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PubMed2001

[Murine models for human diseases].

This article is a bibliographic review concerning mouse mutations, spontaneous, induced or genetically engineered, as models of human genetic diseases. Since the beginning of the last century, mouse models have been instrumental in the understanding of the pathogenesis of many diseases and designing of new therapies. A number of recent technological advances in embryo manipulation and many large-scale mutagenesis screens will dramatically increase the availability of new mouse models in the near future. In the "post-genomic" era, mouse mutants will have a significant role as a model system for functional genome analysis of the upcoming whole-genome information of the human and mouse genomes projects.

Animals↗
PubMed2005

RNA interference: a novel and physiologic mechanism of gene silencing with great therapeutic potential.

The post-genomics scientific era has evolved rapidly while achieving advanced understanding of the structure and function of the genes responsible for both the phenotypic characteristics of higher organisms and the pathophysiology of several genetic diseases. Researchers in the fields of oncology and infectious diseases have become more convinced of the great potential of molecular biology approaches to further develop highly specific diagnostic and less toxic therapeutic strategies. During the last two decades, approaches for the specific silencing of essential viral genes and cellular oncogenes were evaluated with optimism for developing directed therapies. However, there were drawbacks in the use of antisense oligonucleotides as a practical mechanism of achieving gene silencing both in vitro and in vivo. Recently, a novel role for post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) was discovered in the experimental model of C. elegans. This mechanism, termed RNA interference (RNAi) has also been found in other eukaryotes, from plants to mammals, including humans. RNAi is presently being explored both in vitro and in vivo in functional genomics studies and possible therapeutic uses due to its highly specific and physiologic mode of gene silencing. This article focuses on the most current information available regarding the RNAi mechanism and its uses in models of cancer and infectious diseases.

Animals↗
PubMed2026

A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum.

Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.

CRISPR/Cpf1↗
PubMed2006

Validation and functional annotation of expression-based clusters based on gene ontology.

BACKGROUND: The biological interpretation of large-scale gene expression data is one of the paramount challenges in current bioinformatics. In particular, placing the results in the context of other available functional genomics data, such as existing bio-ontologies, has already provided substantial improvement for detecting and categorizing genes of interest. One common approach is to look for functional annotations that are significantly enriched within a group or cluster of genes, as compared to a reference group. RESULTS: In this work, we suggest the information-theoretic concept of mutual information to investigate the relationship between groups of genes, as given by data-driven clustering, and their respective functional categories. Drawing upon related approaches (Gibbons and Roth, Genome Research 12:1574-1581, 2002), we seek to quantify to what extent individual attributes are sufficient to characterize a given group or cluster of genes. CONCLUSION: We show that the mutual information provides a systematic framework to assess the relationship between groups or clusters of genes and their functional annotations in a quantitative way. Within this framework, the mutual information allows us to address and incorporate several important issues, such as the interdependence of functional annotations and combinatorial combinations of attributes. It thus supplements and extends the conventional search for overrepresented attributes within a group or cluster of genes. In particular taking combinations of attributes into account, the mutual information opens the way to uncover specific functional descriptions of a group of genes or clustering result. All datasets and functional annotations used in this study are publicly available. All scripts used in the analysis are provided as additional files.

Algorithms↗
PubMed2002

The complexity of immune and alloimmune response.

Alloimmune response induced by foreign histoincompatible alloantigens is a complex phenomenon possessing mechanisms, characteristics to innate and adoptive immune response. It is also modified by various immunregulating exocrine and autocrine factors. Starting the new time period of functional genomics the knowledge of human genes' structure needs a more clear insight not only about the function and contribution of genes but their historical background, origin and importance in the phylogenesis. Comparative immunology comes into focus of interest helping to understand the complexity of immune and alloimmune response. It is almost unbelievable that immune functions as phagocytosis and cytokine production like IL-1 and TNF have already emerged 700 million years ago in starfishes and sponges. Functions--including recruitment of coelomocytes, killing of micro-organisms by lysosome-like enzyme activity, opsonization by complement analogous proteins and oxidative burst function--remained unchanged during phylogenesis and could be found not only in insects but in mammals as well as representatives of innate immunity. The importance of these molecules is reflected in homology of conservative regions. One of the biggest evolutionary steps happened 500 million years ago when fish developed a jaw in the Placoderms species. This fact led to the development of gut associated immune system. The system was the basis to create the genetic material for recombination and mutation to establish variability and diversity of proteins, as immunoglobulins. It is interesting to lean how diversity of immunglobulins in sharks is insured by joining of blocks of V, D, J and C genes, in contrast to humans, where those genes are located on different chromosome regions. These differences are associated with an immediate production of specific immunglobulin or a slower one combined with immunologic memory. Similar development could be found in T cell antigen specific receptors, too. Concerning the establishment of adoptive immunity by emergence of genetic recombination, which allowed the production of a huge diversity of specific antigen binding proteins, another structure developed parallel from the histoglobin molecule. This protein was created to catch peptide particles which split from the proteins originating from microorganisms, viruses or foreign cell compartments. The cave-like groove capturing the different peptides represented a huge variability. These histocompatibility molecules emerged from this ancient structure for more than 300 million years ago. The genetic family responsible for their synthesis became the most complex gene family including many other genes involved in the immune response. The polymorphic character of the histocompatibility protein is responsible for the capture of the relevant peptides fitting best to the allotype-determined groove. In certain species the same function could be filled by different ancient molecules with the same success. Dendritic cells and their importance in differentiation and antigen presentation became in the focus of interest in the last decade. They have lymphoid and myeloid origin, mature and less differentiated subtypes with characteristic CD markers and cytokine profile. Their function and origin from the stem cell subpopulation is an important example how nature influences the development of immunity to the accommodation and survival to the always changing environment. The new molecular techniques will help to get closer to understand the function of genes regulating immune response and modify them.

Animals↗
PubMed2025

Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32).

Single-stranded DNA binding protein (gp32) serves as the central regulatory component of the multi-subunit T4 bacteriophage DNA replication system by coordinating the system's three functional sub-assemblies, resulting in phage DNA synthesis in T4-infected E. coli cells at the high speeds (~1,000 nts s-1) and the high fidelity (< 1 error per 107 nts) required for genomic function within this cellular eco-system. Gp32 proteins continuously bind to, slide as cooperatively-linked clusters on, and un-bind from transiently exposed single-stranded (ss) DNA templates to carry out their coordinating functions, as well as to protect genomic sequences from nuclease activity and block the formation of interfering secondary structures. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within ssb clusters, but the roles of the disordered C-terminal domains (CTD) in the nucleation of gp32-ssDNA filaments at ss-dsDNA junctions are less well understood. We here present microsecond-resolved single-molecule F&#xf6;rster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine lattices of varying lattice length and polarity near model ss-dsDNA junctions. These data are analyzed to define the molecular steps and related free energy surfaces involved in initiating gp32 cluster formation, which show that the nucleation mechanisms and regulatory interactions driven by gp32 proteins at ss-dsDNA junctions are significantly directed by lattice polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at lattice positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTD domains might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32-ssDNA filaments involved in phage DNA replication and related processes.

Journal Article↗
PubMed2003

An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics.

The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.

AGAMOUS Protein, Arabidopsis↗
PubMed2001

Object-oriented approach to drug design enabled by NMR SOLVE: first real-time structural tool for characterizing protein-ligand interactions.

As a result of genomics efforts, the number of protein drug targets is expected to increase by an order of magnitude. Functional genomics efforts are identifying these targets, while structural genomics efforts are determining structures for many of them. However, there is a significant gap in going from structural information for a protein target to a high affinity (K(d) < 100 nM) inhibitor, and the problem is multiplied by the sheer number of new targets now available. nature frequently designs proteins in classes that are related by the reuse, through gene duplication events, of cofactor binding domains. This reuse of functional domains is an efficient way to build related proteins in that it is object-oriented. There is a growing realization that the most efficient drug design strategies for attacking the mass of targets coming from genomics efforts will be systems-based approaches that attack groups of related proteins in parallel. We propose that the most effective drug design strategy will be one that parallels the object-oriented manner by which nature designed the gene families themselves. IOPE (Integrated Object-Oriented PharmacoEngineering) is such an approach. It is a three-step technology to build focused combinatorial libraries of potential inhibitors for major families and sub-families of enzymes, using cogent NMR data derived from representatives of these protein families. The NMR SOLVE (Structurally Oriented Library Valency Engineering) data used to design these libraries are gathered in days, and data can be obtained for large proteins (> 170 kDa). Furthermore, the process is fully object-oriented in that once a given bi-ligand is identified for a target, potency is retained if different cofactor mimics are swapped. This gives the drug design process maximum flexibility, allowing for the more facile transition from in vitro potency to in vivo efficacy.

Binding Sites↗
PubMed2002

Novel retroviral vectors to facilitate expression screens in mammalian cells.

As tools for functional genomics, expression profiling and proteomics provide correlative data, while expression cloning screens can link genes directly to biological function. However, technical limitations of gene transfer, expression, and recovery of candidate genes have limited wider application of genome-wide expression screens. Here we describe the pEYK retroviral vectors, which maintain high titers and robust gene expression while addressing the major bottleneck of expression cloning--efficient candidate gene recovery. By exploiting schemes for enhanced PCR rescue or strategies for direct isolation of proviral DNA as plasmids in bacterial hosts, the pEYK vectors facilitate cDNA isolation from selected cells and enable rapid iteration of screens and genetic reversion analyses to validate gene candidates. These vectors have proven useful to identify genes linked to cell proliferation, senescence and apoptosis.

Animals↗
PubMed2006

Nuclear architecture in the light of gene expression and cell differentiation studies.

It is evident that primary DNA sequences, that define genomes, are responsible for genome functions. However, the functional properties of chromatin are additionally regulated by heritable modifications known as epigenetic factors and, therefore, genomes should be also considered with respect to their 'epigenomes'. Nucleosome remodelling, DNA methylation and histone modifications are the most prominent epigenetic changes that play fundamental roles in the chromatin-mediated control of gene expression. Another important nuclear feature with functional relevance is the organization of mammalian chromatin into distinct chromosome territories which are surrounded by the interchromatin compartment that is necessary for transport of regulatory molecules to the targeted DNA. The inner structure of the chromosome territories, as well as the arrangement of the chromosomes within the interphase nuclei, has been found to be non-randomly organized. Therefore, a specific nuclear arrangement can be observed in many cellular processes, such as differentiation and tumour cell transformation.

Cell Differentiation↗
PubMed2002

Computational comparison of two mouse draft genomes and the human golden path.

BACKGROUND: The availability of both mouse and human draft genomes has marked the beginning of a new era of comparative mammalian genomics. The two available mouse genome assemblies, from the public mouse genome sequencing consortium and Celera Genomics, were obtained using different clone libraries and different assembly methods. RESULTS: We present here a critical comparison of the two latest mouse genome assemblies. The utility of the combined genomes is further demonstrated by comparing them with the human 'golden path' and through a subsequent analysis of a resulting conserved sequence element (CSE) database, which allows us to identify over 6,000 potential novel genes and to derive independent estimates of the number of human protein-coding genes. CONCLUSION: The Celera and public mouse assemblies differ in about 10% of the mouse genome. Each assembly has advantages over the other: Celera has higher accuracy in base-pairs and overall higher coverage of the genome; the public assembly, however, has higher sequence quality in some newly finished bacterial artificial chromosome clone (BAC) regions and the data are freely accessible. Perhaps most important, by combining both assemblies, we can get a better annotation of the human genome; in particular, we can obtain the most complete set of CSEs, one third of which are related to known genes and some others are related to other functional genomic regions. More than half the CSEs are of unknown function. From the CSEs, we estimate the total number of human protein-coding genes to be about 40,000. This searchable publicly available online CSEdb will expedite new discoveries through comparative genomics.

Animals↗
PubMed2005

Bioinformatics analysis of alternative splicing.

Over the past few years, the analysis of alternative splicing using bioinformatics has emerged as an important new field, and has significantly changed our view of genome function. One exciting front has been the analysis of microarray data to measure alternative splicing genome-wide. Pioneering studies of both human and mouse data have produced algorithms for discerning evidence of alternative splicing and clustering genes and samples by their alternative splicing patterns. Moreover, these data indicate the presence of alternative splice forms in up to 80 per cent of human genes. Comparative genomics studies in both mammals and insects have demonstrated that alternative splicing can in some cases be predicted directly from comparisons of genome sequences, based on heightened sequence conservation and exon length. Such studies have also provided new insights into the connection between alternative splicing and a variety of evolutionary processes such as Alu-based exonisation, exon creation and loss. A number of groups have used a combination of bioinformatics, comparative genomics and experimental validation to identify new motifs for splice regulatory factors, analyse the balance of factors that regulate alternative splicing, and propose a new mechanism for regulation based on the interaction of alternative splicing and nonsense-mediated decay. Bioinformatics studies of the functional impact of alternative splicing have revealed a wide range of regulatory mechanisms, from NAGNAG sites that add a single amino acid; to short peptide segments that can play surprisingly complex roles in switching protein conformation and function (as in the Piccolo C2A domain); to events that entirely remove a specific protein interaction domain or membrane anchoring domain. Common to many bioinformatics studies is a new emphasis on graph representations of alternative splicing structures, which have many advantages for analysis.

Algorithms↗
PubMed2006

The nature of systems biology.

The advent of functional genomics has enabled the molecular biosciences to come a long way towards characterizing the molecular constituents of life. Yet, the challenge for biology overall is to understand how organisms function. By discovering how function arises in dynamic interactions, systems biology addresses the missing links between molecules and physiology. Top-down systems biology identifies molecular interaction networks on the basis of correlated molecular behavior observed in genome-wide "omics" studies. Bottom-up systems biology examines the mechanisms through which functional properties arise in the interactions of known components. Here, we outline the challenges faced by systems biology and discuss limitations of the top-down and bottom-up approaches, which, despite these limitations, have already led to the discovery of mechanisms and principles that underlie cell function.

Cell Physiological Phenomena↗
PubMed2005

Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast.

BACKGROUND: Quantification of intracellular metabolite fluxes by 13C-tracer experiments is maturing into a routine higher-throughput analysis. The question now arises as to which mutants should be analyzed. Here we identify key experiments in a systems biology approach with a genome-scale model of Saccharomyces cerevisiae metabolism, thereby reducing the workload for experimental network analyses and functional genomics. RESULTS: Genome-scale 13C flux analysis revealed that about half of the 745 biochemical reactions were active during growth on glucose, but that alternative pathways exist for only 51 gene-encoded reactions with significant flux. These flexible reactions identified in silico are key targets for experimental flux analysis, and we present the first large-scale metabolic flux data for yeast, covering half of these mutants during growth on glucose. The metabolic lesions were often counteracted by flux rerouting, but knockout of cofactor-dependent reactions, as in the adh1, ald6, cox5A, fum1, mdh1, pda1, and zwf1 mutations, caused flux responses in more distant parts of the network. By integrating computational analyses, flux data, and physiological phenotypes of all mutants in active reactions, we quantified the relative importance of 'genetic buffering' through alternative pathways and network redundancy through duplicate genes for genetic robustness of the network. CONCLUSIONS: The apparent dispensability of knockout mutants with metabolic function is explained by gene inactivity under a particular condition in about half of the cases. For the remaining 207 viable mutants of active reactions, network redundancy through duplicate genes was the major (75%) and alternative pathways the minor (25%) molecular mechanism of genetic network robustness in S. cerevisiae.

Carbon↗
PubMed2005

Advances in genome-wide protein expression using the wheat germ cell-free system.

In the current post-genomic era, cell-free translation platforms are gaining importance in structural as well as functional genomics. They are based on extracts prepared from Escherichia coli cells, wheat germ, or rabbit reticulocytes, and when programmed with any mRNA in the presence of energy sources and amino acids, can synthesize the respective protein in vitro. Among the cell-free systems, the wheat germ-based translation system is of special interest due to its eukaryotic nature and robustness. This chapter outlines the existing protein production platforms and their limitations, and describes the basic concept of the wheat germ-based cell-free system. It also demonstrates how the conventional wheat germ system can be improved by eliminating endogenous inhibitors, by using an expression vector specially designed for this system and polymerase chain reaction-directed protein synthesis directly from cDNAs in a bi-layer translation system. Finally, a robotic procedure for translation based on the wheat germ extract and bi-layer cell-free translation is described.

3' Untranslated Regions↗
PubMed2004

Large-scale heterospecific segregation distortion in Populus revealed by a dense genetic map.

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

Chromosome Mapping↗
PubMed2003

High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system.

A novel transposon mutagenesis system for the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. campestris (Xcc) was developed using a Tn5-based transposome. A highly efficient transformation up to 10(6) transformants per microg transposon DNA was obtained. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses of Tn5 insertion sites suggested a random mode of transposition. The transposition was stable in the transformants for 20 subcultures. Eighteen thousand and 17000 transformants for Xoo and Xcc, respectively, were generated, corresponding to 4X ORF coverage of the genomes. The libraries will facilitate the identification of pathogenicity-related genes as well as functional genomic analysis in Xoo and Xcc.

Blotting, Southern↗
PubMed2001

High-throughput three-dimensional protein structure determination.

In the wake of finished genomic sequencing projects, high-throughput analysis techniques are being developed in various fields of functional genomics. Of special interest in this regard is the three-dimensional structure analysis of proteins by X-ray crystallography and NMR spectroscopy, which has been characterized by distinctly low-throughput in the past. A number of recent advances in instrumentation and software are promising to radically change this situation, leaving the production of suitable protein samples as the sole rate-limiting step in structural analyses.

Crystallography, X-Ray↗