title: Epigenetically Silenced Genes Found Using Methylated DNA Immunoprecipitation in Colon Cancer Cells Deficient in DNMTs

status: Public on Jan 01 2008

city: Madrid

first: Filipe

middle: Vicente

last: Jacinto

state: Madrid

address: C/ Melchor Fernández Almagro, 3,

department: Molecular Pathology

country: Spain

institute: CNIO

type: Methylation profiling by genome tiling array

summary:
CpG island promoter hypermethylation of tumor suppressor genes is a common hallmark of human cancer, and new large-scale epigenomic technologies might be useful in our attempts to define the complete DNA hypermethylome of tumor cells. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKOcells). Using methylated DNA immunoprecipitation (MeDIP) methodology in conjunction with promoter microarray analyses we found that DKO cells experience a significant loss of hypermethylated CpG islands. Further characterization of these candidate sequences demonstrates CpG island promoter hypermethylation and silencing of genes with potentially important roles in tumorigenesis, such as the Ras guanine-nucleotide release factor RASGRF2, the apoptosis-associated basic helixloop transcription factor BHLHB9, and the homeobox gene HOXD1. Hypermethylation of these genes occurs already in premalignant lesions and accumulates during tumorigenesis. Thus, our results demonstrate the usefulness of DNMT genetic disruption strategies combined with MeDIP in searching for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use.
Keywords: Human Proximal Promoter Array; DNA Methylation

overall_design: Comparative experiment: methylated DNA immunoprecipitated/INPUT(Total Genomic DNA) from the colon cancer cell line HCT116 wild type vs methylated DNA imonoprecipitated/INPUT from the colon cancer cell line HCT116 DNMT1/3b doubleknockout (DKO)

first: Manel

last: Esteller


title: Human colon expression in healthy, CD, treated CD, and UC

city: Cincinnati

first: Lee

last: Denson

state: OH

address: 3333 Burnet Avenue

country: USA

institute: CCHMC

summary:
Activation of inflammatory pathways in human IBD
IL-6:STAT3 pathways are activated in the affected colon in IBD.  However, the functional implications of this are not known.  We hypothesized that pro-inflammatory IL-6:STAT3 dependent networks would be up regulated in the colon of pediatric patients with Crohn Disease (CD) and Ulcerative Colitis (UC), and that these would regulate leukocyte survival, proliferation, and recruitment to the gut.  These experiments tested differential colon gene expression relative to these pathways in healthy, CD, and UC samples.  Colon biopsy samples were obtained from CD and UC patients at diagnosis, CD patients during therapy, and healthy controls.  The global pattern of gene expression was determined using GeneSpring software, and biological networks were identified using Ingenuity software.  Data suggested that two IL-6:STAT3 dependent networks are up regulated in pediatric IBD both at diagnosis and during therapy which regulate leukocyte recruitment and survival.  The degree of up regulation of these genes compared to healthy controls was remarkably conserved across the two CD groups and the UC groups, suggesting common mechanisms of mucosal inflammation.
Keywords: Single time point in CD, UC, and healthy controls.

overall_design:
Colon RNA was isolated from biopsies obtained from CD at diagnosis and during therapy, UC at diagnosis, and healthy controls.  Samples were obtained from the most proximal affected segment of colon.  Microarray experiments were performed as described in the CCHMC microarray core, and data was analyzed as described above in the summary.

The '107' internal control CEL files (for batches 1-4) used for normalization of the Sample VALUEs are linked below as supplementary files.


title: Expression comparison between MGAS2221 and isogenic spyCEP mutant

status: Public on Jan 05 2008

city: Reno

first: Paul

last: Sumby

state: NV

address: 1554 N. Virginia St.

department: Microbiology & Immunology

institute: University of Nevada

summary:
Expression analysis was performed to confirm that spyCEP is the only gene differentially regulated between parental strain MGAS2221 and isogenic spyCEP mutant 2221DELTACEP.
Keywords: expression comparison

overall_design: 4 cultures of each strain were analyzed.


title: Transcriptional response of the marine pennate diatom Phaeodactylum tricornutum to Fe limitation

city: La Jolla

first: Andrew

middle: E

last: Allen

state: CA

address: 11149, North Torrey Pines Road, Suite 220

department: Deptartment of Environmental Genomics

institute: J. Craig Venter Institute

summary:
Diatoms, which are responsible for up to 40% of the 45 to 50 billion metric tons of organic carbon production each year in the sea, are particularly sensitive to Fe stress. Here we describe the transcriptional response of the pennate diatom Phaeodactylum tricornutum to Fe limitation using a partial genome microarray based on EST and genome sequence data.  Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport and nitrate assimilation are down-regulated to cope with the reduced cellular iron quota. This retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and storage carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electron s by mitochondrial alternative oxidase (AOX), augmented Fe-independent oxidative stress responses, and sensitized iron capture mechanisms.
Keywords: Marine phytoplankton, pinnate diatom

overall_design: Wild-type Phaeodactylum tricornutum was grown under Fe replete (10,000 nM) and Fe limiting (5nM) conditions. Partial genome gene expression analysis of iron-inducible genes was conducted using a two-color competitive hybridization microarray.

first: Pascal

middle: J

last: Lopez

first: Chris

middle: P

last: Bowler

first: Julie

last: LaRoche


title: Peritoneal Carcinomatosis of gastric cancer

status: Public on Jan 31 2008

city: Tübingen

first: Kay

last: Nieselt

state: DE

address: Sand 14

department: Department for Computer Science

country: Germany

institute: Institute for Bioinformatics and Medical Informatics

summary:
Peritoneal carcinomatosis is a frequent finding in patients with primary gastric cancer, and it is associated with a poor prognosis. A major mechanism in peritoneal carcinomatosis is the dissemination of cancer cells into the abdominal cavity, mainly in diffuse gastric adenocarcinoma. The features that enable diffuse primary gastric tumours to develop peritoneal dissemination have been little investigated and are only incompletely understood. We therefore compared the gene expression profile in patients with diffuse primary gastric cancer with and without peritoneal carcinomatosis.
Specimens from consecutive gastric cancer patients with and without peritoneal carcinomatosis were investigated using oligonucleotide microarrays.
Keywords: Disease state analysis

overall_design: Double loop design with dye swap

first: Derek

last: Zieker

first: Bettina

last: Knapp


title: Time-series of transcriptome analysis of Bacillus subtilis response to 3 mM potassium sorbate

status: Public on Jan 07 2008

city: Leiden

first: Anna

middle: Magdalena

last: Zakrzewska

address: Wassenaarseweg 64

department: IBL

country: Netherlands

institute: University of Leiden

summary:
The weak organic acid sorbic acid is a commonly used food preservative, as it inhibits the growth of bacteria, yeasts and molds. We have used genome-wide transcriptional profiling of Bacillus subtilis cells during mild sorbic acid stress to reveal the growth inhibitory activity of this preservative and to identify potential resistance mechanisms. Our analysis demonstrated that sorbic acid stressed cells induce responses normally seen upon nutrient limitation. This is indicated by the strong de-repression of the CcpA, CodY and Fur regulon, the induction of TCA cycle genes, SigL and SigH mediated genes, and the stringent response. Intriguingly, these conditions did not lead to the activation of sporulation, competence, or the general stress response (GSR). The fatty acid biosynthesis (fab) genes and BkdR regulated genes are upregulated, which may indicate plasma membrane remodeling. This was further supported by the reduced sensitivity towards the fab inhibitor cerulenin upon sorbic acid stress. We are the first to present a comprehensive analysis of the transcriptional response of B. subtilis to sorbic acid stress.
Keywords: time-series, potassium sorbate response

sample_organism: Bacillus subtilis subsp. subtilis str. 168

overall_design: Time-series of B.subtilis response to 3 mM potassium sorbate during controlled growth in batch-fermentors in minimal defined medium. Samples for microarrays were taken from both the treated and control cultures at 0, 10, 20, 30, 40, and 50 min after addition of sorbate. Two biologically independent experiments were performed.

first: Alex

last: Ter Beek

first: Bart

last: Keijser

first: Andre

last: Boorsma

first: Rick

last: Orij

first: Gertien

last: Smits

first: Stanley

last: Brul


title: Array-Based Genomic Comparative Hybridization Analysis of Field Strains of Mycoplasma hyopneumoniae

city: Ames

last: Minion

state: IA

address: 1130 Vet Med

department: VMPM

institute: Iowa State University

summary:
Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97cilium adhesin within different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breed and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined potential variation of fourteen field strains in comparison to strain 232 on which the array was based. Genomic DNA was obtained, amplified with TempliPhi™, and labeled indirectly with Alexa dyes. Post genomic hybridization, the arrays were scanned and data analyzed using a linear statistical model. Results indicate that genetic variation could be detected in all fourteen field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in M. hyopneumoniae genome where spatial clustering of variability between the field strains and strain 232 exists.
Keywords: CGH, Mycoplasma Hyopneumoniae

overall_design: TempliPhi™ amplified DNA samples from field isolates were compared to control strain 232 using a two-color experimental microarray design. Independent samples from one isolate labeled with one dye were paired with control samples labeled with the alternate dye; the samples were mixed and hybridized to the microarray. For nine of the fourteen isolates (95MP1501, 95MP1502, 95MP1503, 95MP1504, 95MP1508, 95MP1509, 97MP0001, 00MP1301, and 05MP2301), four independent field isolate DNA samples were paired with four independent DNA samples from control 232. In two of the four arrays, the control sample was labeled with Alexa 555 dye and compared to the field isolate sample labeled with Alexa 647 dye (Molecular Probes, Inc., Eugene, Ore.). The dye assignment to control and treated samples was reversed for the other two arrays (dye swap). The arrays were hybridized under identical conditions as described below. This procedure was repeated for isolates 95MP1510 for a total of four arrays, where the control sample was labeled with Alexa 647 dye in three of the arrays and Alexa 555 dye for the fourth array. For isolates 95MP1505, 95MP1506 and 95MP1507, a total of five arrays each, including two dye swaps, were done; and for isolate 00MP1502, a total of six arrays were done with the control labeled with Alexa 555 dye for four of the arrays and Alexa 647 for the other two arrays.

first: Melissa

middle: L

last: Madsen

first: Michael

last: Oneal

first: Stuart

middle: W

last: Gardner

first: Erin

last: Strait

first: Dan

last: Nettleton

first: Eileen

last: Thacker

first: F

middle: C


title: Transcriptome Changes in Mycoplasma hyopneumoniae During Infection

first: Chris

summary:
Mycoplasma hyopneumoniae causes swine pneumonia and contributes significantly to porcine respiratory disease complex. The mechanisms of pathogenesis are difficult to address since there is a lack of genetic tools, but microarrays can be used to study transcriptional changes that occur during colonization and disease in pigs. This approach has the potential to identify genes important to virulence. This study sought to identify genes that change transcript levels during infection. To accomplish this, organisms collected from bronchial alveolar lavages were compared to that of broth grown organisms. Bronchial alveolar lavage was performed on pigs 28 days post infection with M. hyopneumoniae, and organisms were isolated by differential centrifugation. Mycoplasma RNA enriched preparations were then obtained from total RNA by subtracting eucaryotic ribosomal and messenger RNAs. cDNA was generated with mycoplasma ORF-specific primers, fluorescently labeled with Cy3 and Cy5, and used to interrogate microarrays. Arrays were scanned and analyzed using a mixed linear statistical model. Nine biological replicates were analyzed in this fashion.
Our analysis indicated that 33 Mhyo genes were up-regulated and 46 genes were down-regulated (p<0.01) during disease in the pig lung at a false discovery rate < 2.7%. Of the down-regulated genes, 27 of 46 (59%) lacked assigned function, and 20 of 33 (61%) of the up-regulated genes were hypothetical genes. Four down-regulated and two up-regulated genes were putative lipoproteins. secA (mhp295; p = 0.003), and two glycerol transport permeases (potA (mhp380); p = 0.006 and ugpA (mhp381); p = 0.003) were up-regulated in vivo. Elongation factor EF-G (fusA (mhp083); p = 0.002), rpoC (mhp635; p = 0.003), adenylate kinase (adk (mhp208); p = 0.001), prolyl aminoacyl tRNA synthetase (proS (mhp397); p = 0.009) and cysteinyl-tRNA synthetase (cysS (mhp661); p < 0.001) were down-regulated in vivo.
Keywords: RNA, spotted DNA/cDNA

overall_design: Nine independent BALF samples were processed for total RNA along with nine in vitro cultures. Each in vivo BALF sample was paired with an independent in vitro culture sample for hybridization on nine two color arrays. For five of the arrays, the control in vitro sample was labeled with Alexa Fluor 555 and compared to Alexa Fluor 647-labeled BALF sample. The dye assignment to control samples was reversed for the other 4 arrays. The slides were hybridized under identical conditions as described under hyb protocol.

first: Supraja

last: Puttamreddy

middle: D

last: Carruthers

middle: F


title: WT macrophage response to transfected bacterial ligands

city: Chicago

first: Jess

last: Leber

state: IL

address: 920 E. 58th Street CLSC 615

department: Microbiology

institute: The University of Chicago

summary:
Transcriptional profiling of WT macrophages transfected with L. monocytogenes genomic DNA, synthetic MDP, or both
Keywords: Ligand comparison.

overall_design: Experiment in WT background, with different combinations of bacterial ligands.  Multiple biological replicates (see array names), with one replicate per array.

middle: H

first: Gregory

last: Crimmins

first: Sridharan

last: Raghavan

first: Jeffery

middle: S

last: Cox

first: Daniel

middle: A

last: Portnoy


title: Primary macrophage response to L. monocytogenes and bacteria-derived ligands

summary:
Transcriptional profiling of macrophages (of various genetic backgrounds) infected with L. monocytogenes or transfected with purified ligands
Keywords: Timecourse, cell type comparison, bacteria strain comparison, drug comparison, ligand comparison.

overall_design: Timecourse experiment in different genetic backgrounds, with both WT and mutant bacteria, and with different combinations of purified ligands.  Multiple biological replicates (see array names), with one replicate per array.


title: WT macrophage response to WT L. monocytogenes, with or without CAPE treatment

summary:
Transcriptional profiling of WT macrophages infected with WT L. monocytogenes for 180 minutes, with or without CAPE treatment
Keywords: Drug comparison.

overall_design: Experiment in WT background, with WT bacteria, with or without drug treatment.  Multiple biological replicates (see array names), with one replicate per array.


title: WT and irf3-/- macrophage response to WT L. monocytogenes

summary:
Transcriptional profiling of WT and irf3-/- macrophages infected with WT L. monocytogenes for 180 minutes
Keywords: Cell type comparison.

overall_design: Experiment in different genetic backgrounds, with WT bacteria.  Multiple biological replicates (see array names), with one replicate per array.


title: WT and myd88-/- macrophage response to WT and hly- L. monocytogenes

summary:
Transcriptional profiling of WT and myd88-/- macrophages infected with WT and hly- L. monocytogenes for 30, 60, 120, or 180 minutes
Keywords: Timecourse, cell type comparison, bacteria strain comparison.

overall_design: Timecourse experiment in different genetic backgrounds, with both WT and mutant bacteria.  Multiple biological replicates (see array names), with one replicate per array.


title: Soleus muscle response to level running

city: Buffalo

first: Kathleen

middle: M

last: McCormick

state: NY

address: 3435 Main Street

institute: University at Buffalo

summary:
This study surveyed expression of genes which might be involved in regulating the satellite cell response to non-damaging exercise.
Low density cDNA arrays were used to survey expression of selected cytokines and growth factors in the soleus muscle following 60 minutes of level running.
Keywords: Exercise response; pre versus 24-hr post

overall_design: Five-mo-old male Fisher 344 rats were assigned to an unexercised control group (n = 7) or to an exercise group (n = 5).  The exercise group performed 60 minutes of level treadmill running (19 m/min) and were sacrificed 24 hours later.

last: Savage

last: Brazeau


title: Zebrafish response to Mycobacterium marinum infection (Compugen series)

city: Chapel Hill

first: John

last: Rawls

state: NC

address: 111 Mason Farm Rd.

department: Department of Cell and Molecular Physiology

institute: University of North Carolina at Chapel Hill

summary:
Mycobacteria infect macrophages that aggregate with additional macrophages and lymphocytes to form granulomas. We have used a functional genomics approach to identify immune response genes expressed during granuloma formation in Mycobacterium marinum-infected transparent zebrafish larvae where individual infection steps can be viewed in real time. We assessed RNA expression profiles from zebrafish larvae that were either infected with Mycobacterium marinum or mock-infected. Zebrafish infections were performed at 1 day post-fertilization (dpf), and samples were derived from pools of 6dpf zebrafish larvae.
Keywords: host response to infection

overall_design: Total RNA was purified from pooled intact zebrafish larvae (154-180 larvae/pool, 2 biological replicate pools/condition) and digested with DNase I using an Absolutely RNA Microprep Kit (Stratagene). RNA quality was confirmed using an Agilent 2100 Bioanalyzer. Total RNA from each replicate pool (5 μg/replicate) was used as template for independent reverse transcription reactions utilizing the 3DNA Array 350 Kit (Genisphere) and modified oligo-dT primers containing a 5’ fluorophore/dendrimer-specific sequence. Competing cDNA samples for each hybridization were paired, purified, and hybridized to epoxy-coated glass slides (MWG Biotech) containing the Zebrafish Oligonucleotide Library (Compugen, Sigma-Genosys). Cy3 and Cy5 labeling of respective dendrimer-specific sequences was performed using using the complementary capture reagents provided in the 3DNA Array 350 Kit (Genisphere). Raw Cy3 and Cy5 expression values were normalized (LOWESS) and expression ratios were transformed to log2 values using ScanArray software (Perkin Elmer).

first: Tamara

last: Pozos

first: Lalita

last: Ramakrishnan


title: Chromosome 20q amplification regulates in vitro response to Kinesin-5 inhibitor

city: Carlsbad

first: Aimee

last: Jackson

address: 1896 Rutherford Rd

institute: Regulus Therapeutics

summary:
We identified gene expression signatures predicting responsiveness to a Kinesin-5 (kinesin spindle protein, KIF11) inhibitor (Kinesin-5i) in cultured colon tumor cell lines.  Genes predicting resistance to Kinesin-5i were enriched for those from chromosome 20q, a region of frequent amplification in a number of tumor types.  siRNAs targeting genes in this chromosomal region identified AURKA, TPX2 and MYBL2 as genes whose disruption enhances response to Kinesin-5i.  Taken together, our results show functional interaction between these genes, and suggest that overexpression of AURKA, TPX2 and MYBL2 is functionally involved in resistance to Kinesin-5i.  Furthermore, our results suggest that patients whose tumors overexpress AURKA due to amplifications of 20q will be more likely to resist treatment with Kinesin-5 inhibitor, and that inactivation of AURKA may sensitize these patients to treatment.
Keywords: Cell line comparison

overall_design: A series of colon cancer cell lines was analyzed for gene expression signatures.  These gene expression signatures were then correlated with in vitro responsiveness of each cell line to a KSP inhibitor (Kinesin-5i).  Transcripts whose expression was correlated with response (>0.5 or <-0.5) were identified as reporters of cellular response to this inhibitor.  Reporters were further tested by RNAi for functional role in response to this inhibitor.

first: Mao

last: Mao

first: Sumire

last: Kobayashi

first: Teresa

last: Ward

first: Matthew

last: Biery

first: Hongyue

last: Dai

first: Steven

middle: R

last: Bartz

first: Peter

last: Linsley


title: Impact of CSN1S1 polymorphism on mammary transcriptome of lactating goats

status: Public on Jan 03 2008

city: St Genes-Champanelle

first: Christine

last: Leroux

address: Centre INRA de Theix

department: PHASE

country: France

institute: INRA

summary:
Genetic (animal species, breed and genotype) has a considerable effect on milk composition. In particular, goats present a remarkable polymorphism at the alpha-S1-casein (CSN1S1) locus which results in large differences in milk protein content and indirectly in milk fat content and its fatty acids composition. In order to decipher the  mammary metabolic pathways involved, we examined the effect of CSN1S1 polymorphism on the expression of 8,379 genes in caprine mammary gland using a bovine oligonucleotide microarray. Six lactating goats fed ad libitum were assigned to 2 groups based on their genotype at the CSN1S1 locus: High vs. Low genotype goats carrying, respectively, two reference alleles associated with high CSN1S1 synthesis and two defective alleles associated with low CSN1S1 synthesis.
Keywords: Genotype comparison

overall_design: 6 slides were performed for a total of 3 independent comparisons (Comp1, Comp2, Comp3): each microarray was co-hybridized with one High CSN1S1 genotype goat sample and one Low CSN1S1 genotype goat sample; each hybridization was repeated in a dye-swap manner for a total of 4 spots per oligonucleotide (2 intra- and 2 inter-slides).

first: Séverine

last: Ollier

first: Stéphanie

last: Chauvet

first: Patrice

last: Martin

first: Yves

last: Chilliard


title: Plasmablasts and germinal center founder cells developing in vivo

city: Birmingham

first: Kai-Michael

last: Toellner

address: Medical School

department: Immunity and Infection

country: United Kingdom

institute: University of Birmingham

summary:
A range of cellular interactions and cytokines influence immunoglobulin class switch recombination (CSR), which occurs both in extrafollicular antibody responses and germinal centers. This study addresses where CSR occurs during extrafollicular responses, allowing identification of the cellular interactions and cytokine-producing cells that influence CSR in vivo. The response in mice of B cells specific for 4-hydroxy-3-nitrophenyl-acetyl (NP) to NP-Ficoll was analyzed. Immunohistology and flow cytometry confirm responding NP-binding cells move to the outer T zone of the spleen and start dividing there on the second day. During the third day they move either to follicles as germinal center founding cells, or extrafollicular foci as plasmablasts. Analysis of expression of AID, Bcl-6 and Blimp-1 mRNA in single NP-binding cells indicates a proportion of T zone B blasts express AID transiently without Bcl-6 and these cells are shown to undergo CSR. By contrast Blimp-1+ cells emerging on the third day and were universally AID- and hence unable to initiate CSR. Bcl-6 mRNA without protein, expressed by 40% of naïve NP-binding cells, is rapidly downregulated. Bcl-6 and AID are coexpressed by germinal centre founding cells only on moving to follicles during the third day after immunization.
Keywords: TaqMan Low Density Array

overall_design: C57BL/6 mice with one knockin of NP-specific immunoglobulin heavy chain from quasimonoclonal (QM) mice were immunized with NP-Ficoll. This induces germinal center founder cells and plasmablasts within 3 days. B cells were FACS-sorted in 24 hour intervals according to NP-binding, and B220 CD138 expression levels.

first: Jennifer

last: Marshall

first: Lalit

last: Pallan

first: Mei-Chi

last: Hsu

first: Francesco

last: Falciani

first: Ian

last: MacLennan


title: Microarray profiles of radiation response in the NCI60 cell lines

city: New York

first: Sally

last: Amundson

department: Center for Radiological Research

institute: Columbia University

summary:
The 60 cell lines of the NCI Anti-Cancer Drug Screen (NCI60) constitute the most extensively characterized in vitro cancer cell model, and have been tested for sensitivity to over 100,000 potential chemotherapy agents.  We have used the NCI60 cell lines and three additional lymphoblast lines to develop a database of responses of cancer cells to ionizing radiation.  We compared clonogenic survival, apoptosis induction, and gene expression response by microarray analysis.  While several studies have measured relative basal gene expression in the NCI60, this is the first comparison of large-scale gene expression changes in response to genotoxic stress.  We found genes differentially regulated in cells with low survival after 2 or 8 Gy γ-rays.  In contrast to reported basal gene expression patterns, little tissue-of-origin effect was detected in the radiation response pattern of gene expression, with the exception of lymphoblastoid cell lines.  The most striking patterns in the radiation data were a set of genes upregulated preferentially in the p53 wild-type lines, and a set of cell-cycle regulatory genes strongly down-regulated across the entire NCI60 panel.  The response of these genes to γ-rays appears to be unaffected by the myriad of genetic differences across this very diverse cell set, and represents the most universal gene expression response to ionizing radiation yet observed.
Keywords: radiation response

overall_design: Cells were exposed to 8Gy and gene expression ratios between untreated (Cy5) cells and exposed cells (Cy3) were measured four hours later


title: Cell line dependent gene expression profiles of two mouse embryonic stem cells

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum München GmbH

summary:
Molecular phenotyping studies carried out so far on different embryonic stem cells (ESC) have focused mainly on the identification of molecular markers responsible for pluripotency. Unlike these, the goals of our study were to compare and functionally characterize the gene expression profiles of R1 ESC established from F1 (129X1/SvJ x 129S1) blastocyst (Nagy et al. 1993 PNAS 90: 8424-8428) and HM-1 ESC established from inbred strain (Selfridge et al. 1992 Somat Cell Mol Genet 18: 325-336), as our earlier study showed performance variations between these cells. ES cells were cultured on mitomycin C treated mouse embryonic fibroblast feeder cell layer. Cells were grown in standard ES cell medium changed daily [high glucose DMEM (Gibco-Invitrogen, Carlsbad, CA) supplemented with Na-pyruvate (0.11% w/w; Gibco-Invitrogen), 0.1mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), fetal bovine serum (15% v/v; HyClone; Logan,UT), 1000 U/ml murine-LIF (CHEMICON International, Temecula, CA) and antibiotics (penicillin: 50 U/ml, streptomycin: 50 μg/ml (Sigma-Aldrich)]. Total RNA was isolated from aliquots of R1 ES cells at passage 13 and HM-1 ES cells at passage 23 using RNeasy Midi kit (Qiagen, Düsseldorf, Germany) procedures. 15 µg of total RNA each from the contrasting samples were used for reverse transcription and labeling either with Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). The labeled samples were dissolved in hybridization buffer and added to the cDNA arrays (custom produced at GSF) containing over 21,000 sequences and hybridized for 17 hours at 42°C. The microarray results of four independent hybridizations were analyzed and differentially regulated genes were identified. Finally, the results of four randomly selected genes were verified by real time PCR analysis. The analysis revealed 55 transcripts that showed significant variation (p < 0.01) between the two ES cell lines. Of these, 8 transcripts were up regulated and the rest down regulated in the HM-1 ES cells. Most of these genes were overrepresented in important biological processes like growth and development (21%), cell organization and biogenesis (11%), regulatory roles (21%), and organogenesis (14%). Moreover, the verification analysis using real-time PCR has confirmed the results of microarray. Thus based on the detailed analysis, and confirmation of the results with independent analysis, it is possible to conclude that the expression profile reflected the true molecular variations between the two ES cell lines, and the identified transcripts can serve as molecular markers that explain biological differences between the two ES cell lines.
Keywords: ES cell lines, murine, nuclear embryo derived, Nocodazol,

overall_design: HM-1 and R1 ES cell lines comparision before and after Nocodazol treatment. 8 microarray experiments including 50% dye swap experiments

first: Beckers

last: Johannes

first: Marion

last: Horsch


title: Reprogramming human somatic cells to a pluripotent state

city: Madison

first: Victor

last: Ruotti

state: WI

address: 425 Henry Mall

department: Biotechnology

institute: UW Madison

summary:
Here we compare the global gene expression of induced pluripotent stem cell (iPS) cells with those of normal human embryonic stem cells and parental mesenchymal cells.
Keywords: reprogramming, iPS

overall_design: We used 1 chip/sample (3 iPS clones for each mesenchymal clone - 12 iPS clones; 4 mesenchymal clones and 5 normal human ES cell lines).


title: Regulation of nitrogen acquisition (NO3-, NH4+ et N2) by nitrogen status

status: Public on Dec 31 2007

city: Beaucouze

first: sandrine

last: balzergue

address: Rue G. Morel

summary:
affy_nitrogen_medicago - affy_nitrogen_medicago - Experiment has been designed to characterize the molecular expression patterns associated to a contrasted modification of the nitrogen status of the whole plant. The systemic effects of nitrogen status modifications are investigated and compared on non nodulated plant supplied with NO3, NH4 or nodulated plants (Sinorhizobium meliloti 2011) supplied with air. The root systems were separated in two compartments of unequal sizes (split root system). Two treatments were applied on the larger compartment in order to modulate the nitrogen status of the plant: for the S treatment, roots are supplied with nutrient solution containing 10 mM NH4NO3,, whereas for the C treatment, roots are supplied with nitrogen free medium. In the case of N2 fixing plants, N limitation was obtained by replacing air by a mixture of Ar and O2 80 per cent and 20 per cent. The effects of these treatments were investigated on roots of the minor compartment supplied continuously with either NO3 1 mM, NH4 1 mM or air (N2) and on the shoots. We were also interested in the molecular expression patterns associated to the roots deprived of N.-The root system of non-nodulated (NO3- and NH4+) or nodulated (N2) plants is split into two unequal parts and each one is installed in a separate compartment. For the S treatement, the major root part is supplied with NH4NO3 10 mM whereas the minor part is supplied with either NO3- 1mM, NH4+ 1mM or N2. For the C treatement, the major root part is supplied with nitrogen-free nutrient solution whereas the minor part is supplied with either NO3- 1mM, NH4plus 1mM or N2. Each treatement is four days long. Samples of roots of six biological types (NO3S, NO3C, NH4S, NH4C, N2S and N2C) were collected. Two biological repeats per biological types have been analyzed. The effect of the S and C treatments were investigated for each N sources by comparing Affymetrix transcriptomes (NO3C vs NO3S, NH4C vs NH4S, N2C vs N2S).
Keywords: treatement (nitrogen-sufficient) vs treatement (nitrogen-limited)

platform_organism:
Sinorhizobium meliloti
Medicago sativa
Medicago truncatula

overall_design: 26 arrays - medicago

first: Sandrine

last: Balzergue

first: Marc

last: Lepetit

last: Ruffel

first: Marie-Laure

last: Martin-Magniette


title: Estrogen's and progesterone's impact on gene expression in the mouse lacrimal and meibomoan gland.

city: Boston

first: David

last: Sullivan

state: MA

address: 20 Staniford Street

institute: Schepens Eye Research Institute

summary:
PURPOSE: The hypothesis tested in the study was that the effect of estrogen and progesterone on the lacrimal gland is mediated through specific receptors and that hormonal effects involve the regulation of gene expression and protein synthesis. METHODS: Lacrimal glands were collected from young adult, ovariectomized mice, that were treated with 17beta-estradiol, progesterone, 17beta-estradiol plus progesterone or vehicle for 2 weeks. Glands were pooled according to treatment, processed for the isolation of RNA, and evaluated for differentially expressed mRNAs by using gene microarrays. Bioarray data were analyzed with sophisticated bioinformatics and statistical programs. The expression of selected genes was verified by using gene chips and quantitative real-time PCR methods. RESULTS: The results demonstrate that 17beta-estradiol, progesterone, or both hormones together significantly influences the expression of hundreds of genes in the mouse lacrimal gland. Sex steroid treatment led to numerous alterations in gene activities related to transcriptional control, cell growth and/or maintenance, cell communication, signal transduction, enzyme catalysis, immune expression, and the binding and metabolism of nucleic acids and proteins. A number of the 17beta-estradiol, progesterone or 17beta-estradiol plus progesterone effects on gene expression were similar, but most were unique to each treatment. Of particular interest was the finding that these hormones seem to contribute little to the known sex-related differences in gene expression of the lacrimal gland. CONCLUSIONS: These results support the hypothesis that estrogen's and progesterone's action on the lacrimal gland involves the regulation of numerous genes. However, these hormone effects do not appear to represent a major factor underlying the sexual dimorphism of gene expression in lacrimal tissue.
Keywords: Placeco vs Hormone Treatment

overall_design: Ovariectomized mice were treateated with placebo, 17beta-estradiol, progesterone or 17beta-estradiol plus progesterone for 14 days.  Each treatment was pooled into 3 biological replicates that were hybridized to separate microarrays.

first: T

last: Suzuki

first: F

last: Schirra

first: S

last: Richards

first: N

last: Treister

first: M

last: Lombardi

first: P

last: Rowley

first: R

middle: V

last: Jensen

first: D


title: LongSAGE during the initiation of ligninolytic enzymes production in Phanerochaete chrysosporium

city: Hikone-City

first: Masahiko

last: Minami

state: Shiga

address: 2500, Hassaka-cho

department: Department of Biological Resources Management

country: Japan

institute: University of Shiga Prefecture

summary:
White rot fungi are able to degrade woody lignin and other persistent organic compounds including artificial chemicals (e.g. chlorinated dioxin) in secondary metabolism. This ability has potential in a wide range of biotechnological applications including remediation of organopollutants and the industrial processing of paper and textiles. Ligninolytic fungi secondarily secrete extracellular oxidative enzymes thought to play an important role in these compounds decay. However, detail of metabolic pathway and initiation signals of the degradation system is unclear. To investigate genes directly and indirectly related to it, we constructed long serial analysis of gene expression (Long SAGE) library from the most studied white rot fungus, Phanerochaete chrysosporium.
Keywords: transcriptome profiling

overall_design: To analyze the transcriptome profile during the initiation of manganese peroxidase (MnP) and lignin peroxidase (LiP) production in Phanerochaete chrysosporium, we constructed the day 3 culture (just started the enzyme production) library and the day 2 culture (the activity of enzymes is not detected) library.


title: Genome and transcriptome divergence in Bacillus subtilis after 6,000 generations of evolution

city: Vancouver

first: Heather

last: Maughan

state: BC

address: Life Sciences Centre

department: Zoology

country: Canada

institute: University of British Columbia

type:
Expression profiling by array
Genome variation profiling by array

summary:
Ten populations were evolved for 6,000 generations. Five had strong selection for sporulation, imposed partially by their cultivation in sporulation-inducing medium, while the other five populations had relaxed selection for sporulation, by cultivating them in sporulation-repressing medium.  Batch cultures were diluted 1:100 daily for approximately 892 days. In the five populations with relaxed selection for sporulation, sporulation ability was eventually lost.
Keywords: comparative genome hybridization and transcriptome divergence

overall_design:
Genomic DNA from all ten populations, and ancestors, was isolated, labeled and hybridized to microarrays.  Only one hybridization was performed for each experimental population, with evolved and ancestral DNA hybridized to the slide.

RNA hybridizations were done by chosing one strain that was evolved in sporulation-repressing medium and one strain that was evolved in sporulation-inducing medium.  Two biological replicates were performed, as well as a dye swap for each biological replicate. Evolved and ancestral strains were grown in identical medium and RNA was isolated at the onset of stationary phase.

first: Carl

last: Birky

first: Wayne

last: Nicholson


title: Global responses of a C. jejuni single domain globin mutant to nitric oxide releasing compounds

city: Sheffield

first: Robert

last: Poole

state: South Yorkshire

address: Firth Court, Western Bank

department: Molecular Biology & Biotechnology

institute: University of Sheffield

summary:
A single-domain globin (cgb) mutant strain of Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate  (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 10 uM NOC-5 & -7 (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis.

Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.
Keywords: Stress-response of a cgb mutant to NO

overall_design:
A C. jejuni NCTC11168 cgb mutant was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 μl sample onto both nutrient agar plates incubated at 37 °C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 °C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier.

cDNA synthesis was carried out using 12 µg of starting material, which was primed with 9 µg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 µl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 °C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 °C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 °C.

Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 °C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner.

The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of ≤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed

first: Claire

last: Monk

middle: K


title: biosensor-series

city: Eggenstein-Leopoldshafen

first: Jessica

last: Legradi

state: Baden-Wuerttemberg

address: Hermann-von-Helmholtz-Platz 1

department: Institute of Toxicology and Genomics ITG

institute: Karlsruhe Institute of Technology

summary:
Early life stages are generally most sensitive to toxic effects. Our knowledge on the action of man-made chemicals on the developing vertebrate embryo is, however, rather limited. We exposed zebrafish embryos to 11 of environmental toxicants and measured the changes in gene expression profiles by microarray. Our results demonstrate that the genome of the zebrafish embryo responds to toxicant exposure in a highly sensitive and specific manner.
Keywords: toxicants response genes expression profiles

overall_design: Microarray analysis were performed, first, with 6 model toxicants, such as, 4-chloroaniline, Cadmium, Methyl mercury, DDT, TCDD, and Valproic acid, to compare gene expression patterns at exposure embryos from 4 to 24 hpf, from 24 to 48 hpf, and from 96 to 120hpf; Second, with added 5 model toxicants, including Acrylamide, Arsenic, Lead, PCB, and t-BHQ, to compare total 11 toxicants gene expression profile at exposure embryos from 96 to 120 hpf; Third, with different toxicants concentration to compare response gene profiles at 96 to 120 hpf; Fourth, with four toxicants mixture, including Cadmium, Mercury, Lead, and Arsenic to compare the chemicals synergistic effects. Last, we did the blind testing to figure out different toxicants according response gene profiles.


title: Transcrip and physiological responses of chemostat-grown Campylobacter jejuni to S-nitrosoglutathione

summary:
A single-domain globin (cgb) mutant strain of Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate  (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 0.25 mM GSNO (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis.
Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.
Keywords: Stress-response to GSNO

overall_design:
A C. jejuni cgb mutant was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 μl sample onto both nutrient agar plates incubated at 37 °C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 °C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, GSNO (final concentration 0.25 mM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier.

cDNA synthesis was carried out using 12 µg of starting material, which was primed with 9 µg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 µl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 °C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 °C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 °C.

Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 °C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner.

The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of ≤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

first: Bruce

last: Pearson

first: Francis

last: Mulholland


title: Transcriptome analysis of GSNO treated batch cultures of C. jejuni

summary:
Batch cultures of Wild-type C. jejuni NCTC11168 were grown in 150 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % O2, 10 % CO2, 80 % N2) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 10 minute exposure samples of both treated and untreated cells were harvested into phenol/ethenol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis.
Keywords: Stress-response of batch cultures to GSNO


title: Meiotic DNA double strand breaks in the yeast Saccaromyces cerevisiae

city: Bethesda

first: Michael

last: Lichten

state: MD

address: 37 Convent Dr

department: LBMB

institute: National Cancer Institute

type: Genome binding/occupancy profiling by genome tiling array

summary:
DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination.
Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose

overall_design: We use two different strategies to map the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) based approach that targets the Spo11p protein, which remains covalently attached to DSB ends in the rad50S mutant background. The second approach involves BND cellulose enrichment of the single strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11p removal. We use dmc1 and dmc1 rad51 mutants that accumulates meiotic single strand DNA intermediates

first: Cyril

last: Buhler

first: Valérie

last: Borde


title: Cardiac and Soleus muscles following exposure of rats to heat acclimation and exercise training

city: Jerusalem

first: Michal

last: Horowitz

country: Israel

institute: The hebrew University

summary:
The processes of adaptation to environmental heat and aerobic exercise training improve efficiency in various body systems and bring about acclimatory homeostasis. In order to examine the global genomic responses of the soleus and heart following exposure of rats to these stressors, nylon cDNA Atlas Array was used.

Male rats were exposed to one of the following stressors: heat acclimation, aerobic training (treadmill), and combined heat acclimation and aerobic training for short (2, 3 days) and long (1 mo) time period.

The study comprised seven experimental groups: Controls-untreated. Heat acclimated groups (2dac, Acc)– exposure to environmental heat at 34C for 2 or 30 days. Exercise groups (3dex, Ex)– graduated training protocol under normothermic conditions for 3 and 30 days at 24C. Exercise training and heat acclimation – (3dexac, ExAc)- exposed to both environmental heat and aerobic exercise as above.

The Series data tables appended below:
1) Heart - normalized log2 ratio of geomeans defined as treatment/control
2) Soleus - normalized log2 ratio of geomeans defined as treatment/control
Keywords: stress response

overall_design:
21 samples, 3 pool each, of:
1) Control untreated rats
2) Long-term heat acclimated rats
3) Long-term aerobic-exercised trained rats.
4) Rats exposed to long-term heat acclimation and exercise training.
5) Short term heat acclimated rats.
6) Short term aerobic exercised trained rats
7) Rats exposed to short-term heat acclimation and exercise training.

first: Einat

last: Kodesh


title: Identification of sexually dimorphic gene expression in the brain of adult zebrafish (Danio rerio)

city: Exeter

first: Eduarda

last: Santos

state: Devon

address: Prince of Wales Road

department: School of Biosciences

institute: University of Exeter

summary:
Sexual differentiation in vertebrates is initiated during embryogenesis and leads to the development of individuals into phenotypic males or females. The molecular pathways showing sexual dimorphic patterns have been principally investigated in the gonads. In other organs, much less is known about the gender-associated pattern of gene expression. The brain plays a coordinating role on sexual function in both genders including regulation of reproductive development and maturation through the synthesis of neuropeptide hormones and the regulation of sexual behavior in both males and females. In this study, we aimed at identifying molecular pathways regulated in a gender specific manner in breeding zebrafish in order to generate a greater understanding of the gender specific physiology of the brain. To do so, we measured the gene expression profiles of individual brains of 6 males and 5 females using a 17k oligonucleotide microarray, and interrogated the dataset for genes showing a gender-specific gene expression profile. 42 genes were found to be differentially expressed between males and females from which 18 genes were over-expressed in males and 24 genes were over-expressed in females.
Keywords: gender specific gene expression in the zebrafish brain

overall_design: Sexually mature fish (6 males and 6 females) were kept in a 15L tank and allowed to breed naturally. After a 20-day period, fish were sacrificed humanly and the brain was extracted, fixed in RNAlater (Ambion) and stored at -20oC until required for microarray analysis. RNA was extracted using the TRI reagent method followed by a LiCl precipitation (Sigma) according to the manufacturer's instructions. Total RNA was amplified and labeled using the Amino Allyl MessageAmp aRNA amplification kit (Ambion). A reference design was used for the array hybridizations. Array analysis was performed according to established protocols (described in Santos et al., 2007) and the data was extracted and queried for differences in gene expression profiles between genders.

first: Peter

last: Kille

first: Victoria

last: Workman

first: Charles

last: Tyler


title: Transcriptional profiling of aluminum toxicity and tolerance responses in maize roots

status: Public on Jan 29 2008

city: Ithaca

first: Lyza

middle: G

last: Maron

address: Tower Rd., Cornell University

department: USDA - ARS

institute: Robert W. Holley Center for Agriculture and Health

summary:
Aluminum (Al) toxicity is a major factor limiting crop yields on acid soils. In maize, Al tolerance is a complex phenomenon involving multiple genes and physiological mechanisms yet uncharacterized. To begin elucidating the molecular basis of maize Al toxicity and tolerance, we performed a detailed temporal analysis of root gene expression under Al stress using microarrays with an Al-tolerant and an Al-sensitive maize genotype. Seedlings of both genotypes were grown in hydroponics in a full nutrient solution containing 39uM of free Al3+ activity. Root samples were collected at 'time zero', and after 2, 6 and 24 hours of treatment.
Keywords: stress treatment, time course

overall_design:
Time course experiment with 0h, 2h, 6h and 24 hours of Al treatment in two maize genotypes contrasting for Al tolerance. The experimental design used for the microarray study was of two interconnected loops, and utilized a total of 16 slide sets. Samples collected from each genotype at one time point were contrasted with the previous and subsequent time points in a loop (4 time points x 2 genotypes = 8 slide sets). Additionally, the loops were interconnected (with dye swap) so that the two genotypes were contrasted directly at each time point (4 time points x 2 replicates = 8 slide sets). The design was balanced for dye distribution throughout samples and biological replicates. Each sample was labeled twice with Cy3 and twice with Cy5; each biological replicate had four samples labeled with Cy3 and four samples labeled with Cy5.

The Sample signal intensity values were log2 transformed and analyzed by two interconnected ANOVA mixed models using PROC MIXED in SAS (Jin et al., 2001; Wolfinger et al., 2001). The normalization ANOVA model  yij = µ + Ai + Dj + (A x D)ij + eij was applied to account for experiment-wide sources of variation associated with array (Ai, degrees of freedom (df) = 15, random effect), dye (Dj, df = 1, fixed effect), and their interactions. The residuals (eij) were treated as normalized values and analyzed in the following ANOVA model (gene model), where effects were evaluated for each gene individually: rikl = µ + Ai + Gk + Tl + (G x T)kl + ekl. Gk is the kth genotype (i.e. C100-6 or L53; df = 1) and represents the effect of each genotype on the expression of every gene, Tl represents the lth treatment (i.e. 0, 2, 6 or 24 hours of Al treatment; df = 3), and (G x T)kl the interaction between genotype and treatment (df = 3). Array (Ai) was included as a random effect to control for spot effects (Jin et al., 2001). Least square means were calculated, and estimates of differential expression were calculated as the difference between least-square means for each of the terms in the model.
ANOVA results linked below as Supplementary files.

first: L

last: Kochian


title: Genome-wide studies of histone Me2 SWIRM and polyamine oxidase domain homologues of mammalian LSD1

status: Public on Dec 30 2007

city: Stockholm

first: Karl

last: Ekwall

address: Alfred Nobels Alle 7

country: Sweden

institute: Karolinska Inst

summary:
In order to gain a more global view of the activity of histone demethylases we report here studies of the two fission yeast SWIRM and polyamine oxidase domain homologues of mammalian LSD1.  Consistent with previous work (Nicolas et al., 2006) we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex.  We find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) to up- and down-regulate expression of different genes.  Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene.  Using hyper-geometric probability comparisons we uncover links between lysine specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1.
Keywords: chip on chip

overall_design: Genome-wide studies of histone demethylation catalysed by the fission yeast SWIRM and polyamine oxidase domain homologues of mammalian LSD1

last: Opel

first: David

last: Lando

first: Carolina

last: Bonilla

first: Sarah

last: C. Trewick

first: Abdelhalim

last: Boukaba

first: Julian

last: Walfridsson

first: James

last: Cauwood

first: Petra

last: J.H. Werler

first: Natasha

last: Murzina

first: Antony

last: M. Carr

first: Tony

last: Kouzarides

first: Robin

last: C. Allshire

first: Ernest

last: D. Laue


title: Circadian regulation of a limited set of microRNAs in Drosophila

status: Public on Dec 29 2007

first: maocheng

last: yang

address: 10 Center Drive

department: NCI

institute: National Institutes of Health

summary:
We used a microarray platform to survey the daily levels of 78 D. melanogaster miRNAs in adult heads of wildtype control flies and the arrhythmic clock mutant cyc01.  At least 2 miRNAs were identified that showed robust daily abundance changes in control flies but not the cyc01 mutant.
Keywords: microRNA, Circadian regulation

platform_organism:
Caenorhabditis elegans
Drosophila melanogaster
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: yw and yw;cyc01   flies were maintained in standard media.  For collections, approximately 50-100 young (2-5 day old) flies were placed in vials that were incubated at 25oC for four days in standard 12hr light-12hr dark cycles [LD; where zeitgeber time 0 (ZT0) is defined as lights-on].  Flies were collected by freezing in dry ice during the fourth LD cycle at the following times; ZT1, 7, 13 and 19.  Subsequently low molecular RNAs were extracted for microarray analysis using a mirMAX microarray.

first: Maocheng

last: Yang

first: JungEun

last: Lee

first: Richard

last: Padgett

first: Isaac

last: Edery


title: Transcriptional signature of wounded keratinocytes reveals selective roles for ERK1/2, P38 and PI3K signalling pathways

city: NICE

first: GEORGIOS

last: FITSIALOS

address: avenue de Valombrose

institute: INSERM

summary:
Covering denuded dermal surface after injury requires migration, proliferation and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38[MAPK] and PI3 kinases, demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. P38[MAPK] inhibition only delays “healing”, probably in line with the control of genes involved in the propagation of injury-initiated signalling. In contrast, PI3 kinase inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF and Ets1. Our results define in vitro human keratinocyte wound closure as a reparation process resulting from a fine balance between positive signals controlled by ERK and p38[MAPK], and negative ones triggered off by PI3 kinase. The perturbation of any of these pathways might lead to dysfunction in the healing process, as those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids.
Keywords: Transcriptome of healing keratinocytes

overall_design:
The DNA microarray analysis concerning the identification of the genes altered in response to cell scratching was performed on 3 independent experiments, derived from 3 distinct cell cultures. Results were generated after 2 independent microarray analyses, meaning that each time point was the result of 6 independent analyses. The data relative to the effect of U0126, SB203580 and LY294002 on the transcriptomal healing response were obtained from 2 independent experiments performed using 2 distinct cell cultures. Results were generated after 2 independent microarray analyses.
The cDNA microarray contained 4.200 distinct cDNA probes. It has been previously described in Moreilhon et al (11). Gene selection was based on relevance to inflammation, infection, differentiation, ion transport, cell signaling, cell migration, proliferation etc… A large fraction of the probes also corresponded to transcripts encoding membrane proteins.
The list of the 4.200 probes is available at http://www.microarray.fr/IPMC/cDNA_microarray5k.html, and the microarray is archived in GEO under reference GSE1853. The cDNA probes were PCR amplified from cDNA derived from Universal Human Reference RNA (Stratagene) by reverse transcription. Probes had the following properties: 1) they have a normalized length of 250 +/- 19 bp; 2) they have a normalized GC content of 52 +/- 8%; 3) they were specific for a unique human gene; and 4) they were controlled by DNA sequencing. PCR products were purified by using QIAquick 96 PCR Purification Kit (Qiagen), resuspended in 3SSC with an average spotting concentration of 200 ng/μl. Microarrays were printed with a SDDC-2 (Bio-Rad) on homemade aldehyde-coated glass microscope slides. Data presented into the present manuscript only refer to sequence-verified probes. Each PCR product was spotted four times on each slide (2 independent clusters of 2 spots spatially separated), to reduce positional bias of the fluorescence readout.
The CyDye-labelled first-strand cDNAs were generated with the CyScribe First-Strand cDNA Labelling Kit (RPN 2600, Amersham Pharmacia Biotech), as described in Moreilhon et al, using ten micrograms of total RNA as template. Unincorporated CyDye-nucleotides were removed using the Nucleotide Removal Kit (Qiagen).
Data Collection and Analysis :
Data Collection :
Microarrays were scanned either on GenePix4000B or on ScanArray5000 (Perkin Elmer). Both machines provided similar results (not shown). 16-bits TIF images were quantified with the corresponding softwares (GenePix Pro 5.0 program (Axon Instruments) for the GenePix, and Quantarray for the ScanArray). Intensities (either total or background values) were defined as average intensities for each spot. Negative controls (“neg”) were spotted on each slide. They corresponded to non mammalian mRNA sequences with no significant identity with any human sequences. Such spots were used as controls. Data was normalized using a dye-swap method as described by Moreilhon et al.. We found that this method, while requiring duplication of experiments, improves the reproducibility of the quantification.
Scratched (S) and non-scratched (NS) RNA samples were reverse transcribed in the presence of known amounts of the corresponding non mammalian “control” RNAs namely DmdNaC and DGNaC added at a respective S/NS ratio of 5, 0.2. Only experiments showing a good correlation between the experimental and true ratios for these controls were kept for analysis. Results are expressed as average ratios of intensities in “scratched cells” over intensities in “non-scratched cells”.
Data was then analyzed and/or visualized with MEV , or with the stand-alone program GeneANOVA, and the SAM’s Excel™ plugin . Lists of genes significantly down and up-regulated under the different experimental conditions were selected by analysis of variance or using SAM. For the analysis of variance, signal was modelled as proposed by Kerr and Churchill (17) according to: yijkg = μ + Ai + Dj + Tk + Gg + (TG)kg + (AG)ig + (DG)jg + εijkg (1), where yijkg is the fluorescent intensity of array i and dye j representing treatment k and gene g. μ, Ai, Dj, Tk, and Gg are factors representing average signals among the whole experiment, one array, one dye, one treatment, or one gene, respectively. (TG)kg, (AG)ig, and (DG)jg are two-factor interactions between treatment and gene, array and gene (spot effects), dye and gene, respectively. εijkg represents a residual noise component, modelled as a Gaussian distribution with mean 0 and variance σ². (TG)kg denotes differences in expression for particular treatment and gene combinations that are not explained by the average effects on these treatments and genes. They represent the effects of interest, which were modelled using GeneANOVA. SAM analysis was performed as described in Tusher et al. (16). Ontologies attached to each gene were then used to classify altered genes according to main biological themes. Annotation of our probes was also accessible through MEDIANTE, our own microarray database which stores annotations derived from public databases. Additional statistical analyses, including principal component analysis (PCA) were performed with GeneANOVA.

first: Giorgos

last: Fitsialos

first: Manal

last: Dayem

first: Kevin

last: Lebrigan

first: Chimene

last: Moreilhon

first: Guerrino

last: Meneguzzi

first: Roser

last: Busca

first: Bernard

last: Mari

first: Laurent

last: Turchi

first: Amandine

last: Chassot

first: Pascal

last: Barbry

first: Gilles

last: Ponzio


title: Radiation-induced genes commonly expressed in lung and skin among three strains of mice.

city: Chiba

first: Shuhei

last: Noda

state: Chiba

address: Anagawa 4-9-1

department: Frontier research center

institute: National institute of radiological sciences

summary:
Gene expression profiling  was used to identify genes that display radiation-induced transcriptional change over mouse strain and tissue differences.
Keywords: mouse strain comparison, irradiation,  time course, lung, skin

overall_design: Inbred A/J, C57BL/6J and C3H/HeMs female mice were irradiated by gamma-rays as local irradiation in single doses to the thorax or hind leg. The animals were sacrificed at 1, 12 and 72 hours after irradiation. Non-irradiated mice served as the controls. The lungs and skin were dissected and used for expression analysis. Each sample including non-irradiated control was hybridized with global control that derived from mixture of commercially available total RNA from ten organs.

first: Mayumi

last: Iwakawa


title: Expression profiling of the solute carrier gene family in the chicken intestine

city: Blacksburg

first: Huifeng

last: Li

state: VA

address: 3570 Litton Reaves Hall

department: Animal and Poultry Sciences

institute: Virginia Tech

summary:
Intestinal development during late embryogenesis and early posthatch has a long term influence on digestive and absorptive capacity in chickens.  The objective of this research was to obtain a global view of intestinal gene expression from late embryogenesis until 2 weeks posthatch with a focus on genes involved in nutrient uptake.
Keywords: developmental time course

overall_design: Small intestine from male chicks from two different lines(A and B) were collected on embryonic days 18 (e18) and 20 (e20), day of hatch, and days 1, 3, 7, and 14 posthatch. There are two sample pools designed for lines and different times.

first: Elizabeth

last: Gilbert

first: Yan

last: Zhang

first: Oswald

last: Crasta

first: Derek

last: Emmerson

first: Kenneth

middle: E

last: Webb Jr

first: Eric

last: Wong


title: A new multi-sample hybridized microarray platform for quantitative gene expression on a small scale

summary:
For increasing sample size in microarry experiments, we have developed a new oligonucleotide microarray technology.
This microarray platform can greatly increase the sample size for increasing the veracity of gene expression and meeting the data analysis requirement.
Keywords: Gene expression, Oligonucleotide microarray, Sample size

overall_design: Limited by SpotBot microarrayer,we printed multiple copies(48)of sub-array which contained oligonucleotide probes of relatively small number of genes (20)and controls(12) on one slide. Multiple labeled samples hybridized individually on each sub-array at same time.

first: Guiqin

last: Wu

first: Jigang

first: Ning

last: Yang


title: Clostridium acetobutylicum ATCC 824 (pLib1) butanol stresses with repeated transfers

city: Newark

first: Eleftherios

middle: Terry

last: Papoutsakis

address: 15 Innovation Way

department: Chemical Engineering

institute: University of Delaware

summary:
Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, with a variety of genetic resistance mechanisms previously identified.  Using random genomic libraries and DNA microarrays, library inserts were preferentially enriched by culturing in media containing butanol, with successive transfers into fresh media containing incrementally increasing butanol concentrations.
Keywords: genomic library preferential enrichment

overall_design: plasmid DNA samples were taken from individual transfers (4th, 7th, 10th, and 13th transfers) of butanol challenged cultures and hybridized against plasmid DNA isolated from the culture used to inoculate the first challenge culture. Two biological replicate challenge experiments were performed and respective microarrays hybridized.

first: Jacob

last: Borden

first: Carlos

middle: J

last: Paredes

middle: T


title: Origin of replication for Pyrococcus abyssi by ChIP-chip

city: Gif-sur-Yvette

first: LAWRENCE

last: AGGERBECK

address: Avenue de la Terrasse

department: Centre de Génétique Moléculaire

institute: Centre National de la Recherche Scientifique

summary:
Recent advances in the study of archaeal DNA replication have uncovered defined replication origins (oriC) and demonstrated specific binding of the Cdc6/Orc1 protein and Mcm helicase to oriC in vivo and/or in vitro. The oriC of the hyperthermophilic archaeon Pyrococcus abyssi is characterized by 13 bp repeats, AT-rich regions and an inverted repeat whose precise roles remain unclear. We report here that the 13 bp repeats are widespread in the three Pyrococcus genomes. Nevertheless, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip), we found that binding of P. abyssi Cdc6/Orc1 to oriC in vivo was highly specific both in exponential and stationary phases, allowing oriC to be distinguished in the 1.8 M bp genome. ChIP-chip analysis also indicated that a single 13 bp repeat is not sufficient for stable binding of Cdc6/Orc1. Purified Cdc6/Orc1 binds a DNA fragment containing the inverted repeat of oriC with a relatively low affinity, suggesting that multiple clusters of the 13 bp repeat discovered in this study contribute to the stable binding of Cdc6/Orc1 to oriC. Our ChIP-chip analysis revealed that Mcm also binds oriC only in proliferating cells, consistent with its role as a licensing factor. Finally, we found that both Cdc6/Orc1 and Mcm have one additional target site. Notably, Mcm binds constitutively to a GC-rich region containing two rRNA genes and a tRNA gene, suggesting a role of archaeal Mcm in DNA replication and/or transcription of this peculiar region.
Keywords: ChIP-chip

overall_design: Experiments were done using at least three (Cdc6 in exponential and stationary phases, MCM in stationary phase) or four (MCM in exponential phase) independently prepared samples.

first: Fujihiko

last: Matsunaga

first: Annie

last: Glatigny

first: Marie-Hélène

last: Mucchielli

first: Nicolas

last: Agier

first: Hervé

last: Delacroix

first: Laetitia

last: Marisa

first: Takashi

last: Meno

first: Yoshizumi

last: Ishino

first: Lawrence

last: Aggerbeck

first: Patrick

last: Forterre


title: Gene Expression Profiling of Destructured Pig Semimembranosus Muscle

city: saint-gilles

first: marie

last: damon

address: domaine de la prise

institute: INRA

summary:
The destructured meat syndrome is clearly detrimental to cooked ham industry but still poorly understood. The affected muscles lose their fibrous aspect and give a soft whitened muscular mass without any organised structure. Normal (n=7) and destructured (n=7) groups of pigs were allotted 36 h post-mortem based on meat L* values. Transcriptome analysis of Semimembranosus muscles was conducted to identify genes potentially involved in this syndrome. Fifty nine genes were differentially expressed (p < 0.01) and 90% of them were upregulated in the affected muscles. Myofibrillar proteins involved in the contractile properties (TNNC2 and TNNT3) or in the integrity (TMOD4) of the sarcomere were increased in the affected muscle. Moreover, eight genes encoding enzymes of glycolysis (ALDOA, ENO3, GAPDH, LDHA, PGAM2, PKM2) or glycogenolysis (PGM1, PYGM) were upregulated in the destructured muscles. These results suggest that both myofibrillar and sarcoplasmic proteins might be involved in the etiology of this syndrom.
Keywords: meat quality comparison

overall_design: Fourteen pigs were included in this study and alloted into two groups according to meat quality measurment. Pigs with low L* value  were considered as normal and unaffected pigs (n=7) whereas pigs with high L* value were considered as affected pigs leading to destructured meat. Total RNA was isolated from each of the 14 semimembranosus muscle samples  and controlled for quality and concentration using an AGILENT 2100 bioanalyzer. cDNA was synthesized and labelled from 5 µg total RNA by simultaneous reverse transcription of mRNA using SuperScriptTM II RNase H- Reverse Transcriptase and 33P-deoxy-CTP. Each muscle sample mRNA was hybridized at 68°C during 48 h to one array containing two duplicated parts (sample names ending by 1 correspond to the left part whereas sample names ending by 2 correspond to the right part). The arrays were then exposed to radioisotopic-sensitive imaging plates that were scanned thereafter with a phosphor imaging system at a 25 µm resolution. Hybridization images were quantified using a semi-automated software through a grid process with a fixed spot diameter (BZscan). The output was then subjected to the data analysis process.

first: Marie

last: DAMON

first: Laurence

last: LIAUBET

last: VINCENT

first: Pascale

last: LEROY

first: Jerome

last: GLENISSON

first: Pierre

last: CHEREL


title: Vibrio cholerae: Wild type El Tor biotype versus crp mutant

city: Atlanta

first: Jorge

middle: Antonio

last: Benitez

state: GA

address: 720 Westview Dr., SW

department: Microbiology

institute: Morehouse School of Medicine

summary:
Transcriptional profiling of an El Tor biotype crp mutant
The virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991) and an isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein were grown in LB medium to optical density at 600 nm of 1.5. The cultures were chilled in ice, cells quickly collected by centrifugation and total RNA imediately extracted. RNAwas extracted and purified using the Trizol plus RNA purification system (Invitrogen) followed RNEasy miniElute cleanup (Qiagen). RNA samples were conserved at - 80 C and used within a week.
Keywords: Genetic modification

overall_design: 4 replicates

first: Weili

last: Liang

first: Anisia

last: Silva

first: Alberto

middle: P

last: Montano


title: Mutant p53 Increases Incidence and Promotes Progression of Mammary Carcinomas in Mice

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

institute: Vrije Universiteit Medical Center

summary:
In human breast cancer, mutations in the p53 gene are associated with poor prognosis, suggesting a mutant p53 “gain of function”. To study this phenomenon, we co-expressed mutant p53R270H in mice in which SV40 early proteins initiate the development of mammary adenocarcinomas in descendants of mammary epithelial cells that survived involution (WAPT-mice). This model allows the characterization of mammary carcinomas that developed in the absence or presence of mutant p53, while the endogenous wild-type p53 is functionallycompromised. Co-expression of mutant p53 significantly aggravated the phenotype of bitransgenic mice, as evidenced by a higher tumor incidence, a worse clinical staging and histological grading, as well as an enhanced invasiveness of the tumors, and more frequent pulmonary metastasis. Our data indicate that mutant p53 does not exert this effect by increasing genomic instability. On the contrary, lower grade tumors in WAP-T mice generally accumulated more amplifications of pro-survival genes than the corresponding tumors in bitransgenic animals, suggesting that mutant p53 can compensate for such genetic alterations by alternative mechanisms, possibly by its transcriptional activities, and thereby facilitate tumor formation. As a common feature and associated with their enhanced invasiveness, high grade tumors showed high level amplification of the Met-locus.
Keywords: mutant p53, SV40 early proteins, tumor progression, tumor grade, tumor stage, mammary carcinoma, animal model, Met

overall_design: Analysis of 34 mammary carcinomas of different histological grades derived from WAP-T and WAP-T x WAP-mutp53 transgenic mice.

first: Christina

last: Heinlein

first: Frauke

last: Krepulat

first: Jürgen

last: Löhler

first: Bauke

last: Ylstra

first: Thijs

last: Krugers

first: Genrich

last: Tolstonog

first: Wolfgang

last: Deppert


title: Global responses of Campylobacter jejuni to nitric oxide releasing compounds

first: Robert

last: Poole

summary:
Wild-type Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate  (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 10 uM NOC-5 & -7 (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis.
Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.
Keywords: Stress-response of a cgb mutant to NOC-5 & -7

overall_design:
C. jejuni NCTC11168 was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 μl sample onto both nutrient agar plates incubated at 37 °C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 °C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier.

cDNA synthesis was carried out using 12 µg of starting material, which was primed with 9 µg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 µl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 °C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 °C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 °C.

Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 °C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner.

The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of ≤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

first: Claire

last: Monk


title: Clostridium acetobutylicum ATCC 824 butanol stress (50 mM)

first: Eleftherios

last: Papoutsakis

state: DE

summary:
Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, but such effects are not well understood at the genomic level.  Using DNA microarrays, the Clostridium acetobutylicum transcriptional stress response to butanol was analyzed.
Keywords: stress response

overall_design: RNA samples were taken from both butanol challenged  (50 mM) and unchallenged bottles at 10, 30, 45, 60 (1h), 120 (2h), 360 (6h), 720 (12h), and 1440 min (24h) poststress. Two slides per timepoint were hybridized on a dye swap configuration, except for the 720 min. point where both slides have the same dye configuration.

first: Keith

last: Alsaker

first: Carlos

last: Paredes


title: Genome-Wide High-Resolution Mapping of Exosome Substrates Reveals Hidden Features in the Arabidopsis Transcriptome

status: Public on Dec 28 2007

city: La Jolla

first: Joseph

middle: R.

last: Ecker

state: CA

address: 10010 N. Torrey Pines Road

department: Genomic Analysis Laboratory

institute: The Salk Institute

type: Expression profiling by genome tiling array

summary:
The exosome complex plays a central role in RNA metabolism, and each of its core subunits is essential for viability in yeast  However, comprehensive studies of exosome substrates and functional analyses of its subunits in multi.CELlular eukaryotes are lacking  Here we show that, in sharp contrast to yeast and metazoan exosome complexes, individual subunits of the plant exosome core are functionally specialized  Using whole-genome oligonucleotide tiling microarray analyses of csl4 null mutant plants and conditional genetic depletions of RRP4 and RRP41, we uncovered  unexpected functional plasticity in the plant exosome core as well as generated a set of high-resolution genome-wide maps of Arabidopsis exosome targets  These analyses provide evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants and reveal novel aspects of stable  structural RNA metabolism  Finally, numerous novel exosome substrates were discovered, including a select subset of mRNAs, miRNA processing intermediates,  and hundreds of noncoding RNAs, the vast majority of which have not been previously described This large collection of RNAs belong to a Òdeeply hiddenÓ layer  of the transcriptome that is tightly repressed and can only be visualized upon inhibition of exosome activity These first genome-wide maps of exosome  substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes
Keywords: Strand-specific gene expression analysis using whole-genome oligonucleotide tiling microarray analyses of three exosome subunits, using csl4 null mutant plants and conditional genetic depletions of RRP4 and RRP41.

overall_design: Two biological replicates were done for each sample on each chromosomal strand (Watson and Crick) which meant a total of four whole-genome oligonucleotide tiling microarrays were used per sample. We studied a total of 8 biological samples for this project Overall, a total of 32 whole-genome oligonucleotide tiling microarrays were used for this study

first: B

last: Gregory

first: J

last: Chekanova

first: S

last: Reverdatto

first: H

last: Chen

first: R

last: Kumar

first: T

last: Hooker

last: Yazaki

first: P

last: Li

first: N

last: Skiba

first: Q

last: Peng

last: Alonso

first: V

last: Brukhin

first: U

last: Grossniklaus

first: D

last: Belostotsky


title: Clostridium acetobutylicum ATCC 824 butyrate stress (29 mM)

last: Papoutsakis

summary:
Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, but such effects are not well understood at the genomic level.  Using DNA microarrays, the Clostridium acetobutylicum transcriptional stress response to butyrate was analyzed.
Keywords: stress response

overall_design: RNA samples were taken from both butyrate challenged  (29 mM) and unchallenged bottles at 10, 30, 45, 60 (1h), 120 (2h), 360 (6h) and 720 (12h) poststress. Two slides per timepoint were hybridized on a dye swap configuration.

last: Alsaker


title: HOXA13 Chromatin Immunoprecipitation on human HG18 Nimblegen 2 array promoter set

city: Stanford

first: John

middle: Louis

last: Rinn

address: 2155 CCSR 269 Campus Dr

department: Dermatology

institute: Stanford University

summary:
HOXA13 Binding sites were located genome wide in primary human foreskin fibroblasts
Keywords: Tiled oligo arrays (Nimblegen)

overall_design: Tiled promoters with a probe per 100bp of refseq known genes 3kb upstream and 700 bp downstream of transcriptional start


title: Clostridium acetobutylicum ATCC 824 acetate stress (43 mM)

first: Eleftherios

summary:
Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, but such effects are not well understood at the genomic level.  Using DNA microarrays, the Clostridium acetobutylicum transcriptional stress response to acetate was analyzed.
Keywords: stress response

overall_design: RNA samples were taken from both acetate challenged  (43 mM) and unchallenged bottles at 10, 30, 45, 60 (1h), 120 (2h), 360 (6h), 720 (12h), and 1440 min (24h) poststress. Two slides per timepoint were hybridized on a dye swap configuration, except for the 720 min. point where both slides have the same dye configuration.

last: Paredes


title: Mutant2

status: Public on Dec 27 2007

city: Albuquerque

first: Sushmita

last: Roy

state: NM

institute: Biology, University of New Mexico

summary:
Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and non-quiescent (NQ) fractions prior to entering stationary phase.  To identify genes involved in this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity.  Twenty-one mutants were identified, including 7 that affected reproductive capacity of both cell types.  Thirteen affected only Q or NQ cells, indicating significant differentiation of these cell types.  doa4 strains, lacking ubiquitin hydrolase, affected viability and reproductive capacity in both cell types.  More than 1300 mRNAs differentiating Q and NQ cell fractions were identified by microarray analysis.  Gene-ontology analysis of Q-cell mRNAs showed significant increases in protein-encoding mRNAs involved in membrane maintenance, oxidative stress response, and signal transduction.  NQ-cell mRNAs encode proteins involved in Ty-element transposition and DNA recombination, consistent with apoptosis in these cells.  Consistent with preparation for rapid response to environmental stimuli, approximately 2000 protease-labile mRNAs were identified in Q cells.  The differentiation of these cell types and the ability of genes to selectively affect the survival of Q or NQ cells in yeast are relevant to chronological aging, cell-cycle, genome-evolution, and stem-cell research and provides insight into complex responses that even simple organisms have to starvation.
Keywords: Cell tyep comparison

overall_design: Yeast deletion mutants (BY4742 background) were grown to stationary-phase (7 days) and then separted into Q and NQ cells using Percoll density gradients.  Microarrays were carried out on these different cell fractions.

first: Anthony

middle: D

last: Aragon

first: Margaret

last: Werner-Washburne