of the wealth of information provided by the genome sequence and its translation into the design of novel therapies for the disease . All the major developments in the field of tuberculosis research after the sequencing of the M . tuberculosis genome will be discussed in this review .

Mol Cell Proteomics, 2004 Aug, 3(8), 834 - 40 Epub 2004 Jun 04.
High-throughput functional genomics identifies genes that ameliorate toxicity due to oxidative stress in neuronal HT-22 cells: GFPT2 protects cells against peroxide; Zitzler J et al.; We describe a novel genetic screen that is performed by transfecting every individual clone of an expression clone collection into a separate population of cells in a high-throughput mode . We combined high-throughput functional genomics with experimental validation to discover human genes that ameliorate cytotoxic responses of neuronal HT-22 cells upon exposure to oxidative stress . A collection of 5,000 human cDNAs in mammalian expression vectors were individually transfected into HT-22 cells, which were then exposed to H(2)O(2) . Five genes were found that are known to be involved in pathways of detoxification of peroxide (catalase, glutathione peroxidase-1, peroxiredoxin-1, peroxiredoxin-5, and nuclear factor erythroid-derived 2-like 2) . The presence of those genes in our "hit list" validates our screening platform . In addition, a set of candidate genes was found that has not been previously described as involved in detoxification of peroxide . One of these genes, which was consistently found to reduce H(2)O(2) -induced toxicity in HT-22, was GFPT2 . This gene is expressed at significant levels in the central nervous system (CNS) and encodes glutamine-fructose-6-phosphate transaminase (GFPT) 2, a rate-limiting enzyme in hexosamine biosynthesis . GFPT has recently also been shown to ameliorate the toxicity of methylmercury in Saccharomyces cerevisiae . Methylmercury causes neuronal cell death in part by protein modification as well as enhancing the production of reactive oxygen species (ROS) . The protective effect of GFPT2 against H(2)O(2) toxicity in neuronal HT-22 cells may be similar to its protection against methylmercury in yeast . Thus, GFPT appears to be conserved among yeast and men as a critical target of methylmercury and ROS-induced cytotoxicity.

Mol Cells, 2004 Apr 30, 17(2), 377 - 80
A method of high frequency virus-induced gene silencing in chili pepper (Capsicum annuum L . cv . Bukang); Chung E et al.; Using a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system, expression of phytogene desaturase (PDS) and ribulose-1,5-bisphosphate carboxylase small-subuit (rbcS) genes was suppressed in Nicotiana benthamiana and pepper plants (Capsicum annuum L . cv . Bukang) . The silenced phenotypes of pale yellow (rbcS), and photobleached leaves (PDS), were invariably obvious 2 weeks after inoculation with the TRV-based vector . In a parallel experiment, the same set of genes was silenced in N . benthamiana and yielded identical phenotypes to pepper 1 week after inoculation . Northern blot analyses showed that the endogenous levels of CarbcS and CaPDS transcripts were dramatically reduced in the silenced leaf tissues . These observations confirm that the silenced phenotype is closely correlated with the pattern of tissue expression . To our knowledge, this is the first high frequency VIGS method in pepper plants . It should provide a tool for large-scale gene silencing studies in pepper functional genomics.

Ann Acad Med Singapore, 2004 May, 33(3), 392 - 7
Refining clinical practice: transforming science research into the art of medicine; Chee YC; This article traces the development of modern day breast cancer treatment from 1896 when observations were made on the positive response of patients to oophorectomy . The oestrogen receptor was defined and tamoxifen was discovered to be an effective anti-oestrogen . The genes related to breast cancer, BRCA1 and BRCA2, were found to confer high risks of breast and ovarian cancer on women with these genes . The application of functional genomics to breast tumours would result in a more accurate classification of cancers and hopefully more specific therapy and better clinical outcomes . An important off-shoot of anti-oestrogen research has resulted in a new class of drugs called selective oestrogen receptor modulators for treatment of osteoporosis and dyslipidemia.

Genome Res, 2004 Jun, 14(6), 1130 - 6
Automatic identification of subcellular phenotypes on human cell arrays; Conrad C et al.; Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization . Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments . Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images . However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes . Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods . Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins . Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells .

J Mol Med, 2004 Apr, 82(4), 214 - 22
Human genome research in China; Qiang B; Significant progress in human genome research has been made in China since 1994 . This review aims to give a brief and incomplete introduction to the major research institutions and their achievements in human genome sequencing and functional genomics in medicine, with emphasis on the "1% Sequencing Project", the generation of single nucleotide polymorphism and haplotype maps of the human genome, disease gene identification, and the molecular characterization of leukemia and other diseases . Chinese efforts towards the sequencing of pathogenic microbial genomes and of the rice (Oryza sativa ssp . Indica) genome are also described .

Theor Appl Genet, 2004 Aug, 109(3), 451 - 63 Epub 2004 May 27.
Large-scale heterospecific segregation distortion in Populus revealed by a dense genetic map; Yin TM et al.; We report the most complete genetic map to have been constructed for the genus Populus . This map includes 544 markers mapped onto 19 linkage groups, equivalent to the Populus chromosome number, with all markers displaying internally consistent linkage patterns . We estimate the genome length to be between 2,300 and 2,500 cM, based both on the observed number of crossovers in the maternal haplotypes, as well as the total observed map length . Genome coverage was estimated to be greater than 99.9% at 20 cM per marker . We did not detect obvious recombination repression in the maternal tree (a hybrid of Populus trichocarpa Hooker x P . deltoides Marsh.) compared to the paternal tree (pure P . deltoides) . Finally, most markers exhibiting segregation distortion were derived from the donor parent in this backcross, and generally occurred in large contiguous blocks on two linkage groups . We hypothesize that divergent selection has occurred on chromosomal scales among the parental species used to create this pedigree, and explore the evolutionary implications of this observation . This genetic linkage map provides the most comprehensive view of the Populus genome reported to date and will prove invaluable for future inquiries into the structural and functional genomics, evolutionary biology, and genetic improvement of this ecologically important model species .

Nat Genet, 2004 Jun, 36(6), 559 - 64
A probabilistic view of gene function; Fraser AG et al.; Cells are controlled by the complex and dynamic actions of thousands of genes . With the sequencing of many genomes, the key problem has shifted from identifying genes to knowing what the genes do; we need a framework for expressing that knowledge . Even the most rigorous attempts to construct ontological frameworks describing gene function (e.g., the Gene Ontology project) ultimately rely on manual curation and are thus labor-intensive and subjective . But an alternative exists: the field of functional genomics is piecing together networks of gene interactions, and although these data are currently incomplete and error-prone, they provide a glimpse of a new, probabilistic view of gene function . We outline such a framework, which revolves around a statistical description of gene interactions derived from large, systematically compiled data sets . In this probabilistic view, pleiotropy is implicit, all data have errors and the definition of gene function is an iterative process that ultimately converges on the correct functions . The relationships between the genes are defined by the data, not by hand . Even this comprehensive view fails to capture key aspects of gene function, not least their dynamics in time and space, showing that there are limitations to the model that must ultimately be addressed.

FEBS Lett, 2004 Jun 1, 567(1), 55 - 62
Regulating eukaryotic gene expression with aptamers; Toulme JJ et al.; Aptamers are RNA or DNA oligonucleotides identified within a randomly synthesized library, through an in vitro selection procedure . The selected candidates display a pre-determined property of interest with respect to a given target . Successful selection has been carried out against targets ranging from small (amino acids, antibiotics) to macro-molecules (proteins, nucleic acids) . They generally show an affinity in the nanomolar range and a high specificity of target recognition . Interestingly, aptamers selected against purified targets in the test tube retain their properties within cells . RNA aptamers can be generated in situ from an appropriate DNA construct or delivered as nuclease-resistant oligonucleotide analogues . For example, aptamers recognizing RNA structure through loop-loop interactions modulate the trans-activation of in vitro transcription mediated by the TAR RNA element of human immunodeficiency virus type 1 . Consequently, they constitute both exquisite tools for functional genomics analysis and promising prototypes of therapeutic agents . Natural aptameric motifs have been identified within mRNA sequences, which upon binding to a metabolite control the expression of the encoded gene, which is generally involved in the biosynthesis of this particular metabolite.

Gene Ther, 2004 Aug, 11(15), 1195 - 204
Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells; Inoue R et al.; The expression of genes from genomic loci can be relatively complex, utilizing exonic, intronic and flanking sequences to regulate tissue and developmental specificity . Infectious bacterial artificial chromosomes (iBACs) have been shown to deliver and express large genomic loci (up to 135 kb) into primary cells for functional analyses . The delivery of large genomic DNA inserts allows the expression of complex loci and of multiple splice variants . Herein, we demonstrate for the first time that an iBAC will deliver and correctly express in human glioma cells the entire CDKN2A/CDKN2B genomic region, which encodes for at least three important cell-cycle regulatory proteins (p16(INK4a), p14(ARF) and p15(INK4b)) . Two of these proteins are expressed from overlapping genes, utilizing alternative splicing and promoter usage . The delivered locus expresses each gene at physiological levels and cellular responses (apoptosis versus growth arrest) occur dependent on cellular p53 status, as expected . The work further demonstrates the potential of the iBAC system for the delivery of genomic loci whose expression is mediated by complex splicing and promoter usage both for gene therapy applications and functional genomics studies.

Mol Psychiatry, 2004 Jun, 9(6), 550 - 6
Hormonal symphony: steroid orchestration of gene modules for sociosexual behaviors; Mong JA et al.; Genes induced by estrogens in the mammalian forebrain influence a variety of neural functions . Among them, reproductive behavior mechanisms are very well understood . Their functional genomics provide a theoretical paradigm for linking genes to neural circuits to behavior . We propose that estrogen-induced genes are organized in modules: Growth of hypothalamic neurons; Amplification of the estrogen effect by progesterone; Preparative behaviors; Permissive actions on sex behavior circuitry; and Synchronization of mating behavior with ovulation . These modules may represent mechanistic routes for CNS management of successful reproduction . Moreover, new microarray results add estrogen-dependent genes, including some expressed in glia, suggesting possible hormone-dependent neuronal/glial coordination.

Brief Funct Genomic Proteomic, 2004 Apr, 3(1), 26 - 34
Caenorhabditis elegans functional genomics: omic resonance; Astin J et al.; The nematode Caenorhabditis elegans is widely used as a model organism for studying many fundamental aspects of development and cell biology, including processes underlying human disease . The genome of C . elegans encodes over 19,000 protein-coding genes and hundreds of non-coding RNAs . The availability of whole genome sequence has facilitated the development of high throughput techniques for elucidating the function of individual genes and gene products . Furthermore, attempts can now be made to integrate these substantial functional genomics data collections and to understand at a global level how the flow of genomic information that is at the core of the central dogma leads to the development of a multicellular organism.

Brief Funct Genomic Proteomic, 2004 Apr, 3(1), 15 - 25
Plenty more fish in the sea: comparative and functional genomics using teleost models; Elgar G; Biology has collaborated with evolution to create an enormous repertoire of animal variation . This in turn has provided experimental biologists with models that can be used in the lab to simulate more complex systems . Amongst the organisms that have been used in this way are fish, where a large number of species have been utilised in a variety of different ways . Fish possess the smallest genomes of any vertebrate, making them ideal as models for genome analysis and gene discovery . Fish are also easy to maintain in a laboratory environment and can be bred easily . Fish often have well-defined physiology and respond well to many experimental procedures . Finally, fish are of great economic importance in their own right, as one of the world's largest sources of protein . In this review, the relationship between fish species is examined along with the role of different fish models in a wide range of biological disciplines.

Biochem Biophys Res Commun, 2004 Jun 18, 319(1), 264 - 74
Improved and automated prediction of effective siRNA; Chalk AM et al.; Short interfering RNAs are used in functional genomics studies to knockdown a single gene in a reversible manner . The results of siRNA experiments are highly dependent on the choice of siRNA sequence . In order to evaluate siRNA design rules, we collected a database of 398 siRNAs of known efficacy from 92 genes . We used this database to evaluate previously proposed rules from smaller datasets, and to find a new set of rules that are optimal for the entire database . We also trained a regression tree with full cross-validation . It was however difficult to obtain the same precision as methods previously tested on small datasets from one or two genes . We show that those methods are overfitting as they work poorly on independent validation datasets from multiple genes . Our new design rules can predict siRNAs with efficacy >/= 50% in 91% of cases, and with efficacy >/=90% in 52% of cases, which is more than a twofold improvement over random selection . Software for designing siRNAs is available online via a web server at or as a standalone version for high-throughput applications.

Biochem Biophys Res Commun, 2004 Jun 18, 319(1), 256 - 63
Many commonly used siRNAs risk off-target activity; Snove O Jr et al.; Using small interfering RNA (siRNA) to induce sequence specific gene silencing is fast becoming a standard tool in functional genomics . As siRNAs in some cases tolerate mismatches with the mRNA target, knockdown of genes other than the intended target could make results difficult to interpret . In an investigation of 359 published siRNA sequences, we have found that about 75% of them have a risk of eliciting non-specific effects . A possible cause for this is the popular BLAST search engine, which is inappropriate for such short oligos as siRNAs . Furthermore, we used new special purpose hardware to do a transcriptome-wide screening of all possible siRNAs, and show that many unique siRNAs exist per target even if several mismatches are allowed . Hence, we argue that the risk of off-target effects is unnecessary and should be avoided in future siRNA design.

Hum Antibodies, 2003, 12(4), 99 - 112
Predicting antigenic peptides suitable for the selection of phage antibodies; Pavlik P et al.; Selection from phage antibody libraries can be considered to be an in vitro immune system in which the antibody response is reduced to the bare minimum of antigen recognition . Using selections of antibodies on peptides from a phage antibody library, we investigated what constitutes peptide antigenicity in the context of the antibody-protein binding site . We selected polyclonal antibodies in a high throughput format against 44% of 90 overlapping peptides derived from three different proteins . Of these, 33% of peptides (epitopic peptides) were able to select antibodies that recognized the protein from which the peptides were derived . Although no algorithm was able to predict all epitopic peptides, solvent accessibility was the best predictor in this cell-free antibody selection context . We subsequently applied solvent accessibility to successfully predict epitopic peptides from p53 and Znf217, and showed that such peptide selected single-chain antibodies were able to recognize soluble p53 in ELISA and Znf217 in a western blot . This is likely to have considerable utility in functional genomics and proteomics where it should be possible to select antibodies against gene products on the basis of deduced amino acid sequence in a high throughput fashion.

Plant Physiol, 2004 Jun, 135(2), 630 - 6 Epub 2004 May 21.
TILLING . Traditional mutagenesis meets functional genomics; Henikoff S et al.; Most of the genes of an organism are known from sequence, but most of the phenotypes are obscure . Thus, reverse genetics has become an important goal for many biologists . However, reverse-genetic methodologies are not similarly applicable to all organisms . In the general strategy for reverse genetics that we call TILLING (for Targeting Induced Local Lesions in Genomes), traditional chemical mutagenesis is followed by high-throughput screening for point mutations . TILLING promises to be generally applicable . Furthermore, because TILLING does not involve transgenic modifications, it is attractive not only for functional genomics but also for agricultural applications . Here, we present an overview of the status of TILLING methodology, including Ecotilling, which entails detection of natural variation . We describe public TILLING efforts in Arabidopsis and other organisms, including maize (Zea mays) and zebrafish . We conclude that TILLING, a technology developed in plants, is rapidly being adopted in other systems.

Methods Mol Biol, 2004, 270, 219 - 36
Gene expression studies using self-fabricated parasite cDNA microarrays; Hoffmann KF et al.; DNA microarray platforms represent a functional genomics technology that uses structured information obtained from genomic sequencing efforts as a means to study transcriptional processes in a systematic and high-throughput manner . Specifically in this chapter, we outline the ordered processes involved in large-scale parasite gene expression studies including complementary (cDNA) microarray fabrication, total RNA isolation, cDNA labeling using fluorochromes, and DNA:DNA hybridization . Methods described herein were adapted for the study of schistosome sexual maturation and developmental biology but could be easily modified for the study of any additional parasitological system.

Int J Med Microbiol, 2004 Apr, 293(7-8), 583 - 8
Bordetella pertussis from functional genomics to intranasal vaccination; Locht C et al.; Whooping cough still represents a major health problem, despite the use of effective vaccines for several decades . Being classically a typical childhood disease, whooping cough in young adults is now more common than it used to be, suggesting that protection after vaccination wanes during adolescence . As an alternative to the current vaccines, we wish to develop live attenuated vaccines to be delivered by the nasal route, such as to mimic the natural route of infection and to induce long lasting immunity . Bordetella pertussis, the etiological agent of whooping cough, produces a number of virulence factors, including toxins . Its recently determined genome sequence makes it now possible to apply functional genomics, such as transcriptomics and systematic knock-out mutagenesis . The expression of most known B . pertussis virulence genes is controlled by the two-component system BvgA/S . DNA microarray analyses have led to the identification of novel genes in the BvgA/S regulon, some of which are activated by BvgA/S and others are repressed by BvgA/S . In addition, some genes appear to be differentially modulated by nicotinic acid and MgSO4, both known to modulate the expression of BvgA/S-regulated genes . Among others, the functional genomics approach has uncovered two strongly BvgA/S-activated genes, named hotA and hotB (for 'homolog of toxin'), the products of which show high sequence similarities to pertussis toxin subunits . The identification of the full array of virulence factors, as well as an integrated understanding of the bacterial physiology should allow us to design attenuated B . pertussis strains useful for intranasal vaccination . A first generation of attenuated strains has already shown full protection in mice after a single intranasal administration . Such strains may also serve as vaccine carriers for heterologous antigens, in order to vaccinate against several different pathogens simultaneously.

Bioinformatics, 2004 Jul 10, 20(10), 1583 - 90 Epub 2004 May 14.
An object model and database for functional genomics; Jones A et al.; MOTIVATION: Large-scale functional genomics analysis is now feasible and presents significant challenges in data analysis, storage and querying . Data standards are required to enable the development of public data repositories and to improve data sharing . There is an established data format for microarrays (microarray gene expression markup language, MAGE-ML) and a draft standard for proteomics (PEDRo) . We believe that all types of functional genomics experiments should be annotated in a consistent manner, and we hope to open up new ways of comparing multiple datasets used in functional genomics . RESULTS: We have created a functional genomics experiment object model (FGE-OM), developed from the microarray model, MAGE-OM and two models for proteomics, PEDRo and our own model (Gla-PSI-Glasgow Proposal for the Proteomics Standards Initiative) . FGE-OM comprises three namespaces representing (i) the parts of the model common to all functional genomics experiments; (ii) microarray-specific components; and (iii) proteomics-specific components . We believe that FGE-OM should initiate discussion about the contents and structure of the next version of MAGE and the future of proteomics standards . A prototype database called RNA And Protein Abundance Database (RAPAD), based on FGE-OM, has been implemented and populated with data from microbial pathogenesis . AVAILABILITY: FGE-OM and the RAPAD schema are available from along with a set of more detailed diagrams . RAPAD can be accessed by registration at the site.

Infect Dis Clin North Am, 2004 Jun, 18(2), 207 - 18
Schistosomiasis (bilharziasis): from antiquity to the present; Mahmoud AA; The history of schistosomiasis is a continuous saga of discovery and disappointments . A lot is known and functional genomics bring the hope for better tools, but the infection and its disease sequelae still sap the energy of millions worldwide . The successes in its control are few, and the failures are enormously challenging to the scientific and public health communities and to decision makers globally . This article examines the fundamental milestones in understanding the parasite-host interaction over approximately 5 millennia of recorded history and sketches the main features of progress in the past few decades without attempting a detailed assessment.

News Physiol Sci, 2004 Jun, 19, 114 - 9
Proteomic strategies and their application in studies of renal function; Cutillas P et al.; Proteomics is a promising new tool for functional genomics . In addition to two-dimensional gel electrophoresis, other methods that are based on liquid chromatography and mass spectrometry are now available to study proteins . In this brief article, we review the strengths and limitations of the proteomic approaches currently available to the researcher, and we provide examples of how proteomics has been, and can in the future be, used to study the kidney.

Comb Chem High Throughput Screen, 2004 May, 7(3), 231 - 8
Making sense of molecular signatures in the immune system; Davies NJ et al.; The development of Functional Genomics technologies has opened new avenues to investigate the complexity of the immune system . Microarray technology has been particularly successful because of its relatively low cost and high genome coverage . Consequently to our ability to monitor the expression of a significant proportion of an organism genome, our understanding of the molecular dynamics behind cell differentiation and cell response has greatly improved . Molecular signatures associated to immune cells have provided important tools to investigate the molecular basis of diseases and have been often associated to diagnostic and prognostic markers . The availability of such large collection of data has stimulated the application of complex machine learning techniques in the attempt to link molecular signatures and cell physiology . Here we review the most recent developments in the analysis of molecular signatures in the immune system.

Appl Bioinformatics, 2002, 1(2), 101 - 5
APBioNet: the Asia-Pacific regional consortium for bioinformatics; Ranganathan S et al.; Bioinformatics and computational biology, along with the related fields of genomics, proteomics, functional genomics and systems biology are new wave scientific disciplines that harness composite computational power across networks to advance biological knowledge at the most basic level and to direct traditional laboratory-based research efforts in the biomedical sciences . 'Fostering the growth of bioinformatics and allied disciplines in the Asia-Pacific region' is the motto of the first regional bioinformatics society, the Asia-Pacific Bioinformatics Network (APBioNet) . APBioNet addresses the issues of hardware, software, databases and networks pertaining to bioinformatics, with the additional layer of pertinent education, training and research . Recent milestones achieved include hosting an international bioinformatics symposium in Asia and setting up large-scale regional grid-computing projects.

Appl Bioinformatics, 2003, 2(2), 67 - 77
Support vector machine applications in bioinformatics; Byvatov E et al.; The support vector machine (SVM) approach represents a data-driven method for solving classification tasks . It has been shown to produce lower prediction error compared to classifiers based on other methods like artificial neural networks, especially when large numbers of features are considered for sample description . In this review, the theory and main principles of the SVM approach are outlined, and successful applications in traditional areas of bioinformatics research are described . Current developments in techniques related to the SVM approach are reviewed which might become relevant for future functional genomics and chemogenomics projects . In a comparative study, we developed neural network and SVM models to identify small organic molecules that potentially modulate the function of G-protein coupled receptors . The SVM system was able to correctly classify approximately 90% of the compounds in a cross-validation study yielding a Matthews correlation coefficient of 0.78 . This classifier can be used for fast filtering of compound libraries in virtual screening applications.

Appl Bioinformatics, 2003, 2(3 Suppl), S3 - 10
Protein structure prediction and analysis as a tool for functional genomics; Baker EN et al.; Bioinformatic analyses of whole genome sequences highlight the problem of identifying the biochemical and cellular functions of the many gene products that are at present uncharacterised . Determination of their three-dimensional structures, either experimentally or by prediction, provides a powerful tool to address function, since it is at this level that biological activity is expressed . Here, we discuss the current approaches to protein structure prediction from sequence data, including the ab initio prediction of new folds, methods of fold recognition and comparative modelling based on homology . The value and limitations of such models are also explored . A major factor for the future will be the growth of the database of experimentally determined protein structures, through structural genomics projects . The prospects for this approach are also discussed, together with our experience in a pilot structural genomics project focused on proteins from Mycobacterium tuberculosis, the cause of tuberculosis (TB).

Sarcoidosis Vasc Diffuse Lung Dis, 2004 Mar, 21(1), 10 - 8
Functional genomics and prognosis in sarcoidosis--the critical role of antigen presentation; Rutherford RM et al.; BACKGROUND: Sarcoidosis is a systemic disorder of unknown cause, highly variable phenotype and unpredictable outcome . Antigen processing, inflammatory response and immunomodulation appear critical to development and prognosis of the disease . METHODS: We performed a comprehensive genomic analysis, applying high-density human GeneChip probe arrays (HUG95A, Affymetrix Inc.) for gene expression profiling from peripheral blood of patients with acute pulmonary sarcoidosis (n = 12) and matched healthy controls (n = 12), mean age 36 +/- 12 and 33 +/- 10 years respectively . RESULTS: At follow-up (18 {15-24} months), 7 patients had self-limited disease and 5 had persistent disease . Significantly different expression comparing patients and controls was identified for 1,860 (14.9%) and 729 (5.8%) gene products at p = 0.05 and p = 0.01 levels respectively . Genes closely associated with persistent disease included HLA-DRB1*1501 DQB1*0602, TNFA, NFKB, cyclic AMP-responsive element modulator (CREM) and T-cell activation marker CD69 . IL1B, IL8, growth related (GRO)-beta/-gamma and CCR 2,5,6 were closely associated with self-limited disease . CONCLUSION: We hypothesize that, in self-limited disease, greater effector cell activation leads to successful antigen elimination/tolerance, whereas HLA-DRB1*1501 DQBI*0602-mediated, probably defective/partial T-lymphocyte activation results in an inefficient primary immune response, antigen intolerance and persistent disease.

J Mol Neurosci, 2004, 23(1-2), 49 - 60
Molecular genetics approaches in yeast to study amyloid diseases; Outeiro TF et al.; The occurrence of protein aggregates in ordered fibrillar structures known as amyloid, found inside and outside of brain cells, is a feature shared by many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases . Although the molecular mechanisms that underlie neurodegeneration will ultimately have to be tested in neuronal and animal models, there are several distinct advantages in using model organisms to elucidate fundamental aspects of protein aggregation, amyloid formation, and toxicity . Here, we review recent studies indicating that amyloid formation by disease-causing proteins can be faithfully recapitulated in simple yeast-based models in Saccharomyces cerevisiae . These studies have already contributed to our basic understanding of molecular chaperone function/dysfunction in Huntington's disease, and functional genomics approaches being undertaken currently will likely bear novel insights into the genes and pathways that modulate neuronal cell dysfunction and death in these devastating diseases . A final advantage of using yeast to study amyloid formation and toxicity is the ease and rapidity with which large-scale drug-screening efforts can be conducted in this model organism.

J Bacteriol, 2004 May, 186(10), 3254 - 8
Genome-wide analysis of lipoprotein expression in Escherichia coli MG1655; Brokx SJ et al.; To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics . The mRNA expression levels of each of these genes under three growth conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by using both Affymetrix GeneChip E . coli antisense genome arrays and real-time PCR (RT-PCR) . Many genes showed significant changes in expression level . The RT-PCR results were in very good agreement with the microarray data . The results of this study represent the first insights into the possible roles of unknown lipoprotein genes and broaden our understanding of the composition of the cell envelope under different environmental conditions . Additionally, these data serve as a test set for the refinement of high-throughput bioinformatic and global gene expression methods.

Pediatr Transplant, 2004 Jun, 8 Suppl 5, 67 - 82
Treatment of advanced Ewing tumors by combined radiochemotherapy and engineered cellular transplants; Burdach S; This review will focus primarily on own recent work on the treatment of advanced Ewing tumors (AETs) and will attempt, in addition, to give a comprehensive overview of novel developments . The field under review has been shaped by investigators from both Europe and the United States of America in a scientific debate evolving over more than a decade at the meetings of the International Society of Pediatric Oncology and other scientific meetings . In the light of this debate, most oncologists will agree that patients with AETs are facing the worst prognosis of all patients with this disease and include both: (i) patients with primary metastatic disease with the worst prognosis as well as (ii) patients with relapse with the worst prognosis . The contributions of various investigators have lead to the identification of specific risk stratification criteria to overcome the heterogeneity of patients within the conventionally defined clinical stages of localized metastatic and relapsed disease . This review will address the following issues of treatment of AETs: (i) a definition of AET; (ii) risks and benefits of allogeneic vs . autologous stem cell transplantation; (iii) the role of total body irradiation; (iv) the number of involved bones as a risk factor in multifocal bone disease in AET; (v) the development of immunogene therapy in AET; (vi) the matching of radiochemo- and immunotherapy in AET; (vii) the future perspective of functional genomics and targeted therapy.

Methods, 2004 Jun, 33(2), 151 - 63
New non-viral method for gene transfer into primary cells; Gresch O et al.; The availability of genetically altered cells is an essential prerequisite for many scientific and therapeutic applications including functional genomics, drug development, and gene therapy . Unfortunately, the efficient gene transfer into primary cells is still problematic . In contrast to transfections of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires a careful selection of gene transfer techniques . Whereas viral strategies are time consuming and involve safety risks, non-viral methods proved to be inefficient for most primary cell types . The Nucleofector technology is a novel gene transfer technique designed for primary cells and hard-to-transfect cell lines . This non-viral gene transfer method is based on a cell type specific combination of electrical parameters and solutions . In this report, we show efficient transfer of DNA expression vectors and siRNA oligonucleotides into a variety of primary cell types from different species utilizing the Nucleofector technology, including human B-CLL cells, human CD34+ cells, human lymphocytes, rat cardiomyocytes, human, porcine, and bovine chondrocytes, and rat neurons .

Mutat Res, 2004 May 18, 549(1-2), 65 - 78
Functional genomics of UV radiation responses in human cells; Koch-Paiz CA et al.; The gene expression responses of MCF-7, a p53 wild-type (wt) human cell line, were monitored by cDNA microarray hybridization after exposure to different wavelengths of UV irradiation . Equitoxic doses of UVA, UVB, and UVC radiation were used to reduce survival to 37% . The effects of suramin, a signal pathway inhibitor, on the gene expression responses to the three UV wavelengths were also compared in this model system . UVB radiation triggered the broadest gene expression responses, and 172 genes were found to be consistently responsive in at least two-thirds of independent UVB experiments . These UVB radiation-responsive genes encode proteins with diverse cellular roles including cell cycle control, DNA repair, signaling, transcription, protein synthesis, protein degradation, and RNA metabolism . The set of UVB-responsive genes included most of the genes responding to an equitoxic dose of UVC radiation, plus additional genes that were not strongly triggered by UVC radiation . There was also some overlap with genes responding to an equitoxic dose of UVA radiation, although responses to this lower energy UV radiation were overall weaker . Signaling through growth factor receptors and other cytokine receptors was shown to have a major role in mediating UV radiation stress responses, as suramin, which inhibits such receptors, attenuated responses to UV radiation in nearly all the cases . Inhibition by suramin was greater for UVC than for UVB irradiation . This probably reflects the more prominent role in UVB damage response of signaling by reactive oxygen species, which would not be affected by suramin . Our results with suramin demonstrate the power of cDNA microarray hybridization to illuminate the global effects of a pharmacologic inhibitor on cell signaling.

BMC Bioinformatics . 2004 Apr 29;5(1):48.
Visualising very large phylogenetic trees in three dimensional hyperbolic space; Hughes T et al.; BACKGROUND: Common existing phylogenetic tree visualisation tools are not able to display readable trees with more than a few thousand nodes . These existing methodologies are based in two dimensional space . RESULTS: We introduce the idea of visualising phylogenetic trees in three dimensional hyperbolic space with the Walrus graph visualisation tool and have developed a conversion tool that enables the conversion of standard phylogenetic tree formats to Walrus' format . With Walrus, it becomes possible to visualise and navigate phylogenetic trees with more than 100,000 nodes . CONCLUSION: Walrus enables desktop visualisation of very large phylogenetic trees in 3 dimensional hyperbolic space . This application is potentially useful for visualisation of the tree of life and for functional genomics derivatives, like The Adaptive Evolution Database (TAED).

BMC Bioinformatics . 2004 Mar 18;5(1):31.
Defining transcriptional networks through integrative modeling of mRNA expression and transcription factor binding data; Gao F et al.; BACKGROUND: Functional genomics studies are yielding information about regulatory processes in the cell at an unprecedented scale . In the yeast S . cerevisiae, DNA microarrays have not only been used to measure the mRNA abundance for all genes under a variety of conditions but also to determine the occupancy of all promoter regions by a large number of transcription factors . The challenge is to extract useful information about the global regulatory network from these data . RESULTS: We present MA-Networker, an algorithm that combines microarray data for mRNA expression and transcription factor occupancy to define the regulatory network of the cell . Multivariate regression analysis is used to infer the activity of each transcription factor, and the correlation across different conditions between this activity and the mRNA expression of a gene is interpreted as regulatory coupling strength . Applying our method to S . cerevisiae, we find that, on average, 58% of the genes whose promoter region is bound by a transcription factor are true regulatory targets . These results are validated by an analysis of enrichment for functional annotation, response for transcription factor deletion, and over-representation of cis-regulatory motifs . We are able to assign directionality to transcription factors that control divergently transcribed genes sharing the same promoter region . Finally, we identify an intrinsic limitation of transcription factor deletion experiments related to the combinatorial nature of transcriptional control, to which our approach provides an alternative . CONCLUSION: Our reliable classification of ChIP positives into functional and non-functional TF targets based on their expression pattern across a wide range of conditions provides a starting point for identifying the unknown sequence features in non-coding DNA that directly or indirectly determine the context dependence of transcription factor action . Complete analysis results are available for browsing or download at http://bussemaker.bio.columbia.edu/papers/MA-Networker/.

Poult Sci, 2004 Apr, 83(4), 552 - 73
Proteomics in the chicken: tools for understanding immune responses to avian diseases; Burgess SC; The entire chicken genome sequence will be available by the time this review is in press . Chickens will be the first production animal species to enter the "postgenomic era." This fundamental structural genomics achievement allows, for the first time, complete functional genomics approaches for understanding the molecular basis of chicken normo- and pathophysiology . The functional genomics paradigm, which contrasts with classical functional genetic investigations of one gene (or few) in isolation, is the systematic holistic genetic analyses of biological systems in defined contexts . Context-dependent gene interactions are the fundamental mechanics of all life . Functional genomics uses high-throughput large-scale experimental methods combined with statistical and computational analyses . Projects with expressed sequence tags in chickens have already allowed the creation of cDNA microarrays for large-scale context-dependant mRNA analysis (transcriptomics) . However, proteins are the functional units of almost all biological processes, and protein expression very often bears no correlation to mRNA expression . Proteomics, a discipline within functional genomics, is the context-defined analysis of complete complements of proteins . Proteomics bridges the "sequence-to-phenotype gap;" it complements structural and other functional genomics approaches . Proteomics requires high capital investment but has ubiquitous biological applications . Although currently the fastest-growing human biomedical discipline, new paradigms may need to be established for production animal proteomics research . The prospective promise and potential pitfalls of using proteomics approaches to improve poultry pathogen control will be specifically highlighted . The first stage of our recently established proteomics program is global protein profiling to identify differentially expressed proteins in the context of the commercially important pathogens . Our trials and tribulations in establishing our proteomics program, as well some of our initial data to understand chicken immune system function, will be discussed.

Acad Radiol, 2004 Apr, 11(4), 448 - 61
A primer on molecular biology for imagers: III . Proteins: structure and function; Pandit SD et al.; This article along with the first 2 in this series (4,12) completes the discussion on the key molecules and process inside the cell namely, DNA, RNA, and proteins . These 3 articles provide a very basic foundation for understanding molecular biology concepts and summarize some of the work of numerous scientists over the past century . We understand these processes far better now than we did in the past, but clearly this knowledge is by no means complete and a number of basic scientists are working hard to elucidate and understand the fundamental mechanisms that operate within a cell . Genes and gene products work with each other in complex, interconnected pathways, and in perfect harmony to make a functional cell, tissue, and an organism as a whole . There is a lot of cross-talk that happens between different proteins that interact with various other proteins, DNA, and RNA to establish pathways, networks, and molecular systems as a team working to perfection . The past 15 years have seen the rapid development of systems biology approaches . We live in an era that emphasizes multi-disciplinary, cross-functional teams to perform science rather than individual researchers working on the bench on a very specific problem . Global approaches have become more common and the amount of data generated must be managed by trained bioinformatics personnel and large computers . In our subsequent articles, we will discuss these global approaches and the areas of genomics, functional genomics, and proteomics that have revolutionized the way we perform science.

In Silico Biol, 2004, 4(2), 195 - 208
Efficient prediction of alternative splice forms using protein domain homology; Hiller M et al.; Alternative splicing can yield manifold different mature mRNAs from one precursor . New findings indicate that alternative splicing occurs much more often than previously assumed . A major goal of functional genomics lies in elucidating and characterizing the entire spectrum of alternative splice forms . Existing approaches such as EST-alignments focus only on the mRNA sequence to detect alternative splice forms . They do not consider function and characteristics of the resulting proteins . One important example of such functional characterization is homology to a known protein domain family . A powerful description of protein domains are profile Hidden Markov models (HMM) as stored in the Pfam database . In this paper we address the problem of identifying the splice form with the highest similarity to a protein domain family . Therefore, we take into consideration all possible splice forms . As demonstrated here for a number of genes, this homology based approach can be used successfully for predicting partial gene structures . Furthermore, we present some novel splice form predictions with high-scoring protein domain homology and point out that the detection of splice form specific protein domains helps to answer questions concerning hereditary diseases . Simple approaches based on a BLASTP search cannot be applied here, since the number of possible splice forms increases exponentially with the number of exons . To this end, we have developed an efficient polynomial-time algorithm, called ASFPred (Alternative Splice Form Prediction) . This algorithm needs only a set of exons as input.

Z Orthop Ihre Grenzgeb, 2004 Mar-Apr, 142(2), 241 - 7
{cDNA-microarrays in cartilage research - functional genomics of osteoarthritis}; Aigner T et al.; Functional genomics represents a new challenging approach in order to analyze complex diseases such as osteoarthritis on a molecular level . The characterization of the molecular changes of the cartilage cells, the chondrocytes, enables a better understanding of the pathomechanisms of the disease . In particular, the identification and characterization of new target molecules for therapeutic intervention is of interest . Also, potential molecular markers for diagnosis and monitoring of osteoarthritis contribute to a more appropriate patient management . The DNA-microarray technology complements (but does not replace) biochemical and biological research in new disease-relevant genes . Large-scale functional genomics will identify molecular networks such as yet identified players in the anabolic-catabolic balance of articular cartilage as well as disease-relevant intracellular signaling cascades so far rather unknown in articular chondrocytes . However, at the moment it is also important to recognize the limitations of the microarray technology in order to avoid over-interpretation of the results . This might lead to misleading results and prevent to a significant extent a proper use of the potential of this technology in the field of osteoarthritis.

Oligonucleotides, 2004 Spring, 14(1), 49 - 64
Use of antisense oligonucleotides in functional genomics and target validation; Ravichandran LV et al.; With the completion of sequencing of the human genome, a great deal of interest has been shifted toward functional genomics-based research for identification of novel drug targets for treatment of various diseases . The major challenge facing the pharmaceutical industry is to identify disease-causing genes and elucidate additional roles for genes of known functions . Gene functionalization and target validation are probably the most important steps involved in identifying novel potential drug targets . This review focuses on recent advances in antisense technology and its use for rapid identification and validation of new drug targets . The significance and applicability of this technology as a beginning of the drug discovery process are underscored by relevant cell culture-based assays and positive correlation in specific animal disease models . Some of the antisense inhibitors used to validate gene targets are themselves being developed as drugs . The current clinical trials based on such leads that were identified in a very short time further substantiate the importance of antisense technology-based functional genomics as an integral part of target validation and drug target identification.

Curr Opin Rheumatol, 2004 May, 16(3), 238 - 45
Functional genomics of fibroblasts; Neumann E et al.; PURPOSE OF REVIEW: Successful analysis of the pathophysiology of rheumatoid arthritis requires the functional understanding of interactions between different cell types and the cell matrix, intracellular signaling pathways, as well as between cartilage, bone, and synovium in rheumatoid arthritis . During the review period, molecular biology has provided and used a growing number of tools to screen the genome such as gene and protein chips, haplotype analysis, and single nucleotide polymorphism analysis, resulting in various novel findings with considerable impact on the overall understanding of rheumatoid arthritis . RECENT FINDINGS: Key issues that have been addressed and elucidated by numerous research groups are the regulation and modulation of synovial fibroblast metabolism and activation by proinflammatory cytokines and chemokines . In addition, examination of adhesion processes and neoangiogenesis has revealed new insights into the interaction network between rheumatoid synovial fibroblasts and the surrounding matrix and cells . Finally, a more detailed view of activation of these fibroblasts has been obtained by analysis of the molecular balance between cellular activation and regulation of apoptosis . SUMMARY: Although high throughput molecular analysis methods provided an ample amount of novel data, it needs to be stressed that a one-method approach of gene expression (eg, by array analysis) is not sufficient to validate the gene/gene product as a new therapeutic target . Therefore, the next steps are the so-called functional genomics or functionomics, which intend to reveal relations between the obtained data and to unveil their interactions for a better understanding of the pathogenesis and the mechanisms that are operative in rheumatoid arthritis.

Expert Opin Ther Targets, 2004 Apr, 8(2), 165 - 70
G-Protein-coupled receptors: new approaches to maximise the impact of GPCRS in drug discovery; Davey J; IBC's Drug Discovery Technology Series is a group of conferences highlighting technological advances and applications in niche areas of the drug discovery pipeline . This 2-day meeting focused on G-protein-coupled receptors (GPCRs), probably the most important and certainly the most valuable class of targets for drug discovery . The meeting was chaired by J Beesley (Vice President, European Business Development for LifeSpan Biosciences, Seattle, USA) and included 17 presentations on various aspects of GPCR activity, drug screens and therapeutic analyses . Keynote Addresses covered two of the emerging areas in GPCR regulation; receptor dimerisation (G Milligan, Professor of Molecular Pharmacology and Biochemistry, University of Glasgow, UK) and proteins that interact with GPCRs (J Bockaert, Laboratory of Functional Genomics, CNRS Montpellier, France) . A third Keynote Address from W Thomsen (Director of GPCR Drug Screening, Arena Pharmaceuticals, USA) discussed Arena's general approach to drug discovery and illustrated this with reference to the development of an agonist with potential efficacy in Type II diabetes.

Curr Opin Biotechnol, 2004 Feb, 15(1), 58 - 63
High-throughput phenomics: experimental methods for mapping fluxomes; Sauer U; Many technologies have been developed to help explain the phenotypic consequences of genetic and/or environmental modifications in areas like functional genomics, pharmaceutical research and metabolic engineering . The missing link in contemporary functional analyses that focus on the analysis of cellular components is the capacity to directly observe functional units . By linking genes and proteins to higher level biological functions, the molecular fluxes through metabolic networks (the fluxome) determine the cellular phenotype . Quantitative monitoring of such whole network operations by methods of metabolic flux analysis, thus bridges the gap by providing a global perspective of the integrated regulation at the transcriptional, translational and metabolic level . This review highlights recent developments towards high-throughput flux analysis.

Acta Biochim Pol, 2004, 51(1), 1 - 8
DNA microarrays, a novel approach in studies of chromatin structure; Widlak P; The DNA microarray technology delivers an experimental tool that allows surveying expression of genetic information on a genome-wide scale at the level of single genes - for the new field termed functional genomics . Gene expression profiling - the primary application of DNA microarrays technology - generates monumental amounts of information concerning the functioning of genes, cells and organisms . However, the expression of genetic information is regulated by a number of factors that cannot be directly targeted by standard gene expression profiling . The genetic material of eukaryotic cells is packed into chromatin which provides the compaction and organization of DNA for replication, repair and recombination processes, and is the major epigenetic factor determining the expression of genetic information . Genomic DNA can be methylated and this modification modulates interactions with proteins which change the functional status of genes . Both chromatin structure and transcriptional activity are affected by the processes of replication, recombination and repair . Modified DNA microarray technology could be applied to genome-wide study of epigenetic factors and processes that modulate the expression of genetic information . Attempts to use DNA microarrays in studies of chromatin packing state, chromatin/DNA-binding protein distribution and DNA methylation pattern on a genome-wide scale are briefly reviewed in this paper.

Curr Opin Nephrol Hypertens, 2004 Jan, 13(1), 59 - 65
Revisiting sodium and water reabsorption with functional genomics tools; Firsov D; PURPOSE OF REVIEW: The kidney plays an essential role in maintaining sodium and water balance, thereby controlling the volume and osmolarity of the extracellular body fluids, the blood volume and the blood pressure . The final adjustment of sodium and water reabsorption in the kidney takes place in cells of the distal part of the nephron in which a set of apical and basolateral transporters participate in vectorial sodium and water transport from the tubular lumen to the interstitium and, finally, to the general circulation . According to a current model, the activity and/or cell-surface expression of these transporters is/are under the control of a gene network composed of the hormonally regulated, as well as constitutively expressed, genes . It is proposed that this gene network may include new candidate genes for salt- and water-losing syndromes and for salt-sensitive hypertension . A new generation of functional genomics techniques have recently been applied to the characterization of this gene network . The purpose of this review is to summarize these studies and to discuss the potential of the different techniques for characterization of the renal transcriptome . RECENT FINDINGS: Recently, DNA microarrays and serial analysis of gene expression have been applied to characterize the kidney transcriptome in different in-vivo and in-vitro models . In these studies, a set of new interesting genes potentially involved in the regulation of sodium and water reabsorption by the kidney have been identified and are currently under detailed investigation . SUMMARY: Characterization of the kidney transcriptome is greatly expanding our knowledge of the gene networks involved in multiple kidney functions, including the maintenance of sodium and water homeostasis.

J Neuroendocrinol, 2004 Apr, 16(4), 383 - 9
Functional genomics of social recognition; Choleris E et al.; Although various types of group living are widespread in mammals, including humans, the study of the hormonal and genetic underpinnings of nonsexual social behaviour, is in its infancy compared to the analysis of sexual behaviour mechanisms . Oxytocin, vasopressin and gonadal hormones certainly play an important role . Social recognition, where animals identify and recognize other individual conspecifics, is a crucial prerequisite for the occurrence of a wide range of social behaviours . Social recognition is also important for coping with one major cost of life in a group: the increased risk of exposure to parasites and infection . We review recent functional genomic studies on the involvement of oxytocin and oestrogen-receptor genes in the regulation of social recognition in mice and in the ecologically relevant context of parasite recognition and avoidance . Based on quantitative studies of social recognition with gene-knockout mice and with antisense DNA, we propose a four-gene micronet contributing to social recognition . This micronet involves the genes coding for oestrogen receptors alpha (ER-alpha), beta (ER-beta), oxytocin and the oxytocin receptor . In this model, circulating oestrogens promote transcription of (i) oxytocin in the paraventricular nucleus of the hypothalamus through ER-beta and (ii) oxytocin receptor in the amygdala through ER-alpha . This model forms the core around which increasingly complex genetic, hormonal and neural interactions associated with social behaviours and recognition can be organized.

Theor Appl Genet, 2004 Jun, 109(1), 10 - 22 Epub 2004 Apr 14.
Annotation of a 95-kb Populus deltoides genomic sequence reveals a disease resistance gene cluster and novel class I and class II transposable elements; Lescot M et al.; Poplar has become a model system for functional genomics in woody plants . Here, we report the sequencing and annotation of the first large contiguous stretch of genomic sequence (95 kb) of poplar, corresponding to a bacterial artificial chromosome clone mapped 0.6 centiMorgan from the Melampsora larici-populina resistance locus . The annotation revealed 15 putative genetic objects, of which five were classified as hypothetical genes that were similar only with expressed sequence tags from poplar . Ten putative objects showed similarity with known genes, of which one was similar to a kinase . Three other objects corresponded to the toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat class of plant disease resistance genes, of which two were predicted to encode an amino terminal nuclear localization signal . Four objects were homologous to the Ty1/ copia family of class I transposable elements, one of which was designated Retropop and interrupted one of the disease resistance genes . Two other objects constituted a novel Spm-like class II transposable element, which we designated Magali.

Parasitol Int, 2004 Jun, 53(2), 183 - 92
Schistosoma mansoni genome project: an update; LoVerde PT et al.; A schistosome genome project was initiated by the World Health Organization in 1994 with the notion that the best prospects for identifying new targets for drugs, vaccines, and diagnostic development lie in schistosome gene discovery, development of chromosome maps, whole genome sequencing and genome analysis . Schistosoma mansoni has a haploid genome of 270 Mb contained on 8 pairs of chromosomes . It is estimated that the S . mansoni genome contains between 15000 and 25000 genes . There are approximately 16689 ESTs obtained from diverse libraries representing different developmental stages of S . mansoni, deposited in the NCBI EST database . More than half of the deposited sequences correspond to genes of unknown function . Approximately 40-50% of the sequences form unique clusters, suggesting that approximately 20-25% of the total schistosome genes have been discovered . Efforts to develop low resolution chromosome maps are in progress . There is a genome sequencing program underway that will provide 3X sequence coverage of the S . mansoni genome that will result in approximately 95% gene discovery . The genomics era has provided the resources to usher in the era of functional genomics that will involve microarrays to focus on specific metabolic pathways, proteomics to identify relevant proteins and protein-protein interactions to understand critical parasite pathways . Functional genomics is expected to accelerate the development of control and treatment strategies for schistosomiasis.

Mol Genet Genomics, 2004 Jun, 271(5), 566 - 76 Epub 2004 Apr 07.
Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments; Tanaka N et al.; Despite recent progress in sequencing the complete genome of rice ( Oryza sativa), the proteome of this species remains poorly understood . To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells . The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching . In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure . The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes . Edman degradation revealed that 60-98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed . The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.

Theor Appl Genet, 2004 Apr, 108(6), 1010 - 6 Epub 2003 Nov 20.
EST-derived microsatellites from Actinidia species and their potential for mapping; Fraser LG et al.; To increase the speed and reduce the cost of constructing a genetic map of Actinidia species (kiwifruit), for use in both breeding and functional genomics programmes, we sampled microsatellites from expressed sequence tags (ESTs) to evaluate their frequency of occurrence and level of polymorphism . Perfect dinucleotide repeats were the microsatellites selected, and these were found to be numerous in both the 5' and 3' ends of the genes represented . The microsatellites were of various lengths, the majority being repeats with the pattern (CT)(n)/(GA)(n) . One hundred and fifty microsatellites, each with more than 10 dinucleotide repeat units, were chosen as possible markers, and when these were amplified, 93.5% were found to be polymorphic and segregating in a mapping population, with 22.6% amplifying more than one locus . Four marker categories were identified . Fully informative markers made up 27% of the total, 36.2% were female informative, 25.8% were male informative and 10% partly informative . The mapping population was an intraspecific cross in the diploid species Actinidia chinensis, with parents chosen for their diversity in fruit and plant characteristics, and for their geographical separation . Linkage was tested using the software 'Joinmap' and a LOD value of 3 . The distribution of the EST-based markers over the linkage groups obtained appeared to be random, taking into consideration the small sample size, that the number of linkage groups (31) exceeded the chromosome number of n=29, and that a number of markers were not assigned to any group . Some microsatellite markers which amplified more than one locus mapped to separate linkage groups . According to our study in A . chinensis, EST-derived microsatellites give large numbers of possible markers very quickly and at reasonable cost . The markers are highly polymorphic, segregate in the mapping population, and increase the value of the genomic map by providing some functional information.

Differentiation, 2004 Mar, 72(2-3), 81 - 91
High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics; Sugimoto A; The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans, in which introduction of double-stranded RNA causes specific inactivation of genes with corresponding sequences . Technical advances in RNAi methodology and the availability of the complete genome sequence have enabled the high-throughput, genome-wide RNAi analysis of this organism . Several groups have used large-scale RNAi to systematically examine every C . elegans gene for knock-down phenotypes, providing basal information to be mined in more detailed studies . Now, in addition to functional genomic RNAi analyses, high-throughput RNAi is also routinely used for rapid, genome-wide screens for genes involved in specific biological processes . The integration of high-throughput RNAi experiments with other large-scale data, such as DNA microarrays and protein-protein interaction maps, enhances the speed and reliability of such screens . The accumulation of RNAi phenotype data dramatically accelerates our understanding of this organism at the genetic level.

BMC Bioinformatics . 2004 Apr 02;5(1):35.
PASS2: an automated database of protein alignments organised as structural superfamilies; Bhaduri A et al.; BACKGROUND: The functional selection and three-dimensional structural constraints of proteins in nature often relates to the retention of significant sequence similarity between proteins of similar fold and function despite poor sequence identity . Organization of structure-based sequence alignments for distantly related proteins, provides a map of the conserved and critical regions of the protein universe that is useful for the analysis of folding principles, for the evolutionary unification of protein families and for maximizing the information return from experimental structure determination . The Protein Alignment organised as Structural Superfamily (PASS2) database represents continuously updated, structural alignments for evolutionary related, sequentially distant proteins . DESCRIPTION: An automated and updated version of PASS2 is, in direct correspondence with SCOP 1.63, consisting of sequences having identity below 40% among themselves . Protein domains have been grouped into 628 multi-member superfamilies and 566 single member superfamilies . Structure-based sequence alignments for the superfamilies have been obtained using COMPARER, while initial equivalencies have been derived from a preliminary superposition using LSQMAN or STAMP 4.0 . The final sequence alignments have been annotated for structural features using JOY4.0 . The database is supplemented with sequence relatives belonging to different genomes, conserved spatially interacting and structural motifs, probabilistic hidden markov models of superfamilies based on the alignments and useful links to other databases . Probabilistic models and sensitive position specific profiles obtained from reliable superfamily alignments aid annotation of remote homologues and are useful tools in structural and functional genomics . PASS2 presents the phylogeny of its members both based on sequence and structural dissimilarities . Clustering of members allows us to understand diversification of the family members . The search engine has been improved for simpler browsing of the database . CONCLUSIONS: The database resolves alignments among the structural domains consisting of evolutionarily diverged set of sequences . Availability of reliable sequence alignments of distantly related proteins despite poor sequence identity and single-member superfamilies permit better sampling of structures in libraries for fold recognition of new sequences and for the understanding of protein structure-function relationships of individual superfamilies . PASS2 is accessible at http://www.ncbs.res.in/~faculty/mini/campass/pass2.html

BMC Bioinformatics . 2004 Feb 16;5(1):14.
Comparison of computational methods for identifying translation initiation sites in EST data; Nadershahi A et al.; BACKGROUND: Expressed Sequence Tag (EST) sequences are generally single-strand, single-pass sequences, only 200-600 nucleotides long, contain errors resulting in frame shifts, and represent different parts of their parent cDNA . If the cDNAs contain translation initiation sites, they may be suitable for functional genomics studies . We have compared five methods to predict translation initiation sites in EST data: first-ATG, ESTScan, Diogenes, Netstart, and ATGpr . RESULTS: A dataset of 100 EST sequences, 50 with and 50 without, translation initiation sites, was created . Based on analysis of this dataset, ATGpr is found to be the most accurate for predicting the presence versus absence of translation initiation sites . With a maximum accuracy of 76%, ATGpr more accurately predicts the position or absence of translation initiation sites than NetStart (57%) or Diogenes (50%) . ATGpr similarly excels when start sites are known to be present (90%), whereas NetStart achieves only 60% overall accuracy . As a baseline for comparison, choosing the first ATG correctly identifies the translation initiation site in 74% of the sequences . ESTScan and Diogenes, consistent with their intended use, are able to identify open reading frames, but are unable to determine the precise position of translation initiation sites . CONCLUSIONS: ATGpr demonstrates high sensitivity, specificity, and overall accuracy in identifying start sites while also rejecting incomplete sequences . A database of EST sequences suitable for validating programs for translation initiation site prediction is now available . These tools and materials may open an avenue for future improvements in start site prediction and EST analysis.

Biotechnol Bioeng, 2004 Apr 20, 86(2), 117 - 24
Digital quantitative measurements of gene expression; Mikkilineni V et al.; One of the primary goals of functional genomics is to provide a quantitative understanding of gene function . However, the success of this enterprise is dependent on the accuracy and precision of the functional genomic data . A novel approach, digital analysis of gene expression (DAGE) described herein, is an accurate and precise technology for measuring digital gene expression on a relative or absolute scale by simply counting the number of transcripts of a gene being expressed at a given time . The result is a greatly improved technology sensitive enough for identifying and quantifying small (but biologically important and statistically relevant) changes in gene expression . Fourteen genes involved in galactose metabolism in Saccharomyces cerevisiae were analyzed for their expression levels in glucose and galactose minimal media . The quantitative expression results were characterized in terms of distributional and accuracy attributes; they were also in general agreement (in terms of direction of change) with corresponding results obtained using microarray technology . DAGE is likely to have profound implications in the field of functional genomics because the gene expression measurements are digital in nature and therefore more accurate than any other technologies .

Semin Oncol, 2004 Feb, 31(1 Suppl 3), 39 - 53
Molecular therapeutics: promise and challenges; Kohn EC et al.; The ability to analyze the genetic and epigenetic aberrations present in a particular patient's tumor on a global basis is rapidly maturing . The emerging fields of functional genomics and functional proteomics offer the opportunity to translate these advances into a full comprehension of the pathophysiology of cancer . Linking these approaches to chemical genomics and molecular therapeutics should provide an expanding repertoire of targeted therapeutics for clinical evaluation . Novel clinical trial designs that can determine the efficacy of targeted therapeutics in patients selected for aberrations in the target are needed to evaluate the wealth of new drugs becoming available . The promise of these technologies and advances in our understanding of cancer is immense, making it our responsibility to see them to fruition . These technologies should lead to a new era of individualized molecular medicine, wherein we will treat each patient with a "prescription" based on the genetic changes in each patient's tumor and their own genetic make-up, resulting in more effective and less toxic therapy.

What Is Genome?, What Is Bioremediation?, What Is Botulism?, What Is Biotechnology?, What Is Pcr?, i, Microorganism, i, Bacterium, n, Bacteria, n, Microbe, n, Microorganisms, n, Sepsis, e, Biological treatment, r, Antibiotics, s, Meningococcus, e, Ps. putida, o, Escherichia coli, o, Cryptococci, n, Lactococci, e, Salmonella typhimurium, c, Microorganism, i, Bacteroides, c, Antibiotics, e, Escherichia coli, a, Escherichia coli, c, Growth media, o, Antibiotics, s, Fermentations, n, Antibiotics, r, Escherichia coli, n, Fermentations, n, Bacillus

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005