Gene Knockout Analysis Research Articles

MetaNetX.org: a website and repository for accessing, analysing and manipulating metabolic networks

Summary: MetaNetX.org is a website for accessing, analysing and manipulating genome-scale metabolic networks (GSMs) as well as biochemical pathways. It consistently integrates data from various public resources and makes the data accessible in a standardized format using a common namespace. Currently, it provides access to hundreds of GSMs and pathways that can be interactively compared (two or more), analysed (e.g. detection of dead-end metabolites and reactions, flux balance analysis or simulation of reaction and gene knockouts), manipulated and exported. Users can also upload their own metabolic models, choose to automatically map them into the common namespace and subsequently make use of the website’s functionality.Availability and implementation: MetaNetX.org is available at http://metanetx.org. Contact: help@metanetx.org

Two Transporters Essential for Reassimilation of Novel Cholate Metabolites by Rhodococcus jostii RHA1

The bacterial uptake of steroids and their metabolites remains poorly understood. We investigated two transporters associated with cholate catabolism in Rhodococcus jostii RHA1. Reverse transcriptase quantitative-PCR indicated that an ATP-binding cassette (ABC) transporter and a major facilitator superfamily (MFS) transporter were upregulated 16.7- and 174-fold, respectively, during the exponential phase of growth on cholate compared to growth on pyruvate. Gene knockout analysis established that these transporters are required for the reassimilation of distinct metabolites that accumulate during growth on cholate. The ABC transporter, encoded by camABCD, was essential for uptake of 1β(2'-propanoate)-3aα-H-4α(3"(R)-hydroxy-3"-propanoate)-7aβ-methylhexahydro-5-indanone and a desaturated analog. The MFS transporter, encoded by camM, was essential for uptake of 3,7(R),12(S)-trihydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oate. These metabolites differ from cholate metabolites reported to be excreted by proteobacteria in that they retain an isopropanoyl side chain at C-17. The uptake of these metabolites was necessary for maximal growth on cholate: a ΔcamB mutant lacking the permease component of the ABC transporter and a ΔcamM mutant lacking the MFS transporter grew to 74% and 77%, respectively, of the yield of the wild type. This study demonstrates for the first time the requirement for specific transporters for uptake of cholate metabolites and highlights the importance and complexity of transport processes associated with bacterial steroid catabolism.

Engineering of Corynebacterium glutamicum to Enhance L-ornithine Production by Gene Knockout and Comparative Proteomic Analysis

Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine. Effect of the inactivation of 2-oxoglutarate dehydrogenase complex (ODHC) on L-ornithine production was also investigated. It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production. The engineered C. glutamicum ATCC13032 (ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L−1 from 0.24 g·L−1 of the wild-type strain. In order to understand the mechanism of L-ornithine production in C. glutamicum ATCC13032 (ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production, the comparative proteome between the wild-type and the engineered strain was analyzed. L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway. The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further increase ornithine production in the engineered C. glutamicum and the ornithine synthesis enzymes (ArgCJBD) may not be the limiting enzymes in the engineered C. glutamicum.

Effect of sporophytic PIRL9 genotype on post-meiotic expression of the Arabidopsis pirl1;pirl9 mutant pollen phenotype

Plant intracellular ras-group-related leucine-rich repeat proteins (PIRLs) are a novel class of plant leucine-rich repeat (LRR) proteins structurally related to animal ras-group LRRs involved in cell signaling and gene regulation. Gene knockout analysis has shown that two members of the Arabidopsis thaliana PIRL gene family, PIRL1 and PIRL9, are redundant and essential for pollen development and viability: pirl1;pirl9 microspores produced by pirl1/PIRL1;pirl9 plants consistently abort just before pollen mitosis I. qrt1 tetrad analysis demonstrated that the genes become essential after meiosis, during anther stage 10. In this study, we characterized the phenotype of pirl1;pirl9 pollen produced by plants heterozygous for pirl9 (pirl1;pirl9/PIRL9). Alexander's staining, scanning electron microscopy, and fluorescence microscopy indicated that pirl1;pirl9 double mutants produced by pirl9 heterozygotes have a less severe phenotype and more variable morphology than pirl1;pirl9 pollen from pirl1/PIRL1;pirl9 plants. Mutant pollen underwent developmental arrest with variable timing, often progressing beyond pollen mitosis I and arresting at the binucleate stage. Thus, although the pirl1 and pirl9 mutations act post-meiosis, the timing and expressivity of the pirl1;pirl9 pollen phenotype depends on the pirl9 genotype of the parent plant. These results suggest a continued requirement for PIRL1 and PIRL9 beyond the initiation of pollen mitosis. Furthermore, they reveal a modest but novel sporophytic effect in which parent plant genotype influences a mutant phenotype expressed in the haploid generation.

Dlk1 Is Necessary for Proper Skeletal Muscle Development and Regeneration

Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-κB and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy.

Deinococcus radiodurans PprI Switches on DNA Damage Response and Cellular Survival Networks after Radiation Damage

Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extremely radioresistant bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to gamma radiation, we performed comparative proteomics analyses on wild type (R1) and a pprI knock-out strain (YR1) under conditions of ionizing irradiation. Results of two-dimensional electrophoresis and MALDI-TOF MS or MALDI-TOF/TOF MS indicated that in response to low dose gamma ray exposure 31 proteins were significantly up-regulated in the presence of PprI. Among them, RecA and PprA are well known for their roles in DNA replication and repair. Others are involved in six different pathways, including stress response, energy metabolism, transcriptional regulation, signal transduction, protein turnover, and chaperoning. The last group consists of many proteins with uncharacterized functions. Expression of an additional four proteins, most of which act in metabolic pathways, was down-regulated in irradiated R1. Additionally phosphorylation of two proteins was under the control of PprI in response to irradiation. The different functional roles of representative PprI-regulated genes in extreme radioresistance were validated by gene knock-out analysis. These results suggest a role, either directly or indirectly, for PprI as a general switch to efficiently enhance the DNA repair capability and extreme radioresistance of D. radiodurans via regulation of a series of pathways.

Comparative Aspects of Placental Development.

The placenta is composed of several different trophoblast cell subtypes that have been defined histologically with distinct anatomical locations, ultrastructural features and physiological functions ranging from nutrient transport to endocrine. The placenta is well known for its production of diverse hormones and growth factors and yet there is wide species variation in the types of hormones that are produced. Indeed there are several examples of large families of placental-specific hormone genes that have evolved independently in different species. For example, the IFN-tau genes have evolved from type I interferons and yet are present only in ruminant species. Rodents, by contrast, have a huge family of prolactin-related genes and primates have an expansion of growth hormone-related genes. We have tried to address the functional significance of such large placental-specific genes limited to a few species using detailed in micegene expression and gene knockout analysis. As a first case, we have performed detailed temporal and spatial gene expression analysis, using northern blotting and detailed in situ hybridization analysis, in the mouse placenta for the 24 genes in the Prolactin-like protein (PLP)/placental lactogen (PL) gene family which sits in a 1 Mb base of chromosome 13. As a group, the genes appear to be exclusively expressed in the placenta and yet, while there is some overlap of some genes, there are at least 14 different patterns of gene expression. There is no obvious correlation between gene expression and position in the gene locus. Surprisingly also, even close paralogues that are the most evolutionarily related often do not have identical expression. Bioinformatic analysis of promoter regions from genes with common subtype expression patterns can identify conserved regulatory elements that may direct trophoblast subtype-specific gene expression. These data suggest that the PLP/PL genes each have unique functions and that the various trophoblast cell subtypes have different physiological roles. These data raise questions about how the physiology of pregnancy is regulated in those species that do not have these placental hormones.

Steady‐state transposon mutagenesis in inbred maize

We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.

Gene knockout analysis of two γ-tubulin isoforms in mice

γ-Tubulin regulates the nucleation of microtubules, but knowledge of its functions in vivo is still fragmentary. Here, we report the identification of two closely related γ-tubulin isoforms, TUBG1 and TUBG2, in mice, and the generation of TUBG1- and TUBG2-deficient mice. TUBG1 was expressed ubiquitously, whereas TUBG2 was primarily detected in the brain. The development of TUBG1-deficient ( Tubg1 −/− ) embryos stopped at the morula/blastocyst stages due to a characteristic mitotic arrest: the mitotic spindle was highly disorganized, and disorganized spindles showed one or two pole-like foci of bundled MTs that were surrounded by condensed chromosomes. TUBG2 was expressed in blastocysts, but could not rescue the TUBG1 deficiency. By contrast, TUBG2-deficient ( Tubg2 −/− ) mice were born, grew, and intercrossed normally. In the brain of wild-type mice, TUBG2 was expressed in approximately the same amount as TUBG1, but no histological abnormalities were found in the Tubg2 −/− brain. These findings indicated that TUBG1 and TUBG2 are not functionally equivalent in vivo, that TUBG1 corresponds to conventional γ-tubulin, and that TUBG2 may have some unidentified function in the brain.

The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage ϕWMY: Cloning, sequencing, expression, and mutational analysis in Escherichia coli

From the genome library of Staphylococcus warneri M, the two successive cell-lysis genes ( holWMY and lytWMY) were cloned and characterized. The lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and p I were 54 kDa and 8.95, respectively. When overproduced in Escherichia coli, lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative N-acetylmuramoyl- l-alanine amidases encoded by the staphylococcal phages ϕ11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for d-alanyl-glycyl endopeptidase), amidase ( l-muramoyl- l-alanine amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys–His–Asn triad is crucial for the enzymatic function. On the other hand, the holWMY gene encoded a protein (HolWMY) with molecular mass and p I of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the S. aureus phage, ϕ11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (ϕWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the holWMY–lysWMY genes in the phage genome.

Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.

We report a remarkably high UV-radiation resistance in the extremely halophilic archaeon Halobacterium NRC-1 withstanding up to 110 J/m2 with no loss of viability. Gene knockout analysis in two putative photolyase-like genes (phr1 and phr2) implicated only phr2 in photoreactivation. The UV-response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2400 genes during light and dark repair. In addition to photoreactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes, and two proteases for eliminating damaged proteins. Moreover, a UV-induced down-regulation of many important metabolic functions was observed during light repair and seems to be a phenomenon shared by all three domains of life. The systems analysis has facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB. Finally, the systems analysis has raised, through the integration of experimentally determined and computationally inferred data, many experimentally testable hypotheses that describe the metabolic and regulatory networks of Halobacterium NRC-1.

Increased expression of the FIGLA transcription factor is associated with primordial follicle formation in the human fetal ovary.

The process of primordial follicle formation is central to the determination of a woman's reproductive lifespan, and in humans occurs towards the end of mid-gestation. Gene knockout analysis in the mouse has shown that Figla, a transcription factor specifically expressed in germ cells, is essential for oocytes to survive and form primordial follicles. Our objective was to investigate whether a human homologue present in the genome database plays a similar role in human ovary development. Standard and real-time RT-PCR demonstrated that the human FIGLA gene is expressed in the fetal ovary but not by a range of other tissues, and that expression increases across mid-gestation, rising some 40-fold by the time of primordial follicle formation. The entire coding sequence was cloned and new exonic sequences identified. Electrophoretic mobility shift assays with in vitro-expressed human FIGLA protein showed that, as in the mouse, FIGLA can heterodimerize with E12 protein and bind to the E-box of the human ZP2 promoter. Similar mobility shifts were identified in human fetal ovary extracts. These results suggest that FIGLA is involved in continued oocyte survival as primordial follicles form in the human as in the rodent ovary.

Linking genotype to phenotype: production and large scale functional analysis of gene knockouts in human cancer cells

Linking genotype to phenotype: production and large scale functional analysis of gene knockouts in human cancer cells

RNA interference of peroxisome-related genes in C. elegans: a new model for human peroxisomal disorders.

RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and peroxins for the development of Caenorhabditis elegans and to compare the roles of these proteins in the nematode to their roles in yeasts and humans. The nematode counterparts of the human ATP-binding cassette half-transporters, the enzymes alkyldihydroxyacetonephosphate synthase and Delta(3,5)-Delta (2,4)-dienoyl-CoA isomerase, the receptors for peroxisomal membrane and matrix proteins (Pex19p and Pex5p), and components of the docking and translocation machineries for matrix proteins (Pex13p and Pex12p) are essential for the development of C. elegans. Unexpectedly, RNAi silencing of the acyl-CoA synthetase-mediated activation of fatty acids, the alpha- and beta-oxidation of fatty acids, the intraperoxisomal decomposition of hydrogen peroxide, and the peroxins Pex1p, Pex2p, and Pex6p had no apparent effect on C. elegans development. The described analysis of functional gene knockouts through RNAi provides a basis for the use of C. elegans as a valuable model system with which to study the molecular and physiological defects underlying the human peroxisomal disorders.

Novel double promoter approach for identification of transgenic animals: A tool for in vivo analysis of gene function and development of gene-based therapies.

Advances in vertebrate genetics have allowed studies of gene function in developing animals through gene knockout and transgenic analyses. These advances have encouraged the development of gene-based therapies through introduction of exogenous genes to enhance and/or replace dysfunctional or missing genes. However, in vertebrates, such analyses often involve tedious screening for transgenic animals, such as PCR-based genotype determinations. Here, we report the use of double-promoter plasmids carrying the transgene of interest and the crystallin-promotor-driven Green fluorescent protein (GFP) in transgenic Xenopus laevis tadpoles. This strategy allows a simple examination for the presence of GFP in the eyes to identify transgenic animals. PCR-based genotyping and functional characterization confirms that all animals expressing GFP in the eyes indeed carry the desired promoter/transgene units. Thus, the use of this and other similar vectors should dramatically improve current transgenesis protocols and reduce the time and cost for identifying transgenic animals.

Splicing signals and factors in plant intron removal

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

Probing the performance limits of the Escherichia coli metabolic network subject to gene additions or deletions.

An optimization-based procedure for studying the response of metabolic networks after gene knockouts or additions is introduced and applied to a linear flux balance analysis (FBA) Escherichia coli model. Both the gene addition problem of optimally selecting which foreign genes to recombine into E. coli, as well as the gene deletion problem of removing a given number of existing ones, are formulated as mixed-integer optimization problems using binary 0-1 variables. The developed modeling and optimization framework is tested by investigating the effect of gene deletions on biomass production and addressing the maximum theoretical production of the 20 amino acids for aerobic growth on glucose and acetate substrates. In the gene deletion study, the smallest gene set necessary to achieve maximum biomass production in E. coli is determined for aerobic growth on glucose. The subsequent gene knockout analysis indicates that biomass production decreases monotonically, rendering the metabolic network incapable of growth after only 18 gene deletions. In the gene addition study, the E. coli flux balance model is augmented with 3,400 non-E. coli reactions from the KEGG database to form a multispecies model. This model is referred to as the Universal model. This study reveals that the maximum theoretical production of six amino acids could be improved by the addition of only one or two genes to the native amino acid production pathway of E. coli, even though the model could choose from 3,400 foreign reaction candidates. Specifically, manipulation of the arginine production pathway showed the most promise with 8.75% and 9.05% predicted increases with the addition of genes for growth on glucose and acetate, respectively. The mechanism of all suggested enhancements is either by: 1) improving the energy efficiency and/or 2) increasing the carbon conversion efficiency of the production route.

Splicing signals and factors in plant intron removal

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

The Age of 'Omes

The Genomic Age is upon us. Vidal discusses strategies for using genomic information to change how scientific questions are addressed. He suggests that data from expression profiling, structural and biochemical genomics, protein interaction mapping, protein localization studies, and global gene knockout analysis need to be integrated into functional maps, and these should be organized into a biological atlas analogous to geographical maps and atlases. He emphasizes that the information needs to be available to all researchers along with tools to aid in the analysis. His view is very positive, yet still presents the pitfalls of global approaches to biological questions. M. Vidal, A biological atlas of functional maps. Cell 104 , 333-339 (2001). [Online Journal]

All about Arabidopsis.

Figure 1Arabidopsis thaliana has grown from a lowly weed into a model organism of lofty stature and spawned a rapidly growing field of research. A wealth of on-line resources has arisen to coordinate the multicenter Arabidopsis sequencing project. The Arabidopsis Genome Initiative (AGI) provides up-to-date information about ongoing efforts to sequence the five chromosomes of A. thaliana. As of March 2000, the AGI has sequenced roughly 86.5% of the genome. Several sites provide access to pooled genome information, but you might also want to visit the individual sequencing centers, including Cold Spring Harbor Laboratory’s Arabidopsis Genome Analysis, the Washington University School of Medicine’s Genome Sequencing Center Arabidopsis thaliana Page, and the Institute for Genomic Research’s Arabidopsis thaliana Database. The Arabidopsis Information Resource (TAIR) will replace the Arabidopsis thaliana Database Project (AtDB); but until TAIR’s first full release, projected for September 2000, users can still access AtDB’s database. With such view and search options as genetic maps, physical maps, and alleles, researchers should have no trouble finding information on this resource. TAIR already has its BLAST service up and running. Selecting the “New GenBank (DNA)” dataset runs a homology search against all sequences submitted within the last month. The “CSHL/WASHU Preliminary (DNA)” data set will search the nightly submissions made by these two sequencing centers. The Kazusa DNA Research Institute, a member of the AGI, has developed the Kazusa Arabidopsis Data Opening Site (KAOS). Visitors can use KAOS’s Arabidopsis Genome Displayer to view the pooled annotated sequence data collected from the AGI. The graphical interface for a particular clone shows BLASTN and BLASTX results, plus predicted coding regions and any corresponding GenBank annotations. The visual alignment of all these data makes it easy to pick out potential genes of interest. The European Scientists Sequencing Arabidopsis (ESSA) coordinates sequencing efforts across several laboratories in the European Union. The Munich Information Centre for Protein Sequences (MIPS) then gathers and analyzes these sequence data, and places them on its web site: the MIPS Arabidopsis thaliana Database (MATDB). In a separate effort from the Arabidopsis Genome Initiative, the University of Minnesota and Michigan State University have taken a different tack with the Arabidopsis cDNA Sequence Analysis Project. These groups are building a database of expressed sequence tags (ESTs) from A. thaliana. Visitors can search sequence annotation at the Search of EST Similarity Analysis Reports, or at Searching Plant ESTs Using BLASTN. To make complete sense of the plethora of sequence data, researchers must undertake the daunting task of functional analyses. The Arabidopsis Functional Genomics Consortium (AFGC) — another collaborative effort — has already begun that work. The AFGC grew out of two technologies: gene knockout and microarray analysis. The Arabidopsis Knockout Facility at the University of Wisconsin Biotechnology Center, offers a fee-based service for researchers. Using their collection of 60,480 Arabidopsis lines that have T-DNA insertions, they will do PCR reactions on pooled lines with your primers. The AFGC Microarray Services is another fee-based tool for functional analyses. The consortium expects their year 2000 arrays to have 7,000–10,000 nonredundant ESTs. The wealth of Arabidopsis web sites coordinates research efforts and facilitates an unprecedented exchange of information. When you see how much Arabidopsis has to offer, you might just turn green with envy. Tabled 1Arabidopsis Genome Initiativehttp://www.arabidopsis.org/agi.htmlArabidopsis Genome Analysishttp://nucleus.cshl.org/protarab/Genome Sequencing Center: Arabidopsis thalianahttp://genome.wustl.edu/gsc/Arabidopsis thaliana Databasehttp://www.tigr.org/tdb/ath1/htmls/index.htmlArabidopsis Information Resourcehttp://www.arabidopsis.org/Kazusa Arabidopsis Data Opening Sitehttp://www.kazusa.or.jp/kaos/MIPS Arabidopsis thaliana Databasehttp://www.mips.biochem.mpg.de/proj/thal/Arabidopsis cDNA Sequence Analysis Projecthttp://www.cbc.med.umn.edu/ResearchProjects/Arabidopsis/Arabidopsis Functional Genomics Consortiumhttp://afgc.stanford.edu/Arabidopsis Knockout Facilityhttp://www.biotech.wisc.edu/Arabidopsis/AFGC Microarray Serviceshttp://afgc.stanford.edu/afgc-array.html Open table in a new tab