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At least 1,387 records · Page 77Linked to original sources
PubMed2001

From Arabidopsis to rice genomics: a survey of French programmes.

During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases. The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases. This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation. French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing. In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants. This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement.

Arabidopsis↗
PubMed2004

High-throughput generation of P. falciparum functional molecules by recombinational cloning.

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

Animals↗
PubMed1998

Predicting function: from genes to genomes and back.

Predicting function from sequence using computational tools is a highly complicated procedure that is generally done for each gene individually. This review focuses on the added value that is provided by completely sequenced genomes in function prediction. Various levels of sequence annotation and function prediction are discussed, ranging from genomic sequence to that of complex cellular processes. Protein function is currently best described in the context of molecular interactions. In the near future it will be possible to predict protein function in the context of higher order processes such as the regulation of gene expression, metabolic pathways and signalling cascades. The analysis of such higher levels of function description uses, besides the information from completely sequenced genomes, also the additional information from proteomics and expression data. The final goal will be to elucidate the mapping between genotype and phenotype.

Bacterial Proteins↗
PubMed2026

High-Content CRISPR Screening: Methods and Applications.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening has become a central technology in functional genomics, enabling genome-scale interrogation via pooled perturbations. Early CRISPR screens employed survival or simple phenotypic readouts to identify essential genes and drug resistance mechanisms. However, as biological questions have shifted toward understanding regulatory networks, cellular heterogeneity, and context-dependent gene functions, there has been increasing demand for screening strategies capable of capturing complex cellular phenotypes beyond cell fitness. Recent advances in single-cell sequencing, high-content imaging, and spatial transcriptomics have expanded the resolution of CRISPR screening by enabling multidimensional phenotypic characterization following genetic perturbation. By integrating pooled perturbations with diverse readouts, these approaches systematically map targeted gene edits to transcriptional states, cellular phenotypes, and microenvironmental contexts. Meanwhile, innovations in library design, delivery, and computational pipelines have further improved the robustness and interpretability of high-content screening platforms. This review synthesizes the methodological evolution of CRISPR screening, emphasizing advances in perturbation strategies, delivery systems, and multimodal readouts. Representative applications spanning oncology, immunotherapy, developmental biology, neurobiology, and infectious diseases are delineated to demonstrate refined gene network annotations. Additionally, existing technical bottlenecks, such as scalability, cost constraints, and in vivo limitations, are critically assessed. Finally, future directions are proposed to facilitate the development of precise medicine.

CRISPR screening↗
PubMed2006

Clinical proteomics: a novel diagnostic tool for the new biology of preterm labor, part I: proteomics tools.

The molecular mechanisms regulating myometrial contractility and preterm premature rupture of the membranes leading to preterm birth are poorly understood. The completion of the human genome sequence led to the development of functional genomics and gene array technology to simultaneously identify candidate genes potentially involved in regulation of human parturition. However, the study of living systems can now be expanded past genomics based on the rationale that it is the protein products of the genes, not simply gene expression, that have effects and cause disturbances at the cellular level. Therefore, identification of disease biomarkers, followed by a description of their functional networks, has the potential to significantly aid the development of new strategies for the prediction, diagnosis, and prevention of preterm birth. Interest in mass spectrometry and its use as a new clinical diagnostic tool has grown rapidly and is poised to become an important medical field for the next century.

Biomarkers↗
PubMed2005

System, trends and perspectives of proteomics in dicot plants Part I: Technologies in proteome establishment.

The first 3 years of the 21st century have seen the impact of plant proteomics on functional genomics that has enhanced our understanding, not only on the plant genome(s), but also more importantly, on the functional aspect of proteins. This is mainly due to availability of the complete genome sequence of the Arabidopsis thaliana-a dicotyledoneous (dicot) model plant-and technological advancements in proteomics. Proteomic analyses of a variety of dicot plants, including both Arabidopsis and the model legume species, barrel medic (Medicago truncatula), have greatly helped in an efficient separation, identification and cataloguing of a large number of proteins, and thereby defining their proteomes. Therefore, we have composed an inclusive review on dicot plant materials, as of February 2004, that provides system, trends and perspectives of proteomics in growth and development and the environment. The review is summarized and discussed as three individual, but interlinked, entities: Part I, technologies in proteome establishment (this review), Part II, proteomes of the complex developmental stages [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)], and Part III, unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)]. This review deals with the diverse proteomic technologies being used in proteome development of different dicot plants.

Arabidopsis↗
PubMed1997

Common genomic variation in the APOC3 promoter associated with variation in plasma lipoproteins.

We hypothesized that common genomic variation that affected the expression and/or function of the products of the APOC3, APOE, FABP2, and PON1 genes would be associated with variation in biochemical phenotypes in a previously unstudied human sample. We determined genotypes of functional genomic variants of APOC3, APOE, FABP2, and PON1 in 509 adult aboriginal Canadians from an isolated community in Northern Ontario. We tested for genotype associations with plasma lipoprotein traits. We found that (1) common variation at nucleotide -455 of the APOC3 promoter was associated with variation in plasma triglycerides (P = .006) and (2) common variation of APOE determining plasma isoforms of apo E was associated with variation in plasma apo B (P = .009). Analysis of subjects classed by APOC3 markers showed that homozygosity for presence of a C at nucleotide -455 and a T at nucleotide -482 was associated with significantly increased plasma triglycerides in both men and women. Furthermore, this allele was approximately twice as frequent in subjects within the highest quartile of plasma triglycerides as in subjects within the lowest quartile. Since the DNA variation detected by the APOC3 markers affects in vitro expression of the gene product, it is possible that the marker itself caused the associations. However, the associations could also have resulted from linkage disequilibrium with other functional variants in APOC3 or the closely linked APOA1 and/or APOA4 genes.

Adolescent↗
PubMed2026

Architectural logic of the 3D genome: mechanisms of dysregulation and emerging cancer therapeutics.

The three-dimensional (3D) genome provides an essential layer of organization that shapes genome function in space and time. Chromatin compartments and topologically associating domains (TADs) arise from the interplay between intrinsic properties of chromatin and architectural factors, including cohesin and CTCF. Despite substantial progress in defining these structural features, whether 3D genome architecture plays a causal role in regulating processes such as transcription, DNA replication, and DNA repair, or instead reflects underlying regulatory activity, remains unresolved. Here, we use the distinction between chromatin-intrinsic features and architectural factors as a framework to evaluate evidence for causality in genome structure-function relationships. We extend this framework to cancer, where both intrinsic alterations (including noncoding mutations, structural variants, and changes in chromatin state) and architectural factor perturbations (such as mutations in architectural proteins and dysregulation of transcriptional machinery) disrupt genome organization and contribute to disease progression. These findings suggest that alterations in genome structure can, in some contexts, actively reshape oncogenic programs. A major limitation in applying 3D genome insights to cancer biology is the cost and complexity of omics assays. Recent advances in artificial intelligence (AI) and machine learning (ML) enable inference and prediction of 3D genome organization from sequence and epigenomic features, providing insight into the extent to which genome folding is encoded intrinsically versus dynamically regulated in architectural factors. This perspective provides a unified view of how genome structure is established, how it relates to function, and how its disruption contributes to tumorigenesis.

3D genome↗
PubMed2002

High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library.

BACKGROUND: The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant. RESULTS: As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics. CONCLUSIONS: The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.

Bryopsida↗
PubMed2000

Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm.

The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as 'functional genomics'. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein-protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein-protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.

Animals↗
PubMed2006

The reactive oxygen species network pathways:an essential prerequisite for perception of pathogen attack and the acquired disease resistance in plants.

Availability of complete Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genome sequences, together with molecular recourses of functional genomics and proteomics have revolutionized our understanding of reactive oxygen species (ROS) signalling network mediating disease resistance in plants. So far, ROS have been associated with aging, cellular and molecular alteration in animal and plant cells. Recently,concluding evidences suggest that ROS network is essential to induce disease resistance and even to mediate resistance to multiple stresses in plants. ROS are obligatory by-products emerging as a result of normal metabolic reactions. They have the potential to be both beneficial and harmful to cellular metabolism. Their dual effects on metabolic reactions are dosage specific. In this review we focus our attention on cellular ROS level to trigger beneficial effects on plant cells responding to pathogen attack. By exploring the research related contributions coupled with data of targeted gene disruption, and RNA interference approaches, we show here that ROS are ubiquitous molecules of redox-pathways that play a crucial role in plant defence mechanism. The molecular prerequisites of ROS network to activate plant defence system in response to pathogen infections are here underlined. Bioinformatic tools are now available to scientists for high throughput analysis of cellular metabolisms. These tools are used to illustrate crucial ROS-related genes that are involved in the defence mechanism of plants. The review describes also the emerging findings of ROS network pathways to modulate multiple stress resistance in plants.

Animals↗
PubMed2004

A one-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation.

Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies. Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation. We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC). Using Agrobacterium tumefaciens, these BIBACs can be efficiently transformed to virtually all organisms, including plants, fungi, yeasts and human cells. As the strategy is based on in vivo recombineering and does not depend on restriction sites, it is applicable to any vector. To show the feasibility of the method five BACs, containing 0-75 kb of fungal DNA, were converted into BIBACs. These were subsequently transformed to the plant pathogenic fungus Fusarium oxysporum f.sp. lycopersici and to Aspergillus awamori, a filamentous fungus often used for large-scale protein production. Molecular characterisation of the transformants showed that the BIBACs were efficiently transferred to the fungi and stably integrated into their genomes.

Chromosomes, Artificial, Bacterial↗
PubMed2005

A single nucleotide polymorphism based approach for the identification and characterization of gene expression modulation using MassARRAY.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. Their abundance and the ease with which they can be assayed have lead to their use in applications beyond simple genotyping. One such application is the quantitative determination of transcript levels associated with distinct alleles or haplotypes found in promoters and coding regions of genes. These changes in expression due to allelic variation are often associated with additional genomic or transcript modifications such as DNA methylation or RNA editing. Here, we describe the use of an integrated genetic analysis platform, based on matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to first, discover coding SNPs (cSNPs); second, use these cSNPs to identify and analyze allele-specific expression; and third, from this knowledge to further analyze methylation patterns as a putative cause for the allele-specific expression. An established model involving allele-specific expression profiles of the human tumor protein 73 (TP73) gene is presented as an example to outline and validate data obtained from the MassARRAY platform. The availability of a single integrated platform to assay stable and dynamic variation at the genomic and transcript level greatly simplifies complex functional genomic studies.

5' Untranslated Regions↗
PubMed2005

Linker histone H1 per se can induce three-dimensional folding of chromatin fiber.

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.

Chromatin↗
PubMed2003

Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats.

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.

Base Sequence↗
PubMed2002

Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework.

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.

Amino Acid Sequence↗
PubMed2003

Structures of trinucleotide repeats in human transcripts and their functional implications.

Among the goals of RNA structural and functional genomics is determining structures and establishing the functions of a rich repertoire of simple sequence repeats in transcripts. These repeats are present in transcripts from their 'birth' in the nucleus to their 'death' in cytoplasm and have the potential of being involved in many steps of RNA regulation. The knowledge of their structural features and functional roles will also shed more light on the postulated mechanisms of RNA pathogenesis in a growing list of neurological diseases caused by simple sequence repeat expansions. Here, we discuss several different lines of research to support the hypothesis that the mechanism of RNA pathogenesis may be a more common phenomenon triggered or modulated also by abundant long normal repeats. We propose structures of the repeat regions in transcripts of genes involved in Triplet Repeat Expansion Diseases. We have classified the polymorphic repeat alleles of these genes according to their ability to form hairpin structures in transcripts, and describe the distribution of different structural forms of the repeats in the human population. We have also reported the results of a systematic survey of the human transcriptome to identify mRNAs containing triplet repeats and to classify them according to structural and functional criteria. Based on this knowledge, we discuss the putative wider role of triplet repeat RNA hairpins in human diseases. A hypothetical model is proposed in which long normal RNA hairpins formed by the repeats may also be involved in pathogenesis.

Alleles↗
PubMed2004

MIPS Arabidopsis thaliana Database (MAtDB): an integrated biological knowledge resource for plant genomics.

Arabidopsis thaliana is the most widely studied model plant. Functional genomics is intensively underway in many laboratories worldwide. Beyond the basic annotation of the primary sequence data, the annotated genetic elements of Arabidopsis must be linked to diverse biological data and higher order information such as metabolic or regulatory pathways. The MIPS Arabidopsis thaliana database MAtDB aims to provide a comprehensive resource for Arabidopsis as a genome model that serves as a primary reference for research in plants and is suitable for transfer of knowledge to other plants, especially crops. The genome sequence as a common backbone serves as a scaffold for the integration of data, while, in a complementary effort, these data are enhanced through the application of state-of-the-art bioinformatics tools. This information is visualized on a genome-wide and a gene-by-gene basis with access both for web users and applications. This report updates the information given in a previous report and provides an outlook on further developments. The MAtDB web interface can be accessed at http://mips.gsf.de/proj/thal/db.

Arabidopsis↗