title: Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A

status: Public on Aug 09 2011

first: Jean-Marc

last: Daran

summary: The pattern of gene transcription in Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. To investigate the requirement of PKA for glucose control of gene expression, we have analyzed global transcription in strains devoid of PKA activity. In S. cerevisiae three genes, TPK1, TPK2, TPK3, encode catalytic subunits of PKA and the triple mutant tpk1 tpk2 tpk3 is unviable. We have worked, therefore, with two strains, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4, that bear suppressor mutations,. We have identified different classes of genes that can be induced, or repressed, by glucose in the absence of PKA. Among these genes, some are also controlled by a redundant signalling pathway involving PKA activation, while others do not respond to an increase in cAMP concentration. On the other hand, among genes which do not respond to glucose in the absence of PKA, some show a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways.

overall_design: The goal of the present study was to investigate the occurrence of PKA-independent glucose signalling in S. cerevisiae. To this end, we have used global transcription analysis to study the effects of glucose on yeast strains completely devoid of PKA activity. In S. cerevisiae three genes TPK1, TPK2,and TPK3 encode catalytic subunits of PKA. While strains expressing only one of these genes grow normally, a triple null mutant (tpk1 tpk2 tpk3) is not viable (Toda et al 1987). Identification of different mutations able to suppress the growth defect of the triple mutant (Garrett and Broach 1989, Reinders et al 1998, Smith et al 1998) has allowed to determine what is the crucial function of PKA. As shown in Fig.1, PKA is needed to counteract the negative effect of the protein kinase Yak1 on yeast growth (Hartley et al 1994, Moriya et al 2001). In the presence of PKA the protein kinase Rim15 (Reinders et al 1998) and the transcription factors Msn2 and Msn4 (Görner et al 1998) can be phosphorylated and exported to the cytoplasm, transcription of the YAK1 gene, which is activated by Msn2/Msn4 (Smith et al 1998), is reduced, Yak1 levels remain low and growth is not hindered. In the absence of PKA, Rim15 remains in the nucleus where it can activate Msn2/Msn4 (Cameroni et al 2004) that turn on YAK1 transcription, thus blocking growth. This explains why strains lacking Rim15, Msn2/Msn4 or Yak1 no longer require PKA for growth. In this work we have used two isogenic strains lacking PKA and carrying the suppressor mutations msn2 msn4 or yak1. Two different suppressor mutants were used with the aim to enable a dissection of effects of the lack of PKA and effects of the suppressor mutations themselves.

first: Daniela

last: Livas

first: Juana

last: Gancedo


title: Expression data of Pseudomonas aeruginosa isolates from Cystic Fibrosis patients in Denmark

status: Public on Aug 06 2011

city: Lyngby

first: Liang

last: Yang

address: Building 301, Room 150

country: Denmark

institute: Technical University of Denmark

summary:
CF patients suffer from chronic and recurrent respiratory tract infections which eventually lead to lung failure followed by death. Pseudomonas aeruginosa is one of the major pathogens for CF patients and is the principal cause of mortality and morbidity in CF patients.
Once it gets adapted, P. aeruginosa can persist for several decades in the respiratory tracts of CF patients, overcoming host defense mechanisms as well as intensive antibiotic therapies.

overall_design:
P. aeruginosa CF strains isolated from different infection stage were selected for RNA extraction and hybridization on Affymetrix microarrays.
Two batch of P. aeruginosa CF isolates are chosen :
1) isolates from a group of patients since 1973-2008 as described in ref (PMID: 21518885);
2) isolates from a group of newly infected children as described in ref (PMID: 20406284).

first: Lei

first: Martin

middle: H

last: Rau


title: Expression data of wild-type C. elegans shifted from 23 degrees to 17 degrees

status: Public on Jun 23 2011

city: Kyoto

first: Takuma

last: Sugi

address: Yoshida Honmachi

country: Japan

institute: Kyoto University

summary:
C. elegans exhibits thermotaxis, where most of the animals that had been cultivated at a particular temperature ranging from 15°C to 25°C for a few hours with a food source and then placed on a thermal gradient for an hour migrate to the cultivation temperature. In addition, animals that were previously conditioned to migrate to a certain temperature are capable of migrating to a new cultivation temperature a few hours after the cultivation temperature was shifted to the new temperature
To gain the detailed molecular insight into thermotactic behavior, the genome-wide microarray analysis during behavioral conditioning was performed. We compared the transcriptional profile of animals conditioned to migrate to the new temperature 17°C with that of animals conditioned to migrate to the previous temperature 23°C

overall_design: Synchronized adult wild-type animals cultivated at 23 degrees were selected for RNA extraction and hybridization on Affymetrix microarrays.

first: Ikue

last: Mori


title: The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal

status: Public on Jun 27 2011

city: Lausanne

first: Sylvain

last: Pradervand

address: Genopode

department: CIG

country: Switzerland

institute: UNI Lausanne

summary:
Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum sensing regulators in P. aeruginosa.  Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS is induced by the oxygen-responsive regulator ANR when the oxygen supply decreases. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.

Keywords: genetic modification

overall_design: Comparative transcriptome analysis with the delta-phrS strains PAO6671(pJT19) and PAO6671(pJTphrS), whereby the first strain harboured the parental vector and the latter a plasmid-borne inducible phrS gene

first: Elisabeth

last: Sonnleitner

first: Nicolas

last: Gonzalez

first: Dieter

last: Haas

first: Udo

last: Blaesi


title: Expression data of Pseudomonas aeruginosa cells in planktonic or biofilm mode of growth

status: Public on Jun 17 2011

city: Bordeaux

first: Bertrand

last: Garbay

address: 146 rue Leo Saignat

country: France

institute: Université Bordeaux2

summary:
Biofilm formation by Pseudomonas aeruginosa relies on specific changes in gene expression. Some of these genes, for instance, control antibiotic resistance.
We used microarrays to detail the global programme of gene expression underlying biofilm formation and identified distinct classes of up-regulated genes during this process.

overall_design: Pseudomonas aeruginosa PAO1 cells were grown as planktonic cells in LB broth for 4 hours (PC4) or 24 hours (PC24) and sessile cels for 24 hours.

first: Patricia

last: Costaglioli

first: Sébastien

last: Vilain

first: Christophe

last: Barthe


title: Alpha Factor arrest cell cycle synchronization time-course microarray in Galactose

status: Public on Jun 11 2011

city: Stanford

first: Umut

last: Eser

state: CA

address: 337 Campus Drive

department: Biology

country: USA

institute: Stanford University

summary: Alpha Factor arrest cell cycle syncronization time-course microarray in Galactose. Time course from 0 to 40 minute.

overall_design: Time-course experiment. Samples are hybridized onto the first timepoint.

first: Melody

last: Fallaure-Fettig

first: Jan

middle: M

last: Skotheim


title: Transcriptional profiling of C. elegans DAF-19 uncovers a ciliary base-associated protein and a CDK/CCRK/LF2p-related kinase required for intraflagellar transport.

status: Public on Jul 26 2011

city: Huddinge

first: Peter

last: Swoboda

state: Stockholm

address: Hälsovägen 7

department: Department of Biosciences and Nutrition

country: Sweden

institute: Karolinska Institutet

summary:
Cilia are ubiquitous cell surface projections that modulate various sensory- and motility based processes and are implicated in a growing number of multi-organ genetic disorders termed ciliopathies. As new components required for cilium biogenesis and function remain unidentified, we sought to further define and validate the transcriptional targets of the ciliogenic C. elegans RFX transcription factor DAF-19. To this end, transcriptional profiling of daf-19 mutants (which do not form cilia) and wild-type animals was performed using selectively staged embryos where ciliogenesis occurs in most ciliated sensory neurons
(CSNs). Statistical comparisons between the two populations revealed 881 differentially regulated genes with 1.5-fold change or greater. A subset of these was confirmed by quantitative RT-PCR. Transgenic worms expressing transcriptional-GFP fusions revealed CSN-specific expression patterns for 9 of 12 candidate genes. We show that two uncharacterized candidate genes, which we term dyf-17 and dyf-18 because their
corresponding mutants display dye-filling (Dyf) defects, are important for ciliogenesis. DYF-17 localizes at the base of cilia and interestingly, is specifically required for building the distal segment of sensory cilia. DYF-18 is an evolutionarily conserved CDK-7/CCRK-related serine-threonine kinase that is necessary for the proper function of intraflagellar transport (IFT), a process critical for cilium biogenesis. Together, our comparative microarray study identifies targets of the evolutionarily conserved RFX transcription factor, DAF-19, providing a rich dataset from which to uncover—in addition to DYF-17 and DYF-18—cellular components important for cilium formation and function.

first: Prasad

last: Phirke

first: Evgeni

last: Efimenko

first: Swetha

last: Mohan

last: Burghoorn

first: Filip

last: Crona

first: Mathieu

middle: W

last: Bakhoum

first: Maria

last: Trieb

first: Kim

last: Schuske

first: Erik

last: Jorgensen

first: Brian

middle: P

last: Piasecki

first: Michel

middle: R

last: Leroux


title: Cell cycle and G1 cyclins

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

overall_design: Refer to individual Series


title: Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans

status: Public on Jul 08 2011

city: Palo Alto

first: Eric

middle: Lieberman

last: Greer

address: Alway M314 300 Pasteur Drive

department: Genetics

summary: The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family1, 2, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals3. Regulators of histone methylation have been associated with ageing in worms4, 5, 6, 7 and flies8, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex9, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex—ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2—extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation—a mark associated with active chromatin—is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

overall_design: There are 23 samples in total. ASH-2 knock-down increases lifespan in a germline dependent manner. We examined ASH-2 regulated genes that are dependent on the presence of an intact germline using WT or glp-1(e2141ts) mutant worms which develop only 5-15 meiotic germ cells treated with either empty vector (EV) or ash-2 RNAi. We examined gene expression at the L3 stage (when we observe changes in H3K4me3) and at a mid life stage (day 8). The majority of ASH-2 controlled genes were regulated in a germline dependent manner. Samples were collected in triplicate for each condition (but Day 8 N2 (WT) E.V. #3 was excluded from all analysis due to quality issues).

middle: L

first: Anne

last: Brunet


title: Lack of G1 cyclin arrest cell cycle synchronization time-course microarray in Glucose

last: Eser

summary: Lack of G1 cyclin arrest cln1,2,3-delta MET3pr-CLN2 cells  Cell cycle syncronization time-course microarray in Glucose.


title: Transcriptional and proteomic response of Pseudomonas aeruginosa PAO1 to spaceflight conditions involves Hfq regulation  and reveals a role for oxygen

status: Public on Jun 28 2011

city: Mol

first: Rob

last: Van Houdt

address: Boeretang 200

department: Interdisciplinary Biosciences

country: Belgium

institute: SCK-CEN

summary: Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further, this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo, and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat, Pseudomonas aeruginosa. Overall, P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium, Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA), involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed, among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth, in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments, in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions, were hypothesized to be at the origin of these observations. Collectively, our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen, results that are of importance for infectious disease risk assessment and prevention, both during spaceflight missions and in the clinic.

overall_design: This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end, P. aeruginosa cultures were grown in space, and the expression profile was compared with ground control samples (both in biological triplicate). Two RWV samples also examined (did not re-analyze them, only compared the outputs).

first: Aurélie

last: Crabbé

first: Michael

middle: J

last: Schurr

first: Pieter

last: Monsieurs

first: Lisa

last: Morici

first: Jill

first: James

last: Wilson

first: Mark

middle: C

last: Ott

first: George

last: Tsaprailis

first: Heidi

last: Stefanyshyn-Piper

first: Cheryl

middle: A

last: Nickerson


title: Pseudomonas aeruginosa two-component regulator BfmR controls bacteriophage lysis and DNA release during biofilm development through PhdA

status: Public on Jun 01 2011

city: Aurora

state: CO

department: Microbiology

institute: University of Colorado School of Medicine

summary: Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of polysaccharides, proteins, and extracelluar (e)DNA, with eDNA being required for the formation and integrity of biofilms. Here we demonstrate that the spatial and temporal release of eDNA is regulated by BfmR, a regulator essential for Pseudomonas aeruginosa biofilm development. The expression of bfmR coincided with localized cell death and DNA release, with high eDNA concentrations localized to the outer part of microcolonies in the form of a ring and as a cap on small clusters. Additionally, eDNA release and cell lysis increased significantly following bfmR inactivation. Genome-wide transcriptional profiling indicated that bfmR was required for repression of genes associated with bacteriophage assembly and bacteriophage-mediated lysis. In order to determine which of these genes were directly regulated by BfmR, we utilized chromatin immunoprecipitation (ChIP) analysis to identify the promoter of PA0691, termed here phdA, encoding a previously undescribed homologue of the prevent-host-death (Phd) family of proteins. Lack of phdA expression coincided with impaired biofilm development, increased cell death and bacteriophage release, a phenotype comparable to ΔbfmR. Expression of phdA in ΔbfmR biofilms restored eDNA release, cell lysis, release of bacteriophages, and biofilm formation to wild type levels. Moreover, overexpression of phdA rendered P. aeruginosa resistant to lysis mediated by superinfective bacteriophage Pf4 which was only detected in biofilms. The expression of bfmR was stimulated by conditions resulting in membrane perturbation and cell lysis. Thus, we propose that BfmR regulates biofilm development by controlling bacteriophage-mediated lysis and thus, cell death and eDNA release, via PhdA.

overall_design:
For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and ΔbfmR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009).  Cells were treated with RNAprotect (Qiagen), and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions.  RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment.  Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.

Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton & D. G. Davies, (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184: 1140-1154.
Sauer, K., M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies & P. Gilbert, (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186: 7312-7326.
Petrova, O. E. & K. Sauer, (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathogens 5: e1000668.

first: Olga

middle: E

last: Petrova

first: Karin

last: Sauer


title: Caenorhabditis elegans infection with Candida albicans

status: Public on Jun 04 2011

city: Boston

first: Read

last: Pukkila-Worley

state: MA

address: 55 Fruit Street

department: Molecular Biology and Medicine

institute: Massachusetts General Hospital

summary: N2 young adult animals were analyzed four hours after exposure to wild-type Candida albicans DAY185, heat-killed C. albicans DAY185 and heat-killed Escherichia coli OP50, all on Brain Heart Infusion (BHI) agar.  It was necessary to use heat-killed E. coli OP50 as a control for these experiments because live E. coli OP50 (the normal nematode food source) is pathogenic to nematodes on BHI agar.  These data identify the C. elegans genes that are differentially regulated during nematode infection with a human fungal pathogen.

overall_design: There are nine samples total that comprise three biological replicates of animals exposed to live C. albicans, heat-killed C. albicans and heat-killed E. coli.  For a given biological replicate, young adult N2 C. elegans were exposed to live C. albicans, heat-killed C. albicans and heat-killed E. coli in parallel to each other.

first: Frederick

last: Ausubel

first: Eleftherios

last: Mylonakis


title: Two Forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway - I

status: Public on Jun 06 2011

city: Bethesda

first: Shaad

middle: M.

last: Ahmad

state: MD

address: Building 10, Room 7N311, 10 Center Drive MSC 1654

department: Systems Biology Center

institute: National Heart Lung and Blood Institute

summary: The development of a complex organ requires the specification of appropriate numbers of each of its constituent cell types, as well as their proper differentiation and correct positioning relative to each other.  During Drosophila cardiogenesis, all three of these processes are controlled by jumeau (jumu) and Checkpoint suppressor homologue (CHES-1-like), two genes encoding forkhead transcription factors that we discovered utilizing an integrated genetic, genomic and computational strategy for identifying novel genes expressed in the developing Drosophila heart.  Both jumu and CHES-1-like are required during asymmetric cell division for the derivation of two distinct cardiac cell types from their mutual precursor, and in symmetric cell divisions that produce yet a third type of heart cell.  jumu and CHES-1-like control the division of cardiac progenitors by regulating the activity of Polo, a kinase involved in multiple steps of mitosis.  This pathway demonstrates how transcription factors integrate diverse developmental processes during organogenesis.

overall_design: GFP-positive and GFP-negative cells were profiled from Stage 11 TinD-GFP Drosophila embryos.

first: Terese

last: Tansey

first: Brian

last: Busser

middle: T

last: Nolte

first: Neal

last: Jeffries

first: Stephen

middle: S

last: Gisselbrecht

first: Nasser

last: Rusan

first: Alan

last: Michelson


title: Alpha Factor arrest cell cycle synchronization time-course microarray in Glucose

first: Umut

last: Eser

summary: Alpha Factor arrest cell cycle syncronization time-course microarray in Glucose. Time course from 0 to 60 minute.

first: Melody

last: Fallaure-Fettig

first: Jan

last: Skotheim


title: Peroxireduxin-4 is over-expressed in colon cancer and its down-regulation leads to apoptosis

city: Vienna

first: Norbert

last: Schweifer

address: Dr. Boehringer Gasse 5-11

country: Austria

institute: Boehringer Ingelheim Austria

summary: The objective of this study was to gain insights into the biological basis of colon cancer progression by characterizing gene expression differences between normal colon epithelium, corresponding colorectal primary tumors and metastases. We found a close similarity in gene expression patterns between primary tumors and metastases, indicating a correlation between gene expression and morphological characteristics. PRDX4 was identified as highly expressed both in primary colon tumors and metastases, and selected for further characterization. Our study revealed that Prdx4 (PrxIV, AOE372) shows functional similarities to other Prx family members by negatively effecting apoptosis induction in tumor cells. In addition, our studies link Prdx4 with Hif-1α, a key regulatory factor of angiogenesis. Targeting Prdx4 may be an attractive approach in cancer therapy, as its inhibition is expected to lead to induction of apoptosis and blockade of Hif-1α-mediated tumor angiogenesis.

overall_design: mRNA expression profiling of normal colon epithelium (5), primary colon tumors (12 with 1 replicate) and either lymph node metastases (9 with 2 replicates) or liver metastases (2) and cell lines (4) from twelve colon cancer patients.

first: Sandra

last: Leydold

first: Michael

last: Seewald

first: Christian

last: Stratowa

first: Klaus

last: Kaserer

first: Wolfgang

last: Sommergruber

last: Kraut


title: The YJR127C/ZMS1 gene product is involved in glycerol-based respiratory growth of the yeast Saccharomyces cerevisiae.

status: Public on May 26 2011

city: Detroit

first: George

middle: Gordon

last: Roberts III

state: MI

address: 540 E. Canfield

department: Immunology/Microbiology

institute: Wayne State University

summary: A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the DeltaYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions.

overall_design:
S cerevisiae strain BY4741 and the BY4741 Δrsf2* mutant were grown in medium containing glycerol as the sole carbon source. mRNA was prepared at A600=0.6. Three biological replicates of the wild-type and two of the mutant were prepared.

*this locus has also been designated YJR127C and ZMS1

first: Lin

last: Lu

middle: G

last: Roberts

first: Cynthia

last: Oszust

last: Hudson


title: Genome wide transcriptional analysis of P. aeruginosa PAO1, response to phosphate limitation

status: Public on Jul 27 2011

city: Chicago

first: Olga

middle: Y

last: ZABORINA

state: IL

address: 5841 S Maryland

department: Surgery

institute: University of Chicago

summary:
P. aeruginosa PAO1 grown as lawns on Nematode Growth Medium prepared without supplementation (NGM Pi<0.1 mM) has high killing ability against C. elegans, however, no mortality in worms has been observed during 48 hrs when feeding on PAO1 lawns grown on phosphate supplemented full NGM Pi 25 mM, pH 6.0 medium.
We used a microarray to define the virulence-related genes in P. aeruginosa grown as lawns in NGM Pi<0.1 mM vs NGM Pi25 mM pH 6.0

overall_design: All samples for gene expression analysis were prepared in biological triplicate. P. aeruginosa MPAO1 cells collected from lawns grown on NGM/[Pi]25 mM, pH 6.0 or  NGM/Pi<0.1 mM were used for RNA isolation. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility

first: John

last: Alverdy

last: Zaborina


title: Genome-wide expression analysis during aging in C. elegans

status: Public on May 11 2011

city: Cambridge

first: Matthew

last: Youngman

address: 77 Massachusetts Ave. (68-440D)

institute: MIT

summary: Aging has been shown to be under genetic control in C. elegans. We performed Affymetrix micorarray-based transcriptional profililng of wild type C. elegans strain Bristol N2 during aging to detect temporal changes in gene expression.

overall_design: RNA was prepared for hybridization to Affy microarrays from synchronized populations of C. elegans at three points during aging: 1) the late larval L4 stage, 2) day 6 of adulthood, and 3) day 15 of adulthood.

first: Dennis

middle: H

last: Kim


title: Expression data from C. elegans (wild type vs. tag-24)

status: Public on Apr 21 2011

city: Durham

first: Alejandro

last: Aballay

state: North Carolina

address: 268 Jones

department: Molecular Genetics and Microbiology

institute: Duke University

summary: To provide insights into the mechanism underlying the enhanced immunity of tag-24/octr-1  animals, we used genome microarrays to find clusters of genes commonly misregulated in tag-24 relative to wild-type animals grown on live P. aeruginosa.

overall_design: Gravid adult wild-type and tag-24/octr-1 animals were lysed using a solution of sodium hydroxide and bleach (ratio of 5:2), washed and the eggs were synchronized for 22 hours in S basal liquid medium at room temperature. Synchronized L1 larval animals were placed onto NGM plates seeded with E. coli OP50 and grown at 20°C until the animals had reached the L4 larval stage. Animals were collected and washed with M9 buffer before transferring to NGM plates containing P. aeruginosa PA14 for 4 hours at 25°C. After 4 hours, animals were collected and washed with M9 buffer, and RNA was extracted using TRIzol reagent (Invitrogen). Residual genomic DNA was removed by DNase treatment (Ambion Austin, TX).


title: Transcriptome analysis of arabinose fermentation by engineered Saccharomyces cerevisiae

status: Public on Mar 01 2011

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

summary: Saccharomyces cerevisiae IMS0002 which, after metabolic and evolutionary engineering, ferments the pentose sugar arabinose. Glucose and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor IMS0001 were subjected to transcriptome analysis to identify key genetic changes contributing to efficient arabinose utilization by strain IMS0002.

overall_design: Glucose- and arabinose limited anaerobic chemostat cultivation of strains IMS0002 and glucose limited IMS0001 at D= 0.03 h-1

first: Wouter

last: Wisselink

first: Chiara

last: Cipollina

first: Bart

last: Oud


title: Transcriptional response on ccw12 mutant from S. cerevisiae

status: Public on Mar 16 2011

city: Heidelberg

first: Sabine

last: Strahl

address: Im Neuenheimer Feld 360

department: Heidelberg Institute for Plant Sciences

country: Germany

institute: University of Heidelberg

summary: Ccw12p is a cell surface mannoprotein required for cell wall stability. To investigate the compensation mechanism after CCW12 deletion we analysed the global gene expression in ccw12 mutant cells.

overall_design: SEY6210 and MEY12A (SEY6210 deleted for CCW12) yeast strains were growth on YPD medium supplemented with adenine. RNA was extracted from two biological replicates of each strain after identical culturing. The RNA samples were analyzed using Affymetrix YG S98 DNA microarrays and transcriptional profiles of MEY12A vs SEY6210 were compared

first: E

last: Ragni

first: H

last: Piberger

first: C

last: Neupert

first: J

middle: M

last: Garcia-Cantalejo

first: L

last: Popolo

last: Arroyo

first: M

last: Aebi

first: S


title: Germline and embryo gene expression of wild-type vs. mutants in lin-54, a component of the C. elegans DRM complex

status: Public on Apr 09 2011

city: Worcester

first: Kirsten

last: Hagstrom

address: 377 Plantation street

department: Molecular Medicine

institute: University of Massachusetts, Medical School

summary: DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we perform microarray expression profiling analysis of lin-54, a DNA-binding member of the DRM complex. To identify genes regulated by LIN-54 in soma and germline, we analyzed wild-type and lin-54 mutant C. elegans embryos and isolated germlines. We chose embryos because they consist primarily of somatic cells, at a developmental stage with both active cell divisions and dynamic developmental gene expression programs. Since lin-54 null animals are sterile, embryos were obtained from a strain carrying the partial loss-of-function allele lin-54(n2990). Germlines were dissected from lin-54(n3423) null adults that lack detectable  transcript and protein. The results revealed conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, genomics and cytological analyses show that DRM binding, a DRM binding motif, and LIN-54-regulated genes are all autosome-enriched. One paradoxical exception occurs the germline, where DRM binds autosomes but genes down-regulated in DRM mutants are enriched on X chromosomes.

overall_design: We compared embryonic or germline gene expression profile of lin-54 mutants with that of wild-type N2 C. elegans. Embryos were obtained from a strain carrying the partial loss-of-function allele lin-54(n2990) grown at 25C for one generation. Germlines were isolated from lin-54(n3423) null adults that lack detectable lin-54 transcript and protein. We isolated the germline region from the tip until late pachytene stage of meiosis, because nuclei in this region are morphologically similar between wild-type and mutant and are all undergoing X chromosome silencing. 3 biological replicates of each genotype/tissue were examined.

first: K


title: Transcriptome profiling of Saccharomyces cerevisiae during a transition from fermentative to glycerol-based respiratory growth reveals extensive metabolic and structural remodeling.

first: George

last: Roberts III

summary: Transcriptome analyses using a wild-type strain of Saccharomyces cerevisiae were performed to assess the overall pattern of gene expression during the transition from glucose-based fermentative to glycerol-based respiratory growth. These experiments revealed a complex suite of metabolic and structural changes associated with the adaptation process. Alterations in gene expression leading to remodeling of various membrane transport systems and the cortical actin cytoskeleton were observed. Transition to respiratory growth was accompanied by alterations in transcript patterns demonstrating not only a general stress response, as seen in earlier studies, but also the oxidative and osmotic stress responses. In some contrast to earlier studies, these experiments identified modulation of expression for many genes specifying transcription factors during the transition to glycerol-based growth. Importantly and unexpectedly, an ordered series of changes was seen in transcript levels from genes encoding components of the TFIID, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and SLIK (Saga LIKe) complexes and all three RNA polymerases, suggesting a modulation of structure for the basal transcriptional machinery during adaptation to respiratory growth. In concert with data given in earlier studies, the results presented here highlight important aspects of metabolic and other adaptations to respiratory growth in yeast that are common to utilization of multiple carbon sources. Importantly, they also identify aspects specific to adaptation of this organism to growth on glycerol as sole carbon source.

overall_design: A time-series illustrating the transition from fermentation using glucose (dextrose) as sole carbon source to respiration using glycerol as sole carbon source. Time points of 15 minutes, 30 minutes and 60 minutes as well as growth on dextrose, glycerol or ethanol as sole carbon source from a starter culture. Three biological replicates of the 30 minute time point and growth on glycerol from starter culture.

first: Alan

last: Hudson


title: Chromosome-biased binding and gene regulation by the C. elegans DRM complex

status: Public on Apr 27 2011

type: Genome binding/occupancy profiling by genome tiling array

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


title: The effect of IAN on P. aeruginosa virulence

status: Public on Feb 20 2011

city: Gyeongsan-Si

first: Jintae

last: Lee

state: Gyeongsangbuk-Do

address: 214-1 Daedong

department: Chemical engineering

country: South Korea

institute: Yeungnam University

summary: Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence.  However, indole and bacterial indole derivatives were unstable in microbial community due to the widespread of diverse oxygenases that could quickly degrade them.  Hence, we sought to identify novel non-toxic, stable, and potent indole derivatives from plant sources for inhibiting biofilm formation of E. coli O157:H7 and P. aeruginosa PAO1.  Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth.  IAN inhibited biofilms more effectively than indole for both E. coli and P. aeruginosa.  Additionally, IAN decreased the production of virulence factor pyocyanin in P. aeruginosa.  DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, while IAN induced indole-related genes and prophage genes in E. coli.  It appears that IAN inhibits biofilm formation of E. coli by reducing curli formation and inducing indole production.  Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa.

overall_design: For the microarray experiments, P. aeruginosa were inoculated in 25 ml of LB medium in 250 ml shake flasks with overnight cultures that were diluted 1:100.  IAN (100 μg/ml) dissolved in 25 μl DMSO or 25 μl DMSO alone as a control was added.  Cells were cultured in LB at 37C with 250 rpm shakingfor 5 hrs.  Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C.  RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA).  RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).

first: Jin-Hyung


title: Fed L1 larvae total RNA levels by microarray

city: Berkeley

first: Barbara

middle: J.

last: Meyer

address: 16 Barker Hall #3204

department: MCB

institute: HHMI/UCB

summary: Here we exploit the essential process of X‐chromosome dosage compensation to elucidate  basic mechanisms that control the assembly, genome‐wide binding, and function of gene  regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription.

overall_design: Total RNA was extracted from fed L1 worms in order to compare total RNA levels with that of mixed embryos.

first: Rebecca

last: Pferdehirt

first: William

last: Kruesi


title: Expression data from transgenic Drosophila melanogaster adults which contain a knockdown effector of cyp6g1, compared to control flies

status: Public on Feb 17 2011

city: RTP

first: Sheetal

last: Shah

state: NC

address: 3054E Cornwallis Road

institute: Syngenta

summary:
To test whether other genes were being silenced in the Cyp6g1 knockdown strain due to off-target RNAi effects, and whether other gene expression changes were contributing to the altered susceptibility to imidacloprid in these knockdown flies. A comparison between w;Act5C-GAL4/CyO; UAS:RNAi_Cyp6g1Hp2/TM3Sb and the genetic control w;Act-GAL4/CyO;+/TM3Sb was performed. Ten 2-3 day old adult males or females were transferred to sugar-agar plates and then collected at various time points (0, 2, 5, 8 hours). The RNA samples for up to three independent experiments per timepoint for each genotype were then pooled, in equal concentrations, before hybridisation to the Affymetrix Drosophila GeneChip 1.
As a preliminary analysis provided evidence for assuming that there is no interaction between genotype and duration in the sugar-agar plate, the  knockdown and control chips for each of the five timepoints provided some degree of replication for the comparison between the knockdown flies and control flies.  Differential expression that was consistently observed between knockdown and control over each of the five timepoints was regarded as providing evidence of a genuine difference.

overall_design: 2-3 day old adult flies of the appropriate genotype (Cyp6g1 knockdown or its genetic control) were transferred to bioassay plates and collected after specified timepoints in the assay plate, i.e. 0, 2, 5 or 8 hours. Each experiment contained ≥3 replicates of each genotype/timepoint, and a different number of independent experiments (1-3) were pooled per microarray sample as described below.   An experiment is defined as treatments set up from the same cohort of flies on a single day. Independent experiments are set up using distinct cohorts of flies, on distinct days.   We included one pair of tests to compare samples from single experiments with pooled samples from independent experiments (DS003001 to DS003004, DS003002 to DS003005), and also a single pair of samples to compare males to females (DS003002 to DS003003).  As it turned out - in our analysis - the timepoints selected were not discriminative, in that there were no differences detected that could be attributed to an effect of time in the bioassay.

first: Carol

last: Yarrow


title: An MLL/COMPASS subunit functions in the C. elegans dosage compensation complex to target X chromosomes for transcriptional regulation of gene expression

overall_design: Refer to individual Series


title: Using Model Organism Saccharomyces Cerevisiae to Evaluate the Differential Effects of ELF-MF and RF-EMF exposure on Global Gene Expression

status: Public on Feb 15 2011

city: Hangzhou

first: Guangdi

last: Chen

state: Zhejiang

address: 388 Yuhangtang

department: School of Medicine

country: China

institute: Zhejiang University

summary:
The potential health hazard of exposures to electromagnetic fields (EMF) continues to cause much public concern. However, the biological and health effects of exposures to EMF remain controversial and their biophysical mechanisms are unclear.
In the present study, we used Saccharomyces cerevisiae to identify genes responding to extremely low frequency magnetic fields (ELF-MF) and to radiofrequency (RF) EMF exposures. The expression of genes was analyzed by microarray screening and confirmed by real-time reverse transcription -polymerase chain reaction. In confirmation experiments, we found that there was no statistically significant change in three of the ELF-MF responsive candidate genes (P>0.05). On the other hand, out of the forty genes that responded to RF-EMF, the confirmation experiments found that only five were affected: structural maintenance of chromosomes 3-gene (SMC3), aquaporin 2 –gene (AQY2), halotolerance protein 9 –gene (HAL9), yet another kinase 1 -gene (YAK1) and one of unknown function gene (open reading frame: YJL171C) (P<0.05). Overall, this study has demonstrated that the yeast cells did not respond to 50 Hz ELF-MF and that the response to RF-EMF is limited to only five genes. The biological consequences of the observed gene expression changes induced by RF-EMF await further investigation.

overall_design: The yeast cells were exposed to 0.4 mT 50 Hz ELF-MF or 1800 MHz RF-EMF at a specific absorption rate of 3.5 W/kg for 6 hours.

first: Zhengping

last: Xu


title: Gene expression profile of pellicle cells of Pseudomonas aeruginosa

status: Public on Mar 31 2011

city: Bunkyo-ku

first: Hiroyuki

last: Arai

state: Tokyo

address: Yayoi 1-1-1

institute: The University of Tokyo

summary: To investigate the gene expression profile of pellicle cells of Pseudomonas aeruginosa, microarray analysis was performed. Transcriptome profiles of pellicle cells and planktonic cells grown in LB medium were determined by Affymetrix GeneChip. Gene expression pattern that is specific to pellicle cells was evaluated by comparing the data set with that of planktonic cells.

overall_design: Pseudomonas aeruginosa wild type (PAO1ut) strain was cultivated aerobically in LB in Erlenmeyer flasks under static or shaking conditions, and total RNAs were extracted at 24 hours (static culture) and early stationary phase (OD600 = 1.4, shaking culture). The experiment was performed in duplicate independent cultures.

first: Kyosuke

last: Yamamoto


title: Loss of subcellular lipid transport due to ARV1 deficiency disrupts organelle homeostasis and activates the unfolded protein response

city: New York

first: Stephen

last: Sturley

state: NY

address: 630 West 168th Street, PH1512 East

department: Department of Pediatrics

institute: Columbia University

summary: The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the UPR. Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest a component of UPR induction in arv1? strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, C/EBP homologous protein (CHOP) and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis resulting in a disruption of ER integrity, one consequence of which is induction of the UPR.

overall_design: Yeast strains were grown to mid-logarithmic stage (A600=0.5-0.6) for RNA extraction and hybridization on Ye6100 or S98 Affymetrix gene chips. The control array (sample name C) was carried out in duplicate.  The ARV1 mutant array (sample name A) was carried out in triplicate.  Both ARV1 mutants Ye6100 arrays were compared to the same control Ye6100 array. Strains represent different isolates of the same genotype, mating type and genetic background (w303). Sturley lab collection (SCY) strains include SCY328 and SCY2004 for controls and SCY820 and SCY840 for ARV1 mutants.


title: RNA immunoprecipitation of GLD-1 followed by microarray analysis of the co-IP'ed mRNAs

status: Public on Feb 04 2011

city: Basel

first: Dimos

last: Gaidatzis

address: Maulbeerstrasse 66

institute: Friedrich Miescher Institute

type:
Other
Expression profiling by array

summary: RNA-binding proteins (RBPs) are critical regulators of gene expression and elucidating the interactions of RBPs with their RNA targets is necessary to understand how combinations of RBPs control transcriptome expression. The Quaking-related (QR) sub-family of STAR domain RBPs includes developmental regulators and tumor suppressors such as C. elegans GLD-1, which functions as a master regulator of germ line development. To understand how GLD-1 interacts with the transcriptome, we identified GLD-1 associated mRNAs by a ribonomic approach. The scale of GLD-1 mRNA interactions allowed us to determine rules governing GLD-1 target selection and to derive a predictive model where GLD-1 association with mRNA is based on the number and strength of 7-mer GLD-1 binding elements (GBEs) within UTRs. GLD-1/mRNA interactions were quantified, and predictions were verified both in vitro and in live animals, including by ‘transplantation’ experiments where ‘weak’ and ‘strong’ GBEs imposed translational repression of increasing strength on a non-target mRNA.Importantly, this study provides a unique quantitative picture of how an RBP interacts with its mRNA targets. As combinatorial regulation by multiple RBPs is thought to regulate gene expression, quantification of RBP/mRNA interactions should be a way to predict and potentially modify biological outcomes of complex posttranscriptional regulatory networks, and our analysis suggests that such an approach is possible.

overall_design: Wild-type (N2) or gld-1(q485); ozIs2 [gld-1::gfp::flag] worms were synchronized and harvested as young adults. To identify GLD-1 associated transcripts, two independent IP strategies were performed: 1. Comparison of anti-FLAG IP to anti-MYC IP on the gld-1(q485); ozIs2 [gld-1::gfp::flag] worm extract, 2. Comparison of anti-FLAG IP on the gld-1(q485); ozIs2 [gld-1::gfp::flag] worm extract to anti-FLAG IP on wild type extract. All IPs were performed in triplicate (12 IPs).

first: Jane

last: Wright

first: Mathias

last: Senften

first: Eric

last: Westhof

first: Rafal

last: Ciosk


title: Expression Data from Ectopic expression of SUMO-1 in C. elegans

status: Public on Jan 31 2011

city: Kuopio

first: Garry

last: Wong

department: Biosciences

country: Finland

institute: Eastern Finland University

summary: Transgenic C. elegans strains that express human SUMO-1 under the control of pan-neuronal (aex-3) or pan muscular (myo-4) promoters were assayed for gene expression changes.

overall_design: Control N2 non transgenic worms were compared to aex-3::His-SUMO-1 and myo-4::His-SUMO-1 worms in 3 biological replicate populations

first: Miia

last: Rytinki

first: Merja

last: Lakso

first: Vuokko

last: Aarnio

first: Kaja

last: Reisner

first: Suvi

last: Asikainen

first: Petri

last: Pehkonen

first: Jorma

last: Palvimo


title: Lyngbyoic Acid, a "Tagged" Fatty Acid from a Marine Cyanobacterium, Disrupts Quorum Sensing in Pseudomonas aeruginosa

status: Public on Jan 24 2011

city: Gainesville

first: Jason

middle: Christopher

last: Kwan

state: FL

address: 2000 SW Archer Road

department: Medicinal Chemistry

institute: University of Florida

summary: Quorum sensing (QS) is a mechanism of bacterial gene regulation in response to increases in population density.   Production of small molecule QS signals, their accumulation within a diffusion-limited environment and their binding to the LuxR-type receptor trigger QS-controlled gene regulatory cascades. QS pathways mediated by acylhomoserine lactones (AHLs) in Gram-negative bacteria are the best studied.  In Pseudomonas aeruginosa, for example, binding of AHLs to their cognate receptors (LasR, RhlR) controls production of virulence factors, pigments, antibiotics and other behaviors important for its interactions with eukaryotic hosts and other bacteria.  We isolated a new small cyclopropane-containing fatty acid, lyngbyoic acid (1), as a major metabolite of the marine cyanobacterium, Lyngbya sp., collected off Fort Pierce, Florida.  The structure of 1 was determined by NMR, MS and optical rotation.  We screened 1 against four reporters based on AHL receptors from Vibrio fischeri (LuxR), Aeromonas hydrophila (AhyR), Agrobacterium tumefaciens (TraR) and P. aeruginosa (LasR) and found that 1 most strongly affected LasR.  We show, by using a defined set of reporters, that compound 1 acts both through the AHL-binding site of LasR and independent of it.   We also show that 1 reduces pyocyanin and LasB, both on the protein and transcript level, in wild-type P. aeruginosa, and that 1 directly inhibits LasB enzymatic activity.  Conversely, dodecanoic acid (11) increased pyocanin and LasB, demonstrating that 1 is a “tagged” fatty acid potentially resistant to β-oxidation.

overall_design: 1 mL Cultures of P. aeruginosa PAO1 were grown for 6h at 37 °C with shaking either in the presence of lyngbyoic acid (1 mM) or EtOH alone (10 μL) as a control.  RNA samples were obtained from one treated and one control culture, and were reverse transcribed, labeled and fragmented according to Affymetrix's protocol.  The two samples were hybridized in duplicate.

first: Theresa

last: Meickle

first: Dheran

last: Ladwa

first: Max

last: Teplitski

first: Valerie

last: Paul

first: Hendrik

last: Luesch


title: Contribution of CgPDR1-regulated genes in enhanced virulence of azole-resistant Candida glabrata (part 1)

status: Public on Dec 31 2010

city: Lausanne

first: Dominique

last: Sanglard

address: Rue Bugnon 48

institute: University of Lausanne and University Hospital Center

summary: In this study, we aimed to determine genome-wide changes in gene expression driven by seven individual CgPDR1 hyperactive alleles as compared to wild-type allele to identify i) the CgPdr1p target genes differentially expressed in presence of CgPDR1 hyperactive alleles and ii)  potential virulence factor(s) regulated by CgPDR1 hyperactive alleles. Microarray experiments revealed a high number of genes (ranging from 80 to 400 genes) differentially regulated by individual CgPDR1 hyperactive alleles.

overall_design: Gene expression was measured in 7 C. glabrata laboratory strains (SFY101, SFY103, SFY105, SFY109, SFY111, SFY115, SFY116) expressing different CgPDR1 hyperactive alleles, in 1 strain (SFY114) expressing a wild-type CgPDR1 allele and in one strain deleted for CgPDR1. The one-color system was used. 3 independent experiments were performed using 3 biological replicates for each strain.

first: Sélène

last: Ferrari


title: Saccharomyces cerevisiae bottom of the fermentor vs. fermentation

status: Public on Jan 14 2011

city: Ribeirão Preto

first: Patricia

middle: Alves

last: de Castro

state: São Paulo

address: Av. do Café

department: Pharmaceuticals Sciences

country: Brazil

institute: USP

summary: The main objective was to identify genes regulated during different stages of fermentation: bottom of the fermentor, feeding and the fermentation stage. The experiment was further validated by microbiological assays.

overall_design: The control refers to the bottom of the fermentor. For analysis, fermentation was compared to the control for two strains: CAT and PE-2.


title: Contribution of CgPDR1-regulated genes in enhanced virulence of azole-resistant Candida glabrata


title: Saccharomyces cerevisiae bottom of the fermentor vs. feeding and fermentation


title: Contribution of CgPDR1-regulated genes in enhanced virulence of azole-resistant Candida glabrata (part 2)

overall_design: Gene expression was measured in 2 C. glabrata clinical isolates (DSY717 and DSY2317). DSY2317 is azole-susceptible and contains a wild-type CgPDR1 allele. DSY717 is azole-resistant and contains a CgPDR1 allele with the gain-of-function mutation L1081F.


title: Loss of mitochondrial functions in Candida glabrata results in enhanced virulence in mice

summary: Two C. glabrata related isolates recovered from the same patient undergoing azole therapy were characterized. The first isolate, BPY40, is azole-susceptibleand the second, BPY41, is azole-resistant. To determine whether the petite mutation conferred a selective advantage during host infection, the virulence of BPY40 and BPY41 was assessed in mice. Surprisingly, the petite mutant, even if showing in vitro growth deficiency as compared to BPY40, was more virulent than BPY40 both in intravenous and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice as compared to its parental isolate. Genome-wide changes in gene expression driven by the petite mutation were analyzed by microarrays. Enrichment of specific biological processes (oxido-reductive metabolism, stress response) was observed in BPY41, all consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall genome-wide changes in gene expression driven by the petite mutation were analyzed by microarrays. Enrichment of specific biological processes (oxido-reductive metabolism, stress response) was observed in BPY41, all consistent with mitochondrial dysfunction.

overall_design: Gene expression was measured in 2 related C. glabrata clinical isolates (BPY40 and BPY41). The one-color system was used . 3 independent experiments were performed using 3 biological replicate for each strain.


title: Saccharomyces cerevisiae bottom of the fermentor vs. feeding

overall_design: The control refers to the bottom of the fermentor. For analysis, feeding was compared to the control for two strains: CAT and PE-2.


title: miRNA array after p63 knock down in human squamous cell carcinoma

status: Public on Nov 23 2010

city: Boston

first: benjamin

middle: robert

last: ory

address: 55 fruit street

institute: Mas General Hospital Cancer Center

summary: A central challenge in human cancer therapy is the identification of pathways that control tumor cell survival and chemosensitivity in the absence of functional p53. The p53-related transcription factors p63 and p73 exhibit distinct, p53-independent roles in development and cancer: p73 promotes genome stability and mediates chemosensitivity, while p63 largely lacks these p53-like functions and instead promotes proliferation and cell survival. Here, we identify a new and physiologically important mechanism of p63/p73 cross-talk which governs the balance between pro-survival and pro-apoptotic programs in both human and murine squamous cell carcinoma. Through comprehensive profiling of p63-regulated microRNAs (miRs), we identified a subset which target p73 for inhibition, including miR-193a-5p, a direct endogenous transcriptional target repressed by p63 and activated by pro-apoptotic p73 isoforms in both normal cells and tumor cells in vivo. Consequently, chemotherapy treatment causes p63/p73-dependent induction of this miR, thereby limiting chemosensitivity due to miR-mediated feedback control of p73. We demonstrate that interrupting this feedback by inhibiting miR-193a suppresses tumor cell viability and induces dramatic chemosensitivity both in vitro and in vivo. Thus, we have identified a direct, miR-dependent regulatory circuit mediating inducible chemoresistance, whose inhibition provides a new therapeutic opportunity in p53-deficient tumors.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus
Human alphaherpesvirus 1
Human betaherpesvirus 5
Murid betaherpesvirus 1
human gammaherpesvirus 4
JC polyomavirus
Human immunodeficiency virus 1
Murid gammaherpesvirus 4
Human gammaherpesvirus 8
Betapolyomavirus hominis
Betapolyomavirus macacae

overall_design: Knockdown of endogenous p63 by p63-directed or control lentiviral shRNA in JHU-029 human SCC cells at 48h, in duplicate experiments. Array analysis showing the fold-change and direction of change for all miRs regulated > 1.5-fold in p63-ablated versus control samples

first: Benjamin

last: Ory


title: Transcriptional effects of LMP3 in MSC

city: Rome

first: camilla

last: bernardini

address: L.go F.Vito 1

department: Human Anatomy

country: Italy

institute: UCSC

summary: Lim Mineralization Protein, splice variant-3 (LMP3) is known to induce osteoblast differentiation as a result of active modulation of selected molecules involved in the osteogenic cascade. LMP3 seems to act as an upstream modulator of osteogenic differentiation, although the complete network of molecular events involved in this process are still largely unknown. In particular, LMP3 seems to act, at least in part, through the upregulation of bone morphogenetic proteins (BMPs) which are known to exert multiple functions, leading to the regulation of diverse cellular processes. Thus, LMP actions inside the cell are likely to involve the activation of main signaling pathways, suggesting a possible wider role of the molecule in cell homeostasis. The aim of this study is to analyze the gene expression profiles exhibited by human bone marrow mesenchymal stem cells (MSCs) expressing the LMP3 gene in order to investigate the early transcriptional events preceding the acquisition of a differentiated phenotype. For this purpose, MSCs were transduced using a replication-defective adenoviral vector carrying the human LMP3 gene. Gene expression profiling was then carried out by microarray analysis one day after cell transduction. The results obtained in this study suggest that in addition to activating the TGFB1-signaling pathway, LMP3 acts on the fine balance between proliferation and differentiation of mesenchymal cells. Gene modulation directly related to the reprogramming of the transcriptional machinery induced by the viral vector is also discussed.

overall_design: For microarray analysis, cells were plated in T75 flasks and transduced with 100 MOI of either AdLMP3 or Adψ5 (mock-transduction), while untransduced cells served as negative controls. The experiment was carried out in three independent replicates, thus an overall number of 9 samples were analyzed.

first: wanda

last: lattanzi


title: AMPK and calcineurin induced longevity is mediated by CRTC-1 and CREB

status: Public on Nov 20 2010

city: La Jolla

first: Ana

middle: PC

last: Rodrigues

address: 10010 North Torrey Pines Road

institute: Salk Institute for Biological Studies

summary:
AMPK (AAK-2) and calcineurin (TAX-6) mediate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC (CREB regulated transcriptional coactivator).
We performed microarrays to examine the transcriptional responses elicited by the pro-longevity: activation of AMPK, deactivation of calcineurin, and decrease of CREB (CRH-1) activity.

overall_design: Gene expression profiles for crh1 (nn3315) and tax-6 (ok2065) mutants, aak-2c (aa1-321) overexpressers and WT (N2) controls were obtained by measuring RNA levels in replicate pools of 3000 synchronized L4 worms. Three replicate pools of each strain were prepared on separate days.

first: Will

last: Mair

first: Ianessa

last: Morantte

middle: P

first: Gerard

last: Manning

first: Marc

last: Montminy

first: Reuben

last: Shaw

first: Andrew

last: Dillin


title: Physiology of Pseudomonas aeruginosa in Biofilms Revealed by Comparative Transcriptomic Analysis.

city: Bozeman

first: Kate

last: McInnerney

state: MT

address: 109 Lewis Hall

department: Microbiology

institute: Montana State University

summary: Abstract: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and published data sets.

overall_design: Two drip flow biofilm conditions with three replicates each:  (1) baseline control at 72hrs, (2) no treatment for 12 hours past baseline. Data from these two conditions were pooled

first: James

last: Folsom

first: Lee

last: Richards

first: Betsey

last: Pitts

first: Frank

last: Roe

first: Garth

middle: D

last: Ehrlich

first: Albert

last: Parker

first: Aurelien

last: Mazurie

first: Philip

last: Stewart


title: Expression analysis in yeast model of Huntington's disease (HD)

status: Public on Nov 09 2010

city: Leicester

first: Eran

last: Tauber

address: University Road

department: Genetics

country: United Kingdom

institute: University of Leicester

summary:
Expressing a mutant fragment of huntingtin (Htt) in yeast produces several HD-relevant phenotypes.  We used microarrays to study global change in expression induced by this mutant htt fragment.
Expression was also analysed in three suppressor deletion strains expressing a toxic mutant htt fragment: bna4Δ, mbf1Δ, and ume1Δ.

overall_design: Yeast samples expressing either a wild-type or mutant htt fragment (Htt25Q or Htt103Q, respectively), and  suppressor deletion strains expressing Htt103Q: bna4Δ, mbf1Δ, and ume1Δ.

first: Flaviano

last: Giorgini


title: Comparative genomic hybridisation data from Pseudomonas aeruginosa strains isolated from cystic fibrosis lung infections

status: Public on Nov 04 2010

city: Belfast

first: Sally

middle: Helene

last: Pattison

address: MBC 97 Lisburn Road

department: Centre for Infection & Immunity

institute: Queen's University Belfast

summary:
Pseudomonas aeruginosa is a common pathogen in the lungs of the cystic fibrosis patients.  As infection develops the organism progressively adapts to its environment and its mode of pathogenesis alters, frequently including the loss of quorum sensing (QS) regulated virulence factors.
We used microarrays to determine genomic differences by comparative genome hybridisation between two P. aeruginosa isolates from CF patients, one of which exhibited an active quorum sensing (QS) system (UUPA38) typical of early acute infection while the other was QS-compromised (UUPA85) typical of chronic CF-adapted infection.

overall_design: Genomic DNA was harvested from the two isolates, fragmented using DNase I and hybridized to Affymetrix microarrays. We aimed to identify genes not present in both isolates.

first: Elena

last: Delgianni

first: John

last: Moore

first: J

last: Elborn

last: Dooley


title: Novel virulence factors in host-adapted Pseudomonas aeruginosa infection of the Cystic Fibrosis airways

type:
Expression profiling by array
Genome variation profiling by array

summary:
Pseudomonas aeruginosa airway infection is the primary cause of death in Cystic Fibrosis (CF). During early infection P. aeruginosa produces multiple virulence factors, which cause acute pulmonary disease and are largely regulated by quorum sensing (QS) intercellular signalling networks. Longitudinal clinical studies have observed the loss, through adaptive mutation, of QS and QS-related virulence in late chronic infection. Although the mechanisms are not understood, infection with QS mutants has been linked to a worse outcome for CF patients. By comparing QS-active and QS-inactive P. aeruginosa CF isolates, we have identified novel virulence factors and pathways associated with QS disruption. In particular, we noted factors implicating increased intra-phagocyte survival. Our data present novel targets as candidates for future CF therapies. Some of these targets are already the subject of drug development programmes for the treatment of other bacterial pathogens and may provide cross-over benefit to the CF population.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series.

middle: E


title: Suppression of statin effectiveness by copper and zinc in yeast and human cells

status: Public on Oct 29 2010

city: Seattle

first: Douglas

middle: McKay

last: Fowler

state: WA

address: 1705 NE Pacific St, Foege Building S313

department: Genome Sciences

institute: University of Washington

summary: Lovastatin and other statins inhibit HMG-CoA reductase, which carries out an early step in the sterol biosynthesis pathway.  Statins lower cholesterol and are widely prescribed to prevent heart disease, but like many drugs, they can interact with nutritionally acquired metabolites.  To probe these interactions, we explored the effect of a diverse library of metabolites on statin effectiveness using a Saccharomyces cerevisiae model. In yeast, treatment with lovastatin results in reduced growth.  We combined lovastatin with the library of metabolites and found that copper and zinc ions impaired the ability of the statin to inhibit yeast growth.  Using an integrated genomic and metabolomic approach, we found that lovastatin plus metal synergistically upregulated some sterol biosynthesis genes.  This altered pattern of gene expression resulted in greater flux through the sterol biosynthesis pathway and an increase in ergosterol levels.  Each sterol intermediate level was correlated with expression of the upstream gene.  Thus, the ergosterol biosynthetic response induced by statin is enhanced by copper and zinc.  In cultured mammalian cells, these metals also rescued statin growth inhibition.  Because copper and zinc impair the ability of statin to reduce sterol biosynthesis, dietary intake of these metals could have clinical relevance for statin treatment in humans.

overall_design: There are 23 total samples comprising replicates of combinations of statin (5 ug/mL), CuSO4 (1mM), and ZnSO4 (2mM) treatments.  There are three biological replicates.  Within each biological replicate set, there are two conditions that were randomly chosen to be duplicated (i.e., technical replicates). An RNA sample consisting of a mix of RNA from all 6 experimental conditions was used as the reference.

first: Stanley

last: Fields


title: Developmental Roles of MEC and NuRD Complexes in Caenorhabditis elegans.

status: Public on Oct 31 2010

city: Fribourg

first: Claude-Olivier

last: Marti

address: Chemin du Musée 10

department: Biology

institute: University of Fribourg, Zoology

summary:
Expression data from Caenorhabditis elegans let-418(RNAi), mep-1(RNAi) and gfp(RNAi) L1 larvae.
The C. elegans genome encodes two homologs of the human protein Mi-2, namely LET-418 and CHD-3. LET-418 plays an essential role during development; its depletion leads to a pleiotropic and lethal phenotype that includes larval arrest, an everted vulva and sterility. Without maternal contribution, let-418 mutants stop their development at the L1 larval stage (von Zelewsky et al., 2000). We further characterized this arrest and showed that it is very similar to the L1 diapause induced by starvation; both germline and somatic cells remain in a quiescent state in let-418 L1 arrested larvae, indicating that LET-418 activity is required to bypass the L1 arrest in presence of food. The let-418 L1 larvae express ectopically the P granule component PGL-1 in somatic cells (Unhavaithaya et al., 2002). Interestingly, the phenotype of mep-1 mutants is remarkably similar to that of let-418: RNAi targeting mep-1 also induced an L1 arrest phenotype; furthermore, MEP-1 and LET-418 have been shown to physically interact (Unhavaithaya et al., 2002 and M. Passannante). The null allele mep-1(q660) is temperature sensitive and shows a more severe phenotype at higher temperatures. At 20°C, about 10% of mep-1 homozygotes derived from heterozygous mothers arrest as young larvae, whereas the remaining 90% develop into sterile adults (Belfiore et al., 2002). Later in development, the somatic gonad is affected in mep-1(q660) mutants. This results in an abnormal and disorganized gonad, a phenotype also observed in let-418(s1617) mutants. Both let-418 and mep-1 mutants produce a very limited number of oocytes and have pseudovulvae derived from P8.p (Belfiore et al., 2002; von Zelewsky et al., 2000 and C. Wicky, personal communication).
Preliminary quantitative real-time PCR revealed that the expression of genes coding for P granule components was deregulated in both mep-1(RNAi) and let-418(RNAi) L1 larvae (data not shown). To further investigate this issue, we performed a complete gene expression analysis. Given the fact that mep-1(q660) mutants are sterile, we used RNA interference to generate mep-1 depleted worms. Bacteria expressing gfp dsRNA (pPE128.110 in HT115) were used as reference, since RNA interference may induce gene expression changes by itself.

overall_design: C. elegans L1 larvae treated with RNA interference were selected for RNA extraction and hybridization on Affymetrix microarrays. Synchronized wild type L4 animals were grown at 25° on bacteria expressing either gfp, let-418 or mep-1 dsRNA. Eggs were collected by bleaching gravid adults and allowed to hatch in the absence of food at 25°C. Newly hatched L1 larvae were fed on bacteria expressing the different dsRNA for three hours to recover from starvation. Three replicates per RNAi.

first: Marti

last: Claude-Olivier

first: Catharine

last: Aquino

first: Stefan

last: Zoller


title: RIP-chip analysis of the C. elegans GLD-2 and RNP-8 protein

status: Public on Sep 21 2010

city: Madison

first: Judith

last: Kimble

state: WI

address: 433 Babcock Drive

department: HHMI / Biochemistry

institute: University of Wisconsin-Madison

summary:
C. elegans GLD-2 forms an active PAP with multiple RNA-binding partners to regulate diverse aspects of germline and early embryonic development. One GLD-2 partner, RNP-8, was previously shown to influence oocyte fate specification. To identify transcripts selectively associated with both GLD-2 and RNP-8, we employ a genomic approach using the method of RNA immunoprecipitation followed by microarray analysis (RIP-chip).
We used microarrays to identify mRNAs selectively associated with either GLD-2 or RNP-8.

overall_design: Worm extracts were prepared from synchronized adult C. elegans (15 h after L4 stage). For GLD-2 IP, an immoblized anti-GLD-2 antibody was then used to purify the GLD-2 complexes from either wild-type (N2) or gld-2(RNAi) worm extracts. RNA was then extracted from the pellets and analyzed on C.elegans Affymetrix genechip.  Four biological replicates were performed, each sample processed in parallel. For RNP-8 IP, an immoblized anti-RNP-8 antibody was then used to purify the RNP-8 complexes from either wild-type (N2) or rnp-8(q784) worm extracts and three biological replicates were performed. For wt or gld-2(RNAi) samples, total RNA was extracted from worm extracts and hybridized on C.elegans Affymetrix genechip.

first: Kyung

middle: W

last: Kim


title: Gene expression data from Pseudomonas aeruginosa strains isolated from cystic fibrosis lung infections

summary:
Pseudomonas aeruginosa is a common pathogen in the lungs of the cystic fibrosis patients.  As infection develops the organism progressively adapts to its environment and its mode of pathogenesis alters, frequently including the loss of quorum sensing regulated virulence factors.
We used microarrays to detail differences between two P. aeruginosa isolates from CF patients, one of which (UUPA38) exhibited an active quorum sensing system (QS+) typical of early acute infection while the other (UUPA85) was QS-compromised (QS-) typical of chronic CF-adapted infection.

overall_design: Bacterial cell biomass was harvested from triplicate biofilm and planktonic cultures of each of 2 strains of P. aeruginosa.  RNA was extracted, converted to cDNA and hybridized to Affymetrix microarrays. We aimed to identify genes which were differentially transcribed between the 2 isolates during both modes of growth.

middle: S

first: James


title: Transcriptional profile of pmt1 pmt4 mutants

status: Public on Sep 30 2010

city: Madrid

first: Javier

last: Arroyo

state: Madrid

address: Plaza Ramon y Cajal S/N

department: Microbiologia II

country: Spain

institute: Facultad de Farmacia (UCM)

summary: We did transcription profiling on the effect of pmt1 pmt4 deletion, genes involved in the O-glycosylation process. These mutants cells show inhibition of mating, filamentation and induction of cell wall compensatory mechanism.

overall_design:
Two biological samples were analyzed for each condition, and a microarray experiment were carried out for each sample
1. wild type cells SEY6210
2. pmt1 pmt2 cells from SEY6210 background


title: Transcriptional profile of pmt1 pmt2 mutants

summary: We did transcription profiling on the effect of pmt1 pmt2 deletion. These genes are involved in the O-glycosylation process. These mutants cells show inhibition of mating, filamentation and induction of cell wall compensatory mechanism.


title: Light/dark- and temperature-regulated transcriptional rhythms in adult Caenorhabditis elegans

status: Public on Sep 01 2010

city: Reno

first: Alexander

middle: Martinus

last: van der Linden

state: NV

address: 1664 N. Virginia St.

institute: University of Nevada

summary:
Most organisms have an endogenous circadian clock that is synchronized to environmental signals such as light and temperature. Although circadian rhythms have been described in the nematode C. elegans at the behavioral level, these rhythms appear to be relatively non-robust. Moreover, in contrast to other animal models, no circadian transcriptional rhythms have been identified. Thus, whether this simple nematode contains a bona fide circadian clock remains an open question.
We used microarray experiments to identify light- and temperature-regulated transcriptional rhythms in C. elegans, and show that subsets of these transcripts are regulated in a circadian manner. In addition, we find that light and temperature also globally drive the expression of many genes, indicating that C. elegans exhibits systemic responses to these stimuli.

overall_design: Populations of growth-synchronized wild-type C. elegans L1 larvae were entrained for 5 days until adulthood to 12:12 hr light/dark (LD) cycles (500-1000 lux) at a constant temperature of 18°C, or for 4 days to 12:12 hr temperature cycles (25:15°C - warm/cold or WC) in constant darkness. RNA was collected every 4 hrs during the last entrainment and the subsequent free-running days and analyzed via hybridization of Affymetrix GeneChips. L4 larvae were transferred to FUDR-containing plates to inhibit embryonic development.

first: Matthew

last: Beverly

first: Sebastian

last: Kadener

first: Joseph

last: Rodriguez

first: Sara

last: Wasserman

first: Michael

last: Rosbash

first: Piali

last: Sengupta


title: Response of Pseudomonas aeruginosa PAO1 to low shear modeled microgravity

status: Public on Jul 05 2010

city: Mol

first: Rob

last: Van Houdt

address: Boeretang 200

department: Interdisciplinary Biosciences

institute: SCK-CEN

summary: Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety, health and performance of the crewmembers. As a ubiquitous environmental organism that is occasionally part of the human flora, Pseudomonas aeruginosa could pose a health hazard for the immuno-compromised astronauts. In order to gain insights in the behavior of P. aeruginosa in spaceflight conditions, two spaceflight-analogue culture systems, i.e. the rotating wall vessel (RWV) and the random position machine (RPM), were used. Microarray analysis of P. aeruginosa PAO1 grown in the low shear modeled microgravity (LSMMG) environment of the RWV compared to the normal gravity control (NG), revealed a regulatory role for AlgU (RpoE). Specifically, P. aeruginosa cultured in LSMMG exhibited increased alginate production and up-regulation of AlgU-controlled transcripts, including those encoding stress-related proteins. This study also shows the involvement of Hfq in the LSMMG response, consistent with its previously identified role in the Salmonella LSMMG- and spaceflight response. Furthermore, cultivation in LSMMG increased heat- and oxidative stress resistance and caused a decrease in the culture oxygen transfer rate. Interestingly, the global transcriptional response of P. aeruginosa grown in the RPM was similar to that in NG. The possible role of differences in fluid mixing between the RWV and RPM is discussed, with the overall collective data favoring the RWV as the optimal model to study the LSMMG-response of suspended cells. This study represents a first step towards the identification of specific virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions, and could direct future research regarding the risk assessment and prevention of Pseudomonas infections for the crew in flight and the general public.

overall_design: The wild type P. aeruginosa PAO1 strain (ATCC 15692) was used in this study and all cultures were grown in Lennox L Broth Base (LB) (Life Technologies) at 28 °C. An overnight shaking culture (125 r.p.m.) of P. aeruginosa in LB was washed and diluted in 0.85% NaCl solution to an OD600 of 1. This bacterial suspension was used to inoculate fresh LB medium at a final concentration of 10-4 CFU/ml. Synthecon Rotating Wall Vessel bioreactors (RWV) (50 ml or 10 ml) were filled with inoculated medium so that no headspace (i.e. no bubbles) was present. Other than for stress resistance assays, for which 10 ml capacity bioreactors were used, RWV bioreactors with a capacity of 50 ml were adopted for all experiments. Identical bioreactors were mounted in triplicate on (i) a RWV device in vertical position (LSMMG) (Cellon), (ii) a RWV device in horizontal position (NG) and (iii) the center of the inner Random Positioning Machine (RPM) frame (RG) (Fokker Space), and placed in a large humidified (70%-80% relative humidity) culture chamber, to avoid evaporation of culture medium through the gas-permeable membrane at the back of each vessel (Figure 1). A 25 r.p.m. rotation speed was adopted for the RWV cultures, while RPM-cultures were randomly rotated at 10 r.p.m. (60°/s). Bacteria were grown in the three described test conditions for 24 hours. After 24 hours of cultivation, the contents of every bioreactor was gently mixed by pipetting and divided into several aliquots. Ten millilitres of culture from each growth condition was immediately fixed with RNA Protect Reagent (Qiagen), following the manufacturer's instructions, and fixed cell pellets were frozen at -20 °C until RNA extraction. Samples were immediately exposed to different stresses.

first: Aurélie

last: Crabbé

first: Benny

last: Pycke

first: Pieter

last: Monsieurs

first: Cheryl

last: Nickerson

first: Natalie

last: Leys

first: Pierre

last: Cornelis


title: Expression data from the PVD and OLL neurons in C. elegans

status: Public on Jun 16 2010

city: Nashville

first: William

middle: Clay

last: Spencer

state: TN

address: 465 21st Ave S

department: Cell & Developmental Biology

institute: Vanderbilt University

summary:
Nociceptive neurons develop a complex dendritic arbor to sense noxious stimuli, which enables animals to react to environmental insults and perform self-protective behaviours. The genetic programs controlling neuronal dendritic morphogenesis are poorly understood. In C. elegans, the PVD sensory neuron generates a complex dendritic arbor that envelops the body of the animal. This nociceptive neuron enables study of dendrite formation in vivo.
We used expression arrays to identify genes highly enriched in the PVD neuron while undergoing dendritic morphogenesis.  These genes could function in pathways controlling dendrite formation.

overall_design: We employ the mRNA-tagging method to isolate poly(A) RNA from the PVD and OLL neurons by expressing a 3X FLAG-tagged poly(A) binding protein PAB-1 in PVD and OLL under control of the ser-2prom3B promoter.  The enriched poly(A) RNA is amplified using the NuGEN WT-Pico amplification system and applied to Affymetrix C. elegans gene expression microarrays. A reference sample was obtained by isolating total RNA from whole animals at the L3/L4 stages and processed as above.

last: Watson

first: Cody

middle: J

last: Smith

middle: C

first: Tim

last: O'Brien

first: Byeong

last: Cha

first: Adi

last: Albeg

first: Millet

last: Treinin

first: David

last: Miller III


title: Expression data from P. aeruginosa colonizing the Murine GI Tract

status: Public on Jul 03 2010

city: Dallas

first: Andrew

last: Koh

state: TX

address: 5323 Harry Hines Boulevard

department: Pediatrics

institute: University of Texas Southwestern Medical Center

summary:
P. aeruginosa bacteremia in cancer and bone marrow transplant patients transpires when P. aeruginosa colonizes the GI tract and translocates when the host undergoes immunosuppression.  We used microarrays to analyze gene expression when P. aeruginosa transitions from being in the drinking water to when it colonizes the murine GI tract.
to analyze gene expression changes in P. aeruginosa as it transitions from living in the drinking water to when it colonizes the murine GI tract.

overall_design: P. aeruginosa was recovered from the drinking water administered to adult C3H/HeN mice and also from murine cecums, and RNA was extracted and hybridized on Affymetrix microarrays.

middle: Y

first: Gerald

middle: B

last: Pier


title: Expression data from Caenorhabditis elegans exposed to albendazole

status: Public on Jun 14 2010

city: Glasgow

first: Steven

middle: Thomas

last: Laing

address: 464 Bearsden Road

department: Veterinary Medicine

institute: University of Glasgow

summary:
We have investigated how the model nematode Caenorhabditis elegans responds to and metabolizes albendazole; an important anthelmintic for human and animal parasite control.
The transcriptional response of the mutant strain CB3474 ben-1(e1880)III, which is highly resistant to benzimidazoles due to a null mutation in the β-tubulin drug target, was examined. This approach was successful in minimizing transcriptional responses associated with non-specific stress or with the drug mode of action, resulting in only in a small subset of genes showing differential expression in response to drug exposure.

overall_design: Matched cultures of synchronised C. elegans were grown to young adult stage in liquid culture. The nematodes were then exposed to 300 µg/ml albendazole (ABZ) or  exposed only to the DMSO excipient used to deliver the albendazole (CONT) for 4 hours. RNA was extracted from three biological replicates and hybridised to Affymetrix arrays.

middle: T

first: Al

last: Ivens

first: John

last: Gilleard


title: Definition of the Pseudomonas aeruginosa Anr and Dnr Regulons

status: Public on Jun 22 2010

city: Braunschweig

first: Max

last: Schobert

address: Spielmannstr. 7

department: Institute of Microbiology

institute: Technische Universitaet Braunschweig

summary:
The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box.
The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators.

overall_design:
We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments.

Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 µM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed.

First comparison:
Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions.

Second comparison:
Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions.

Third comparison:
Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.

first: Katharina

last: Trunk

first: Beatrice

last: Benkert

first: Nicole

last: Quäck

first: Martin

last: Eschbach

first: Richard

last: Münch

first: Lothar

last: Jänsch

first: Matthias

last: Trost

first: Jan

last: Buer

first: Jürgen

last: Wehland

first: Martina

last: Jahn

first: Maurice

last: Scheer

first: Jahn

last: Dieter


title: Gene expression levels in immortalized B cells from members of 45 CEPH-Utah pedigrees

city: Philadelphia

first: Renuka

last: Nayak

state: PA

address: 3516 Civic Center Blvd

institute: HHMI, University of Pennsylvania

summary:
We used microarrays to examine gene expression levels from members of 45 CEPH-Utah pedigrees.

Keywords: array-based gene expression

overall_design:
We measured gene expression levels in immortalized B cells from members of 45 CEPH-Utah pedigrees.  For some cell lines, RNA extracts were hybridized twice, representing technical replicates. For these cell lines, we take the average signal intensity as a measure of relative abundance of a gene. These gene expression levels were then used in genome-wide linkage analyses to identify genetics determinants of gene expression.

CEL files were lost for 3 Samples GSM420949, GSM421101, and GSM421103.

first: Vivian

middle: G

last: Cheung

middle: R

first: Isabel

middle: X

last: Wang

first: Susannah

last: Elwyn

first: Michael

last: Morley

last: Spielman


title: Expression data from human skeletal muscle

city: Birmingham

first: Jelai

state: AL

address: 1665 University Blvd, Ryals 327

department: Biostatistics

institute: University of Alabama at Birmingham

summary:
Insulin is a potent pleiotropic hormone that affects processes such as cellular growth, differentiation, apoptosis, ion flux, energy expenditure, and carbohydrate, lipid, and protein metabolism.
We used microarrays to detail the global programme of gene expression underlying the influence of insulin in human skeletal muscle collected from different human individuals including 20 insulin sensitive, 20 insulin resistant and 15 diabetic patients. We identified distinct classes of up-regulated and down-regulated genes during these processes.
The pathophysiology of obesity represents an imbalance between a high energy intake and/or low energy expenditure. Resting energy expenditure (REE) comprises 60-75% of total energy expenditure. The respiratory quotient (RQ) is used to estimate fuel partitioning between fat and carbohydrate as preferred substrates for energy generation, and fuel preferences to generate REE also exhibit individual variation. Genes influencing REE and RQ could represent candidate genes for obesity, Metabolic Syndrome, and Type 2 Diabetes due to the involvement of these traits in energy balance and substrate oxidation.
We used microarrays to explore the molecular bases for individual variation in REE and fuel partitioning as reflected by RQ. We performed microarray studies in human vastus lateralis muscle biopsies from 40 healthy subjects with measured REE and RQ values. We identified genes significantly correlated with REE and RQ, respectively.

overall_design: Human skeletal muscle samples were biopsied from different individuals before and after insulin treatment for RNA extraction and hybridization on Affymetrix microarrays.

first: Timothy

last: Garvey

first: Lidia

last: Maianu

first: Mitchell

last: Martin

first: Xuxia

last: Wu

first: Grier

middle: P

last: Page

first: David

last: Allison


title: The Role of the Rad4-Rad23 Complex and Rad4 Ubiquitination in UV-Responsive Transcription

status: Public on Aug 02 2010

city: CARDIFF

first: Simon

last: Reed

address: Heath Park

department: School of Medicine

institute: Cardiff University

summary: The Rad23/Rad4 protein complex plays a major role in DNA damage recognition during nucleotide excision repair (NER) in yeast. We recently showed that two distinct pathways contribute to efficient NER in yeast. The first operates independently of de novo protein synthesis and requires a nonproteolytic function of the 19S regulatory complex of the 26S proteasome and Rad23. The second pathway requires de novo protein synthesis, and relies on the activity of a newly identified Rad7-containing E3 ubiquitin ligase that ubiquitinates Rad4 in response to UV. Surprisingly, we found that cells deleted of either Rad23 or Rad4 caused reduced Rad4 and Rad23 mRNA levels respectively. We considered the possibility of an unexpected role of Rad23 and Rad4 in regulating the expression of genes involved in the transcriptional response to DNA damage. Gene expression profiling has suggested that Rad23 and Rad4 may function as a complex to affect transcription of a small subset of genes in response to UV damage. To determine how Rad4 and Rad23 contribute to the regulation of these genes, we have examined the occupancy of Rad4/Rad23 in their promoter regions by chromatin immunoprecipitation (ChIP), both in the presence and absence of UV damage. Our preliminary ChIP data suggests that the Rad4/Rad23 complex regulates a set of genes in response to UV light. We also proposed that the transcriptional regulatory activity of the Rad4-Rad23 complex required Rad4 ubiquitination. These arrays test this theory using the psocs mutant strain, which is unable to facilitate Rad4 ubiquitination after UV irradiation.

overall_design:
*** This Series represents the gene expression component of the study.

Expression analysis was performed on the pRAD7 strain, which served as the WT control, and harboured the WT RAD7 sequence on the pRS313 plasmid, the psocs strain, which harboured the same plasmid with 2 point mutations in the RAD7 sequence that prevented post-UV Rad4 ubiquitination. Expression analysis was also conducted on a pRAD7 and psocs Strain with RAD23 deleted. Analysis was performed using untreated strains, and strains 15 minutes and 60 minutes after 100Jm-2 UV irradiation. mRNA was extracted from logaritmically growing cells.

first: Neil

last: Humphryes