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At least 433 records · Page 24Linked to original sources
PubMed2003

Single-nucleotide mutations for plant functional genomics.

In the present genomics era, powerful reverse-genetic strategies are needed to elucidate gene and protein function in the context of a whole organism. However, most current techniques lack the generality and high-throughput potential of descriptive genomic approaches, such as those that rely on microarray hybridization. For example, in plant research, effective insertional mutagenesis and transgenic methods are limited to relatively few species or are inefficient. Fortunately, single-nucleotide changes can be induced in any plant by using traditional chemical mutagens, and progress has been made in efficiently detecting changes. Because base substitutions in proteins provide allelic series, and not just knockouts, this strategy can yield refined insights into protein function. Here, we review recent progress that has been made in genome-wide screening for point mutations and natural variation in plants. Its general applicability leads to the expectation that traditional mutagenesis followed by high-throughput detection will become increasingly important for plant functional genomics.

Arabidopsis↗
PubMed2005

Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris.

Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.

Bacterial Proteins↗
PubMed2005

Subfamily hmms in functional genomics.

The limitations of homology-based methods for prediction of protein molecular function are well known; differences in domain structure, gene duplication events and errors in existing database annotations complicate this process. In this paper we present a method to detect and model protein subfamilies, which can be used in high-throughput, genome-scale phylogenomic inference of protein function. We demonstrate the method on a set of nine PFAM families, and show that subfamily HMMs provide greater separation of homologs and non-homologs than is possible with a single HMM for each family. We also show that subfamily HMMs can be used for functional classification with a very low expected error rate. The BETE method for identifying functional subfamilies is illustrated on a set of serotonin receptors.

Animals↗
PubMed2002

"A system biology" approach to bioinformatics and functional genomics in complex human diseases: arthritis.

Human and other annotated genome sequences have facilitated generation of vast amounts of correlative data, from human/animal genetics, normal and disease-affected tissues from complex diseases such as arthritis using gene/protein chips and SNP analysis. These data sets include genes/proteins whose functions are partially known at the cellular level or may be completely unknown (e.g. ESTs). Thus, genomic research has transformed molecular biology from "data poor" to "data rich" science, allowing further division into subpopulations of subcellular fractions, which are often given an "-omic" suffix. These disciplines have to converge at a systemic level to examine the structure and dynamics of cellular and organismal function. The challenge of characterizing ESTs linked to complex diseases is like interpreting sharp images on a blurred background and therefore requires a multidimensional screen for functional genomics ("functionomics") in tissues, mice and zebra fish model, which intertwines various approaches and readouts to study development and homeostasis of a system. In summary, the post-genomic era of functionomics will facilitate to narrow the bridge between correlative data and causative data by quaint hypothesis-driven research using a system approach integrating "intercoms" of interacting and interdependent disciplines forming a unified whole as described in this review for Arthritis.

Animals↗
PubMed2004

Functional genomic analysis of the ADP-ribosylation factor family of GTPases: phylogeny among diverse eukaryotes and function in C. elegans.

ADP-ribosylation factor (Arf) and Arf-like (Arl) proteins are a family of highly conserved 21 kDa GTPases that emerged early in the evolution of eukaryotes. These proteins serve regulatory roles in vesicular traffic, lipid metabolism, microtubule dynamics, development, and likely other cellular processes. We found evidence for the presence of 6 Arf family members in the protist Giardia lamblia and 22 members in mammals. A phylogenetic analysis was performed to delineate the evolutionary relationships among Arf family members and to attempt to organize them by both their evolutionary origins and functions in cells and/or organisms. The approximately 100 protein sequences analyzed from animals, fungi, plants, and protists clustered into 11 groups, including Arfs, nine Arls, and Sar proteins. To begin functional analyses of the family in a metazoan model organism, we examined roles for all three C. elegans Arfs (Arf-1, Arf-3, and Arf-6) and three Arls (Arl-1, Arl-2, and Arl-3) by use of RNA-mediated interference (RNAi). Injection of double-stranded RNA (dsRNA) encoding Arf-1 or Arf-3 into N2 hermaphrodites produced embryonic lethality in their offspring and, later, sterility in the injected animals themselves. Injection of Arl-2 dsRNA resulted in a disorganized germline and sterility in early offspring, with later offspring exhibiting an early embryonic arrest. Thus, of the six Arf family members examined in C. elegans, at least three are required for embryogenesis. These data represent the first analysis of the role(s) of multiple members of this family in the development of a multicellular organism.

ADP-Ribosylation Factors↗
PubMed2005

Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19.

The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Arabidopsis↗
PubMed2003

Chromatin as a tool for the study of genome function in cancer.

The transition to malignancy requires an extensive reconfiguration of the genome's expression program that does not result entirely from actual changes in primary DNA sequence-i.e., mutation. Epigenetic-meta-DNA-gene expression states result from an assembly over a given locus of a poorly understood nucleoprotein entity that includes histones and other architectural components of chromatin, nonhistone DNA-bound regulators, and additional chromatin-bound polypeptides. This structure is rapidly reestablished in the wake of the DNA replication fork, thus ensuring its persistence in rapidly proliferating cells and thereby yielding an exceptionally stable mode of gene expression. Chromatin is the perfect vehicle for enabling such genome control. During S phase both covalently modified histones and histone-associated regulatory proteins distribute to the newly synthesized daughter chromatids in a form of "molecular dowry" inherited from the G(1) state of the genome, and impose a specific mode of function on the underlying DNA. An extensively studied example of chromatin-based epigenetic inheritance connects DNA methylation to the targeting of chromatin remodeling and modification. In a broad sense, however, genome reprogramming in cancer is associated with the remodeling of a multitude of regulatory DNA stretches-e.g., promoters, enhancers, locus control regions (LCRs), insulators, etc.-into a specific chromatin architecture. This architectural entity provides a general molecular signature of the cancer epigenome that complements and significantly expands its DNA methylation-based component.

Chromatin↗
PubMed2003

Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster.

Assigning functional significance to completed genome sequences is one of the next challenges in biological science. Conventional genetic tools such as deficiency chromosomes help assign essential complementation groups to their corresponding genes. We describe an F2 genetic screen to identify lethal mutations within cytogenetic region 61D-61F of the third chromosome of Drosophila melanogaster. One hundred sixteen mutations were identified by their failure to complement both Df(3L)bab-PG and Df(3L)3C7. These alleles were assigned to 14 complementation groups and 9 deficiency intervals. Complementation groups were ordered using existing deficiencies, as well as new deficiencies generated in this study. With the aid of the genomic sequence, genetic and physical maps in the region were correlated by use of PCR to localize the breakpoints of deficiencies within a 268-kb genomic contig (GenBank accession No. AC005847). Six essential complementation groups were assigned to specific genes, including genes encoding a porphobilinogen deaminase and a Sac1-like protein.

Alleles↗
PubMed2005

[Cell microarrays and functional genomics].

With the complete sequencing of the human genome, research priorities have shifted from the identification of genes to the elucidation of their function. Methods currently used by scientists to characterize gene function, such as knock-out mice, are based upon loss of protein function and analysis of the resulting phenotypes to infer a potential role for the protein under scrutiny. Until now, these methods have been successful but time consuming and only a few genes at a time could be analyzed. Cell microarrays allow to simultaneously transfect thousands of different nucleic acid molecules, RNA or DNA, into adherent cells. It is then possible to analyze a large pallet of resulting phenotypes in clusters of transfected cells. We are currently manufacturing cell microarrays with collections of full-length cDNA cloned in expression vectors (gain of function analyses) or siRNA (loss of function studies) to unravel function of genes involved in differentiation and proliferation of human cells. Although there are still some technological difficulties to overcome, the potential for cell microarrays to speed up functional exploration of genomes is very promising.

Cells, Cultured↗
PubMed2005

Methods for the functional genomic analysis of ubiquitin ligases.

Ubiquitin ligases (E3s) are critical components of the ubiquitin-proteasome system as they are the major determinants of specificity in ubiquitin conjugation. The number of predicted E3s in the mammalian genome is exceeding 400 and is represented by two major subfamilies: HECT domain-containing E3s and RING finger-type E3s. Given the size of this protein family and lack of knowledge on the functions of most of these 400 proteins, their functional annotation should benefit from modern genomic tools. This article presents a methodology consisting of the use of a cDNA expression library to identify suppressors of polyglutamine (polyQ)-mediated protein aggregate formation in cells, as an example of a genomic approach to assign functions to E3s. In this screen, we identified novel RING finger-type E3s exhibiting suppressor activity among >50% of all the potential E3s in the mouse and human genomes. This method could be adapted easily to identify E3s that function in other processes and signaling pathways.

Algorithms↗
PubMed2003

Nutrigenes, functional genomics and systems biology.

Traditionally, the classic reductionist approach attributes functions to individual genes. For instance, this has involved the analysis of motifs or the amino acid sequences of single gene products. It is unclear how the products of particular collections genes act together to provide higher order functionality in health and disease. To address this higher order problem, the function of collections of genes, as opposed to "one gene at a time" has to be studied. Accordingly, a model system is needed to test systems biology. In our studies, we used the homocysteine-folate metabolism as a model system.

Animals↗
PubMed2005

Survey of current protein family databases and their application in comparative, structural and functional genomics.

The last two decades have witnessed significant expansions in the databases storing information on the sequences and structures of proteins. This has led to the creation of many excellent protein family resources, which classify proteins according to their evolutionary relationship. These have allowed extensive insights into evolution and particularly how protein function mutates and evolves over time. Such analyses have greatly assisted the inheritance of functional annotations between experimentally characterised and uncharacterised genes. Moreover, the development of bioinformatics tools acts as a companion to the new technologies emerging in biology, such as transcriptomics and proteomics. The latter enable researchers to analyse gene expression profiles and interactions on a genome-wide scale, generating vast datasets of proteins, many of which include experimentally uncharacterised proteins. Protein family/function databases can be used to help interpret this data and allow us to benefit more fully from these technologies. This review aims to summarise the most popular sequence- and structure-based protein family databases. We also cover their application to comparative genomics and the functional annotation of the genomes.

Biological Evolution↗
PubMed1998

Functional genomics in the mouse: phenotype-based mutagenesis screens.

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.

Animals↗
PubMed2006

Artificial chromosome-based transgenes in the study of genome function.

The transfer of large DNA fragments to the mouse genome in the form of bacterial, yeast or phage artificial chromosomes is an important process in the definition of transcription units, the modeling of inherited disease states, the dissection of candidate regions identified by linkage analysis and the construction of in vivo reporter genes. However, as with small recombinant transgenes, the transferred sequences are usually integrated randomly often with accompanying genomic alterations and variable expression of the introduced genes due to the site of integration and/or copy number. Therefore, alternative methods of integrating large genomic transgenes into the genome have been developed to avoid the variables associated with random integration. This review encourages the reader to imagine the large variety of applications where artificial chromosome transgenes can facilitate in vivo and ex vivo studies in the mouse and provides a context for making the necessary decisions regarding the specifics of experimental design.

Animals↗
PubMed2001

Functional genomics of oxidant-induced lung injury.

In summary, acute lung injury is a severe (>40% mortality) respiratory disease associated with numerous precipitating factors. Despite extensive research since its initial description over 30 years ago, questions remain about the basic pathophysiological mechanisms and their relationship to therapeutic strategies. Histopathology reveals surfactant disruption, epithelial perturbation and sepsis, either as initiating factors or as secondary complications, which in turn increase the expression of cytokines that sequester and activate inflammatory cells, most notably, neutrophils. Concomitant release of reactive oxygen and nitrogen species subsequently modulates endothelial function. Together these events orchestrate the principal clinical manifestations of the syndrome, pulmonary edema and atelectasis. To better understand the gene-environmental interactions controlling this complex process, we examined the relative sensitivity of inbred mouse strains to acute lung injury induced by ozone, ultrafine PTFE, or fine particulate NiSO4 (0.2 microm MMAD, 15-150 microg/m3). Measuring survival time, protein and neutrophils in bronchoalveolar lavage, lung wet: dry weight, and histology, we found that these responses varied between inbred mouse strains, and susceptibility is heritable. To assess the molecular progression of NiSO4-induced acute lung injury, temporal relationships of 8734 genes and expressed sequence tags were assessed by cDNA microarray analysis. Clustering of co-regulated genes (displaying similar temporal expression patterns) revealed the altered expression of relatively few genes. Enhanced expression occurred mainly in genes associated with oxidative stress, anti-proteolytic function, and repair of the extracellular matrix. Concomitantly, surfactant proteins and Clara cell secretory protein mRNA expression decreased. Genome wide analysis of 307 mice generated from the backcross of resistant B6xA F1 with susceptible A strain identified significant linkage to a region on chromosome 6 (proposed as Aliq4) and suggestive linkages on chromosomes 1, 8, and 12. Combining of these QTLs with two additional possible modifying loci (chromosome 9 and 16) accounted for the difference in survival time noted in the A and B6 parental strains. Combining these findings with those of the microarray analysis has enabled prioritization of candidate genes. These candidates, in turn, can be directed to the lung epithelium in transgenic mice or abated in inducible and constitutive gene-targeted mice. Initial results are encouraging and suggest that several of these mice vary in their susceptibility to oxidant-induced lung injury. Thus, these combined approaches have led to new insights into functional genomics of lung injury and diseases.

Animals↗
PubMed2006

Functional genomics of Buchnera and the ecology of aphid hosts.

In many animal groups, mutualistic bacterial symbionts play a central role in host ecology, by provisioning rare nutrients and thus enabling specialization on restricted diets. Among such symbionts, genomic studies are most advanced for Buchnera, the obligate symbiont of aphids, which feed on phloem sap. The contents of the highly reduced Buchnera genomes have verified its role in aphid nutrition. Comparisons of Buchnera gene sets indicate ongoing, irreversible gene losses that are expected to affect aphid nutritional needs. Furthermore, almost all regulatory genes have been eliminated, raising the question of whether and how gene expression responds to environmental change. Microarray studies on genome-wide expression indicate that Buchnera has evolved some constitutive changes in gene expression: homologues of heat stress genes have elevated transcript levels in Buchnera (relative to other bacteria) even in the absence of stress. Additionally, the microarray results indicate that responses to heat stress and to amino acid availability are both few and modest. Observed responses are consistent with control by the few ancestral regulators retained in the genome. Initial studies on the role of host genes in mediating the symbiosis reveal distinctive expression patterns in host cells harbouring Buchnera. In the near future, a complete genome of pea aphid will accelerate progress in understanding the functional integration of aphid and Buchnera genomes. Although information for other insect symbioses is relatively limited, studies on symbionts of carpenter ants and tsetse flies indicate many similarities to Buchnera.

Animal Feed↗
PubMed2005

'PACLIMS': a component LIM system for high-throughput functional genomic analysis.

BACKGROUND: Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the approximately 11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required. RESULTS: The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured. CONCLUSION: Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.

Algorithms↗
PubMed2003

When negative is positive in functional genomics.

The post-genomic era has caused classical approaches to analyse gene function to be reviewed and refined. Conventional reverse genetic approaches to predict gene function have drawbacks in terms of genetic redundancy and being limited mainly to well-characterized genomes. The relatively recent use of dominant-negative transgenes has been successful in elaborating certain pathways and it is now possible to extend this technique to convert transcription factors into dominant repressor functions by fusion to repressor domains such as ENGRAILED from Drosophila. This methodology opens up new possibilities to overcome genetic redundancy, to identify novel gene functions and also to apply information on conserved protein domains between species to repress heterologous protein function.

Animals↗