title: Transcriptome Analysis of Agmatine and Putrescine Catabolism in Pseudomonas aeruginosa PAO1

status: Public on Apr 07 2008

city: Atlanta

first: Han Ting

last: Chou

state: GA

address: 1 University Park Drive

institute: Georgia State University

summary:
Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of P. aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into GABA and succinate before entering the TCA cycle in support of cell growth as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were identified to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown indispensable for polyamine utilization. The newly identified dadRAX locus, encoding the regulator, alanine transaminase and racemase respectively, coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintain alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, the alternative gamma-glutamylation pathway for the conversion of putrescine into GABA was also discussed. Subsequently, GabD, GabT and PA5313 were identified for GABA utilization. Growth defect of PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA was also demonstrated in vitro. Polyamine utilization in general was proven independent of the PhoPQ two-component system even the expression of which was induced by polyamines. Multiple potent catabolic pathways as depicted in this study could serve pivotal roles in control of intracellular polyamine levels.
Keywords: Metabolism, polyamines, agmatine, putrescine, glutamate, phoPQ.

overall_design: P. aeruginosa PAO1 was grown aerobically in minimal medium P with 350 rpm shaking at 37C, in the presence of L-glutamate alone or with the addition of putrescine, agmatine or GABA at 20 mM. Cells were harvested when the optical density at 600 nm reached 0.5~0.6 by centrifugation for 5 minutes at 4C. Total RNA samples were isolated by RNeasy purification kit following instructions of the manufacturer (Qiagen). Reverse transcription for cDNA synthesis, fragmentation by DNase I treatment, cDNA probe labeling and hybridization were performed according to the instructions of GeneChip manufacturer (Affymetrix). Data were processed by Microarray Suite 5.0 software normalizing the absolute expression signal values of all chips to a target intensity of 500. GeneSpring software (Silicon Genetics) was used for expression pattern analysis and comparison. Only genes showing consistent expression profiles in duplicates were selected for further analysis.

first: Chung-Dar

last: Lu

first: Han-Ting


title: Identification of Molecular Pathways Affected by Pterostilbene, a Natural Dimethylether Analog of Resveratrol

status: Public on Mar 05 2008

city: University

first: Ameeta

last: Agarwal

state: MS

address: NCNPR, Room 2049

department: National Center for Natural Products Research

country: USA

institute: University of Mississippi

summary:
Background:  Pterostilbene, a naturally occurring phenolic compound produced by agronomically important plant genera such as Vitis and Vacciunium, is a phytoalexin exhibiting potent antifungal activity.  Additionally, recent studies have demonstrated several important pharmacological properties associated with pterostilbene.  Despite this, a systematic study of the effects of pterostilbene on eukaryotic cells at the molecular level has not been previously reported.  Thus, the aim of the present study was to identify the cellular pathways affected by pterostilbene by performing transcript profiling studies, employing the model yeast Saccharomyces cerevisiae.  Methods:  S. cerevisiae strain S288C was exposed to pterostilbene at the IC50 concentration (70 uM) for one generation (3 h).  Transcript profiling experiments were performed on three biological replicate samples using the Affymetrix GeneChip Yeast Genome S98 Array.  The data were analyzed using the statistical methods available in the GeneSifter microarray data analysis system.   To validate the results, eleven differentially expressed genes were further examined by quantitative real-time RT-PCR, and S. cerevisiae mutant strains with deletions in these genes were analyzed for altered sensitivity to pterostilbene.  Results: Transcript profiling studies revealed that pterostilbene exposure significantly down-regulated the expression of genes involved in methionine metabolism, while the expression of genes involved in mitochondrial functions, drug detoxification, and transcription factor activity were significantly up-regulated.  Additional analyses revealed that a large number of genes involved in lipid metabolism were also affected by pterostilbene treatment.  Conclusions:  Using transcript profiling, we have identified the cellular pathways targeted by pterostilbene, an analog of resveratrol.  The observed response in lipid metabolism genes is consistent with its known hypolipidemic properties, and the induction of mitochondrial genes is consistent with its demonstrated role in apoptosis in human cancer cell lines. Furthermore, our data show that pterostilbene has a significant effect on methionine metabolism, a previously unreported effect for this compound.
Keywords: Transcript profiling, S. cerevisiae, pterostilbene

overall_design: S. cerevisiae S288C cells at OD 0.2 were treated with either pterostilbene (Ptero) at IC50 concentration (70 uM), or solvent (0.25% DMSO), allowed to grow to OD 0.5, then harvested and frozen.  Three biological replicate samples were analyzed for each treatment.

first: Zhiqiang

last: Pan

middle: K

first: Tao

last: Xu

first: Qin

last: Feng

first: Scott

middle: R

last: Baerson

first: Stephen

middle: O

last: Duke

first: Agnes

middle: M

last: Rimando


title: Expression Profiling of the Developing Mouse Lung

city: Rochester

first: Soumyaroop

last: Bhattacharya

state: NY

address: 601 Elmwood Avenue Box 850

department: Pediatrics

institute: University of Rochester Medical Center

summary:
Mammalian lung development is a complex morphogenetic process, which initiates near mid-gestation and continues through early postnatal life. The lung arises as two lateral buds that emerge from the ventral foregut endoderm at ~ 9 days after fertilization (in mouse) and undergo numerous rounds of dichotomous branching to form the bronchial tree. This stage of development is referred to as the pseudoglandular phase, histologically characterized by loose mesenchyme surrounding undifferentiated epithelial tubes.
We have undertaken a comprehensive gene expression profiling of the entire process of murine lung development using oligonucleotide-based microarrays. Our data reveals the expression pattern of ~ 11,000 genes throughout the morphologic stages of lung development. Examination of the data confirms previously known patterns of expression for extracellular matrix genes and provides new information regarding relationships in temporal expression among groups of these genes. Large-scale cluster analysis reveals associations in the expression profile of specific genes with defined developmental processes.
Keywords: time course

overall_design: RNA was isolated from individual (E18, P1, P4, P7, P10, P14, P21, and adult) or pooled (E12, E14, E16) whole lungs using a modified guanidinium:phenol extraction method.Ten micrograms of total RNA was used to generate target cRNA for hybridization to Affymetrix Mu11K chipset subA and subB oligonucleotide microarrays. A single chip set was used for each time-point. Before target generation, individual RNA samples were pooled so that each target was derived from a minimum of three individual lungs.

first: Thomas

last: Mariani


title: Tobramycin Treatment of P. aeruginosa Biofilms Grown on CFBE41o- Cells

status: Public on Jun 10 2008

city: Hanover

first: George

last: O'Toole

state: NH

address: 505 Vail Building

department: Department of Microbiology and Immunology

institute: Dartmouth Medical School

summary:
We grew Pseudomonas aeruginosa biofilms on CFBE41o- human airway cells in culture, and we treated these biofilms with tobramycin.  Microarray analysis was performed to gain an understanding of the global transcriptional changes that occur during antibiotic treatment.
Keywords: Antibiotic Response

overall_design: We compared three untreated control samples with three tobramycin-treated samples.  Biofilms were grown on CFBE41o- cells in culture for 9 hours in MEM/0.4% arginine.  Replicate samples were then incubated in the presence or absence of 500 μg/mL tobramycin for 30 minutes.

first: Gregory

middle: G

last: Anderson

first: Sophie

last: Moreau-Marquis

first: Bruce

middle: A

last: Stanton


title: Adaptation of S. cerevisiae to fermentative conditions

status: Public on Jun 16 2008

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

summary:
The capacity of respiring cultures of Saccharomyces cerevisiae to instantaneously switch to fast alcoholic fermentation upon a transfer to anaerobic sugar-excess conditions is a key characteristic of Saccharomyces cerevisiae in many of its industrial applications. This transition was studied by exposing aerobic glucose-limited chemostat cultures grown at a low specific growth rate to two simultaneous perturbations: oxygen depletion and relief of glucose limitation. This shift towards fully fermentative conditions caused a massive transcriptional response, where one third of all genes within the genome were transcribed differentially. During the first 30 min, most of these changes were driven by relief from glucose limitation. An anaerobic induction response was only observed after the initial response to glucose excess. By comparing this study with public datasets representing dynamic and steady conditions, 14 up-regulated and 11 down-regulated genes were determined to be anaerobiosis specific and can therefore be use as “signature” transcripts for anaerobicity under dynamic as well as under steady state conditions
Keywords: global transcriptional time-dependent profile

overall_design: To invoke rapid and full induction of fermentative capacity, respiratory, aerobic glucose-limited chemostat cultures (D=0.1•h-1) were shifted to fully fermentative conditions by sudden depletion of oxygen and addition of glucose. The glucose was added two min after sparging the continuous culture with pure nitrogen, when the dissolved oxygen concentration had decreased from 75-80% to 10-15% of air saturation. Samples for micro-arrays were taken for each time point after the perturbation (5, 10, 30, 60 and 120 min) from two independently cultured replicates, while steady state data were taken from three independent chemostats. The complete dataset therefore comprised 13 samples.

first: J

last: van den Brink

middle: T

last: Pronk

first: P

last: Daran-Lapujade

middle: H

last: de Winde


title: Transcriptome profiling of slr-2, C.elegans C2H2 Zn-finger

status: Public on Mar 30 2008

city: Laramie

first: David

last: Fay

state: WY

address: 1000 E. University Dept. 3944

department: MOLB

institute: University of Wyoming

summary:
Our slr-2 dataset showed strong overrepresentation of genes previously identified in a serial analysis of gene expression (SAGE) intestinal library (McGhee et al., 2006) (p << 0.01); 812 genes were common to both data sets. Consistent with the deregulation of intestinal genes, we observed repression of several important metabolic pathways, including the TOR and insulin signaling networks, suggesting that slr-2(ku297) mutants experience metabolic stress. We also compared differentially regulated genes in slr-2 and lin-35 single mutants. Again, we saw a statistically significant overlap (p-value < 0.01); 261 genes were present in both data sets. Strikingly, > 75% of genes common both datasets showed expression changes in the same direction, although the common dataset contained an approximately equal mixture of up and downregulated genes. Furthermore, more than fifty genes common to the lin-35 and slr-2 datasets are known to have intestinal-associated functions. That some of these common intestinal genes were absent from the gut SAGE library could be due to differences in the developmental stage of the animals assayed (adults versus L1s) as well as experimental approaches (SAGE versus microarrays)
Keywords: analysis of staged mutant worms

overall_design:
We compared transcriptome profiles of slr-2 mutants and control N2 worms at L1 stage stage.
Each comparison was done in triplicate with independently grown and isolated animals.
Data for N2 L1 stage were from our previous experiments (Series GSE6547)
To achieve higher accuracy, RMA values were calculated on the basis of 21 microarray experiments (including data from Series GSE6547)

middle: S

first: Natalia

middle: V

last: Kirienko


title: P. aeruginosa PAO1 PA2663 mutant vs. wild type strain

status: Public on Feb 22 2008

city: College Station

first: Can

last: Attila

state: TX

address: 524 Jack E. Brown Bldg.

department: Chemical Eng

institute: Texas A&M University

summary:
P. aeruginosa PAO1 wild type and PA2663 mutant strains expression in biofilm cells relative to P. aeruginosa PAO1 wild type strain expression in biofilm cells. All samples cultured in LB with glass wool
Keywords: Biofilm

overall_design:
Strains: P. aeruginosa PAO1 wild-type, PA2663 mutant
Medium: LB
Biofilm grown on glass wool
Time: 24 h
Cell Type: biofilm

last: Wood


title: Effect of loss of CREB and CREM on kainate-induced gene expression in the hippocampus (MG-U74C).

status: Public on Apr 03 2008

city: Heidelberg

first: Jan

last: Rodriguez Parkitna

address: Im Neuenheimer Feld 581

country: Germany

institute: German Cancer Research Center

summary:
Ablation of the Creb1 gene in forebrain neurons was performed using the Cre/loxP system, with the recombinase expressed from the Camk2alfa promoter. Mice were crossed into the Crem KO background to prevent compensation of CREB loss by CREM overexpression.
Our goal was to analyze how loss of CREB will affect acitivity-regulated transcription induced by strong stimulation, i.e. kainate.
Keywords: Treatment x Genotype

overall_design: Animals were injected i.p. with either 20 mg/kg kainate or saline and sacrificed after one hour by cervical dislocation. Expression profiling was performed using total RNA isolated from the hippocampus.

last: Rodriguez-Parkitna

last: Lemberger


title: Effect of loss of CREB and CREM on kainate-induced gene expression in the hippocampus (MG-U74B).


title: Pairing competitive and topologically distinct regulatory modules enhances patterned gene expression

status: Public on Feb 01 2008

city: Haifa

first: Itai

last: Yanai

address: Technion City

department: Biology

country: Israel

institute: Technion - Israel Institute of Technology

summary:
Biological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time-series, whole-genome microarray analyses to systematically perturb and characterize components of a C. elegans lineage-specific transcriptional regulatory network.  These data are supported by select reporter gene analyses and comprehensive yeast-one-hybrid and promoter sequence analyses.  Based on these results we define and characterize two modules composed of muscle- and epidermal-specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross-inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage.  The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches.
Keywords: Gene expression response of RNAi knockdowns

overall_design:
To study the function of the C-lineage gene regulatory network we used RNAi to knockdown expression of the 13 TFs expressed early in the C lineage, and then used whole-genome microarrays to assess each perturbation for changes in mRNA abundance. RNAi has several advantages as a method to perturb gene function: it reduces both maternal and zygotic transcripts, thus avoiding maternal rescue effects; for essential genes it allows collection of fully affected progeny; and it enables comparison between perturbations. Measuring transcript levels on microarrays is also advantageous, because it is highly parallel, allowing direct comparisons between genes within each perturbation, and it is comprehensive, thus enabling discovery of regulated genes.  In addition, it allows direct comparison to previous efforts to characterize the pal-1 regulatory network (Baugh et al, 2005a; Baugh et al, 2005b).  To increase sensitivity and specificity, we used mex-3 mutant mothers whose progeny are primarily composed of C-like lineages (Draper et al, 1996).  In each case, RNA was collected from embryos two and three cell cycles after the initial zygotic expression of the targeted TF mRNA (Figure 1). We note that our sampling of only two time points might underestimate the magnitude of the observed effect of RNAi on mRNA levels, particularly if the expression level of the assayed mRNA peaks before or after the selected time points. The paralogous tbx-8 and tbx-9 genes, which were previously demonstrated to be functionally redundant (Pocock et al, 2004b; Baugh et al, 2005a), were simultaneously targeted by RNAi in order to assay a more penetrant effect on gene expression.

mex-3 (zu155) mutant worms (JJ518) were used in this experiment. RNAi was administered by soaking and RNA was collected from embryos, both as previously described (Baugh 2005; Baugh et al, 2005b). Cohorts of ten embryos were used for each RNA preparation, and half of the RNA was used for linear amplification. The Artus “ExpressArt mRNA Amplification Kit” was used for amplification. The manufacturer’s protocol was modified in round one with a ten-fold increase in concentration of primers A, B, and C and a four-fold decrease in cDNA synthesis reaction volumes. Microarray hybridization onto Affymetrix C. elegans GeneChip, scanning, data reduction and analysis were done as previously described (Baugh et al, 2003). Expression values were normalized using RMA (Bolstad et al, 2003).

first: Ryan

middle: L

last: Baugh

first: Jessica

middle: J

last: Smith

first: Casey

last: Roehrig

first: Shai

last: Shen-Orr

first: Julia

last: Claggett

first: Andrew

last: Hill

first: Donna

last: Slonim

first: Craig

middle: P

last: Hunter


title: Expression data from Drosophila melanogaster E2f/Rb genetic pathway manipulation

status: Public on Apr 27 2008

city: Durham

first: William

last: Ward

state: NC

address: 109 TW Alexander Dr

department: NHEERL

institute: United States EPA

summary:
We demonstrate that the cell cycle regulators, E2f /Dp and Rb, control the transcription of ribosomal proteins (RP) in Drosophila embryos.  Mutation of E2f1 or Dp and over expression of Rbf1 increase and reduce RP transcription, respectively.  Although E2f/Dp/Rb might exert this effect through a repressor complex, the regulatory regions of RP genes do not show an enrichment of canonical E2f binding sites.  In addition, E2f1, Dp and Rbf1 also regulate the expression of RACK1, a ribosomal component and a negative regulator of cell cycle progression.  These findings strengthen the coupling of cell cycle regulation to protein biosynthesis.
Keywords: genotype response

overall_design: Our study examines the genomic response of intact Drosophila embryos to the genetic manipulation of E2F/Rb pathway molecules.  For over-expression experiments, Arm-Gal4 stocks were crossed to UAS-gene stocks for the following genes: E2f1/Dp, E2f2/Dp, Rbf1, and E2f1336-805 (a dominant negative form of E2f1 lacking the DNA binding domain) [16].  The E2f17172 and E2f191 null alleles were balanced by TM3[Kr-GFP].  Dpa2 and Dpa4 null alleles were balanced by CyO[Kr-GFP].  14-16 hr E2f17172/E2f191 null mutant embryos were hand selected based on the absence of fluorescent Bolwig’s organs in Kr-GFP balanced stocks using a Zeiss stereomicroscope equipped with epifluorescence.  E2f17172/E2f191 and Dpa2/Dpa4 null mutant embryos (8-10h) were selected using the COPASTM SELECT system for automated sorting of multi-cellular organisms. Eighteen RNA samples, two from each cross, were obtained from approximately 200 to 700 embryos per sample.  For each cross, 2 h embryo collections were aged for 8 to 14 h at 25oC, quick frozen in an ethanol/dry ice mixture, and stored at -80 oC.  The RNA was prepared for microarray analysis in accordance with Affymetrix instructions.

first: Harry

last: Hurd

first: Robert

last: Duronio


title: Plasticity of the systemic inflammatory response to acute infection during critical illness

status: Public on Feb 12 2008

city: St. Louis

first: J

middle: Perren

last: Cobb

state: MO

address: Campus Box 8109, 660 South Euclid Avenue

institute: Washington University in St. Louis

summary:
Diagnosis of acute infection in the critically ill remains a challenge. Changes in physiologic parameters and existing molecular diagnostics are not specific and microbiologic confirmation of infection can take days. As cellular first responders, preclinical studies indicate that circulating leukocytes are activated in response to bacterial infection, manifesting infection-specific transcriptional programs.  We hypothesized that circulating leukocyte transcriptional profiles can be used to diagnose infection and monitor response to therapy in the critically ill.
Keywords: ventilator associated pneumonia, predictor, microarray, timecourse, human, murine

overall_design: A translational research approach was employed.  Fifteen mice underwent intratracheal (i.t.) injections of live P. aeruginosa, P. aeruginosa endotoxin, live S. pneumoniae, or normal saline. At 24 hours after injury, GeneChip microarray analysis of circulating buffy coat RNA identified 219 genes that distinguished between the pulmonary insults and differences in 7-day mortality. Similarly, buffy coat microarray expression profiles were generated from 20 mechanically ventilated patients every two days for up to three weeks; 11 of these patients developed pneumonia.  Principal components analysis of the expression levels of the human orthologs for the mouse genes resulted in gene expression trajectories that describe the cohort of 11 patients as they develop, are treated for, and convalesce from ventilator-associated pneumonia (VAP).  When the VAP microarray profiles were analyzed independently, 85 genes were identified with consistent changes in abundance during the seven days bracketing the diagnosis of VAP.  The estimated prediction accuracy in this small patient cohort was 91%.  The abundance of select transcripts was validated using real-time PCR. Principal components analysis of the average microarray-measured abundances of these genes defined a general trajectory (riboleukogram) for the onset of acute infection in this patient population. Individual riboleukograms demonstrated that the onset and course of infection are specific for each individual, but as patients recover from infection the riboleukograms converge.  The gene ontologies of the co-expression networks indicate that leukocyte chemokines and granule maturation are especially informative for pneumonia diagnostics.

first: Jonathan

middle: E

last: McDunn

first: Kareem

middle: D

last: Husain

first: Ashoka

last: Polpitiya

first: Anton

last: Burykin

first: Jianhua

last: Rua

first: Qing

last: Li

last: Schierding

last: Lin

last: Dixon

first: Weixiong

last: Zhang

last: Coopersmith

first: Michael

middle: W

last: Dunne

first: Bijoy

last: Ghosh

first: Gary

last: Stormo

first: Perren


title: Efficient generation of continuous cell lines from Drosophila embryos by expression of oncogenic Ras

status: Public on Mar 12 2008

city: Columbus

first: Amanda

last: Simcox

state: OH

address: 484 W. 12th Avenue

department: Molecular Genetics

institute: The Ohio State University

summary:
Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene, RasV12 (a constitutively activated form of Ras), profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The cells have undergone more than 90 population doublings and therefore constitute continuous cell lines. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was silenced. We successfully created several cell lines and found these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful mechanism to promote proliferation in Drosophila primary culture and serves as an efficient means to generate continuous cell lines. This offers an opportunity to create cell lines of specific genotypes and potentially of a given cell type.
Keywords: Cell type comparison

overall_design: Cells from a RasV12 line at passage 12, CL8 cell line and L2 cell line were grown to 70% confluence and RNA was extracted (Quiagen RNeasy). Three samples of RasV12, four samples of CL8 and one sample of L2 cell line, derived from independent T-flasks were processed. Targets were generated and hybridized to DrosGenome 1 Affymetrix gene chips using standard procedures (Affymetrix.com). All analyses were done using the Bioconductor suite of packages (www.bioconductor.org) in R (www.r-project.org). Expression values were calculated using the GC Robust Multiarray Average (GCRMA) method and statistical tests for differential expression were done using the 'limma' package. Clustering was performed on the top 20% of genes ranked by standard deviation, using 1-correlation as the distance measure and an average linkage. For class discrimination analysis, the 'pamr' package was used.

first: Ting

last: Chen

first: Jon

last: Butchar

first: Steven

last: Justiniano


title: Microarray Analysis of Toxicogenomic Effects of Ortho-Phenylphenol on Pseudomonas aeruginosa

status: Public on Apr 01 2008

city: ft. meade

first: Hyeung-Jin

last: Jang

state: MD

address: 701 Mapes Road

department: OPP/BEAD

institute: USEPA

summary:
In the present study, we employed Affymetrix Pseudomonas aeruginosa GeneChip arrays to investigate the dynamics of global gene expression profiles during the cellular response of Pseudomonas aeruginosa to ortho-phenylphenol, which involved initial growth inhibition and metabolism.
Keywords: Time course

overall_design: We conducted four independent microarray experiments (biological replicates) in the absence (control) and the presence (experimental) of ortho-phenylphenol. We calculated fold change as the ratio between the signal averages of four untreated (control) and four ortho-phenylphenol-treated (experimental) cultures for 0, 20 and 60 min exposures.

first: Chantal

last: Nde

first: Freshteh

last: Toghrol

first: William

last: Bentley


title: Investigations of the effects of subMIC antibiotics on the P. aeruginosa transcriptome

status: Public on Feb 28 2008

city: Kgs. Lyngby

first: Mette

middle: E.

last: Skindersoe

address: Matematiktorvet 1. build. 301

country: Denmark

institute: Technical University of Denmark

summary:
Antibiotics in low concentrations may have an array of effects. We have exposed growing P. aeruginosa cells to different classes of antibiotics in order to investigate this hypothesis.
We have used DNA microarrays to investigate the effect of low concentrations of antibiotics (azithromycin, ceftazidime and ciprofloxacin) on the gene expression of P. aeruginosa aiming to elucidate general trends.
Keywords: Treatment with antibiotics

overall_design: Exponentially growing P. aeruginosa cells were exposed to antibiotics in concentrations not affecting growth, grown for 2 hours and harvested. Untreated cell were grown likewise for reference.

first: M

last: Alhede

first: R

last: Phipps

first: L

last: Yang

first: P

last: Jensen

first: T

middle: B

last: Rasmussen

last: Bjarnsholt

last: Tolker-Nielsen

first: N

last: Høiby

last: Givskov


title: H. pylori induces an antimicrobial host response in rhesus macaques in a Cag Pathogenicity Island-Dependent Manner

city: Davis

first: Michael

last: Hornsby

state: CA

address: County Road 98 and Hutchison Drive

institute: University of California, Davis

summary:
We used the rhesus macaque model to study the effects of the cag pathogenicity island (cag PAI) on the H. pylori host-pathogen interaction. Specific pathogen free (SPF) monkeys with no prior exposure to H. pylori were experimentally challenged with wild type (WT) H. pylori strain J166 (N=4) or its cag PAI isogenic knockout (KO, N=4). Animals underwent endoscopy before and 1, 4, 8, and 13 wks after challenge.  Gastric biopsies were collected for quantitative culture, histopathology, and host gene expression. Quantitative cultures showed that all experimentally challenged animals were infected with WT H. pylori or its isogenic cag PAI KO. Histopathology demonstrated that inflammation and expansion of the lamina propria were attenuated in animals infected with KO compared to WT.  Microarray analysis was performed on challenged animals before and 1 and 13 wks after challenge, and on unchallenged control animals (N=4). Of the 119 up-regulated genes in the WT-infected animals, several encode innate antimicrobial
effector proteins, including elafin, siderocalin, DMBT, DUOX2, and several novel paralogues of human defensin-2. Quantitative RT-PCR analysis showed that high level induction of each of these genes was dependent upon the presence of the cag PAI.  Immunohistochemistry  confirmed increased defensin epithelial cell staining in animals challenged with WT H. pylori compared to either KO-challenged or uninfected control animals. We propose that one function of the cag PAI is to induce an antimicrobial host response that serves to increase the competitive advantage of H. pylori in the gastric niche.
Keywords: Transcript profiling , Wild type H. pylori, cag-PAI KO H. pylori

overall_design: Twelve male and female rhesus macaques (Macaca mulatta) housed at the California National Primate Research Center (CNPRC) were hand raised by nursery staff to obtain Specific Pathogen (Helicobacter pylori) Free (SPF) experimental animals.  At six months of age the animals were documented to be uninfected with H. pylori by serology, histology, and cultures of gastric biopsies using methods reported previously (Solnick et al., 1999).  Two experimental groups of four SPF animals were challenged with either wild type (WT) H. pylori J166 or an isogenic PAI knockout (KO).  A third group of four SPF animals served as uninoculated controls. Monkeys were housed indoors in separate cages throughout the duration of the experiment.  All experiments were approved by the Research Advisory Committee of the CNPRC and the Institutional Animal Care and Use Committee at the University of California.Antrum and corpus stomach biopsies from rhesus macaques infected with wild type Helicobacter pylori J166, isogenic cag-PAI knockout strain, or mock infected were pooled.  Biopsies were taken prior to bacterial inoculation and at 1 and 13 weeks post infection.  Control biopsies were taken according to the same time line.  Four animals were used for each experimental condition.

first: Jennifer

last: Juff

first: Robert

last: Kays

first: Don

middle: R

last: Canfield

first: Charles

last: Bevins

first: Jay

last: Solnick


title: Expression data of myofibroblast isolated from patients with idiopathic pulmonary fibrosis

status: Public on Jan 17 2008

city: Fukushima

first: Yoshimi

last: Homma

state: Fukushima

address: 1 Hikarigaoka

department: Department of Biomolecular Science

country: Japan

institute: Fukushima Medical University

summary:
Idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease of unknown etiology, is characterized by the expansion of myofibroblasts and abnormal deposition of extracellular matrix in the lung parenchyma.  To elucidate the molecular mechanisms that lead to IPF, we analyzed myofibroblasts established from patients with IPF by oligonucleotide microarrays.  Gene expression profiles revealed a novel pathophysiologic function of myofibroblasts as a generator of reactive oxygen species, and a self-defense mechanism against oxidative stress of their own generating.
Keywords: cell type comparison, disease state analysis

overall_design: We isolated two myofibroblast cell culture from patients with idiopathic pulmonary fibrosis.  Embryonic pulmonary fibroblast was used for the reference.

first: Y

last: Kabuyama


title: Gene expression response to the antifungal compound sampangine

status: Public on Jan 09 2008

first: Ameeta

last: Agarwal

summary:
Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus.  In the present study, transcriptional profiling experiments coupled with the analysis of mutants were performed in an effort to elucidate its mechanism of action.  Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low oxygen conditions.  Several additional lines of evidence obtained suggest that these responses could involve effects on heme.   First, the hem1 deletion mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells.  In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine.  Furthermore, spectral analysis of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells.  Transcriptional profiling experiments were also performed in C. albicans to investigate the response of a pathogenic fungal species to sampangine.  Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles suggestive of heme-related defects.  Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.

This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Saccharomyces cerevisiae
Candida albicans

overall_design: Refer to individual Series


title: Expression profile analysis of menin1 mutants

status: Public on Jan 28 2008

city: Bethesda

first: Brian

last: Oliver

address: 50 South Drive

department: LBG

institute: NIDDK, NIH

summary:
Expression analysis of Drosophila embryos with mnn overexpression, and adults with mnn deletion.
Keywords: Expression analysis

overall_design: Described in Cerrato et al, Dev Biol. 2006 Oct 1;298(1):59-70.

first: David

middle: M

last: Sturgill

first: Aniello

last: Cerrato

first: Sonia

last: Santa Anna

last: Parisi

first: Scott

last: Eastman

last: Doctolero

first: Settara

last: Chandrasekharappa

first: Stephen

last: Marx

first: Francis

last: Collins


title: CD4+ T Cell Decline is Predicted by Differential Expression of Genes in HIV seropositive patients

status: Public on Mar 25 2008

city: Rockville

first: Maryanne

last: Vahey

address: 1600 East Gude Drive

institute: Walter Reed Army Institute of Research

summary:
Patterns of expressed genes examined in cryopreserved peripheral blood mononuclear cells (PBMCs) of seropositive persons electing to stop antiretroviral therapy in the AIDS Clinical Trials Group Study A 5170, were scrutinized to identify markers capable of predicting the likelihood of CD4+ T cell depletion after cessation of ART.
Two cohorts were matched for clinical characteristics in a study of the effects of therapy interruption (TI) in ART-treated patients with immunological preservation. Disease progression was determined by CD4+ T cell count decline 24 weeks after TI.Gene Set Enrichment Analysis (GSEA) identified a set of genes in the Ras signaling pathway, associated with the downregulation of apoptosis, as significantly upregulated in the good outcome group at cessation of ART.
Keywords: Disease state analysis

overall_design: There are 96 samples in this study. 24 patients with good outcome results and 24 matched patients with poor outcome results. Each patient has two time points: week0 and week 24


title: Transcript-specific translational regulation in the unfolded protein response of Saccharomyces cerevisiae

status: Public on Jan 07 2008

city: Norwich

first: Colin

last: Hanfrey

state: Norfolk

address: Norwich Research Park

country: United Kingdom

institute: Institute of Food Research

summary:
The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) causes stress and induces the unfolded protein response (UPR) which is characterised in part by the transcriptional induction of genes involved in assisting protein folding. Translational responses to ER stress have been less well described and here we report on a genome-wide analysis of translational regulation in the response to the ER stress-inducing agent dithiothreitol (DTT) in Saccharomyces cerevisiae. Although the observed polysome profiles were similar under control and ER stress conditions microarray analysis identified transcipt-specific translational regulation. Genes with functions in ribosomal biogenesis and assembly were translationally repressed under ER stress. In contrast mRNAs for known UPR genes, including the UPR transcription factor HAC1, the ER-oxidoreductase ERO1 and the ER-associated protein degradation (ERAD) gene DER1 were enriched in polysomal fractions under ER stress conditions. In addition, we show that splicing of HAC1 mRNA is required for efficient ribosomal loading and that Gcn2p is required for normal HAC1 splicing, so shedding light on the role of this protein kinase in the UPR pathway.
Keywords: stress response, translational analysis

overall_design: Polyribosomes were extracted from S. cerevisiae cells treated with 2 mM DTT or water (control), and fractionated according to ribosome loading. Following RNA purification from these fractions, for each sub-polysomal and polysomal RNA sample, fractions from three independent extracts per treatment (DTT/control) were pooled.

first: Tom

last: Payne

first: Amy

last: Bishop

first: Anthony

last: Michael

first: Simon

last: Avery

last: Archer


title: Courtship-exposed male flies

status: Public on Jan 02 2008

first: Ginger

last: Carney

address: 3258 TAMU

summary:
I hypothesized that social interactions, such as those involved in reproductive behaviors, would lead to immediate and assayable changes in gene expression that may have important effects on individual reproductive success and fitness through alterations in physiology or via short-term or long-term changes in nervous system function.
Keywords: exposed compared to unexposed treatment

overall_design: Four-day males were exposed or mock-exposed to a virgin female fly for 5 min. Exposed males that courted as well as mock-exposed males were collected for RNA extraction. Three samples for each treatment were hybridized to Affymetrix Drosophila Genome arrays for a total of 6 arrays.


title: Whole body transcriptional response of female fruitflies to juvenile hormone

status: Public on Dec 31 2007

city: Guelph

first: Andreas

last: Heyland

state: ON

address: 50 Stone Rd East

department: Integrative Biology

country: Canada

institute: University of Guelph

summary:
Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, and reproduction in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the expression of antimicrobial peptides (AMPs) in Drosophila melanogaster. In S2* cells challenged with bacterial peptidoglycans, 20E induces promoter activity and expression of AMPs in a dose-dependent manner, while JH III and its synthetic analogs (JHa) methoprene and pyriproxyfen abolish this 20E-dependent response. Using microarrays and GFP reporter gene assays in adult flies, we confirm that JH is a hormonal immuno-suppressor in vivo. When silencing both partners of the ecdysone receptor (EcR ) / ultraspiracle (USP) heterodimer with RNAi in S2* cells, 20E fails to activate Diptericin (Dpt) expression, suggesting that 20E regulates expression of this gene through EcR / USP signaling. In contrast, silencing methoprene-tolerant (MET), a candidate JH receptor, does not impair the immuno-suppressive action of JH III and JHa, indicating that in this context MET does not function as a JH receptor. Our results suggest that the balance of 20E and JH is a major determinant of immune homeostasis in insects.
Keywords: Topical hormone treatment of whole flies

overall_design: To examine the transcriptional response of y, w flies to treatment with exogenous JH, we performed a microarray experiment on females treated with JH or solvent (control). Flies were grown on regular yeast diet, switched to no-yeast food within one hour of eclosion, and yeast-starved for 5 days posteclosion to lower their endogenous JH titer and to synchronize their physiology (cf. Tu and Tatar, 2003; Gershman et al., 2007). Subsequently, we anesthetized flies on ice and topically treated them with 0.1 ul of 187 mM JH III in acetone or with 0.1 ul 100% acetone (control) using a 1 ul Hamilton syringe with a repeating dispenser. 12 hours after hormone administration, samples were snap-frozen in liquid nitrogen and stored at -80°C. RNA was isolated from samples (2 JH samples, 2 control samples, each with 30 females) by lysis, as described in Gershman et al. (2007). cDNA products were hybridized at the Brown University Genomics Core Facility to Affymetrix GeneChip Drosophila_1 Genome Arrays (2 replicate chips per treatment). The dataset consisted of 14,009 probe sets, with 6,142 probe sets annotated. Expression data were analyzed for significant over- or underrepresentation of gene ontology (GO) terms with the web application FatiGO (Al-Shahrour et al., 2004), using a two-fold change criterion. To test whether JH treatment significantly suppresses expression of AMPs we used t-tests implemented in JMP IN 5.1.

first: Thomas

last: Flatt

first: Florentina

last: Rus

first: Ermelinda

last: Porpiglia

first: Chris

last: Sherlock

first: Rochele

last: Yamamoto

first: Alina

last: Garbuzov

first: Subba

last: Palli

first: Marc

last: Tatar

first: Neal

last: Silverman


title: mef2 activity levels differentially affect gene expression during Drosophila muscle development

city: Cardiff

first: Michael

middle: Victor

last: Taylor

state: S. Glam

address: Main Building, Park Place

department: School of Biosciences

institute: Cardiff University

summary:
The conserved Mef2 transcription factor is a major regulator of gene expression and differentiation.  Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis.  However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression.  The aim of this project was to investigate whether there are genes with different mef2-requirements for their expression during muscle differentiation in vivo during the development of Drosophila melanogaster.
Keywords: allelic series comparison

overall_design: We used microarrays in conjunction with a mef2 allelic series to determine the gene expression profile in developing embryos at five different levels of mef2 activity.  The allelic series extends from a null mutation, mef222.21, through to the control, via three hypomorphic alleles, mef2113, mef2424, and mef265.  The series corresponds to different levels of Mef2 in the following order: control > 65 > 424 > 113 > 22.21.  The control stock for the microarray analysis was dp cn a px sp, the stock used for the mutagenesis that produced mef265, mef2424 and mef2113. For the microarrays, 30 minute collections of control and mutant embryos for each mef2 allele were individually staged and pools of 150 embryos were then processed at mid stage 13.  This corresponds to the early differentiation phase of muscle development and the expression of multiple muscle sarcomeric protein genes.  Quadruplicate samples were assayed using Affymetrix® Genechips by the Flychip Drosophila microarray resource.

first: Stuart

last: Elgar

first: Jun

last: Han


title: Gene expression response to the antifungal compound sampangine in S. cerevisiae

city: University

first: Ameeta

last: Agarwal

address: NCNPR, Room 2049

institute: University of Mississippi

summary:
Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus.  In the present study, transcriptional profiling experiments coupled with the analysis of mutants were performed in an effort to elucidate its mechanism of action.  Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low oxygen conditions.  Several additional lines of evidence obtained suggest that these responses could involve effects on heme.   First, the hem1 deletion mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells.  In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine.  Furthermore, spectral analysis of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells.  Transcriptional profiling experiments were also performed in C. albicans to investigate the response of a pathogenic fungal species to sampangine.  Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles suggestive of heme-related defects.  Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.
Keywords: antifungal compound, transcriptional profiling, S. cerevisiae

overall_design: S. cerevisiae S288C cells at OD 0.2 were treated with either sampangine (SMP) at IC50 concentration (1.17 ug/ml), or solvent (0.25% DMSO), allowed to grow to OD 0.5, then harvested and frozen.  Three biological replicate samples were analyzed for each treatment.

middle: K

first: Tao

last: Xu

first: Melissa

last: Jacob

first: Qin

last: Feng

middle: C

last: Lorenz

first: Larry

middle: A

last: Walker

first: Alice

last: Clark


title: Transcriptional response to MnSOD over-expression in Drosophila melanogaster

status: Public on Jan 01 2008

city: Los Angeles

first: Diana

last: Abdueva

address: 443 S San Pedro St, STE 403

institute: USC

summary:
Profiled the transcriptional response to over-expression Manganese-SOD (MnSOD) in adult Drosophila.  A doxycycline-regulated system was employed to induce MnSOD over-expression,  resulting in mean and maximal lifespan increases of ~ 20%. Examination of the genome-wide response to this lifespan intervention provided key insights into the molecular basis of aging.
Keywords: genetic modification; MnSOD over-expression

overall_design: 20 Affymetrix DrosGenome1 arrays were employed with four replicates for each of five conditions. Cohorts of treated and untreated MnSOD transgenic flies were sampled at the same chronological age (~50% survival of -DOX flies, day 73) as well as, at the same physiological age (~50% survival for +DOX and -DOX flies, day 83 and day 73, respectively). The effect of DOX was controlled for by sampling control flies treated with or without DOX at the same chronological age (~50% survival of -DOX flies, day 78).

first: Christina

last: Curtis

first: Gary

middle: N

last: Landis

first: Donna

last: Folk

first: Nancy

last: Wehr

first: Nicholas

last: Hoe

first: Morris

last: Waskar

first: Dmitriy

last: Skvortsov

first: Daniel

last: Ford

first: Allan

last: Luu

first: Ananth

last: Badrinath

first: Rodney

last: Levine

first: Timothy

last: Bradley

first: Simon

last: Tavaré

first: John

last: Tower


title: Overlapped gene expression alterations in heavy metals- and carcinogen-exposed human hepatoma (Hep G2) cells

city: Sapporo

first: Akinobu

last: Ito

address: North-13, West-8, Kitaku

department: Graduate School of Engineering

institute: Hokkaido University

summary:
From the result of comparative the gene expression analyses of human hepatoma cell line, HepG2 following exposures of three heavy metals; arsenic, cadmium and nickel and three carcinogens; N-dimethylnitrosoamine (DMN), 12-O-tetradecanoylphorbol-13-acetate (TPA) and tetrachloroethylene (TCE),  31-55% of the genes altered by As, Cd and Ni exposure were overlapped with those by three model carcinogen exposures in our experiments.  In particular, three heavy metals shared certain characteristics with TPA and TCE in remarkable up-regulations of the genes associated with progression of cell cycle, which might play a central role in heavy metal carcinogenesis.  In addition, this characteristic of gene expressions alteration was counteracted by intracellular accumulation of vitamine C in As-exposed cells but not in Cd- and Ni-exposed cells.  These results suggest that the cell proliferative responses are caused by reactive oxygen species mainly in As exposure, while other mechanisms would be involved in these responses in Cd and Ni exposures.
Keywords: other

overall_design: In this study, we examined the gene expression alteration in human hepatoma cell line, HepG2 following exposures of three heavy metals; arsenic, cadmium and nickel and three carcinogens; N-dimethylnitrosoamine (DMN), 12-O-tetradecanoylphorbol-13-acetate (TPA) and tetrachloroethylene (TCE) using DNA microarray with 8795 human genes. Furthermore, we also performed the DNA microarray analyses for the heavy metal exposed-cells that were loaded with vitamine C beforehand to examine the effects of antioxidant molecule to heavy metal exposures.

first: Koji

last: Kawata

first: Ryuhei

last: Shimazaki

first: Satoshi

last: Okabe


title: PAO1-psrA::Tn expression compared to PAO1 in LB

status: Public on Feb 14 2008

city: Honolulu

first: Tung

last: Hoang

state: HI

address: 1800 East-West Road

department: Microbiology

institute: University of Hawaii at Manoa

summary:
b-Oxidative enzymes for fatty acid degradation (Fad) of long-chain fatty acid (LCFA), a component of lung surfactant phosphatidylcholine, are induced in vivo during lung infection in cystic fibrosis patients, which could contribute to nutrient acquisition and pathogenesis of Pseudomonas aeruginosa. In addition, fatty acid biosynthesis (Fab) is essential for the syntheses of two virulence controlling acylated-homoserine-lactone molecules in this organism. We mapped the promoter regions of the fadBA5-operon (PA3014 and PA3013) and a fadE homologue (PA2815) involved in Fad and the fabAB-operon involved in Fab. Focusing on the transposon mutagenesis of strain PAO1 carrying the PfadBA5-lacZ fusion, we identified a regulator for the fadBA5-operon to be PsrA (PA3006). Transcriptome analysis of the DpsrA mutant indicates its importance in regulating b-oxidative enzymes, which confirms a previous proteomic study. We further showed that induction of the fadBA-operon responds to LCFA signals, and this induction requires the presence of PsrA, suggesting that PsrA binds to LCFA to derepress fadBA5. Electrophoresis mobility shift assay indicate specific binding of PsrA to the fadBA5-promoter region. This binding is disrupted by specific LCFA (C18:1D9, C16:0, and to a lesser extent C14:0), but not by the first intermediate of b-oxidation, acyl-CoA. We proposed that PsrA is a Fad-regulator that binds and responds to LCFA signals in Pseudomonas aeruginosa.
Keywords: Pseudomonas aeruginosa, wild-type PAO1 compared to PAO1-psrA::Tn

overall_design: PAO1 and PAO1-psrA::Tn cultures grown in LB and cells were harvested at mid-log phase. Total RNA was isolated from both samples, and used for cDNA synthesis. And then, the cDNA for both samples were fragmented and labeled. The cDNA of PAO1 was used for 2 GeneChips, and PAO1-psrA::Tn cDNA was used for three GeneChips.

first: Yun

last: Kang

first: David

middle: T

last: Nguyen

first: Mike

middle: S

last: Son


title: Classification of ovarian tumor samples

city: Montreal

first: Jaroslav

middle: Petr

last: Novak

state: Quebec

address: 740, Dr Penfield Avenue

department: Microarray Platform

institute: McGill University and Genome Quebec Innovation Centre

summary:
We have previously described differences in the expression profiles of tumors by comparing tumors of two known classes: low malignant potential versus highly malignant (invasive). This report presents an effort to discover unknown classes within a large heterogeneous set of ovarian tumors, using unsupervised learning approaches.
We were able to define four classes in a set of 74 ovarian cancer tumors. Groups identified as A1 and A2 were closely related and correlated with the “invasive” pathology class while the group identified as B2 was correlated with low malignant potential tumors; group B1 consisted of a mixture of low potential and invasive samples. We selected characteristic candidate genes, which were validated by quantitative-PCR and by comparison to other published studies.
Keywords: Unsupervised classification

overall_design: We used unsupervised classification methods to identify unknown classes in a set of 74 cancer samples

first: Veronique

last: Ouellet

first: Cecile

last: Le Page

first: Kristina

last: Martinu

first: Andre

last: Ponton

first: Dimcho

last: Bachvarov

first: Abdelali

last: Filali-Mouhim

first: Diane

last: Provencher

first: Patricia

last: Tonin

first: Thomas

middle: J

last: Hudson

first: Anne-Marie

last: Mes-Masson


title: Transcript and Proteomic Analyses of Wild-Type and GPA2 Mutant Saccharomyces cerevisiae Strains

status: Public on Dec 21 2007

city: Washington

first: Dzung

last: Thach

state: DC

address: 4555 Overlook Ave SW

department: Center for Biomolecular Science and Engineering

institute: Naval Research Laboratory

summary:
In response to limited nitrogen and abundant carbon sources, diploid Saccharomyces cerevisiae strains undergo a filamentous transition in cell growth as part of pseudohyphal differentiation. Use of the disaccharide maltose as the principal carbon source, in contrast to the preferred nutrient monosaccharide glucose, has been shown to induce a hyper-filamentous growth phenotype in a strain deficient for GPA2 which codes for a Galpha protein component that interacts with the glucose-sensing receptor Gpr1p to regulate filamentous growth. In this report, we compare the global transcript and proteomic profiles of wild-type and Gpa2p deficient diploid yeast strains grown on both rich and nitrogen starved maltose media. We find that deletion of GPA2 results in significantly different transcript and protein profiles when switching from rich to nitrogen starvation media.  The results are discussed with a focus on the genes associated with carbon utilization, or regulation thereof, and a model for the contribution of carbon sensing/metabolism-based signal transduction to pseudohyphal differentiation is proposed.
Keywords: Saccharomyces cerevisiae, nitrogen starvation, maltose, pseudohyphal differentiation, yeast, expression profiling

overall_design: For transcriptome profiling, there were 12 Affymetrix Yeast S98 microarrays total.  There were four conditions: wildtype MLY61 and gpa2 deletion mutant MLY132 grown in YPM media or transferred to low nitrogen media SLAM.  Each condition was done in triplicate, starting with triplicate yeast cultures.  Four conditions done in triplicates resulted in 12 samples that went onto 12 microarrays.

first: Igor

middle: L

last: Medintz

first: Gary

last: Vora

first: Amir

last: Rahbar


title: D.m.Third_instar_larval_midgut_normal

status: Public on Dec 18 2007

city: Urbana

first: Hongmei

last: LI

state: IL

address: 505 S. Goodwin Ave

department: Entomology

institute: University of Illinois at Urbana-Champaign

summary:
Oligoarray analysis was used to determine the number and nature of genes expressed in third-instar Drosophila melanogaster larval midguts.  The majority of transcripts were associated with protein synthesis and metabolism.  Serine proteases were the main proteolytic enzymes detected.
Keywords: stress response

overall_design: Three replicates for each sample.  We used Affy chip Drosophila 1.

last: Li

first: Barry

last: Pittendrigh

first: Larry

last: Murdock


title: CA/AA time course of y w; tim01

status: Public on Dec 09 2007

city: Southampton

first: Herman

last: Wijnen

state: Hampshire

address: Life Sciences Building 85 - Highfield Campus

department: Centre for Biological Sciences

institute: University of Southampton

summary:
Circadian clocks are temporally aligned to the environment via signals, or Zeitgebers, such as daily light and temperature cycles, food availability, and social behavior. In this study, we show that genome-wide expression profiles from temperature-entrained flies show a dramatic difference in the presence or absence of a thermocycle. Whereas transcription appears to be modified globally by changes in temperature, there is a specific set of transcripts that continue to oscillate in constant conditions following temperature entrainment. These transcripts show a significant overlap with a previously defined set of transcripts oscillating in response to a photocycle. Further, these overlapping transcripts maintain the same mutual phase relationships after entrainment by temperature or light. Comparison of the collective temperature- and light-entrained circadian phases indicates that natural environmental light and temperature cycles cooperatively entrain the circadian clock. These findings suggest that a single transcriptional clock in the adult fly head is able to integrate information from both light and temperature.
Keywords: circadian time course

overall_design:
y w; tim01 flies that had been reared in constant darkness initially at 25 C and later in a 12-hr 18 C/ 12-hr 25 C thermocycle for more than 4 days were harvested every four hours during an additional 18 C/25 C thermo cycle (also indicated as a cold/ambient or CA cycle) or an additional day at constant 25 C (also indicated as ambient/ambient or AA) . Relative to time CA0 as the time of the onset of the 18 C cryophase or AA0 as the time of the onset of the subjective cryophase during constant 25 C, samples were collected in a CA2-
CA6-CA10-CA14-CA18-CA22 and AA2-AA6-AA10-AA14-AA18-AA22 schedule. Heads
were isolated by breaking up frozen flies and passing them through a set of
sieves. RNA was prepared using guanidine-thiocyanate extraction followed by
purification over a CsCl gradient. Additional purification of the RNA samples was
achieved by applying them to Rneasy columns (Qiagen). Biotin-labeled cRNA
probe was generated from 25 μg of purified RNA and hybridized as described
previously (Wijnen H, Naef F, and Young MW, Methods Enzymol. 2005; 393: 341-365).
For more information see also http://biorhythm.rockefeller.edu

first: Catharine

last: Boothroyd

first: Felix

last: Naef

first: Lino

last: Saez

first: Michael

last: Young


title: Expression data from four cconsecutive stages of human B cell development

city: Munich

first: Reinhard

last: Hoffmann

address: Trogerstr. 30

institute: Institute for medical microbiology

summary:
Cells from four develppmental stages were purified by FACS from human bone marrow samples
Keywords: cell development

overall_design: Cells were sorted as follows: PB1: CD34+VpreB+ PB2: VpreB-CD34+ LAR: kappa/lambda-VpreB-large SMA: kappa/lambda-VpreB-small. All cells were CD19+

first: Fritz

last: Melchers


title: AA1/AA2 time course of cn bw

overall_design:
cn bw flies that had been reared in constant darkness initially at 25 C and later in a 12-hr 18 C/ 12-hr 25 C thermocycle for more than 4 days were harvested every four hours during an additional two days at constant 25 C (also indicated as ambient/ambient or AA days 1 and 2) . Relative to time AA0 as the time of the onset of the subjective cryophase during constant 25 C, samples were collected in a AA2-AA6-AA10-AA14-AA18-AA22-AA26-AA30-AA34-AA38-AA42-AA46 schedule. Heads
were isolated by breaking up frozen flies and passing them through a set of
sieves. RNA was prepared using guanidine-thiocyanate extraction followed by
purification over a CsCl gradient. Additional purification of the RNA samples was
achieved by applying them to Rneasy columns (Qiagen). Biotin-labeled cRNA
probe was generated from 25 μg of purified RNA and hybridized as described
previously (Wijnen H, Naef F, and Young MW, Methods Enzymol. 2005; 393: 341-365).
For more information see also http://biorhythm.rockefeller.edu


title: CA/AA time course of y w

overall_design:
y w flies that had been reared in constant darkness initially at 25 C and later in a 12-hr 18 C/ 12-hr 25 C thermocycle for more than 4 days were harvested every four hours during an additional 18 C/25 C thermo cycle (also indicated as a cold/ambient or CA cycle) or an additional day at constant 25 C (also indicated as ambient/ambient or AA) . Relative to time CA0 as the time of the onset of the 18 C cryophase or AA0 as the time of the onset of the subjective cryophase during constant 25 C, samples were collected in a CA2- CA6-CA10-CA14-CA18-CA22 and AA2-AA6-AA10-AA14-AA18-AA22 schedule. Heads were isolated by breaking up frozen flies and passing them through a set of sieves. RNA was prepared using guanidine-thiocyanate extraction followed by purification over a CsCl gradient. Additional purification of the RNA samples was achieved by applying them to Rneasy columns (Qiagen). Biotin-labeled cRNA probe was generated from 25 μg of purified RNA and hybridized as described previously (Wijnen H, Naef F, and Young MW, Methods Enzymol. 2005; 393: 341-365).
For more information see also http://biorhythm.rockefeller.edu


title: second CA/AA time course of y w

overall_design:
y w flies that had been reared in constant darkness initially at 25 C and later in a 12-hr 18 C/ 12-hr 25 C thermocycle for more than 4 days were harvested every four hours during an additional 18 C/25 C thermo cycle (also indicated as a cold/ambient or CA cycle) or an additional day at constant 25 C (also indicated as ambient/ambient or AA) . Relative to time CA0 as the time of the onset of the 18 C cryophase or AA0 as the time of the onset of the subjective cryophase during constant 25 C, samples were collected in a CA2-
CA6-CA10-CA14-CA18-CA22 and AA2-AA6-AA10-AA14-AA18-AA22 schedule. Heads
were isolated by breaking up frozen flies and passing them through a set of
sieves. RNA was prepared using guanidine-thiocyanate extraction followed by
purification over a CsCl gradient. Additional purification of the RNA samples was
achieved by applying them to Rneasy columns (Qiagen). Biotin-labeled cRNA
probe was generated from 25 μg of purified RNA and hybridized as described
previously (Wijnen H, Naef F, and Young MW, Methods Enzymol. 2005; 393: 341-365).
For more information see also http://biorhythm.rockefeller.edu


title: Toxicogenomic analysis of sodium sodium hypochlorite antimicrobial mechanisms in Pseudomonas aeruginosa

status: Public on Dec 04 2007

city: Singapore

first: Matthew

middle: Wook

last: Chang

address: 62 Nanyang Drive

department: School of Chemical and Biomedical Engineering

country: Singapore

institute: Nanyang Technological University

summary:
In the present study, we employed Affymetrix Pseudomonas aeruginosa GeneChip arrays to investigate global gene expression profiles during the cellular response of Pseudomonas aeruginosa to sodium hypochlorite
Keywords: Antimicrobial response

sample_organism:
Pseudomonas aeruginosa
Pseudomonas aeruginosa PAO1

overall_design: We calculated fold change as the ratio between the signal averages of four untreated (control) and five sodium hypochlorite-treated (experimental) cultures for 20 min exposure.

first: David

last: Small

first: Wook

first: Freshteh

last: Toghrol

first: William

middle: E

last: Bentley


title: Comparison of global gene expression between Pseudomonas aeruginosa strains 383 and 2192

status: Public on Nov 20 2007

city: Charlottesville

first: Jayasimha

last: Rao

state: VA

address: 1300 Jefferson Park Avenue

institute: University of Virginia

summary:
At mid-log phase (OD600 of 0.5), unique gene expression patterns were observed between these two strains with 3.4% of the transcripts (188/5570) expressed differentially.
Of the 188 significantly varied (>1.8 fold) genes, 115 were up-regulated in 383 while 73 were up-regulated in 2192.
Keywords: Different strains of bacterium

overall_design: The goal of this experiment was to identify the differentially expressed genes from two genetically similar but phenotypically distinct P. aeruginosa strains 383 and 2192. Two strains were isolated two days apart from the sputum of the same cystic fibrosis patient. Following proper culture RNA was extracted from the two strains. Affymetrix GeneChip Pseudomonas aeruginosa was used to examine the gene expression paterns of the two strains.

first: J

first: Joanna

last: Goldberg


title: Circadian time course

status: Public on Dec 07 2007

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series

middle: W


title: Expression data from eye antennal imaginal disc expressing either activated Ras or secreted Spitz

status: Public on Nov 14 2007

city: Bronx

first: Lucy

middle: Catherine

last: Firth

state: NY

address: 1300 Morris Park Avenue

institute: Albert Einstein College of Medicine

summary:
We have used microarrays to identify genes expressed and required for the second mitotic wave (SMW) during eye development. Eye discs expressing Spitz under the control of GMR Gal4 have no SMW as Spitz promotes G1 arrest, ectopic differentiation also occures. To control for the ectopic differentiation, Spi expressing eye antennal discs were compared to eye antennal discs expressing activated RasV12. In discs expresseding RasV12 under the control of GMRGal4 the SMW takes place normally prior to any ectopic differentiation.
We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process.
Keywords: Comparison of eye antennal discs of two different over expression genotypes

overall_design: Drosophila eye antennal imaginal discs expressing either UAS RasV12 or UAS Spi under the control of GMRGal4 were dissected from 3rd instar larvae for RNA extraction and hybridization on Affymetrix microarrays.

first: Nicholas

last: Baker


title: SFP1 dependent transcription

status: Public on Nov 10 2007

city: Delft

first: Chiara

last: Cipollina

address: Julianalaan 67

department: Biotechnology

institute: Delft University of Technology

summary:
In Saccharomyces cerevisiae growth, size control and cell cycle progression are strictly coordinated and regulated according to the nutritional conditions. In particular, ribosome biogenesis appears a key event in this regulatory network. SFP1 encodes a zinc-finger protein promoting the transcription of a large cluster of genes involved in ribosome biogenesis. It has been suggested that Sfp1 is a cell size modulator acting at Start. To better study the regulatory role of Sfp1 and its putative involvement in cell size and cycle control, we analysed the behaviour of an sfp1 null mutant strain and of an isogenic reference strain growing in chemostat cultures. This approach allowed us to analyze both strains at the same specific growth rate, thus eliminating the secondary effects due to the slow growing phenotype that the sfp1 null mutant shows in shake flask. We studied glucose(anaerobic)- and ethanol(aerobic)-limited cultures, as paradigms of two different metabolic states. Major alterations of the transcriptional profile were observed during growth on glucose, while no significant differences were observed when comparing ethanol growing cultures. In particular, in the former growth condition, Sfp1 appears involved in the control of ribosome biogenesis but not of ribosomal protein gene expression.
Keywords: global transcriptional profile, genetic modification, ribosome biogenesis

overall_design: The reference S. cerevisiae strain CEN.PK113.7D and the isogenic sfp1 null mutant were grown in chemostat cultures under ethanol(aerobic)- and glucose(anaerobic)- limitation. The dilution rate was set at 0.10 h-1 and 0.05 h-1 for ethanol- and glucose-limited cultures, respectively. A genome-wide transcriptional analysis was performed for the reference and the sfp1 null mutant strains for each growth condition. All data presented in this work were derived from three independent chemostat cultures (12 samples in total were analysed).

first: Joost

last: van den Brink

first: Pascale

last: Daran-Lapujade

first: Jack

last: Pronk

first: Marina

last: Vai

first: Johannes

middle: H

last: de Winde


title: Characterization of five novel Pseudomonas aeruginosa cell-surface signaling systems

status: Public on Nov 24 2007

city: Amsterdam

first: Marian

last: Llamas

address: van der Boechorststraat 7

department: Medical Microbiology

institute: VU Medical Center

summary:
Cell-surface signaling is a sophisticated regulatory mechanism used by gram-negative bacteria to sense signals from outside the cell and transmit them into the cytoplasm. This regulatory system consists of an outer membrane-localized TonB-dependent receptor (TonB-dependent transducer), a cytoplasmic membrane-localized anti-sigma factor and an extracytoplasmic function (ECF) sigma factor. By microarray analysis we have identified the regulons of four novel P. aeruginosa signaling systems. For that, the ECF sigmas PA0149, PA2050, PA2093 and PA4896 have been overexpressed in P. aeruginosa and their target gene candidates have been identified using DNA microarray.
Keywords: Overexpression of ECF sigma factors

overall_design: Five different samples are analyzed namely P. aeruginosa (pMMB67EH) (control/reference sample), P. aeruginosa (pMMB-PA4896) (overexpressing the PA4896 ECF sigma factor), P. aeruginosa (pMMB-PA0149) (overexpressing the PA0149 ECF sigma factor), P. aeruginosa (pMMB-PA2050) (overexpressing the PA2050 ECF sigma factor), and P. aeruginosa (pMMB-PA2093) (overexpressing the PA2093 ECF sigma factor). Two different replicates are included for the three first samples and one for the two last samples.

first: Wilbert

last: Bitter


title: Drosophila Myb-MuvB/dREAM

city: Berkeley

first: Soyeon

last: Ahn

address: University of California, Berkeley

department: Statistics

institute: UC Berkeley

type: Genome binding/occupancy profiling by genome tiling array


title: The embryonic muscle transcriptome of C. elegans

status: Public on Nov 08 2007

city: Bethesda

first: WeiPing

last: Chen

address: Bldg 8, Room 1A11, NIDDK/NIH

department: GCL

institute: NIDDK/NIH

summary:
Background:

The force generating mechanism of muscle is evolutionarily ancient; the fundamental structural and functional components of the sarcomere are common to motile animals throughout phylogeny. Recent evidence suggests that the transcription factors that regulate muscle development are also conserved. Thus, a comprehensive description of muscle gene expression in a simple model organism should define a basic muscle transcriptome that is also expressed in animals with more complex body plans.  To this end, we have applied Micro-Array Profiling of C. elegans (MAPCeL) to muscle cell populations extracted from developing C. elegans embryos.

Results:

Fluorescence Activated Cell Sorting (FACS) was used to isolate myo-3::GFP-positive muscle cells, and their cultured derivatives, from dissociated early C. elegans embryos.  Microarray analysis identified 6,693 expressed genes, 1,324 of which are enriched in the myo-3::GFP positive cell population relative to the average embryonic cell.  The muscle-enriched gene set was validated by comparisons to known muscle markers, independently derived expression data, and GFP reporters in transgenic strains.  These results confirm the utility of MAPCeL for cell type-specific expression profiling and reveal that 60% of these transcripts have human homologs.

Conclusions:

This study provides a comprehensive description of gene expression in developing C. elegans embryonic muscle cells.  The finding that over half of these muscle-enriched transcripts encode proteins with human homologs suggests that mutant analysis of these genes in C. elegans could reveal evolutionarily conserved models of muscle gene function with ready application to human muscle pathologies.
Keywords: time course, muscle cells, HLH-1 induction

overall_design: Approximately 80 one- and two-cell stage embryos were collected from either heat-shock hlh-1 (KM267) or heat-shock pal-1(JA1179) worms and were incubated for 20 min (hs pal-1) or 60 min (hs hlh-1) at room temperature prior to a heat pulse at 34oC for 30min.  After the heat pulse, embryos were incubated at room temperature for 2, 4, and 6 hours (hs hlh-1) or 2, 4, 6, and 8 hours (hs pal-1) prior to total RNA isolation. Embryos were also collected prior to heat shock as a control (0 hour).  Triplicate independent samples were prepared for all time points.  All hs pal-1 experiments were performed on embryos derived from skn-1, pop-1 double RNAi injected animals.  The isolation of total RNA from embryos followed the procedures in (Baugh et al. 2003). Total RNA was used with the Super Smart PCR cDNA synthesis kit (BD Clontech) in 20 cycles with a modified primer (SMART7T27 Primer: 5'-TGAAGCAGTGGTAACAACGCAGAGTAATACGACTCACTATAGGGAGAAGC(T)27VN -3') prior to one cycle target labeling reaction by standard procedures (Affymetrix).  Affymetrix C. elegans gene chips (P/N900383, ~22,500 transcript probes) were processed according to manufactures protocol using the NIDDK Genomics Core Laboratory.  Microarray data was normalized by MAS5 prior to analysis using GeneSpring software (Silicon Genetics).

first: Rebecca

last: Fox

first: Joan

last: McDermott

first: Thomas

last: Brodigan

first: Tetsunari

last: Fukushige

first: Michael

last: Krause

first: David

last: Miller


title: Changes in gene expression in response to heme deficiency and hypoxia

status: Public on Nov 06 2007

first: Olga

last: Protchenko

address: 10 Center Dr, Bldg.10-Rm.9B-05

department: NIDDK

institute: National Institutes of health

summary:
We addressed changes in gene expression profile in response to
reduced oxygen tension in wild type or heme depletion caused by
deletion of heme biosynthesis gene hem1
Keywords: Response to heme depletion and hypoxia

overall_design:
I.Heme depletion.The hem1 delta strain was grown for 6 hrs in YPD media supplemented with 250 uM ALA. Cells were washed three times with water to remove excess ALA. Cells were then inoculated in duplicates into 15 ml cultures of
YPD media that was supplemented either with or without ALA. Cultures were induced for 16 h before 5 ml of cells were harvested and washed
three times in DEPC water. The culture density at the time of harvest
was approximately OD600=0.5.
II.Hypoxia. BY45742 strain was grown for 6 hrs in
YPD medium and then inoculated in 15 ml the same medium and grown
under normoxic or hypoxic conditions for 12 hrs. To create hypoxic
conditions, cultures were incubated in an anaerobic chamber using a
BBL-GasPak (BD Biosciences).

first: Caroline

last: Philpott


title: Transcriptional profiling of ribosomal protein knockouts

city: Boston

first: Suzanne

last: Komili

state: MA

address: 44 Binney St.

institute: Harvard University

summary:
Duplicated genes escape gene loss by conferring a dosage benefit or evolving diverged functions.  The yeast Saccharomyces cerevisiae contains many duplicated genes encoding ribosomal proteins.  Prior studies have suggested that these duplicated proteins are functionally redundant and affect cellular processes in proportion to their expression.  In contrast, through studies of ASH1 mRNA in yeast, we demonstrate paralog-specific requirements for the translation of localized mRNAs.  Intriguingly, these paralog-specific effects are limited to a distinct subset of duplicated ribosomal proteins.  Moreover, transcriptional and phenotypic profiling of cells lacking specific ribosomal proteins reveals differences between the functional roles of ribosomal protein paralogs that extend beyond effects on mRNA localization.  Finally, we show that ribosomal protein paralogs exhibit differential requirements for assembly and localization. Together, our data indicate complex specialization of ribosomal proteins for specific cellular processes, and support the existence of a ribosomal code.
Keywords: genetic modification

overall_design:
We used Affymetrix arrays to analyze the transcriptional profiles of cells lacking certain duplicated ribosomal protein genes in order to determine if paralogous ribosomal proteins have differing cellular effects and roles.
Two biological replicates were performed for each strain.  Each ribosomal protein deletion was then compared to the isogenic wild-type strain.

first: Natalie

middle: G

last: Farny

first: Frederick

middle: P

last: Roth

first: Pamea

last: Silver


title: Saccharomyces cerevisiae TPP 2-oxo acid decarboxylases

status: Public on Nov 15 2007

first: Jean-Marc

last: Daran

department: Department of Biotechnology

summary:
The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases.
Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbonâ€“limited chemostat with phenylalanine as nitrogen source indicated that:
i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate Î±-keto acid decarboxylase.
ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and
iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any Î±-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway.
Keywords: Strain comparison

overall_design:
Comparison of glucose limited chemostat cultivations with phenylalanine as sole nitrogen sources of the following strains:
- CENPK113-7D MATa MAL2-8c SUC2
- CENPK555-4A MATa MAL2-8c SUC2 ydr380w delta
- CENPK5632-3B MATalpha MAL2-8c SUC2 ydr380w delta ydl080c delta
- CENPK609-11A MATa MAL2-8c SUC2 pdc1, 5, 6 delta ydr380w delta

first: JM

first: J

last: Pronk


title: Upf1p-associated transcripts in S. cerevisiae

status: Public on Nov 01 2007

city: Worcester

first: Marcus

last: Johansson

address: 55 Lake Avenue North

department: Molecular Genetics and Microbiology

institute: University of Massachusetts Medical School

summary:
The goal of this experiment was to identify transcripts associated with the S. cerevisiae Upf1 protein.
Keywords: RNA IP

overall_design: The RNA population from four independent pairs of Upf1p-TAP and mock affinity selections were analyzed by using Affymetrix Yeast Genome S98 Arrays.


title: cRNA amplification methods enhance microarray identification of transcripts expressed in the nervous system

city: Nashville

state: TN

address: 465 21st Avenue South

department: Cell and Developmental Biology

institute: Vanderbilt University

summary:
Background: DNA microarrays provide a powerful method for global analysis of gene expression. The application of this technology to specific cell types and tissues, however, is typically limited by small amounts of available mRNA, thereby necessitating amplification. Here we compare microarray results obtained with two different methods of RNA amplification to profile gene expression in the C. elegans larval nervous system.   Results: We used the mRNA-tagging strategy to isolate transcripts specifically from C. elegans larval neurons. The WT-Ovation Pico System (WT-Pico) was used to amplify 2 ng of Pan-neural RNA to produce labeled cDNA for microarray analysis. These WT-Pico-derived data were compared to microarray results obtained with a labeled aRNA target generated by two rounds of In Vitro Transcription (IVT) of 25 ng of Pan-neural RNA. WT-Pico results in a higher fraction of Present calls than IVT, a finding consistent with the proposal that DNA-DNA hybridization results in lower mismatch signals than the RNA-DNA heteroduplexes produced by IVT amplification. Microarray data sets from these samples were compared to a Reference profile of all larval cells to identify transcripts with elevated expression in neurons. These results were validated by the high proportion of known neuron-expressed genes detected in these profiles and by promoter-GFP constructs for previously uncharacterized genes in these data sets. Together, the IVT and WT-Pico methods identified 2,173 unique neuron-enriched transcripts. Only about half of these transcripts (1,044), however, are detected as enriched by both IVT and WT-Pico amplification.
Conclusion: We show that two different methods of RNA amplification, IVT and WT-Pico, produce valid microarray profiles of gene expression in the C. elegans larval nervous system with a low rate of false positives.  However, our results also show that each method of RNA amplification detects a unique subset of bona fide neural-enriched transcripts and thus a wider array of authentic neural genes are identified by the combination of these data sets than by the microarray profiles obtained with either method of RNA amplification alone. With its relative ease of implementation and greater sensitivity, WT-Pico is the preferred method of amplification for cases in which sample RNA is limiting.
Keywords: expression profile

overall_design: We profiled gene expression throughout the nervous system of the model organism Caenorhabditis elegans using two different amplification methods, WT-Pico and IVT (in vitro transcription). Since postembryonic cells are unattainable via this method, we used mRNA-tagging to isolate pan-neural RNAs from larval animals. In short, an epitope tagged poly-A binding protein (FLAG-PAB-1) was driven in all neurons using the F25B3.3 promoter.  We then amplified harvested RNA using IVT or WT-Pico.  Data generated from this study reveals significant differences between the two amplification methods.  However, both seem to identify bona fide neural transcripts.  Our work suggests that using two amplification methods, when possible, will provide one with a more accurate view of transcription in a particular cell type.

first: Joseph

middle: D

last: Watson

first: Shenglong

last: Wang

first: Stephen

last: Von Stetina

first: W

last: Spencer

first: Shawn

last: Levy

first: Phillip

last: Dexheimer

first: Nurith

last: Kurn

first: Joe

last: Heath

middle: M

last: Miller III


title: Cisplatin-induced gene expression changes in A549 NSCLC cells

status: Public on Dec 01 2007

city: Leicester

first: Gabriela

middle: M.

last: Almeida

address: Biocentre, University Road

department: Cancer Studies and Molecular Medicine

institute: University of Leicester

summary:
Affymetrix high-density oligonucleotide microarray analysis was performed to analyse cisplatin-induced gene expression changes in A549 NSCLC cells. Cells were treated with 50 µM of cisplatin for 1 hour and incubated for a further 10 hours in drug-free media before the gene expression changes were investigated. Results show that cisplatin induced changes in the expression of genes involved in apoptosis, cell cycle control, DNA repair and transcription.
Keywords: drug response

overall_design: Gene expression changes in response to cisplatin were analysed by Microarray technology in A549 NSCLC cells. Cells were treated with 50 µM of cisplatin (or drug-free media) for 1 hour and incubated for a further 10 hours in drug-free media before the cisplatin-induced gene expression changes were investigated. Control and cisplatin-treated samples were collected from three independent experiments.

first: Tiago

last: Duarte

first: George

last: Jones


title: A SAGA-Independent Function of SPT3 Mediates Transcriptional Deregulation in a Mutant of the Ccr4-Not Complex

status: Public on Oct 26 2007

city: Geneva

first: Martine

last: Collart

address: Rue Michel-Servet 1, Centre Médical Universitaire

department: Microbiology and Molecular Medicine

country: Switzerland

institute: University of Geneva

summary:
The conserved multi-subunit Ccr4-Not complex regulates gene expression in diverse ways. In this work, we characterize the suppression of temperature sensitivity associated with a mutation in the gene encoding the scaffold subunit of the Ccr4-Not complex, NOT1, by the deletion of SPT3.
We determine that the deletion of SPT3, but not the deletion of genes encoding other subunits of the SAGA complex, globally suppresses transcriptional defects of not1-2. We find that transcriptional activation in not1-2 is associated with increased binding of TFIID and SAGA at promoters of upregulated genes, and this is suppressed by the deletion of SPT3. Interestingly, Spt3p-dependent activation of transcription occurs in not1-2 even if the SAGA complex is disrupted by the deletion of SPT7 that encodes a subunit of SAGA required for its integrity. Consistent with a SAGA-independent function of Spt3p, the deletion of SPT3 displays synthetic phenotypes when combined with a deletion of SPT7. Taken together, our results provide a new view of the Spt3 protein by identifying a SAGA-independent function of this protein that is functionally linked to the Ccr4-Not complex.
Keywords: genetic modification

overall_design: 4 strains were analyzed: the wild type reference strain, two simple mutants (not1-2 and spt3) and one double mutant (not1-2 spt3). All strains were in duplicate.

first: Nicole

last: James

middle: A


title: Creation of Drosophila cell lines by overexpression of Ras

status: Public on Oct 19 2007

city: Columbus

first: Amanda

last: Simcox

address: 484 W. 12th Avenue

department: Molecular Genetics

institute: The Ohio State University

summary:
RasV12 was used to drive the generation of cell lines from primary cultures.  Lines have been passaged for over a year, indicating that they are true lines.
Keywords: genetic modification, cell type comparison

overall_design: RasV12 drove the creation of cell lines from primary culture.  The array datasets were created from passage 11 cells and compared to datasets of established cell lines, as well as embryonic, imaginal disc and whole-fly sets.

first: Wei

last: Jing

first: Jonathan

last: Butchar


title: GAL-NMD2

first: Feng

last: He

address: 368 Plantation Street

department: Microbiology and Physiological Systems

summary:
The goal of this set of experiments was to identify transcripts that are differentially expressed upon reactivation of NMD in an nmd2::HIS3 strain by galactose-induced expression of the NMD2 gene.
Keywords: Genetic modification and time course

overall_design: Yeast strains harboring nmd2::HIS3 and the GAL1-NMD2 allele on a plasmid or the empty vector were grown in SC-medium containing raffinose but lacking uracile. Genome-wide expression profiles of the GAL-NMD2 and nmd2 deletion strains were analyzed over a 60-minute time course after adding galactose to the cell cultures. Four independent replicates were carried out for this experiment.

first: PPhyllis

last: Spatrick


title: Whole genome microarray analysis of C. elegans rrf-3 and eri-1 mutants

city: Stockholm

first: Suvi

middle: Tuulia

last: Asikainen

state: Huddinge

address: Karolinska University Hospital

department: CLINTEC

country: Sweden

institute: Karolinska Institutet

summary:
RRF-3 and ERI-1 are first identified proteins required for accumulation of at least some endogenous secondary siRNAs in C.elegans. Genome wide gene expression analysis was performed on L4 stage rrf-3 and eri-1 mutant C. elegans to study effects caused by loss of these proteins. Mutant rrf-3 and eri-1 strains exhibited similar expression patterns when compared to N2 wild type, while 72 transcripts were found to be co-overexpressed and 4 transcripts co-underexpressed (> 2-fold, p< 0.05). Ontology analysis indicated many of the gene products were associated with protein phosphorylation and sperm function. These results provide additional support for the hypothesis that RRF-3 and ERI-1 act together in a siRNA pathway and may indicate biological processes that are related to endo-siRNAs.
Keywords: Mutant vs. wild type -comparison

overall_design: rrf-3(pk1426) mutant worms (NL2099), eri-1 (mg366) mutant worms (GR1373), and wild type worms (N2) were grown on culturing plates containing NGM agar media with OP50 Escherichia coli (E. coli). Worms were grown at 20°C and harvested at L4 stage. RNA isolation was made using Ribopure Total RNA isolation -kit (Ambion). Animals were grown and RNA isolated separately for each biological replicate. Total RNA was labeled for chip experiments using the cRNA protocol (Affymetrix, Palo Alto, CA). DNA from each mutant was hybridized to Gene Chip C. elegans whole genome arrays containing 22,500 transcripts (Affymetrix, Palo Alto, CA) as three biological replicates (3 chips each for N2, rrf-3, eri-1). Microarray hybridization and scanning was performed at the Biomedicum Biochip Center (Helsinki, Finland).

first: Markus

last: Storvik

first: Merja

last: Lakso

first: Garry

last: Wong


title: Drosophila Myb-MuvB/dREAM (Expression data)

first: Soyeon

last: Ahn

summary:
Myb-MuvB (MMB)/dREAM is a nine subunit complex first described in Drosophila as a repressor of transcription, dependent upon E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent upon DNA sites and developmental context.
Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNAi and micro-array expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupies thousands of chromosomal sites where a substantial number are proximal to repressed genes that are normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression whereas at other non-overlapping sites, Myb was critical for repression. These data highlight that the MMB factors are utilized in a combinatorial way for targeting gene regulation. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants.
Keywords: Drosophila Myb-MuvB/dREAM, RNAi, ChIP-chip

overall_design: RNAi to deplete Lin-52, Mip40, Myb, Mip120, Mip130, E2F2, both RBFs (RBF1 and RBF2) and L(3)MBT were performed in triplicate. RNAi with a nonspecific RNA derived from a pBSK+ plasmid (named SK+) was used as control. Total RNA was extracted from RNAi-transfected cells after 4 days using RNeasy Mini Kit (QIAGEN).

first: Daphne

last: Georlette

first: David

last: MacAlpine

first: Evelyn

last: Cheung

first: Peter

last: Lewis

first: Eileen

last: Beall

first: Stephen

last: Bell

first: Terry

last: Speed

first: John

middle: R

last: manak

first: Michael

last: Botchan


title: Trasncriptional response of Saccharomyces cerevisiae to nitrogen limitation in chemostat culture

status: Public on Oct 16 2007

first: Jean-Marc

last: Daran

summary:
Zinc is indispensable for the catalytic activity and structural stability of many proteins, and its deficiency can have severe consequences for microbial growth in natural and industrial environments. For example, Zn depletion in wort negatively affects beer fermentation and quality. Several studies have investigated yeast adaptation to low Zn supply, but were all performed in batch cultures, where specific growth rate depends on Zn availability. The transcriptional responses to growth-rate and Zn availability are then intertwined, which obscures result interpretation. In the present study, transcriptional responses of Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under Zn limitation and excess in chemostat culture. To investigate the context-dependency of this transcriptional response, yeast was grown under several chemostat regimes resulting in various carbon (glucose), nitrogen (ammonium) and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified and enabled the definition of a Zn-specific Zap1 regulon comprising of 26 genes and characterized by a broader ZRE consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large amount of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified
Keywords: Chemostat based transcriptome analysis

first: L

last: Hazelwood

first: J

middle: T

last: Pronk

first: JM

first: P

last: Daran-Lapujade


title: Expression data for Saccharomyces cerevisiae oxidative stress response

status: Public on Oct 24 2007

city: Blacksburg

first: Vladimir

last: Shulaev

address: Virginia Bioinformatics Inst., Virginia Tech, Washington st.

institute: Virginia Bioinformatics Institute

summary:
Oxidative stress is a harmful condition in a cell, tissue, or organ, caused by an imbalnace between reactive oxygen species and other oxidants and the capacity of antioxidant defense systems to remove them. The budding yeast S. cerevisiae has been the major eukaryotic model for studies of response to oxidative stress.
We used microarrays to study the genome-wide temporal response of the yeast S. cerevisiae to oxidative stress induced by cumene hydroperoxide.
Keywords: time course

overall_design: The effects of oxidative stress induced by CHP on the transcriptional profile of S. cerevisiae was studied from a dynamical perspective. Yeast cultures were grown in controlled batch conditions, in 1 L fermentors. Three replicate cultures in mid-exponential phase were exposed to 0.19 mM CHP, while three non-treated cultures were used as controls. Samples were collected at t=0 (immediately before adding CHP) and at 3,6,12,20,40,70 and 120 min after adding the oxidant. Samples were processed for RNA extraction and profiled using Affymetrix Yeast Genome S98 arrays.

first: Wei

last: Sha

first: Ana

last: Martins

first: Reinhard

last: Laubenbacher

first: Pedro

last: Mendes