title: Stress response of Saccharomyces cerevisiae to exposure to natural product pyocyanin.

status: Public on Jan 22 2007

first: Coran

middle: MH

last: Watanabe

address: Texas A&M Dept. Chemistry MS 3255

department: Chemistry

institute: Texas A*M University

summary:
Pyocyanin has been shown to engage in redox transfer of electrons from NADPH to oxygen to generate superoxide radicals.  Transcriptional response to oxygen stress has been characterized in yeast and should be observable upon exposure to pyocyanin if this is the true mode of action.
We used microarrays to detail the global programme of gene expression induced in the stress response to pyocyanin.
Keywords: stress response

overall_design: Saccharomyces cerevisiae cells were exposed to 25 and 250 ug/ml of pyocyanin for a period of 6 hours incubation for RNA extraction and hybridization on Affymetrix microarrays. Results were compared to transcription levels in untreated cells under the same incubations conditions. The data indicated that transcriptional changes were consistent with an oxidative stress response, as observed by other researchers investigating the mode of action of pyocyanin toxicity.

first: Scott

last: Angell

first: Bennie

last: Bench

first: Howard

last: Williams

first: Kim

last: Liu


title: C. elegans gene expression in response to the pathogenic P. aeruginosa strain PA14.

status: Public on Feb 08 2007

city: La Jolla

first: Emily

last: Troemel

state: CA

department: Cell and Developmental Biology

country: USA

institute: University of California, San Diego

summary:
Analysis of differential gene expression in C. elegans adults exposed to three different bacteria: E. coli strain OP50, wild-type P. aeruginosa PA14 and gacA mutant PA14. Samples were analyzed at 4 hours and 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection.
Keywords: Time course, response to pathogen infection

overall_design: Three independent biological replicates were isolated for each treatment. All treatments were performed in parallel.

middle: R

first: Dennis

middle: H

last: Kim

first: Fred

middle: M

last: Ausubel


title: Whole genome expression profile for cbc2-delta cells

status: Public on Jan 17 2007

city: Fort Worth

first: Yiwei

last: Jiang

state: TX

address: 3420 darcy Street

department: Cell biology and genetics

institute: University of North Texas Health Science Center

summary:
The goal of this experiment is to identify transcripts regulated by Cbc2, the small subunit of nuclear cap binding complex (CBC) in Saccharomyces cerevisiae.
Keywords: Genetic modification

overall_design: Six independent expression profiling experiments were carried out for isogenic wild-type (Y255) and cbc2Δ (Y2685) strains using Affymetrix Yeast Genome S98 Arrays. Three repeats were applied to each strain.

first: XIAOFENG

last: WU

first: YIWEI

last: JIANG


title: Rap1 and Abf1 DNA-binding ts mutants and wild type after 1 hr at 37 C

status: Public on Oct 24 2006

city: Albany

first: Randall

middle: Heywood

last: Morse

state: NY

institute: Wadsworth Center

summary:
Abf1 and Rap1 are General Regulatory Factors that contribute to transcriptional activation of a large number of genes, as well as to replication, silencing, and telomere structure in yeast.  In spite of their widespread roles in transcription, the scope of their functional targets genome-wide has not been previously determined.  We have used microarrays to examine the contribution of these essential GRFs to transcription genome-wide, by using ts mutants that dissociate from their binding sites at 37 C.  We combined this data with published ChIP-chip studies and motif analysis to identify probable direct targets for Abf1 and Rap1.  We also identified a substantial number of genes likely to bind Rap1 or Abf1, but not affected by loss of GRF binding.  Interestingly, the results strongly suggest that Rap1 can contribute to gene activation from farther upstream than can Abf1.   Also, consistent with previous work, more genes that bind Abf1 are unaffected by loss of binding than those that bind Rap1.  Finally, we showed for several such genes that the Abf1 C-terminal region, which contains the putative activation domain, is not needed to confer this peculiar "memory effect" that allows continued transcription after loss of Abf1 binding.
Keywords: Genetic modification (ts mutant versus wild type)

overall_design: Three replicates each of rap1 ts and abf1 ts yeast were grown at 25 C and shifted to 37 C, along with matched wild type controls, prior to RNA isolation.  The ts mutants lose DNA binding at 37 C.

first: Arunadevi

last: Yarragudi

first: Laura

middle: W

last: Parfrey


title: Expression analysis of edc3 cell

status: Public on Jan 06 2007

city: Worcester

first: Feng

last: He

state: MA

address: 368 Plantation Street

department: Microbiology and Physiological Systems

institute: University of Massachusetts Medical School

summary:
The goal of this experiment is to identify transcripts regulated by Edc3p, an activator of mRNA decapping.
Keywords: Genetic modification

overall_design: Five independent expression profiling experiments were carried out for isogenic wild-type (HFY114) and edc3Δ (SYY14) strains using Affymetrix Yeast Genome S98 Arrays.

first: Chunfang

last: Li

first: Shuyun

last: Dong


title: Targets of pharmacological histone deacetylase inhibition in human umbilical vein endothelial cells

status: Public on Sep 20 2006

city: Toronto

first: Philip

middle: A.

last: Marsden

state: Ontario

address: 1 King's College Circle

department: Department of Medicine

country: Canada

institute: University of Toronto

summary:
We and others have previously reported the paradoxical downregulation of endothelial nitric oxide synthase (eNOS, NOS3) steady-state mRNA by pharmacological histone deacetylase (HDAC) inhibitors. This microarray experiment was designed to identify novel targets of an HDAC inhibitor (trichostatin A (TSA), 500 nM, 24h, Sigma) in primary human endothelial cells (human umbilical vein endothelial cells, HUVEC).
Keywords: gene expression profiling, paired samples, dichotomous conditions

overall_design: HUVEC were isolated and cultured from three individuals (n=3, biological replicates). Paired samples were then treated with TSA (500nM, 24h, Sigma) or a vehicle-control. After sample preparation, hybridization to Affymetrix Human Genome Focus Arrays, image acquisition and the evaluation of several quality control metrics, summary expression measures were computed using the MAS5.0, RMA and GCRMA pre-processing strategies, respectively. The values were generated using the affy and gcrma packages of the Bioconductor Project.

middle: A

first: Charles

middle: C

last: Matouk

first: Jason

middle: E

last: Fish


title: Mast Cell_time series

status: Public on Aug 21 2006

city: Singapore

first: Jayapal

last: Manikandan

country: Singapore

institute: NUS

summary:
Human umbilical cord blood derived mast cells IgE sensitized followed by crosslinking receptor at different time points
Keywords: time-course

first: Manikandan

last: Jayapal

first: Renji

last: Reghunathan

first: Zhi

last: Liang

first: Hwee

middle: K

last: Tay

first: Alirio

last: Melendez

first: Reghunathan

last: Renji


title: Axon Guidance Study in Drosophila embryos

status: Public on Dec 14 2006

city: Coimbra

first: Tiago

last: Magalhaes

address: Departamento Zoologia, Universidade Coimbra

department: Centro Neurociências

country: Portugal

institute: Universidade de Coimbra

summary:
During the development of the Drosophila central nervous system the process of midline crossing is orchestrated by a number of guidance receptors and ligands.  Many key axon guidance molecules have been identified in both invertebrates and vertebrates, but the transcriptional regulation of growth cone guidance remains largely unknown. One open question is whether transcriptional regulation plays a role in midline crossing, or if local translation can account for the necessary fine tuning of protein levels. To investigate this issue, we conducted a genome wide analysis of transcription in Drosophila embryos using wild type and a number of well-characterized Drosophila guidance mutants and transgenics. We also analyzed a publicly available microarray time course of Drosophila embryonic development with an axon guidance focus. Using hopach, a novel clustering method which is well suited to microarray data analysis, we identified groups of genes with similar expression patterns across guidance mutants and transgenics. We then systematically characterized the resulting clusters with respect to their relevance to axon guidance using two complementary controlled vocabularies: the Gene Ontology (GO) and anatomical annotations of the Atlas of Pattern of Gene Expression (APoGE) in situ hybridization database. The analysis indicates that regulation of gene expression does play a role in the process of axon guidance in Drosophila. We also find a strong link between axon guidance and hemocyte migration, a result that agrees with mounting evidence that axon guidance molecules are co-opted in vertebrate vascularization. Cell cyclin activity in the context of axon guidance is also suggested from our array data. RNA and protein patterns of cell cyclin in axon guidance mutants and transgenics support this possible link. This study provides important insights into the regulation of axon guidance in vivo and suggests that transcription does play a role in control of axon guidance.
Keywords: Mutant Analysis

overall_design: Several mutants and transgenics were analyzed, totalizing 17 distinct conditions. Individual descriptions are included in each microarray. For each condition there are 3 or 4 replicates. The file's name indicates the replicates, e.g., comm.a reflects the replicate a of mutant commissureless. The experiment design covers a range of mutants and transgenics of key axon guidance mutans, in different dosages. The protocol was the standard Affymetrix protocol.

last: Magalhães

first: Jessica

last: Palmer

first: Corey

middle: S

last: Goodman


title: RNAs associated with LARK in Drosophila pharate adult brain

status: Public on Dec 06 2006

city: Boston

first: Yanmei

last: Huang

address: 136 Harrison Ave, A305B

department: Neuroscience

institute: Tufts University School of Medicine

summary:
Circadian behaviors are regulated by intrinsic biological clocks consisting of central molecular oscillators and output pathways.  Despite significant progress in elucidating the central timekeeping mechanisms, the molecular pathways coupling the circadian pacemaker to overt rhythmic behavior and physiology remain elusive.  The Drosophila LARK RNA-binding protein is a candidate for such a coupling factor.  Previous research indicates that LARK functions downstream of the clock to mediate behavioral outputs.  To better understand the roles of LARK in the Drosophila circadian system, we sought to identify RNA molecules associated with LARK in vivo, using a novel strategy that involves capturing the RNA ligands by immunoprecipitation, visualizing the captured RNAs using whole gene microarrays, and identifying functionally relevant targets through genetic screens.
Keywords: Association with RNA-binding protein

overall_design:
LARK-containing ribonucleoprotein complexes (LARK-RNPs) were precipitated from lysates of hand-dissected pharate adult brains using an affinity-purified anti-LARK antibody (around 1000 brains were used per immunoprecipitation experiment).  A portion of each lysate was saved prior to immunoprecipitations (IPs) in order to measure the relative abundance of transcripts in a total RNA sample.  RNAs extracted from the LARK-RNP and total RNA samples were labeled and hybridized to Drosophila whole-genome gene microarrays; signal intensities for individual genes were compared between samples to identify those RNAs that were enriched by immunoprecipitation (relative to their abundances in total RNA).  RNAs that were selectively enriched in the LARK-RNP samples were considered to be potential targets of the RNA-binding protein.

Due to the difficulty to dissect large amount of fly brains, only two such immunoprecipitation experiments were performed, each generating an IP RNA sample and a total RNA (control) sample. The amount of RNAs obtained from IP is very small thus only one array is used for each sample - i.e. there are only biological replicates and no technical replicate.

first: Rob

last: Jackson


title: Airway epithelium, large airways, phenotypically normal smokers vs non-smokers, MAS5 (HuGeneFL)

status: Public on Nov 21 2006

city: New York

first: Yael

last: Strulovici-Barel

address: 1300 York Avenue

department: Department of Genetic Medicine

institute: Weill Cornell Medical College

summary:
Upregulation of Expression of the Ubiquitin Carboxyl Terminal Hydrolase L1 Gene in Human Airway Epithelium of Cigarette Smokers
The microarray data deposited here is from 44 HuGeneFL GeneChips, from 9 normal non-smokers and 13 phenotypic normal smokers, large airways, 2 samples per individual, one from the right lung and one from the left lung. These samples were previously described in Hackett NR, Heguy A, Harvey BG, O'Connor TP, Luettich K, Flieder DB, Kaplan R, Crystal RG. Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers. Am J Respir Cell Mol Biol. 2003 29:331-43 and in Heguy A, Harvey BG, O'Connor TP, Hackett NR, Crystal RG. Sampling-dependent up-regulation of gene expression in sequential samples of human airway epithelial cells. Mol Med. 2003 9:200-8. These data are part of a study aimed at  understanding how cigarette smoking modifies neuroendocrine cells, in which microarray analysis with TaqMan confirmation was used to assess airway epithelial samples obtained by fiberoptic bronchoscopy from 81 individuals (normal nonsmokers, normal smokers, smokers with early COPD and smokers with established COPD). Of 11 genes considered to be neuroendocrine cell-specific, only ubiquitin C-terminal hydrolase L1(UCHL1), a member of the ubiquitin proteasome pathway, was consistently upregulated in smokers compared to nonsmokers. Up-regulation of UCHL1 at the protein level was observed with immunohistochemistry of bronchial biopsies of smokers compared to nonsmokers. Interestingly, however, while UCHL1 expression was present only in neuroendocrine cells of the airway epithelium in nonsmokers, UCHL1 expression was also expressed in ciliated epithelial cells in smokers, an intriguing observation in light of recent observations that ciliated cells can are capable of transdifferentiating to other airway epithelium. In the context that UCHL1 is involved in the degradation of unwanted, misfolded or damaged proteins within the cell and is overexpressed in >50% of lung cancers, its overexpression in chronic smokers may represent an early event in the complex transformation from normal epithelium to overt malignancy.
Keywords: smokers vs non-smokers

overall_design: comparison of gene expression in airway epithelial cells of the large airways of phenotypic normal smokers vs normal non-smokers

first: Brendan

last: Carolan

first: Adriana

last: Heguy

first: Ben-Gary

last: Harvey

first: Phil

last: Leopold

first: Barbara

last: Ferris

first: Ronald

last: Crystal


title: Expression data from yeast treated with enediynes compared to gamma radiation

status: Public on Oct 16 2006

city: Chapel Hill

first: Rebecca

middle: Catherine

last: Fry

address: 1213 MHRC

department: Environmental Sciences and Engineering

institute: UNC-Chapel Hill

summary:
We are investigating the transcriptional response of yeast to treatment with enediynes or gamma radiation, which generate different extents of double or single strand breaks in DNA.
We used microarrays to detail the global programme of gene expression underlying the DNA damage response in yeast
Keywords: dose

overall_design: Yeast were grown to mid log phase and treated with enediynes or gamma radiation (in biological triplicate) resulting in similar extents of cell killing.  The responses were compared to each other and we have deciphered a gene expression profile that is specific for double and single strand breaks in DNA.

first: Fry

last: Rebecca

first: DeMott

last: Michael

first: Cosgrove

middle: P

last: Joseph

first: Begley

middle: J

last: Thomas

first: Samson

middle: D

last: Leona

first: Dedon

last: Peter


title: Expression profile of MRP14(S100A9)-positive and -negative mouse bone marrow granulocytes under different stimulations

status: Public on Aug 08 2006

city: Muenster

first: Martin

last: Eisenacher

state: NRW

address: von-Esmarch-Str. 56

department: Integrierte Funktionelle Genomik (IFG)

country: Germany

summary:
MRP14 is tissue-specific expressed only in granulocytes, monocytes and in inflammation-activated keratinocytes. Its existence in the early phase of inflammation may be a hint towards a participation in immune response. Intra- and extracellular functions have been described yet. An antibacterial effect is caused by chelation of zinc, which then cannot be used for bacterial growth. The experiments were performed to shed light on the function of the SA100 protein MRP14.
Keywords: cell type comparison, response to stimulus

overall_design: MRP14(SA100A9)-positive and -negative mouse bone marrow granulocytes are measured unstimulated as control (2 repeats each at time point 0h, 5 repeats each at time point 4h) and then stimulated for 4h with LTB-4 (1 repeat each), A23187 (a calcium ionophore, 4 repeats each), LPS (part of gram negative bacteria, 3 repeats each) and PMA (1 repeat each); MRP14(SA100A9)-positive and -negative mouse spleen tissue (1 repeat each) for comparison; another data set contains all experiments of this data set with Mu11KsubA instead of Mu11KsubB arrays

first: Thomas

last: Vogl


title: Genetic programming and gene expression profiling for molecular discrimination and characterization of lung cancers

status: Public on Nov 01 2006

city: Duesseldorf

first: Michael

last: Rosskopf

address: Universitaetsstrasse 1

department: Bioinformatics

institute: University of Duesseldorf

summary:
Lung cancers are a heterogeneous group of diseases with respect to biology and clinical behavior. So far, diagnosis and classification are based on histological morphology and immunohistological methods for discrimination between two main histologic groups: small cell lung cancer (SCLC) and non-small cell lung cancer which account for 20% and 80% of lung carcinomas, respectively. While SCLCs express properties of neuroendocrine cells, NSCLCs, which are divided into the three major subtypes adenocarcinoma, squamous cell carcinoma and dedifferentiated large cell carcinoma, show different characteristics such as the expression of certain keratins or production of mucin and lack neuroedocrine differentiation. The molecular pathogenesis of lung cancer involves the accumulation of genetic und epigenetic alterations including the activation of proto-oncogenes and inactivation of tumor suppressor genes which are different for lung cancer subgroups. The development of microarray technologies opened up the possibility to quantify the expression of a large number of genes simultaneously in a given sample. There are several recent reports on expression profiling on lung cancers but the analysis interpretation of the results might be difficult because of the heterogeneity of cellular components. A contamination of the tumor sample with normal epithelia, blood vessels, stromal cells, leucocytes and tumor necrosis may confound the true expression profile of the tumor. The use of laser capture microdissection (LCM) greatly improves the sample preparation for microarray expression analysis. Consequently, we used advanced technology including LCM and microarray analysis.
In detail, we examined gene expression profiles of tumor cells from 29 previously untreated patients with lung cancer (10 adenocarcinomas (AC), 10 squamous cell carcinomas (SCC), 9 small cell lung cancer (SCLC)) in comparison to normal lung tissue (LT) of 5 control patients without tumor. Bronchoscopical biopsies from the primary lung tumor were taken before treatment. Biopsies were cut into 8µm sections and from each section cancer cells were isolated using laser capture microdissection in order to obtain pure samples of tumor cells. Total RNA was extracted, reversely transcribed, in-vitro transcribed, labelled and hybridized to the array. For expression analysis, microarrays covering 8793 defined genes (Human HG Focus Array, Affymetrix) were used. Following quality control, array data were normalized and analysed for significant differences using variance stabilizing transformation (VSN) and significance analysis of microarrays (SAM), respectively. Based on differentially expressed genes cancer samples could be clearly separated from non cancer samples using hierarchical clustering. Comparing AC, SCC and SCLC with normal lung tissue, we found 205, 335 and 404 genes, respectively, that were at least 2-fold differentially expressed with an estimated false discovery rate < 2.6%. Each histological subtype showed a distinct expression profile. Further, using a genetic programming approach we constructed a classificator to discriminate AC, SCC, NT and SCLC. To this end, the 50 genes with the greatest signal-to-noise ratio were selected to train the classificator. By leave-one-out cross validation all 34 samples were correctly classified in this training set. In order to validate the 50-gene-classificator on a test set, further 13 microdissected lung cancer samples were used and correctly classified in concordance to pathologic finding.
In conclusion, the different lung cancer subtypes have distinct molecular phenotypes which reflect biological characteristics of the tumor cells and which might be the basis for development of targeted therapy. Moreover, gene expression profiling and genetic programming is a suitable tool for classification and discrimination of different histological subtypes in lung cancer in comparison to normal lung tissue.
Keywords: ordered

overall_design: Comparison of gene expression profiles of normal lung tissues, adenocarcinomas, squamous cell carcinomas and small cell lung cancers.

first: Ulrich

last: Rohr

first: Astrid

last: Rohrbeck

last: Steidl

first: Helene

last: Geddert

first: Slavek

last: Kliszewski

first: Andreas

last: Schwalen

first: Helmut

last: Gabbert

first: Rainer

last: Haas

first: Ralf

last: Kronenwett


title: Drosophila heat stress response time series analysis

status: Public on Aug 01 2006

city: Aarhus

first: Morten

middle: Muhlig

last: Nielsen

address: Palle Juul-Jensens Bouleward 99

department: Department of Molecular Medicine

country: Denmark

institute: Aarhus University

summary:
gene expression was measured in control and heat resistance selected adult female flies before and at 8 time points after heat stress for 1h @ 36 degrees

Abstract The availability of full genome sequences has allowed the construction of microarrays, with which screening
of the full genome for changes in gene expression is possible. This method can provide a wealth of information about
biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various
processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster
females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on
up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening
(368C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological
replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term
heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved
in the stress response. We identified 3 main groups of stress responsive genes that were early–upregulated, early–
downregulated, and late–upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons
with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of
heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis,
which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in
general. The majority of stress responsive genes identified in this and other studies were downregulated, and the
degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat
and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an
expected exception, heat shock genes were generally found to be upregulated by stress in general.
Keywords: Time series


title: Transcriptome analysis of PAO1 cultures supplemented with PQS

status: Public on Dec 12 2006

city: Braunschweig

first: Florian

last: Bredenbruch

address: Mascheroderweg 1

department: Cellbiology

institute: GBF-Braunschweig

summary:
Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organised quorum sensing (QS) systems via small acyl-homoserine lactone (AHL) signal molecules. Recently, a third bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the QS systems. To further dissect the various independent regulation levels for many QS induced genes and to evaluate the impact of PQS on the QS circuitry, we performed a transcriptome analysis of PAO1 cultures supplemented with PQS. The global transcriptional profile in response to PQS revealed a marked up-regulation of genes belonging to the tightly interdependent functional groups of iron acquisition and oxidative stress response. Remarkably, not only most of the differentially regulated genes but also the induction of a lacZ transcriptional fusion of rhlR could be traced back to a iron chelating effect of PQS. Nevertheless, although iron deficiency per se induced rhlR, there seems to be PQS specific effects that are independent of the PQS effect on P. aeruginosa iron homeostasis
Keywords: PQS response

overall_design: For RNA extraction bacteria were harvested at early logarithmic, late logarithmic and stationary phase of growth. Three independent cultures of PQS-treated and untreated PAO1 each were pooled and the RNA was immediately stabilized with RNAprotect Bacteria Reagent (Qiagen, Valencia, CA). RNA was isolated using the RNeasy kit (Qiagen), treated with DNaseI (Roche) for 30 min at 37°C and re-purified with the RNeasy spin column. The subsequent steps of cDNA generation and Biotin-ddUTP terminal labelling were performed as described in the manufacturer’s instructions for the P. aeruginosa GeneChipÒ. Fragmented and labelled cDNA was hybridised to a GeneChip at 50 °C for 16 h. The washing steps, staining and scanning of the microarrays were performed using the Affymetrix GeneChip system. Data analysis was performed using the Affymetrix Microarray Suite Software 5.0 with Affymetrix default parameters. As expression analysis was performed in duplicate, a total of two GeneChips per culture condition was scanned at 570 nm, 3 µm resolution in an Affymetrix GeneChip scanner. The signals were multiplied by a scaling factor to make the average signal for all the arrays equivalent. The data were imported into a Microsoft Access database capable of searching for genes, which were found in all four pairings defined by the Affymetrix Microarray Suite Software as having significant changes in their signal intensities. Data were combined with the latest annotation from the website of the P. aeruginosa PAO1 sequence and the community annotation project provided at www.pseudomonas.com.

first: Susanne

last: Häussler

first: Robert

last: Geffers

first: Jan

last: Buer


title: Temperature sensitive eIF5A mutant shows accumulation of transcripts targeted to the Nonsense Mediated Decay pathway

status: Public on Jul 12 2006

city: Martinsried

last: Schrader

state: Bavaria

address: Am Klopferspitz 18

department: Proteinanalytics

institute: Max-Planck-Institut for Biochemistry

summary:
The highly conserved protein eIF5A found in archaea and all eucaryotes uniquely contains the posttranslationally formed amino acid hypusine. Despite being essential the functions of this protein and its modification remain unclear. To gain more insight into these functions temperature sensitive mutants of the human EIF5A1 were characterized in the yeast Saccharomyces cerevisiae.
EIF5A is proposed to be an RNA binding protein. Thus, we used microarrays to identify special RNA subsets of up- and downregulation that are influenced by a limited eIF5A function in strains expressing a point mutated form of eIF5A.
Keywords: two sets of four biological replicates of wild-type and mutant strains

overall_design: Wild-type and mutant yeast strains were grown at 25°C following total RNA extraction and hybridization on Affymetrix microarrays. cRNA probes derived from RNA preparations were obtained from four isogenic, haploid strains from each of the wild-type and the mutant. This ensured that changes in the transcriptome could be attributed to the impaired HYP function of V81G rather than to differences in the genetic background. Therefore, two groups of 4 datasets (per wild-type W303-WT and mutant W303-V81G strains) were generated after hybridisation to the micro arrays.

first: Craig

last: Young

first: Detlef

last: Kozian

first: Reinhard

last: Hoffmann

first: Friedrich

last: Lottspeich


title: Response of yeast to saponin treatment

status: Public on Jul 28 2006

city: Bracknell

first: Michael

last: Csukai

state: Berkshire

address: Jealott's Hill

country: United Kingdom

institute: Syngenta

summary:
Genome-wide expression profiling was used to identify genes that showed altered expression in response to treatment with the saponins tomatidine, tomatine and the sterol biosynthesis inhibitory compound fenpropimorph
Growth inhibition by the compounds was monitored by measuring the OD600nm. Cells were pelleted and frozen in liquid nitrogen. Acid-washed glass beads (0.5 mm diameter; Sigma) were added and the cells disrupted using two 20 s cycles at speed setting 6 in the Savant Bio 101 Fast Prep FP120. Total RNA was isolated using the Qiagen RNeasy kit (Qiagen, Inc., Valencia, CA, USA). Microarray hybridisation was performed using the Affymetrix GeneChip® Yeast genome S98 array using protocols described by Affymetrix, Inc. (Santa Clara, CA, USA) (as previously described (Zhu et al., 2001). Data were analyzed using the RMA algorithm using Expressionist Pro (GeneData Inc., Switzerland).
Keywords: response to abiotic stress

overall_design: Overnight batch cultures of strain S288C were diluted to an optical density at 600nm of 0.1. After 1 hour of growth at 30°C, the compounds were added and the cultures were incubated for an additional 5 hours prior to harvesting. Control cultures were treated with the solvent dimethylformamide (DMF). Tomatidine treated cultures were grown in the presence of 0.625 ppm tomatidine, 13.9 ppm Tomatine and 0.313 ppm Fenpropimorph, all dissolved in DMF. Each of the three replicates of the cultures produced on 3 independent days.

first: Veronika

last: Simons

first: John

last: Morrissey

first: Maita

last: Latijnhouwers

first: Adam

last: Cleaver

first: Carol

last: Yarrow

first: Anne

last: Osbourn


title: A Genome-Wide Survey of Drosophila Mushroom Body Transcripts with Chemical Ablation

status: Public on Jul 01 2006

city: Basel

first: Lydia

last: Michaut

address: Klingelbergstrasse 70

department: Cell Biology

country: Switzerland

institute: Biozentrum

summary:
Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain.  To get insights of the repertoire of MB genes that control initiation and maintenance of neural differentiation as well as the repertoire of neural factors that may have functions in the synaptic plasticity of MB neurons during learning and memory, we compared the transcript profiles between wild type and MB-ablated brains using a Drosophila whole-genome microarray.  Newly hatched larvae were briefly administered with a DNA-synthesis inhibitor, hydroxyurea, and raised to adults, from which total brain RNA was analyzed.
Keywords: Chemical Ablation of Mushroom bodies from Drosophila brain

overall_design:
Two conditions analyzed: Control Brains and Musroom Body-ablated brains.
Each condition was analyzed in triplicate.

first: Masatomo

last: Kobayashi

first: Ayako

last: Ino

first: Indrayani

last: Ghangrekar

first: Taiki

last: Nakajima

first: Kuniaki

last: Takahashi

first: Ryu

last: Ueda

first: Kaoru

last: Saigo

first: Walter

last: Gehring

first: Katsuo

last: Furukubo-Tokunaga


title: Gene expression and survival changes in Saccharomyces cerevisiae during suspension culture.

status: Public on Jun 15 2006

city: New Orleans

first: Timothy

last: Hammond

state: LA

address: 1430 Tulane Ave

department: Department of Medicine, Nephrology Section, SL-45

institute: Tulane University Health Sciences Center

summary:
This study explores the connection between changes in gene expression and the genes that determine strain survival  during suspension culture, using the model eukaryotic organism, Saccharomyces cerevisiae.  The Saccharomyces cerevisiae homozygous diploid deletion pool, and the BY4743 parental strain were grown for 18 hours in a rotating wall vessel, a suspension culture device optimized to minimize the delivered shear. In addition to the reduced shear conditions, the rotating wall vessels were also placed in a static position or in a shaker in order to change the amount of shear stress on the cells.
Keywords: shear stress, time course

overall_design:
The parental and Homozygous diploid pool were each pipetted into 6 RWVs per strain. For this study, the type of RWV used was the 10 mL HARV, which is designed to provide continuous O2-CO2 gas exchange across the membrane. Residual air was removed through the syringe port after loading.
All RWVs were then transferred to a 30 °CC incubator.  Within the incubator, two RWVs per strain were placed on rotators set at 30 rpm, two RWVs were placed in a gyrorotatory shaker incubator agitated at 90 rpm coincidental with the gravity vector, and the remaining two RWVs were allowed to remain static in a horizontal position.  All RWVs were grown for 18 hours before harvet.  RNA was extracted from the BY4743 samples, DNA from the deletion pool and used to generate probes for gene expression or DNA Tag3 arrays.

first: Kelly

last: Johanson

first: Patricia

middle: L

last: Allen

first: Romer

last: Gonzalez-Villalobos

first: Chasity

middle: B

last: Baker

first: Riccardo

last: D'Elia

middle: G


title: Impact of Nonsense-mediated mRNA Decay on the Global Expression Profile of Budding Yeast

status: Public on Aug 31 2006

city: Madison

first: Michael

last: Culbertson

state: WI

address: 1525 Linden Drive

department: Genetics

institute: University of Wisconsin

summary:
Isogenic UPF1+ or upf1- yeast strains were treated with 10 ug/ml thiolutin to inhibit global transcription.  Targets were obtained from 16 time points: 0, 2, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60 minutes after transcription inhibition. Three biological replicates of each were generated and the expression profiles were determined using Affymetrix YG-S98 arrays.  Comparisons between the sample groups allow the identification of genes with differential expression over time between UPF1+ and upf1-.
Keywords: time course

overall_design: Sample from 2 genotypes, 16 time points, 3 biological replicates were analyzed


title: Transcriptional profiling of copper-stressed Pseudomonas aeruginosa.

status: Public on Oct 04 2006

city: Chicago

first: Gail

middle: M

last: Teitzel

state: IL

department: MGCB

institute: University of Chicago

summary:
Anthropogenic pollution has increased the levels of heavy metals in the environment.  Bacterial populations continue to thrive in highly polluted environments and bacteria must have mechanisms to counter heavy metal stress.  We chose to examine the response of the environmentally-relevant organism Pseudomonas aeruginosa to two different copper treatments.  A short, 45 min exposure to copper was done in the Cu shock treatment to examine the immediate transcriptional profile to Cu stress.  The Cu adapted treatment was designed to view the transcriptional profile of cells that were actively growing in the presence of Cu.
Keywords: stress response

overall_design: We analyzed 2 biological replicates of Pseudomonas aeruginosa exposed to a 45 min Cu shock as compared to a control that was exposed to HCl to bring the pH to similar levels.  We analyzed 2 biological replicates of Pseudomonas aeruginosa that were grown in the presence of Cu for approx. 6h (Cu adapted) as compared to an untreated control.

first: Matthew

middle: R

last: Parsek


title: Gene Expression in Unstaged Head Hair Follicles Plucked from Men and Women

status: Public on Jun 30 2006

city: RTP

first: David

last: Dix

state: NC

address: E. HWY54  US EPA

department: NHEERL

institute: US EPA

summary:
Investigation of Gene Expression Profiling in Unstaged Head Hair Follicles Plucked from Men and Women
Keywords: Gene Expression Profiling of Normal Hair Follicles

overall_design: Total RNA was extracted from 1-3 unstaged follicles plucked from the scalp of each of 36 adult human volunteers.  The average quantifiable yield of RNA per follicle was 112.5 ng.  Ten samples were selected, based on ribosomal ratio, relative integrity number and total yield, for expression profiling.  By preamplifying the extracted RNA (starting with as little as 30 ng), sufficient labeled RNA was generated to conduct Afymetrix-based gene expression analysis.

first: SungJae

last: Kim

first: Kary

last: Thompson

first: Rachel

middle: N

last: Murrell

first: Judith

last: Schmid

first: Jane

last: Gallagher

first: John

last: Rockett


title: PCP shock of P. aeruginosa

status: Public on Sep 08 2006

city: Blacksburg

first: Nancy

middle: G.

last: Love

state: VA

address: 418 Durham Hall

department: Civil and Environmental Engineering

institute: Virginia Tech

summary:
Through chemical contamination of natural environments, microbial communities are exposed to many different types of chemical stressors; however, research on whole genome responses to this contaminant stress is limited.  This study examined the transcriptome response of a common soil bacterium, Pseudomonas aeruginosa, to the common environmental contaminant pentachlorophenol (PCP).  Cells were grown in chemostats at a low growth rate to obtain substrate-limited, steady-state, balanced-growth conditions. The PCP stress was administered as a continuous increase in concentration, and samples taken over time were examined for physiological function changes with whole cell acetate uptake rates (WAUR) and cell viability, and for gene expression changes using Affymetrix GeneChip technology and RT-PCR.  Cell viability, measured by heterotrophic plate counts, showed a moderately steady decrease after exposure to the stressor, but WAURs did not change in response to PCP.  In contrast to the physiological data, the microarray data showed significant changes in the expression of several genes.  In particular, genes coding for multi-drug efflux pumps, including MexAB-OprM, were strongly upregulated.  The upregulation of these efflux pumps protected the cells from the potentially toxic effects of PCP, allowing the physiological whole-cell function to remain constant.
Keywords: stress response to pentachlorophenol

overall_design: Cells of P. aeruginosa were grown in steady-state nutrient-limiting conditions using a minimal medium with acetate as the sole carbon source.  PCP was added as a continuous input, and the samples were taken at timepoints corresponding to approximately 0.5, 1, and 2 generation times. Reactors were run at least in triplicate, and samples from duplicate WT reactors and a single RpoS- reactor were used for the microarrays.  Gene expression is reported as the average fold-change associated with gene expression of PCP-shocked cells compared with the pre-PCP timepoint (0 hours) for the WT.

first: Jocelyn

last: Fraga Muller

first: Ann

last: Stevens

first: Johanna

last: Craig


title: mating response genes

status: Public on Nov 03 2006

city: Athens

first: Paul

last: Mack

state: GA

address: 1057 Green St.

institute: University of Georgia

summary:
Male-derived accessory gland proteins (Acps) that are transferred to females during mating have profound effects on female reproductive physiology including increased ovulation, mating inhibition, and effects on sperm utilization and storage. The extreme rates of evolution seen in Acps may be driven by sperm competition and sexual conflict, processes which may ultimately drive complex interactions between female- and male-derived molecules and sperm. However, little is known of how gene expression in female reproductive tissues changes in response to the presence of male molecules and sperm. To characterize this response, we conducted parallel genomic and proteomic analyses of gene expression in the reproductive tract of 3-day-old unmated and mated female Drosophila melanogaster. Using DNA microarrays, we identified 539 transcripts that are differentially expressed in unmated vs. mated females and revealed a striking peak in differential expression at 6 hrs postmating and a marked shift from primarily down-regulated to primarily up-regulated transcripts within 3 hrs after mating. Combining two-dimensional gel electrophoresis and liquid chromatography mass spectrometry analyses, we identified 84 differentially expressed proteins at 3 hrs postmating, including proteins which appeared to undergo post-translational modification. Together, our observations define transcriptional and translational response to mating within the female reproductive tract and suggest a bimodal model of postmating gene expression initially correlated with mating and the final stages of female reproductive tract maturation and later with the declining presence of male reproductive molecules and with sperm maintenance and utilization.
Keywords: keywords: reproduction, reproductive tract, accessory gland proteins, sperm, timecourse

overall_design: Three-day-old mated and unmated females were dissected to remove the lower reproductive tract (upper uterus, sperm-storage organs, and accessory glands). Mated females were dissected either immediately following mating (0 hr) or at 3, 6, or 24 hrs following the termination of mating. Tracts of 12-40 females of like category were pooled and total RNA extracted via a TRIzol-based protocol. Processing and labeling of transcript was performed by the Molecular Biology Core Facility at the Medical College of Georgia. Arrays from mated females at the different timepoints were compared to unmated females.

first: Anat

last: Kapelnikov

first: Yael

last: Heifetz

last: Bender


title: Microarray analysis of male flies selected for increased aggressive behavior

status: Public on Aug 18 2006

city: San Diego

first: Herman

middle: Albert

last: Dierick

address: 10640 John J. Hopkins Drive

institute: The Neurosciences Institute

summary:
We generated 4 separate subpopulations of selected flies from 1 starting population of a mixture of Canton-S flies.  Two of the populations (AggrI and AggrII) were selected for increased aggression by picking males from a population cage that engaged in the most intense fighting (known as escalation behavior).  Thirty males were selected every generation (from a total of 120 males per cage) per line and mated with random virgin females from that same generation.  Two control populations (NeutrI and NeutrII) were selected by picking random males from the population cage after 15-30 aggressive males were removed.  After 21 generations of selection males from the Aggr lines were dramatically more aggressive than males from the Neutr lines.  The Neutr lines did not significantly differ from the starting population although a trend of decreased aggression was visible.
Keywords: selection for male aggression

overall_design: Four populations were analyzed.  Two were selected for increased aggression and two were reference groups, selected for a decrease but with no significant effect.

middle: A

first: Ralph

last: Greenspan


title: Molecular signature of CD34+ hematopoietic stem and progenitor cells in chronic phase CML

status: Public on Aug 17 2006

first: Michael

last: Rosskopf

summary:
Chronic myelogenous leukaemia (CML) is a malignant disorder of the hematopoietic stem cell, which is characterized by the reciprocal translocation between chromosomes 9 and 22 (t(9;22)(q34;q11)) The translocation results in the formation of the BCR-ABL fusion oncogene encoding a protein with constitutive activated tyrosine kinase activity which plays a central role in the pathogenesis of the disease. There are still several open questions with respect to BCR-ABL-induced malignant transformation. A large limitation of the existing data about BCR-ABL effects is that they are derived to a great proportion from human hematopoietic cell lines, BCR-ABL-transformed murine cell lines or fibroblasts and mouse models, which might not be representative for chronic phase CML. A suitable cell population for studies on CML biology are primary hematopoietic stem and progenitor cells from patients with CML. Therefore, we provide in this study a genome-wide expression signature of highly enriched CD34+ cells from bone marrow (BM) of untreated patients with CML in chronic phase. Gene expression profiles of immunomagnetically enriched BM CML CD34+ cells (n=9) were compared with those of normal BM CD34+ cells (n=8) using microarrays covering 8.746 genes. Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays. Following quality control and normalization differentially expressed genes were identified by significance analysis of microarrays (SAM). Comparing both groups 918 genes were significantly differentially expressed (q value <0.1%; fold change > 1.3). Several of the BCR-ABL-induced effects described in cell lines and BCR-ABL-transduced cells could also be found in primary CML progenitor cells as for example the transcriptional activation of the classical MAPK pathway and the PI3 kinase/AKT pathway and the down-regulation of the pro-apoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include an up-regulation of components of the TGFb signalling pathway and the non-canonical Wnt/Ca2+ pathway, a transcriptional activation of fetal haemoglobin genes and genes associated with early hematopoietic stem cells (HSC) such as HoxA9 and MEIS1 and up-regulation of genes involved in fatty acid metabolism, of the thrombin receptor PAR1 and the neuroepithelial cell transforming gene 1. Differential expression of differentiation-associated genes suggested an alteration of the composition of the CD34+ cell population in CML. This was confirmed by immunophenotypical subset analyses of chronic phase CML CD34+ cells showing an increase of erythroid progenitors and a decrease of granulocyte-macrophage progenitor cells while the proportion of HSC was similar in normal and CML CD34+ cells. In conclusion, our results give novel insights into the biology of CML hematopoietic stem and progenitor cells and could be the basis for identification of new targets for therapy.
Keywords: ordered

overall_design: Comparison of gene expression profiles of CD34+ hematopoietic stem and progenitor cells from bone marrow of patients with untreated chronic myelogenous leukemia (CML) in chronic phase with those from bone marrow of healthy volunteers.

first: E

last: Diaz-Blanco

first: Ingmar

last: Bruns

first: Ralf

last: Kronenwett


title: Stress response of Saccharomyces cerevisiae to exposure to natural product Azinomycin B.

status: Public on May 30 2006

city: College Station

first: Coran

middle: MH

last: Watanabe

address: Texas A&M Dept. Chemistry MS 3255

department: Chemistry

institute: Texas A*M University

summary:
Stress responses to DNA damaging agents show characteristic responses. The effect of Azinomycin B upon cultured yeast cells in growth conditions appears to be significant.
We used microarrays to detail the global programme of gene expression induced in the stress response to Azinomycin B.
Keywords: stress response

overall_design: Saccharomyces cerevisiae cells were exposed to selected doses of Azinomycin B for a period of 6 hours incubation for RNA extraction and hybridization on Affymetrix microarrays. We sought to compare stress response states of 100 and 10 ug/ml to control growth.

first: Gilbert

middle: T

last: Kelly

first: Rader

last: Jensen

first: Robert

last: Coleman

first: Kim

last: Liu


title: Changes in gene expression when Nab3 is non-functional.

city: Baltimore

first: Jeff

last: Corden

state: MD

address: 725 N. Wolfe St.

department: Molecular Biology and Genetics

institute: Johns Hopkins School of Medicine

summary:
Nrd1 and Nab3 are two yeast RNA binding proteins which have been shown to be involved in transcription termination of non poly(A) genes.  We have used expression profiling of a Nab3 mutant to discover novel RNA targets of the Nrd1 and Nab3 transcription termination pathway.  Failure to terminate RNA polymerase II by Nab3 leads to continued transcription well beyond the correct termination sites, altering the expression of adjacent downstream genes.  Using this concept, our microarray uncovered the up-regulation of numerous genes that are located downstream of “cryptic unstable transcripts”, transcripts that are transcribed, terminated and rapidly degraded by Nrd1, Nab3, and the nuclear exosome.
Keywords: yeast temperature sensitive mutant 25C vs 37C

overall_design: Four yeast total RNA samples where analyzed in this study.  These RNAs come from two separate strains of yeast, each strain at either the permissive temperature (25C) or the non-permissive temperature (37C).  One of these strains is a wild type strain of yeast used as a control.  The other strain has a mutation in the Nab3 protein that confers temperature sensitivity at the non-permissive temperature of 37C.  After two hours at the non-permissive temperature, we observe disruptions in the Nrd1/Nab3 transcription termination pathway and this is the scheme that we followed for our microarray experiment.  Our goal was to observe the global expression changes after 2 hours without Nab3 function.

first: John

last: Arigo

first: Jeffry


title: Pleiotropic fitness effects of the Tre1/Gr5a region in Drosophila

status: Public on May 31 2006

city: Raleigh

first: Stephanie

last: Rollmann

address: Department of Zoology

institute: North Carolina State University

summary:
The abundance of transposable elements and DNA repeat sequences in mammalian genomes raises the question whether such insertions represent passive evolutionary baggage or may influence the expression of complex traits.  We addressed this question in Drosophila melanogaster where the effects of single transposable elements on complex traits can be assessed in genetically identical individuals reared in controlled environments.  Here we demonstrate that single P-element insertions in the intergenic region between the Gustatory receptor 5a (Gr5a) and Trapped in endoderm 1 (Tre1), which encodes an orphan receptor, exert complex pleiotropic effects on fitness traits, including selective nutrient intake, resistance to starvation and heat stress, and life span.  Mutations in this region interact epistatically with downstream components of the insulin signaling pathway.  Transposon-induced sex-specific and sex-antagonistic effects further accentuate the complex influences intergenic transposable elements can contribute to complex phenotypes.
Keywords: Transcriptional profiles of P-element insertion lines in the Tre1/GR5a region of Drosophila

overall_design: BG02514, BG02257 and Canton S (B) lines were reared simultaneously at 25 C under a 12 h light/dark cycle and 70% humidity.  At 5-7 days post-eclosion, we removed the heads of two replicate groups of 100 males and 100 females for each line.  Total RNA was isolated from each replicate and biotinylated cRNA probes were hybridized to high density oligonucleotide microarrays (Affymetrix, Inc.) and visualized with a streptavidin-phycoerythrin conjugate, as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000), using internal references for quantification.  The quantitative estimate of expression of each probe set is the Signal (Sig) metric, as described in the Affymetrix Microarray Suite, Version 5.0.  Sig values were analyzed by two-way ANOVA according to the model Y = μ + L + S + LxS + E, where L is the effect of line, S is the effect of sex and E the error variance

last: Magwire

first: Theodore

last: Morgan

first: Ergi

last: Ozsoy

first: Akihiko

last: Yamamoto

first: Trudy

middle: F

last: Mackay

last: Anholt


title: Establishing and Characterizing a Cell Culture Model for M Cells

status: Public on Aug 04 2006

city: Muenster

first: Martin

last: Eisenacher

summary:
M cells are special cells in the epithelium of the small intestinal bowel and play an important role during the initiation of a primary, fast immune response. On a Costar-Transwell filter cells of a colon adeno carcinoma cell line (Caco-2) are growing until differentiation. Then on the other side of the Caco cell layer lymphocytes from human venous blood are added. Pores in the filter membrane allow contact between lymphocytes and Caco cells. Therefore some cells of the Caco cell layer are differentiating to "M cell like" cells, showing the morpholgy and function of natural M cells.
Keywords: cell type comparison, induction response

overall_design: Two replicates of non-induced filter-grown Caco cells and three replicates of lymphocyte-induced filter-grown Caco cells (including lymphocyte expression) for the identification of regulated genes; one experiment of bottle-grown Caco cells and one experiment of bottle-grown lymphocytes for baseline expression profiling and control reasons.

first: Christoph

last: Cichon


title: Ecdysone- and stress-induced expression profile in Kc167 cells

status: Public on Aug 05 2006

city: Dallas

last: Abrams

address: 5323 Harry Hines Blvd

institute: University of Texas Southwestern Medical Center

summary:
Kc167 cells were mock-treated/treated with combinations of steroid hormone ecdysone and gamma-irradiation, and harvested.  The expression profiles were determined using Affymetrix Drosophila Genechip 1 arrays.  Comparisons between the sample groups allow the identification of genes with ecdysone- and/or radiation-responsive expression patterns.
Keywords: response to hormone, stress response

overall_design: 1 set of control, ecdysone, gamma-irradiated, ecdysone + irradiated samples was analyzed.

first: Anna

middle: C

last: Christich

first: Naoko

last: Sogame

first: Joseph

last: Chapo


title: Differential expression in heat resistance selected adult female Drosophila melanogaster

status: Public on Aug 01 2006

city: Aarhus

first: Morten

last: Nielsen

summary:
gene expression was measured in five independent heat resistance selected replicate lines and five control lines.
The genetic architecture underlying heat resistance remains partly unclear despite the well documented involvement of heat shock proteins (Hsps). It was previously shown that factors besides Hsps are likely to play an important role for heat resistance. In this study, gene expression arrays were used to make replicate measurements of gene expression before and up to 64h after a mild heat stress treatment, in flies selected for heat resistance and unselected control flies, to identify genes differentially expressed in heat resistance selected flies. We found 108 genes upregulated and 10 downregulated using the Affymetrix gene expression platform. Among the upregulated genes, a substantial number are involved in the phototransduction process. Another group of genes upregulated in selected flies is characterized by also responding to heat shock treatment several hours after peak induction of known Hsps revert to non-stress levels. These findings suggest phototransduction genes to be critically involved in heat resistance, and support a role for components of the phototransduction process in stress sensing mechanisms. In addition the results suggest yet uncharacterized genes responding to heat stress several hours after treatment to be involved in heat stress resistance. These findings mark an important increase in the understanding of heat resistance.
Keywords: control versus selected


title: Molecular phenotype of G-CSF- or pegylated G-CSF-mobilized CD34+ cells

status: Public on May 03 2006

city: Duesseldorf

first: Michael

last: Rosskopf

institute: University of Duesseldorf

summary:
Peripheral  blood stem and progenitor cells (PBSC) are widely used for autologous and allogeneic hematopoietic stem cell transplantation. PBSC can be mobilized into the peripheral blood using cytokines, cytotoxic chemotherapy or a combination of both. Granulocyte colony stimulating factor (G-CSF) has become the most commonly administered cytokine for PBSC mobilization because of its high potency and lack of serious toxicity. Recently, a modified form of recombinant human G-CSF has been introduced. This new compound is pegylated G-CSF (Peg-G-CSF) which possesses a substantially longer half-life than the unconjugated drug because of its reduced renal excretion and therefore provides the basis for continuous G-CSF serum-levels after a single injection. The use of Peg-G-CSF in patients who had received a cytotoxic chemotherapy was accompanied by mobilization kinetics different from those observed in patients who had received unconjugated G-CSF. In particular, more rapid leukocyte recovery and occurrence of CD34-positive cells in the peripheral blood was seen. These findings are presumably related to the more even and continuously high serum level of G-CSF maintained by Peg-G-CSF.
Using microarray technology and functional assays, we examined whether pegylation of G-CSF and its different pharmacokinetics results in addition to an earlier leucocyte recovery in transcriptional and functional changes in CD34+ cells obtained from patients who had received either G-CSF or Peg-G-CSF following a standard cytotoxic chemotherapy for PBSC mobilisation. CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays. Following quality control and normalization differentially expressed genes were identified by significance analysis of microarrays (SAM). Comparing both groups 339 genes were significantly differentially expressed (q value <5%; fold change > 1.2). Peg-G-CSF-mobilized CD34+ cells showed an expression pattern of more early progenitor cells as early stem cell markers such as HOX genes were higher expressed and differentiation-associated genes were lower expressed in comparison with G-CSF-mobilized cells. Moreover, Peg-G-CSF-mobilized CD34+ cells had a greater expression level of cell-cycle-promoting genes suggesting a greater cycle activity of these cells. In conclusion, despite the similar active drug component different pharmacokinetics of Peg-G-CSF and G-CSF result in distinct molecular phenotypes reflecting different functional characteristics.
Keywords: ordered

overall_design: CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). From each patient only one sample was analyzed. Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays according to manufacturer's instructions. Following quality control and normalization 9 samples of G-CSF-mobilized CD34+ cells were compared with 7 samples of PEG-G-CSF-mobilized cells were compared by significance analysis of microarrays (SAM). The aim was to figure out transcriptional differences resulting from various pharmacokinetics of the same drug (G-CSF: fluctuating serum levels; PEG-G-CSF: continously high serum levels)

first: Ingmar

last: Bruns

first: Ulrich

last: Steidl

first: Ralf

last: Kronenwett


title: Azithromycin-treated PAO1 vs untreated PAO1

status: Public on May 10 2006

city: Braunschweig

first: Yusuf

last: Nalca

state: Niedersachsen

address: Mascheroder Weg 1

department: ZIB

institute: GBF Braunschweig

summary:
Experimental Design

Type of experiment:
In order to evaluate the influence of subinhibitory concentrations of the macrolide antibiotic Azithromycin (AZM) on the global Pseudomonas aeruginosa gene expression, we performed a comparative gene expression analysis of AZM-treated versus untreated P. aeruginosa PAO1 cultures.

Experimental factors:
P. aeruginosa PAO1 cultures with and without addition of 2 µg/ ml AZM were grown until early stationary phase. Total RNA was extracted when the cultures reached an OD600 of 2.8.

The transcriptomes of the untreated cultures were taken as reference values. Both of the AFFYMETRIX Chips of AZM-treated PAO1 (GSM45590, GSM45591) were compared with each of the two chips of the untreated PAO1 (GSM45588, GSM45589).

Hybridization procedures and parameters:
Hybridization of the probes was carried out according to the manufacturer’s “Expression Analysis Protocol (p. 25: Modified Fluidic Protocol for P. aeruginosa cDNA Assay)”, see http://www.affymetrix.com:

Hybridization for 16 h at 50°C and 60 rpm.

- Post Hyb Wash 1: 10 cycles of 2 mixes/cycle with Wash Buffer A at 25°C
- Post Hyb Wash 2: 4 cycles of 15 mixes/cycle with Wash Buffer B at 50°C
- Stain: Stain the probe array for 10 minutes in Streptavidin Solution Mix at 25°C
- Post Stain Wash: 10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C
- 2nd Stain Wash: Stain the probe array for 10 minutes in antibody solution at
25°C
- 3rd Stain Wash: Stain the probe array for 10 minutes in SAPE solution at 25°C
- Final Wash: 15 cycles of 4 mixes/cycle with Wash Buffer A at 30°C. The
holding temperature is 25°C.

Measurement of data and specifications:
Data transformation and selection procedures:
·	The entire signals were scaled to 150 (4% capped median); scaling factors of the arrays were within 3.4 fold of each other, as the % genes called present were in the range of 23.7% and 38.1% for three arrays (GSM455889, GSM45589, GSM45591) and 74,4% for one array (GSM45590).
The background levels (Average Background) were similar for all arrays (in a range of 47.20 and 55.64). The Q scores were comparable (in a range of 2.38 and 2.81).
The Average Signals of the four arrays were similar (between 187.8 and 228.7).
·	Scanning hardware: AFFYMETRIX Scanner; software: AFFYMETRIX MAS 5 (Statistical Algorithms Reference Guide, http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf), MICRO DB 3 and DMT 3
·	Image analysis software: AFFYMETRIX MAS 5

Data selection:
·	Threshold of Signal Log ratio: -1/1; change is I (increased), MI (marginally increased), D (decreased), MD (marginally decreased); change p-value >0.999 or <0.001; only genes that are present (Detection=P) in at least one of the compared chipsets.
·	Only following genes were considered: Minimal threshold of regulation ±2 fold; minimal change p-values of 0.999 and 0.001, respectively; minimal difference of signal intensities of 200.
·	The final gene expression data table used by the authors to make their conclusions after data selection and transformation will be published as supplementary data.
Keywords: other

first: Lothar

last: Jaensch

first: Florian

last: Bredenbruch

first: Robert

last: Geffers

first: Jan

last: Buer

first: Susanne

last: Haussler

last: Jansch


title: MDA-MB-468 Smad4 positive/negative clones treated with TGF-ß

status: Public on Apr 25 2006

city: Berlin

first: xiaoqing

last: wu

state: Berlin

address: Robert-Roessle-strasse 10

department: Molecular Cell biolgoy

institute: Institute for biology

summary:
By performing oligonucleotide microarray analysis the role of Smad4 in response to TGF-ß was evaluated in established MDA-MB-468 Smad4 negative and positive clones that were treated with TGF-ß for different time points.
Keywords: time-course

first: Xiaoqing

last: Wu

first: Guenter

last: Cichon

first: Atakan

last: Aydin

first: Wolfgang

last: Uckert

last: Herwig


title: Radiation-induced Dmp53-dependent expression profile

status: Public on Jun 06 2006

first: John

last: Abrams

summary:
Embryos were collected, aged, mock-treated/treated with 40Gy gamma radiation, and allowed to recover for 1.5 hours.  Targets from 3 sets of wild type (yw, w1118) and 2 sets of mutant (Dmp53NS) biological replicates were generated and the expression profiles were determined using Affymetrix Drosophila Genechip 1 arrays.  Comparisons between the sample groups allow the identification of genes with radiation-responsive and Dmp53-dependent expression patterns.
Keywords: stress response, genetic modification

overall_design: 3 sets of paired control and irradiated Wild Type and 2 sets of paired control and irradiated Mutant (Dmp53NS) biological replicates were analyzed

first: Anna

last: Christich

first: Naoko

last: Sogame

first: Joseph

last: Chapo


title: Heterozygous carriers of Nijmegen Breakage Syndrome Have a Distinct Gene Expression Phenotype.

status: Public on Jul 26 2006

city: Philadelphia

first: Michael

middle: Patrick

last: Morley

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Penn Cardiovascular Institute

institute: Perelman School of Medicine at the University of Pennsylvania

summary:
By comparing the expression levels of genes between carriers of Nijmegen Breakage Syndrome and non-carriers, we showed that NBS carriers have a distinct gene expression phenotype.
Keywords: Cell Line Comparison

overall_design: Gene expression analysis using Affymetrix Human Focus arrays; comparison of expression levels of genes using t-statistic and identification of genes that allow classification of individuals as carriers or non-carriers by linear discriminant analysis.

first: Vivian

last: Cheung

first: Warren

last: Ewens


title: Histone H2B^30-37

status: Public on Apr 08 2006

city: Pullman

middle: J.

last: Wyrick

state: WA

address: Biotechnology Life Sciences 241

department: School of Molecular Biosciences

institute: Washington State University

summary:
Histone H2B was mutated to give H2B^30-37.  Total RNA from three replicate cultures of wild-type and mutant was isolated and the expression profiles were determined using Affymetrix arrays.  Comparisons between the sample groups allow the identification of genes regulated by H2B HBR domain.
Keywords: repeat

overall_design: mutant compared to wild-type, 3 replicates

last: Parra

middle: J


title: Defining the Pseudomonas aeruginosa SOS Response and its Role in the Global Response to the Antibiotic Ciprofloxacin

city: La Jolla

first: Floyd

last: Romesberg

state: CA

address: 10550 N. Torrey Pines Road

institute: The Scripps Research Institute

summary:
Pseudomonas aeruginosa infections can be virtually impossible to eradicate and the evolution of resistance during antibiotic therapy is a significant concern.  In this study, we use DNA microarrays to characterize the global transcriptional response of P. aeruginosa to clinical-like doses of the antibiotic ciprofloxacin and also to determine the component that is regulated by LexA cleavage and the SOS response.  We find that genes involved in virtually every facet of metabolism are down-regulated in response to ciprofloxacin.  The LexA-controlled SOS regulon identified by microarray analysis includes only fifteen genes, but does include several genes that encode proteins involved in recombination and replication, including two inducible polymerases known to play a role in mutation and the evolution of antibiotic resistance in other organisms.  The data suggests that the inhibition of LexA cleavage during therapy might help combat this pathogen by decreasing its ability to adapt and evolve resistance.
Keywords: Time course of response of P. aeruginosa to the antibiotic ciprofloxacin

overall_design: Sample preparation and data analysis.  For each strain (PAO1 and the lexA uninducible mutant), 5 clones were inoculated in LB and grown 18 h.  Cultures were diluted 1:500 and grown to mid-log phase (OD600 ~0.4-0.5) at which point ciprofloxacin was added to a final concentration of 1 μg/ml.  At 0, 30 and 120 minutes following ciprofloxacin addition, appropriate volumes from each of the 5 cultures per strain were pooled and added to 2 volumes of RNAprotect reagent (Qiagen); cell pellets were stored at 4 ºC until RNA extraction.  Total RNA was extracted using the RNeasy Mini kit (Qiagen) at the end of the sample collection period.  This procedure was repeated three independent times to generate three samples each just prior to and 120 minutes post ciprofloxacin addition.

first: Ryan

last: Cirz

first: Bryan

last: O'Neill

first: Jennifer

last: Hammond

first: Steven

last: Head

middle: E


title: Histone H2B^3-37

summary:
Histone H2B was mutated to give H2B^3-37.  Total RNA from three replicate cultures of wild-type and mutant was isolated and the expression profiles were determined using Affymetrix arrays.  Comparisons between the sample groups allow the identification of genes regulated by H2B N-terminal domain.
Keywords: repeat


title: Histone H2B K->G

summary:
Histone H2B was mutated to give H2B K6,11,16,17,21,22G.  Total RNA from three replicate cultures of wild-type and mutant was isolated and the expression profiles were determined using Affymetrix arrays.  Comparisons between the sample groups allow the identification of genes regulated by H2B N-terminal acetylated lysine residues.
Keywords: repeat


title: Histone H2B^3-32

summary:
Histone H2B was mutated to give H2B^3-32.  Total RNA from three replicate cultures of wild-type and mutant was isolated and the expression profiles were determined using Affymetrix arrays.  Comparisons between the sample groups allow the identification of genes regulated by H2B N-terminal domain.
Keywords: repeat


title: Histone H2B^3-32, H2B K->G, H2B^3-37, and H2B^30-37 mutations

summary:
Histone H2B was mutated to give H2B^3-32, H2B K->G, H2B^3-37, and H2B^30-37.  Total RNA from three replicate cultures of wild-type and mutant was isolated and the expression profiles were determined using Affymetrix arrays.  Comparisons between the sample groups allow the identification of genes regulated by H2B HBR domain.

This SuperSeries is composed of the SubSeries listed below.

overall_design:
mutants compared to wild-type, 3 replicates
This reference Series links data in the following related Series:
GSE3802 Histone H2B^3-32
GSE3803 Histone H2B K->G
GSE3804 Histone H2B^3-37
GSE3805 Histone H2B^3-37


title: Common features and differences in the transcriptome of large cell anaplastic lymphoma and classical Hodgkin lymphoma

status: Public on Mar 31 2006

city: Essen

first: Ludger

last: Klein-Hitpass

address: Virchowstr. 173

department: Universitaetsklinikum

institute: Institut fuer Zellbiologie

summary:
Analysis of differential gene expression in human non-Hodgkin`s lymphoma cell lines and a primary leukaemic tumor sample of large cell anaplastic type in comparison with Hodgkin`s lymphoma cell lines and other non-Hodgkin`s lymphoma samples and non-neoplastic lymphocytes
Keywords: cell type comparison

overall_design: Samples were analyzed to be compared to publically available data sets

first: Klaus

last: Willenbrock

first: Ralf

last: Küppers

first: Christoph

last: Renné

first: Verena

last: Distler

first: Susan

last: Eckerle

first: Eckhart

last: Weidmann

first: Andreas

last: Bräuninger

first: Martin-Leo

last: Hansmann


title: Human macrophages exposed to hypoxia

status: Public on Apr 11 2006

city: Göteborg

first: Per-Arne

last: Svensson

country: Sweden

institute: Internal Medicine

summary:
The aim of this study was to investigate the effects of hypoxia on human macrophages
Keywords: Effect of treatment

overall_design: Cultured macrophages from 4 donors were exposed to normoxia (21% oxygen) or hypoxia (0% oxygen) for 24 h. Two μg RNA from 4 donors incubated at normoxia and 4 donors at hypoxia was pooled respectively and these 2 pools were used for the target preparation and analyzed on duplicate DNA microarrays. Comparisons were made between the expression profile from macrophages exposed to hypoxia and the expression profile from the control normoxic macrophages to identify genes regulated during hypoxia.

first: Ellen

middle: K

last: Rydberg

first: Lillemor

last: Hultén

first: Margareta

last: Jernås

first: Pontus

last: Boström

first: Sven-Olof

last: Olofsson

first: Olov

last: Wiklund


title: Fast and chronic ethanol tolerance - time course

status: Public on Mar 24 2006

city: Houston

first: Zhiyong

last: Yang

state: TX

address: One Baylor Plaza

institute: Baylor College of Medicine

summary:
To better understand the molecular mechanisms underlying ethanol action we have developed an assay system to study sensitivity and fast and chronic tolerance to the sedative effects of ethanol.  Flies developed fast ethanol tolerance after single exposure to 50% of ethanol vapor for 40 min. Flies can also develope chronic ethanol tolerance after 5x 40 min exposure to 50% ethanol vapor interspersed with 4x 80 min exposure to 10% ethanol vapor.
We used microarray analysis to identify genes involved in the development of fast and chronic ethanol tolerance in Drosophila. More than 600 hundred genes were found to have changed their expression level in response to different ethanol treatment. Among these genes, Homer was picked for further study and is demonstrated that its expression in ellipsoid body is critical for the normal sensitivity and fast toleance to ethanol.
Keywords: time course

overall_design: Whole heads of 3 groups of  Drosophila were separated for RNA extraction and hybridization on Affymetrix microarrays. These 3 groups of flies were treated with different ethanol treatment. Naive group of flies (GSM101531, GSM101532, GSM101533, GSM101534, GSM101535, GSM101536) received only 50 min of humidified air. Fast group of flies (GSM101537, GSM101538, GSM101539, GSM101540, GSM101541, GSM101542) received humidified air for 50 min followed by a single exposure of 50% ethanol vapor for 40 min. Chronic group of flies (GSM101543, GSM101544, GSM101545, GSM101546, GSM101547, GSM101548) ) received humidified air for 50 min followed by 5 exposures of 50% ethanol vapor for 40 min.

first: Nancy

middle: L

last: Urizar

first: Howard

last: Edenberg

first: Ronald

last: Davis


title: Drosophila whole testis gene expression

status: Public on Mar 15 2006

first: Natalie

last: Terry

address: 421 Curie Blvd

department: Department of Cell and Developmental Biology

institute: University of Pennsylvania

summary:
Whole testes were dissected from adult males.  RNA from five or six biological replicates were generated and the expression profiles were determined using Affymetrix Drosophila Genechip 1 arrays.  Comparisons between the bgcn- and Os+bgcn- groups allowed for the identification of stem cell genes.
Keywords: repeat

overall_design: 5 bgcn-, 6 Os+bgcn-, and 5 wild-type biological replicates were analyzed.

first: Steve

last: DiNardo


title: Dysregulation of Gene Expression in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Lesioined Mouse Substantia Nigra

status: Public on May 12 2006

city: Rochester

last: Federoff

state: NY

address: 601 Elmwood Ave Box 645

department: Center for Aging and Developmental Biology

institute: University of Rochester

summary:
Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death.
Keywords: Parkinson's disease; MPTP; gene expression; microarray; cytoskeleton; mouse

overall_design: The demonstration of a shared pathogenic cascade among the differently triggered forms of PD requires a characterization of the molecular changes accompanying the temporal evolution of midbrain dopamine injury, dysfunction, and cell death. Identification of consensus molecular responses is aided by the application of robust exploratory methods that permit cataloging of changes in gene expression. Here, mRNA profiling by two leading oligonucleotide array systems, Affymetrix GeneChips and Amersham CodeLink, was used to reveal candidate molecules that are dysregulated during MPP+-induced injury of the mouse SN in a chronic MPTP model of PD. Different time points were examined to discriminate temporal patterns of gene expression in this model. Applying three separate data mining methods, we found consistent downregulation of genes encoding proteins functioning in the maintenance of the cytoskeleton, neurotransmitter release, and trophic signaling in the SN. Additionally, decline in cell cycle regulatory genes is suggestive of abortive mitosis and death of postmitotic neurons.

first: Renee

last: Miller

first: Linda

last: Callahan

first: Cindy

last: Casaceli

first: Linlin

last: Chen

first: Gretchen

last: Kiser

first: Buena

last: Chui

first: Tamma

last: Kaysser-Kranich

first: Timothy

last: Sendera

first: Chockalingam

last: Palaniappan


title: Interspecies expression profiling in Leishmania

status: Public on Mar 10 2006

city: West Lafayette

last: Holzer

state: IN

address: 175 S. University St.

department: Biochemistry

institute: Purdue University

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

sample_organism:
Leishmania major
Leishmania mexicana

overall_design: Refer to individual Series


title: Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells

status: Public on Mar 01 2006

city: Cambridge

first: Cheng Cheng

last: Zhang

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute

summary:
Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of critical questions of stem cell biology. Microarray studies showed that the HSC supportive mouse fetal liver CD3+ cells specifically expressed angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). When highly enriched HSCs were cultured in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors for 10 days, a 24 or 30 fold net expansion of long-term HSCs was observed by reconstitution analysis. The coiled-coil domain of Angptl2 mediated the effects of Angptl2 on HSC expansion. Furthermore, angiopoietin-like 5, angiopoietin-like 7, and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.
Keywords: Cell type comparison

overall_design:
Cultured 3 different cell types
-> Total RNA isolation -> dscDNA -> Biotinylated cRNA -> hybridization to Affymetrix U74Bv2 and U74Cv2 mouse chips

first: Chengcheng

first: Megan

last: Kapa

first: Guangtao

last: Ge

first: Kathleen

last: Xie

first: Wei

last: Tong

first: Christopher

last: Hug

first: Harvey

last: Lodish


title: Whole genome gene expression profiles of migratory cells in the Drosophila ovary

status: Public on Apr 04 2006

city: Baltimore

first: Xuejiao

last: Wang

address: WBSB413, 725 N. Wolfe Str.

department: Biological Chemistry

institute: Johns Hopkins Univ.

summary:
Cell migration contributes to normal development and homeostasis as well as to pathological processes such as inflammation and tumor metastasis.  Previous genetic screens have revealed a few major signaling pathways that govern follicle cell migrations in the Drosophila ovary, several of which elicit transcriptional responses.  However few downstream targets of the critical transcriptional regulators, such as the C/EBP homolog SLBO, have been identified.  To characterize the gene expression profile of two migratory cell populations and identify SLBO targets, we employed a magnetic bead based cell separation approach to purify border cells and centripetal cells expressing the mouse CD8 antigen, and carried out whole genome microarray analysis.
Keywords: cell type comparison, genetic modification

overall_design: 12 samples were analyzed to compare gene expression between different cell types or gene types. Three biological repeats were done for each experiments.

first: Jinyan

last: Bo

first: Tina

last: Bridges

first: Katherine

middle: D

last: Dugan

first: Tien-chi

last: Pan

first: Lewis

last: Chodosh

first: Denise

last: Montell


title: Wild type versus delta vts1

status: Public on Feb 15 2006

city: Los Angeles

first: Guillaume

middle: Francois

last: Chanfreau

address: 607 Charles E. Young Drive East

department: Chemistry and Biochemistry

institute: UCLA

summary:
The experiment was designed to look for genes up/downregulated in the absense of Vts1p compared to wild-type cells grown in steady state conditions.
Keywords: VTS1

overall_design: 3 chips delta VTS1 vs. 2 wild type control chips

first: Michael

last: Janis

first: Albert

last: Lee


title: NO2 study

city: Bethesda

first: Robert

last: Danner

address: 10 Center Drive

department: Critical Care Medicine Department

institute: Clinical Center

summary:
NO effects on gene expression, independent of cGMP, were examined globally. Differentiated human U937 cells that lack soluble guanylate cyclase were exposed to Snitrosoglutathione, a NO donor, or glutathione without or with dibutyryl-cAMP (Bt2cAMP). NO regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO, Bt2cAMP was associated with ARE-containing transcripts. Cell cycle genes induced by NO were G1/S phase associated (7/8) including E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO, as was E2F1 binding to E2F promoter elements. NO activated p38 MAPK, stabilizing p21 mRNA and increasing p21 protein. Subsequent increases in protein binding to CDE/CHR sites repressed key G2/M phase genes and increased the proportion of cells in G2/M. Thus, NO triggers a specific and coordinated program of cell cycle arrest independent of cGMP. Stress kinase pathways and mRNA stability are major mechanisms by which NO regulates the transcriptome.
Keywords: other

first: Xiaolin

last: Cui

first: Jianhua

last: Zhang

first: Penglin

last: Ma

first: Daniela

last: Myers

first: Ilana

middle: G

last: Goldberg

first: Kelly

last: Sittler

first: Jennifer

last: Barb

first: Peter

last: Munson

first: Ana

last: del Pilar Cintron

first: Shuibang

first: J

last: Philip McCoy


title: Enigma (CG9006) RNAi vs control RNAi in Drosophila Kc-167 cells.

status: Public on Feb 04 2006

city: Charlestown

first: Spyros

last: Artavanis-Tsakonas

address: 7308 Building 149, 13th Street

department: MGH Cancer Research Center

institute: Massachusetts General Hospital

summary:
5 day RNAi treatment to knockdown Enigma, CG9006, a Drosophila mitochondrial protein with homology to acyl-CoA dehydrogenases.
Keywords: Genetic modification

overall_design: Kc-167 cells, 5 day treatment with Enigma-specific RNAi or beta-lactamase control RNAi. Specifics:1.6X10^6 cells in 0.5mL of serum-free Sang's M3 media incubated for 1 hour with 20ug of dsRNA, subsequently equal volume of M3 media containing 10% serum was added. Cells harvested after 5 days. RNAi efficiency measured by immunoblot for Enigma.

first: Philippos

last: Mourikis

first: Gregory

last: Hurlbut

first: Spryos


title: Effect of Sleep Deprivation on the Whole Brain of Drosophila

status: Public on Feb 27 2006

first: John

last: Zimmerman

address: 125 South 31st Street

department: Center for Sleep & Respiratory Neurobiology

institute: University of Pennsylvania School of Medicine

summary:
To gain insight into the dynamic molecular processes that are altered during prolonged wakefulness and during sleep. We performed an RNA expression profiling study examining temporal changes in the brain of Drosophila in relationship to the duration of prior sleep or wakefulness.  Our experimental design allowed us to determine whether genes identified as differentially regulated between sleep and wakefulness were up- or down-regulated in these states.
Because stimulation of the experimental animal during the normal sleep period is used to prolong wakefulness in most experimental paradigms, the interpretation of the effects of prolonged wakefulness is confounded by the effect of the perturbation stimulus itself on the animal’s biology.  We controlled for this effect in our experimental paradigm by examining gene expression changes in response to identical stimulation but during the animal’s normal wakefulness. The design of our study also allowed us to control for circadian variation in gene expression, since we compared sleeping and sleep deprived flies at the same diurnal time.
Keywords: sleep deprivation, time course, stress response

overall_design:
Gene expression was measured in brains of virgin Canton-S female Drosophila kept awake during their normal consolidated sleep period by sleep deprivation (Deprived) and in brains of time-matched control flies allowed to sleep normally (SLEEP). Activity of populations of flies was monitored by measuring the number of times infrared beams were interrupted. Rest/activity patterns of all populations of flies evaluated were within previously established normal ranges for this genotype, gender, and age. Sleep deprivation was produced by manually stimulating the population monitors that housed the flies and was verified by both visual observation of the population of flies and by analysis of population activity. RNA was isolated from a pool of 139-193 brains dissected from flies after sleep deprivation durations of 0, 2, 4, or 6 hours starting at the beginning of the consolidated sleep period (ZT 14), and from brains of groups of undisturbed control flies sacrificed at the same diurnal time as the respective experimental groups.The inclusion of a 0-hr time point allowed us to determine the temporal direction of change for genes that were differentially expressed between the SLEEP and SD groups. For each group at each time-point, there were 3 biological replications. Gene expression was evaluated using Drosophila 1.0 Affymetrix gene chips (Affymetrix, Santa Clara, CA). Normalization was carried out using the robust multi-array average (RMA) method (Irizarry et al., 2003) to form one expression measure for each gene on each array. Differences in gene expression between the SLEEP and SD groups at 2, 4, and 6 hours were analyzed using a one-way ANOVA based on the Fs statistic (Cui et al., 2005). P-values were calculated by permuting model residuals 1000 times and subsequently adjusted using a false discovery rate (FDR) algorithm (Storey, 2002). At a FDR of 10%, 578 genes were determined to be differentially expressed between SLEEP and SD groups, and at an FDR of 5%, 252 genes were differentially expressed.   We used the more stringent 5% FDR for subsequent analysis.
In addition, to control for the effect of handling the flies during the sleep deprivation, we manually stimulated the flies during their active period from ZT10-ZT14. Gene expression in brains isolated from these active period stimulation (APS) flies was compared to gene expression in brains isolated from unperturbed flies sacrificed at the same diurnal time (WAKE). We chose to identify genes differentially expressed between APS and WAKE by analyzing only the subset of expression data from the active period stimulation experiment for the 252 genes identified in the sleep deprivation experiment since this was our primary analysis. Here, we used the ANOVA analysis without correcting for multiple testing due to the small number of tests (252) (P<0.01) and to avoid underestimating the effects of manual stimulation.

first: Wendy

last: Rizzo

first: David

middle: M

last: Raizen

first: Miroslaw

last: Mackiewicz

first: Keith

middle: R

last: Shockley

first: Gary

last: Churchill

first: Allan

middle: I

last: Pack


title: LD/DD time course of y w; tim01 Drosophila #2

status: Public on Feb 03 2006

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:
Background. The transcriptional circuits of circadian clocks control physiological
and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by
entraining the clock circuits and (2) via clock-independent molecular pathways. In
this study we examine the relationship between autonomous transcript
oscillations and light-driven transcript responses
Methodology/Principal Findings. Transcript profiles of wild-type and arrhythmic
mutant Drosophila were recorded both in the presence of an environmental
photocycle and in constant darkness. Systematic autonomous oscillations in the
12-48 hr period range were detectable only in wild-type flies and occurred
preferentially at the circadian period length. However, an extensive program of
light-driven expression was confirmed in arrhythmic mutant flies. Many lightresponsive
transcripts are preferentially expressed in the adult compound eye
and the phospholipase C component of phototransduction, NO RECEPTOR
POTENTIAL (NORPA), is required for their light-dependent regulation.
Conclusions/Significance. Although there is growing evidence for the existence
of multiple molecular clock circuits in plants and fungi, Drosophila appears to
possess only one such system. The sustained photic expression responses
identified here are partially coupled to the circadian clock and may reflect a
mechanism for flies to modulate functions such as visual sensitivity and synaptic
transmission in response to seasonal changes in photoperiod.
Keywords: Time course

overall_design:
y w; tim01 flies that had been kept in a 12-hr light/ 12-hr dark cycle for three days were harvested every four hours during an additional light/dark day (ZT) and a subsequent day in constant darkness (CT). Relative to Zeitgeber time 0 (ZT0) as the time of lights on
during the LD cycle and Circadian time 0 (CT0) as the time corresponding to
subjective lights-on during freerun in DD, time courses were collected in a ZT2-
ZT6-ZT10-ZT14-ZT18-ZT22-CT2-CT6-CT10-CT14-CT18-CT22 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: Michael

middle: W

last: Young


title: Functional Genomic Analysis of Commercial Baker’s Yeast during Initial Stages of Model Dough-Fermentation

status: Public on Feb 24 2006

city: Tsukuba

first: Jun

last: Shima

state: Ibaraki

address: 1-2-12 Kannondai

department: Applied Microbiology

country: Japan

institute: National Food Research Institute

summary:
Gene expression profiles of baker’s yeast during initial dough-fermentation were investigated using liquid fermentation media to obtain insights at the molecular level into rapid adaptation mechanisms of baker’s yeast. Results showed that onset of fermentation caused drastic changes in gene expression profiles within 15 min. Genes involved in the tricarboxylic acid (TCA) cycle were down-regulated and genes involved in glycolysis were up-regulated, indicating a metabolic shift from respiration to fermentation. Genes involved in ethanol production (PDC genes and ADH1), in glycerol synthesis (GPD1 and HOR2), and in low-affinity hexose transporters (HXT1 and HXT3) were up-regulated at the beginning of model dough-fermentation. Among genes up-regulated at 15 min, several genes classified as transcription were down-regulated within 30 min. These down-regulated genes are involved in messenger RNA splicing and ribosomal protein biogenesis, in zinc finger transcription factor proteins, and in transcriptional regulator (SRB8, MIG1). In contrast, genes involved in amino acid metabolism and in vitamin metabolism, such as arginine biosynthesis, riboflavin biosynthesis, and thiamin biosynthesis, were subsequently up-regulated after 30 min. Interestingly, the genes involved in the unfolded protein response (UPR) pathway were also subsequently up-regulated. Our study presents the first overall description of the transcriptional response of baker’s yeast during dough-fermentation, and will thus help clarify genomic responses to various stresses during commercial fermentation processes.
Keywords: fermentation

overall_design: Saccharomyces cerevisiae T128 was used as a model of typical commercial baker’s yeast used in Japan. After 18 h cultivation, cells in stationary phase were collected by centrifugation (2,700g for 5 min). Some of the cell pellets were suspended in 900 ml of sterilized water. Cells for no-fermentation control were harvested after the fed-butch cultivation and stored until RNA extraction. Cell pellets (11,700 OD units) were suspended in 390 ml of lequid fermentation (LF) medium in a 500-ml flask and then fermented for 300 min. To investigate gene expression profiles during initial stages of dough-fermentation, cell samples for DNA microarray analysis were obtained from each culture medium at 15 min, 30 min, and 60 min. Cells in stationary phase were then collected by centrifugation (2,700g for 5 min), and stored until RNA extraction.

first: Fumiko

last: Tanaka

first: Akira

last: Ando

first: Toshihide

last: Nakamura

first: Hiroshi

last: Takagi


title: LD/DD time course of y w; tim01 Drosophila #1


title: LD/DD time course of y w Drosophila #3

summary:
Background. The transcriptional circuits of circadian clocks control physiological
and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by
entraining the clock circuits and (2) via clock-independent molecular pathways. In
this study we examine the relationship between autonomous transcript
oscillations and light-driven transcript responses
Methodology/Principal Findings. Transcript profiles of wild-type and arrhythmic
mutant Drosophila were recorded both in the presence of an environmental
photocycle and in constant darkness. Systematic autonomous oscillations in the
12-48 hr period range were detectable only in wild-type flies and occurred
preferentially at the circadian period length. However, an extensive program of
light-driven expression was confirmed in arrhythmic mutant flies. Many lightresponsive
transcripts are preferentially expressed in the adult compound eye
and the phospholipase C component of phototransduction, NO RECEPTOR
POTENTIAL (NORPA), is required for their light-dependent regulation.
Conclusions/Significance. Although there is growing evidence for the existence
of multiple molecular clock circuits in plants and fungi, Drosophila appears to
possess only one such system. The sustained photic expression responses
identified here are partially coupled to the circadian clock and may reflect a
mechanism for flies to modulate functions such as visual sensitivity and synaptic
transmission in response to seasonal changes in photoperiod. For more information see also http://biorhythm.rockefeller.edu.
Keywords: Time course

overall_design:
y w flies that had been kept in a 12-hr light/ 12-hr dark cycle for three days were harvested every four hours during an additional light/dark day (ZT) and a subsequent day in constant darkness (CT). Relative to Zeitgeber time 0 (ZT0) as the time of lights on
during the LD cycle and Circadian time 0 (CT0) as the time corresponding to
subjective lights-on during freerun in DD, time courses were collected in a ZT2-
ZT6-ZT10-ZT14-ZT18-ZT22-CT2-CT6-CT10-CT14-CT18-CT22 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: LD/DD time course of y w; tim01, cn bw, and y w Drosophila