Unknown,Transcriptomics,Genomics,Proteomics

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Functional genomic analysis of a nitrate reductase knockdown relative to wild-type in model-ecosystems (mesocosm)

ABSTRACT: Current advances in genomics and computational biology have afforded novel insight as to how the phenotype is generated from the genotype – systems biology. We argue that systems biology, when viewed through an ecological lens, provides an unprecedented opportunity to understand how genes cascade through multiple levels of biological organization to alter ecosystem function. To test this approach, we established six monocultures of Arabidopsis thaliana ‘Columbia’- wild-type plants, six monocultures of a single gene variant (mutant) to the wild-type, and six mixtures with equal density plantings of each genotype in mesocosm chambers (50 x 50 x 45 cm). The mutant harbored a T-DNA insertion in the main nitrate reductase gene (nia2). This is the gateway enzyme for N metabolism, which resulted in activity levels that were 38% of the wild-type. Mesocosms were instrumented to monitor soil and air temperature, water and humidity status, and CO2 differentials. Transcript expression profiles were generated for each of the monoculture populations by collecting and processing 100 leaves per mesocosm at generation 2 and 4. Design: Expression profiles were generated for each monoculture population by pooling 100 leaves per mesocosm into one sample. This resulted in 6 biological replicates for each genotype per generation. Thus, a total of 48 samples were generated (6 wild-type + 6 nia2 mutants x 2 generations x 2 CO2 treatments) and hybridized onto microarrays. We used a direct loop design analyzing generations separately (diagrams of hybridization designs are linked as supplementary PDF files).

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: David Weston

PROVIDER: E-GEOD-16068 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The existence of two separate lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that one of these lineages (lineage I) might be more pathogenic towards human hosts. We have previously shown that the more pathogenic lineage expresses higher levels of Shiga toxin 2 (Stx2) than the non-pathogenic lineage II. To evaluate why lineage 2 isolates do not express appreciable levels of toxin, two lineage 2 isolates (FRIK966 and FRIK2000) were chosen as representatives of lineage 2 and whole genome microarrays were performed using Agilent microarrays using the E. coli O157:H7 EDL933 lineage I clinical type isolate as a reference. Microarray results were utilized to evaluate what genes and pathways might be missing or differentially expressed. Quantitative RT-PCR was utilized to validate the microarray data. Based upon the transcriptome of Escherichia coli O157:H7 EDL933 an oligonucleotide microarray, made up of 60 mers was designed. A total of 4873 genes in an 8 x 15K Agilent microarray design. Designed using a custom script, specifications for gene specific oligos were based upon various design characteristics such as temperature of melting, 3’ location, specificity, lack of repeat nucleotides, etc. (Charbonnier et al., 2005). Arrays were manufactured using Agilent Sure-print technology. Each array consisted of duplicate elements for each gene randomly distributed with Agilent control elements included. All procedures were performed according to respective manufacturer protocols. Lineage I and lineage II strains were grown overnight as described, a total of 10e7 cells were washed twice in fresh media, normalized based upon optical density, inoculated into fresh media, incubated at 37oC shaking 120 x g for 3 hours and then suspended in RNAprotect bacteria reagent (Qiagen Inc., Valencia, CA.). Total RNA was extracted using RNeasy Bacteria Mini Kit (Qiagen Inc., Valencia, CA.) and trace amounts of DNA were removed using RNase-Free DNase Set (Qiagen Inc., Valencia, CA.). RNA was quantified using Nanodrop system (NanoDrop Technologies, Wilmington, DE) and quality confirmed by electrophoresis on a Bio-rad Experion system (BioRad XXX). For each sample, 10 ug of RNA were labeled with either CyDye3-dCTP or CyDye5-dCTP (Perkin Elmer) using the LabelStar kit (Qiagen Inc., Valencia, CA.) and Random nonamers (Integrated DNA Technologies ). Labeled cDNA were hybridized to the microarray using Agilent Hi-RPM hybridization solution in an Agilent Hybridization chamber (Agilent.). A total of 8 arrays, each with duplicate elements for each gene, alternating dye swap for each replicate (4 biological replicates), were analyzed to obtain genes that were consistently and differentially regulated in comparison to EDL933, while limiting false discover rate (FDR) below a stringent 5% (Benjamini and Hochberg, 1995).

Project description:Several recent reports show that the cerebral cortex in humans and animals with altered expressions of Wnt/cadherin network-associate molecules display cytoarchitectural abnormalities reminiscent of cortical dysplasias seen in some (mouse-, rat-, and monkey-based) animal models of prenatal cocaine exposure. Therefore, we employed oligo microarrays followed by real-time RT-PCR to compare expressions of genes involved in Wnt and cadherin systems in the cerebral wall of 18-day-old (E18) fetuses from cocaine-treated (20 mg/kg cocaine, s.c., b.i.d., E8-18) and drug-naive (saline, s.c.) mice. The pregnant mice chronically treated with cocaine in the above-described manner represent one of the animal models producing offspring with widespread cortical dysplasias. Out of more than 150 relevant genes in the arrays, 32 were upregulated and 9 were downregulated in cocaine-exposed fetuses. The majority of these genes (30 out of 41) were similarly affected in the frontal and occipital regions of the cerebral wall. We also used Western immunoblotting to examine the ability of cocaine to regulate the protein levels of beta-catenin, the key functional component of both Wnt and cadherin systems. While the total cell levels of beta-catenin were increased throughout the cerebral wall of cocaine-exposed fetuses, its nuclear (gene-transcription driving) levels remained unaltered. This suggests a transcription-unrelated role for cocaine-induced upregulation of this protein. Overall, our findings point to an intriguing possibility that that cerebral cortical dysplasias observed in several animal models of prenatal cocaine exposure may be at least in part related to alterations in the Wnt/cadherin molecular network. It has been demonstrated that exposure to cocaine increases cell death in the fetal CNS. To examine the molecular mechanisms of this effect, we employed mouse oligo microarrays followed by real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) to compare expressions of apoptosis-related genes in the cerebral wall of 18-day-old (E18) fetuses from cocaine-treated (20 mg/kg cocaine, s.c., b.i.d., E8th-E18th) and drug-naive (saline, s.c.) mice. Out of approximately 400 relevant genes in the arrays, 53 showed alterations in expression in cocaine-exposed fetuses. Upregulation was observed in 35 proapoptotic and 8 antiapoptotic genes; 4 proapoptotic and 6 antiapoptotic genes were down-regulated. The affected genes encode a wide range of apoptosis-related proteins, including death receptors (NTF-R1, NTF-R2, DR3, DR5, LTbeta-R, GITR, P57 TR-1) and their adaptor and regulatory proteins (MASGE-D1, TRAF-2, SIVA, MET, FLIP, FAIM, IAP1, ATFA), members of transcription regulatory pathways (JNK, NF-kappaB, P53), members of BCL-2 family of proteins (BID, BAD, BAX, BIK, NIP21, NIP3, NIX, BCL-2), DNA damage sensor (PARP-1), caspases and their substrates and regulatory proteins (caspases 8, 4, 9, and 3, ACINUS, CIDE-A, CIDE-B, GAS2), mitochondrially released factors (cytochrome c, AIF, PRG3), specific endoplasmic reticulum- and oxidative stress-associated factors (BACH2, ABL1, ALG2, CHOP), members of cell survival AKT and HSP70 pathways (PIK3GA, PTEN, HSP70, BAG1, BAG2), and others. This suggests that cocaine affects survival of developing cerebral cells via multiple apoptosis-regulating mechanisms. Experiment Overall Design: Animals Experiment Overall Design: Timed pregnant Swiss Webster (CFW; Charles River Experiment Overall Design: Lab. Wilmington, MA) dams were maintained in individual Experiment Overall Design: cages in a climate-controlled room on a 12/12-h light/ Experiment Overall Design: dark cycle. They were divided into two groups. The first Experiment Overall Design: (experimental) group received subcutaneous (at the dorsum Experiment Overall Design: of the neck) injections of 20 mg/kg cocaine hydrochloride Experiment Overall Design: (Research Technology Branch, National Institute Experiment Overall Design: of Drug Abuse, Rockville, MD) dissolved in 200 mkl of Experiment Overall Design: 0.9% saline, twice a day (at 8:00 AM and 8:00 PM) from Experiment Overall Design: 8th through 18th day of pregnancy (E8âE18). The second Experiment Overall Design: (control) group was subjected to the same schedule of Experiment Overall Design: 0.9% saline only injections. The cocaine treatment was Experiment Overall Design: designed to replicate the one capable of reducing cerebral Experiment Overall Design: cortical mass in mouse offspring. Also, the Experiment Overall Design: relatively protracted period the chronic treatment was Experiment Overall Design: chosen to maximize the changes in the tissue expression Experiment Overall Design: of cocaine-regulated genes of interest. Throughout the Experiment Overall Design: treatment, all mice were weighed daily, and, from E8, the Experiment Overall Design: control and experimental animals were pair-fed, with the Experiment Overall Design: daily amount of food (Mouse Chow; Ralston Purina Saint Experiment Overall Design: Louis, MO) provided to each control dam being matched Experiment Overall Design: to that consumed by the paired experimental dam. Water Experiment Overall Design: was available ad libitum. We found that this feeding Experiment Overall Design: regiment resulted in similar weight gains from E8 to E18 Experiment Overall Design: in both experimental and control animal groups Experiment Overall Design: (46.41F0.45% and 44.99F0.59% respectively). On E18, Experiment Overall Design: 1 h after the morning treatment, the animals were Experiment Overall Design: anesthetized by peritoneal injection of 50 mg/kg sodium Experiment Overall Design: pentobarbital (Abbott Lab., Abbott Park, IL) and their Experiment Overall Design: uteri were removed. The frontal cerebral wall (containing Experiment Overall Design: cerebral cortex and underlying transient cortical zones Experiment Overall Design: anterior to the striatal level) and the occipital cerebral wall Experiment Overall Design: (containing cerebral cortex and underlying transient Experiment Overall Design: cortical zones posterior to the level of the hippocampus) Experiment Overall Design: were dissected from the fetuses and stored in liquid Experiment Overall Design: nitrogen prior to analysis. The animal experimentations Experiment Overall Design: used in this study were approved by the University of Experiment Overall Design: Maryland Animal Care and Use Committee. Experiment Overall Design: Microarray Experiment Overall Design: Tissue from two control and two experimental fetuses Experiment Overall Design: were used in the processing of a single microarray slide Experiment Overall Design: (Part G4121A, Agilent Technol). Five slides were processed Experiment Overall Design: per region of the fetal cerebral wall. For a given region, the Experiment Overall Design: tissue for each microarray slide was obtained from a Experiment Overall Design: separate set of control and experimental litters. Total RNA Experiment Overall Design: was isolated using TRIzol Reagent (Invitrogen, Carlsbad, Experiment Overall Design: CA). The yield of total RNA was determined by absorbance Experiment Overall Design: at 260 nm on a DU 640 Spectrophotometer (Beckman Experiment Overall Design: Coulter, Fullerton, CA). The 260/280 nm ratios of the Experiment Overall Design: samples were >1.8. For each assay, 5 mkg of RNA from Experiment Overall Design: control tissue and 5 mkg of RNA from experimental tissue Experiment Overall Design: were reverse transcribed using 3DNA Array Detection Kit Experiment Overall Design: (Genesphere, Hatfield, PA) with primers containing different Experiment Overall Design: dcaptureT sequences for control and experimental Experiment Overall Design: samples. SuperScript II Reverse Transcriptase for these Experiment Overall Design: reactions was purchased from Gibco BRL (Gaithersburg, Experiment Overall Design: MD). The resultant cDNAs were hybridized to microarray Experiment Overall Design: slides for 16 h in Agilent Hybridization Buffer (Agilent Experiment Overall Design: Technol) at 60 8C. Upon completion of the hybridization, Experiment Overall Design: the slides were washed for 10 min with 0.005% Triton in Experiment Overall Design: 6xsodium chloride/sodium citrate buffer (pH 7; SSC) at Experiment Overall Design: room temperature and then for five more min with 0.005% Experiment Overall Design: Triton in 0.1xSSC and 1 more minute in 0.1xSSC (pH 7), Experiment Overall Design: both at 4 8C. Washed slides were air-dried for 30 s. For Experiment Overall Design: fluorescent labeling of microarray-bound cDNA, the slides Experiment Overall Design: were incubated for 3 h at 65 8C with Capture Reagent Experiment Overall Design: Hybridization Mixture from 3DNA Array Detection Kit Experiment Overall Design: (Genesphere). In this mixture, Alexa 546 fluorochromeincorporating Experiment Overall Design: dendrimers contained single-stranded arms Experiment Overall Design: complimentary to the dcaptureT sequences used in the Experiment Overall Design: reverse transcription of RNA from control tissue, while Experiment Overall Design: Alexa 647 fluorochrome-incorporating dendrimers contained Experiment Overall Design: arms complimentary to the capture sequences used Experiment Overall Design: in reverse transcription of RNA from experimental tissue. Experiment Overall Design: The labeling reaction was terminated by washing of the Experiment Overall Design: microarray slides at room temperature for 10 min with Experiment Overall Design: 0.005% Triton in 2xSSC and for another 10 min with in Experiment Overall Design: 0.2xSSC. After that, the slides were air-dried for 30 s. The Experiment Overall Design: processed microarray slides were scanned at 10 Am Experiment Overall Design: resolution on a GenePix 4100A scanner (Axon Instr., Union Experiment Overall Design: City, CA) with the laser excitation at 532 nm [emission filter Experiment Overall Design: 575DF35 (green); photomultiplier voltage 550] for Alexa Experiment Overall Design: 546 (control samples) and the laser excitation at 635 nm Experiment Overall Design: [emission filter 670DF40 (red); photomultiplier voltage Experiment Overall Design: 695] for Alexa 647 (experimental samples). The signals Experiment Overall Design: were converted into 16-bits-per pixel resolution images, Experiment Overall Design: providing color depth of 65,536 levels. The densitometry Experiment Overall Design: was performed with GenePix Pro 4.1 software (Axon Instr.). Experiment Overall Design: Background was subtracted using the dlocal background Experiment Overall Design: correctionT procedure available in the software. Quality control utilized 255 positive controls, 161 negative controls Experiment Overall Design: and 646 QC-spots present on Agilentâs arrays. For Experiment Overall Design: replicates within a slide, median signal values were Experiment Overall Design: calculated. For each array, the data were normalized in Experiment Overall Design: Acuity 3.1 software (Axon Instr.) by applying locally Experiment Overall Design: weighted scatterplot smoothing (LOWESS) transformation. Experiment Overall Design: The between array scale normalization Experiment Overall Design: was done using intensity log10 ratio distribution box plots (GeneSight Experiment Overall Design: software, BioDiscovery, El Segundo, CA) for verification. Experiment Overall Design: Statistical analysis of the normalized data was conducted Experiment Overall Design: by the Significant Analyses for Microarray Algorithm Experiment Overall Design: (SAM) using Excel macros available at the Stanford SAM website. Experiment Overall Design: In the analyses of both frontal and occipital regions of the fetal cerebral wall, Experiment Overall Design: the cut-off thresholds were set to identify the maximum Experiment Overall Design: number of cocaine exposure-regulated genes at the minimal Experiment Overall Design: false discovery rate (FDR) allowed by SAM for a given set Experiment Overall Design: of microarray data. For both regions, the FDR rates were Experiment Overall Design: <0.5%. The apoptosis and Wnt pathway-related genes were identified with Pathway Assist 2.0 software (Stratagene, La Jolla, CA). Experiment Overall Design: This was followed by manual review of literature for each Experiment Overall Design: identified gene.

Project description:We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis. Genomic array design Microarrays were designed as described below and manufactured by Agilent Technologies (www.agilent.com). Further information on design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of transcription start sites and identification of promoter sequences We interrogated 5 databases: Ensembl, VEGA, Refseq, ZGC full length clones and a database provided by Dr. Leonard Zon (Harvard Medical School, Boston, USA) in order to assemble an extensive list of zebrafish transcripts. The Zon lab database is a hand-curated database of zebrafish genes that have homologues in other species. We included all transcripts that appeared in the manually annotated databases (VEGA, Zon) and in the ZGC full length database. We also identified genes present in any 2 of the 5 databases and included those not already selected. The transcripts were mapped to the zebrafish genome (Zv4, June 2004) obtained from UCSC Bioinformatics (http://genome.ucsc.edu) and the transcription start site (TSS) for each transcript was determined. Transcripts with TSSs within 500bp were clustered into a transcriptional unit (TU) and promoter regions were identified relative to the most upstream TSS. This resulted in the identification of 13,413 TUs and corresponding promoter regions. Each promoter region was extracted and masked for repetitive sequence by RepeatMasker. If the promoter region contained a gap the upstream sequence was also masked. Information on the transcriptional units that were included in the final design can be found at http://jura.wi.mit.edu/bioc/gbell/zfish_chip/. Selection of oligonucleotides 60-mer oligonucleotide probes representing the region between 1.5kb upstream and 0.5kb downstream of the annotated TSS of each transcriptional unit were then designed. Although transcription factors and other DNA binding proteins are known to regulate genes from distances of greater than -1.5kb or + 0.5kb, much information can be gained from regions close to the TSS [45], and the H3K4Me3 mark studied in this paper is found at the most 5â end of a gene, close to the TSS. Selection of 60-mers for the microarrays was essentially as described in [14] using the Zv4 build of the zebrafish genome and a locally customized version of ArrayOligoSelector. 60-mers were chosen so that promoter regions contained approximately one probe every 250bp with a maximum distance between probes for each promoter region set at 600bp. In cases where only one probe could be designed for a particular TU these were not included in the final design. This process yielded 80,839 probes for 11,171 promoter regions We also incorporated several sets of control probes, both positive and negative. On each array there are 1090 probes designed against âgene desertâ regions, which are genomic regions that are unlikely to be bound by transcriptional regulators, and 270 probes designed against Arabidopsis thaliana genes, which are not present in the zebrafish genome (by BLAST). In addition, because our main motivation for making these microarrays is to identify mesodermally-regulated genes we included 7 genes expressed in mesoderm during gastrulation as positive controls (wnt11, flh, vent, msgn1, myod, fgf8, pcdh8). Probes designed against these promoters, which flank from 3-4kb around each TSS, are arrayed 2-4 times on each slide. Since these genes are expressed at gastrula stages to varying degrees, they also serve as a positive controls in this study. Finally there are 2256 controls added by Agilent and a variable number of blank spots. These probes were divided between two microarray slides each with 44,290 features. We refer to these two microarray slides as the âproximal promoter setâ. A proximal promoter set based on these designs as well as an expanded set of 9 slides which contain regions from â9kb to + 3kb relative to the TSS, are available by contacting Agilent (www.agilent.com) or by downloading the design files from http://jura.wi.mit.edu/bioc/gbell/zfish_chip/ for self-manufacture.

Project description:Anopheles gambiae mosquitoes exhibit an endophilic, nocturnal blood feeding behavior. Despite the importance of light as a regulator of malaria transmission, our knowledge on the molecular interactions between environmental cues, the circadian oscillators and the host seeking and feeding systems of the Anopheles mosquitoes is limited. In the present study, we show that the blood feeding behavior of mosquitoes is under circadian control and can be modulated by light pulses, both in a clock dependent and in an independent manner. Short light pulses (~2-5 min) in the dark phase can inhibit the blood-feeding propensity of mosquitoes momentarily in a clock independent manner, while longer durations of light stimulation (~1-2 h) can induce a phase advance in blood-feeding propensity in a clock dependent manner. The temporary feeding inhibition after short light pulses may reflect a masking effect of light, an unknown mechanism which is known to superimpose on the true circadian regulation. Nonetheless, the shorter light pulses resulted in the differential regulation of a variety of genes including those implicated in the circadian control, suggesting that light induced masking effects also involve clock components. Light pulses (both short and longer) also regulated genes implicated in feeding as well as different physiological processes like metabolism, transport, immunity and protease digestions. RNAi-mediated gene silencing assays of the light pulse regulated circadian factors timeless, cryptochrome and three takeout homologues significantly up-regulated the mosquito's blood-feeding propensity. In contrast, gene silencing of light pulse regulated olfactory factors down-regulated the mosquito's propensity to Our study suggests that the mosquitoâs feeding behavior is under circadian control. Long and short light pulses can induce inhibition of blood-feeding through circadian and unknown mechanisms, respectively, that involve chemosensory factors. A series of assays were performed to assess transcriptomic changes in mosquitoes upon light stimulation and blood feeding in order to assess relationships between photic stimulation and modulation of feeding behavior.

Project description:Background: The mosquito Anopheles gambiae is a major vector of human malaria. Increasing evidence indicates that blood cells (hemocytes) comprise an essential arm of the mosquito innate immune response against both bacteria and malaria parasites. To further characterize the role of hemocytes in mosquito immunity, we undertook the first genome-wide transcriptomic analyses of adult female An. gambiae hemocytes following infection by two species of bacteria and a malaria parasite. Results: We identified 4047 genes expressed in hemocytes, using An. gambiae genome-wide microarrays. While 279 transcripts were significantly enriched in hemocytes relative to whole adult female mosquitoes, 959 transcripts exhibited immune challenge-related regulation. The global transcriptomic responses of hemocytes to challenge with different species of bacteria and/or different stages of malaria parasite infection revealed discrete, minimally overlapping, pathogen-specific signatures of infection-responsive gene expression; 105 of these represented putative immunity-related genes including anti-Plasmodium factors. Of particular interest was the specific co-regulation of various members of the Imd and JNK immune signaling pathways during malaria parasite invasion of the mosquito midgut epithelium. Conclusion: Our genome-wide transcriptomic analysis of adult mosquito hemocytes reveals pathogen-specific signatures of gene regulation and identifies several novel candidate genes for future functional studies. In order to identify hemocyte-specific and immune-responsive transcripts, we first compared transcripts expressed in hemocytes from one day old sugar-fed mosquitoes to transcripts detected in whole mosquitoes of the same age and feeding status. This resulted in identification of the hemocyte-enriched transcriptome. We then compared hemocytes from 1 day old mosquitoes, 1 hour after immune challenge with heat-killed Escherichia coli or Micrococcus luteus, to control female mosquitoes injected with sterile PBS to determine the bacteria challenge responsive transcriptomes. We used heat-killed bacteria in these assays, because our primary interest was in identifying the bacterial responsive transcriptome and to avoid the potentially confounding effects of altered gene expression due to the lethal effects of a systemic infection associated with injection of living bacteria. Lastly, we compared hemocytes from mosquitoes at 24 hours and 19 days after ingestion of a blood meal infected with Plasmodium berghei to mosquitoes of the same age fed a non-infected blood meal to determine the ookinete and sporozoite infection responsive transcriptomes, respectively. This design resulted in a total of five experimental treatments. The following samples are not included in this submission: Hemo E coli vs. hemo unchallenged A Hemo E coli vs. hemo unchallenged B Hemo m luteus vs. hemo unchallenged A Hemo m luteus vs. hemo unchallenged B

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Functional genomic analysis of a nitrate reductase knockdown relative to wild-type in model-ecosystems (mesocosm)

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