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PubMed1999

Genetics of Methanococcus: possibilities for functional genomics in Archaea.

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

Anaerobiosis↗

PubMed2006

Functional genomics identifies TOR-regulated genes that control growth and division.

BACKGROUND: The TOR (target of rapamycin) ser/thr protein kinase is the central component of a eukaryotic signaling pathway that regulates growth and is the direct target of the clinically useful drug rapamycin. Recent efforts have identified at least two multiprotein complexes that contain TOR, but little is known in higher eukaryotes about the genes downstream of TOR that control growth. RESULTS: By combining the use of a small molecule inhibitor (rapamycin), transcriptional profiling, and RNA interference in Drosophila tissue culture cells, we identified genes whose expression responds to Drosophila TOR (dTOR) inhibition and that regulate cell size. Several of the dTOR-regulated genes that function in cell size control have additional roles in cell division. Most of these genes are conserved in mammals and several are linked to human disease. This set of genes is highly enriched for regulators of ribosome biogenesis, which emphasizes the importance of TOR-dependent transcription in building the protein synthesis machinery in higher eukaryotes. In addition, we identify two dTOR-regulated genes, CG3071 and CG6677, whose human orthologs, SAW and ASH2L, are also under TOR-dependent transcriptional control and encode proteins with conserved functional roles in growth. CONCLUSIONS: We conclude that combining RNA interference with genomic analysis approaches, such as transcriptional profiling, is an effective way to identify genes functioning in a particular biological process. Moreover, this strategy, if applied in model systems with simpler genomes, can identify genes with conserved functions in mammals.

Animals↗

PubMed2005

Diverse aging rates in metazoans: targets for functional genomics.

Aging, or senescence, has typically been measured by demographic analysis, which has its merits but is blind to key aspects of functional development and deterioration. If one uses demographic analyses, however, the approach providing most insight is the analysis of age-specific mortality. The continuing increase in DNA sequencing power combined with emerging computational techniques will allow in the near future detailed investigation of mechanisms of aging in diverse species beyond the typical laboratory bestiary. A comparative approach of this sort needs to consider, in addition to simple longevity, the effects of phylogeny and body size on the species in question. Insight may be gained from the study of species exhibiting accelerated aging relative to more "typical" species. These naturally short-lived species, such as several small shrews and marsupials, avoid the worry inherent in "accelerated aging" genotypes of common models, which is that they are only short-lived because of some idiosyncratic pathology unrelated to general aging. A case of special interest that has yet to be seriously investigated is the domestic dog, in which selective breeding has produced phenotypes within the same species that age at two-fold different rates. Exceptionally long-lived species offer exceptional opportunities to discover whether there are few or many ways to create long-lived organisms. Slow-aging species with the most to offer include bats and naked mole-rats. Perhaps no fundamental question in biology is more intriguing that why and how nature has produced such a dazzling array of aging rates. The development of functional genetics over the next several decades promises to lead us toward an answer.

Animals↗

PubMed2003

Functional genomics and radioisotope-based imaging procedures.

After the sequencing of the human genome has been completed, non-invasive imaging studies are needed to assess the function of new genes in living organisms. The evaluation of genetically manipulated animals or new designed biomolecules will require a thorough understanding of physiology, biochemistry and pharmacology, and the experimental approaches will involve many new technologies including in vivo imaging with single photon emission computed tomography (SPECT) and positron emission tomography (PET). Nuclear medicine procedures may be applied for the determination of gene function and regulation using established and new tracers or using in vivo reporter genes such as enzymes, receptors, antigens or transporters. Pharmacogenomics will identify new surrogate markers for therapy monitoring which may represent potential new tracers for imaging. Also, drug distribution studies for new therapeutic biomolecules are needed at least during preclinical stages of drug development. Clinical gene therapy needs non-invasive tools to evaluate the efficiency of gene transfer. These informations can be used for therapy planning, follow-up studies in treated tumors and as an indicator of prognosis. Therapy planning is performed by the assessment of gene expression for example using radio-labeled specific substrates to determine the activity of suicide enzymes such as the Herpes Simplex Virus thymidine kinase. Follow-up studies with single photon emission tomography or positron emission tomography may be done to evaluate early or late effects of gene therapy on tumor metabolism or proliferation. Finally, new biomolecules will be developed by bioengineering methods which may be used for isotope-based diagnosis and treatment of disease.

Gene Expression Regulation↗

PubMed2003

Structural proteomics: toward high-throughput structural biology as a tool in functional genomics.

Structural proteomics is the determination of atomic resolution three-dimensional protein structures on a genome-wide scale in order to better understand the relationship between protein sequence, structure, and function. Here we describe our ongoing structural proteomics project on the nonmembrane proteins of the archeaon, Methanobacterium thermoautotrophicum. This article provides a snapshot of an ongoing pilot project in an emerging area of multidisciplinary research that involves bioinformatics, molecular biology, biochemistry, and instrumental methods such as NMR spectroscopy and X-ray crystallography. An assessment of the technical challenges in this type of large-scale project along with a comparison of the efficiency of sample production for both X-ray crystallography and NMR spectroscopy will be discussed. Examples of new insights into protein function and the relationship between structure and sequence will also be presented.

Bacterial Proteins↗

PubMed2002

Maize individualized chromosome and derived radiation hybrid lines and their use in functional genomics.

The duplicated and rearranged nature of plant genomes frequently complicates identification, chromosomal assignment and eventual manipulation of DNA segments. Separating an individual chromosome from its native complement by adding it to an alien genetic background together with the generation of radiation hybrids from such an addition line can enable or simplify structural and functional analyses of complex duplicated genomes. We have established fertile disomic addition lines for each of the individual maize chromosomes, except chromosome 10, with oat as the host species; DNA is available for chromosome 10 in a haploid oat background. We report on instability and transmission in disomic additions of maize chromosomes 1, 5, and 8; the chromosome 2, 3, 4, 6, 7, and 9 additions appear stable. The photoperiodic response of the two recovered maize chromosome 1 addition lines contrasts to the long-day flowering response of the oat parents and the other addition lines. Only when grown under short days did maize chromosome 1 addition lines set seed, and only one line transmitted the maize chromosome 1 to offspring. Low resolution radiation hybrid maps are presented for maize chromosomes 2 and 9 to illustrate the use of radiation hybrids for rapid physical mapping of large numbers of DNA sequences, such as ESTs. The potential of addition and radiation hybrid lines for mapping duplicated sequences or gene families to chromosome segments is presented and also the use of the lines to test interactions between genes located on different maize chromosomes as observed for ectopic expression of cell fate alterations.

Avena↗

PubMed2001

High efficiency gene transfer is an efficient way of defining therapeutic targets: a functional genomics approach.

BACKGROUND: Dendritic cells are the most potent antigen presenting cells and key to many aspects of the immune function. Studying the intracellular signalling mechanism used by dendritic cells would provide an insight into the functioning of these cells and give clues to strategies for immunomodulation. METHOD: Highly efficient adenoviral infection of dendritic cells for the delivery of transgenes was obtained. These viral vectors were used to introduce IkappaB alpha into dendritic cells for the inhibition of NF-kappaB. This was used to investigate the role of NF-kappaB in dendritic cell function. RESULTS: By blocking the NF-kappaB function a potent inhibition of the expression of costimulating molecules by dendritic cells with the concomitant loss of T cell stimulating function was demonstrated. CONCLUSION: The use of adenoviral vectors may be a useful way of studying the role of genes in dendritic cell function.

Adenoviridae↗

PubMed2003

CD1d-restricted NKT regulatory cells: functional genomic analyses provide new insights into the mechanisms of protection against Type 1 diabetes.

Deficiencies in NKT cell number and function mediate the development of Type 1 diabetes (TID). NKT cell activation with the CD1d ligand alpha-galactosylceramide (alpha-GalCer) corrects these deficiencies and prevents the onset and recurrence of T1D in NOD mice. To investigate how alpha-GalCer accomplishes this, we conducted three sets of studies. First, gene microarray analyses showed that alpha-GalCer treatment decreases interleukin (IL)16 and increases IL10 and MIP1beta gene expression in the spleen. Anti-IL16 antibody treatment protects NOD mice against insulitis and T1D, and neutralization of MIP1beta abrogates IL4 induced protection from T1D. Second, alpha-GalCer treatment of NOD.ILA(-/-) mice demonstrated that IL4 expression is required for prevention of T1D but not for NKT cell development. Third, we found that diabetes resistance in three novel congenic NOD.B6Idd4 mouse strains is associated with an increased number of NKT cells in pancreatic lymph nodes (PLNs). This increase was not evident in the spleen or PLNs of NOD.MIP1a(-/-) mice after alpha-GalCer treatment. Our data suggest that MIP1beta is a candidate gene in Idd4 that regulates NKT cell function and diabetes susceptibility. By controlling the expression and activity of IL16 and MIP1beta alpha-GalCer treatment may modulate the number, localization and function of NKT cells and regulate susceptibility to T1D.

Adoptive Transfer↗

PubMed1999

A web-based research tool for functional genomics of the microcirculation: the leukocyte adhesion cascade.

OBJECTIVE: Currently, microvascular data are almost exclusively deposited in scholarly journals. With the increasing availability of molecular data and the construction of genomic databases, a need arises to organize physiological data in a more accessible format. The microcirculation is a functional system that spans all organ systems and shares certain characteristics of organization with metabolic pathways, which have successfully been organized around genomic information for several prokaryotes. Here, we present a web-based research and teaching tool (http:@hsc.Virginia.EDU/medicine/basic-sci/biomed/l ey/) that covers a small aspect of microcirculatory physiology, the leukocyte adhesion cascade. METHODS: Currently, the site is organized in a flat-text mode with hypertext links to GenBank, Medline, and online journals where available. RESULTS: The web-based research and education tool is useful for graduate student education, and as a research resource for genetic researchers interested in gene function and for physiologists and biomedical engineers interested in the molecular basis of the leukocyte adhesion cascade. Our effort is intended to be a beginning toward a distributed database for the microcirculation (microcirculation physiome. see http://www.bme.jhu.edu/news/microphys). CONCLUSIONS: We invite all microvascular researchers to provide annotations and comments to make the research and teaching tool more useful to the scientific community.

Algorithms↗

PubMed2002

FUGOID: functional genomics of organellar introns database.

FUGOID is a web-based, taxonomically broad organelle intron database that collects and integrates various functional and structural data on organellar (mitochondrial and chloroplast) introns. The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism. It is implemented in a relational database management system, allowing sophisticated, user-friendly searching, data entry and revision. Users can access the database by any common web browser using a variety of operating systems. The main page of the database is available at http://wnt.cc.utexas.edu/~ifmr530/introndata/main.htm.

Animals↗

PubMed2001

Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants.

With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of approximately 2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

Arabidopsis↗

PubMed2001

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Antifungal Agents↗

PubMed2004

Gene ontology application to genomic functional annotation, statistical analysis and knowledge mining.

While a massive amount of biomolecular information is increasingly accumulating in different databanks, on the other hand high-throughput technologies are generating a great quantity of data that need to be annotated with the genomic information available, and interpreted. To this aim, the use of specific ontologies can greatly help either in integrating different information stored within heterogeneous databanks, or in identifying and clustering sequence data sharing common characteristics. In the molecular biology domain, the Gene Ontology (GO) is the most developed and widely used ontology. To demonstrate its great utility in the annotation and biological interpretation of gene sets obtained by means of high-throughput experiments, we implemented the web application here described. It enables functional annotations of a given gene set on a genomic scale and across different species. Within our application the annotations provided by the GO vocabulary allow either to easily bind several information from different resources, or to cluster annotated genes according to their biological characteristics. Through the GO structure it is also possible to represent biological concepts with different specificity levels, from very general to very precise concepts. Furthermore, the statistical evaluation of the categorizations provided by the GO annotations enables to highlight the most significant biological characteristics of a gene set, and therefore to mine knowledge from data. Our created tool meets the need to manage a vast quantity of biological data with a simple user interface adapt also for users with limited informatics knowledge, leading them to evaluate the functional significance of experiment's results with graphical views and statistical indexes in a well-known web browser user interface.

Genomics↗

PubMed2006

Genetic transformation of Populus trichocarpa genotype Nisqually-1: a functional genomic tool for woody plants.

We report here the Agrobacterium-mediated genetic transformation of Nisqually-1, a Populus trichocarpa genotype whose genome was recently sequenced. Several systems were established. Internodal stem segments from vigorously growing greenhouse plants are the explants most amenable to transformation. For the most efficient system, approximately 40% of the stem segments infected with pBI121-containing Agrobacterium tumefaciens C58 produced transgenic calli, as confirmed by beta-glucuronidase (GUS) staining. The regeneration efficiency of independent transgenic plants was approximately 13%, as revealed by genomic Southern analysis. Some transgenic plants were produced in as little as 5 months after co-cultivation. This system may help to facilitate studies of gene functions in tree growth and development at a genome level.

Agrobacterium tumefaciens↗

PubMed2000

Identification of eIF2Bgamma and eIF2gamma as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach.

The 5'-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bgamma) and 2 (eIF2gamma), respectively. The involvement of eIF2Bgamma and eIF2gamma in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

5' Untranslated Regions↗

PubMed2025

Charting Postnatal Heart Development Using In Vivo Single-Cell Functional Genomics.

The transition at birth, marked by increased circulatory demands and rapid growth, necessitates extensive remodeling of the heart's structure, function, and metabolism. This transformation requires precise spatial and temporal coordination among diverse cardiac cell types; central to this process is cardiomyocyte maturation, yet the regulatory mechanisms driving these changes remain poorly understood. Here, we present a temporal and spatial atlas of postnatal hearts by integrating single-nucleus transcriptomics with image-based spatial transcriptomics, which uncovers the dynamic regulatory networks of cardiomyocyte maturation. To functionally interrogate candidate regulators in vivo , we developed Probe-based Indel-detectable Perturb-seq (PIP-seq), a high-throughput platform that uses probe-based chemistry to directly capture sgRNA expression, perturbation status, and transcriptomic profiles at single-nucleus resolution. Applying PIP-seq to postnatal cardiac development identified 21 novel regulators of cardiomyocyte maturation, highlighting critical nodal points in this process. Our study establishes a high-resolution framework for dissecting postnatal heart development, underscoring the integrative and highly ordered roles of microenvironment and intercellular communication in cardiomyocyte maturation. Importantly, PIP-seq enables systematic, high-throughput exploration of gene function and networks underlying complex biological processes in their native in vivo context.

Journal Article↗

PubMed2007

Functional genomics in Trypanosoma brucei identifies evolutionarily conserved components of motile flagella.

Cilia and flagella are highly conserved, complex organelles involved in a variety of important functions. Flagella are required for motility of several human pathogens and ciliary defects lead to a variety of fatal and debilitating human diseases. Many of the major structural components of cilia and flagella are known, but little is known about regulation of flagellar beat. Trypanosoma brucei, the causative agent of African sleeping sickness, provides an excellent model for studying flagellar motility. We have used comparative genomics to identify a core group of 50 genes unique to organisms with motile flagella. These genes, referred to as T. brucei components of motile flagella (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein function we used RNA interference to target 41 TbCMF genes. Sedimentation assays and direct observation demonstrated clear motility defects in a majority of these knockdown mutants. Epitope tagging, fluorescence localization and biochemical fractionation demonstrated flagellar localization for several TbCMF proteins. Finally, ultrastructural analysis identified a family of novel TbCMF proteins that function to maintain connections between outer doublet microtubules, suggesting that they are the first identified components of nexin links. Overall, our results provide insights into the workings of the eukaryotic flagellum, identify several novel human disease gene candidates, reveal unique aspects of the trypanosome flagellum and underscore the value of T. brucei as an experimental system for studying flagellar biology.

Amino Acid Sequence↗

PubMed2005

Development of a functional genomics platform for Sinorhizobium meliloti: construction of an ORFeome.

The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.

Bacterial Proteins↗