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At least 109 records · Page 6Linked to original sources
PubMed2005

Gene knockdown by large circular antisense for high-throughput functional genomics.

Single-stranded genomic DNA of recombinant M13 phages was tested as an antisense molecule and examined for its usefulness in high-throughput functional genomics. cDNA fragments of various genes (TNF-alpha, c-myc, c-myb, cdk2 and cdk4) were independently cloned into phagemid vectors. Using the life cycle of M13 bacteriophages, large circular (LC)-molecules, antisense to their respective genes, were prepared from the culture supernatant of bacterial transformants. LC-antisense molecules exhibited enhanced stability, target specificity and no need for target-site searches. High-throughput functional genomics was then attempted with an LC-antisense library, which was generated by using a phagemid vector that incorporated a unidirectional subtracted cDNA library derived from liver cancer tissue. We identified 56 genes involved in the growth of these cells. These results indicate that an antisense sequence as a part of single-stranded LC-genomic DNA of recombinant M13 phages exhibits effective antisense activity, and may have potential for high-throughput functional genomics.

Animals↗
PubMed2005

Synchrotron radiation circular dichroism spectroscopy of proteins and applications in structural and functional genomics.

The technique of Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy and its advantages over conventional circular dichroism spectroscopy are described in this tutorial review, as well as recent applications of the technique in structural and functional genomics. Circular dichroism (CD) spectroscopy is a well-established method in biological chemistry and structural biology, but its utility can be limited by the low flux of the light source in the far ultraviolet and vacuum ultraviolet wavelength regions in conventional CD instruments. The development of synchrotron radiation circular dichroism (SRCD), using the intense light of a synchrotron beam, has greatly expanded the utility of the method, especially as a tool for both structural and functional genomics. These applications take advantage of the enhanced features of SRCD relative to conventional CD: the ability to measure lower wavelength data containing more electronic transitions and hence more structural information, the higher signal-to-noise hence requiring smaller samples, the higher intensity enabling measurements in absorbing buffers and in the presence of lipids and detergents, and the ability to do faster measurements enabling high throughput and time-resolved spectroscopy.This article discusses recent developments in SRCD instrumentation, software, sample preparation and methods of analyses, with particular emphasis on their applications to the study of proteins. These advances have led to new applications in structural genomics (SG), including the potential for fold recognition as a means of target selection and the examination of membrane proteins, a class of proteins usually excluded from SG programmes. Other SG uses include detection of macromolecular interactions as a screen for complex formation, and examination of glycoproteins and sugar components. In functional genomics (FG) new applications include screening for ligand binding as a means of identifying function, and examination of structural differences in mutant proteins as a means of gaining insight into function.

Circular Dichroism↗
PubMed2006

Use of geminiviral vectors for functional genomics.

Virus-induced gene silencing (VIGS) can be used to study the function of a gene by downregulating its expression and analyzing the resulting phenotype. VIGS is a handy tool that is less time consuming and labor intensive than other methods for generating mutants. Geminiviruses are particularly convenient and valuable choices as VIGS vectors in functional genomics. The small size of their DNA genome, the simplicity of the methods for inoculation, their wide host range and their conserved genome organization are just a few of the advantageous characteristics that this group of viruses has to offer. Geminivirus-based vectors have proved to be very efficient in VIGS systems, and further development of these systems will most probably permit their application in studies of the functional genomics of important crops that are recalcitrant to other forms of analysis.

Geminiviridae↗
PubMed2003

Functional genomics in the study of autoimmune diseases.

Functional genomics can be defined as a series of new biotechnological tools based on whole-genome information that can be used to understand basic mechanisms of gene regulation, structure and function. Most of these tools have been used and modeled in research of lower organisms such as the yeast C. Elegans. The time has come for their use in human genome research and there is hope that the new technologies and possibilities will provide with new knowledge on disease pathogenesis and the development of new drugs.

Autoimmune Diseases↗
PubMed2002

Functional genomics as applied to mapping transcription regulatory networks.

The sequencing of the human genome and the entire genomes of many model organisms has resulted in the identification of many genes. Many large-scale experiments for generating gene disruptions and analyzing the phenotypes are underway to ascertain gene function. A future challenge will be to determine interaction and regulation of all the genes of an organism. Recent advances in functional genomic technology have begun to shine light on such gene network problems at both transcriptomic and proteomic levels. Functional genomics will not only elucidate what the genes do, but will also help determine when, where and how they are expressed as an orchestrated system. In this review, we discuss the functional genomics approaches to extract knowledge about transcription regulatory mechanisms from combinations of sequence data, microarray data and ChIP data. We focus in particular on the budding yeast Saccharomyces cerevisiae.

Gene Expression Regulation, Fungal↗
PubMed2000

A novel functional genomics approach identifies mTERT as a suppressor of fibroblast transformation.

As a tool for functional genomics, a hairpin ribozyme gene library with randomized target recognition sequences was constructed in a retroviral vector. This library has the potential to target and cleave any possible RNA substrate. Mouse fibroblasts transduced with this ribozyme gene vector library were selected in a focus formation assay to isolate in vivo functional ribozymes that promote cell transformation in tissue culture. After two successive rounds of selection by focus formation assay, a transforming ribozyme (Rz007) was identified. The sequence of this ribozyme was used to identify the putative target genes responsible for the transformation. A candidate gene target for Rz007 encodes telomerase reverse transcriptase (mTERT). Both mRNA level and enzymatic activity of mTERT were down-regulated in Rz007-transformed cells. Furthermore, newly designed ribozymes, recognizing other potential ribozyme cleavage sites unique to the mTERT mRNA, also cause cell transformation, thus validating the role of mTERT in suppressing the transformation phenotype. These surprising results suggest that the commonly accepted role of telomerase in maintaining cellular immortalization is more complicated than previously thought. These studies also demonstrate the utility of this novel 'reverse' functional genomics approach, enabling the targeted discovery of genes, whether previously known or not, that are involved in any selectable phenotype.

3T3 Cells↗
PubMed2006

Functional genomic methodologies.

The ability to form tenable hypotheses regarding the neurobiological basis of normative functions as well as mechanisms underlying neurodegenerative and neuropsychiatric disorders is often limited by the highly complex brain circuitry and the cellular and molecular mosaics therein. The brain is an intricate structure with heterogeneous neuronal and nonneuronal cell populations dispersed throughout the central nervous system. Varied and diverse brain functions are mediated through gene expression, and ultimately protein expression, within these cell types and interconnected circuits. Large-scale high-throughput analysis of gene expression in brain regions and individual cell populations using modern functional genomics technologies has enabled the simultaneous quantitative assessment of dozens to hundreds to thousands of genes. Technical and experimental advances in the accession of tissues, RNA amplification technologies, and the refinement of downstream genetic methodologies including microarray analysis and real-time quantitative PCR have generated a wellspring of informative studies pertinent to understanding brain structure and function. In this review, we outline the advantages as well as some of the potential challenges of applying high throughput functional genomics technologies toward a better understanding of brain tissues and diseases using animal models as well as human postmortem tissues.

Animals↗
PubMed2006

Functional genomics in Dictyostelium: MidA, a new conserved protein, is required for mitochondrial function and development.

Genomic sequencing has revealed a large number of evolutionary conserved genes of unknown function. In the absence of characterized functional domains, the discovery of the role of these genes must rely on experimental approaches. We have selected 30 Dictyostelium discoideum genes of unknown function that showed high similarity to uncharacterized human genes and were absent in the complete proteomes from Saccharomyces cerevisiae and S. pombe. No putative functional motifs were found in their predicted encoded proteins. Eighteen genes were successfully knocked-out and three of them showed obvious phenotypes. A detailed analysis of one of them, midA, is presented in this report. Disruption of midA in Dictyostelium leads to pleiotropic defects. Cell size, growth rate, phagocytosis and macropinocytosis were affected in the mutant. During development, midA- cells showed an enhanced tendency to remain at the slug stage, and spore viability was compromised. The expression of MidA fused to GFP in midA- strain rescued the phenotype and the fused protein was located in the mitochondria. Although cellular oxygen consumption, mitochondrial content and mitochondrial membrane potential were similar to wild type, the amount of ATP was significantly reduced in the mutant suggesting a mitochondrial dysfunction. Metabolomic analysis by natural-abundance 13C-nuclear magnetic resonance has shown the lack of glycogen accumulation during growth. During starvation, mutant cells accumulated higher levels of ammonia, which inhibited normal development. We hypothesize that the lack of MidA reduces mitochondrial ATP synthetic capacity and this has an impact in some but not all energy-dependent cellular processes. This work exemplifies the potential of Dictyostelium as a model system for functional genomic studies.

Amino Acid Sequence↗
PubMed2005

Agrobacterium-mediated transformation as a tool for functional genomics in fungi.

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi.

Bacterial Proteins↗
PubMed2003

The modular era of functional genomics.

A report on the Keystone Symposium 'Functional Genomics: Global Analysis of Complex Biological Systems', Santa Fe, USA, 20-24 February 2003.

Gene Expression Regulation↗
PubMed2002

Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics?

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.

Amides↗
PubMed2002

Functional genomics: high-throughput mRNA, protein, and metabolite analyses.

A tremendous amount of DNA sequence information is now available to scientists and engineers. These DNA sequences provide the foundation for studying how the genome of an organism is functioning and they are particularly useful for metabolic engineers interested in manipulating plants for the production of chemicals and enzymes. Functional genomics relies on high-throughput techniques for measuring the mRNA (the transcriptome), protein (the proteome), and metabolite (the metabolome) components of plants as well as their organs and tissues. Microarray technologies, recent advances in protein mass spectrometry, and high-throughput metabolite analyses are beginning to provide detailed information on the total mRNA, protein, and metabolite components of plants. This knowledge will allow scientists to monitor changes in proteins and metabolites in plants. Ultimately, it may allow them to discover new metabolic pathways and to model metabolic and regulatory networks in plants.

Base Sequence↗
PubMed2004

Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae.

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.

Animals↗
PubMed2003

The German cDNA network: cDNAs, functional genomics and proteomics.

Among the greatest challenges facing biology today is the exploitation of huge amounts of genomic data, and their conversion into functional information about the proteins encoded. For example, the large-scale cDNA sequencing project of the German cDNA Consortium is providing vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterization we have tagged over 500 of these with the green fluorescent protein (GFP), and examined the subcellular localizations of these fusion proteins in living cells. These data have allowed us to classify the proteins into subcellular groups which determines the next step towards a detailed functional characterization. To make further use of these GFP-tagged constructs, a series of functional assays have been designed and implemented to assess the effect of these novel proteins on processes such as cell growth, cell death, and protein transport. Functional assays with such a large set of molecules is only possible by automation. Therefore, we have developed, and adapted, functional assays for use by robotic liquid handling stations and reading stations. A transport assay allows to identify proteins which localize to distinct organelles of the secretory pathway and have the potential to be new regulators in protein transport, a proliferation assay helps identifying proteins that stimulate or repress mitosis. Further assays to monitor the effects of the proteins in apoptosis and signal transduction pathways are in progress. Integrating the functional information that is generated in the assays with data from expression profiling and further functional genomics and proteomics approaches, will ultimately allow us to identify functional networks of proteins in a morphological context, and will greatly contribute to our understanding of cell function.

Cloning, Molecular↗
PubMed2006

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation.

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.

Animals↗
PubMedDate not supplied

[From molecular genetics to functional genomic and physiome].

After a brief overview on the advances in the molecular genetics knowledge the paper present new concepts of functional genomics and physiom. First, the results of a group of cell and molecular biology researchers in the filed of genic expression of biologic active endogenous substances, ion receptors and channels are discussed, underlining their influence on actual genomic trend of physiological sciences. Discussions continues on theoretical basis of gene therapy and in the end are presented the researches that made the transition from functional genom to the integrative concepts of physiom, cardiom and proteom, fulfilling the content of the classical notion of normal and pathologic physiology.

Gene Expression↗
PubMed2024

A functional genomic framework to elucidate novel causal metabolic dysfunction-associated fatty liver disease genes.

BACKGROUND AND AIMS: Metabolic dysfunction-associated fatty liver disease (MASLD) is the most prevalent chronic liver pathology in western countries, with serious public health consequences. Efforts to identify causal genes for MASLD have been hampered by the relative paucity of human data from gold standard magnetic resonance quantification of hepatic fat. To overcome insufficient sample size, genome-wide association studies using MASLD surrogate phenotypes have been used, but only a small number of loci have been identified to date. In this study, we combined genome-wide association studies of MASLD composite surrogate phenotypes with genetic colocalization studies followed by functional in vitro screens to identify bona fide causal genes for MASLD. APPROACH AND RESULTS: We used the UK Biobank to explore the associations of our novel MASLD score, and genetic colocalization to prioritize putative causal genes for in vitro validation. We created a functional genomic framework to study MASLD genes in vitro using CRISPRi. Our data identify VKORC1 , TNKS , LYPLAL1 , and GPAM as regulators of lipid accumulation in hepatocytes and suggest the involvement of VKORC1 in the lipid storage related to the development of MASLD. CONCLUSIONS: Complementary genetic and genomic approaches are useful for the identification of MASLD genes. Our data supports VKORC1 as a bona fide MASLD gene. We have established a functional genomic framework to study at scale putative novel MASLD genes from human genetic association studies.

Humans↗
PubMed2005

Functional genomics in antibacterial drug discovery.

Antibacterial drug discovery has experienced a paradigm shift from phenotypic screening for antibacterial activity to rational inhibition of preselected targets. Functional genomics techniques are implemented at various stages of the early drug discovery process and play a central role in target validation and mode of action determination. The spectrum of methods ranges from genetic manipulations (e.g. knockout studies, mutation analyses and the construction of conditional mutants) to transcriptome and proteome expression profiling. Functional genomics supports antibacterial drug discovery by improving knowledge on gene function, bacterial physiology and virulence and the effects of antibiotics on bacterial metabolism.

Animals↗