title: Synaptic activity-regulated gene expression_embryonic day 18

city: Amsterdam

first: Jildau

last: Bouwman

address: de Boelelaan 1085

department: Functional genomics

country: Netherlands

institute: CNCR, Vrije Universiteit

summary:
Activity-dependent changes in synapses rely on functional changes in resident proteins and on gene expression. We addressed the relationship between synapse activity and the expression of synaptic genes by comparing RNA levels in the neocortex of normal mice versus synaptically silent munc18-1 null mutants, using microarray expression analysis, quantitative PCR and Northern blotting. We hypothesized that genes under control of synaptic activity are differentially expressed between mutants and controls and found that few synaptic signaling genes were differentially expressed. However, all neuropeptide genes with detectable expression on the microarray were differentially expressed, being 3-20 fold higher in control cortex. Genes encoding their receptors and most other synaptic components were not differentially expressed. Differential expression of the neuropeptide genes was confirmed by qPCR analysis. In situ hybridization indicated that the difference in neuropeptide expression was uniform and not due to the loss of specific cells in the mutant. In primary sensory neurons, which do not depend on synaptic activity for their input, the differential expression of neuropeptides was not observed. These data reveal no simple relation between activity of synapses and expression of their resident proteins, but instead identified a unique feature of neuropeptide gene expression, which appears to depend on synaptic activity.
Keywords: munc18-1 null mutant mice are characterized by the complete loss of both evoked and spontaneous transmitter release

overall_design: 11 biological replicates of munc18-1 null mutants, hybridized with 11 pooled biological replicates. On six arrays the mutant sample was labeled with cy3 and on the other five with cy5.

first: Sabine

last: Spijker

first: Desirée

last: Schut

first: Betty

last: Wächtler

first: Bauke

last: Ylstra

first: August

last: Smit

first: Matthijs

last: Verhage


title: E. coli Transcriptome Dynamics during the Transition from Anaerobic to Aerobic Conditions

status: Public on Jul 28 2006

city: Sheffield

first: Jonathan

last: Partridge

address: Firth Court, Western Bank

department: Molecular Biology and Biotechnology

country: United Kingdom

institute: The University of Sheffield

summary:
Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen.  Several previous transcript-profiling experiments have compared separate anaerobic and aerobic cultures.  Here, for the first time, the process of adaptation was investigated by determining changes in transcript profiles when anaerobic steady-state cultures were perturbed by the introduction of air.
Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow 1000 fermentation vessels (1.8 l capacity) with culture agitation speed constant at 400 rpm and the temperature maintained at 37 °C. Oxygen levels were monitored using galvanic oxygen electrodes while the pH was maintained at 7.2 ±0.2 by automatic titration with sterile KOH. Evans defined medium was used as the growth medium with glucose (30 mM) as the carbon source with the dilution rate being 0.2 h-1.  Anaerobic cultures were maintained by sparging the chemostat with oxygen-free nitrogen (95 %) and carbon dioxide (5 %) (0.2 l min-1).  The switch to aerobic conditions was achieved by sparging the culture with air (2.0 l min-1) in place of the above gas mix. After a period of 5, 10, 15 and 60 min exposure to air, cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA before total RNA purification using Qiagen’s Rneasy Midi kit as recommended by manufacturer’s instructions.
Keywords: time course response to air

overall_design:
Analysis used anaerobic steady state RNA as control samples for comparison to the experimental samples taken at 5, 10, 15 and 60 min. Indirect comparisons were made across multiple arrays with raw data pulled from different channels for data analysis and comparison to the control data. Data analysis was carried out using Imagene, version 5.1 and Genesight version 4 (Biodiscovery Inc). The mean values from each channel were log2 transformed and normalized using the LOWESS algorithm to remove intensity dependent effects within the calculated values.  Normalised values were used to calculate the Cy3/Cy5 fluorescence ratios from experimental and biological repeats (seperate chemostat runs)with all replicates combined. Data was compiled from two aerobic and two anaerobic cultures each with a dye-swap replicate, thus providing two biological repeats within four technical repeats.
Presented is the LOG2 normalised data comparing the control (anaerobic) with the identified experimental (5, 10, 15, 60 min air exposure) with supplementary material provided in the form of Raw array files.

middle: D

first: Colin

last: Scott

first: Yue

last: Tang

first: Robert

middle: K

last: Poole

first: Jeffrey

last: Green


title: Transcriptional Profile of Haemophilus influenzae: Effects of Iron and Heme

city: Oklahoma City

first: Timothy

middle: M

last: VanWagoner

state: OK

address: 940 SL Young Blvd, BMS340

department: Pediatrics

country: USA

institute: University of Oklahoma Health Sciences Center

summary:
Haemophilus influenzae frequently causes human disease, and humans are it’s sole niche. This bacterium has an absolute requirement for both a porphyrin and an iron source for aerobic growth, and exogenous heme can satisfy both requirements.  Heme and iron can be acquired by H. influenzae from free or human protein-bound sources. The ability to selectively regulate the acquisition of heme and iron from physiological sources is a major virulence determinant for this microorganism. We utilized whole genome arrays to identify the full set of H. influenzae Rd KW20 iron and heme regulated genes.  Condition specific RNA was derived from cells starved for both heme and iron and cells from the same culture 20 mins after the addition of exogenous iron and heme. The results identified 162 genes with a change in transcription ≥ 1.5 fold. Eighty genes in 42 operons were preferentially expressed under iron/ heme starvation; 82 genes in 50 operons were preferentially expressed under iron/heme replete conditions.  In each case, all genes contained within the operon were co-regulated. The former group included genes encoding proteins known to have a role in iron and heme uptake as well as several hypothetical ORFs.  Enzymes involved in the gluconeogenesis pathway and glycogen biosynthesis were also upregulated.  The genes showing increased transcription immediately after the addition of iron and heme primarily encode proteins involved with aerobic respiration and protein biosynthesis, consistent with a relaxation of starvation.  Genomic transcriptional profiling provides a more complete understanding of the effects of iron and heme availability.
Keywords: Transcription analyses

overall_design: Three replicate cultures were grown in iron and heme depleted media for 90 minutes and samples taken for expression analyses. Fe and heme were supplemented to the remaining cultures and the cultures allowed to incubate an additional 20 minutes. Microarray analyses were used to determine changes in gene expression resulting from transfer from FeHm-deplete to FeHm-replete conditions.

first: Paul

middle: W

last: Whitby

first: Thomas

last: Seale

first: Daniel

middle: J

last: Morton

first: Terrence

middle: L

last: Stull


title: The kinetics of transcriptomic changes induced by cigarette smoke in rat lungs

city: Cologne

first: Bernhard

last: Gerstmayer

address: Stockheimer Weg 1

country: Germany

institute: Miltenyi Biotec GmbH

summary:
Gene expression profiling in animal models exposed to cigarette mainstream smoke (CS) shapes up as a promising tool for investigating the molecular mechanisms involved in the onset and development of CS-related disease and may aid in the identification of disease candidate genes. Here we report on differential gene expression in lungs of rats exposed for 2, 7, and 13 weeks to 300 and 600 µg total particulate matter (TPM)/l CS with sacrifice 2, 6, or 20 h after the last exposure. The results showed a distinct, mostly transient, expression pattern of differentially expressed genes encoding antioxidant and xenobiotic-metabolizing enzymes, which confirmed and extended in qualitative and quantitative terms the findings of a former study, where rats were exposed to CS either acutely (once) or short-term (3 weeks). With increasing length of exposure, there was a distinct, mostly sustained, expression of genes implicated in innate and adaptive immune responses, clearly pointing to an emerging inflammatory response. Notably, this inflammatory response included the expression of lung disease-related genes not yet linked to CS exposure, such as osteopontin, arginase 1, and chitinase, as well as genes encoding proteolytic enzymes. Finally, our experiments also revealed a CS exposure-dependent shift in the cyclical expression of genes involved in controlling the circadian rhythm. Altogether, these results provide further insight into the molecular mechanisms of CS-dependent disease onset and development and thus may also be useful for defining CS-specific molecular biomarkers of disease.
Keywords: gene expression profiling, cigarette smoke-related disease

overall_design: Four rats per group were nose-only exposed in flow-past exposure chambers (type FPC1-132) to diluted CS (300 or 600 µg TPM/l) or to conditioned fresh air (control) for 2, 7, or 13 weeks (2 times 1 h/day with a 30 min fresh air break in between, 5 days/week) and sacrificed at 2, 6, or 20 hours after the last exposure. Whole lung tissue was used for total RNA preparation, was frozen in liquid nitrogen immediately after dissection and stored at -70°C. After quantification and checking for integrity, equal mounts of RNA from the 4 animals per group were pooled, and, for hybridization on PIQOR microarrays, subjected to linear amplification and labeling. Two micrograms of RNA was labeled by reverse transcription via Cy5-dCTP incorporation (CS-exposed tissue) and Cy3-dCTP incorporation (unexposed tissue). Each labeled sample was then divided in half and hybridized on 2 customized PIQOR microarrays.

first: S.

last: Gebel

first: B.

first: P.

last: Kuhl

first: J.

last: Borlak

first: K.

last: Meurrens

first: T.

last: Mueller


title: Signature of human hepatocytes treated with Aroclor1254

status: Public on Aug 01 2006

city: Hannover

first: Jürgen

address: Nikolai-Fuchs-Strasse 1

institute: Fraunhofer Institut

summary:
In order to determine molecular rules for transcriptional regulation of targeted genes to explain in part the pleiotropic effect observed in animals and humans upon exposure to Aroclor 1254 we treated human hepatocytes with Aroclor1254 and analysed RNA expression using a Nimblegene human custom array.
Keywords: Human hepatocytes, Aroclor1254 treatment, 20µM, 72h

overall_design: Human hepatocytes isolated from surgical material were treated with 20µM Aroclor1254 for 72h. Non-treated cells served as control. Total RNA was isolated using QIAGEN´s RNeasy total RNA isolation kit according to the manufacturer’s recommendations. Oligonucleotide microarrays were hybridized and washed according to the manufacturer’s recommendations.


title: Expression profiles of hrpRS- and hrpL- mutants

city: manhattan

first: Xiaoyan

email: xtang@ksu.edu, llan@ksu.edu

state: KS

address: 4702 Throckmorton

department: Plant Pathology

institute: Kansas State University

summary: Pseudomonas syringae pv. tomato DC3000 is a model pathogen infecting tomato and Arabidopsis plants. Genes encoding the type III secretion system and substrate proteins (collectively called TTSS genes) of this bacterium are induced in plants and in minimal medium (MM). The induction of TTSS genes is mediated by HrpL, an alternative sigma factor recognizing the hrp box in the promoter of TTSS genes. The transcription of hrpL is activated by HrpR and HrpS, two homologous DNA binding proteins encoded by the hrpRS operon. Microarray analysis was conducted to evaluate the DC3000 genes regulated by hrpL and hrpRS in MM. The analysis identified a number of novel hrpLactivated genes with a putative TTSS-independent function. Genes regulated by hrpL were mostly regulated by hrpRS in the same manner, but a large number of genes regulated by hrpRS were hrpL-independent, indicating that hrpL represents one branch of the regulatory pathways downstream of hrpRS. The induction of the TTSS genes was associated with down-regulation of the house-keeping genes, indicating that the activation of the TTSS has a cost on the basic cellular activities. The novel genes/pathways identified by the microarray provide new insight into the bacterial functions coordinating with the TTSS.

sample_organism: Pseudomonas syringae pv. tomato str. DC3000

overall_design: Three samples (biological replicates) of each condition and mutation were analyzed, except for hrpRS-, which has two.  11 Samples analyzed.

first: Lefu

last: Lan


title: Analysis of gene expression in Nicotiana benthamiana following NbCD1- or NbCD3-overexpression using SuperSAGE Array1

city: Ueda

first: Hideo

last: Matsumura

state: Nagano

address: Tokita 3-15-1

department: Gene Research Center

country: Japan

institute: Shinshu University

summary:
We developed a novel platform for genome-wide gene expression analysis in any eukaryotic organism, that we coin SuperSAGE Array. The SuperSAGE Array is a microarray onto which oligonucleotides of 26 nucleotides corresponding to SuperSAGE tag sequences are directly synthesized.  A SuperSAGE Array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology.  This technology was also applied to the detailed study of expressed genes identified by SuperSAGE in Nicotiana benthamiana, an organism without available DNA database.
Keywords: transgene overexpression, transient expression

overall_design: Using SuperSAGE, we first explored N. benthamiana genes that were up- or down-regulated by NbCD1- or NbCD3-overexpression, respectively.   N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens carrying plasmids harboring genes for NbCD1, NbCD3 and GFP (control), respectively, in the glucocorticoid-inducible gene expression cassette GVG9.  Two days after agroinfiltration, the leaves were treated with dexamethasone (DEX) to induce expression of the transgenes.   Four hours after DEX treatment, leaves were harvested, and RNA was isolated for SuperSAGE analysis.    For the identification of genes whose expression is altered by NbCD1-overexpression, 10,269 and 10,884 tags (6,659 and 6,796 genes) were compared between GFP- and NbCD1-overexpressing leaves.  Similarly, 10,063 tags (5,564 genes) from GFP-overexpressing leaves and 10,677 tags (5,858 genes) from NbCD3-overexpressing leaves were compared.  We selected those tags that show more than four-fold representational differences between NbCD1- or NbCD3-, and GFP-overexpressing leaves, respectively .   These tags were altogether used to establish a SuperSAGE Array.    In total, 154 tags corresponding to the genes up- or down-regulated by either NbCD1- or NbCD3-overexpression were selected for an oligonucleotide array .


title: Oncostatin M Regulation of Gene Expression in Breast Tumour Cells.

city: Birmingham

first: Nick

last: Underhill-Day

address: Edgbaston

department: Biosciences

institute: University of Birmingham

summary:
Oncostatin m (OSM) induces potent growth inhibitory and morphogenic responses in several different tumour cell types but the genetic events are not well understood. OSM can signal through two separate heterodimeric receptor complexes, gp130/LIFRα and gp130/OSMRβ. In this investigation we utilised cytokines, oncostatin M, interleukin-6 (IL-6) and leukaemia inhibitory factor (LIF) and LIF receptor antagonist, LIF-05, to help identify patterns of gene expression elicited by the different IL-6 receptor complexes in breast tumour cell line, T47D . We have tried to identify an OSM gene signature common to multiple breast tumour-derived cell lines (T47D, MCF-7 and MDA-MB-231) and identified OSM-gene regulation at time-points which coincide with the onset of OSM-induced biological effects. These findings identify a core transcriptional mechanism specific to the OSMRβ in breast tumour cells.
Keywords: Comparative, timecourse, cell line-specific

overall_design:
The first aim was to identify OSM-specific gene expression events at early time-points (30 and 60 minutes) in T47D breast tumour cells and compare these against gene expression regulated by IL-6 (30 and 60 minutes) and LIF (60 minutes) respectively. Arrays performed included:

(A) unstimulated versus OSM + LIF-05
(B) unstimulated versus OSM
(C) unstimulated versus IL-6
(D) unstimulated versus LIF
(E) unstimulated versus LIF-05

This allowed gene expression arising from stimulation of gp130/OSMRβ (A) or gp130/LIFRα and gp130/OSMRβ (B) to be assessed and compared with expression arising from stimulation of gp130/gp130 (C) and gp130/LIFRα (D) receptor complexes. The LIF receptor antagonist, which blocks OSM-induced gp130/LIFRα signalling, was also assessed for aberrant gene activity (E). For each condition two replicate arrays were performed (dye swap). This gives a total of 16 arrays.

Secondly, gene expression resulting from OSM stimulation was identified at extended time-points (180 minutes, 1 day, 3 days and 6 days) and also included dye-swap replicate arrays. A further 8 arrays were performed.

Finally, gene expression resulting from 60 minutes OSM stimulation of T47D, MCF-7 and MDA-MB-231 breast tumour cell lines was compared (including dye-swap replicates). A further 4 arrays were performed (MCF-7 and MDA-B-321).

first: Nicholas

first: John

last: Heath


title: Effect of gas-substrate limitation on barophilic growth of Methanocaldocccus jannaschii at 500 atm.

city: Berkeley

first: Boonchai

last: Boonyaratanakornkit

state: CA

address: 201 Gilman Hall #1462

department: Chemical Engineering

institute: UC Berkeley

summary:
Barophilic growth of the hyperthermophilic methanarchaeon Methanocaldococcus jannaschii occurred when gas-substrate availability did not limit growth. In contrast, when growth was limited by gas transfer, no enhancement of growth was evident and a stress response was exhibited at both high and low pressure. A pressure-induced transcriptional response was evident, regardless of whether growth was enhanced by pressure. High-pressure adaptation of a barophilic organism can thus occur at the transcriptional level, even though the cells are stressed by low substrate availability and do not exhibit accelerated growth.
Keywords: stress response, gas substrate limitation, bioreactor volume, high pressure

overall_design: All RNA samples were extracted in the exponential phase of growth. The cDNA arrays that were used contained three technical replicate spots for each ORF in the genome of M. jannaschii. Samples from cells grown in 200 ml of medium at 500 atm and 88C were hybridized against samples from cells grown in 200 ml of medium at 7 atm and 88C in four different pairs of biological replicates (including two pairs of dye-swapped samples). All other samples were hybridized in three different pairs of biological replicates (including one pair of dye-swapped samples).

middle: B

first: Jesus

last: Cordova

first: Chan-Beum

last: Park

first: Douglas

middle: S

last: Clark

first: Anjana

last: Simpson

first: Najib

last: El-Sayed

first: Vivian

last: Peng


title: Translation State Array Analysis of TNF Stimulated Human Endothelial Cells

status: Public on Jul 27 2006

city: Salt Lake City

middle: I.

last: Schmid

state: UT

department: HMBG

institute: University of Utah

summary:
Confluent human umbilical vein endothelial cells (HUVECs) were exposed to TNF-alpha (100 units/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, TNF monosome and TNF polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (TNF polysome/TNF monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study.
Keywords: Translation State Array Analysis

overall_design:
HUVECs were stimulated with TNF-alpha (100 U/mL) for 2 hours.  Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes).  The supernatant was removed and cells were carefully resuspended in 375 micL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice.  125 micL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting.  The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4 deg C) to remove nuclei and cellular debris.  The supernatant was transferred to a new tube containing 50 micL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 micL 5M NaCl.  This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB).  Gradients were spun at 43,700 rpm for 90 minutes (4 deg C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles.  Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer.  Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen).  These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters.
	Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters.  To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter.  Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array.  Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1.  Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter.  The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction:  TS=P/M.  The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls.  In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1.  Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.

middle: I

first: Huimiao

last: Jiang

first: L

last: Kraiss


title: Synaptic activity-regulated gene expression_embryonic day 16

first: Jildau

last: Bouwman

overall_design: 4 biological replicates of munc18-1 null mutants, hybridized with 4 pooled biological replicates. On one array the mutant sample was labeled with cy3 and on the other three with cy5.

first: Sabine

last: Spijker

first: Desirée

last: Schut

first: Betty

last: Wächtler

first: Bauke

last: Ylstra

first: August

last: Smit

first: Matthijs

last: Verhage


title: Pseudomonas putida expression profiles at different stress conditions

first: Burkhard

last: Tummler

address: Carl-Neuberg-Str. 1

institute: Medizinische Hochschule Hannover

summary:
The metabolically versatile Pseudomonas putida strain KT2440 is the first Gram-negative soil bacterium certified as a biosafety strain and is being used for applications in agriculture, biotechnology and bioremediation. P. putida has to cope in its niche with numerous abiotic stresses. The stress response to 4°C, pH 4.5, 0.8 M urea or 45 mM sodium benzoate, respectively, was analyzed by the global mRNA expression profile and screening for stress-intolerant Tn5 transposon mutants. In total we identified 49 gene regions to be differentially expressed and 32 genes in 22 operons to be indispensable for growth during exposure to one or the other abiotic stresses. We propose that stress is sensed by the outer membrane proteins OmlA and FepA and the inner membrane constituents PtsP, PhoPQ and CbrAB. The metabolic response is regulated by the cyo operon, the RelA/SpoT modulon, PcnB and VacB that control mRNA stability and BipA that exerts transcript-specific translational control. The adaptation of the membrane barrier, the uptake of phosphate, the maintenance of intracellular pH and redox status and the translational control of metabolism are the indispensable key mechanisms of the P. putida stress response.
Keywords: functional genomics

overall_design:
Purified DNA of some 4600 overlapping shotgun clones in vector pCR-BluntII-TOPO (Invitrogen, Groningen, The Netherlands) was used as template in standard PCR amplification with the vector-specific primer pair d(TCGGATC CACTAGTAACG) and d(GGCCGCCAGTGTGATG). Shotgun cloning had been performed according to the procedures described by the vector's manufacturer. Each PCR reaction was individually checked by agarose gel electrophoresis. Prior to spotting onto microarrays, the DNA concentration of the fragments was adjusted to about 300 ng μl1. Spotting was done using an SDDC-2 DNA Micro-Arrayer from Engineering Services Inc. (Toronto, Canada) and SMP3 pins (TeleChem International Inc., Sunnyvale, USA) onto glass slides coated with poly L-lysine as described in detail earlier (Diehl et al., 2001).
For labelling DNA-samples, random priming (Feinberg and Vogelstein, 1983) was performed on 500 ng genomic Pseudomonas DNA in the presence of 15 μM each of dATP, dGTP and dTTP, and 25 μM Cy3- or Cy5-labelled dCTP (Amersham-Pharmacia, Amersham, UK). Hybridization of such samples to the microarrays was done as described (Diehl et al., 2001). Fluorescence signals were detected on a ScanArray5000 unit (Packard, Billerica, USA) and analysed with the GenePix software package (Axon Instruments, Union City, USA).

first: Diana

last: Stjepandic

first: Kerstin

last: Bohm

first: Christian

last: Weinel

first: Oleg

last: Reva

first: Jorg

last: Hoheisel


title: Septic shock patients: outcome at D1 (24 hours after inclusion)

city: Paris

first: Didier

last: Payen de la Garanderie

address: 2 rue Ambroise Pare

department: DAR-ICU

country: France

institute: Hopital Lariboisiere

summary:
Microarray was performed on PBMC with an inflammatory dedicated slide of 340 genes, and target samples labelled with Cy3 for day 0 (reference) and with Cy5 for the other days. Changes in expression levels were evaluated by the ratio Dx/D0. According to outcome and time evolution, a two-dimensional clustering was performed.
Keywords: dead vs alive septic shock patients at D1 post-inclusion

overall_design: The day of inclusion corresponded to the first blood sampling (15 ml) quoted day 0. The following blood samples were performed at days 1, 7, and 28. Since the pre-sepsis expression of the genes as a control cannot be obtained, the day 0 sample was used patient per patient as a reference value for the next days samples for micro-array study. Patients outcome (dead or alive of evolution) was considered, as the time evolution when the patients survived.

first: Yulia

last: Belikova

first: Anne-Claire

last: Lukasciewicz

first: Nicolas

last: Sevenet

last: Payen


title: Synaptic activity-regulated gene expression_embryonic day 14

first: Jildau

last: Bouwman

overall_design: 6 biological replicates of munc18-1 null mutants, hybridized with 6 pooled biological replicates. On three arrays the mutant sample was labeled with cy3 and on the other three with cy5.

last: Spijker


title: Potato autopolyploidy

status: Public on Aug 02 2006

city: Rockville

first: Jia

last: Liu

state: MD

address: 9712 Medical Center Drive

institute: Plant Genomics

summary:
A synthetic autopolyploidy plant series O37 was developed from chromosome doublings of a monoploid (12 chromosome) plant derived from a Solanum phureja background. Monoploid (1x), diploid (2xR3 and 2xR5) and tetraploid (4x) plants were used for gene expression profiling. Total RNA was isolated from growing leaflets of six plants of each genotype at 20 day after planting. Total RNA was isolated and amplified from growing root tips of each genotype at 7 days after planting.
Keywords: Genotype,  Tissue type,  Biorep, Loop design

overall_design: Microarray hybridizations were performed in a loop design for three biological replicates.

first: Robert

last: Stupar


title: Array-Based Comparative Genome Hybridization in Clinical Genetics

city: Tel-Aviv

first: Anat

last: Bar-Shira

address: 6 Weizmann St.

department: Genetic Institute

country: Israel

institute: Tel-Aviv Sourasky Medical Center

type: Genome variation profiling by genome tiling array

summary:
Abnormalities in DNA copy number are frequently found in patients with multiple anomaly syndromes and mental retardation. Array-CGH is a high resolution whole-genome technology which improves detection of submicroscopic aberrations underlying these syndromes. Eight patients with mental disability, multiple congenital anomalies and dysmorphic features were screened for submicroscopic chromosomal imbalances using the GenoSensor Array 300 Chip. Subtelomeric aberrations previously detected by FISH analysis were confirmed in two patients, and accurate diagnosis was provided in two previously undiagnosed complex cases. Microdeletions at 15q11.2-q13 in a newborn with hypotonia, cryptorchidsm and hypopigmentation were detected with few discrepancies between the array results and FISH analysis. Contiguous microdeletion of GSCL, HIRA and TBX1 genes at 22q11.2 was identified in a previously undiagnosed boy with an unusual presentation of the VCF/DiGeorge spectrum. In a newborn with aniridia, a borderline false negative WT1 deletion was observed, most probably because of differences between the size of the genomic deletion and the microarray probe. A false positive rate of 0.2% was calculated for clone-by-clone analysis, while the per patient false positive rate was 20%. Array-based CGH is a powerful tool for the rapid and accurate detection of genetic disorders associated with copy number abnormalities, and can significantly improve clinical genetic diagnosis and care.
Keywords: comparative genome hybridization (CGH)

overall_design: High-molecular weight genomic DNA was purified from patients' ’peripheral blood leukocytes. Test DNA and normal reference DNA (from an individual of the opposite sex of the test sample, except for N1 and N2) was labeled to incorporate Cy3 or Cy5 fluorophores. Equal aliquots of labeled test and reference DNA were combined and hybridized to GenoSensor Array 300 (GPL3709). This array contains 287 genomic clones, including those for each human telomere, as well as all of the known microdeletion syndromes and additional selected loci representing each chromosome arm. The GenoSensor CGH Array 300 clone list is available at http://www.vysis.com/PDF/GenoSensor300ClonesAndKey_July2004.pdf. Hybridization signal images in three colors, Cy3, Cy5 and DAPI blue were then analyzed by the GenoSensor reader system based on DAPI staining that identified target spots and their location on the grid. By analyzing the set of Cy3/Cy5 ratios (test-to-reference ratios) on all targets, the GenoSensor Array 300 Reader Software (version 1) calculates the ratio most representative of the modal DNA copy number of the sample DNA. For each target the normalized ratio, relative to the modal DNA copy number is calculated. This normalized ratio of the target indicates the degree of gain or loss of copy number, and for each clone the copy number changes are presented. For each target, a P value is calculated automatically by the GenoSensor Array 300 Reader Software (Abbott Vysis), and a P value of 0.005 or less was considered significant.


title: Signalling molecules in MEFs/FL

city: Baltimore

first: Kenneth

middle: R.

last: Boheler

address: 5600 Nathan Shock Drive

department: GRC

institute: National Institute on Aging, NIH

summary:
In silico analysis identified signalling molecules related to Wnt (Wnt1, 2, 5a, 5b, 6, 11, 14, Wisp1, 2, sFRP1 and 2, Dkks 2 and 3) and BMP (BMP1, 4, 5) pathways.  Many of these tags matched multiple transcripts in GenBank, while other potential molecules could not be detected because the transcripts either lacked an anchoring enzyme site or the site was immediately adjacent to the polyA+ sequence.
Keywords: SAGE

overall_design: To identify potential signalling molecules (secreted or cell-cell interacting) implicated in the formation of INPs, we constructed a SAGE library from MEFs


title: Ethylene-insensitive5 encodes a 5'->3' exoribonuclease required for regulation of EIN3-targeting F-box proteins EBF1/2

status: Public on Jul 26 2006

city: La Jolla

first: Joseph

last: Ecker

address: 10010 N. Torrey Pines Road

department: Genomic Analysis Laboratory

institute: The Salk Institute

type: Expression profiling by genome tiling array

summary:
thylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2 (1-3). Here, we report the identification of ETHYLENE INSENSITIVE5 as the 5'->3' exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response.
Keywords: Arabidopsis growth regulation signal transduction

overall_design: For the oligonucleotide tiling array experiments 10 days-old light-grown Arabidopsis ein5-1 seedlings were transferred to a Petri-plate containing incubation buffer (1mM Pipes, 1mM sodium citrate, 1mM KCl, 15mM sucrose), and maintained in agitation at 75 rpm for 30 min covered with foil. At this point vacuum was applied for exactly 15 seconds and cordycepin (Sigma Chemical Co., St. Louis) was added to a final concentration of 150 ug/ml. The seedlings were then incubated for 2 more hours in the dark at room temperature and kept in agitation at 75 rpm in Petri-plates. The seedlings were then briefly blotted to on filter paper to dry and kept in liquid nitrogen. Total RNA was extracted from the samples using the RNAeasy plant kit (Qiagen, Valencia, CA). Biotinylated target RNA was prepared from 120 ug of total RNA from each sample using the GeneChip® Expression Analysis system (Affymetrix, Santa Clara, CA). Each sample of the purified and fragmented labeled target cRNA was hybridized to one set of Arabidopsis genome tiling arrays consisting of twelve custom made arrays (Yamada, K et al., 2003). Hybridization, washes and the staining were carried out as described above for the Affymetrix array ATH1. Cell files were obtained using the GCOS software (Affymetrix, Santa Clara, CA) and the ChipViewer software was used to visualize and analyze the tiling chip data (Yamada et al. 2003. Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome. Science 302, 842-846). Data submitted by Huaming Chen.

first: Gabriela

last: Olmedo

first: Hongwei

last: Guo

first: Brian

last: Gregory

first: Saeid

last: Nourizadeh

first: Laura

last: Aguilar-Henonin

first: Hongjiang

last: Li

first: Fengying

last: An

first: Plinio

middle: R

last: Guzman


title: NHGRI Menin ChIP-Chip

status: Public on Jul 25 2006

city: Bethesda

first: Sean

last: Davis

address: 37 Convent Drive, Room 6138

institute: National Cancer Institute

type: Genome binding/occupancy profiling by genome tiling array

summary:
Multiple endocrine neoplasia type I (MEN1) is a familial cancer syndrome characterized primarily by tumors of multiple endocrine glands. The gene for MEN1 encodes a ubiquitously expressed tumor suppressor protein called menin. Menin was recently shown to interact with several components of a trithorax family histone methyltransferase complex including ASH2, Rbbp5, WDR5, and the leukemia proto-oncoprotein MLL. To elucidate menin's role as a tumor suppressor and gain insights into the endocrine-specific tumor phenotype in MEN1, we mapped the genomic binding sites of menin, MLL1, and Rbbp5, to approximately 20,000 promoters in HeLa S3, HepG2, and pancreatic islet cells using the strategy of chromatin-immunoprecipitation coupled with microarray analysis. We found that menin, MLL1, and Rbbp5 localize to the promoters of thousands of human genes but do not always bind together. These data suggest that menin functions as a general regulator of transcription.
Keywords: ChIP/chip

overall_design: NHGRI Menin ChIP-Chip

first: Peter

last: Scacheri


title: Identification of Francisella tularensis genes affected by iron limitation

city: Dallas

first: Eric

middle: J.

last: Hansen

state: TX

address: 5323 Harry Hines Blvd.

department: Microbiology

institute: UT Southwestern Medical Center

summary:
Three pairs of total RNA preparations from F. t. LVS grown under iron-replete and iron-restricted conditions were used in the DNA microarray.  Seven arrays were used to examine the genes affected by iron limitation.
Keywords: Dual Channel (Cy5/Cy3) arrays

overall_design: Oligonucleotides designed according to F. t. Schu4 sequence were spotted in triplicate on the arrays.  cDNA generated from F. t. LVS total RNA were labeled and hybridized with the arrays.

first: Kaiping

last: Deng

last: Blick


title: No accelerated rate of protein evolution in male biased Drosophila pseudoobscura genes

city: Wien

first: Schlötterer

last: Christian

address: Josef Baumann Gasse 1

department: Institut für Tierzucht und Genetik

country: Austria

institute: Veterinärmedizinische Universität Wien

summary:
Sexually dimorphic traits are subject to diversifying selection. Also genes with a male biased gene expression are probably affected by sexual selection and have a high rate of protein evolution. We used SAGE to measure sex biased gene expression in Drosophila pseudoobscura. Consistent with previous results from D. melanogaster, a larger number of genes were male biased (402 genes) than female biased (138 genes). About 34% of the genes changed the sex related expression pattern between D. melanogaster and D. pseudoobscura. Combining gene expression with protein divergence between both species, we observed a striking difference in rate of evolution for genes with a male biased gene expression in one species only. Contrary to expectations, D. pseudoobscura genes in this category showed no accelerated rate of protein evolution, while D. melanogaster genes did. If sexual selection is driving molecular evolution of male biased genes, our data imply a radically different selection regime in D. pseudoobscura.
Keywords: SAGE

overall_design: Male and female SAGE libraries of D. pseudoobscura were developed for analyzing the gene expression pattern.

first: Muralidhar

last: Metta

first: Rambabu

last: Gudavalli

first: Jean-Michel

last: Gibert

first: Christian

last: Schlötterer


title: Transcriptional Profiling Implicates Novel Interactions between Abiotic Stress and Hormonal Responses in Thellungiella

status: Public on Jul 22 2006

city: Waterloo

first: Barbara

last: Moffatt

state: Ontario

address: 200 University Ave West

department: Biology

country: Canada

institute: University of Waterloo

summary:
Thellungiella, an Arabidopsis-related halophyte, is an emerging model species for studies designed to elucidate molecular mechanisms of abiotic stress tolerance. Using a cDNA microarray containing 3628 unique sequences derived from previously reported libraries of stress-induced cDNAs of the Yukon ecotype of Thellungiella, we obtained transcript profiles of its response to drought, cold, high salinity and re-watering after drought. A total of 153 transcripts were found to be significantly differentially regulated under the conditions studied. Only six of these genes responded to all three stresses of drought, cold and salinity. Unlike in Arabidopsis, there were relatively few transcript changes in response to high salinity in this halophyte. Furthermore, drought responsive-transcripts in Thellungiella provided a link between the down-regulation of defense-related transcripts and the increase of endogenous abscisic acid during drought. This antagonistic interaction between drought and biotic stress response may potentially be beneficial for survival under drought stress. Intriguingly, changes of transcript abundance in response to cold implicate the involvement of jasmonic acid in the cold acclimation of Thellungiella. Taken together, our results provide useful starting points for more in depth analysis of Thellungiella’s extreme stress tolerance.
Keywords: Abiotic stress response

overall_design:
We used a custom cDNA microarray to perform an initial analysis of the molecular response of the Yukon ecotype of Thellungiella subjected to cold, drought, or drought followed by re-watering or salinity treatments. The 4 different sets of microarray exp are :
1) Control versus Cold
2) Control versus Drought
3) Drought versus Rewatering
4) Control versus Salinity

In each set of microarray exp, we have performed 3 biological replicates (A, B, C) and technical replicate (dye swap) resulting in a total of 24 hybridizations.

first: C

middle: E

last: Wong

first: Y

first: A

last: Labbe

first: P

last: Nuin

last: Diaz-Camino

first: B

last: Whitty

first: D

last: Guevara

first: G

last: Golding

first: E

middle: A

last: Weretinylk

first: M

last: Griffith


title: Heart Failure, DITPA and Captopril

status: Public on Jul 21 2006

city: Tucson

first: Niranjan

last: Maitra

state: AZ

address: 1501 North Campbell Avenue

department: Sarver Heart Center

institute: University of Arizona

summary:
Three weeks after infarction, age matched rats with large infarction by ECG criteria were randomly assigned to one of the three groups: 1) Treatment with Captopril for 21 days and a daily subcutaneous dose of DITPA for the last 10 days of treatment, 2) Treatment for 10 days with DITPA alone, 3) Control heart failure and 4) Sham-operated.
Keywords: Drug Effects on Heart Failure

overall_design: Total RNA was prepared from left ventricle using TRIzol and later Poly A+ RNA was isolated with Oligotex beads. About 0.5ug Poly A+ RNA was reverse transcribed and labeled with Alexa Fluor 546 and 647. Labeled probed were hybridized with Operon's rat Unigene oligo library printed on glass slides. After 2 lows and 1 high stringency washes, Prolong Antifade was added to the slides to prevent any bleaching effect. The experiments were repeated 2-4 times by dye-swaping. Slides were scanned in Array Worx scanner with dual wave length. The data ratio was normalized using a location and intensity dependent Lowess formula.

first: Kevin

last: Greer

first: Eugene

last: Morkin


title: Expression in Primary Effusion Lymphoma (PEL) BCBL-1 compared to BJAB (BL cell line) and  L-428 (cHD cell line)

status: Public on Jul 14 2006

city: Montreal

first: Eduardo

last: Hernandez

state: Quebec

address: 3755 Cote-Sainte-Catherine Road

department: Molecular Oncology Group

institute: Lady Davis Institute for Medical Research (JGH)

summary:
Infection of B lymphocytes by HHV-8 is associated with the development of Primary Effusion Lymphoma (PEL). By microarray we identify differential expressions in three pro-inflammatory factors; LTA4H, TSP-1 and IL-16 in the BCBL-1 in comparison to BJAB (BL cell line)

By microarray we identify differential expressions in three pro-inflammatory factors; LTA4H, TSP-1 and IL-16 in the BCBL-1 in comparison to L-428 a Classical Hodgkin’s disease cell line (cHD) as compared to BJAB a BL cell line.
Keywords: Direct cell to cell comparison

overall_design:
Primary Effusion Lymphoma (PEL) was compared to BJAB (BL cell line). A direct comparison microarray was designed to reveal any differently expressed genes between those cell lines.

Primary Effusion Lymphoma (PEL) BCBL-1 was compared to L-428 a Classical Hodgkin’s disease cell line (cHD). A direct comparison microarray was designed to reveal any differently expressed genes between those cell lines in comparison to BJAB, a BL cell line.


title: Antagonism between two mechanisms of antifungal drug resistance

status: Public on Jul 15 2006

city: Mississauga

first: James

last: Anderson

address: 3359 Mississauga Road N.

institute: University of Toronto

summary:
Genome-wide assays to test the interaction between two independently evolved mechanisms of fluconazole resistance
Keywords: Comparative genomic hybridization

overall_design: Three populations at 100 and 400 generations of evolution in increasing concentrations of fluconazole [experiment 1 - (Anderson et al.,2003 Genetics,163:1287-98, PMID: 12702675, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed&term=12702675)], a single double-mutant (progeny strain from each of the three crosses of the strains from generation 100, and the erg3r mutant strain, were all assayed for gene expression against sce13 the ancestor strain.

first: Caroline

last: Sirjusingh


title: Expression Profiling of Spalax ehrenbergi: bioprospecting for hypoxia tolerance

city: Urbana

first: Mark

middle: Robert

last: Band

state: IL

address: 1201 W. Gregory Dr

department: Biotechnology

institute: University of Illinois

summary:
This study compares response to hypoxia in muscle between 2 hypoxia tolerant species of Spalax ehrenbergi, blind subterranean mole rat .
The blind subterranean mole rat (Spalax ehrenbergi superspecies) is an excellent model of hypoxic tolerance, living underground and exposed to fluctuating O2 and CO2 levels. Unique structural and functional adaptations of the cardiovascular and respiratory systems allow survival at severely reduced oxygen tension. In this study we have compared expression profiles of muscle tissue between two hypoxia tolerant species of Spalax ehrengergi at normoxic (21%) and hypoxic (3%) levels of oxygen concentration by cross species hybridization using a mouse cDNA array containing 15,000 gene elements. Results uncover numerous genes involved in angiogenesis, apoptosis, and oxidative stress and responding in a species specific manner to hypoxia. Among the most striking, cardiac ankyrin repeat protein (Carp) and activating transcription factor 3 (Atf3), LIM and cysteine-rich domains 1 (Lmcd1) and syndecan 2 (Sdc2). These results support the hypothesis that Spalax is variably adapted to fluctuating oxygen tension. Differences may involve very specific metabolic pathways and functional adaptations at the structural and molecular levels. Elucidation of the natural variation and evolutionary changes under hypoxia within this superspecies may have biomedical applications in ischemic syndromes and cancer.
Keywords: hypoxia, Spalax, cDNA microarray

sample_organism:
Nannospalax ehrenbergi
Nannospalax judaei
Nannospalax galili

platform_organism:
Mus musculus
Nannospalax ehrenbergi

overall_design: 12 animalsin total, 2 species (s52;S. galili, s60; S. judaei), 2 conditions (normoxia, hypoxia), 3 biological replicates. Loop design with dye swap.

first: Aaron

last: Avivi

first: Leonid

last: Brodsky

first: Eviatar

last: Nevo


title: Crassostrea array characterization

status: Public on Jul 13 2006

city: Charleston

first: MarineGenomics

last: Project

state: SC

address: 331 Fort Johnson Rd

summary:
Determination of tissue-specific expression profiles and heterologous hybridization by multiple Crassostrea spp.
Keywords: Gill versus Hepatopancreas

sample_organism:
Crassostrea virginica
Magallana gigas
Magallana ariakensis

platform_organism:
Crassostrea virginica
Magallana gigas

overall_design: 3 replicate hybridizations, tissue pooled from three individuals

first: Matthew

last: Jenny

first: Annalaura

last: Mancia

first: David

last: McKillen

first: Harold

middle: F

last: Trent

first: Yian

last: Chen

first: Jonas

last: Almeida

first: Robert

last: Chapman

first: Gregory

last: Warr


title: 5'DFUR treatment in MCF7. Effect at 24 hours

city: Barcelona

first: Miriam

last: Molina-Arcas

address: Av. Diagonal, 645

department: Bioquimica i Biologia Molecular

country: Spain

institute: Universitat de Barcelona

summary:
MCF7 cells were treated during 90 minuts with 5'DFUR 250uM in presence or absence of NBTI 100nM. RNA from two independent experiments was isolated and hybridization was performed in duplicate in all cases, using reciprocal labelling
Keywords: 5'DFUR effect. Time 24h

overall_design: MCF7 cells were treated during 90 min with 5’-DFUR 250 mM either in the presence or the absence of NBTI 100 nM. After 24 hours cells were trypsinized and total RNA was isolated and amplified. To ensure the reproducibility of the technique cDNA hybridization was performed in duplicate in all cases, using reciprocal labelling. Moreover,  RNA from two independent experiments was isolated, thus, four microarray were hybridized for each condition.

first: Míriam

first: Gema

last: Moreno-Bueno

first: Héctor

last: Hernández-Vargas

first: F. Javier

last: Casado

first: José

last: Palacios

first: Marçal

last: Pastor-Anglada


title: 5'DFUR treatment in MCF7. Effect at 4 hours

summary:
MCF7 cells were treated during 90 min with 5'DFUR 250 uM in presence or absence of NBTI 100nM. The effect was analyzed 4 hours after the beginning of treatment. RNA from two independent experiments was isolated and hybridization was performed in duplicate in all cases, using reciprocal labelling.
Keywords: 5'DFUR effect. Time 4h

overall_design: MCF7 cells were treated during 90 min with 5’-DFUR 250 mM either in the presence or the absence of NBTI 100 nM.  After 4 hours from the beginning of the treatment cells were trypsinized and total RNA was isolated and amplified. To ensure the reproducibility of the technique cDNA hybridization was performed in duplicate in all cases, using reciprocal labelling. Moreover,  RNA from two independent experiments was isolated, thus, four microarray were hybridized for each condition.


title: Detection of gene expression in Anopheles gambiae whole adults

city: Chicago

first: Joelle

last: Perusse

address: 920 E. 58th Street

department: Department of Genetics

institute: Yale University

summary:
RNA from adult mosquitoes was aminoallyl-labelled and directly hybridized to glass high density microarrays. Intensity values are used in a study of detection of transcription above background.
Keywords: High density oligonucleotide array; detection of gene transcription

overall_design: The five direct hybridizations of male and female biological replicate samples were done with dye swap.

first: Gabor

last: Halasz

first: Marinus

last: van Batenburg

first: Sujun

last: Hua

first: Kevin

middle: P

last: White

first: Harmen

last: Bussemaker


title: Expression data from Escherichia coli

status: Public on Jul 07 2006

city: Ithaca

first: Dr Zhijun

last: Wang

email: zw37@cornell.edu, zjwang@shmu.edu.cn

state: NY

address: Uptown road

department: Department of Population Medicine and Diagnostic Sciences

institute: Cornell University

summary:
Transcription profiles in BL21, BL21/pOri1 and BL21/pOri2 were analysed using DNA microarray technology. BL21, BL21/pOri1 or BL21/pOri2 strains were cultured at chemostat status and harvested after the cultivation arrived steady status.
Keywords: Effects of plasmid DNA on Escherichia coli metabolism

overall_design: BL21, BL21/pOri1 and BL21/pOri2 were cultured at the exponential growth status or at the same growth rate status, respectively. E. coli BL21, BL21/pOri1 and BL21/pOri2 RNAs were reverse-transcripted into cDNAs, The RNAs from the BL21, BL21/pOri1, BL21/pOri2 were labelled with biotin. The labelled cDNAs were mixed and then hybridized on the microarray slides. Experiments were repeated four times. Most of them have no significant difference comparing plasmid-carrying E. coli and plasmid-free E. coli

first: Zhijun

first: Li

last: Xiang

first: Junjie

last: Shao

first: Grzegorz

last: Wegrzyn


title: Global gene expression profiling of developing maize anthers

city: Ames

first: Patrick

middle: S.

last: Schnable

state: IA

address: 2035B Roy J Carver Co-Lab

institute: Iowa State University

summary:
The maize spikelet contains two florets, each of which contains three anthers.  Morphologically, the anthers in the upper and lower florets proceed through the same developmental program. To test for global differences in gene expression between these anthers and to identify genes that are coordinately regulated during development, RNA isolated from upper and lower floret anthers at six developmental stages was used to hybridize cDNA microarrays.  Approximately 9% of the tested genes exhibited statistically significant differences in gene expression between anthers in the upper and lower florets.  This suggests that several basic biological processes are differentially regulated between upper and lower floret anthers, including photosynthesis, translation, and metabolism.  To identify genes that are coordinately regulated during anther development, cluster analyses were performed.  A variety of patterns of gene expression were observed.  Five genes involved in programmed cell death were up-regulated at the time the tapetum begins to degenerate (i.e., the early microspore stage).  This finding strongly supports the hypothesis that tapetal degeneration occurs via programmed cell death.  In addition, many genes involved in protein synthesis exhibited differential expression patterns during anther development. Each microarray slide was scanned multiple times.  This approach led to the identification an additional 30 to 40% of statistically significant differences in gene expression as compared to a single-scan strategy.
Keywords: time course

overall_design: Experimental aims: 1)  To develop a cDNA microarray scanning approach that utilizes multiple scans of different intensities, thus maximizing the power to detecting statistically significant differences in gene expression; 2) To apply the multiple scan approach to identify differentially expressed genes in:  a) the upper floret of (N)Ky21 at the selected stages of anther development; b) the lower floret of (N)Ky21 at the selected stages of anther development; and c) the upper vs. lower floret at each selected stage of anther development.


title: Sex-specific gene expression in the human left ventricle

status: Public on Jul 09 2006

city: Berlin

first: Joerg

last: Isensee

address: Hessische Str. 3-4

department: Center for Cardiovascular Research

institute: Charite Berlin

summary:
In this study cDNA microarrays were used to identify genes with sex-biased expression in the human left ventricle of two age classes (<40 years and 50-65 years of age).
Keywords: cDNA microarray hybridization, sex-specific expression

overall_design: Total RNA was isolated from human left ventricular myocardial tissue from explanted hearts of <40 years old women (n=3), <40 years old men (n=4), 50-65 years old women (n=5) and 50-65 years old men (n=5). Total RNAs were pooled according to sex and age and hybridized on Agilent cDNA microarrays (age-matched, male vs. female or vice versa). Four technical replicates were carried out with exchanged dye-labelled RNA probes (dye-swap) on two of them.

first: Henning

middle: w

last: Witt

first: Patricia

last: Ruiz


title: Determine the extent of inter-species hybridization in adult parasites.

city: Forest Hill

first: Russell

middle: Leigh

last: McInnes

state: Vic

address: 347 Burwood Hwy

department: LSCA

country: Australia

institute: Agilent Technologies

summary:
We report the design, fabrication, and validation of a microarray covering the majority of the Schistosoma japonicum and Schistosoma mansoni transcriptomes. The oligonucleotide microarray contains 12,166 S. mansoni and 7055 S. japonicum target sequences. A confidence threshold of 0.001 (P value) was used in all analyses. The cross hybridization potential of the microarray was representative of 10,362 probes detected in both channels, while 12,052 probes hybridised to S. japonicum and 11,254 probes hybridised to S. mansoni. Differential hybridisation consisted of 3422 probes to S. mansoni mRNA and 3103 probes to S. japonicum mRNA. Important biological functions, such as transport, metabolism, immune evasion and host?parasite immunological interplay, cell communication, and sexual maturation are represented on this microarray. This is the first microarray commercially manufactured for studying schistosomes, and the large size and verified quality of the resource demonstrate its power for characterising the schistosome transcriptome.
Keywords: Interspecies Comparison

sample_organism:
Schistosoma japonicum
Schistosoma mansoni

overall_design: Adult mixed sex S. mansoni V adult mixed sex S. japonicum

first: Geoff

last: Gobert

first: Don

last: McManus

first: Luke

last: Moertel

first: Nelson

last: Charles

first: Jones

last: Malcolm

first: Hu

last: Wei


title: Tetracyclines Specifically Target the Apicoplast of the Malaria Parasite Plasmodium falciparum

status: Public on Jul 11 2006

city: San Francisco

first: Joseph

last: DeRisi

department: Biochemistry and Biophysics

institute: University of California San Francisco

summary:
Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum. Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect.
Keywords: Plasmodium falciparum treated with Doxycycline

platform_organism:
Saccharomyces cerevisiae
Plasmodium falciparum

overall_design: We analyzed a series of 12 microarrays covering 55 hours of Plasmodium falciparum treated with doxycycline and 12 microarrays covering the same 55 hours with no doxycycline treatment

first: Erica

last: Dahl

first: Jennifer

last: Shock

first: Bhaskar

last: Shenai

first: Jiri

last: Gut

first: Philip

middle: J

last: Rosenthal


title: Analysis of gene expression profiling of normal retina and brain cortices

status: Public on Jul 06 2006

city: Philadelphia

first: Gerardo

middle: L.

last: Paez

state: PA

address: 3900 Delancey Street

department: Clinical Studies Philadelphia

institute: University of Pennsylvania

summary:
Using a canine custom retinal cDNA microarray our aim is to identify normal gene expression profiling for retina and frontal, occipital and temporal brain cortices
Keywords: retina-brain comparisons

overall_design: Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) and further purified by Rneasy mini kit (Qiagen, Valencia, CA). Purity and RNA quality were evaluated by absorbance at 260 nm and by denaturing formaldehyde agarose gel electrophoresis. High quality RNAs with A260/280 ratio over 1.8 and intact 28S and 18S RNA bands were used for microarray analysis. To generate an RNA reference sample for microarray hybridizations, we pooled equal amounts of total RNA from the occipital, temporal, and frontal brain regions collected from three 16-week-old beagles to achieve a homogeneous pool of transcripts. The pooled RNA was divided into aliquots (2μg/μl) and stored at -80 °C until use. For the purpose of this work, four tissue groups have been established, containing five biological replicates for normal retina and three for each respective brain region. After initial validation of reproducibility, only one microarray experiment was used for each sample.

middle: L

first: Barbara

last: Zangerl

first: Gustavo

middle: D

last: Aguirre

first: Kimberley

last: Sellers


title: Global transcriptional analysis of the obligate intracellular bacteria Rickettsia conorii

city: Geneva

first: FRANCOIS

last: Patrice

state: Ge 4

address: Rue Gabrielle-Perret-Gentil, 4

department: Service of Infectious Diseases

country: Switzerland

institute: Genomic Research Laboratory

summary:
Rickettsia conorii are obligate intracellular bacteria responsible for the Mediterranean spotted fever. Their adaptation to highly divergent environments ranging from the arthropod vector to the human beings is essential to survival and virulence. Through a combination of total RNA purification and random prokaryotic cDNA amplification, we successfully analyzed transcription profiles by microarrays starting from 100 ng of R. conorii RNA. Real-time quantitative PCR results performed on unpurified total RNA was consistent with microarray measurements. Here, from the selected targets spotted on our microarray, we showed that in R. conorii, nutrient stress is accompanied by over-expression of the virB operon. Regulation of these genes could be mediated by the stringent response through ppGpp, consistent with the observed up-regulation of spoT and gmk. Exposure of R. conorii to a nutrient stress paradoxically reduced the expression of both GroEL and its transcriptional regulator RpoH. This shows that the over-expression of this chaperonin is a part of the natural life of intracellular growing rickettsiae. Our findings also evidenced that, unexpectedly, many atypical sequences, including small-size split genes, ORFans genes and highly conserved intergenic regions contains expressed sequences that are differentially regulated. The notable deficiency of transcriptional regulators within R. conorii genome does not reflect its incapability to undergo transcriptional changes but rather the existence of an alternative adaptation strategy.
Keywords: Global transcriptional analysis

overall_design:
Rickettsia conorii were grown in confluent monolayers of African green monkey kidney cells (Vero cell, ATCC C1587).
Combination of total RNA and random prokaryotic cDNA; measurements performed in triplicate.

first: My-Van

last: La

first: Patrice

last: Francois

first: Clarisse

last: Rovery

first: Sylvianne

last: Robineau

first: Pascal

last: Barbry

first: Jacques

last: Schrenzel

first: Didier

last: Raoult

first: Patricia

last: Renesto


title: Stress-specific role of the fission yeast Gcn5 acetylatransferase in programming a subset of stress-response genes

city: Huddinge

first: Anthony

last: Wright

address: Blickagången 6

country: Sweden

institute: Karolinska Institute

summary:
We have found Gcn5 is specifically requried for KCl and CaCl2 mediated stress in Schizosaccharomyces Pombe. We have charaterised the genome wide gene expression responses to KCl stress and show the Gcn5 in involved in the regulation of sebset of stress response genes.
Keywords: stress response

overall_design: Four subsets is included: 1 gcn5 mutant compared to wt under rich medium without treatement, 2 gcn5 mutant compared to wild type both under KCl treatment, 3 wild type 15min treatment with KCl compared to non-treated, 4 Wild type 60min KCl treatement compared to non-treated. Gene expression profiling experiment in which gene expression in logarithmically growing cultures of test group was compared to that control in rich medium at 25C.RNA was extracted and subjected to cDNA expression profiling analysis using the established protocols (Xue et al, 2004). Independent RNA preparations were used to hybridise to microarray slides with dye swap. Data normalized with 'Lowess' per chip per spot normalization.

first: Anna

last: Johnsson

first: Yongtao

last: Xue-Franzen


title: Examine strain and gender associated gene expression in Schistosoma japonicum

first: Russell

last: McInnes

summary:
We utilised a 22,575 feature custom oligonucleotide DNA microarray designed from public domain databases of schistosome expressed sequence tags to explore differential gene expression between the Philippine (SJP) and Chinese (SJC) strains of S. japonicum, and between male and female S. japonicum. We found that 593, 664 and 426 probes were differentially expressed between the two geographical strains when we compared mix sexed adults, male worms and female worms. Additionally, the study revealed that 1,163 male- and 1,016 femaleassociated probes were differentially expressed in SJP whereas 1,047 male- and 897 female-associated probes were differentially expressed in SJC.
Keywords: Strain and Gender Comparison

platform_organism:
Schistosoma japonicum
Schistosoma mansoni

overall_design: Five comparisons, each with dye-swapped replicates were done.  Design 1 used four arrays to compare mixed sex adult S. japonicum (Strain Philippine) to mixed sex adult S. japonicum (Strain Chinese).  Design 2 used two arrays to compare adult male S. japonicum (Philippine strain) to adult male S. japonicum (Chinese strain).  Design 3 used two arrays to compare adult female S. japonicum (Chinese strain) to adult female S. japonicum (Philippine Strain).  Design 4 used two arrays to compare adult male S. japonicum (chinese strain) to adult female S. japonicum (chinese strain).  Design 5 used two arrays to compare adult male S. japonicum (philippine strain) to adult female S. japonicum (philippine strain).

first: Geoff

last: Gobert

first: Don

last: McManus

first: Luke

last: Moertel

first: Terrence

last: Piva

first: Lauren

last: Young


title: Obesity impact on the left ventricular transcriptome of 4 and 10 months old SHHF rats

status: Public on Jul 05 2006

city: Toulouse

first: Philippe

middle: V

last: Rouet

address: 1 Ave Jean Poulhes

department: Cardiology

institute: INSERM U1048

summary:
Impact of the obesity on RNA total levels of male SHHF left ventricular aged from 4 and 10 months old.
Keywords: Comparative cDNA hybridization

overall_design: Comparison of two groups : 5 obese SHHF rats and 5 lean SHHF rats from each age group, with cDNA macroarrays on radioactive membranes


title: Impact of the obesity on the right atrium transcriptom of SHHF rats

summary:
Impact of the obesity on RNA total levels of male SHHF right atrium aged from 10 months old.
Keywords: Comparative cDNA hybridization

overall_design: Comparison of two groups : 4 obese SHHF rats and 4 lean SHHF rats, with oligo-microarrays on glass slides


title: Profiling limb muscle degeneration by inducible dominant negative thyroid hormone receptor transgene over-expression

status: Public on Jul 08 2006

city: Bethesda

first: Biswajit

last: Das

address: 18 Library Drive, Building 18T, Room 106

department: Laboratory of Gene Regulation and Development

institute: National Institutes of Health/National Institute of Child Health and Human Development

summary:
Premetamorphic Xenopus laevis tadpoles limbs develop in response to thyroid hormone by proliferation and subsequent differentiation. We have previously reported that inhibiting the thyroid hormone action using a dominant negative receptor in limbs of developing tadpoles inhibits its growth and the tadpole limb has almost no muscle. The goal of this experiment is to study the role of thyroid hormone in the maintenance of limb muscle using transgenic tadpoles carrying a doxycyline inducible dominant negative thyroid receptor transgene expressed exclusively in the muscle using a muscle specific promoter and identify the genes involved in the degenerative process . F1 progeny of transgenic Xenopus frogs and their nontransgenic siblings (at stages NF55 and NF66) were treated with 50µg doxycycline hyclate in 0.1 X MMR solution for 2 weeks or 6 weeks to induce the over-expression of the transgene. Limbs from the tadpoles were dissected at the end of the experiment.
Keywords: development or differentiation design,reference design,replicate design,time series design

overall_design: We analyzed 3 replicate arrays for each sample type. NF66 juvenile frogs serve as a endpoint control whereas 2 week doxycycline treatment of nontransgenic siblings serve as age-matched control for 2 week treatment of transgenic tadpoles.

first: Liquan

last: Cai

first: Donald

last: Brown


title: Transcriptional response to nitrogen availability in yeast

city: Brussels

first: Bart

last: Scherens

address: Av. E. Gryson, 1

country: Belgium

institute: IRMW-ULB

summary:
We used cDNA microarray technology to compare the genome-wide expression profiles of a wild type strain (BY4700) (E02, E04, E06) or the isogenic strain deleted of GLN3 and GAT1 genes (E01, E03, E05) grown in YNB medium with glutamine as nitrogen source (M.Gln) against the wild type strain grown in M.Gln after addition of rapamacyn (20 min) (E01, E02), or M.proline (M.Pro) (E03, E04) or after a two hours shift from M.Gln to M.Pro (E05, E06), all growth conditions known to modify the expression of genes involved in nitrogen utilization. These microarrays allowed to identify the set of genes that were up or down-regulated in response to the quality of the nitrogen source. To evaluate whether the majority of genes responding to the nitrogen source were dependent on Gln3 and Gat1, we compared the expression profiles of the wild type strain and of the isogenic strain deleted of GLN3 and GAT1 genes (03167b: ura3, gln3∆, gat1∆), when both strains were grown on M.Gln + rapamycin (E07), or M.Pro (E08) or after a shift from M.Gln to M.Pro (E09). We also used an independent means of identifying Gln3-Gat1 regulated genes by comparing the expression profiles in wild type and ure2∆ (4709∆URE2) strains on M.Gln medium (E10).
Keywords: nitrogen availability, knock-out response

overall_design: The hybridization signal was measured using a GSM418 laser scanner. Image analysis for each array was processed using the GenePix Pro 4.0 (Axon Instruments, Inc.) software package, which measures fluorescence intensity pairs for each gene. Following image acquisition, a visual inspection of the individual spots on each microarray (size, signal-to-noise ratio, background level, and spot uniformity) completed the flagging (present/not present, good/bad) of the data. To maximize sensitivity two scans were made, one at high laser power and high PMT (Photo Multiplier Tube) gain to detect the faintest spots, and a second one using low laser power and a PMT gain avoiding saturation. The values for spots presenting ≥ 5% saturation in the first scan were calculated based on an extrapolation after linear regression analysis of the intensities from both scans. These data were then imported in the GeneSpring 7.1 (Silicon Genetics) software package, applying a per spot per chip intensity-dependent (Lowess) normalization for further analysis.

first: André

last: Feller

first: Fabienne

last: Vierendeels

first: Francine

last: Messenguy

first: Evelyne

last: Dubois


title: CGH analysis of Candida albicans strains

status: Public on Jun 30 2006

city: Boston

first: Anna

last: Selmecki

state: MA

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

summary:
Aneuploidy and the evolution of aneuploid karyotypes of Candida albicans strains was identified using aCGH.  Whole chromosome and segmental aneuploidies, (specifically on the left arm of chromosome 5 - shown to be due to isochromosome formation) are associated with the appearance of resistance to the antifungal drug fluconazole.
Keywords: Comparative Genomic Hybridization

overall_design: Hybridization of all strains was compared to the hybridization of SC5314, the sequenced laboratory strain.

middle: M

first: Anja

last: Forche

first: Judith

last: Berman


title: The kidney collecting duct acid-base regulon

status: Public on Jul 01 2006

city: Paris

first: Alain

last: Doucet

institute: Institut des Cordeliers

summary:
Kidneys are essential for acid-base homeostasis, especially when organisms cope with changes in acid or base dietary intake. Because collecting ducts constitute the final site for regulating urine acid-base balance, we undertook to identify the gene network involved in acid-base transport and regulation in the mouse outer medullary collecting duct (OMCD). For this purpose, we combined kidney functional studies and quantitative analysis of gene expression in OMCDs, by transcriptome and candidate gene approaches, during metabolic acidosis. Furthermore, to better delineate the set of genes concerned with acid-base disturbance, the OMCD transcriptome of acidotic mice was both compared to that of normal mice and mice undergoing an adaptative response through potassium depletion. Metabolic acidosis, achieved through a NH4Cl-supplemented diet for 3 days, not only induced acid secretion, but also stimulated the aldosterone and vasopressin systems and triggered cell proliferation. Accordingly, metabolic acidosis increased the expression of genes involved in acid-base transport, sodium transport, water transport and cell proliferation. In particular, more than 25 transcripts encoding proteins involved in urine acidification (subunits of H-ATPase, kidney anion exchanger, chloride channel Clcka, carbonic anhydrase 2, aldolase) were co-regulated during acidosis. We propose that these trancripts cooperating to a same function and co-regulated during acidosis constitute a functional regulon.
Keywords: SAGE; Kidney collecting duct; Mouse; Metabolic acidosis; V-ATPase

overall_design: Approximately 650 OMCDs were microdissected, as previously described from collagenase-treated kidneys of 6 mice fed the NH4Cl-supplemented diet for 3 days. 20960 tags were sequenced in the acidosis SAGE library. After withdrawing duplicate ditags and linker sequences, the 19146 tags remaining corresponded to 8847 different tags.This acidosis library is similar in size and diversity to the normal mouse OMCD libraries previously generated (GEO accession number : GSM23478). The tag abundance in the two library was compared and discussed.

first: Lydie

last: Cheval

first: Luciana

last: Morla

first: Jean-Marc

last: Elalouf


title: Signature of mesenchymal cell lines

city: tours

first: pierre

last: charbord

address: 10 bd tonnellé

institute: Laboratoire d'hématologie de tours

summary:
We have analysed tes adipocytic, osteoblastic, chondrocytic and vascular smooth muscle differentiation of the stromal potential of 5 mouse mesenchymal lines derived from hematopoietic sites (bone marrow and fetal liver). The sole line, AFT024, that provide both quadripoential and capable of maintening hematopoiesis in vitro, has served as reference for genomic studies aimed at establishing the signature of the multipotential stromal mesenchymal stem cell (MSC).
Keywords: molecular signature of MSC

overall_design: Competitive hybridisation using AFT024 cells as reference and 2018, BFC012, BMC9 or BMC10  cells as sample. 8 replicates constituted by 4 dye-swaps for each experiment.

first: sebastien

last: Chateauvieux

first: Jean-Laurent

last: Ichante

first: Vincent

last: Frouin

first: Genevieve

last: Pietu

first: Michelle

last: Martin

first: Kateri

middle: a

last: Moore

first: Pierre

last: Charbord


title: Planarian stem cells

status: Public on Jul 19 2006

city: Pisa

first: Leonardo

last: Rossi

address: Via Volta 4

department: Morfologia Umana e Biologia Applicata

country: Italy

institute: University of Pisa

summary:
Difficulty of experimental accessing makes in vivo stem cell studies not easy in mammals and model systems with regenerative capability represent an alternative for the characterization of stem cell-specific genes. Planarians retain a population of pluripotent stem cells, the neoblasts, throughout their adult life.  Neoblasts can be selectively destroyed by X-ray allowing us to compare stem cell-less organisms with wild-type worms. We have undertaken a genomic approach producing an
Keywords: X-ray, dose response, stem cells, stress response


title: Gene expression profiling of livers from cafestol-fed APOE3Leiden mice

city: Maastricht

first: Marten H.

last: Hofker

address: P.O. Box 616

department: Molecular Genetics

institute: University Maastricht

summary:
Unfiltered coffee markedly increases serum lipid levels in humans and mice. The responsible compounds are the fat-soluble diterpenes cafestol and kahweol. Cafestol is responsible for more than 80% of the effect on serum lipids and is the most potent cholesterol-elevating compound known in the human diet. Aim of these microarray studies was to identify novel genes and regulatory pathways determining the cholesterol raising effect of cafestol by genome-wide expression studies.
Keywords: Transgenic APOE3Leiden_Mouse_liver_diet response_cafestol

overall_design: Transgenic APOE3Leiden (E3L) mice of the N21th generation (>99% C57BL6/JIco) were used. Mice were 10 weeks of age at the start of experiments, and received control or cafestol diet for 4 weeks (n=7 and 8 respectively). As control diet, diet W (Hope Farms) was used, a semi-synthetic diet (18.2 MJ/kg) enriched with saturated fat (15g/100g) and cholesterol (0.25g/100g). This diet was supplemented with 0.04% (wt/wt) cafestol and 0.02% (wt/wt) kahweol for the cafestol diet. Microarray experiments were performed in duplicate: the same RNAs were labeled, divided into two different pools and hybridized onto two different GEMs 2.03 (Incyte Genomics, Palo Alto, CA). Duplicates are indicated by the same SY or TP number in the sample titles.


title: Characterization of the piRNA Complex from Rat Testes

status: Public on Jul 03 2006

city: Waltham

first: Nelson

middle: C

last: Lau

address: 415 South St MS029

institute: Brandeis University

type: Non-coding RNA profiling by high throughput sequencing

summary:
This data series contains the sequences and read counts of Piwi-interacting RNAs and other small RNAs from mouse and rat testes extracts.  In a search for different classes of small RNAs that might be involved in transcriptional gene silencing, we encountered a novel class of small RNAs within mammalian testes.  This study reports the identification of small RNAs and their relative abundance levels to each other based on the deep coverage of the cDNA library.
Keywords: High-throughput pyrosequencing by the 454 Life Sciences Genome Sequencer 20™ System

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: We constructed a cDNA library from small RNAs purified from Q column fractions of testes extracts.  Briefly, gel-purified small RNAs from the column fractions were ligated with adaptor oligos on both 3' and 5' ends of the small RNAs.  Reverse-transcription and PCR amplification was then performed on the ligated small RNAs to yield a cDNA library.  This cDNA library was finally converted by PCR into a single-stranded DNA library compatible for coupling to beads on the 454 Life Sciences Genome Sequencer 20™ System.  Reads and sequences of the small RNAs were processed from linker sequences, examined for matches to the mouse or rat genomes, and tallied.

first: Anita

middle: G

last: Seto

first: Jinkuk

last: Kim

first: Satomi

last: Kuramochi-Miyagawa

first: Toru

last: Nakano

first: David

last: Bartel

first: Robert

last: Kingston


title: JM43_Glucose_Air_N2_Air_Shift

city: Urbana

first: Kurt

middle: E

last: Kwast

address: 407 S. Goodwin Ave.

department: Molecular & Integrative Physiology

institute: University of Illinois at Urbana-Champaign

summary:
The yeast cells were grown aerobically on glucose medium. At time zero (generation 0) the saprge gas was switched from air to O2-free N2 and samples were harvested after 0 (aerobic control), 0.04, 0.08, 0.13, 0.19, 0.25, 0.38, 0.5, 1, 2, 3, 4, 5 and 6 generations of anaerobic growth. After six generations, the saprge gas was switched back to air and samples were harvested at 6 (anaerobic control), 6.03, 6.06, 6.1, 6.13, 6.2, 6.3, 6.4, 6.6, 6.8, and 7.6 generations.
Keywords: time-course

first: Liang-Chuan

last: Lai

first: Alexander

last: Kosorukoff

first: Patricia

last: Burke


title: JM43_Galactose_Air_N2_Air_Shift

summary:
The yeast cells were grown aerobically on galactose medium. At time zero (generation 0) the saprge gas was switched from air to O2-free N2 and samples were harvested after 0 (aerobic control), 0.04, 0.08, 0.13, 0.19, 0.25, 0.38, 0.5, 1, 2, 3, 4, 5 and 6 generations of anaerobic growth. After six generations, the saprge gas was switched back to air and samples were harvested at 6 (anaerobic control), 6.03, 6.06, 6.1, 6.13, 6.2, 6.3, 6.4, 6.6, 6.8, and 7.6 generations.
Keywords: time-course


title: Determine the extent of similarity of gene expression in in-vitro compared to in-vivo grown Schistosomes.

first: Russell

last: McInnes

summary:
The schistosomulum is the main target of vaccine- induced protective immunity; however, most studies have utilized schistosomula produced by mechanical transformation of infective larvae followed by in vitro culture rather than larvae isolated directly from the lungs of infected mammals.  Using transmission electron microscopy, we demonstrated that there was little difference in the ultrastructure of Schistosoma japonicum schistosomula obtained by the two methods. However, significant differences in gene expression profiles were apparent when we used an oligonucleotide microarray to compare the gene expression profiles of schistosomula obtained in vivo from lung tissue with those maintained in vitro, and with adult worms of S. japonicum.
Keywords: Comparison of culturing conditions

overall_design: Two comparisons with replicates were done.  Design 1 used two dye-swapped arrays to compare In vivo grown S. japonicum lung schistosomula (Chinese strain) to adult mixed sex schistosome (Chinese strain).  Design 2 used three replicated arrays to compare In vivo grown S. japonicum lung schistosomula (Chinese strain) to in vitro cultured S. japonicum schistosomula (Chinese strain).

first: Geoff

last: Gobert

first: Don

last: McManus

first: Luke

last: Moertel

first: Alex

last: Loukas

first: MK

last: Jones

first: M

last: Tran


title: cAMP Ca2+ NFAT signalling in RPTEC control vs. CAA

city: Munich

first: Andreas

last: Benesic

state: Bavaria

address: Marchioninistr. 15

department: Dept. of medicine 2

institute: University Hospital Grosshadern, Munich

summary:
The nephrotoxic ifosfamide-metabolite chloroacetaldehyde (CAA) induces changes in intracellular Ca2+-concentration of human RPTEC. This effect is due to a PKA-dependent inhibition of Na+/Ca2+ exchange (Benesic, A., Schwerdt, G. at. al. (2005) Kidney Int 68, 2029-2041). Thus,  possible effects of CAA-exposure on cAMP and Ca2+ regulated genes as well the Ca2+/NFAT pathway were investigated by cDNA-microarray analysis. Both microarrays (cAMP/Ca2+ and Ca2+(NFAT) were performed twice. After 24 h CAA exposure a marked downregulation of c-fos and junB was observed in all arrays performed. In the cAMP/Ca2+-arrays downregulation of FOS, ADRB1, INHBA, CDK5, IL2, PLN, SRF, GCG, SGK, JUNB, BDNF, S100A6, CCNA1, MIF, HK2, SST and  EGR2 by CAA was observed in two independent experiments. From the AP-1 family expression of junD and fosB were not altered. Overexpression by CAA of CREB and DDIT3 were only observed in one of two experiments. JUND, FOSB, FOSL1, CREM and CREBBP expression was not influenced in reproducible manner, as well as all members of the NFAT-family. In the Ca2+/NFAT-Arrays, PPP3CB, VAV3, NFATC1, IL8, IL6, FASLG, IL3, IFNB1, FOS, BCL2, EGR3, CD3G, HRAS, EP300, IL4, CALM3, RELA, EGR2, NPPB, IFNG, NFATC4, KPNA5, CAMK2BNFKB2 , VEGF, IL8RA, IL2,GATA4, MEF2A, PTPRC, ICOS, NFKBIE, CDKN1A, CSNK2B, ACTB, MEF2B and JUNB mRNA´s were repeatedly reduced by CAA exposure.  The only gene with upregulation in both experiments was found to be IFN-alpha. Thus CAA exposure for 24 h leads to a downregulation of of the AP-1 members JUNB and FOS, whereas there is small positive effect on CREB mRNA. CAA reproducibly lowers the expression of IL-2, 3, 4, 6 and 8, whereas the levels of TNFalpha and TGFbeta1 and 3 mRNA  were not altered. IFN-alpha mRNA levels were reproducibly increased (>2-fold). In conclusion, CAA seems to interact with Ca2+ and cAMP signalling pathways, with a strong impact on AP-1 proteins and inflammatory signalling.
Keywords: stress response to toxic agent

overall_design:
Human proximal tubule epithelial cells hRPTEC in primary culture were serum starved for 24 h and incubated 24 h with either control medium or medium containing 15 µmol/L CAA.
Cells were lysed and mRNA extracted using the ArrayGrade mRNA extraction Kit (www.superarray.com). Arrays were performed strictly according to the manufacturer´s instructions

first: Sigrid

last: Mildenberger

first: Michael

last: Gekle


status: Public on Jun 23 2006

city: Bunkyo-ku

first: Katsuhiko

last: Shirahige

state: Tokyo

address: 1-1-1 Yayoi

department: Research Center for Epigenetic Disease

institute: The University of Tokyo

summary:
The SMC protein complexes safeguard genomic integrity through their functions in chromosome segregation and repair. The chromosomal localization of the budding yeast Smc5/6 complex here determined reveals that the complex works specifically on the duplicated genome in differently regulated pathways. One controls the association to centromeres and chromosome arms in unchallenged cells, the second regulates the association to DNA breaks, and the third directs the complex to the chromosome arm that harbors the ribosomal DNA arrays. The chromosomal interaction pattern predicts a function that becomes more important with increasing chromosome length, and that the complex's role in unchallenged cells is independent of DNA damage. Additionally, localization of Smc6 to collapsed replication forks indicates an involvement in their rescue. Altogether this shows that the complex maintains genomic integrity in multiple ways, and evidence is presented that the Smc5/6 complex is needed during replication to prevent the accumulation of branched chromosome structures.
Keywords: ChIP-chip analysis

overall_design:
•	The goal of the experiment -
Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways.
•	Keywords, for example, time course, cell type comparison, array CGH (the use of MGED ontology terms is recommended).
Cell cycle, Double Strand Break Induction, time course after break induction, Saccharomyces cerevisiae, Chromosome VI array, Whole Genome Tiling Array, Smc5, Smc6, Nse1, Nse4, Nse5, Scc1, Scc2, Mre11, Rad53
•	Experimental factors
Distribution of the Smc5/6 complex in the cell cycle, distribution of Smc5/6 complex before and after induction of a DNA double strand break on chromosome VI in G2/metaphase cells, distribution of Smc6 in the absence of functional Scc2, Mre11 or Rad53, distribution of Smc6 at collapsed replication forks, and distribution of Cohesin subunit Scc1 in G2/metaphase. Distribution was analyzed on Chromosome VI and/or whole genome arrays. Cells were grown in rich yeast cell extract media.  All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae W303; ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5)
•	Experimental design
ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Cerevisiae chromosome VI array or Whole genome arrays were used. Total number of hybridization was 69. All description about hybridized samples is attached as the separate sheet.
•	Quality control steps taken
Duplication, confirmation using different tags, different subunits of the same complex, and time course after double-strand break induction. Checking of the ChIP fraction by Western blotting. Mock hybridisation of samples immunoprecipitated from cells containing no tag recognized by antibody used.
•	Links to the publication, any supplemental websites or database accession numbers.
http://tilingarray.bio.titech.ac.jp/smc56dsb

Samples used, extract preparation and labelling:
•	The origin of each biological sample
Saccharomyces cerevisiae W303 (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3 RAD5).
•	Manipulation of biological samples and protocols used
Strains containing flag-tagged versions of Smc6, Smc5, Nse1, Nse4, or Nse5 proteins were used. For synchronization in G1, alpha factor was added (Zymo research and Innovagen AB) at 3 µg/ml final concentrations to logarithmically growing cells, and G1 arrest was obtained at indicated temperatures for 2.5 h. For S-phase arrest, cells were grown to log phase and arrested in G1 with alpha factor for 2 h, and then incubated in the presence of 200 mM hydroxyurea (HU) (Sigma) for 30 min. Cells were then washed twice with rich medium containing 100µg/ml pronase (Calbiochem). Finally cells were resuspended in medium containing HU and pronase, and growth continued for indicated time periods. G2/M arrest was achieved 2.5 h after addition of benomyl (Sigma, 80µg/ml), to logarithmically growing cells. G1, S and G2/M arrests were checked microscopically and by FACS. DSBs were induced by activation of the HO endonuclease from the GAL1-10-promoter by addition of 2% galactose to cell cultures initially grown in 2% raffinose.
•	Technical protocols for preparing the hybridization extract
Extract preparation was processed following the Shirahige lab protocol (http://chromosomedynamics.bio.titech.ac.jp ). 5x108 cells were disrupted using Multi-Beads Shocker (MB400U, YASUI KIKAI, Osaka). Whole cell extract was sonicated to obtain 400-600 bp genomic DNA fragments. Anti-Flag monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Dynal, protein A Dynabeads) were used for chromatin immunoprecipitation. The immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization.
•	Hybridization procedures and parameters:
Hybridization, blocking and washing were carried out as previously described (http://chromosomedynamics.bio.titech.ac.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3'biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix).
•	Measurement data and specifications:
S. cerevisiae arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the cel files data and processed data files can be downloaded from GEO database or from our Web sites. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available from K.S. on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp and for whole genome arrays to 100bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-value<=0.01 for chromosome VI and pvalue?0.0025 for whole genome array). Secondly, reliability of binding ratio was judged by change p-value (p-value<=0.01 for chromosome VI and p-value<=0.0025 for whole genome array). Thirdly, clusters consisting of at least 500bp contiguous loci that satisfied the above two criteria were selected, because it is known that a single site of protein-DNA interaction resulted in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. Under these conditions both types of arrays give essentially the same results for the binding profile. Repeated sequences were masked out and not included in the calculation of protein binding profile. The correlation coefficient of Smc6 binding profiles in G2/M from two independent experiments was 0.955, showing that the whole genome ChIP on chip data was reproducible. For comparison of the localization of Smc6 in different experiments and that of Smc6 and Scc1 on whole genome maps, peaks with signal to log ratio higher than 0.6 were chosen, and peaks which were distributed within a 5kb region was recognized as one peak. In the analysis of the number of Smc6 localization sites on each chromosome a 40 kb region spanning all centromeres were excluded. This region in addition to that downstream the rDNA repeats was also excluded from the analysis when comparing Smc6 localization in wild type, scc2-4 and scc1-73 cells.

•	Array Design:
General array design: in situ synthesized arrays by Affymetrix
Chromosome VI S.cerevisiae: rikDACF, P/N# 510636
Whole Genome Array  S.cerevisiae: S.cerevisiae Tiling1.0F Arrays, Part#520286 and Part#520280

first: Hanna

middle: B

last: Lindroos

first: Lena

last: Strom

first: Takehiko

last: Itoh

first: Yuki

last: Katou

first: Camilla

last: Sjogren