title: Saccharomyces cerevisiae chemostat steady state microarray compendium

status: Public on Feb 05 2009

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

institute: Delft University of Technology

summary:
Background
Microorganisms adapt their transcriptome by integrating multiple chemical and physical signals from their environment. Shake-flask cultivation does not allow precise manipulation of individual culture parameters and therefore precludes a quantitative analysis of the (combinatorial) influence of these parameters on transcriptional regulation. Steady-state chemostat cultures, which do enable accurate control, measurement and manipulation of individual cultivation parameters (e.g. specific growth rate, temperature, identity of the growth-limiting nutrient) appear to provide a promising experimental platform for such a combinatorial analysis.
Results
A microarray compendium of 170 steady-state chemostat cultures of the yeast Saccharomyces cerevisiae is presented and analyzed. The 170 microarrays encompass 55 unique conditions, which can be characterized by the combined settings of 10 different cultivation parameters.
By applying a regression model to assess the impact of (combinations of) cultivation parameters on the transcriptome, most S. cerevisiae genes were shown to be influenced by multiple cultivation parameters, and in many cases by combinatorial effects of cultivation parameters. The inclusion of these combinatorial effects in the regression model led to higher explained variance of the gene expression patterns and resulted in higher function enrichment in subsequent analysis.
We further demonstrate the usefulness of the compendium and regression analysis for interpretation of shake-flask-based transcriptome studies and for guiding functional analysis of (uncharacterized) genes and pathways.
Conclusions
Modeling the combinatorial effects of environmental parameters on the transcriptome is crucial for understanding transcriptional regulation. Chemostat cultivation offers a powerful tool for such an approach.
Keywords: chemostat steady state samples

overall_design: The Saccharomyces cerevisiae laboratory reference strain CEN.PK 113-7D MATa was grown at steady state in chemostat cultures under 55 different conditions. A condition can be characterized by a specific configuration of the settings of ten different cultivation parameters. One of these cultivation parameters is the available carbon source. Throughout the compendium five different carbon sources were used, i.e. acetate, ethanol,, galactose, glucose and maltose. Other paramaters as Aeration type, Medium type, C-source(s), N-source, S-source, P-source, Limiting element, Growth rate, Temperature (C), pH, Extra compound, Protocol can be analysed.

first: T

last: Knijnenburg

first: JM

last: van den Broek

first: P

last: Daran-Lapujade

first: J

last: de Winde

middle: T

last: Pronk

last: Reinders

first: L

middle: F

last: Wessels


title: Microsatellite stable (n=38) and instable-high (n=13) human colorectal tumors assayed with Affymetrix HuGeneFL arrays.

status: Public on Apr 10 2009

city: Ann Arbor

first: Rork

last: Kuick

state: MI

address: University of Michigan, SPH II, 1415 Washington Heights, Room M2533

country: USA

institute: University of Michigan

summary:
mRNA assays were performed on 51 samples of human colorectal tumors using Affymetrix HuGeneFL arrays containing 7129 probe-sets.  We compared 38 microsatelite stable (MSS) tumors with 13 microsatellite instable-high (MSI) tumors to form a list of genes differing between the two types.  In order to identify molecular signatures characterizing MSI tumors, we examined only MSI-high cancers and not MSI-low ones.

Keywords: disease state analysis

overall_design:
Human samples of 38 microsatellie stable and 13 microsatellite instable-high colorectal tumors, each from a separate patient, had mRNA assays performed using Affymetrix HuGeneFL arrays, with 7129 probe-sets.

A supplementary Excel file (Colon_HuFL_logs.xls) is attached below that gives the log-transformed probe-set values.  It also gives the p-values from T-tests comparing these values between MSS and MSI samples, as well as an estimated average fold-difference, for which the mathematical functions are explicitly given.

first: Eduardo

last: Vilar

first: Bhramar

last: Mukherjee

first: Leon

last: Raskin

first: David

last: Misek

first: Jeremy

middle: M

last: Taylor

first: Thomas

middle: J

last: Giordano

first: Samir

last: Hanash

first: Eric

middle: R

last: Fearon

first: Gad

last: Rennert

first: Stephen

middle: B

last: Gruber


title: U937 AML1-ETO inducible samples

status: Public on May 01 2009

city: Boston

first: Kimberly

last: Stegmaier

state: MA

address: Harvard Medical School

department: Department of Pediatric Oncology

institute: Dana-Farber Cancer Institute and Children’s Hospital Boston

summary:
U937 AML cells that express an inducible AML1-ETO construct under the control of the tetracycline promoter.
Microarrays used to discover an AML1-ETO signature for a GE-HTS screen to identify AML1-ETO modulators.

overall_design: U937 AML cells that express an inducible AML1-ETO construct under the control of the tetracycline promoter at 0, 12, 24, 48, and 72 hours and corresponding controls (2 replicates each).

first: Steven

last: Corsello

first: Giovanni

last: Roti

first: Kenneth

middle: N

last: Ross


title: Expression data from Pseudomonas aeruginosa exposed to a low concentration of Azithromycin

city: Osaka

first: Toshihiro

last: Kai

state: Osaka

address: 3-1-98 Kasugade-naka, Konohana-ku

department: Genomics Research

country: Japan

institute: Dainippon Sumitomo Pharma Co., Ltd.

summary:
Low-dose macrolides are effective therapy in patients with chronic lung infections, but the mechanisms of action are unclear. We compared global gene expression profiles between P.aeruginosa with and without low-dose Azithromycin (AZM) to study why the low-dose macrolide therapy is effective for cystic fibrosis and diffuse panbronchiolitis.

Keywords: dose-response

overall_design: Overnight culture of P. aeruginosa strain PAO1 was inoculated at 1:100 into fresh LB medium. Various concentrations of AZM (0, 0.5, 2 ug/ml) were added to th culture media. Cells were collected 9 hours after the addition of AZM, and total RNA was isolated for DNA microarray experiments.

first: Kazuhiro

last: Tateda

first: Soichiro

last: Kimura

first: Yoshikazu

last: Ishii

first: Hideaki

last: Ito

first: Hiroaki

last: Yoshida

first: Toru

first: Keizo

last: Yamaguchi


title: Low-intensity microwave irradiation does not substantially alter gene expression in late larval and adult C. elegans

status: Public on Mar 12 2009

city: Loughborough

last: Nottingham Arabidopsis Stock Centre (NASC)

address: Sutton Bonington Campus

department: School of Biosciences, University of Nottingham

country: United Kingdom

summary:
Reports that low-intensity microwave radiation can induce heat-shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by very slight heating.  This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9-3 mW kg-1 for 6-well plates) that minimises the temperature differential between sham and exposed conditions to ≤ 0.1°C.  Comparable measurement and simulation studies of SAR distribution within this exposure system are presented. We compared 5 Affymetrix gene-arrays of pooled triplicate RNA populations from sham-exposed L4/adult worms against 5 gene-arrays of pooled RNA from microwave-exposed worms (taken from the same source population in each run).  Few genes showed consistent expression changes across all 5 comparisons, and all such expression changes appeared modest after applying standard normalisation procedures (≤ 30% up- or down-regulated).  The number of statistically significant differences in gene expression (846) was less than the false-positive rate expected by chance (1131).  As one example, an apparent up-regulation of the vit-3 vitellogenin gene by microwave exposure was not mirrored by similar changes affecting the other co-regulated members of the same vit gene family. We conclude that the pattern of gene expression in L4/adult C elegans is not substantially perturbed by low-intensity microwave radiation, and that the minor changes observed in this study may well be explicable as false positives.  As a check on the sensitivity of the Affymetrix gene-arrays used, we also compared RNA samples from N2 worms subjected to a sub-heat-shock treatment (28ºC) against controls kept  at 26 ºC (but using only 2 gene arrays per condition).  After similar normalisation, many more genes (3712) showed substantial expression changes (i.e. > 2-fold at p < 0.05), including a group of six heat-shock genes which  were strongly but unexpectedly down-regulated (by > 10-fold).  However, further replication and confirmation by real-time RT-PCR would be needed to establish how many of these changes might also be false positives.
Experimenter name: Adam Dawe
Experimenter phone: +27 21 959 2364
adam@sanbi.ac.za
Experimenter institute: South African National Bioinformatics Institute
Experimenter address:  University of Western Cape, Old Chemistry Building, University of Western Cape, Modderdam Road, Bellville 7530, Capetown
Experimenter zip/postal_code: 7530
Experimenter country: South Africa
Keywords: Microwave radiation, gene expression, gene arrays, Caenorhabditis elegans

overall_design: 14 samples were used in this experiment

first: Adam

last: Dawe

first: Rakesh

last: Bodhicharla

first: Neil

last: Graham

first: Sean

last: May

first: Tom

last: Reader

first: Benjamin

last: Loader

first: Andrew

last: Gregory

first: David

last: de Pomerai

first: Mays

last: Swicord

first: Giorgi

last: Bit-Babik


title: Gene Expression Profiling of Whole Blood: Comparison of target preparation methods

status: Public on Jan 30 2009

city: Portland

first: Christina

middle: A

last: Harrington

state: OR

address: 3303 SW Bond Ave

department: Gene Microarray Shared Resource

institute: Oregon Health and Science University

summary:
Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts.  The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes.  Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos.  In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system.

Results: We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method.  Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed.  The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined.  Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples.

Conclusion:  RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles.

Keywords: Whole Blood, Protocol Variation, Expression Profiling, Microarray, globin, PAXGene, PNA

overall_design: Blood samples were collected from healthy, human donors in PAXGene tubes; RNA was isolated either on day of collection or after freezing and storage.  Four different methods of microarray target preparation for whole blood RNA samples were examined.  Three of the methods used total RNA extracted from whole blood as the starting sample for mRNA amplification and target labeling: Affymetrix one-cycle target labeling (Method 1: no depletion_Affymetrix); Affymetrix one-cycle target labeling with globin PNAs added during cDNA synthesis (Method 2: Globin PNAs_Affymetrix); and NuGEN Ovation system v1 (Method 4: no depletion_NuGEN).  In the other method tested, total RNA was treated with Ambion GLOBINclear to reduce globin transcripts prior to labeling with the Affymetrix one-cycle labeling protocol (Method 3: GLOBINclear_Affymetrix).  The Affymetrix one-cycle target labeling protocol produces biotin-labeled, amplified cRNA; the NuGEN protocol produces biotin-labeled, amplified cDNA targets.  The analysis of this study included 24 total samples, with three biological replicates per sample storage state (fresh/frozen) and labeling method (Methods 1 - 4).

first: Kristina

last: Vartanian

first: Rachel

last: Slottke

first: Timothy

last: Johnstone

first: Amanda

last: Casale

first: Stephen

last: Planck

first: Dongseok

last: Choi

first: Justine

last: Smith

first: James

last: Rosenbaum


title: A condensin-like dosage compensation complex acts at a distance to control expression throughout the genome

status: Public on Mar 01 2009

city: Berkeley

first: Barbara

middle: J.

last: Meyer

state: CA

address: 16 Barker Hall #3204

department: MCB

institute: HHMI/UCB

type: Genome binding/occupancy profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Global transcriptional response of Saccharomyces cerevisiae following the deletion of succinate dehydrogenase

status: Public on Dec 16 2008

city: Kongens Lyngby

first: Kiran

middle: Raosaheb

last: Patil

address: Bulding 223, Søltofts Plads

country: Denmark

institute: Technical University of Denmark

summary:
Background
Mitochondrial respiration is an important and widely conserved cellular function in eukaryotic cells. The succinate dehydrogenase complex (SDH-complex) plays an important role in respiration as it connects the mitochondrial respiratory chain to the tricarboxylic acid (TCA) cycle where it catalyzes the oxidation of succinate to fumarate. Cellular response to the SDH-complex dysfunction (i.e. impaired respiration) thus has important implications not only for biotechnological applications but also for understanding cellular physiology underlying metabolic diseases such as diabetes. We therefore explored the physiological and transcriptional response of Saccharomyces cerevisiae to the deletion of SDH3, that codes for an essential subunit of the SDH-complex.
Results
Although the SDH-complex has no direct role in transcriptional regulation and the flux through the corresponding reaction under the studied conditions is very low, deletion of SDH3 resulted in significant changes in the expression of several genes involved in various cellular processes ranging from metabolism to the cell-cycle. By using various bioinformatics tools we explored the organization of these transcriptional changes in the metabolic and other cellular functional interaction networks.
Conclusions
Our results show that the transcriptional regulatory response resulting from the impaired respiratory function is linked to several different parts of the metabolism, including fatty acid and sterol metabolism.

overall_design: Two replicates for sdh3 deletion mutants. Reference strain transcriptome data: Westergaard SL et al., FEMS Yeast Res 2004, 5: 193-204.

first: Donatella

last: Cimini

first: Chiara

last: Schiraldi

first: Jens

last: Nielsen


title: Microarray Analysis of Toxicogenomic Effects of Chlorhexidine diacetate on Pseudomonas aeruginosa

status: Public on Jan 10 2009

city: Fort Meade

first: Chantal

middle: Wheko

last: Nde

state: MD

address: 701 Mapes Road

department: OPP/BEAD

institute: USEPA

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

Keywords: Transcriptome study

overall_design: We conducted three independent microarray experiments (biological replicates) in the absence (control) and the presence (experimental) of Chlorhexidine diacetate. We calculated fold change as the ratio between the signal averages of three untreated (control) and three chlorhexidine diacetate-treated (experimental) cultures for 0, 10 and 60 min exposures.

first: Freshteh

last: Toghrol

first: William

middle: E

last: Bentley


title: Conserved mechanisms across development and tumorigenesis revealed by a mouse development perspective of human cancers.

status: Public on Jan 23 2009

first: Alvin

middle: T.

last: Kho

address: 320 Longwood Avenue

department: Informatics Program

institute: Boston Children's Hospital

summary:
Identification of common mechanisms underlying organ development and primary tumor formation should yield new insights into tumor biology and facilitate the generation of relevant cancer models. We have developed a novel method to project the gene expression profiles of medulloblastomas (MBs)—human cerebellar tumors—onto a mouse cerebellar development sequence: postnatal days 1-60 (P1-P60). Genomically, human medulloblastomas were closest to mouse P1-P10 cerebella, and normal human cerebella were closest to mouse P30-P60 cerebella. Furthermore, metastatic MBs were highly associated with mouse P5 cerebella, suggesting that a clinically distinct subset of tumors is identifiable by molecular similarity to a precise developmental stage. Genewise, down- and up-regulated MB genes segregate to late and early stages of development, respectively. Comparable results for human lung cancer vis-a-vis the developing mouse lung suggest the generalizability of this multiscalar developmental perspective on tumor biology. Our findings indicate both a recapitulation of tissue-specific developmental programs in diverse solid tumors and the utility of tumor characterization on the developmental time axis for identifying novel aspects of clinical and biological behavior.

Keywords: Whole cerebellum gene expression profiling, cerebellar development, mouse.

overall_design: Time series with technical (split aliquots) replicates.

first: Isaac

middle: S

last: Kohane

first: David

middle: H

last: Rowitch


title: Histone H3 K4,36,79G

status: Public on Dec 31 2008

city: Pullman

first: John

last: Wyrick

state: WA

address: Biotechnology Life Sciences 241

department: School of Molecular Biosciences

institute: Washington State University

summary:
Total RNA from two replicate cultures of wild-type and mutant strains was isolated and the expression profiles were determined using Affymetrix arrays. Comparisons between the sample groups allow the identification of genes regulated by histone H3 K4,36,79G mutant.
Keywords: repeat

overall_design: Comparison of genes whose mRNA levels are affected in a histone H3 K4,36,79G mutant yeast strain compared to wild-type yeast strain.

first: Amy

last: Rodrigurez

first: Yi

last: Jin


title: Gene expression in AES-2 clonal isolates of Pseudomonas aeruginosa

city: Sydney

first: Jim

last: Manos

state: NSW

address: Camperdown

country: Australia

institute: University of Sydney

summary:
Pseudomonas aeruginosa (P. aeruginosa) lung infection is a significant cause of mortality in patients with cystic fibrosis (CF). Most CF patients acquire unique P. aeruginosa strains from the environment; however clonal strains have been identified in CF communities in several countries. Two clonal strains infect 10% to 40% of patients in three CF clinics in mainland eastern Australia. The expression profiles of four planktonically-grown isolates of one Australian clonal strain (AES-2), and four non–clonal CF P. aeruginosa isolates were compared to each other and to the reference strain PAO1 using the Affymetrix P. aeruginosa PAO1 genome array, to gain insight into properties mediating the enhanced infectivity of AES-1. The isolates were subsequently grown as 3-day old biofilms and similarly extracted for RNA and compared as above. Data analysis was carried out using BIOCONDUCTOR software.
Keywords: Comparative strain hybridization

overall_design: Four isolates of the AES-2 strain were compared to four non-clonal isolates and to the reference strain PAO1, when grwn both as planktonic and as a biofilm culture.

first: Jonathan

last: Arthur

last: Rose

first: Scott

last: Bell

first: Colin

last: Harbour


title: Dietary restriction in Caenorhabditis elegans

status: Public on Dec 15 2008

city: Kyoto

first: Eisuke

last: Nishida

address: Kitashirakawa, Sakyo-ku

department: Department of Cell and Developmental Biology

institute: Graduate School of Biostudies, Kyoto University

summary: Dietary restriction (DR) is the most effective and reproducible intervention to extend lifespan in divergent species1.  In mammals, two regimens of DR, intermittent fasting (IF) and caloric restriction (CR), have proven to extend lifespan and reduce the incidence of age-related disorders2.  An important characteristic of IF is that it can increase lifespan, even when there is little or no overall decrease in calorie intake2.  The molecular mechanisms underlying IF-induced longevity, however, remain largely unknown.  Here we establish an IF regimen that effectively extends the lifespan of Caenorhabditis elegans, and show that a nutrient-related signalling molecule, the low molecular weight GTPase Cel-Rheb, has a dual role in lifespan regulation; Cel-Rheb is required for the IF-induced longevity, whereas inhibition of Cel-Rheb mimics the CR effects.   We also show that Cel-Rheb exerts its effects in part via the insulin/IGF-like signalling effector DAF-16 in IF, and that Cel-Rheb is required for fasting-induced nuclear translocation of DAF-16.  We find that HSP-12.6, a DAF-16 target, functions to mediate the IF-induced longevity.  Furthermore, our analyses demonstrate that most of fasting-induced upregulated genes require Cel-Rheb function for their induction, and that Cel-Rheb/Cel-TOR signalling is required for the fasting-induced downregulation of an insulin-like peptide, INS-7.  These findings identify the essential role of signalling via Cel-Rheb in IF-induced longevity and gene expression changes, and suggest a molecular link between the IF-induced longevity and the insulin/IGF-like signalling pathway.

overall_design: We examined fasting-induced changes of the gene expression profiles in Caenorhabditis elegans.  We performed the genome-wide analysis by using Affymetrix GeneChip oligonucleotide microarrays, and examined the effect of downregulation of Cel-Rheb and Cel-TOR by RNAi on the expression profiles.  Five independent experiments were performed with wild type N2.  Synchronized worms under six conditions (control-fed, control-fasting, Rheb RNAi-fed, Rheb RNAi-fasting, TOR RNAi-fed, and TOR RNAi-fasting) were collected and frozen with liquid nitrogen at day 4 of adulthood.  Total RNA was extracted with Sepasol(R)-RNA ⅠSuper (Nacalai tesque), and purified with RNeasy Mini Kit (Qiagen), according to manufacture’s instructions.  Synthesis of cDNA, in vitro transcription and biotin labelling cRNA, and hybridization to the C. elegans Genome Array (Affymetrix) were performed according to Affymetrix protocols. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned chip images were analyzed with GeneSpring GX 7.3.1 (Agilent Technologies).

first: Sakiko

last: Honjoh

first: Takuya

last: Yamamoto


title: Gene expression levels in African Americans vary with genetic ancestry at most expressed genes

status: Public on Dec 05 2008

city: philadelphia

first: Dustin

last: Hancks

state: PA

address: 415 curie blvd rm 515 crb

department: Genetics

institute: University of Pennsylvania

summary:
Variation in gene expression is a fundamental aspect of human phenotypic variation.  Several studies have analyzed gene expression levels in populations of different continental ancestry, and concluded that there is variation across populations at a fraction of expressed genes.
Here we analyze gene expression levels in African American cell lines, which differ from previously analyzed cell lines in that samples from this population have variable proportions of continental ancestry.  We show that for most genes examined, gene expression varies with genetic ancestry.
Keywords: Human Gene Expression Study

overall_design: Lymphoblastoid cell lines (LCL) for 60 HapMap CEU, 60 HapMap YRI, and 82 AFA from the Human Variation Panel were obtained from Coriell Cell Repositories. LCLs were grown in culture, total RNA was extracted and hybridized to Affymetrix HG-FOCUS arrays.

first: Alkes

middle: L

last: Price

first: Nick

last: Patterson

middle: C

last: Reich

first: Vivian

middle: G

last: Cheung

first: Richard

last: Spielman


title: Histone H3 K4,9,14,18,23,27,36,79G

last: Wyrick

summary:
Total RNA from two replicate cultures of wild-type and mutant strains was isolated and the expression profiles were determined using Affymetrix arrays. Comparisons between the sample groups allow the identification of genes regulated by histone H3 K4,9,14,18,23,27,36,79G mutant.
Keywords: repeat

overall_design: Comparison of genes whose mRNA levels are affected in a histone H3 K4,9,14,18,23,27,36,79G mutant yeast strain compared to wild-type yeast strain.


title: Nodal points and complexity of Notch-Ras signal integration

status: Public on Jan 06 2009

first: Gregory

middle: D.

last: Hurlbut

address: 240 Longwood Ave,LHRRB-410A

department: Cell Biology

institute: Harvard Medical School

summary:
Metazoans utilize a handful of highly conserved signaling pathways to create a signaling backbone that governs all stages of development, by providing spatial and temporal cues that influence gene expression. How these few signals have such a versatile developmental action is of significance to evolution, development, and disease. Their versatility likely depends upon the larger-scale network they form through integration. Such integration is exemplified by cross-talk between the Notch and the Receptor Tyrosine Kinase (RTK) pathways. We examined the transcriptional output of Notch-RTK cross-talk during Drosophila development and present in vivo data that supports a role for selected mutually-regulated genes as potentially important nodal points for signal integration.  We find the complex interplay between these pathways involves their mutual regulation of numerous core components of RTK signaling in addition to targets that include components of all the major signalling pathways (TGF-β, Hh, Jak/Stat, Nuclear Receptor and Wnt). Interestingly, Notch-RTK integration did not lead to general antagonism of either pathway, as is commonly believed. Instead, integration had a combinatorial effect on specific cross-regulated targets, which unexpectedly included the majority of Ras-responsive genes, suggesting Notch can specify the response to Ras activation.
Keywords: Genetic modification with time course

overall_design: To identify nodal points that interlink the Notch and RTK pathways, we examined the transcriptional output of each during Drosophila embryonic development and identified common transcriptional targets. Pathway activation was achieved through ubiquitous GAL4-mediated expression of activated Notch (UAS-NotchICD) or through an activated form of the RTK signal mediator Ras1 (UAS-Ras1V12), either alone or in combination, under the control of the armadillo promoter, which drives GAL4 at moderate physiological levels. Homozygous armadillo-GAL4 virgin females were crossed either to: 1) homozygous UAS-NotchICD males; 2) males homozygous for both UAS-Ras1V12 and UAS-NotchICD; or 3) homozygous UAS-Ras1V12 males. As a control for GAL4 dependent effects, homozygous armadillo-GAL4 virgin females were crossed to w1118 males. Embryos resulting from these crosses were collected on apple juice plates for one hour, incubated at 25°C for 5 hours and 30 minutes for the first time point and 6 hours and 45 minutes for the second time point; dechorionated by treatment with 50% Chlorox bleach at room temperature for 1 minute with agitation, washed with 0.5X PBS, 1% Tween, rinsed with ddH2O. Embryos were then shock frozen and stored in liquid nitrogen at 5:45-6:45 hours After Egg Lay (AEL) for the first time point and 7-8 hours AEL for the second. All collections were performed in parallel sets but staggered to equalize the length of embryo submersion in liquid. Each collection contained approximately 500 embryos. Frozen embryos were ground by pestle in the Trizol reagent (Invitrogen). RNA was extracted via standard methods, with the inclusion of an additional Trizol extraction prior to RNA precipitation to increase RNA purity. Total RNA was prepared from three independent experiments for each described embryonic genotype at each of the two time points. RNA samples were subjected in triplicate to analysis by Affymetrix high-density oligonucleotide arrays using the DrosGenome1 array (Affymetrix) that contains 14,010 probe sets specific to 13,108 Drosophila genes. Probe synthesis and microarray hybridization were performed according to standard Affymetrix protocols. External standards were included to control for hybridization efficiency and sensitivity. Following washing, the chips were scanned with a Hewlett-Packard GeneArray laser scanner. Signal levels were obtained and statistical analysis performed using GC-Robust Multi-Array expression measure (GC-RMA) and LInear Modes for MicroArray (LIMMA) data packages and the affylmGUI graphical user interface in the R programming environment.

middle: D

first: Mark

middle: W

last: Kankel

first: Spyros

last: Artavanis-Tsakonas


title: Temporal expression analysis of C. elegans larvae hatching in the presence and absence of food.

status: Public on Feb 27 2009

city: Durham

first: L.

middle: Ryan

last: Baugh

state: NC

address: 4314 French Family Science Center

department: Biology

institute: Duke University

summary:
When C. elegans larvae hatch in the absence of food they persist in a stress resistant, developmentally arrested state (L1 arrest).  We characterized mRNA expression genome-wide in a pair of bifurcating time series starting in the late embryo and proceeding through the hatch in the presence and absence of food (E. coli).
We used Affymetrix C. elegans expression arrays to measure gene expression in 18 total timepoint/conditions in three biological replicates.
Keywords: time course; plus/minus food

overall_design: Nematode populations were staged such that they hatched withing a 2.5 hr window, and time point zero (0 hr) corresponds to when 50%  of the population had hatched.  RNA was isolated with TRIzol and 100 ng was used for T7 RNA polymerase-based mRNA amplification and biotin labeling using MessageAmp II-Biotin Enhanced kit (Ambion).  Labeled cRNA was fragmented, hybridized to the Affymetriz array, and scanned according to the manufacturer’s protocol.

first: L

middle: R

first: Paul

last: Sternberg


title: Expression analysis of the Saccharomyces cerevisiae hst3hst4 mutant

status: Public on Dec 04 2008

city: Baltimore

first: Ivana

last: Celic

address: 725 N Wolfe Street

institute: JHSOM

summary: The hst3hst4 strain (FY background) has the HST3 and HST4 genes, encoding putative NAD-dependent deacetylases that regulate histone 3 K56 acetylation, deleted. Expression profiling using Affymetrix microarrays was used to assess the change in the gene expression in this strain in comparison to wild-type under normal growth conditions.

overall_design: Total RNA was extracted from two independent cultures of the hst3hst4 strain and the wt strain, and was hybridized to microarrays. Gene expression levels of each mutant culture was compared to each wt culture.

first: Jef

last: Boeke


title: Specific Roles for the Ccr4-Not Complex Subunits in Expression of the Genome

status: Public on Dec 10 2008

city: Geneva

first: Martine

last: Collart

address: Rue Michel-Servet 1, Centre Médical Universitaire

department: Microbiology and Molecular Medicine

country: Switzerland

institute: University of Geneva

summary:
These Affymetrix data were used to determine the role of each non-essential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast S. cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene de-regulation were observed upon deletion of any given subunit, revealing the specificity of each subunit’s function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome.
Keywords: genetic modification, stress response

overall_design: Wild-type and not2Δ, not3Δ, not4Δ, not5Δ, caf1Δ, caf130Δ and ccr4Δ null strains were grown in glucose rich medium to exponential phase and collected. Wild-type and not2Δ, not4Δ, caf1Δ, caf130Δ and ccr4Δ null strains were also grown in glucose rich medium to glucose depletion and collected 30 min after glucose depletion. Each sample was prepared twice independently. One array from the caf1Δ strain grown to glucose depletion, and one from the not5Δ and not4Δ strains growing exponentially were found to be problematic and discarded. A not5Δ sample was not prepared after glucose depletion, because it flocculates in that condition.

first: Cécile

last: Deluen

first: Grégory

last: Theiler


title: Gene expression in PAO1, clonal AES-1 and non-clonal isolates of Pseudomonas aeruginosa

first: Jim

last: Manos

summary:
Pseudomonas aeruginosa (P. aeruginosa) lung infection is a significant cause of mortality in patients with cystic fibrosis (CF). Most CF patients acquire unique P. aeruginosa strains from the environment; however clonal strains have been identified in CF communities in several countries. Two clonal strains infect 10% to 40% of patients in three CF clinics in mainland eastern Australia. The expression profiles of four planktonically-grown isolates of one Australian clonal strain (AES-1), and four non–clonal CF P. aeruginosa isolates were compared to each other and to the reference strain PAO1 using the Affymetrix P. aeruginosa PAO1 genome array, to gain insight into properties mediating the enhanced infectivity of AES-1. The isolates were subsequently grown as 3-day old biofilms and similarly extracted for RNA and compared as above. Data analysis was carried out using BIOCONDUCTOR software.
Keywords: Comparative strain hybridization

overall_design: Four isolates of AES-1 strain were compared to four non-clonal isolates and to the reference strain PAO1, when grown both as planktonic and as a biofilm culture.

first: Jonathan

last: Arthur

first: Barbara

last: Rose

first: Michelle

last: Curtis

first: Jeremy

last: Webb

first: Staffan

last: Kjelleberg

first: Peter

last: Bye

first: Colin

last: Harbour


title: RNA interference and retinoblastoma related genes are required for repression of endogenous siRNA targets in C. elegans

status: Public on Dec 01 2008

city: Cambridge

first: Sebastian

last: Hoersch

address: 77 Massachusetts Avenue (E18-366)

department: David H. Koch Institute for Integrative Cancer Research at MIT

institute: Massachusetts Institute of Technology

summary:
Expession data from L1-L2 stage nematodes (C. elegans), wild type and four mutants (alg-1, zfp-1, rde-4, lin-35).

In C. elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons.
Based on this microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1 and alg-1 and the Retinoblastoma (Rb) mutant lin-35, we found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated previously in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) upregulated in the rde-4 and zfp-1 mutants and b) upregulated in the lin-35(Rb) mutant, but not the downregulated genes are highly represented in the set of genes with corresponding endogenous short interfering RNAs (endo-siRNAs).

Keywords: wildtype-mutant comparison, RNAi, Rb, endo-siRNA

overall_design: Nematodes were synchronized at L1 stage for RNA preparations. All conditions (WT and 4 mutants) were profiled in triplicate. Replicates were biological replicates (separately grown worm populations), with two exceptions: due to shortage of biological material available for the lin-35(n745) unc-13(e1091) I and zfp-1(ok554) III strains, one of the two biological samples was hybridized twice in order to set up a consistent triplicate structure across the dataset.

first: Alla

last: Grishok

first: Phillip

last: Sharp


title: Molecular profiling of human bone marrow and adipose tissue-derived stromal cells

city: Roma

first: Nathalie

last: Saulnier

address: Largo Francesco Vito,1

country: Italy

institute: Università Cattolica del Sacro Cuore di Roma

summary:
Bone marrow (BM) has been considered so far the reference source for stromal cells (SC) originally called mesenchymal stem cells. Recently human adult adipose tissue (AT) has been reported as a valuable source for the isolation of cells exerting a mesenchymal-like phenotype. Though both BM- and AT- derived stromal cells (BMSC and ATSC) share similar immuno-phenotype and exhibit multi-lineage potential in vitro, a consensual panel of specific markers is still debated and the precise molecular mechanisms governing their differentiated fate are not fully understood. The aim of this study was to compare the genome wide expression profiles of stromal cells isolated from AT and BM and to emphasize the core of MSC stemness properties. Moreover we focused on the molecular characteristics of ATSC, attempting to reveal their specific features.We identified an overlapping dataset of 190 genes commonly regulated by ATSC and BMSC. Among these, we were able to categorize 6 key biological families that could be regarded as a stemness signature  that underlie the self-renewal potential and the ability to generate progenitor cells. In particular, a pivotal role of signalling pathways along with the expression of numerous transcription regulators emerged from this study. Genes specifically modulated in ATSC, suggested that these cells posses anti-oxidative and neuroprotective properties.
Taken together, these results provide new hints towards the understanding of the molecular basis  of MSC maintenance and suggest that ATSC have interesting properties which could be useful for several potential clinical applications.
Keywords: genomic profiling

overall_design: Bone marrow and adipose tissue samples were respectively obtained from four long-term disease-free Hodgkin lymphoma patients, during follow-up investigation and from four patients subjected to partial abdominoplasty. In addition, we used in this study a cell line (MRC-5 cells) as control to rule out genes involved in homeostasis and emphasize genes specifically expressed in both types of mesenchymal stem cells. To have results statistically significant, we enrolled four patients for each tissue (4 chips/tissue) and we performed a technical replicate (triplicate) for the MRC-5 cells.

first: Maria-Ausiliatrice

last: Puglisi

first: Wanda

last: Lattanzi

first: Giovanni

last: Pani

first: Maria-Luisa

last: Novi

first: Manuela

last: Giachelia

first: Sergio

last: Alfieri

first: Fabrizio

last: Michetti

last: Gasbarrini

first: Antonio


title: Molecular profiling of adipose tissue-derived stromal cells cultured in hepatogenic medium

summary: Adipose tissue-derived mesenchymal stromal cells (ATSC) hold great promises in regenerative medicine, due to their easy retrieval, their high proliferative capacity, and overall, their multi-lineages differentiation potential. In the last decade, several studies have reported the plasticity of ATSC toward a hepatic fate. Nonetheless, the molecular mechanisms allowing the conversion from a mesenchymal to an epithelial phenotype remain poorly understood. In this study, we investigated the full genome expression profiles of ATSC cultured for 4 weeks under pro-hepatogenic condition in comparison to control ATSC. Sets of differentially expressed genes were then functionally categorized to understand which pathways trigger the hepatic conversion. We showed that ATSC-derived hepatocyte-like cells overexpress sets of genes associated with hepatic functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted the downregulation of several transcripts involved in stemness maintenance along with genes associated with the epithelial-mesenchymal-transition. Taken together, these data suggest that in vitro hepatogenic differentiation converts ATSC into immature hepatic cells, functionally related to liver progenitor cells.

overall_design: Adipose tissue was obtained from 2 patients (1 male, 1 female, mean age: 47 year old) undergoing partial abdominoplasty. Adipose tissue-derived stromal cells (ATSC) were isolated according to standard procedures, using the in vitro adherence property of these cells. At passage culture 4, ATSC were submitted to an in vitro hepatogenic regimen, consisting of the sequential addition of growth factors. After 1 month of in vitro differentiation, cells were harvested and their transcriptome was compared to control ATSC.

first: Anna

last: Piscaglia

first: Marta

last: Barba

first: Maria


title: Expression data from yeast with Anc1p or without under basal or MMS exposed conditions

status: Public on Nov 01 2008

city: Chapel Hill

first: Rebecca

middle: Catherine

last: Fry

address: 1213 MHRC

department: Environmental Sciences and Engineering

institute: UNC-Chapel Hill

summary:
We are investigating the transcriptional response of Anc1 deficient yeast under basal and MMS exposed conditions
We used microarrays to detail the global programme of gene expression underlying the MMS response in WT and Anc1 deficient yeast

Keywords: dose

overall_design: Yeast strains either WT or Anc1 deficient were exposed or unexposed to MMS

first: Rachel

last: Erlich

first: Thomas

middle: J

last: Begley

first: Danielle

last: Daee

first: Robert

last: Lahue

first: Leona

last: Samson


title: A combinatorial code for pattern formation in Drosophila oogenesis

status: Public on Nov 11 2008

city: Princeton

first: Stas

last: Shvartsman

state: NJ

address: Carl Icahn Lab

department: Chemical Engineering and LSI Genomics

institute: Princeton University

summary:
Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Our analysis of a large number of published gene expression patterns in the follicle cells suggests that they follow a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of inductive signals, provided by the highly conserved epidermal growth factor receptor and Decapentaplegic
(DPP) pathways. We demonstrate the validity of the code by testing it against a set of patterns obtained in a large-scale transcriptional profiling experiment. Using the proposed code, we distinguish 36 distinct patterns for 81 genes expressed in the follicular epithelium and characterize their joint dynamics over four stages of oogenesis. The proposed combinatorial
framework allows systematic analysis of the diversity and dynamics of two-dimensional transcriptional patterns and guides future studies of gene
regulation.

Keywords: EGFR, BMP, gain/loss-of-function

overall_design:
RNA was isolated from hand dissected, stage 9-10 egg chambers.  Five genetic backgrounds were profiled including: wild type, EGFR gain of function, EGFR loss of function, BMP gain of function, and BMP loss of function. Three biological replicates were hybridized for each pathway perturbation. The samples were split across two rounds of hybridization. Each round of hybridizations included three biological replicates for wild type:
The first round included EGFR gof, EGFR lof, BMP gof, and wild type controls A1-A3 (GSM313514-16). For these 12 arrays the extraction, labeling, and hybridization steps were done in parallel.
The second round included the BMP lof and wild type controls B1-B3 (GSM313517-19). For these 6 arrays, the extraction, labeling, and hybridization steps were done in parallel.

first: Nir

last: Yakoby

first: Christopher

middle: A

last: Bristow

last: Gong

first: Xenia

last: Schafer

first: Jessica

last: Lembong

first: Jeremiah

last: Zartman

first: Marc

last: Halfon

first: Trudi

last: Schupbach


title: Gene Expression Patterns Induced by HPV-16 L1 VLP in Leukocytes from Vaccine Recipients

status: Public on Nov 14 2008

city: Frederick

first: Richard

last: Lempicki

address: PO Box B

department: Clinical Services Program

institute: Leidos Biomedical Research, Inc.

summary:
Human papilloma (HPV) virus-like particle (VLP) vaccines were recently licensed.  Though neutralizing antibody titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking.  Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMC from 17 vaccine and 4 placebo recipients before vaccination, and 1 month after receiving the second immunization. Vaccination with HPV-16 L1 VLP was associated with modulation of genes involved in the inflammatory/defense response, cytokine, interferon and cell cycle pathways in VLP-stimulated PBMC. In addition, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing antibody titers were found for cyclin d2 (CCND2) and galectin (LGALS2).  Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with the microarray data was seen for over two-thirds of these probesets.  Up-regulation of immune/defense response genes by VLP, in particular interferon-induced genes was observed in PBMC collected prior to vaccination, with many of these genes being further induced following vaccination. In conclusion, we used gene expression profiling to identify important innate and adaptive response related- genes induced by vaccination with HPV VLP.  Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials.

Keywords: Time course - pre and post HPV immunizaton

overall_design: Microarrays (Affymetrix Human Focus) were used to compare the gene expression signature in PBMCs stimulated for 3 days with media alone,  Sf9/baculovirus insect cell lysate, and HPV-16 L1 VLP expressed from baculovirus-infected Sf9 insect cells from 17 vaccine and 4 placebo recipients before vaccination, and 1 month after receiving the second immunization (2 months after the initial immunization.  Post-vaccination baculovirus sample for Pt078 and pre-vaccination baculovirus sample for Pt082 failed QC.

first: Alfonso

last: García-Piñeres

first: Allan

last: Hildesheim

first: Lori

last: Dodd

first: Troy

last: Kemp

first: Jun

last: Yang

first: Brandie

last: Fullmer

first: Clayton

last: Harro

first: Douglas

last: Lowy

first: Ligia

last: Pinto


title: P. aeruginosa and indole and 7-hydroxyindole

status: Public on Nov 21 2008

city: College Station

first: Jintae

last: Lee

state: TX

department: Chemical Engineering

institute: Texas A&M University

summary:
We used DNA microarrays to investigate the impact of indole and 7-hdroxyindole on P. aeruginosa PAO1.For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures of P. aeruginosa PAO1 diluted that were 1:100.  For P. aeruginosa with 7-hydroxyindole and indole, 500 uM 7-hydroxyindole in 250 uL DMF, 1000 uM indole in 250 uL DMF, or 250 uL DMF alone were added to cells grown in LB.  The cells were shaken at 250 rpm and at 37oC for 7 h to form biofilms on the glass wool, and RNA was isolated from the biofilm cells.

Keywords: comparison with E. coli signal indole on P. aeruginosa PAO1

first: Can

last: Attila

first: Thomas

middle: K

last: Wood


title: Biofilms and type III secretion are not mutually exclusive in Pseudomonas aeruginosa

first: Helga

last: Mikkelsen

address: Building O, Downing Site

department: Department of Biochemistry

institute: University of Cambridge

summary:
Biofilm formation and type III secretion have been shown to be reciprocally regulated in P. aeruginosa, and it has been suggested that factors related to acute infection may be incompatible
with biofilm formation. We investigated how growth conditions influence the production of virulence factors by studying the inter-relationships between colonies, biofilms and planktonic cells.
We found that biofilms in our growth conditions express the type III secretion and these lifestyles are therefore not mutually exclusive in P. aeruginosa.

Keywords: Comparison of different growth modes and growth phases of Pseudomonas aeruginosa

overall_design: Pseudomonas aeruginosa cells grown in five different conditions were analysed with three biological replicates for each sample. The five different conditions were planktonic cells in exponential phase, planktonic cells in stationary phase, colonies on agar plates incubated for 15 or 40 hours and biofilms in a continuous flow system after three days of growth.

first: Nicholas

last: Bond

first: Mette

last: Skindersoe

first: Michael

last: Givskov

first: Kathryn

last: Lilley

first: Martin

last: Welch


title: P. aeruginosa PA14-WT vs pyrF mutant vs pyrF mutant w/uracil

status: Public on Nov 12 2008

first: Akihiro

last: Ueda

address: 3122 TAMU

summary:
Comparative transcriptome analyses of P. aeruginosa PA14 pyrF mutant with PA14 wild type, and with PA14 pyrF mutant with 1 mM uracil supplement and PA14 wild type with 10 mM uracil in biofilm cells. All samples were cultured in LB with glass wool at 37C for 7h.
Keywords: Pseudomonas aeruginosa biofilm pyrF uracil

overall_design:
Strains: P. aeruginosa PA14 wild type, PA14 pyrF mutant
Medium: LB for PA14 wild type and PA14 pyrF mutant, LB with 1 mM uracil for PA14 pyrF mutant, LB with 10 mM uracil for PA14 wild type
Cell type: Biofilm cells grown on glass wool
Time: 7 h
Temperature: 37C


title: Expression data of synovial cells isolated from patients with rheumatoid arthritis

status: Public on Oct 14 2008

city: Fukushima

first: Yoshimi

last: Homma

state: Fukushima

address: 1 Hikarigaoka

department: Department of Biomolecular Science

institute: Fukushima Medical University

summary:
Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone.  To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patient with RA by oligonucleotide microarrays.  Gene expression profiles reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress.
Keywords: cell type comparison, disease state analysis

overall_design: We isolated synovial cell culture from patients with rheumatoid arthritis and osteoarthritis.  Fibroblast from patient with osteoarthritis was used for the reference.

first: Y

last: Kabuyama


title: Arabidopsis thaliana open flowers: Wild type vs. sdg4 mutant (SALK T-DNA line_128444)

status: Public on Oct 27 2008

city: Yokohama

first: Motoaki

last: Seki

address: 1-7-22, Suehiro-cho, Tsurumi

institute: RIKEN CSRS

summary:
Transcriptional profiling in open flowers of Arabidopsis wild type control plants and sdg4 mutant (SALK T-DNA line_128444). The sdg4 mutant is a Arabidopsis T-DNA mutant in which T-DNA is inserted in a SET domain protein, SDG4 (At4g30860). Expression profiling studies indicate that SDG4 might function in modulating the expression of the genes that function in the growth of pollen tubes.
Keywords: epigenetic modification

overall_design: Two-condition experiment, open flowers of wild type control  vs. open flowers of sdg4 mutant (SALK T-DNA line_128444). Two biological replicates: 2 control, 2 mutant, independently grown and harvested. One replicate per array.

first: Joyce

last: Cartagena

first: Sachihiro

last: Matsunaga

first: Kiichi

last: Fukui


title: Mating induces an immune response and developmental switch in the Drosophila oviduct

status: Public on Nov 24 2008

city: Rehovot

first: Yael

last: Heifetz

address: POB 12

country: Israel

institute: The Hebrew university of Jerusalem

summary:
Mating triggers physiological and behavioral changes in females.
To understand how females effect these changes, we used microarray,
to characterize gene expression in oviducts of mated and unmated Drosophila females. The transition from nonegg laying to egg laying elicits a distinct molecular profile in the oviduct.

overall_design: 3 microarrays (3 biologicl repeats) of RNA from oviducts of unmated females (control) and 3 microarrays (3 biological repeats) of RNA from oviducts of mated females

first: Anat

last: Kapelnikov

first: Yuval

last: Gottlieb


title: Chemical genomics study of Snf1 as a gene repressor

city: Pittsburgh

first: Karen

middle: M.

last: Arndt

address: 4249 Fifth Avenue

department: Biological Sciences

institute: University of Pittsburgh

summary: The Snf1 kinase plays a critical role in recalibrating cellular metabolism in response to glucose depletion. Hundreds of genes show changes in expression levels when the SNF1 gene is deleted. However, cells can adapt to the absence of a specific gene when grown in long term culture. Here we apply a chemical genetic method to rapidly and selectively inactivate a modified Snf1 kinase using a pyrazolopyrimidine inhibitor. By allowing cells to adjust to a change in carbon source prior to inhibition of the Snf1 kinase activity, we identified a set of genes whose expression increased when Snf1 was inhibited. Prominent in this set are genes that are activated by Gcn4, a transcriptional activator of amino acid biosynthetic genes. Deletion of Snf1 increased Gcn4 protein levels without affecting its mRNA levels. The increased Gcn4 protein levels required the Gcn2 kinase and Gcn20, regulators of GCN4 translation. These data indicate that Snf1 functions upstream of Gcn20 to regulate control of GCN4 translation.

overall_design: Strains growing in raffinose medium and expressing Snf1 and Snf1-I132G proteins were treated with a pyrazolopyrimidine inhibitor (2NM-PP1) to specifically inhibit Snf1-I132G kinase activity. RNA combined from three individual transformants were processed in duplicate for each strain, for a total analysis of four Affymetrix Yeast Genome S98 arrays.

first: Margaret

last: Shirra

first: Rhonda

last: McCartney

first: Chao

last: Zhang

first: Kevan

middle: M

last: Shokat

last: Schmidt


title: Gene Expression Analysis of Ovarian Samples

status: Public on Jul 31 2008

city: Charlotte

first: Zahra

middle: Bahrani

last: Mostafavi

address: 1542 Garden Terrece

department: Research

institute: Carolinas Medical Center

summary:
The Homeobox (HOX) family of genes encodes transcription factors involved in basic developmental processes, most notably during embryogenesis. A possible HOX gene link between development and oncogenesis has recently been described. Dysregulation of HOX genes may be an early event in malignant transformation likely to induce antibody response and thus provide a potential marker for early diagnosis of cancer. Ovarian cancer is characterized by poor early detection and serves as an excellent model system to develop potential markers for early diagnosis. In this study we begin to characterize HOX gene expression in malignant tumors of the ovary and analyze the potential role of HOX genes as biomarkers for early detection of ovarian cancer.

Microarray analysis of mRNA from human ovarian tissues was performed on 65 samples of normal, benign, borderline malignant and malignant ovarian tissue. These samples were analyzed using the Affymetrix Human Genome Focus GeneChip (HG-Focus) microarray to distinguish the differential pattern of mRNA expression between the four types of samples. Real-time reverse transcription PCR was utilized to confirm up-regulation of HOX genes as determined by microarray analysis.

Our results demonstrate multiple HOX genes to be up-regulated in ovarian cancer. We have shown stepped increase in HOX expression comparing normal, benign neoplastic, and malignant human ovarian tissue samples.  This suggests dysregulation of HOX genes may be an early event in malignant transformation and warrants additional studies to validate HOX gene products as potential markers for early detection of ovarian cancer.
Keywords: Human Ovarian cancer, HOX, Homeobox, microarray, gene expression

overall_design: 67 samples were analyzed. 4 groupings based on pathology (normal, benign, borderline malignant, malignant). An average of normal samples was used as controls. Cell lines were used as ovarian cancer control samples.

first: David

last: Tate

middle: B

first: M.

middle: T


title: Alterations in total and in polysomal mRNA fraction in response to expression of RNA binding motif protein 35A

status: Public on Oct 16 2008

city: Buffalo

first: Yurij

last: Ionov

state: NY

address: Elm and Carlton streets

department: Cancer Genetics

institute: Roswell Park Cancer Institute

summary: The gene coding for RNA binding motif protein 35A (RBM35A) is inactivated by frameshift mutations in an LS180 colon carcinoma cell line and in approximately in 50% of colon tumors with microsatellite instability. To get insight into the mechanism of action of these putative tumor suppressor gene we expressed functional copy of the RBM35A cDNA in the LS180 cells. We analyzed alterations in mRNA profiles in total and in polysomal fraction of mRNA in LS180 cells in response to expressing RBM35A gene under Tet off tetracycline inducible promoter.

overall_design: Tet-off LS180-RBM35A cells with restored expression of RBM35A protein (incubated without Dox for at least six days) and their parental cells maintained in the presence of Dox were grown to no more than 80% confluency to ensure good loading of polysomes.  Total RNA was extracted using TRIZOL reagent (Invitrogen) according to manufacturer instructions. Polysomal fractions of mRNA of cells expressing or not expressing  RBM35A protein were isolated using sucrose density gradient sedimentation. Ten micrograms of polysomal RNA from RBM35+ and RBM35- cells were used to synthesize double-stranded cDNA using a SuperScript II reverse transcriptase kit (Invitrogen).  Biotinylated cRNA was synthesized from double-stranded cDNA using the Enzo Bioarray High Yield transcript labeling kit (EnzoLife Sciences), then purified using the GeneChip sample cleanup module (Affymetrix) and fragmented.  Fragmented cRNA was hybridized to Affymetrix HG-U95Av2 GeneChips.

first: Olga

last: Leontieva


title: Effect of Sirolimus treatment on lupus nephritis in NZB/W F1 female mice

first: Padmalatha

last: Reddy

address: 35 Cambridgepark Drive

institute: WYETH

summary: NZB/WF1 female mice spontaneously develop autoimmune lupus nephritis. Expression profiling of kidney tissue from (a) 12 week NZB/W F1 female mice defined as asymptomatic for lupus nephritis, (b) 36 and 42 week NZB/W F1 female mice defined as diseased/symptomatic for lupus nephritis and (c) 36 and 42 week NZB/W F1 female mice that are diseased/symptomatic for lupus nephritis and treated with Sirolimus was carried out. The goal of the study was to identify genes associated with lupus nephritis and modulated by Sirolimus, an inhibitor of mTOR. In addition,  lupus nephritis genes resistant to Sirolimus therapy were also identfied

overall_design: This series of samples comprises of kidney tissue from (a) 12 week old NZB/W F1 female mice defined as asymptomatic for lupus nephritis (N=4), (b) 36 (N=3) and 42 week (N=3) old NZB/W F1 female mice defined as diseased/symptomatic for lupus nephritis and (c) 36 (N=3)and 42 (N=3) week old NZB/W F1 female mice that are asymptomatic for lupus nephritis on treatment with Sirolimus

first: Holly

last: Legault

middle: S

first: Joseph

middle: P

last: Sypek

first: Mark

last: Collins

first: Elizabet

last: Goad

first: Samuel

last: Goldman

first: Wei

last: Liu

first: Stuart

last: Murray

first: Andrew

last: Dorner

first: Margot

last: O'Toole


title: Transcriptional Signatures in Response to Wheat Germ Agglutinin and Starvation in Drosophila melanogaster Larval Midgut

status: Public on Oct 03 2008

city: Urbana

first: Hongmei

last: LI

state: IL

address: 505 S. Goodwin Ave

department: Entomology

institute: University of Illinois at Urbana-Champaign

summary:
One function of plant lectins is to serve as defenses against herbivorous insects.  The midgut is the critical site affected by dietary lectins such as wheat germ agglutinin (WGA).  We observed marked cellular structural and gene expression changes in Drosophila melanogaster third-instar larval midguts from insects WGA-fed or starved.  Dietary WGA caused shortening, branching, swelling, distortion and in some cases disintegration of the midgut microvilli (Mv). Starvation was accompanied by shortening of the Mv.  Microarray analyses revealed that dietary WGA evolved differential expression of 62 transcripts; seven of which were also differentially expressed in starved insects.  The differentially regulated gene cluster in WGA-fed larvae were associated with (i) cytoskeletal organization, (ii) immune responses, (iii) detoxification reactions, and (iv) energy metabolism.  Four putative transcription factor binding motifs (TFBMs) were associated with the differentially-expressed genes. At least one of these putative TFBMs exhibited substantial similarity to MyoD, a TFBM associated with cellular structures in mammals.  These results are in keeping with the hypothesis that WGA causes a starvation-like effect as well as structural changes of midgut cells of Drosophila third-instar larvae.
Keywords: stress response

overall_design: Three treatments (control, WGA, and Starvation), and three replicates for each treatment. So there are total nine samples. no dye-swap.

last: Li

first: Barry

last: Pittendrigh

first: Larry

last: Murdock


title: Influences of Bwoman-Birk Inhibitor on genomics in Drosophila melanogaster larval midguts

summary:
Bowman-Birk Inhibitor (BBI) has both insecticidal and anti-cancerous properties.  It has been hypothesized that dietary BBI slows insect growth by inhibiting the catalytic activity of digestive enzymes trypsins and chyomotrypsins, resulting in the midgut having reduced access to amino acids needed for growth.  In mammals, BBI was hypothesized to influence cellular energy metabolism.  Thus, we tested the hypothesis that dietary BBI also impacts energy-associated pathways in the midgut of Drosophila melanogaster.  We investigated the impact of dietary BBI on the following parameters in the midguts of third-instar Drosophila larvae: (i) cellular metabolites, (ii) global transcriptome response, (iii) putative transcription factor binding sites (TFBSs) associated with the differentially expressed transcripts, and (iv) epithelial cellular structure.  Dietary BBI caused: (i) a reduction of cellular DHAP, glucose, and succinate; and, (ii) increased Fructuse-6-phosphate; (ii) differential expression of genes associated with the glucose and fatty acid utilization; and, (iii) a shortening of midgut epithelial microvilli, a phenomenon previously associated with glucose starvation.  Additionally, fifty seven percent of the putative TFBSs associated with the differentially expressed transcripts have previously been associated with glucose and insulin activities in mammalian studies.  Collectively these results support the hypothesis that dietary BBI influences energy utilization in the Drosophila midgut.
Keywords: stress response

overall_design: Two treatments (control vs BBI-fed), and three replicates were conducted. There were total six samples.  There was no dye-swap.


title: Global Analysis of the Meiotic Crossover Landscape

status: Public on Sep 16 2008

city: San Francisco

first: Jennifer

last: Fung

address: S316 Genentech Hall,  MC2200  600 16th St.

department: Biochemistry and Biophysics

institute: UCSF

summary:
Using microarrays to genotype the parental origin of progeny resulting from a cross between S96 and YJM789 yeast strains, we mapped the distribution of crossovers that occurred during meiosis. Knowledge of the crossover distribution allowed us to assess changes in crossover control in wild type and mutant strains.
The S96 strain is a S288 derivative and thus its DNA sequence has high homology to the oligo sequences used to create the S98 Affymetrix Gene chip.  The YJM789 strain is ~ 0.6% divergent from S288.

Keywords: wild type and mutant analysis

overall_design: Strains were allowed to undergo sporulation (meiosis) and progeny (spores) from 4-spore viable tetrads were grown and genomic DNA was extracted and hybridzed to S98 Affymetrix Gene chips.  Segregation patterns were analyzed for the location of crossovers which are represented by reciprocal exchange of DNA.

first: Stacy

last: Chen


title: FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast

first: Kevin

middle: J.

last: Verstrepen

address: 52 Oxford Street

department: Harvard FAS Center for Systems Biology

institute: Harvard University

summary:
The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known “green beard genes”, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait.
This dataset contains raw transcriptome data of flocculating cells (that express FLO1 driven by the GAL1 promoter) and non-flocculating cells (that do not express FLO1).

overall_design: Cultures of flocculating and non-flocculating cells were grown for 24 hours in YPGal medium.  Subsequently, RNA was isolated from these cultures, converted into cDNA and analyzed using commercially available Afymetrix S98 arrays.  For each culture, two biological replicates were analyzed.


title: Expression data from wildtype adult Drosophila males and females

status: Public on Aug 01 2008

first: Dean

last: Baker

address: Department of Genetics, Downing Street

summary:
Wild-type laboratory strains of model organisms are typically kept in isolation for many years, with the action of genetic drift and selection on mutational variation causing lineages to diverge with time.  Natural populations from which such strains are established, show that gender-specific interactions in particular drive many aspects of sequence level and transcriptional level variation.  Here, our goal was to identify genes that display transcriptional variation between laboratory strains of Drosophila melanogaster, and to explore evidence of gender-biased interactions underlying that variability.
Keywords: expression analysis; gender and genotype effects

overall_design: Three replicate groups of males and virgin females were collected for each sex and genotype combination.  Total RNA was extracted independently for each of the 30 samples (five lines x two sexes x three replicates)

first: Lisa

last: Meadows

first: Jing

last: Wang

first: Julian

last: Dow

first: Steven

last: Russell


title: Gene expression analysis of commercial baker’s yeast Saccharomyces cerevisiae during an air-drying process

status: Public on Jun 25 2008

city: Tsukuba

first: Yoshinori

last: Murata

state: Ibaraki

address: 1-1 Ohwashi

department: Post-harvest Science and Technology Division

institute: Japan International Research Center for Agricultural Science

summary:
In this study, we focused on air-drying stress and analyzed the changes in gene expression of commercial baker’s yeast during the air-drying process. Changes in gene expression profiles of commercial baker’s yeast during an air-drying process at 37oC that simulated dried yeast production were analyzed using DNA microarrays.
Keywords: Stress response

overall_design: Total RNA was extracted from the stress-treated yeast cells by using a hot phenol method. Poly(A)+ RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan). cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix user’s manual (Affymetrix, Santa Clara, USA). Biotinyated cRNA was fragmented and then used as a probe.Affimetrix Yeast Genome S98 arrays (Affymetrix) were used as DNA microarrays. All experiments were done in duplicate independently.

first: Jun

last: Shima


title: SE2Fs Mediate A Fundamental Cell Cycle Deregulation in High-Grade Serous Ovarian Carcinomas

status: Public on Aug 21 2008

city: Leiden

first: Jan

last: Oosting

address: Albinusdreef 2

department: Pathology

country: Netherlands

institute: Leiden University Medical Center

summary:
Several studies described a role for the E2F/Rb pathway in ovarian serous carcinomas (SCAs). Since E2F/Rb pathway deregulation is a general hallmark of human cancer, it remains unclear whether this deregulation is of particular importance in SCAs or whether it reflects a common oncologic feature.
Here, we attempted to clarify this issue by the examination of microarray expression profiles of SCAs (10 SCA1s, 15 SCA3s) and particularly by the comparison with another, less malignant, ovarian cancer type, serous borderline tumours (13 SBTs). Results were further validated by quantitative RT-PCR, both on the microarray samples and an independent panel. In addition, TP53 mutation analysis was performed.
This integrated analysis revealed a significant increase in the expression of the transcription factors E2F1 and E2F3 in SCAs, when compared to SBTs. This was associated with a vast overexpression of E2F target genes in SCAs compared to SBTs. Overall, at least 45% of those genes with a significantly higher expression in SCAs were E2F targets. When taking into account the different SCA tumour grades, particularly SCA3s exhibited a major deregulated E2F target expression pattern, compared to SBTs. To a lesser extent, this was also the case for SCA1s, although the E2F target expression pattern of several SCA1s appeared to be more similar to SBTs. Generally, overexpression of E2F targets in SCAs appeared to be well-structured since those targets considered as negative regulators of the cell cycle or promoters of apoptosis were usually not overexpressed in SCAs. Similar to E2F target deregulation, TP53 mutations were identified in SCA3s, to a lesser extent in SCA1s, and not in SBTs.
These results suggest that a structured, generally upregulated, E2F transcription factor activity is associated with a global cell cycle disturbance in high grade SCAs, and exceeds typical E2F/Rb pathway disruption in tumours, at least compared with SBTs

overall_design: Expression levels of 13 SBTs, including 2 micropapillary SBTs (MPCs), 10 SCAs grade 1 (SCA1s), and 15 SCAs grade 3 (SCA3s) were compared.

first: Tim

last: de Meyer

first: Ingrid

last: Bijsmans

first: K

last: van de Vijver

first: S

last: Bekaert

first: Wim

last: van Criekinge

first: Manon

last: van Engeland

first: Nathalie

middle: L

last: Sieben


title: Cooperation of two mRNA-binding proteins drives metabolic adaptation to iron deficiency

status: Public on Jul 15 2008

city: Durham

first: Dennis

last: Thiele

address: 3813 Research Drive

department: Pharmacology and Cancer Biology

institute: Duke University

summary:
Expression of the yeast Cth2 protein stimulates degradation of mRNAs encoding proteins with Fe-dependent functions in metabolism, in iron storage and in other cellular processes. We demonstrate that in response to Fe deprivation, the Cth2-homologue, Cth1, stimulates specific degradation of mRNAs involved in mitochondrially localized activities that include respiration and amino acid biosynthesis. Furthermore, yeast cells grown under Fe deprivation accumulate mRNAs encoding proteins that function in glucose metabolism.  These studies demonstrate a reprogramming of cellular metabolism during Fe-starvation dependent on the coordinated activities of two mRNA binding proteins.
Keywords: Messenger RNAs down regulated by Cth1 and Cth2 proteins in response to Fe-limitation

overall_design: cth1cth2 cells independently transformed with pRS416 (V), pRS416-CTH1 (C) or pRS416-CTH2 (T) were grown in triplicate in SC-Ura containing 100 μM BPS until exponential growth phase (approximately 6hrs) at 30 degrees, RNA was extracted using a standard glass beads protocol, labeled and hybridized to Yeast Genome S98 Afffymetrix arrays.

first: Sandra

middle: V

last: Vergara

first: Sergi

last: Puig


title: Transcriptional responses to lactic acid in anaerobic chemostat cultures of Saccharomyces cerevisiae

status: Public on Jul 30 2008

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

institute: Delft University of Technology

summary:
Raw expression values (CHP data) for transcriptional profiling of the response of Saccharomyces cerevisiae to challenges with lactic acid at pH 3 and pH 5.
Keywords: response to lactic acid

overall_design:
The laboratory reference strain CEN.PK 113-7D (MATa) was grown at 30 °C in 1.5-L chemostat fermentors (Applikon, Schiedam, The Netherlands) with a working volume of 1-L using an electronic level sensor to maintain a constant volume.  All cultures, including the reference, were fed with minimal medium as described by Verduyn et al. (1992) with 25 g L-1 glucose as the limiting nutrient and 0.15 ml L-1 silicone antifoam (BDH, Poole, England) to prevent excessive foaming.  The dilution rate was set to 0.10 h-1 and the pH was controlled at 5.0 with the automatic addition (ADI 1031 bio controller, Applikon) of 2 M KOH.  The stirrer speed was set at 800 RPM and anaerobicity was maintained by sparging the fermentor with N2 gas at 500 ml min-1.  To prevent diffusion of oxygen, the fermentor was equipped with Norprene tubing and Viton O-rings and the medium vessel was also flushed with N2 gas.  A comparable degree of weak acid uncoupling was ensured by decreasing the biomass yield to approximately 50% of the reference condition (no organic acids added) with the addition of the appropriate concentration of lactic acid to the reservoir media.
Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications.  Double-stranded cDNA synthesis was carried out using 15 μg of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix).  The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix).  Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 μg of biotinylated cRNA.
Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0.

first: Derek

last: Abbott

first: Erwin

last: Suir

first: Jack

last: Pronk

first: Antonius

last: van Maris


title: Similarity of Gene Expression Patterns in Human Alveolar Macrophages in Response to P. aeruginosa and B. cepacia

status: Public on Jul 26 2008

city: New York

first: Yael

last: Strulovici-Barel

address: 1300 York Avenue

department: Department of Genetic Medicine

institute: Weill Cornell Medical College

summary:
To determine if differences in the severity of pulmonary infection in cystic fibrosis been seen in late isolates od Pseudomonas aeruginosa and Burkholderia cepacia are associated with differences in the initial repsonse of alveolar macrophages (AM) to these pathogens, we assessed gene expression changes in human AM in response to infection with a laboratoty strain, early and late clinical isolates of P. aeruginosa, and B. cepacia.

Keywords: Comparison of gene expression in alveolar macrophages of normal non-smokers and normal smokers.

overall_design:
Alveolar macrophages were obtained from bronchoalveolar lavage.

Two clinical strains isolated from the sputum of an individual with CF, AD2A and AD15B (provided by J. Burns, University of Washington, Seattle). AD2A is an early clinical isolate, and AD15B is a late clinical isolate; both were derived from the same individual.

first: Stefan

last: Worgall

first: Adriana

last: Heguy

first: Karsta

last: Leuttich

first: Timothy

last: O'Connor

first: Ben-Gary

last: Harvey

first: Luis

middle: E

last: Quadri

first: Ronald

middle: G

last: Crystal


title: Expression data from  Pseudomonas aeruginosa infiltrated into resistant and susceptible cultivars of Nicotiana tabacum.

status: Public on Aug 29 2008

city: Fort Collins

first: Tiffany

last: Weir

address: 1173 Campus Delivery

institute: Colorado State University

summary:
Although Pseudomonas aeruginosa is an opportunistic pathogen that does not often naturally infect alternate hosts such as plants, the plant-P. aeruginosa model has become a widely recognized system for identifying new virulence determinants and studying pathogenesis of this organism.  Here we examine how both host factors and P. aeruginosa PAO1 gene expression are affected in planta after infiltration into incompatible and compatible cultivars of tobacco (Nicotiana tabacum L.) Nicotiana tabacum has a resistance gene (N) against tobacco mosaic virus; and although resistance to PAO1 infection correlated to the presence of a dominant N-gene, our data suggests that it is not a factor in resistance against Pseudomonas. We did observe that the resistant tobacco cultivar had higher basal levels of salicylic acid, and a stronger salicylic acid response upon infiltration of PAO1. Salicylic acid acts as a signal to activate defense responses in plants, limiting the spread of the pathogen and preventng access to nutrients. It has also been shown to have direct virulence modulating effects on P. aeruginosa.  We also examined host effects on the pathogen by analyzing global gene expression profiles of bacteria removed from the intracellular fluid of the two plant hosts.  We discovered that the availability of micronutrients, particularly sulfate and Pi, are important factors in in planta pathogenesis, and that the amounts of these nutrients made available to the bacteria may in turn have an effect on virulence gene expression. Indeed, there are several reports suggesting that P. aeruginosa virulence is influenced in mammalian hosts by the availability of iron and by levels of O2.

Keywords: comparative genomic hybridization, disease state analysis

overall_design: Pseudomonas aeruginosa cells were removed from leaf tissue 24 hours post-infiltration and RNA was directly extracted from these cells. Comparisons are between cells removed from a susceptible cultivar of Nicotiana tabacum (cv. Samsun) and a resistant cultivar (cv. Xanthi). Three biological replicates per cultivar were analyzed.

first: Valerie

last: Stull

first: Dayakar

last: Badri

first: Lily

last: Trunck

first: Herbert

last: Schweizer

first: Jorge

last: Vivanco


title: Formaldehyde as source of free-energy during growth of engineered Saccharomyces cerevisiae on glucose

status: Public on Jun 20 2008

first: Jean-Marc

last: Daran

summary:
Previously, it has been demonstrated that formate can be utilized by Saccharomyces cerevisiae as additional energy source using cells grown in a glucose-limited chemostat. Here, we investigated utilization of formaldehyde as co-substrate. Since endogenous formaldehyde dehydrogenase activities were insufficient to allow co-feeding of formaldehyde, the Hansenula polymorpha FLD1, encoding formaldehyde dehydrogenase, was introduced in S. cerevisiae. Chemostat cultivations revealed that formaldehyde was co-utilized with glucose, but the yield was lower than predicted. Moreover, formate was secreted by the cells. Upon co-expression of the H. polymorpha gene encoding formate dehydrogenase, FMD, the levels of secreted formate decreased, but the biomass yield was still lower than anticipated.
Transcriptome comparisons of cells of the engineered FLD1/FMD-expressing S. cerevisiae strain grown with or without formaldehyde feed, suggested that the cells experienced biotin limitation, possibly due to inactivation of biotin by formaldehyde in the feed. When separate feeds were used for formaldehyde and biotin, the engineered S. cerevisiae strain was able to efficiently utilize formaldehyde as additional energy source.
Keywords: response to additional compound

overall_design:
In industrial biotechnology, the cost of feedstocks (often sugars) are crucial for production of both biomass related products, such as proteins, or for the production of commodity chemicals. In many such processes a large fraction of the sugars is dissimilated to either provide free-energy (in the form of ATP) or to provide electrons (in the form of NADH). Co-consumption of methanol, which can be derived from fossil sources (via natural gas) or from biomass (via syngas) , via the above mentioned route can provide NADH and/or ATP, thereby creating the possibility to utilise the sugars more efficiently. It would therefore be advantageous to co-feed S. cerevisiae with methanol during fermentations since it is a cheap alternative to sugars as energy source.
Efficient dissimilation of the intermediate formaldehyde is a crucial first step for utilisation of methanol by S. cerevisiae. Therefore, his study analyses the effect of co-expression of H. polymorpha FLD1 encoding NAD+-dependent formaldehyde and FMD encoding formate dehydrogenases, on tolerance to formaldehyde and co-consumption of formaldehyde by S. cerevisiae.

first: R

last: Baerends

first: A

last: Van Maris

first: I

last: van der Klei

first: M

last: Veenhuis

first: J

first: JM


title: Glucose Pulse to sfp1delta continuous cultures

status: Public on Jun 09 2008

first: Chiara

last: Cipollina

department: Biotechnology

summary:
The Saccharomyces cerevisiae SFP1 is required for proper regulation of ribosome biogenesis and cell size in response to nutrients. A mutant deleted for SFP1 shows specific traits among which a slow growth phenotype, which is particularly evident during growth on glucose. To assess the effects of nutrients on the activity of Sfp1 independent by growth rate related feedback we grew an sfp1Δ mutant and its isogenic reference strain in chemostat cultures, at the same specific growth rate, under glucose/ethanol-limitation. Our data show that Sfp1 is involved in the modulation of cell size and RiBi gene expression and that these two functions are differently influenced by nutrients.
The continuous cultures were then pulsed with a glucose excess generating a situation of batch growth similar to shake flask cultures. The dynamic analysis of the metabolic and transcriptional response following the glucose addition suggested that Sfp1 plays a role at the crossroads of ribosome biogenesis and central carbon metabolism regulation.
Finally, we show that the down-regulation of RP genes, which was observed in an sfp1Δ strain during shake flask growth, cannot be directly ascribed to the absence of Sfp1 but is most probably a secondary effect due to the low growth potential of the mutant strain.
Keywords: time-course (Steady-state based anaerobic glucose pulse)

overall_design:
After ten volume changes, few seconds after the samples for the steady state analysis were collected, the anaerobic glucose pulse experiments were started by sparging the medium reservoir and the fermenter with pure nitrogen gas (airflow of 0.5 L min-1, Hoek-Loos, Schiedam, <5 ppm O2). NorpreneTM tubing and butyl septa were used to minimize oxygen diffusion into the anaerobic culture. Two minutes after nitrogen sparging and just before the addition of glucose, the medium and the effluent pumps were switched off. At this time point (which we will refer to as time T=0) the 200 mM glucose pulse was injected aseptically through a rubber septum.
Sampling from chemostats, total RNA extraction, probe preparation and hybridization to Affymetrix Genechip® microarrays were performed as previously described (1). Samples were collected at steady state and then at 5, 10, 30, 60 and 120 minutes after the pulse. The results relative to steady state samples were derived from three independent cultures, those relative to the time course analysis were derived from two independent cultures.

first: Joost

last: van den brink

first: Pascale

last: Daran-Lapujade

first: Jack

last: Pronk

first: Danilo

last: Porro

first: Johannes

middle: H

last: de Winde


title: Tobramycin Treatment of Pseudomonas aeruginosa

status: Public on Jun 10 2008

city: Hanover

first: George

last: O'Toole

state: NH

address: 505 Vail Building

department: Department of Microbiology and Immunology

institute: Dartmouth Medical School

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

overall_design: Refer to individual Series


title: Metastasized vs. non-metastasized primary head and neck squamous cell carcinomas: pathway-based gene expression analysis

status: Public on May 28 2008

first: Jan

last: Oosting

summary:
Gene expression patterns of 11 metastazised tumors of the head and neck region were compared to 11 non-metastasized tumors
Keywords: disease state analysis

overall_design: 11 metastazised head and neck squamous cell carcinomas from 3 regions (mouth, oropharynx, larynx) were compared to 11 non-metastazised tumors on affymetrix arrays

first: Erik

middle: F

last: Hensen


status: Public on Jun 01 2008

city: Stanford

first: Eric

middle: D

last: Hoopfer

address: 385 Serra Mall

department: Biology

institute: Stanford University

summary:
This study identifies genes that show EcR-dependent gene expression in MB neurons at the onset of axon pruning.
Keywords: nuclear receptor gene regulation

overall_design: A dominant negative form of the ecdysone receptor (EcRDN) was expressed in MB neurons by OK107-GAL4.  Animals were staged at 0 and 5 hours relative to puparium formation.  RNA was isolated from MB neurons labeled with mCD8::GFP driven by OK107-GAL4 by laser capture microdissection, labeled and hybridized to Affymetrix Drosophila Genome Arrays.  These microarrays were compared to RNA isolated from y,w animals at similar time points.

first: Andrea

last: Penton

first: Ryan

middle: J

last: Watts

first: Liqun

last: Luo


title: P. aeruginosa PAO1 WT and PA2663 biofilm cells

status: Public on May 29 2008

city: College Station

first: Can

last: Attila

address: 524 Jack E. Brown Bldg.

department: Chemical Eng

institute: Texas A&M University

summary:
P. aeruginosa PAO1 PA2663-UW expression in biofilm cells relative to P. aeruginosa PAO1 WT-UW expression in biofilm cells. All samples cultured in LB with glass wool.
Keywords: Mutation

overall_design:
Strains: P. aeruginosa PAO1 wild-type, PA2663 mutant
Medium: LB
Biofilm grown on glass wool
Time: 7 h
Cell type: biofilm

first: Thomas

middle: K

last: Wood


summary:
Genomic analysis of axon pruning in Drosophila mushroom body neurons identifies the RNA-binding protein Boule as a negative regulator

This SuperSeries is composed of the SubSeries listed below.


title: Tobramycin Treatment of Planktonic Pseudomonas aeruginosa

summary:
As a comparison to tobramycin-treated P. aeruginosa biofilms, we investigated the response of planktonic P. aeruginosa to tobramycin by microarray.
Keywords: Tobramycin Response

overall_design: We included 2 control (untreated) cultures and 2 tobramycin-treated cultures.  We used mid-exponential phase cultures of P. aeruginosa PA14.  Replicate cultures were incubated in the presence or absence of 5 μg/mL tobramycin for 30 minutes at 37°C.

first: Gregory

last: Anderson

first: Sophie

last: Moreau-Marquis

first: Bruce

middle: A

last: Stanton


title: Timecourse: MB neurons at the onset and early steps of axon pruning

summary:
Drosophila mushroom body (MB) γ neurons undergo axon pruning during metamorphosis through a process of localized degeneration of specific axon branches.  Developmental axon degeneration is initiated at the onset of metamorphosis by the pre-pupal rise in the steroid hormone ecdysone.  This study identifies genes that alter their expression in MB neurons at the onset and early steps of axon pruning.
Keywords: timecourse

overall_design: y,w animals were staged at -18, 0 and 5 hours relative to puparium formation.  RNA was isolated from MB neurons labeled with mCD8::GFP driven by OK107-GAL4 by laser capture microdissection, labeled and hybridized to Affymetrix Drosophila Genome Arrays.


title: The response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation

status: Public on Apr 17 2008

city: Vancouver

first: Hennie

middle: JJ

last: van Vuuren

state: BC

address: 2205 East Mall

country: Canada

institute: UBC

summary:
We used genome-wide expression analyses to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty percent of the yeast genome significantly changed expression levels to mediate long-term adaptation to an environment in which ethanol is both a stressor and a carbon source. Within this set, we identify a group of 223 genes, designated as the Fermentation Stress Response (FSR), that are dramatically and permanently induced; FSR genes exhibited changes ranging from four-to eighty-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, was responsible for entry of yeast cells into stationary phase. Ethanol seems to regulate yeast metabolism through hitherto undiscovered regulatory networks during wine fermentation.
Keywords: time course, stress response, fermentation

overall_design: The main study is based on expression measurements at 0.5,2,3.5,7 and 10 % ethanol (corresponding to roughly 24,48,60,120 and 340 hours). Experiments were done in triplicate (biological replicates). This submission also includes the files at 1 hour and at 12 hours for completeness. (also in triplicate)

first: Virginia

last: D Marks

first: Shannan

last: Ho Sui

first: Daniel

last: Erasmus

last: van der Merwe

first: Jochen

last: Brumm

first: Wyeth

middle: W

last: Wasserman

first: Jennifer

last: Bryan


title: Transcriptional profiling of carotenoid producing S. cerevisiae cells

first: Jean-Marc

last: Daran

summary:
To obtain insight in the genome-wide response of heterologous carotenoid production in Saccharomyces cerevisiae, we have analyzed the transcriptome of S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous. For this purpose, two strains producing different levels of carotenoids were grown in carbon-limited continuous cultures and genome-wide expression was analyzed. The strain producing low carotenoid levels did not exhibit a clear genome-wide transcriptional response, suggesting that low carotenoid levels do not result in cellular stress. Transcriptome analysis of a strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ATP-binding cassette (ABC) type transporters and major facilitator transporters which are involved in secretion of toxic compounds out of cells. Our results suggest that production of high amounts of carotenoids in S. cerevisiae lead to toxicity and that these cells are prone to secrete carotenoids out of the cell. Indeed, secretion of beta-carotene into sunflower oil was observed upon addition of this hydrophobic solvent to the growth medium. Finally, it was observed that deletion of the ABC transporter pdr10, one of the induced PDR transporters, highly decreased the transformation efficiency of an episomal vector containing carotenogenic genes. The few colored transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to control strains transformed with the same carotenogenic genes. These results indicate that Pdr10 might be specifically involved in carotenoid tolerance in S. cerevisiae strains.
Keywords: dose response

overall_design: The genome wide transcriptional response of S. cerevisiae cells that heterologously produce carotenoids might provide information concerning the impact of carotenoid production on yeast physiology and might identify bottlenecks relevant for the production of these compounds. DNA microarray experiments have been proven to be a powerful tool to study the genome wide transcriptional response of S. cerevisiae to changes of the physiological state and the environment (for example 3,. Genomics approaches on cells producing heterologous metabolites to study their impact on yeast physiology have not been reported yet for S. cerevisiae. Additionally, most transcriptome studies with S. cerevisiae have been performed with cells grown in shake flasks cultures. The main drawback of shake flask cultivation is that the environment is continuously changing, which may be of high influence on carotenoid production, and interpretation of transcriptome data . Chemostat cultivation offers advantages for studies with DNA microarrays because it enables cultivation of microorganisms under tightly defined environmental conditions. An interlaboratory comparison of transcriptome data obtained in chemostat cultures has indeed demonstrated that the accuracy and reproducibility of this approach are superior to those obtained in previous studies with shake-flask cultures .

first: Rene

last: Verwall

first: Albert

last: van Ooyen


title: Xylose metabolism in recombinant Saccharomyces cerevisiae

status: Public on Sep 24 2008

city: Espoo

first: Laura

last: Salusjärvi

address: Tietotie 2

country: Finland

institute: VTT Technical Research Centre of Finland

summary: In the present study transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at genome-wide level how signalling and carbon catabolite repression differed in cells grown on either glucose or xylose. The more detailed knowledge about is xylose sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is it rather recognised as a non-fermentable carbon source is important in achieving understanding for further engineering this yeast for more efficient anaerobic fermentation of xylose.

overall_design: Three aerobic batch fermentations were carried out both on 50 g l-1 glucose and on 50 g l-1 xylose to compare the yeast transcriptome and proteome of cells growing on xylose with that of glucose repressed and glucose derepressed cells. Samples of the xylose-grown cells were harvested at 72 h from the start of the xylose cultures with 32 g l-1 of residual xylose present. Samples of the glucose repressed cells were harvested at 5 h from the start of the glucose cultures with 37 g l-1 of residual glucose present. Samples of the glucose derepressed cells were harvested at 24 h from the start of the glucose cultures containing no glucose but 13 g l-1 of accumulated ethanol.

first: Matti

last: Kankainen

first: Rabah

last: Soliymani

first: Juha-Pekka

last: Pitkänen

first: Merja

last: Penttilä

last: Ruohonen