Functional genomics

Genetics, 1999 Sep, 153(1), 135 - 77
The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes; Spradling AC et al.; A fundamental goal of genetics and functional genomics is to identify and mutate every gene in model organisms such as Drosophila melanogaster . The Berkeley Drosophila Genome Project (BDGP) gene disruption project generates single P-element insertion strains that each mutate unique genomic open reading frames . Such strains strongly facilitate further genetic and molecular studies of the disrupted loci, but it has remained unclear if P elements can be used to mutate all Drosophila genes . We now report that the primary collection has grown to contain 1045 strains that disrupt more than 25% of the estimated 3600 Drosophila genes that are essential for adult viability . Of these P insertions, 67% have been verified by genetic tests to cause the associated recessive mutant phenotypes, and the validity of most of the remaining lines is predicted on statistical grounds . Sequences flanking >920 insertions have been determined to exactly position them in the genome and to identify 376 potentially affected transcripts from collections of EST sequences . Strains in the BDGP collection are available from the Bloomington Stock Center and have already assisted the research community in characterizing >250 Drosophila genes . The likely identity of 131 additional genes in the collection is reported here . Our results show that Drosophila genes have a wide range of sensitivity to inactivation by P elements, and provide a rationale for greatly expanding the BDGP primary collection based entirely on insertion site sequencing . We predict that this approach can bring >85% of all Drosophila open reading frames under experimental control.

Hum Mol Genet, 1999, 8(10), 1821 - 32
Computational methods for the identification of differential and coordinated gene expression; Claverie JM; With the first complete 'draft' of the human genome sequence expected for Spring 2000, the three basic challenges for today's bioinformatics are more than ever: (i) finding the genes; (ii) locating their coding regions; and (iii) predicting their functions . However, our capacity for interpreting vertebrate genomic and transcript (cDNA) sequences using experimental or computational means very much lags behind our raw sequencing power . If the performances of current programs in identifying internal coding exons are good, the precise 5'-->3' delineation of transcription units (and promoters) still requires additional experiments . Similarly, functional predictions made with reference to previously characterized homologues are leaving >50% of human genes unannotated or classified in uninformative categories ('kinase', 'ATP-binding', etc.) . In the context of functional genomics, large-scale gene expression studies using massive cDNA tag sequencing, two-dimensional gel proteome analysis or microarray technologies are the only approaches providing genome-scale experimental information at a pace consistent with the progress of sequencing . Given the difficulty and cost of characterizing genes one by one, academic and industrial researchers are increasingly relying on those methods to prioritize their studies and choose their targets . The study of expression patterns can also provide some insight into the function, reveal regulatory pathways, indicate side effects of drugs or serve as a diagnostic tool . In this article, I review the theoretical and computational approaches used to: (i) identify genes differentially expressed (across cell types, developmental stages, pathological conditions, etc.); (ii) identify genes expressed in a coordinated manner across a set of conditions; and (iii) delineate clusters of genes sharing coherent expression features, eventually defining global biological pathways.

Arch Virol Suppl, 1999, 15, 189 - 201
Viruses and gene silencing in plants; Baulcombe D; Genetic engineering of virus resistance in plants may be conferred by transgenes based on sequences from the viral genome . In many instances the underlying mechanism involves the transgenically expressed proteins . However there are other examples in which the mechanism is based on RNA . It appears that this mechanism is related to post transcriptional gene silencing in transgenic plants . This gene silencing is likely to involve antisense RNA produced by the action of a host-encoded RNA dependent RNA polymerase . The natural role of this mechanism is as a genetic immune system conferring protection against viruses . There may also be a genomic role of the process reflected in RNA directed methylation of transgenes . Further understanding of this mechanism has obvious implications for virus resistance in plants . In addition the gene silencing can be used as a component of a new technology with application in functional genomics.

Parasitology, 1999, 118 Suppl, S11 - 4
Status of protozoan genome analysis: trypanosomatids; Blackwell JM et al.; The three trypanosomatid genome projects have employed common strategies which include: analysis of pulsed-field gel electrophoretic chromosomal karyotypes; physical mapping using big DNA (cosmid, pacmid P1, bacterial artificial chromosome, yeast artificial chromosome) libraries; partial cDNA sequence analysis to develop sets of expressed sequence tags (ESTs) for gene discovery and use as markers in physical mapping; genomic sequencing; dissemination of information through development of web-sites and ACeDB-based fully integrated databases; and establishment of functional genomics programmes to maximize useful application of genome data . Highlights of the projects to date have been the demonstration that, despite extensive chromosomal size polymorphisms for diploid homologues within Africa trypanosomes, T . cruzi or Leishmania, the physical linkage groups for markers on each chromosome are retained across all isolates/species studied within each group . For African trypanosomes, detailed analysis of chromosome 1 has demonstrated that repetitive sequences and the two retroposon-like elements RIME and INGI are localized to a defined region at one end of the chromosome, with the bulk of the central region of the chromosome containing genes coding for expressed proteins . Comparative mapping shows that, although subtelomeric changes account for a large proportion of the polymorphism in chromosome size in African trypanosomes, there are significant expansions and contractions in regions across the entire chromosome . The highlight of the genomic sequencing projects has been the demonstration of just 2 putative transcriptional units of chromosome 1 of Leishmania major, extending on opposite strands from a point in the central region of the chromosome . A similar observation made on 93.4 kb of contiguous sequence for T . cruzi chromosome 3 suggests the presence of promoter and regulatory elements at the junctions of large polycistronic transcriptional units . All data obtained from the genome projects are made available through the public domain, which has prompted changing philosophies in how we approach analysis of the biology of these organisms, and strategies that we can employ now in the search for new therapies and vaccines.

Trends Plant Sci, 1999 Sep, 4(9), 340 - 347
Listening to the silent genes: transgene silencing, gene regulation and pathogen control; Kooter JM et al.; By capitalizing on the initially puzzling observations of unpredictable transgene silencing and variable expression, plant scientists have pioneered research into a novel type of epigenetic regulation, termed homology-dependent gene silencing . This silencing process has implications for natural mechanisms of gene expression in plants and other eukaryotes, and has branched out into studies of reversible DNA modifications; RNA metabolism, transport and processing; and host responses to plant viruses, viroids and transposable elements . The analysis of transgene silencing systems has enriched our understanding of other epigenetic phenomena, including paramutation, as well as heterosis and genome evolution . This research is also highly relevant to the biotechnology industry, which is interested in avoiding unwanted transgene silencing in genetically engineered lines and in exploiting various types of silencing to inactivate specific genes . Homology-dependent gene silencing can also be used in high-throughput approaches for functional genomics.

Trends Biotechnol, 1999 Sep, 17(9), 374 - 81
Prospects for drug screening using the reverse two-hybrid system; Vidal M et al.; Rational drug-screening strategies have been limited by the number of available protein targets . The fields of genomics and functional genomics are now merging into 'chemical genomics' approaches, in which large numbers of potential target proteins can be used in standardized high-throughput drug-screening assays . Because protein-protein interactions are critical to most biological processes and can be tested in standardized assays, they may represent optimal targets in the chemical-genomics era . The reverse two-hybrid system appears to have several properties that would be critical for the success of this approach.

Curr Top Microbiol Immunol, 1999, 243, 23 - 36
Antigen sequence- and library-based mapping of linear and discontinuous protein-protein-interaction sites by spot synthesis; Reineke U et al.; The knowledge (antigen-derived peptide scans)- and library (de novo)-based mapping of linear and discontinuous antibody epitopes as well as protein-protein contact sites in general by spot synthesis now is a well established technique . Due to its automation, this technique also promises great potential for applications in functional genomics . It should help to elucidate the complex network of interacting protein molecules involved in signal transduction events (Adam-klages et al . 1996; Hoffmuller et al . 1999) . Although little chemistry is involved in the preparation of peptide scans or libraries and the synthesis procedure is relatively simple, the laboratories of immunologists or molecular biologists are often not equipped to perform spot synthesis . In this case scans or libraries can be purchased from commercial suppliers.

Eur J Pharmacol, 1999 Jun 30, 375(1-3), 327 - 37
Genetically modified animals in pharmacological research: future trends; Rudolph U et al.; The recognition of molecular control elements which govern cell and organ function is essential for the development of novel drug therapies and for an understanding of drug actions . Thus, a major interest is focused on methodologies which permit the identification of novel control elements . This is of particular relevance for the identification of drug targets, the distinction of target isoforms, the differentiation of signalling pathways, the generation of disease models and toxicological testing . In this review, we discuss different classes of genetically modified animals and their potential to elucidate biological processes relevant for pharmacological research including functional genomics . Techniques which permit the time- and tissue-specific inducible regulation of gene expression present an important methodological advance.

Gene Expr, 1999, 7(4-6), 387 - 400
The complexity of radiation stress responses: analysis by informatics and functional genomics approaches; Fornace AJ Jr et al.; Molecular responses to genotoxic stress are complex and are mediated by a variety of regulatory pathways . One key element in cellular response is the stress gene transcription factor p53, which can regulate nearly 100 genes that have already been identified . Although p53 plays a central role in the cellular response to DNA-damaging agents such as ionizing radiation (IR), other pathways can also have important roles . One example is the transcriptional responses associated with IR-induced apoptosis, where induction of some genes is limited to p53 wild-type (wt) cells that also have the ability to undergo rapid apoptosis after irradiation . In contrast, other genes are triggered after IR in lines undergoing rapid apoptosis regardless of p53 status . From this and other examples, it is apparent that the pattern of stress gene expression is cell type specific in both primary and transformed lines . The premise will be developed that such differences in stress gene responsiveness can be employed as molecular markers using a combination of informatics and functional genomics approaches . An example is given using the panel of lines of the NCI anticancer drug screen where both the p53 status and sensitivity to a large collection of cytotoxic agents have been determined . The utility of cDNA microarray hybridization to measure IR-stress gene responses has recently been demonstrated and a large number of additional IR-stress genes have been identified . The responses of some of these genes to IR and other DNA-damaging agents varied widely in cell lines from different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses; this also highlights the need for informatics approaches to discover and prioritize hypotheses regarding the importance of particular cellular factors . The aim of this review is to demonstrate the utility of combining an informatics approach with functional genomics in the study of stress responses.

Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8825 - 6
Functional genomics; Fields S et al.; Complete genome sequences are providing a framework to allow the investigation of biological processes by the use of comprehensive approaches . Genome analysis also is having a dramatic impact on medicine through its identification of genes and mutations involved in disease and the elucidation of entire microbial gene sets . Studies of the sequences of model organisms, such as that of the nematode worm Caenorhabditis elegans, are providing extraordinary insights into development and differentiation that aid the study of these processes in humans . The field of functional genomics seeks to devise and apply technologies that take advantage of the growing body of sequence information to analyze the full complement of genes and proteins encoded by an organism.

Biochem Genet, 1999 Feb, 37(1-2), 23 - 40
Analysis of background-dependent genetic interactions without inbred strains; Kovac I et al.; Experimental analysis of background dependent effects of genetic interactions can be designed using strains generated by introgression of small genetic regions containing identical genotypes at loci in question into different inbred strains . We use a novel multilocus paradigm, denoted conditional intergenic functional association (CIFA), to simulate this procedure, with the trade-off of power for convenience that is affordable when sufficiently strong effects are present . We analyze nine enzyme loci at three chromosomes in groups of D . melanogaster with different developmental rates that showed similar allelic frequencies at individual loci . Results obtained suggest the presence of adaptive interaction between particular alleles at two loci when genetic variation at seven background loci is eliminated . Biochemical considerations show that, in the resulting developmental context, strong interaction between these genes may emerge from shifted control of the pentose phosphate pathway, with cascading effects on the glycolysis, TCA cycle, and biosynthetic pathways: one gene may assume control of the irreversible rate-limiting step in the pentose phosphate pathway, whereas the other gene may assume control of the NADP+ level that regulates the same rate-limiting step as an electron acceptor . The newly developing functional genomics research and the absence of inbreeding make CIFA directly applicable to complex human traits in large samples.

Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8774 - 8
A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations; Beetham PR et al.; Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells . To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene . An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells . COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides . After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196 . To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion . Recovery of fluorescent cells confirmed the deletion correction . Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

Mol Microbiol, 1999 Jul, 33(1), 1 - 7
Genetics of Methanococcus: possibilities for functional genomics in Archaea; Tumbula DL et al.; Although the genomic sequences of a number of Archaea have been completed in the last three years, genetic systems in the sequenced organisms are absent . In contrast, genetic studies of the mesophiles in the archaeal genus Methanococcus have become commonplace following the recent developments of antibiotic resistance markers, DNA transformation methods, reporter genes, shuttle vectors and expression vectors . These developments have led to investigations of the transcription of the genes for hydrogen metabolism, nitrogen fixation and flagellin assembly . These genetic systems can potentially be used to analyse the genomic sequence of the hyperthermophile Methanococcus jannaschii, addressing questions of its physiology and the function of its many uncharacterized open reading frames . Thus, the sequence of M . jannaschii can serve as a starting point for gene isolation, while in vivo genetics in the mesophilic methanococci can provide the experimental systems to test the predictions from genomics.

Science, 1999 Jul 16, 285(5426), 380 - 3
Plant functional genomics; Somerville C et al.; Nucleotide sequencing of the Arabidopsis genome is nearing completion, sequencing of the rice genome has begun, and large amounts of expressed sequence tag information are being obtained for many other plants . There are many opportunities to use this wealth of sequence information to accelerate progress toward a comprehensive understanding of the genetic mechanisms that control plant growth and development and responses to the environment.

Clin Exp Pharmacol Physiol, 1999 Jul, 26(7), 530 - 5
Physiological genetics: application to hypertension research; Jacob HJ; 1 . The rapid advancement of the human genome within the next 5-7 years begins a new era for biological research . The structure of all approximately 100,000 genes will be known, but the function of the majority of these genes will remain unknown . This paper outlines a 'physiological genetics' strategy for determining the genetic basis of hypertension by combining a variety of techniques (e.g . genetics, molecular biology, bioinformatics and physiology), to help identify gene function and the pathways involved in the development of hypertension in the rat . 2 . Using comparative gene mapping, these regions can be used to implicate susceptibility loci for hypertension in humans, resulting in rapid conversion of basic research in animal models to relevant clinical assessment . The present study outlines some new strategies (i.e . whole-animal physiological genetics) as a means to study disease aetiology in polygenic disorders and to facilitate gene identification in the ascent of functional genomics.

Genome Res, 1999 Jun, 9(6), AP1 - 8, insert
A high-density integrated genetic linkage and radiation hybrid map of the laboratory rat; Steen RG et al.; The laboratory rat (Rattus norvegicus) is a key animal model for biomedical research . However, the genetic infrastructure required for connecting phenotype and genotype in the rat is currently incomplete . Here, we report the construction and integration of two genomic maps: a dense genetic linkage map of the rat and the first radiation hybrid (RH) map of the rat . The genetic map was constructed in two F2 intercrosses (SHRSP x BN and FHH x ACI), containing a total of 4736 simple sequence length polymorphism (SSLP) markers . Allele sizes for 4328 of the genetic markers were characterized in 48 of the most commonly used inbred strains . The RH map is a lod >/= 3 framework map, including 983 SSLPs, thereby allowing integration with markers on various genetic maps and with markers mapped on the RH panel . Together, the maps provide an integrated reference to >3000 genes and ESTs and >8500 genetic markers (5211 of our SSLPs and >3500 SSLPs developed by other groups) . {; James and Tanigami, RHdb (); Wilder ();; RATMAP server ()} RH maps (v . 2.0) have been posted on our web sites at or . Both web sites provide an RH mapping server where investigators can localize their own RH vectors relative to this map . The raw data have been deposited in the RHdb database . Taken together, these maps provide the basic tools for rat genomics . The RH map provides the means to rapidly localize genetic markers, genes, and ESTs within the rat genome . These maps provide the basic tools for rat genomics . They will facilitate studies of multifactorial disease and functional genomics, allow construction of physical maps, and provide a scaffold for both directed and large-scale sequencing efforts and comparative genomics in this important experimental organism.

Pac Symp Biocomput . 1999;:41-52.
Linear modeling of mRNA expression levels during CNS development and injury; D'Haeseleer P et al.; Large-scale gene expression data sets are revolutionizing the field of functional genomics . However, few data analysis techniques fully exploit this entirely new class of data . We present a linear modeling approach that allows one to infer interactions between all the genes included in the data set . The resulting model can be used to generate interesting hypotheses to direct further experiments.

FEBS Lett, 1999 Jun 4, 452(1-2), 3 - 6
Plant genomics; Terryn N et al.; The rapidity with which genomic sequences of the model plant Arabidopsis thaliana and soon of rice are becoming available has strongly boosted plant molecular biology research . Here, two main genomic fields will be discussed: the progress in different structural genome projects, such as mapping, sequencing, genome organization and comparative genomics, and the so-called functional genomics approaches to analyze the genome using such molecular tools as transcript profiling, micro-arrays, and insertional mutagenesis . In addition a section on bioinformatics is included.

Chem Biol, 1999 Jun, 6(6), 343 - 51
Cellular delivery of peptide nucleic acids and inhibition of human telomerase; Hamilton SE et al.; BACKGROUND: Human telomerase has an essential RNA component and is an ideal target for developing rules correlating oligonucleotide chemistry with disruption of biological function . Similarly, peptide nucleic acids (PNAs), DNA analogs that bind complementary sequences with high affinity, are outstanding candidates for inducing phenotypic changes through hybridization . RESULTS: We identify PNAs directed to nontemplate regions of the telomerase RNA that can overcome RNA secondary structure and inhibit telomerase by intercepting the RNA component prior to holoenzyme assembly . Relative potencies of inhibition delineate putative structural domains . We describe a novel protocol for introducing PNAs into eukaryotic cells and report efficient inhibition of cellular telomerase by PNAs . CONCLUSIONS: PNAs directed to nontemplate regions are a new class of telomerase inhibitor and may contribute to the development of novel antiproliferative agents . The dependence of inhibition by nontemplate-directed PNAs on target sequence suggests that PNAs have great potential for mapping nucleic acid structure and predictably regulating biological processes . Our simple method for introducing PNAs into cells will not only be useful for probing the complex biology surrounding telomere length maintenance but can be broadly applied for controlling gene expression and functional genomics.

Arch Biochem Biophys, 1999 Jul 1, 367(1), 146 - 50
Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean; Steele CL et al.; The first specific reaction in the biosynthesis of isoflavonoid compounds in plants is the 2-hydroxylation, coupled to aryl migration, of a flavanone . Using a functional genomics approach, we have characterized a cDNA encoding a 2-hydroxyisoflavanone synthase from soybean (Glycine max) . Microsomes isolated from insect cells expressing this cytochrome P450 from a baculovirus vector convert 4', 7-dihydroxyflavanone (liquiritigenin) to 4',7-dihydroxyisoflavone (daidzein), most likely via 2,4',7-trihydroxyisoflavanone which spontaneously dehydrates to daidzein . The enzyme also converts naringenin (4',5,7-trihydroxyflavanone) to genistein, but at a lower rate . 2-Hydroxyisoflavanone synthase transcripts are strongly induced in alfalfa cell suspensions in response to elicitation .

J Biol Chem, 1999 Jun 25, 274(26), 18218 - 30
A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies; de Haard HJ et al.; We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments . Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones . The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested . These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone . In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified . The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore . Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered . The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response . This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

Curr Top Dev Biol, 1999, 45, 1 - 40
Development of the leaf epidermis; Becraft PW; The leaf epidermis is essential to plant survival not only because of its protective role at the interface with the plant's environment but also because of crucial developmental functions . The protoderm is set aside early in embryogenesis, possibly in the zygote . Epidermal identity is determined by the interactions of a complex set of factors, including developmental phase of the plant, regional identity within the leaf, and axiality . For the most part, these characteristics appear to be specified by internal tissues . On the other hand, the epidermis has a key role in regulating organ growth and expansion; thus interactions between the epidermis and internal tissues regulate the overall leaf architecture . Overlying this is the specification of different cell types within the epidermis . Some aspects of this appear to involve interactions with internal tissues but the patterning of many epidermal cell types seems to occur within the two-dimensional field of the epidermis itself and to require both cell signaling and cell lineage dependent mechanisms . Genetic analyses have provided much of the insight into the underlying principles that regulate epidermal development and a number of molecules important for various aspects of the process have been identified . Yet, for the most part, our understanding of the molecular basis for each component of epidermal development is still rudimentary and we have not yet scratched the surface of understanding how these pieces are integrated . The emerging technologies of functional genomics will provide powerful tools for solving these problems and the near future is likely to produce rapid progress.

Nucleic Acids Res, 1999 Jun 1, 27(11), 2354 - 60
A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice; Zheng B et al.; Gene targeting in mouse embryonic stem (ES) cells can be used to generate single gene mutations or defined multi-megabase chromosomal rearrangements when applied with the Cre- loxP recombination system . While single knockouts are essential for uncovering functions of cloned genes, chromosomal rearrangements are great genetic tools for mapping, mutagenesis screens and functional genomics . The conventional approach to generate mice with targeted alterations of the genome requires extensive molecular cloning to build targeting vectors and DNA-based genotyping for stock maintenance . Here we describe the design and construction of a two-library system to facilitate high throughput gene targeting and chromo-somal engineering . The unique feature of these libraries is that once a clone is isolated, it is essentially ready to be used for insertional targeting in ES cells . The two libraries each bear a complementary set of genetic markers tailored so that the vector can be used for Cre- loxP -based chromosome engineering as well as single knockouts . By incorporating mouse coat color markers into the vectors, we illustrate a widely applicable method for stock maintenance of ES cell-derived mice with single gene knockouts or more extensive chromosomal rearrangements.

Drug Discov Today, 1999 Mar, 4(3), 109 - 114
Functional genomics: from genes to new therapies; Dyer MR et al.; The application of genomics in pharmaceutical R&D is presently one of the central issues in the industry . The evolution of functional genomics approaches and their integration into a technology platform for therapeutic discovery is a challenging and complex process . In this review, the authors describe how functional genomics will offer significant opportunities in the search for causal and disease-modifying therapies for better treatment of society's most outstanding medical needs.

Curr Opin Plant Biol, 1999 Apr, 2(2), 109 - 13
Fast forward genetics based on virus-induced gene silencing; Baulcombe DC; Gene expression in plants can be suppressed in a sequence-specific manner by infection with virus vectors carrying fragments of host genes . Recent developments have revealed that the mechanism of this gene silencing is based on an RNA-mediated defence against viruses . It has also emerged that a related mechanism is involved in the post-transcriptional silencing that accounts for between line variation in transgene expression and cosuppresion of transgenes and endogenous genes . The technology of virus-induced gene silencing is being refined and adapted as a high throughput procedure for functional genomics in plants.

Curr Biol, 1999 May 6, 9(9), R338 - 41
Functional genomics: learning to think about gene expression data; Brent R; Three recent studies of gene expression patterns in whole cells provide examples of the inferences one can make from this type of information . They also provide examples of the non-traditional types of reasoning we will need to use to make such inferences.

Int Arch Allergy Immunol, 1999 Feb-Apr, 118(2-4), 282 - 4
Positional candidate gene approach and functional genomics strategy in atopy gene discovery; Nickel RG et al.; As part of our effort in searching for genetic factors contributing to the susceptibility to atopy and asthma, we have focused on a 'positional candidate' approach in identifying CC chemokine gene polymorphisms and their functional correlates . To date, a single-nucleotide polymorphism was found in the RANTES proximal promoter region, and a high degree of sequence variation was identified in the 3'-untranslated region -of the eotaxin gene . Also, we are pursuing a series of functional genomics' studies designed to identify differentially expressed genes in a panel of allergen-specific human Th2 cells and in antigen-induced hyperreactive murine airways . This is performed using a combination of protocols including suppression-subtractive hybridization and cDNA array hybridizations with 18,363 nonredundant sequences . A data base is being generated from a list of subtracted cDNA sequences and array-positive clones to categorize differentially expressed genes . Sequences are being placed in biologically relevant categories on the basis of function (i.e., receptor, signal transduction pathways, transcription, and translation) . With the increasing amount of sequence information compiled by the Human Genome Project, it will be particularly challenging to integrate functional gene-mapping efforts to define and compare aberrant genotypes/phenotypes in atopic diseases.

Nat Biotechnol, 1999 Apr, 17(4), 355 - 9
Linear expression elements: a rapid, in vivo, method to screen for gene functions; Sykes KF et al.; The increasing accumulation of genomic sequence information has accentuated the need for new methods to efficiently assess gene function and to prepare reagents to study these functions . Toward solving this general problem in functional genomics, we report a method by which any PCR-amplified open-reading frame (ORF) can be noncovalently linked to a eukaryotic promoter and terminator, and directly injected into animals to produce local gene expression . We also demonstrate that ORFs can be delivered into mice to produce antibodies specific for the encoded foreign protein by simply attaching mammalian promoter and terminator sequences . This technology makes it possible to screen large numbers of genes rapidly for their functions in vivo or to produce immune responses without the necessity of cloning, bacterial propagation, or protein purification.

Genome Res, 1999 Apr, 9(4), 348 - 59
Functional genomics in Caenorhabditis elegans: An approach involving comparisons of sequences from related nematodes; Thacker C et al.; Comparative genomic analysis was used to investigate the gene structure of the bli-4 locus from two related Caenorhabditis species, C . elegans and C . briggsae . In C . elegans, bli-4 is a complex gene encoding a member of the kex2/subtilisin-like family of proprotein convertases . Genomic sequence comparisons coupled with RT-PCR analysis identified five additional coding exons that had not been identified previously using standard recombinant DNA techniques . The C . briggsae gene was able to rescue both viable blistered and developmentally arrested mutants of C . elegans bli-4, demonstrating functional conservation . In addition, deletion analysis of conserved sequences outside of coding regions, combined with phenotypic rescue experiments, identified regulatory elements that alter the expression of the bli-4 gene . These results demonstrate the utility of genomic sequence comparisons of homologous genes in related species as an effective tool with which to dissect the functional information of complex genes.

Electrophoresis, 1999 Feb, 20(2), 261 - 8
Functional genomics in cancer research: identification of target genes of the Epstein-Barr virus nuclear antigen 2 by subtractive cDNA cloning and high-throughput differential screening using high-density agarose gels; Kaiser C et al.; In the past, the identification and isolation of phenotype-associated genes was a difficult and time-consuming task . However, recent improvements of methods that are designed to isolate differentially expressed genes have remarkably speeded up the process of target gene isolation . The ultimate goal of functional genomics is to apply these technologies to clone phenotype-associated genes irrespective of the availability of probes (e.g., antibodies) and an intimate knowledge of biological background . We demonstrate the use of a novel subtractive cDNA cloning approach for the isolation and characterization of target genes of the Epstein-Barr virus nuclear antigen 2 (EBNA2) . Two different subtractive cDNA libraries specific for two different time periods following activation of a conditional estrogen receptor/EBNA2 (ER/EBNA2) fusion protein were generated . Comparison of the two libraries by cross-hybridization experiments allowed the differentiation between direct and indirect target genes of EBNA2 and led to the identification of a novel direct target gene of EBNA2.

Curr Pharm Des, 1998 Oct, 4(5), 417 - 22
Genomics in the real world; Wiley SR; The term genomics has evolved into a catch-all term for a variety of information intensive biological methodologies . While the promise of genomics in the bio/pharmaceutical industry is great, its impact on the drug discovery pipeline has not yet been realized, excluding a few notable exceptions . As companies acquire several years of experience in working with genomic data, it is likely that the impact on the discovery process will slowly emerge as we learn to integrate these new technologies into individual discovery programs . It is clear that extracting novel biologically valid targets targets from exponentially growing amounts of sequence data requires time and considerable investment in biological research infrastructure . In order to accelerate the process of target validation, a variety of functional genomics technologies are also being developed to try to predict the effect of inhibitory compounds in advance of development . Resources spent on early stage exploratory efforts such as these can pay off by improving the success rate for screening and medicinal chemistry.

Pharmacol Ther, 1999 Feb, 81(2), 91 - 110
An overview of chemotherapeutic targets for antimalarial drug discovery; Olliaro PL et al.; The need for new antimalarials comes from the widespread resistance to those in current use . New antimalarial targets are required to allow the discovery of chemically diverse, effective drugs . The search for such new targets and new drug chemotypes will likely be helped by the advent of functional genomics and structure-based drug design . After validation of the putative targets as those capable of providing effective and safe drugs, targets can be used as the basis for screening compounds in order to identify new leads, which, in turn, will qualify for lead optimization work . The combined use of combinatorial chemistry--to generate large numbers of structurally diverse compounds--and of high throughput screening systems--to speed up the testing of compounds--hopefully will help to optimize the process . Potential chemotherapeutic targets in the malaria parasite can be broadly classified into three categories: those involved in processes occurring in the digestive vacuole, enzymes involved in macromolecular and metabolite synthesis, and those responsible for membrane processes and signalling . The processes occurring in the digestive vacuole include haemoglobin digestion, redox processes and free radical formation, and reactions accompanying haem release followed by its polymerization into haemozoin . Many enzymes in macromolecular and metabolite synthesis are promising potential targets, some of which have been established in other microorganisms, although not yet validated for Plasmodium, with very few exceptions (such as dihydrofolate reductase) . Proteins responsible for membrane processes, including trafficking and drug transport and signalling, are potentially important also to identify compounds to be used in combination with antimalarial drugs to combat resistance.

Trends Biotechnol, 1999 Mar, 17(3), 121 - 7
Proteomics: quantitative and physical mapping of cellular proteins; Blackstock WP et al.; Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function . Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process . The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level . Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.

J Med Chem, 1999 Mar 11, 42(5), 910 - 9
Identification of novel classes of protein kinase inhibitors using combinatorial peptide chemistry based on functional genomics knowledge; Lukas TJ et al.; A discovery approach based on an intramolecular inhibitory mechanism was applied to a prototype calmodulin (CaM)-regulated protein kinase in order to demonstrate a proof-of-principle for the development of selective inhibitors . The overall approach used functional genomics analysis of myosin light chain kinase (MLCK) to identify short autoinhibitory sequences that lack CaM recognition activity, followed by recursive combinatorial peptide library production and comparative activity screens . Peptide 18 (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2), one of several selective inhibitors discovered, has an IC50 = 50 nM for MLCK, inhibits CaM kinase II only at 4000-fold higher concentrations, and does not inhibit cyclic AMP-dependent protein kinase . Analogues of peptide 18 containing conformationally constrained cis-4-aminocyclohexanecarboxylic acid retained affinity and selectivity . The inhibitors add to the armamentarium available for the deconvolution of complex signal transduction pathways and their relationship to homeostasis and disease, and the approach is potentially applicable to enzymes in which the catalytic and regulatory domains are found within the same open reading frame of a cDNA.

Curr Opin Microbiol, 1998 Oct, 1(5), 589 - 97
Bacterial genomics and adaptation to life on plants: implications for the evolution of pathogenicity and symbiosis; Preston GM et al.; Many bacteria form intimate associations with plants . Despite the agricultural and biotechnological significance of these bacteria, no whole genome sequences have yet been described . Plant-associated bacteria form a phylogenetically diverse group, with representative species from many major taxons . Sequence information from genomes of closely related bacteria, in combination with technological developments in the field of functional genomics, provides new opportunities for determining the origin and evolution of traits that contribute to bacterial fitness and interactions with plant hosts.

Chem Biol, 1999 Feb, 6(2), R33 - 7
Ribozymes, genomics and therapeutics; Rossi JJ; Genome-sequencing projects are proceeding at a rapid pace and determining the function of open reading frames is the next great challenge . Ribozymes with site-specific cleaving activity could aid greatly in this process . High-throughput screening methods to identify optimal target sites for ribozyme cleavage will provide tools for functional genomics as well as therapeutic reagents.

Annu Rev Genet, 1998, 32, 601 - 18
Epitope tagging; Jarvik JW et al.; Epitope tagging is a recombinant DNA method by which a protein encoded by a cloned gene is made immunoreactive to a known antibody . This review discusses the major advantages and limitations of epitope tagging and describes a number of recent applications . Major areas of application include monitoring protein expression, localizing proteins at the cellular and subcellular levels, and protein purification, as well as the analysis of protein topology, dynamics and interactions . Recently the method has also found use in transgenic and gene therapy studies and in the emerging fields of functional genomics and proteomics.

Nucleic Acids Res, 1999 Feb 15, 27(4), 1079 - 83
cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes; Candeliere GA et al.; A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development . Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated . Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g . from primitive progenitors, intermediate precursors or mature osteoblasts . EST expression provides insight into possible interrelated physiological functions and putative interacting molecules during differentiation . This method offers a functional genomics approach to isolate differentiation stage-specific genes in samples as small as a single cell.

Br J Pharmacol, 1998 Dec, 125(7), 1387 - 92
Orphan G-protein-coupled receptors: the next generation of drug targets?
Wilson S, Bergsma DJ, Chambers JK, Muir AI, Fantom KG, Ellis C, Murdock PR, Herrity NC, Stadel JM.
The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery . Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets . In most cases the extent of sequence homology is insufficient to assign these 'orphan' receptors to a particular receptor subfamily . Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors . Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections . The functional genomics approach involves the use of 'humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery . Both systems provide an excellent platform for identifying novel receptor ligands . Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.

Microsc Res Tech, 1998 Dec 1, 43(5), 358 - 68
Deconstructing (and reconstructing) cell migration; Maheshwari G et al.; An overriding objective in cell biology is to be able to relate properties of particular molecular components to cell behavioral functions and even physiology . In the "traditional" mode of molecular cell biology, this objective has been tackled on a molecule-by-molecule basis, and in the "future" mode sometimes termed "functional genomics," it might be attacked in a high-throughput, parallel manner . Regardless of the manner of approach, the relationship between molecular-level properties and cell-level function is exceedingly difficult to elucidate because of the large number of relevant components involved, their high degree of interconnectedness, and the inescapable fact that they operate as physico-chemical entities-according to the laws of kinetics and mechanics-in space and time within the cell . Cell migration is a prominent representative example of such a cell behavioral function that requires increased understanding for both scientific and technological advance . This article presents a framework, derived from an engineering perspective regarding complex systems, intended to aid in developing improved understanding of how properties of molecular components influence the function of cell migration . That is, cell population migration behavior can be deconstructed as follows: first in terms of a mathematical model comprising cell population parameters (random motility, chemotaxis/haptotaxis, and chemokinesis/haptokinesis coefficients), which in turn depend on characteristics of individual cell paths that can be analyzed in terms of a mathematical model comprising individual cell parameters (translocation speed, directional persistence time, chemotactic/haptotactic index), which in turn depend on cell-level physical processes underlying motility (membrane extension and retraction, cell/substratum adhesion, cell contractile force, front-vs.-rear asymmetry), which in turn depend on molecular-level properties of the plethora of components involved in governance and regulation of these processes . Hence, the influence of any molecular component on cell population migration can be understood by reconstructing these relationships from the molecular level to the physical process level to the individual cell path level to the cell population distribution level . This approach requires combining experimental, theoretical, and computational methodologies from molecular biology, biochemistry, biophysics, and bioengineering.

Nucleic Acids Res, 1999 Jan 1, 27(1), 373 - 6
The University of Minnesota Biocatalysis/Biodegradation Database: specialized metabolism for functional genomics; Ellis LB et al.; The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, nde x.html) first became available on the web in 1995 to provide information on microbial biocatalytic reactions of, and biodegradation pathways for, organic chemical compounds, especially those produced by man . Its goal is to become a representative database of biodegradation, spanning the diversity of known microbial metabolic routes, organic functional groups, and environmental conditions under which biodegradation occurs . The database can be used to enhance understanding of basic biochemistry, biocatalysis leading to speciality chemical manufacture, and biodegradation of environmental pollutants . It is also a resource for functional genomics, since it contains information on enzymes and genes involved in specialized metabolism not found in intermediary metabolism databases, and thus can assist in assigning functions to genes homologous to such less common genes . With information on >400 reactions and compounds, it is poised to become a resource for prediction of microbial biodegradation pathways for compounds it does not contain, a process complementary to predicting the functions of new classes of microbial genes.

Nucleic Acids Res, 1999 Jan 1, 27(1), 44 - 8
MIPS: a database for genomes and protein sequences; Mewes HW et al.; The Munich Information Center for Protein Sequences (MIPS-GSF), Martinsried near Munich, Germany, develops and maintains genome oriented databases . It is commonplace that the amount of sequence data available increases rapidly, but not the capacity of qualified manual annotation at the sequence databases . Therefore, our strategy aims to cope with the data stream by the comprehensive application of analysis tools to sequences of complete genomes, the systematic classification of protein sequences and the active support of sequence analysis and functional genomics projects . This report describes the systematic and up-to-date analysis of genomes (PEDANT), a comprehensive database of the yeast genome (MYGD), a database reflecting the progress in sequencing the Arabidopsis thaliana genome (MATD), the database of assembled, annotated human EST clusters (MEST), and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database (described elsewhere in this volume) . MIPS provides access through its WWW server to a spectrum of generic databases, including the above mentioned as well as a database of protein families (PROTFAM), the MITOP database, and the all-against-all FASTA database.

Nucleic Acids Res, 1999 Jan 1, 27(1), 39 - 43
The PIR-International Protein Sequence Database; Barker WC et al.; The Protein Information Resource (PIR; edu/pir/) supports research on molecular evolution, functional genomics, and computational biology by maintaining a comprehensive, non-redundant, well-organized and freely available protein sequence database . Since 1988 the database has been maintained collaboratively by PIR-International, an international association of data collection centers cooperating to develop this resource during a period of explosive growth in new sequence data and new computer technologies . The PIR Protein Sequence Database entries are classified into superfamilies, families and homology domains, for which sequence alignments are available . Full-scale family classification supports comparative genomics research, aids sequence annotation, assists database organization and improves database integrity . The PIR WWW server supports direct on-line sequence similarity searches, information retrieval, and knowledge discovery by providing the Protein Sequence Database and other supplementary databases . Sequence entries are extensively cross-referenced and hypertext-linked to major nucleic acid, literature, genome, structure, sequence alignment and family databases . The weekly release of the Protein Sequence Database can be accessed through the PIR Web site . The quarterly release of the database is freely available from our anonymous FTP server and is also available on CD-ROM with the accompanying ATLAS database search program.

Nucleic Acids Res, 1999 Jan 1, 27(1), 35 - 8
The Genome Sequence DataBase: towards an integrated functional genomics resource; Skupski MP et al.; During 1998 the primary focus of the Genome Sequence DataBase (GSDB; ) located at the National Center for Genome Resources (NCGR) has been to improve data quality, improve data collections, and provide new methods and tools to access and analyze data . Data quality has been improved by extensive curation of certain data fields necessary for maintaining data collections and for using certain tools . Data quality has also been increased by improvements to the suite of programs that import data from the International Nucleotide Sequence Database Collaboration (IC) . The Sequence Tag Alignment and Consensus Knowledgebase (STACK), a database of human expressed gene sequences developed by the South African National Bioinformatics Institute (SANBI), became available within the last year, allowing public access to this valuable resource of expressed sequences . Data access was improved by the addition of the Sequence Viewer, a platform-independent graphical viewer for GSDB sequence data . This tool has also been integrated with other searching and data retrieval tools . A BLAST homology search service was also made available, allowing researchers to search all of the data, including the unique data, that are available from GSDB . These improvements are designed to make GSDB more accessible to users, extend the rich searching capability already present in GSDB, and to facilitate the transition to an integrated system containing many different types of biological data.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14628 - 33
Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks; Beerli RR et al.; To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences . Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5'-GNN-3' subset of a 64-member zinc finger alphabet . Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences . A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5'-(GNN)6-3' family of 18-bp DNA sites . To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model . A polydactyl protein specifically recognizing an 18-bp sequence in the 5'-untranslated region of this gene was converted into a transcriptional repressor by fusion with Kr uppel-associated box (KRAB), ERD, or SID repressor domains . Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64 . We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene . We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

Immunol Today, 1998 Nov, 19(11), 524 - 7
Tapping the potential of molecular libraries in functional genomics; Borrebaeck CA; Functional genomics is facing the challenge of functionally identifying thousands of genes generated by the various genome projects during the past decade . Success will require novel high-capacity technologies that can cope with very large numbers of molecules . Here, Carl Borrebaeck reviews recent developments in molecular libraries and their role in rational gene identification.

Bioinformatics, 1998, 14(8), 656 - 64
GeneCards: a novel functional genomics compendium with automated data mining and query reformulation support; Rebhan M et al.; MOTIVATION: Modern biology is shifting from the 'one gene one postdoc' approach to genomic analyses that include the simultaneous monitoring of thousands of genes . The importance of efficient access to concise and integrated biomedical information to support data analysis and decision making is therefore increasing rapidly, in both academic and industrial research . However, knowledge discovery in the widely scattered resources relevant for biomedical research is often a cumbersome and non-trivial task, one that requires a significant amount of training and effort . RESULTS: To develop a model for a new type of topic-specific overview resource that provides efficient access to distributed information, we designed a database called 'GeneCards' . It is a freely accessible Web resource that offers one hypertext 'card' for each of the more than 7000 human genes that currently have an approved gene symbol published by the HUGO/GDB nomenclature committee . The presented information aims at giving immediate insight into current knowledge about the respective gene, including a focus on its functions in health and disease . It is compiled by Perl scripts that automatically extract relevant information from several databases, including SWISS-PROT, OMIM, Genatlas and GDB . Analyses of the interactions of users with the Web interface of GeneCards triggered development of easy-to-scan displays optimized for human browsing . Also, we developed algorithms that offer 'ready-to-click' query reformulation support, to facilitate information retrieval and exploration . Many of the long-term users turn to GeneCards to quickly access information about the function of very large sets of genes, for example in the realm of large-scale expression studies using 'DNA chip' technology or two-dimensional protein electrophoresis . Availability: Freely available at Contact: cards@bioinformatics.weizmann.ac.il

Science, 1998 Oct 23, 282(5389), 682 - 9
New goals for the U.S . Human Genome Project: 1998-2003; Collins FS et al.; The Human Genome Project has successfully completed all the major goals in its current 5-year plan, covering the period 1993-98 . A new plan, for 1998-2003, is presented, in which human DNA sequencing will be the major emphasis . An ambitious schedule has been set to complete the full sequence by the end of 2003, 2 years ahead of previous projections . In the course of completing the sequence, a "working draft" of the human sequence will be produced by the end of 2001 . The plan also includes goals for sequencing technology development; for studying human genome sequence variation; for developing technology for functional genomics; for completing the sequence of Caenorhabditis elegans and Drosophila melanogaster and starting the mouse genome; for studying the ethical, legal, and social implications of genome research; for bioinformatics and computational studies; and for training of genome scientists.

Trends Biotechnol, 1998 Sep, 16(9), 373 - 8
Systematic functional analysis of the yeast genome; Oliver SG et al.; The genome sequence of the yeast Saccharomyces cerevisiae has provided the first complete inventory of the working parts of a eukaryotic cell . The challenge is now to discover what each of the gene products does and how they interact in a living yeast cell . Systematic and comprehensive approaches to the elucidation of yeast gene function are discussed and the prospects for the functional genomics of eukaryotic organisms evaluated.

Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10769 - 73
Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells; Luo G et al.; Mouse has become an increasingly important organism for modeling human diseases and for determining gene function in a mammalian context . Unfortunately, transposon-tagged mutagenesis, one of the most valuable tools for functional genomics, still is not available in this organism . On the other hand, it has long been speculated that members of the Tc1/mariner-like elements may be less dependent on host factors and, hence, can be introduced into heterologous organisms . However, this prediction has not been realized in mice . We report here the chromosomal transposition of the Sleeping Beauty (SB) element in mouse embryonic stem cells, providing evidence that it can be used as an in vivo mutagen in mice.

Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 1998 Aug, 15(4), 195 - 7
{Chinese Human Genome Project--opportunity and challenge}; Chen Z et al.; The Chinese Human Genome Project(HGP) started in 1993 and has since made substantial progress in 3 major aspects: the genome resource conservation and genetic polymorphism studies of multiple Chinese nationalities; the development of an advanced technological system for genome research; the cloning of some desease-related genes and a large number of expressed sequence tags(ESTs) . At the same time a qualified research team has been formed and some centers for genome studies have been founded in the country . The major tasks in the next phase of the project are: (1) Further expanding the genetic material conservation scope including collection of more DNA samples and establishment of more immortalized cell lines; (2) Developing morbid genomics which includes epidemiological survey and registration, collection, maintenance and banking of the data and genetic material resource; making significant progress in mapping, cloning, mutation screening, and large-scale sequencing of the genes related to tumors, inheritable diseases and particularly multigenic diseases in the hope that a number of genes for important diseases will be obtained; (3) Initiation of the functional genomics which includes studies on bioinformatics, expression spectrum of transcription and translation of the genome in physiological and pathological processes; (4) Enhancing the studies on the ethical, legal and social issues related to human genome sciences.

Curr Opin Genet Dev, 1998 Jun, 8(3), 311 - 5
Genomic strategies for defining and dissecting developmental and physiological pathways; Nadeau JH et al.; A major challenge in genetics research is defining and dissecting the diversity of developmental and physiological pathways that lie between genes and traits . New functional genomics methods are transforming these studies by providing comprehensive and systematic approaches that complement traditional methods of formal genetics, biochemistry, and cell biology . Together, these complementary approaches will test whether reductionism can account for the complex web of interactions that lead from genetic variation to morphological, physiological, and behavioral traits in health and disease.

Mutat Res, 1998 May 25, 400(1-2), 25 - 32
Large scale ENU screens in the mouse: genetics meets genomics; Hrabe de Angelis M et al.; The worldwide effort to completely sequence the human and mouse genome will be accomplished within the next years . The focus of current activities within the framework of human genome research is mainly on the assembly of high resolution genetic and physical maps and genomic sequencing . Cloning of new genes is getting more easy using those maps . Nevertheless, it is necessary to work on a systematic analysis of gene function . Results obtained from these efforts will be of enormous value for future biological and biomedical research . However, even the complete sequence will not in all cases reveal the molecular and cellular role of the different genes . Therefore, the next phase of the Human Genome Project will have at its core the functional analysis of genes . Those genes relevant for the diagnosis, prevention and therapy of human diseases are of particular interest . Looking at the history of life sciences, mutants have been the most important tool to obtain insight into the biological function of genes . The mouse is the model of choice for the study of inherited diseases in man . In order to meet the requirements for functional human genome analysis, we need a large number of mouse mutants similar to the collection of mutants available for other model organisms such as flys and worms . To fully apply the power of genetics, multiple alleles of the same gene such as hypomorphs or hypermorphs are required . Efficient production of mouse mutants showing specific phenotypes can be achieved by the use of ethylnitrosourea (ENU) . ENU is the most powerful mutagen known and we currently see a renaissance of ENU mutagenesis . The application of ENU mutagenesis is reviewed and discussed in the context of a new era of functional genomics .

Mutat Res, 1998 May 25, 400(1-2), 3 - 14
Functional genomics in the post-genome era; Woychik RP et al.; As the biomedical research community enters the post-genome era, studying gene expression patterns and phenotypes in model organisms will be an important part of analyzing the role of genes in human health and disease . New technologies involving DNA chips will improve the ability to evaluate the differential expression of a large number of genes simultaneously . Also, new approaches for generating mutations in mice will significantly decrease the cost and increase the rate of generating mutant lines that model human disease .

Genome Res, 1998 Jul, 8(7), 698 - 710
Functional genomics in the mouse: phenotype-based mutagenesis screens; Schimenti J et al.; Significant progress has been made in sequencing the genomes of several model organisms, and efforts are now underway to complete the sequencing of the human genome . In parallel with this effort, new approaches are being developed for the elucidation of the functional content of the human genome . The mouse will have an important role in this phase of the genome project as a model system . In this review we discuss and compare classical genetic approaches to gene function-phenotype-based mutagenesis screens aimed at the establishment of a large collection of single gene mutations affecting a wide range of phenotypic traits in the mouse . Whereas large scale genome-wide screens that are directed at the identification of all loci contributing to a specific phenotype may be impractical, region-specific saturation screens that provide mutations within a delimited chromosomal region are a feasible alternative . Region-specific screens in the mouse can be performed in only two generations by combining high-efficiency chemical mutagenesis with deletion complexes generated using embryonic stem (ES) cells . The ability to create and analyze deletion complexes rapidly, as well as to map novel chemically-induced mutations within these complexes, will facilitate systematic functional analysis of the mouse genome and corresponding gene sequences in humans . Furthermore, as the extent of the mouse genome sequencing effort is still uncertain, we underscore a necessity to direct sequencing efforts to those chromosomal regions that are targets for extensive mutagenesis screens.

FEBS Lett, 1998 Jun 23, 430(1-2), 55 - 8
Functional genomics: the search for novel neurotransmitters and neuropeptides; Civelli O; Functional genomics can be defined as the search for the physiological role of a gene for which only its primary sequence is known . Most of the genes encoding proteins containing seven hydrophobic stretches code for G protein-coupled receptors (GPCRs) . Although many of these have been shown to interact with known natural ligands, several bind ligands which have not been thus far isolated . These are the so-called orphan GPCRs . As an example of functional genomics, an 'orphan receptor strategy' has been developed to identify the natural ligands of orphan GPCRs . The application of this strategy is bound to revolutionize our understanding of the diversity of the primary messengers which modulate synaptic transmission . This review discusses the basic concepts and some of the particular problems associated with the orphan receptor strategy . The strategy's potential is exemplified by its successes which culminated in the discovery of the neuropeptides 'orphanin FQ/nociceptin' and 'orexins/hypocretins' . The steps that led to the characterization of these neuropeptides are discussed as are some of the further studies that have addressed the roles of these neuropeptides . To conclude, some of the implications of the application of the orphan receptor strategy are discussed.

Trends Biotechnol, 1998 Jul, 16(7), 301 - 6
Microarrays: biotechnology's discovery platform for functional genomics; Schena M et al.; Advances in microarray technology enable massive parallel mining of biological data, with biological chips providing hybridization-based expression monitoring, polymorphism detection and genotyping on a genomic scale . Microarrays containing sequences representative of all human genes may soon permit the expression analysis of the entire human genome in a single reaction . These 'genome chips' will provide unprecedented access to key areas of human health, including disease prognosis and diagnosis, drug discovery, toxicology, aging, and mental illness . Microarray technology is rapidly becoming a central platform for functional genomics.

Adv Biochem Eng Biotechnol, 1998, 61, 87 - 115
Molecular biology of hyperthermophilic Archaea; van der Oost J et al.; The sequences of a number of archaeal genomes have recently been completed, and many more are expected shortly . Consequently, the research of Archaea in general and hyperthermophiles in particular has entered a new phase, with many exciting discoveries to be expected . The wealth of sequence information has already led, and will continue to lead to the identification of many enzymes with unique properties, some of which have potential for industrial applications . Subsequent functional genomics will help reveal fundamental matters such as details concerning the genetic, biochemical and physiological adaptation of extremophiles, and hence give insight into their genomic evolution, polypeptide structure-function relations, and metabolic regulation . In order to optimally exploit many unique features that are now emerging, the development of genetic systems for hyperthermophilic Archaea is an absolute requirement . Such systems would allow the application of this class of Archaea as so-called "cell factories": (i) expression of certain archaeal enzymes for which no suitable conventional (mesophilic bacterial or eukaryal) systems are available, (ii) selection for thermostable variants of potentially interesting enzymes from mesophilic origin, and (iii) the development of in vivo production systems by metabolic engineering . An overview is given of recent insight in the molecular biology of hyperthermophilic Archaea, as well as of a number of promising developments that should result in the generation of suitable genetic systems in the near future.

Gene, 1998 Jul 17, 215(1), 143 - 52
Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map; Hua SB et al.; Identification of all human protein-protein interactions will lead to a global human protein linkage map that will provide important information for functional genomics studies . The yeast two-hybrid system is a powerful molecular genetic approach for studying protein-protein interactions . To apply this technology to generate a human protein linkage map, the first step is to construct two-hybrid cDNA libraries that cover the entire human genome . With a homologous recombination-mediated approach, we have constructed a modular human EST-derived yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2 . Quality analysis of this library indicated that the approach of constructing two-hybrid cDNA libraries from individually arrayed human EST clones is feasible, and such a two-hybrid library is suitable for detecting protein-protein interactions . This is also the first time that a comprehensive two-hybrid system cDNA library has been constructed from a collection of individually arrayed EST clones.

Anal Biochem, 1998 Jun 15, 260(1), 1 - 17
The detection of enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Bischoff KM et al.; More than a hundred different enzymes impinging on aspects of cell function ranging from carbohydrate and lipid metabolism to signal transduction and gene expression to biomolecule degradation have been detected by the assay of their enzymatic activities following SDS-PAGE . The strategies by which this has been accomplished are as varied as the enzymes themselves and offer testimony to the creativeness and ingenuity of life scientists . Assay of enzyme activity following SDS-PAGE is well adapted to identifying the source of catalytic activity in a heterogeneous protein mixture or a heterooligomeric protein (20), or determining if multiple catalytic activities reside in a single polypeptide (60) . The alliance of versatile enzyme assay techniques with the molecular resolution of SDS-PAGE offers a powerful means for meeting the increasing demand for the high-throughput screening arising from protein engineering, combinatorial chemistry, and functional genomics.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3731 - 6
Data transferability from model organisms to human beings: insights from the functional genomics of the flightless region of Drosophila; Maleszka R et al.; At what biological levels are data from single-celled organisms akin to a Rosetta stone for multicellular ones? To examine this question, we characterized a saturation-mutagenized 67-kb region of the Drosophila genome by gene deletions, transgenic rescues, phenotypic dissections, genomic and cDNA sequencing, bio-informatic analysis, reverse transcription-PCR studies, and evolutionary comparisons . Data analysis using cDNA/genomic DNA alignments and bio-informatic algorithms revealed 12 different predicted proteins, most of which are absent from bacterial databases, half of which are absent from Saccharomyces cerevisiae, and nearly all of which have relatives in Caenorhabditis elegans and Homo sapiens . Gene order is not evolutionarily conserved; the closest relatives of these genes are scattered throughout the yeast, nematode, and human genomes . Most gene expression is pleiotropic, and deletion studies reveal that a morphological phenotype is seldom observed when these genes are removed from the genome . These data pinpoint some general bottlenecks in functional genomics, and they reveal the acute emerging difficulties with data transferability above the levels of genes and proteins, especially with complex human phenotypes . At these higher levels the Rosetta stone analogy has almost no applicability . However, newer transgenic technologies in Drosophila and Mus, combined with coherency pattern analyses of gene networks, and synthetic neural modeling, offer insights into organismal function . We conclude that industrially scaled robogenomics in model organisms will have great impact if it can be realistically linked to epigenetic analyses of human variation and to phenotypic analyses of human diseases in different genetic backgrounds.

Protein Sci, 1998 Jan, 7(1), 201 - 5
How many membrane proteins are there?
Boyd D, Schierle C, Beckwith J.
One of the basic issues that arises in functional genomics is the ability to predict the subcellular location of proteins that are deduced from gene and genome sequencing . In particular, one would like to be able to readily specify those proteins that are soluble and those that are inserted in a membrane . Traditional methods of distinguishing between these two locations have relied on extensive, time-consuming biochemical studies . The alternative approach has been to make inferences based on a visual search of the amino acid sequences of presumed gene products for stretches of hydrophobic amino acids . This numerical, sequence-based approach is usually seen as a first approximation pending more reliable biochemical data . The recent availability of large and complete sequence data sets for several organisms allows us to determine just how accurate such a numerical approach could be, and to attempt to minimize and quantify the error involved . We have optimized a statistical approach to protein location determination . Using our approach, we have determined that surprisingly few proteins are misallocated using the numerical method . We also examine the biological implications of the success of this technique.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2021 - 6
Functional genomics: probing plant gene function and expression with transposons
Martienssen RA.
Transposable elements provide a convenient and flexible means to disrupt plant genes, so allowing their function to be assessed . By engineering transposons to carry reporter genes and regulatory signals, the expression of target genes can be monitored and to some extent manipulated . Two strategies for using transposons to assess gene function are outlined here: First, the PCR can be used to identify plants that carry insertions into specific genes from among pools of heavily mutagenized individuals (site-selected transposon mutagenesis) . This method requires that high copy transposons be used and that a relatively large number of reactions be performed to identify insertions into genes of interest . Second, a large library of plants, each carrying a unique insertion, can be generated . Each insertion site then can be amplified and sequenced systematically . These two methods have been demonstrated in maize, Arabidopsis, and other plant species, and the relative merits of each are discussed in the context of plant genome research.

Curr Opin Biotechnol, 1998 Feb, 9(1), 35 - 42
The polymerase chain reaction: from functional genomics to high-school practical classes; Taylor GR et al.; After a decade of intensive use as an in vitro alternative to cloning DNA, PCR is now well established as the default method for DNA and RNA analysis . Recent developments have consolidated this position by the introduction of more robust formats, improvements in thermal cyclers and labelling and detection methods . The trend is towards increasing automation, although comparatively few diagnostic kits based on PCR are in wide use . At the same time the applications of PCR are being extended with modifications such as long, accurate PCR and arrayed oligonucleotides or expressed sequences.

Biotechnol Prog, 1998 Jan-Feb, 14(1), 8 - 20
Mathematical modeling and analysis in biochemical engineering: past accomplishments and future opportunities; Bailey JE; This is a personal commentary on the history and future prospects of mathematical modeling and analysis in biochemical engineering . Major transitions in these fields were driven by the appearance of the Aiba, Humphrey, and Millis text, Fredrickson's guidance on conceptualizing mathematical representations of cell populations, and Ramkrishna's development of the cybernetic modeling approach . The value of mathematical models to organize data, to consider interactions in complex systems in a rational way, to correct the conventional wisdom, and to understand essential qualitative features of biological systems has been clearly documented in prior research . The impact of this research in biotechnology discovery has so far been limited, but this will change in the future if we are adept in recognizing emerging opportunities and in integrating new concepts and tools into our research . Mathematical structures and methods, allied with extraordinary contemporary computing power, are essential to the emerging field of functional genomics . Important in this quest is a hierarchy of powerful modeling, analysis, and computational tools which can capture essential quantitative features of available experimental data and use these effectively for analysis and design of metabolism.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 334 - 9
Large-scale temporal gene expression mapping of central nervous system development; Wen X et al.; We used reverse transcription-coupled PCR to produce a high-resolution temporal map of fluctuations in mRNA expression of 112 genes during rat central nervous system development, focusing on the cervical spinal cord . The data provide a temporal gene expression "fingerprint" of spinal cord development based on major families of inter- and intracellular signaling genes . By using distance matrices for the pair-wise comparison of these 112 temporal gene expression patterns as the basis for a cluster analysis, we found five basic "waves" of expression that characterize distinct phases of development . The results suggest functional relationships among the genes fluctuating in parallel . We found that genes belonging to distinct functional classes and gene families clearly map to particular expression profiles . The concepts and data analysis discussed herein may be useful in objectively identifying coherent patterns and sequences of events in the complex genetic signaling network of development . Functional genomics approaches such as this may have applications in the elucidation of complex developmental and degenerative disorders.

Curr Opin Genet Dev, 1997 Dec, 7(6), 777 - 83
Functional genomics: going forwards from the databases; Rastan S et al.; Extrapolating systematically from gene sequence to function is undoubtedly the major challenge facing industry and academia alike as we approach the end of the millennium . Many electronic and laboratory approaches are being developed to meet this challenge but the rate of evolution of these is not keeping pace with the speed of sequence generation.

Nippon Rinsho, 1998 Jan, 56(1), 224 - 32
{Trends of genomic researches for drug discoveries}; Nemoto Y; In drug discoveries it becomes more important to search and validate target genes that control diseases . The development of novel strategies for functional genomics is indispensable for large scale analysis of human genes . (1) Recently, several research projects sequencing 5'-end of full-length enriched cDNA libraries were started because most of expressed sequence tags(ESTs) lack the 5'-end information of mRNA that is necessary for identifying the correct coding region . (2) New key technologies to quantify numerous gene expression levels simultaneously are now in progress . (3) Toward the functional analysis of unpredictable human genes, new strategies by using model animals (mouse, yeast, nematode) are under construction . Advances in molecular genetics and functional genomics will create new challenges for both drug discoveries and clinical evaluations.

Nat Biotechnol, 1997 Nov, 15(12), 1257 - 9
The simplicity of complex MACs; Vos JM; The development of mammalian artificial chromosomes (MACs) would be useful for biotechnology and biomedicine, including their use in functional genomics, transgenic animals and gene therapy . By analogy to large cloning systems in microorganisms, MACs may be engineered using endogenous chromosomal elements such as the yeast-based artificial chromosomes (YACs), or exogenous extra-chromosomal components derived from viruses and other cellular parasites such as the bacterial-based artificial chromosomes (BACs) and p1 artificial chromosomes (PACs).

Electrophoresis, 1997 Aug, 18(8), 1259 - 313
Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12; Link AJ et al.; Mining the emerging abundance of microbial genome sequences for hypotheses is an exciting prospect of "functional genomics" . At the forefront of this effort, we compared the predictions of the complete Escherichia coli genomic sequence with the observed gene products by assessing 381 proteins for their mature N-termini, in vivo abundances, isoelectric points, molecular masses, and cellular locations . Two-dimensional gel electrophoresis (2-DE) and Edman sequencing were combined to sequence Coomassie-stained 2-DE spots representing the abundant proteins of wild-type E . coli K-12 strains . Greater than 90% of the abundant proteins in the E . coli proteome lie in a small isoelectric point and molecular mass window of 4-7 and 10-100 kDa, respectively . We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone . Of the 223 uniquely identified loci, 60% of the encoded proteins are proteolytically processed . As previously reported, the initiator methionine was efficiently cleaved when the penultimate amino acid was serine or alanine . In contrast, when the penultimate amino acid was threonine, glycine, or proline, cleavage was variable, and valine did not signal cleavage . Although signal peptide cleavage sites tended to follow predicted rules, the length of the putative signal sequence was occassionally greater than the consensus . For proteins predicted to be in the cytoplasm or inner membrane, the N-terminal amino acids were highly constrained compared to proteins localized to the periplasm or outer membrane . Although cytoplasmic proteins follow the N-end rule for protein stability, proteins in the periplasm or outer membrane do not follow this rule; several have N-terminal amino acids predicted to destabilize the proteins . Surprisingly, 18% of the identified 2-DE spots represent isoforms in which protein products of the same gene have different observed pI and M(r), suggesting they are post-translationally processed . Although most of the predicted and observed values for isoelectric point and molecular mass show reasonable concordance, for several proteins the observed values significantly deviate from the expected values . Such discrepancies may represent either highly processed proteins or misinterpretations of the genomic sequence . Our data suggest that AhpC, CspC, and HdeA exist as covalent homomultimers, and that IcdA exists as at least three isoforms even under conditions in which covalent modification is not predicted . We enriched for proteins based on subcellular location and found several proteins in unexpected subcellular locations.

Trends Genet, 1997 Sep, 13(9), 370 - 4
Gene trapping and functional genomics; Evans MJ et al.; Classical genetics depends upon investigation of function by random destruction with little information on structure . Modern mapping using random polymorphisms, cloning and sequencing investigates structure without function . The genome projects with their rapid gene discovery are, however, redefining classical genetic approaches . The efficient translation of this wealth of new information into insights in biological function at molecular, cellular and organismal levels requires large-scale approaches to the generation of mutants . Gene trapping in embryonic stem (ES) cells allows an efficient approach to the functional analysis of the murine genome . The usually separate processes of gene discovery, mapping, the observation of the expression pattern and the mutant phenotype in vivo, can be integrated by the use of an indexed library of insertionally mutated ES cell clones . It should be possible to generate mutants for a large proportion of the genes of the mammalian genome.

Nat Genet, 1997 Aug, 16(4), 338 - 44
Functional genomics in mice by tagged sequence mutagenesis; Hicks GG et al.; Most mammalian genes will soon be characterized as cDNA sequences with little information about their function . To utilize this sequence information for large-scale functional studies, a gene trap retrovirus shuttle vector has been developed to disrupt genes expressed in murine embryonic stem (ES) cells . A library of mutant clones was isolated, and regions of genomic DNA adjacent to 400 independent provirus inserts were cloned and sequenced . The flanking sequences, designated 'promoter-proximal sequence tags', or PSTs, identified 63 specific genes and anonymous cDNAs disrupted as a result of virus integration . The efficiency of tagged sequence mutagenesis suggests that many of the 10,000-20,000 genes expressed in ES cells can be targeted, providing defined mutations for the analysis of gene functions in vivo . In addition, PSTs provide the first expressed sequence tags derived from genomic DNA, and define gene features such as exon boundaries and promoters that are missing from cDNA sequences.

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