title: Expression profiling of chickpea responses to drought, cold and high-salinity stresses

status: Public on Jul 24 2007

city: Bundoora

first: Nitin

last: Mantri

state: Victoria

address: Bldg 223, Level 1, Plenty Road

department: Biotechnology & Environmental Biology

country: Australia

institute: RMIT University

summary:
‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea genotype ICC 3996 to drought, cold and high-salinity stresses. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaf/shoot, flower/pod and/or root tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf/flower/root tissues pooled separately) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1.

Overall, 46, 54 and 266 ESTs were identified as DE under drought, cold, and high-salinity stresses, respectively. The important ones DE in response to drought stress include induction of transcripts associated with dehydrin-cognate, lipid-transfer protein precursor, and glutamate-hydrolysing asparagine synthetase, whilst repression of transcripts associated with senescence, photosynthesis/energy metabolism, auxin-repressed protein, starch metabolism, AP2/EREBP1 DNA binding domain, putative ARF1 GTPase activating protein, and histidine-containing phosphotransfer protein ATHP3. The interesting transcripts DE in response to cold-stress included induction of phosphate-induced protein, cationic peroxidase, UDP-glucose 4-epimerase, Avr9/Cf9 rapidly elicited protein, DNA-J like protein involved in intracellular protein transport, and protein kinase, whist repression of transcripts associated with a hypothetical transmembrane protein, membrane related protein CP5, lipid-transfer protein precursor, WD repeat protein and several transcripts associated with cellular metabolism, cell cycle and DNA processing, protein synthesis and photosynthesis/energy metabolism. Under high-salinity stress, several transcripts were DE only in roots at 24 hpt but DE in shoots or both, shoots and roots at 48 hpt, relating to the theory of upward movement of salt to shoots at later stages when roots fail to restrict it (Munns et al., 2002). The important transcripts DE in response to high-salinity stress included induction of aluminum-induced protein, auxin-repressed proteins, metallothionein-like protein, glutamate dehydrogenase, sucrose synthase, UDP-glucose 4-epimerase, xylose isomerase, class 10 pathogenesis related protein, disease resistance protein, SNAKIN 2 antimicrobial peptide, and DNA-J like protein involved in intra-cellular protein transport. Whilst high-salinity stress caused the repression of transcripts associated with cell rescue/death, cell cycle/DNA processing, cellular metabolism, photosynthesis/energy metabolism, and transport facilitation. Several of the above transcripts have been previously implicated to be associated with abiotic stress response in other crops. Hence, the study of more genotypes and transcriptional changes at several time points may provide a better picture of involvement of the genes being interrogated here in drought tolerance/susceptibility. Subsequently, the functionality of candidate tolerance genes detected through this approach could be validated by overexpressing the genes through transgenics or silencing them using knockout-mutants/antisense/RNAi.

Keywords: abiotic stress response, drought, cold, high-salinity, chickpea

platform_organism:
Cicer arietinum
Lathyrus sativus
Lens culinaris

overall_design:
Total RNA was extracted from separately pooled leaf/flower/root tissues at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity and quality of the total RNA samples were assessed by OD260/OD280 ratios and gel electrophoresis respectively. Fluorescent-labeled targets were prepared and hybridized to array slides as described [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Plant Biotechnology Journal. 4(6), 647–666]. All hybridizations were performed with six technical replicates and three biological replicates, incorporating dye-swapping (i.e. reciprocal labelling of Cy3 and Cy5) to eliminate any dye bias.

Slides were scanned at 532 nm (Cy3 green laser) and 660 nm (Cy5 red laser) at 10 µm resolution using an Affymetrix® 428™ array scanner (Santa Clara, CA), and captured with the Affymetrix® Jaguar™ software (v. 2.0, Santa Clara, CA). Image analysis was performed using Imagene™ 5 (BioDiscovery, Marina Del Rey, CA) software. Quantified spot data was then compiled and transformed using GeneSight™ 3 (BioDiscovery, Marina Del Rey, CA). Data transformations consisted of a local background correction (mean intensity of background was subtracted from mean signal intensity for each spot), omitting flagged spots, LOWESS normalisation of the entire population, creating a Cy5/Cy3 mean signal ratio, taking a shifted log (base 2), and combination of duplicated spot data. To identify differentially expressed (DE) genes, expression ratio results were filtered to eliminate genes whose 95% confidence interval for mean fold change (FC) did not extend to 2-fold up or down, followed by Students t test with False Discovery Rate (FDR) multiple testing correction to retain only genes in which expression changes versus untreated control were significant at P < 0.05.

middle: L

first: Edwin

last: Pang

first: Rebecca

last: Ford

first: Tristan

middle: E

last: Coram


title: Active and repressive chromatin in the mammalian genome intersperse without spreading in imprinted gene clusters

status: Public on Jul 01 2007

city: Vienna

first: Denise

middle: P.

last: Barlow

address: Dr. Bohrgasse 9/4

department: Austrian Academy of Sciences

country: Austria

institute: Center of Molecular Medicine (CeMM)

type: Genome binding/occupancy profiling by genome tiling array

type: Expression profiling by genome tiling array

summary:
The Igf2r imprinted cluster is an epigenetic model of cis-acting silencing in which expression of a ncRNA silences multiple genes. Here, we map chromatin profiles on the maternal and paternal chromosome in a 250 kb region. We show that histone modifications associated with expressed and silent genes are mutually exclusive and localize to discrete regions. Expressed genes are modified in two ways; at promoter regions by H3K4me3+H3K4me2+H3K9Ac and on putative regulatory elements by H3K4me2+H3K9Ac. Repressed genes displayed two types of non-overlapping profiles. Type 1 localized to short regions and contained H3K9me3+H4K20me3 and sometimes HP1. Type 2 spanned a large domain containing multiple tissue-specific silent genes and contained H3K27me3 alone. These data identify different forms of repressive chromatin on chromosome arms that resemble constitutive and facultative heterochromatin but are restricted to short regions and interspersed with euchromatin.
Keywords: ChIP-Chip, chromatin profile, genomic imprinting, noncoding RNA

overall_design:
Chromatin immunoprecipitaion (ChIP) was combined with a custom PCR tiling array (Chip) to determine the profile of histone modifications on the maternal and paternal allele of the imprinted Igf2r cluster on mouse chromosome 17. ChIP-Chip experiments were carried out on mouse embryo fibroblast (13.5dpc) cell lines.
The main steps in the array design were (i) design of primers to amplify single copy DNA, (ii) spotting each PCR fragment 8 times on the array.The main steps in the ChIP were: (i) preparation of chromatin by micrococcal nuclease (MNase) digestion, (ii) use of specific antibodies, (iii) amplification of input and ChIP enriched chromatin using two rounds of T7 in vitro transcription, (iv) conversion of amplified RNA to cDNA and labelling with Alexa dyes, (v) hybridisation, washing, scanning. Two biological replicates were performed for each antibody in each cell line. A dye swap was also incorporated in the second hybridisation. A novel method to test gene expression was also employed. PCR fragments lying within the exons of expressed genes gave a significant signal. This method also allowed for the detection of non-coding RNAs. A total of forty hybridizations were performed in the study.

first: Kakkad

last: Regha

first: Mathew

middle: A

last: Sloane

first: Ru

last: Huang

first: Florian

middle: M

last: Pauler

first: Katarzyna

last: Warczok

first: Balazs

last: Melikant

first: Martin

last: Radolf

first: Joost

middle: H

last: Martens

first: Thomas

last: Jenuwein


title: Methylation profiling in pediatric medulloblastoma

status: Public on Jul 31 2007

city: Heidelberg

first: Stefan

last: Pfister

address: INF 580

department: Molecular Genetics

country: Germany

institute: German Cancer Research Center

type: Methylation profiling by genome tiling array

summary:
Whole-genome screening of CpG Island methylation status by array-based profiling of absolute methylation status (array-PAMS).
Keywords: methylation profiling, pediatric medulloblastoma, single sample

overall_design: Methylation-specifically digested DNA (Cy3) was hybridized against methylation-sensitively digested DNA (Cy5) from the same sample. CGI methylation in 20 pediatric medulloblastomas (M) and normal cerebellum (Cb, pool of five unaffected donors, age 25–33 years)

first: Christof

last: Schlaeger

first: Bernhard

last: Radlwimmer

first: Peter

last: Lichter


title: Transciptional profiling of Candida albicans wild-type strains with TRK1 gene modification

status: Public on Jul 18 2007

city: Buffalo

first: Wansheng

last: Li

state: NY

address: 3435 main st.

department: Oral Biology

country: USA

institute: University at Buffalo

summary:
We built 3 strains from Candida albicans wild-type strains, mutant strain with one TRK1 gene deleted(one allele of two genes was deleted), overexpression strain(add one allele of the gene to rp10 locus), and restoration strain(add one allele of the gene to mutant strains).
The goal is study the expression level change of genes among the three strains compared with wild-type strain on a genomic scale.
Keywords: genetic modification

overall_design:
Indirect balanced design
Totally 12 samples/arrays. Three strains(mutant strain, overexpression strain and restoration strain) were hybrized to reference strain wild-type, each has four technical replicates, including 2 dye-swap replicates and 2 duplicate experiments.

first: Slavena

last: Vylkova


title: Myocardial gene expression of hibernating and control tissue from patients with ischemic left ventricular dysfunction

city: Bad Krozingen

middle: G

last: Nuehrenberg

state: Baden-Württemberg

address: Südring 15

department: Klinik für Kardiologie II

institute: Universitäts-Herzzentrum Freiburg • Bad Krozingen

summary:
Objectives: While cardiac scar tissue is damaged irreversibly, hibernating myocardium is characterized by reversible contractile dysfunction. Limited data are available in humans regarding the molecular biology of hibernating myocardium. The aim of this study was to identify new molecular mechanisms distinctive for human hibernating myocardium  by gene expression analysis.
Results: Of 4,171 transcripts examined, we identified 86 to be differentially expressed. Twentyone genes showed an increased and 65 genes a decreased expression in hibernating myocardium. Functional clustering revealed major changes in the expression of genes associated with transcription, protein modification and phosphorylation, regulation of apoptosis and intercellular communication. Besides the reported upregulation of ß-adrenergic receptor kinase-1 in heart failure, we observed new gene expression patterns, such as the upregulation of fas-activated serine/threonine kinase (FAST) or reduced expression of desmoplakin. Downregulation of desmoplakin in cardiomyocytes from hibernating myocardium was also seen on the protein level, consistent with a role of this protein in the development of a dilative cardiomyopathy.
Conclusions: Gene expression analysis of hibernating myocardium provided novel insights into the molecular mechanisms underlying ischemic heart failure. Dysfunction of desmosomes may contribute to impaired contractility and dilative tissue remodeling.
Keywords: disease state analysis

overall_design: Methods: We compared in patients with advanced coronary artery disease and impaired left ventricular function the gene expression profile of myocardial biopsies from hibernating myocardium (n=5), as identified in PET by a mismatch between perfusion and glucose metabolism, with expression profiles of matched biopsies from remote myocardium. Gene-specific PCR of selected genes was used to validate our technical approach. Immunohistochemical staining was performed for one gene of specific interest.

first: Dietlind

last: Zohlnhöfer

last: Nührenberg

first: Felix

last: Haas

first: Frank

last: Bengel

first: Albert

last: Schömig

first: Patrick

last: Baeuerle

first: Markus

last: Schwaiger


title: Transcriptomics of phagocytosis and melanization in Aedes aegypti.

city: Madison

first: Bruce

last: Christensen

state: WI

address: 1656 Linden Drive

department: Pathobiological Sciences

institute: University of Wisconsin-Madison

summary:
As is the case in vertebrates, successful mosquito immune responses depend on a complex and carefully orchestrated network of processes in order to defend the host from a foreign invader and to simultaneously maintain a homeostatic environment.  Although commonalities exist between immune response mechanisms across phyla, invertebrates (e.g., mosquitoes) lack anticipatory, adaptive immune responses.  However, a number of immune response elements, both cellular and humoral, exist to combat infection. Organisms that successfully invade the mosquito body cavity (hemocoel) potentially are subjected to a variety of mosquito defenses including: melanotic encapsulation, the production of antimicrobial peptides (AMPs) or reactive oxygen and nitrogen intermediates, or to engulfment by phagocytic cells.  These arrays are used to temporally compare and contrast transcriptome profiles associated with these responses, as manifest in the cells (hemocytes) that circulate in the mosquito body cavity.   Hemocytes have documented association with phagocytosis and melanization reactions in mosquitoes, and these responses, to a variety of Gram positive and Gram negative bacteria, have been characterized.   Inoculated Escherichia coli elicit a strong phagocytic response, and Micrococcus luteus are rapidly melanized.  For transcriptome profiling, inoculated moquitoes are compared to naïve (uninoculated) mosquitoes.  Data obtained will begin to clarify the interactive role of hemocyte genes and gene products in orchestrating phagocytic, melanization, and AMP responses against invading bacteria.
Keywords: time course

overall_design: Three-day old adult female mosquitoes were inoculated with ~500,000 bacteria from an overnight culture of either E. coli or M. luteus.  For each sample, approximately 100 mosquitoes were inoculated, while an additional 100 from the same cohort were left as naive.  Mosquito hemolymph was then perfused at 1, 8, or 24 hours post-inoculation. cDNA from treated and naive were labeled with Cy3 or Cy5 dye and hybridized on custom two-color chips.  For each time point, and each inoculated species, at least four biological replicates from different cohorts were prepared.  At least two technical replicates of each biological replicate were performed.  to test dye-bias, each time point has at least one dye-swap technical replicate.  A total of 51 chips were analyzed for this study.

first: George

middle: F

last: Mayhew

first: Lyric

middle: C

last: Bartholomay


title: Gene expression changes in BVDV2-infected BL3 cells

status: Public on Jun 30 2007

city: Ames

first: John

middle: D.

last: Neill

state: IA

address: 2300 Dayton Ave.

institute: National Animal Disease Center

summary:
The goal of this study was to study the gene expression changes that occurred in BL3 cells that were infected with type 2 bovine viral diarrhea virus (BVDV2).  What happens within the cell and how the cell responds to the virus is completely unknown.
Keywords: gene expression changes, BVDV2-infected cells, acute infection

overall_design: This study compared the gene expression patterns in noninfected BL3 cells to that in cells that had been infected with BVDV2 for 18 hours.  The 2 SAGE libraries were compared to each other to determine which transcripts were present at altered levels in the infected cells.  This data will give clues to how the viral infection gives rise to the different pathologies observed in infected animals.

middle: D

first: Julia

last: Ridpath


title: Responses in Medicago truncatula to Sinorhizobium meliloti wild type or the succinoglycan-deficient exoY mutant.

city: St Paul

first: Kathryn

last: VandenBosch

state: MN

address: Room 250 BioSci 6022 1445 Gortner Ave

department: Plant Biology

institute: University of Minnesota

summary:
For transcript analysis of responses in Medicago truncatula to its symbiont Sinorhizobium meliloti wild type or the succinoglycan-deficient exoY mutant we compared transcripts from line A17 roots inoculated with 25 mL OD = 0.05 S. meliloti wild type or exoY mutant.
Keywords: 1 line; 2 S. meliloti samples

overall_design:
1 line; 2 S. meliloti samples consists of 6 microarrays.
Each experiment consists of three direct (reps1, 2, 3) and three dye-swapped (reps 4, 5, 6) technical replicates.

last: Jones

first: Natasha

last: Sharopova

first: Graham

last: Walker


title: Bovine ovarian follilce selection

city: Columbia

first: Zhilin

last: Liu

state: MO

address: 920 East Campus Dr., ASRC S140

institute: University of Missouri-Columbia

summary:
Lack of, or abnormal, ovarian follicular development prevents establishment of pregnancy in human and is associated with reduced reproductive efficiency in farm animals. Development of new treatments is limited by our poor understanding of ovarian follicular development.  The objective of this study is to identify changes in gene expression during the selection stage of bovine follicular growth. Follicles of different sizes were collected and intrafollicular concentrations of steroids were measured to identify healthy follicles. Gene expression profiles were obtained using cDNA slides containing ~18,000 bovine EST probes. Using a false discovery rate of 0.05, we identified 976 probes differentially expressed across different follicles with at least 1.4 fold changes. Genes involved in immune system, cell organization and biogenesis, and cell proliferation were significantly over represented in the list, indicating the potential involvement of these biological processes in follicular selection.
Keywords: gene expression, follicle stage comparison

overall_design: RNA samples from the 16 largest follicles of 16 heifers with the follicle size of 8mm to 12 mm were used in the hybridization. A design with two samples hybridized on each array and each samples repeated 3 times (swap dyes) on different arrays was used.

first: Allen

last: Garverick

first: Robert

middle: S

last: Youngquist

first: Eric

last: Antoniou


title: IFN-gamma-inducible gene expression in Toxoplasma-infected human fibroblasts

status: Public on Jul 19 2007

city: Stanford

last: Stanford Microarray Database

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

summary:
Human fibroblasts were infected or not with Toxoplasma gondii (Pru strain) for 18 h at a nominal MOI of 3. Cells were subsequently treated with human recombinant interferon gamma (0.5 ng/ml = 30 pM) for 0, 2, 4 and 8 h before RNA was harvested for cDNA microarray experiments. Uninfected samples were named N0, N2, N4 and N8 (i.e., N2 indicated uninfected cells treated with interferon gamma for 2 h); infected samples were named P0, P2, P4 and P8. Duplicate cultures were analyzed for each condition (indicated as "a" and "b"). The reference channel (ch1, green) contained Universal Human Reference RNA. Using a genome-wide microarray analysis we show here a complete dysregulation of interferon-gamma-inducible gene expression in human fibroblasts infected with Toxoplasma.  Notably, 46 of the 127 interferon-gamma-responsive genes were induced and 19 were suppressed in infected cells before they were exposed to interferon gamma, indicating that other stimuli produced during infection may also regulate these genes. Following interferon-gamma treatment, none of the 127 interferon-gamma-responsive genes could be significantly induced in infected cells.
Groups of assays that are related as part of a time series.
Infection: Toxoplasma gondii (18h post-infection)
Time: time of IFN-gamma treatment
Growth Condition: interferon gamma
Keywords: time_series_design

overall_design: Computed

first: Seon

last: Kim


title: Potato tuber development

city: Rockville

first: Jia

state: MD

address: 9712 Medical Center Drive

institute: Plant Genomics

summary:
An in vivo and in vitro potato tuber development gene expression study.
Keywords: Developmental stage analysis, time course

overall_design: For in vitro tuber development expression analysis, RNA was isolated from in vitro microtubers at 2, 5, 10, 20 and 30 days following observed tuber induction. Two microtuber populations were used as biological replicates for the developmental stages. The RNA from all developmental stages was pooled to generate the reference samples. Ten microarray hybridizations were performed. For in vivo tuber development expression analysis, RNA was isolated from tubers growing in growth chamber conditions. Tissues were divided into six group, according to developmental size: stolon (no tuber formation), 1-5 mm tubers, 6-10 mm tubers, 11-15 mm tubers, 16-25 mm tubers, and 26-35 mm tubers. Two biological replicates of ten plants each were grown sequentially in the same growth chamber. The RNA from all developmental stages was pooled to generate the reference samples. Twelve microarray hybridizations were performed. For all experiments, the RNA was labeled using the indirect labeling method with random hexamer primers. Amplified cRNA was used as labeling template for stolons. Total RNA was used as labeling template in all other labeling reactions.

last: Stupar

first: Jiming

last: Jiang


title: California Vibrio cholerae CGH

status: Public on Jul 17 2007

summary:
Environmental isolates of Vibrio cholerae from California coastal water compared to reference strain N16961.
A genotyping experiment design type classifies an individual or group of individuals on the basis of alleles, haplotypes, SNP's.
Keywords: genotyping_design; array CGH

overall_design: User Defined

first: Michael

last: Miller


title: Low-level MMS treatment of S.cerevisiae

status: Public on Jun 26 2007

first: Ana

last: Kitanovic

address: Im Neuenheimer Feld 364

department: Biology

institute: Institute for Pharmacy and Molecular Biotechnology

summary:
The cellular response to treatment with DNA-damaging substances at low concentrations which are genotoxic but do not have a strong cytotoxic effect are of special interest. In addition, environmental variations that influence growth conditions, e.g. different media, and individual fitness, e.g. different strains, are likely to influence and modulate the adverse effects of individual DNA damaging substances. At sub-cytotoxic levels, DNA damaging substances play an important role in the accumulation of genomic mutations. In longer living organisms, like humans and other mammals, exposure to DNA damaging substances over extended period of time is a critical factor that contributes to the development of various diseases and in particular of tumors. The aim of our work was to study how strain background and growth conditions influence respond to DNA damage caused by low doses of MMS and which part of these changes is responsible for their sensitivity to toxic conditions. We analyzed sensitivity of two yeast strains FF18984 and BY4742 to MMS in media with limited and full nutrient availability.
Keywords: Yeast, S.cerevisiae, MMS, stress response, DNA damage

overall_design: YPD and F1 media were inoculated with overnight pre-cultures and grown at 30°C to mid log-phase (OD600 0.6 to 0.8). Cultures were split into three parts: the first aliquot was mock-treated and used as control; the second and third aliquots were treated with low concentration of MMS (0.00125% and 0.0125%). All cultures were incubated at 30° C. Samples were collected after 30 min and 1h incubation. The total of 14 samples were analysed. With the FF18984 strain experiments were done in minimal (F1) and full (YPD) medium with both MMS concentrations (0.00125% and 0.0125%) for 30 min and 1h. With the BY4742 strain experiments were done in full (YPD) medium with 0.0125% MMS for 1h. As a reference we used mock-treated samples from the same medium and the same time point.

first: Torsten

last: Kroll

first: Larissa

last: Pusch

first: Nicole

last: Hauser

first: Stefan

last: Wölfl


title: GAF pGHost vs mariner

city: Groningen

first: Anne

middle: de

last: Jong

address: Nijenborgh 7

country: Netherlands

institute: university of Groningen

summary:
Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.
Keywords: GAF pGHost vs mariner

overall_design:
Comparison of the pGhT7:ISS1 and marinerT7 transposon libraries
Previous experiments were performed on marinerT7 generated libraries. To test whether the method could also be applied to pGh9T7:ISS1 libraries, and to assess the level of saturation and randomness achieved by each transposon, we compared a large (> 20,000 CFU) marinerT7 library with a pGh9T7:ISS1 one, each grown for approximately 20 generations in GM17. After isolation, the DNA was digested with DdeI and AluI and each detection reaction was performed in duplicate. Microarray data were analyzed as before using the background filter generated with the TIGR4-specific amplicons (the normalized signal data and cyberT output data for these arrays are available at http://molgen.biol.rug.nl/publication/GAF_data). This analysis resulted in the detection of 1,473 genes and the not-detected genes were similar to those not-detected in the analysis of the three marinerT7 digests. The ratio of the majority of the genes was between 0.5 and 2, indicating that they are mutated by both transposons. However, 139 (9%) genes had a ratio lower than 0.5 or higher than 2, indicating that these genes are only, or preferentially, mutagenized by one of the two transposons. These “hotspots” are distributed throughout the genome (Table 3). There were 60 genes preferentially mutated by pGh9T7:ISS1, whereas 79 were preferentially mutated by marinerT7, indicating that it has a slightly higher saturation rate than pGh9T7:ISS1. Anchored PCR using primers located on some of the ORFs that had a ratio close to the cut-off point of 0.5 indicated that these genes were indeed preferentially hit by either transposon and that these ratios were not due to slight differences in, for instance, T7 RNA polymerase efficiency (data not shown).

first: Jetta

last: Bijlsma


title: Kruppel-like transcription factor 13 regulates T lymphocyte survival in vivo.

summary:
Kruppel-like transcription factor (KLF)13, previously shown to regulate RANTES expression in vitro, is a member of the Kruppel- like family of transcription factors that controls many growth and developmental processes. To ascertain the function of KLF13 in vivo, Klf13-deficient mice were generated by gene targeting. As expected, activated T lymphocytes from Klf13-/- mice show decreased RANTES expression. However, these mice also exhibit enlarged thymi and spleens. TUNEL, as well as spontaneous and activation-induced death assays, demonstrated that prolonged survival of Klf13-/- thymocytes was due to decreased apoptosis. Microarray analysis suggests that protection from apoptosis-inducing stimuli in Klf13-/- thymocytes is due in part to increased expression of BCL-XL, a potent antiapoptotic factor. This finding was confirmed in splenocytes and total thymocytes by real-time quantitative PCR and Western blot as well as in CD4+CD8? single-positive thymocytes by real-time quantitative PCR. Furthermore, EMSA and luciferase reporter assays demonstrated that KLF13 binds to multiple sites within the Bcl-XL promoter and results in decreased Bcl-XL promoter activity, making KLF13 a negative regulator of BCL-XL.
Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Keywords: Logical Set

first: Meixia

last: Zhou


title: Ex vivo sorted murine pro-B cells: Wild-type versus Pax5-/-

status: Public on Jul 13 2007

first: Meinrad

last: Busslinger

address: Dr. Bohr-Gasse 7

institute: Research Institute of Molecular Pathology

summary:
Transcriptional profiling of mouse pro-B cells comparing wild-type and Pax5-deficient cells
Keywords: Genetic modification

overall_design: Two-condition experiment. Wild-typre versus Pax5-/- pro-B cells. Biological replicates: 2 wild-type, 2 Pax5-/-, independently sorted.

first: Alexandra

last: Schebesta


title: In vitro cultured murine pro-B cells: Wild-type versus Pax5-/-

overall_design: Two-condition experiment. Wild-typre versus Pax5-/- pro-B cells. Biological replicates: 4 wild-type, 4 Pax5-/-, independently grown and harvested.


title: Shigella CGH

city: Beijing

first: Junping

last: Peng

address: No 6, Rongjing Eastern Street, BDA

country: China

institute: Chinese center for disease control and prevention

summary:
Background
Compelling evidence indicates that Shigella species, the etiologic agents of bacillary dysentery, as well as enteroinvasive Escherichia coli, are derived from multiple origins of Escherichia coli and form a single pathovar. To further understand the genome diversity and virulence evolution of Shigella, comparative genomic hybridization microarray analysis was employed to compare the gene content of E. coli K-12 with those of 43 Shigella strains from all serotypes.

Results
For the 43 strains subjected to CGH microarray analyses, the common backbone of the Shigella genome was estimated to contain more than 1,900 open reading frames, with a mean number of 729 undetectable ORFs. The mosaic distribution of absent regions indicated that insertions and/or deletions have led to the highly diversified genomes of pathogenic strains.
Conclusion
These results support the hypothesis that by gain and loss of functions, Shigella species became successful human pathogens through convergent evolution from diverse genomic backgrounds. Moreover, we also found many specific differences between different lineages, providing a window into understanding bacterial speciation and taxonomic relationships.
Keywords: comparative genomic hybridization

sample_organism:
Escherichia coli
Shigella boydii
Shigella dysenteriae
Shigella flexneri

platform_organism:
Escherichia coli K-12
Shigella flexneri 2a str. 301
Shigella dysenteriae Sd197
Shigella boydii Sb227
Shigella sonnei Ss046

overall_design: There are 39 strains used in comparative genomic hybridization, and competitive hybridization was done at least three times for one strain.

first: Xiaobing

last: Zhang

first: Jian

last: Yang

first: Jing

last: Wang

first: Qi

last: Jin


title: Post-replicative formation of cohesion is required for repair and induced by a single DNA break

city: Bunkyo-ku

first: Katsuhiko

last: Shirahige

state: Tokyo

address: 1-1-1 Yayoi

department: Research Center for Epigenetic Disease

country: Japan

institute: The University of Tokyo

summary:
Sister chromatid cohesion, established during replication by the protein complex Cohesin, is essential for both chromosome segregation and double-strand break (DSB) repair. Normally cohesion formation is strictly limited to the S-phase of the cell cycle, but DSBs can trigger cohesion also after DNA replication has been completed. The function of this damage-induced cohesion remains unknown. In this investigation we show that it is essential for repair in post-replicative cells in yeast. Furthermore, it is established genome-wide after induction of a single DSB, and controlled by the DNA damage response and Cohesin regulating factors. We thus define a cohesion establishment pathway that is independent of DNA duplication and acts together with cohesion formed during replication in sister chromatid-based DSB repair.
Keywords: ChIP-chip analysis

supplemental_files: ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE8nnn/GSE8270/suppl/GSE8270_Binding ratio of protein of interest_Scc1_rH2AX.txt

supplemental_files: ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE8nnn/GSE8270/suppl/GSE8270_Binding ratio of protein of interest_Scc2.txt

overall_design:
Scc2, Scc1, and rH2AX distribution with or without DSB in wild type and mutant strains. DSB was induced by galactose promoter driven HO. All yeast strains were haploid and of W303 origin (ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3, RAD5).
DNA was purified and amplified as previously described by Katou et al. (nature, 2003), and hybridized to SC3456a 52005F or S.cerevisiae Tiling 1.0F arrays, Part#520286 (Affymetrix). Scc2 was C- terminally tagged with 6HIS-3xFLAG, while Mcd1UNCL was marked with 3HA epitopes. Anti-FLAG antibody M2 (Sigma), anti-HA antibody (Babco), or anti γ-H2A-antobodies kindly provided by A. Verreault  were used for immuno-precipitations.

first: Lena

last: Strom

first: Charlotte

last: Karlsson

first: Hanna

middle: B

last: Lindroos

first: Sara

last: Wedahl

first: Yuki

last: Katou

first: Camilla

last: Sjogren


title: Using breast (cancer) cell lines to measure bortezomib therapy response

status: Public on Jul 14 2007

city: Amsterdam

first: Dimitry

last: Nuyten

address: Plesmanlaan 121

department: Radiation Oncology

institute: The Netherlands Cancer Institute

summary:
Revealing targeted therapy for human cancer by gene module maps
A major goal of cancer research is to match specific therapies to molecular targets in cancer. Genome-scale expression profiling has identified new subtypes of cancer based on consistent patterns of variation in gene expression, leading to improved prognostic predictions. However, how these new genetic subtypes of cancers should be treated is unknown. Here we show that a gene module map can guide the prospective identification of targeted therapies for genetic subtypes of
cancer. By visualizing genome-scale gene expression in cancer as combinations of activated and deactivated functional modules, gene module maps can reveal specific functional pathways associated with each subtype that might be susceptible to targeted therapies. We show that in human breast cancers, activation of a poor prognosis “wound signature” is strongly associated with induction of a proteasome
gene module. Inhibition of proteasome activity by bortezomib, a drug approved for human use in multiple myeloma, abrogated wound signature expression and selectively killed breast cells expressing the wound signature. Thus, gene module maps may enable rapid translation of complex genomic signatures in human disease to targeted therapeutic strategies.
Keywords: Gene expression profiling, breast cancer, therapy response, in vitro

overall_design: 8 breast cancer cell lines and a breast epithelial cell line were cultured in standard serum conditions. The breast cancer cell lines were profiled after both standard culture conditions (10% fetal bovine serum; FBS) and serum starvation. The epithelial cells after serum starvation. All cells were also profiled after treatment with a proteosome inhibitor (bortezomib). Changes in gene expression and changes in the previously described wound-response signature were measured.

first: Eran

last: Segal

first: David

last: Wong

first: Meihong

last: Lin

first: Marc

middle: J

last: Van de Vijver

first: Howard

middle: Y

last: Chang


title: Genes differentially expressed in fiber 1 day post anthesis (dpa)

city: Raleigh

first: Earl

middle: William

last: Taliercio

state: NC

address: 3127 Ligon St

institute: USDA/ARS

summary:
A method was developed to isolate RNA from 1 dpa fiber. ESTs derived from this and other cotton mRNAs were sequenced and assembled into contigs. Contigs composed of EST unique to libraries of interest and unique singletons were represented on a microarray. Microarrays were hybridized with labed nucleic acids derived from whole 1 dpa ovules and 1 dpa fiber to identify genes differentially regulated during fiber initiation. Genes with known expression in fiber were included to validate the microarray. Analysis of the gene ontology (GO) of differentially expressed genes suported previously reported aspects of fiber initiation such as an increase in protein biosynthesis. An increase in the "membrane" was indicated by the GO analysis ans supported by increased staining of fiber initial ER. Analysis of individule genes differentially regulated in fiber initials indicated calcium may play a role in fiber deveopment which was also supported by an increase in calcium in fiber initials.
Keywords: comparative hybridization

overall_design:
The microarray represents 11143 gene models. One gene model is represented by 5 different oligonucleotides, 8604 gene models are represented by 4 different oligonucleotides, 1706 gene models are represented by 3 different oligonucleotides, 831 gene models are represented by 3 of the same oligonucleotides and one gene model is represented by 2 different oligonucleotides.

Each micro array is hybridized with a labelled nucleic acid derived form RNA isolated from 1 dpa fiber or 1 dpa ovules harvested on different days from field grown plants. One is a dye swap.

first: Deborah

last: Boykin


title: Identification of genes differentially expressed in 1 dpa and 10 dpa fiber

summary:
A method was developed to isolate RNA from 1 dpa fiber. ESTs derived from this and other cotton mRNAs were sequenced and assembled into contigs. Contigs composed of EST unique to libraries of interest and unique singletons were represented on a microarray. Microarrays were hybridized with labed nucleic acids derived from 10 dpa fiber and 1 dpa fiber to identify genes differentially regulated during fiber initiation and fiber elongation. Genes with known expression in fiber were included to validate the microarray. Analysis of the gene ontology (GO) of differentially expressed genes suported previously reported aspects of fiber initiation such as cell wall remodeling. Transcription factors upregulated in fiber initials and elongating fiber were identified
Keywords: cell type comparison

overall_design:
The microarray represents 11143 gene models. One gene model is represented by 5 different oligonucleotides, 8604 gene models are represented by 4 different oligos, 1706 gene models are represented by 3 different oligonucleotides, 831 gene models are represented by 3 of the same oligonucleotides and one gene model is represented by 2 different oligonucleotides.

Each micro array is hybridized with a labeled nucleic acid derived form RNA isolated from 1 dpa fiber or 10 dpa fiber harvested on different days from field grown plants. One is a dye swap.


title: trxB1 overexpression response to hydrogen peroxide stress in Lactobacillus plantarum WCFS1

status: Public on Jul 12 2007

city: Wageningen

first: L.

middle: Mariela

last: Serrano

address: Nieuwe Kanaal 9A

institute: TI Food and Nutrition

summary:
Two independent chemostat cultivations were performed for both wild type and trxB1 over-expression strain, NZ7602. Three hybridization experiments, all with the same hybridization scheme (see overall design), were performed with samples obtained from these chemostats before and after treatment with hydrogen peroxide (30 minutes 2.5 mM peroxide). Two of these experiments used samples obtained from the same fermentation. In each experiment three arrays were used. Per array two cDNA labeled targets were hybridized on custom designed L. plantarum WCFS1 11K Agilent oligo microarrays (GEO Acc. Nr. GPL4318) using the Agilent 60-mer oligo microarray processing protocol version 4.1. These microarrays contained an average of 3 probes per gene. Dried slides were scanned in the Scan Array Express (PerkinElmer Life Sciences; Packard Bioscience) at 10 microns.  Spot intensity data was quantified (average intensity) in ImaGene version 5.0 (BioDiscovery, Inc., El Segundo, CA). Signal intensities of all probes were corrected
against background and normalized by fitting a plot of M (= 2log [cy5 intensity/cy3 intensity]) against A (= 0.5 • 2log [cy5 intensity • cy3 intensity]) using the lowess algorithm in BASE [32]. The fold change (FC) is defined as 2M. For the statistical analysis we used microarray analysis of variation (R/maanova) [33] In this maanova test we used three variables: fermentation, treatment, and genotype. We tested the model taking into consideration the interaction between genotype and
treatment. Significantly regulated genes were defined as genes whose nominal adjusted pvalues were less than 10% and a FC>1.5. The maanova test resulted only in two sets of interesting data because
the interaction effect did not reveal significant changes in transcript levels.  One dataset representing the transcripts affected as a result of the overproduction of TR and another set representing the transcripts affected due to oxidative stress. These two datasets were denominated
genotype and hydrogen peroxide datasets respectively.
Keywords: hydrogen peroxide stress response

overall_design:
The hybridization scheme contained the following cDNA comparisons:
 (1) wildtype with trxB1 over-expression strain (supplementary files
GSM206844,GSM206845,GSM206846)
(2) wildtype with wildtype treated with hydrogen peroxide (supplementary files GSM206852,GSM206848,GSM206847)
; and (3) wildtype treated with hydrogen peroxide with trxB1 over-expression strain treated with hydrogen peroxide (supplementary files GSM206849,GSM206850,GSM206851).

first: L

first: Douwe

last: Molenaar

first: Michiel

last: Wels

first: Bas

last: Teusink

first: Peter

last: Bron

first: Willem

last: de Vos

first: Eddy

last: Smid


title: Dissecting Gene Expression Networks in Mammalian Organogenesis (MORGEN)

status: Public on Jul 11 2007

city: Vancouver

first: BCGSC

last: BC Cancer Agency

state: BC

address: 675 West 10th Avenue

department: Gene Expression

country: Canada

institute: Canada's Michael Smith Genome Sciences Centre

summary:
Mammalian Organogenesis - Regulation by Gene Expression Networks (MORGEN) Project
Keywords: LongSAGE

overall_design: SAGE libraries sequenced to at least 300,000 tags.


title: Adult ALL (Acute Lymphoblastic Leukemia)

first: Dejan

last: Juric

address: 269 Campus Drive

department: Medicine, Oncology

institute: Stanford University School of Medicine

summary:
A set of 37 BCR-ABL positive (21 and 16 of which expressed p185 and p210, respectively) and 17 BCR-ABL negative adult ALL specimens from the ECOG/MRC intergroup study E2993. Gene expression profiling was performed using the 3DNA Array 900 Expression Array Detection Kit (Genisphere, Hatfield, PA), according to the manufacturer's instructions. Universal Human Reference RNA (Stratagene, La Jolla, CA) was used as a reference RNA. The results provide insights into molecular mechanisms and pathways associated with BCR-ABL oncogenesis.
Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Keywords: Logical Set


title: Detection and characterizing CNVs identified by tiling resolution BAC array CGH.

status: Public on Jun 23 2007

city: Nijmegen

first: Joris

middle: Andre

last: Veltman

address: Geert Grooteplein 10

department: Human Genetics

institute: Radboud University Nijmegen Medical Centre

type: Genome variation profiling by genome tiling array

summary:
In this study 405 patients with unexplained mental retardation and 89 unaffected parents were analyzed for DNA copy-number changes using a tiling-resolution genomewide microarray containing 32,447 BACs. This series contains 367 slides from 315 individuals previously not reported, plus an additional 239 slides from 179 individuals previously reported in the series GSE3191.
Keywords: array CGH

overall_design: A 32k tiling resolution genomic microarray consisting of 32,447 overlapping BAC clones, selected to cover the entire human genome, was used to generate genomic copy number profiles for 494 samples using methods previously described (de Vries et al. 2005). These samples were originally analyzed by this method within a diagnostic setting with the aim to identify copy number changes related to mental retardation. The samples therefore consisted of patients with unexplained mental retardation (n=405, of which 102 were run in replicate with dye-reversal) as well as unaffected parents (n=89, 38 complete trios). In all cases genomic DNA was isolated from uncultured blood leucocytes, excluding culture-induced rearrangements and aneuploidies as described by Redon et al. (2006). Hybridization was performed in a two-color experiment against a reference pool containing equal amounts of genomic DNA from 10 healthy blood donors (either mis-match or match). De novo alterations were validated by FISH and MLPA.

first: Rolph

last: Pfundt

first: Jayne

last: Hehir-Kwa


title: Changes in transcription patterns in BoMac cells in response to PDD spirochetes

status: Public on Jun 22 2007

first: Richard

last: Zuerner

address: P.O. Box 70

department: BDLRU

institute: USDA, ARS, NADC

summary:
Papillomatous digital dermatitis (PDD) is a polymicrobial infection in soft tissue adjacent to the hoof and is the leading cause of lameness in dairy cattle. Treponema phagedenis-like (TPL) spirochetes are a constant feature of PDD lesions and are localized deep in infected tissue.  Host-cell response mechanisms to TPL bacteria are poorly understood. To assess how bovine macrophages respond to cellular constituents of TPL spirochetes, changes in transcription were analyzed using serial analysis of gene expression (SAGE) and real time RT-PCR.  This analysis revealed that bovine macrophages responded to stimulation with TPL-lysates by induction of several pro-apoptotic genes. Although several proinflammatory cytokines (eg. IL-6 and IL-8) are induced in treated macrophages, receptors and their accessory proteins for IL-1, IL-6, and IL-11 are either down regulated or unchanged.  Surprisingly, IL-1beta is not induced and IL-18 is suppressed in treated macrophages. Two genes encoding proteins having negative effects on NFκB, IκB and Siva-1, are significantly induced in stimulated cells.  Combined, these data suggest that the innate immune response of bovine macrophages exposed to TPL cellular constituents are impaired thereby enabling bacteria to resist clearance and induce lesion formation.
Keywords: Bovine macrophage, transcription response, spirochete bacteria

overall_design:
An immortalized bovine macrophage cell line (BoMac) was used throughout this study (supplied by Dr. Judith Stabel, USDA, ARS, NADC, Ames, IA).  Cells were propagated in RPMI 1640 medium (GIBCO, Gaithersburg, MD) containing 10% fetal calf serum (Colorado Serum Company), 100µg/ml streptomycin, and 100U/ml penicillin (Sigma, St. Louis, MO) at 37C with 5% CO2.
PDD spirochetes (Treponema phagedenis-like strain 1A) in the exponential phase of growth were harvested by centrifugation (10,000g for 15 min.) and washed twice with PBS.  Cell lysates were prepared by sonication using short-wave pulses of 30s each (Vibra Cell, Sonics and Materials Inc, Danbury, Connecticut) until no intact cells were observed by dark field microscopy. Protein concentrations of whole cell sonicates (WCS) were determined using the Bradford assay (Bio-Rad, Hercules, CA) following the manufacturers instructions.
Bovine macrophages grown to about 90% confluence were stimulated with WCS lysate of TPL strain 1A (1 µg/ml) suspended in complete RPMI 1040.  Cells were incubated at 37oC for 4, 8, and 18 hrs. Cell monolayers were disrupted with RLT+ lysis solution and total cellular RNA isolated with RNeasy kits (Qiagen) according to the manufacturer’s instructions.  Where appropriate cell suspensions were further disrupted by homogenization using Qiashredder columns (Qiagen).  Residual DNA was removed by DNaseI treatment, followed by RNA purification (Qiagen).
Cellular mRNA was enriched from total RNA using PolyATract mRNA isolation system II (Promega, Madison, WI) and used as starting material for preparation of SAGE libraries.  Two SAGE libraries were prepared from 5 ug ds-cDNA, one each from non-stimulated and WCS-stimulated macrophages (4 hr treatment) following established protocols using NlaIII as the anchoring enzyme.  Concatermerized ditags were size selected by electrophoresis through polyacrylamide gels, then ligated with SphI-digested pZERO-1 (Invitrogen Corp.)  and used to transform E. coli to zeocin resistance.  Transformed colonies were picked at random to 96-well plates and plasmid DNA prepared by alkaline lysis (Qiagen).   Plasmid DNA was sequenced unidirectionally from the T7 primer region using fluorescent dideoxy chain-termination reactions separated in a 3700-sequence analyzer (Applied Biosystems, Inc.).
Sequence data interpreted using Phred and Sequencher (Gene Codes Corporation, Ann Arbor, MI) were combined and edited manually.  Inserts from a total of 2155 and 2131 plasmids were sequenced with average error rates of 0.00435 and 0.00977 from control and stimulated cell libraries, respectively.  Data files for each library were processed with eSAGE software to extract ditags, remove duplicate sequences, and compare the two libraries to determine statistically significant differences in tag frequency.
SAGE data were validated by quantifying transcription levels of selected genes using RNA isolated from three distinct biological replicates each of treated and control samples. Semiquantitative RT-PCR was done in a DNA Engine Opticon 2 detection system (MJ Research, Inc) using QuantiTect™ SYBR Green PCR Kit (Qiagen), gene specific primers (Table 1), with 200 ng/µl cDNA template.  PCR primers were designed using Primer Design 5 software (Sci Ed Central) from sequences obtained from GenBank.  All reactions were performed in triplicate and qRT-PCR data were analyzed by using the 2-(ΔΔCt) method.  The β-actin gene was used as the control gene (ΔCt) and non-stimulated samples were used as the calibrator (ΔΔCt).

middle: L

first: Mohammed

last: Heidari

first: Margaret

last: Elliot

first: David

middle: P

last: Alt

first: John

last: Neill


title: Incipient speciation by divergent adaptation and antagonistic epistasis in yeast

status: Public on Jun 21 2007

city: Mississauga

first: James

last: Anderson

state: Ontario

address: 3359 Mississauga Road N.

institute: University of Toronto

summary:
Understanding the conditions that promote the evolution of reproductive isolation, and thus speciation. Here we empirically test some of the key predictions of speciation theory (Coyne 2004; Kohn 2005) by experimentally evolving the initial stages of speciation in yeast. After allowing replicate populations to adapt to two divergent environments, we observed the consistent, de novo evolution of two forms of postzygotic isolation: reduced rate of mitotic reproduction and reduced efficiency of meiotic reproduction. In general, divergent selection resulted in greater reproductive isolation than parallel selection, as predicted by ecological speciation theory. Our experimental system allowed for the first controlled comparison of the relative importance of ecological and genetic mechanisms of isolation, and the novel ability to quantify the effects of antagonistic epistasis. For mitotic reproduction, hybrid inferiority was conditional upon the selective environments and was both ecological and genetic in basis. In contrast, isolation associated with meiotic reproduction was unconditional and was caused solely by genetic mechanisms. Overall, our results show that adaption to divergent environments promotes the evolution of isolation through antagonistic epistasis, providing evidence of a plausible common avenue to speciation and adaptive radiation in nature (Schluter 2000,2001: Funk 2006)
Keywords: Speciation, antagonistic epistasis, divergent adaptation

overall_design:
We measured gene expression effects due to interaction of divergently
adapted genomes in hybrid cells.  Reference RNA was a mix of parental
lines adapted to the different environments.  Test RNA was from the
hybrid.  There were four independent replicates for each comparison. The goal was NOT to measure the changes in expression
associated with adaptation, which would have used the progenitor as a
source of reference RNA.

first: Jeremy

last: Dettman

first: Caroline

last: Sirjusingh

first: Linda

middle: M

last: Kohn


title: S. coelicolor Wild Type Time-course Study in R5- medium (Culture#1)

status: Public on Jun 19 2007

city: Minneapolis

first: Salim

last: Charaniya

address: 421 Washington Ave SE., 151 Amundson Hall

department: Chemical Engineering and Materials Science

institute: University of Minnesota

summary:
Antibiotic biosynthesis in Streptomyces species is controlled by a complex genetic and biochemical network of global and pathway specific regulators.  Details of their precise interactions in mediating temporal and spatial expression of secondary metabolite genes remain poorly defined.  In this study, we employed whole-genome microarrays to investigate the temporal transcriptome profiles of S. coelicolor A3(2) M145 wild type and disruption mutants of regulatory genes (afsS and absA1) known to affect antibiotic biosynthesis.
Keywords: Time course

overall_design: The time-course study involved analysis of 6 samples (15h, 22h, 24h, 26h, 35h, 43h). Of these, 15h and 22h were analyzed in duplicate arrays. Genomic DNA of S. coelicolor wild type (M145) was used as a reference for all the arrays. cDNA was labeled with Alexa647 and genomic DNA was labeled with Cy3.

first: Wei

last: Lian

first: Karthik

last: Jayapal

first: Wei-Shou

last: Hu


title: Identification of Iron-Inducible Genes in Magnetospirillum magneticum AMB-1 Non-Magnetic Mutant

status: Public on Jul 07 2007

city: Koganei

first: Tadashi

last: Matsunaga

department: Biotechnology

institute: Tokyo University of Agriculture and Technology

summary:
Iron is limiting in the environment, bacteria respond to this deprivation by activating genes required for bacterial iron homeostasis. Transcriptional regulation in response to iron in Gram-negative bacteria is largely mediated by the ferric uptake regulator protein Fur, which in the presence of iron binds to a specific sequence in the promoter regions of genes under its control and acts as a repressor. Here we describe comparative global gene expression analysis using DNA microarray based on the whole genome sequence of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 was conducted between wild type strain and a non-magnetic NMA61 mutant strain, generated by mini-Tn5 transposon mutagenesis which is incapable of assimilating iron to cytoplasm. No induction of the fur genes in NMA61 mutant strain was considered to be due to low intracellular iron concentration. In the iron-replete condition, among 4492 genes, 434 genes were down-regulated and 527 genes were up-regulated in the wild type strain. Among 434 genes down-regulated, 299 genes were not down-regulated in NMA61 mutant strain, indicating these genes are candidates of Fur-regulated.
Keywords: Iron, magnetotactic bacteria

overall_design: A non-magnetic mutant of Magnetospirillum magneticum AMB-1 (NMA61) generated transposon mutagenesis was grown under various iron conditions. Global gene expression analysis of iron-inducible genes was conducted by using DNA microarray.

first: Takeyuki

last: Suzuki

first: Atsushi

last: Arakaki

first: Yoshiko

last: Okamura

first: Haruko

last: Takeyama


title: S. coelicolor Wild Type Time-course Study in R5- medium (Culture#2)

overall_design: The time-course study involved analysis of 9 samples (10h, 13h, 16h, 19h, 22h, 25h, 31h, 35h, 37h). Of these, the 16h sample was analyzed on three arrays (3 technical replicates). All other samples were analyzed on duplicate arrays (2 technical replicates). Genomic DNA of S. coelicolor wild type (M145) was used as a reference for all the arrays. cDNA was labeled with Alexa647 and genomic DNA was labeled with Cy3.


title: Pcs1 and Mde4 are inner centromere proteins required to prevent merotelic kinetochore orientation

status: Public on Jun 18 2007

first: Katsuhiko

last: Shirahige

summary:
Chromosome segregation depends on proper attachment of sister kinetochores to microtubules. Merotelic kinetochore orientation is an error which occurs when a single kinetochore is attached to microtubules emanating form opposite poles. Mechanisms preventing or correcting the merotelic attachment must operate to avoid chromosome missegregation. Pcs1 has been implicated in preventing merotelic attachment in mitosis and meiosis II. We describe here the identification of Mde4 protein which forms a complex with the Pcs1. Both Mde4 and Pcs1 localize to the central core of the centromere. Similarly to the pcs1 mutant, in the absence of mde4 lagging chromosomes are frequently observed during mitosis and meiosis II . We provide the first evidence that the lagging chromosomes in pcs1 and mde4 mutants are due to merotelic kinetochore orientation.
Keywords: ChIP-chip analysis

overall_design: ChIP-chip analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Pombe whole chromosome array was used.

first: Juraj

last: Gregan

first: Christian

last: Riedel

first: Alison

last: Pideaux

first: Yuki

last: Katou

first: Cornelia

last: Rumpf

first: Alexander

last: Schleiffer

first: Robin

last: Allshire

first: Kim

last: Nasmyth


title: Telomerase inhibitor TMPyP4 induced gene expression

status: Public on Jun 20 2007

city: Delhi

first: Anjali

last: Verma

state: Delhi

address: Mall road

department: Structural Biology and Genome Informatics Division

country: India

institute: Institute of Genomics and Integrative Biology

summary:
The effect of TMPyP4, the well established anticancer telomerase inhibitor on genome wide expression of genes was investigated
Keywords: Time course

overall_design: Cells were treated with TMPyP4 for either 24 or 48 hours before RNA isolation.Untreated cells were taken as controls and the TMPyP4 treated cells as treatments. Experiments at each time point were replicated four times.

first: Farhan

last: Mohd.

first: Abhay

last: Sharma

first: Shantanu

last: Chowdhury


title: Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression - Embryo data

city: Saint Paul

first: Nathan

last: Springer

address: 1445 Gortner Ave

summary:
Microarray analysis of gene expression patterns in immature ear, seedling, and embryo tissues from the maize inbred lines B73 and Mo17 identified numerous genes with variable expression. Some genes had detectable expression in only one of the two inbreds; most of these genes were detected in the genomic DNA of both inbreds, indicating that the expression differences are likely caused by differential regulation rather than by differences in gene content. Gene expression was also monitored in the reciprocal F1 hybrids B73xMo17 and Mo17xB73. The reciprocal F1 hybrid lines did not display parental effects on gene expression levels. Approximately 80% of the differentially expressed genes displayed additive expression patterns in the hybrids relative to the inbred parents. The approximately 20% of genes that display nonadditive expression patterns tend to be expressed at levels within the parental range, with minimal evidence for novel expression levels greater than the high parent or less than the low parent.  Analysis of allele-specific expression patterns in the hybrid suggested that intraspecific variation in gene expression levels is largely attributable to cis-regulatory variation in maize. Collectively, our data suggest that allelic cis-regulatory variation between B73 and Mo17 dictates maintenance of inbred allelic expression levels in the F1 hybrid, resulting in additive expression patterns.
Keywords: genotype comparison

overall_design: Affymetrix expression profiling was used to study gene expression in 19 day after pollination embryo tissue of maize. Three biological replicates were performed for four different genotypes; B73, Mo17, B73xMo17 and Mo17xB73.

first: Robert

last: Stupar


title: Serial Analysis of Ribosomal DNA and  the Unexpected Dominance of Rare Members of Microbial Communities

status: Public on Jun 15 2007

city: Tiburon

first: Matthew

last: Ashby

address: 3152 Paradise Drive

institute: Taxon Biosciences, Inc.

summary:
Accurate description of a microbial community is an important first step in understanding the role of its components in ecosystem function.  A method for surveying microbial communities termed Serial Analysis of Ribosomal DNA (SARD) is described here.  Through a series of molecular cloning steps, short DNA sequence tags are recovered from the fifth variable (V5) region of the prokaryotic 16S rRNA gene from microbial communities.  These tags are ligated to form concatemers comprised of 20-40 tags which are cloned and identified by DNA sequencing.  Four agricultural soil samples were profiled with SARD to assess the method’s utility.  A total of 37,008 SARD tags comprising 3,127 unique sequences were identified.  Comparison of duplicate profiles from one soil genomic DNA preparation revealed the method was highly reproducible.  The large numbers of singleton tags together with non-parametric richness estimates indicated a significant amount of sequence tag diversity remained undetected with this level of sampling.  The abundance classes of the observed tags were scale-free and conformed to a power law distribution.  Numerically, the majority of the total tags observed belonged to abundance classes that were each present at less than 1% of the community.  Over 99% of the unique tags individually made up less than 1% of the community.  Therefore, from either numerical or diversity standpoints, low abundant taxa comprised a significant proportion of the microbial communities examined and could potentially make a large contribution to ecosystem function.  SARD may provide a means to explore the ecological role of these rare members of microbial communities in qualitative and quantitative terms.
Keywords: SARD profiles, culture-independent study, microbial community survey, microbial census

overall_design: Four soil samples were profiled with SARD.  The four samples were made up of two pairs of samples:  Pol-W and Pol-NE; and WP43 and WP45.  The members of each pair were separated by about 25 m.  The two pairs of samples were separated by about 7 km.

middle: N

first: Jasper

last: Rine

first: Emmanuel

last: Mongodin

first: Karen

middle: E

last: Nelson

first: Dago

last: Dimster-Denk


title: Life cycle of Ciona intestinalis

city: Sapporo

first: Sorin

middle: Vasile

last: Sabau

address: 5-1-1-1 Minamisawa, Minamiku

institute: Tokai University

summary:
Recent whole-genome studies and in-depth expressed sequence tag (EST) analyses have identified most of the developmentally relevant genes in the urochordate, Ciona intestinalis. In this study, we made use of a large-scale oligo-DNA microarray to further investigate and identify genes with specific or correlated expression profiles. This large-scale oligo-DNA microarray for C. intestinalis should facilitate the understanding of global gene expression and gene networks during the development and ageing of a basal chordate.
Keywords: gene expression, development, ageing, life cycle

overall_design:
We collected 15 test samples for the developmental stages, comprising fertilized eggs (FE), 2-cell, 4-cell, 8-cell, 16-cell, 32-cell, and 64-cell embryos, and early gastrulae (EG), late gastrulae (LG), early neurulae (EN), embryos at the initial tailbud (ITB), early tailbud (ETB), middle tailbud (MTB), late tailbud (LTB) stages, and larvae (LV). For the adult, we collected samples from 4 different stages, comprising juveniles (JN), 1.5 month-old (1.5M) adults, 2.5 month-old (2.5M) adults and 4.0 month-old (4.0M) adults. Juveniles were collected 2 weeks after metamorphosis. At that time, the juveniles have a small body size but most of the adult organs, except for the ovary and testis. Adults of 1.5M are pre-maturation stage; they do not yet have mature gonads. Adults of 2.5M are in the period of reproduction, and have mature gonads, eggs and sperm. Adults of 4.0M still have gonads but have lost their reproductive capacity.
Two different RNA targets for microarray hybridization were used. Namely, for the developmental stages, RNA from fertilized eggs was used as a reference sample and RNA from the 14 stages of embryogenesis was used as test sample. In the adult stages, RNA combinations of adjacent stages were used, i.e. larvae vs. juveniles, juveniles vs. 1.5M adults, 1.5M vs. 2.5M adults, and 2.5M vs. 4.0M adults. In all of the RNA combinations, the hybridization was performed twice by reversing dyes (dye swap).

first: Kaoru

last: Azumi

middle: V

first: Manabu

last: Fujie

first: Takeshi

last: Usami

first: Ryo

last: Koyanagi

last: Kawashima

first: Shigeki

last: Fujiwara

first: Michio

last: Ogasawara

first: Masanobu

last: Satake

first: Masaru

last: Nonaka

first: Hong-Gang

last: Wang

first: Yutaka

last: Satou

first: Nori

last: Satoh


title: Gene expression signatures predict outcome in non-muscle invasive bladder carcinoma - a multi-center validation study

city: Aarhus N

first: Lars

last: Dyrskjot

address: Brendstrupgaardsvej

department: Department of Molecular Medicine

country: Denmark

institute: Aarhus University Hospital, Skejby

summary:
Background
Clinically useful molecular markers predicting the clinical course of patients diagnosed with non-muscle invasive bladder cancer are needed to improve treatment outcome.
Methods
We used custom designed oligonucleotide microarrays to validate four previously reported gene expression signatures for molecular diagnosis of disease stage and carcinoma in situ, and for predicting disease recurrence and progression. We analyzed tumors from 404 patients diagnosed with bladder cancer in hospitals in Denmark, Sweden, England, Spain and France. Molecular classifications were compared to pathological diagnosis and clinical outcome. The median follow-up time for the patients was 3.5 years.
Results
Classification of disease stage using a 52-gene classifier was found to be highly significantly correlated with pathological stage (P<0.001). Furthermore, the classifier added information regarding future disease progression of Ta or T1 tumors (P<0.001). The molecular 77-gene progression classifier was highly significantly correlated with progression free survival (P<0.001) and cancer specific survival (P=0.001). Furthermore, multivariate Cox´s regression analysis showed the progression classifier to be an independently significant variable associated with disease progression after adjustment for age, sex, stage, grade and treatment (hazard ratio 2.4, P=0.005). The diagnosis of carcinoma in situ (CIS) using a 68-gene classifier showed a highly significant correlation with histopathological CIS diagnosis (odds ratio 5.8, P<0.001) in multivariate logistic regression analysis.
Conclusions
We conclude that this multicenter validation study confirms the clinical utility of molecular classifiers to guide treatment decisions for patients initially diagnosed with non-muscle invasive bladder cancer.
Keywords: Multi center validation study of gene expression signatures

overall_design: This microarray study is based on a two channel spotted oligonucleotide microarray with the use of a common reference sample. All oligonucleotides were spotted in duplicate and all 404 bladder tumors were measured twice. The final average log2 values are supplied as supplemental files to this GEO series.


title: Nitric oxide-dependent changes in Schistosoma mansoni gene expression

status: Public on Jul 04 2007

city: Hamilton

first: Andrew

middle: G.

last: McArthur

address: 1280 Main Street West

department: Biochemistry & Biomedical Sciences

institute: McMaster University

summary:
Nitric oxide (NO) is a gaseous intercellular signaling molecule that also plays a role in host-parasite relations. NO acts rapidly, either via regulation of soluble guanylate cyclase, or by direct interactions with enzymes and other proteins, and has also been shown to have effects on gene expression. Here, we use SAGE  (Serial Analysis of Gene Expression) to identify NO-responsive changes in gene expression in Schistosoma mansoni following a 3 hour exposure to sodium nitroprusside, an NO donor. Overall, these results indicate that NO does not rapidly induce large-scale changes in schistosome gene expression, but that expression of particular genes of interest appear to respond to NO.
Keywords: Schistosoma, SAGE, NOS, nitric oxide, gene expression

overall_design: Adult S. mansoni perfused from infected Swiss-Webster female mice (obtained from the NIAID Schistosomiasis Resource Center) 42-49 days postinfection were maintained in culture (RPMI medium) overnight and then exposed for 3 hours to either 1 mM sodium nitroprusside (SNP), a well-characterized NO donor, or to RPMI alone. Worms remained viable and motile following treatment. Total RNA was extracted with Trizol (Invitrogen) and treated with DNAse 1 (Ambion) to remove contaminating genomic DNA, and Long-SAGE libraries constructed.

middle: G

first: Shanta

last: Messerli

first: William

last: Morgan

first: Shanda

middle: R

last: Birkeland

first: Jeremiah

last: Bernier

first: Michael

last: Cipriano

first: Robert

last: Greenberg


title: S. coelicolor YSK3225 R5- Medium Time-course Study (Culture #2)

status: Public on Jun 14 2007

first: Karthik

last: Jayapal

address: 421 Washington Ave SE

summary:
Antibiotic biosynthesis in Streptomyces species is controlled by a complex genetic and biochemical network of global and pathway specific regulators.  Details of their precise interactions in mediating temporal and spatial expression of secondary metabolite genes remain poorly defined.  In this study, we employed whole-genome microarray methods to investigate the temporal transcriptome profiles of S. coelicolor A3(2) ∆absA1::apr mutant strain and compared with the M145 Wild-type strain (series submitted separately).
Keywords: Time course

overall_design: Time-course study involved analysis of 6 samples (18h, 24h, 26h, 28h, 32h and 42h). All arrays used cDNA labeled with Alexa647 and gDNA labeled with Cy3.

first: Wei

last: Lian

first: Wei-Shou

last: Hu


title: S. lividans TK21 - S. coelicolor M145 Comparative Genomic Hybridizations

status: Public on Jun 08 2007

summary:
Background
The genomes of Streptomyces coelicolor and Streptomyces lividans bear a considerable degree of orthology. While S. coelicolor is the model streptomycete for studying antibiotic synthesis and differentiation, S. lividans is almost exclusively considered as the preferred host, among actinomycetes, for cloning and expression of exogenous DNA. We used whole genome microarrays as a comparative genomics tool for identifying the subtle yet crucial differences between these two chromosomes.
Results
We identified five large S. coelicolor genomic islands (≥25 kb) and 18 smaller islets absent in S. lividans chromosome. Many of these regions show anomalous GC bias and codon usage patterns. Six of them are in close vicinity of tRNA genes while nine are flanked with near perfect repeat sequences indicating that these are probable recent evolutionary acquisitions into S. coelicolor. Embedded within these segments are at least four DNA methylases and two probable methyl-sensing restriction endonucleases. Comparison with S. coelicolor transcriptome and proteome data revealed that some of the missing genes are active during the course of growth and differentiation in S. coelicolor. In particular, a pair of methylmalonyl CoA mutase (mcm) genes involved in polyketide precursor biosynthesis, an acyl-CoA dehydrogenase implicated in timing of actinorhodin synthesis and bldB, a developmentally significant regulator whose mutation causes complete abrogation of antibiotic synthesis belong to this category.
Conclusion
Our findings provide tangible hints for elucidating the genetic basis of important phenotypic differences between these two streptomycetes. Importantly, absence of certain genes in S. lividans identified here could potentially explain the relative ease of DNA transformations or the conditional lack of actinorhodin synthesis in S. lividans. Further genetic studies based on these results will enable one to target specific sequences in the genetically well-characterized S. coelicolor to adapt it for industrial processes.
Keywords: Comparative Genomic Hybridization

sample_organism:
Streptomyces coelicolor
Streptomyces lividans

overall_design: Hybridizations were done in triplicates. In all cases, S. coelicolor gDNA was labeled with Cy3 and S. lividans gDNA with Cy5

first: Frank

last: Glod

first: David

middle: H

last: Sherman


title: A defect in the ionotropic glutamate receptor 6 gene (GRIK2) causes autosomal recessive mental retardation

city: Berlin

first: Reinhard

last: Ullmann

department: Human Molecular Genetics

institute: MPIMG

summary:
Non-syndromic mental retardation is one of the most important unresolved problems in genetic health care. Autosomal forms are far more common than X-linked ones, but in contrast to the latter, they are still largely unexplored. Here we report on a complex mutation in the ionotropic glutamate receptor 6 gene (GRIK2, GLUR6), which co-segregates with moderate to severe non-syndromic autosomal recessive mental retardation in a large consanguineous Iranian family1. The predicted gene product lacks the first ligand-binding domain, the two adjacent transmembrane domains and the putative pore-forming loop of the GLUK6 protein, suggesting a complete loss of function, which is supported by electrophysiological data. This finding provides the first irrefutable proof that GLUK6 is indispensable for higher brain functions in man, and future studies of this and other ionotropic kainate receptors will shed more light on the pathophysiology of mental retardation.
Keywords: array CGH

overall_design: Five samples with a homozygous deletion of GLUR6 were analysed by means of  submegabase array CGH on platform GPL5114


title: Characterization of the Giardia intestinalis Transcriptome

status: Public on Jul 03 2007

summary:
Giardia gene expression is being examined using Serial Analysis of Gene Expression (SAGE) to monitor genome-wide levels of messenger RNA (mRNA) expression throughout Giardia's life cycle. Examination of genome-wide gene expression patterns will provide a coherent picture of activation and inactivation of biological pathways. This research will provide a comprehensive understanding of changes in giardial gene expression in response to important host physiological signals and will serve as a valuable model for study of other parasites and complex eukaryotes, such as yeast and animals. It will provide a dynamic framework, in the context of the life cycle, to the annotation of the Giardia genome, including the detection of unpredicted genes via detection of their tags.
Keywords: Giardia, SAGE, trophozoites, encystation, excystation, cysts

overall_design: The Giardia SAGE project detects up- and down-regulation of genes related to giardial infection (excystation), pathogenicity (trophozoites), transmission (encystation), and survival in the environment (cysts).

first: Frances

middle: D

last: Gillin

first: Staffan

last: Svard

first: Barbara

last: Davids

middle: S

last: Reiner

first: Sarah

last: Preheim

first: Daniel

last: Palm


title: S. coelicolor YSK3225 R5- Medium Time-course Study (Culture #1)

overall_design: Time-course study involved analysis of 12 samples (11h, 19h, 22h, 24h, 26h, 28h, 31h, 32h, 35h, 37h, 38h and 41h). All arrays used cDNA labeled with Alexa647 and gDNA labeled with Cy3.


title: Clostridium acetobutylicum ATCC 824 60-mer selection

status: Public on Jun 07 2007

city: Newark

first: Eleftherios

middle: Terry

last: Papoutsakis

state: DE

address: 15 Innovation Way

department: Chemical Engineering

institute: University of Delaware

summary:
Several custom made Agilent arrays were hybridized to select the best 60-mers for the transcriptional profiling of C. acetobutylicum strains.
Keywords: Test of 60-mer performance

overall_design: RNA samples were taken from both C. acetobutylicum ATCC and M5 strains at several points during the exponential phase of growth and the samples for each strain were mixed. For the ATCC824 strain the samples were taken at OD (600 nm) = {1.09, 1.8, 2.6, 2.0} whereas for the the M5 strain samples were taken at OD (600 nm) = {0.868, 1.36, 2.40, 3.20,  4.20}. The total amount of labeled material hybridized was 0.75 micrograms.

first: Carlos

middle: J

last: Paredes

first: Jacob

last: Borden

first: Ryan

last: Sillers

first: Shawn

last: Jones

first: Nathan

last: Cheng

middle: T


title: Effects of the Structural Maintenance of Chromosomes (SMC) protein on gene expression in Bacillus subtilis

status: Public on Jul 02 2007

city: Cambridge

first: Alan

middle: D.

last: Grossman

email: clee2@mit.edu, adg@mit.edu

state: MA

address: 31 Ames Street, 68-530

institute: MIT

summary:
We monitored changes in Bacillus subtilis global gene expression in response to deletion or disruption of smc or ftsY.
Keywords: genetic modification

overall_design: We extracted RNA, reverse transcribed, labeled, and co-hybridized samples to arrays containing PCR products amplified from >99% of B. subtilis genes. We performed at least three independent replicates of each experiment.

first: Robert

middle: A

last: Britton

first: Elke

last: Kuester-Schoeck

first: Thomas

last: Auchtung


title: Cell wide response to low oxygen exposure in Desulfovibrio vulgaris Hildenborough

status: Public on Jul 01 2007

city: Berkeley

first: Adam

middle: P.

last: Arkin

address: 1 Cyclotron Road

department: Physical Biosciences Division

institute: Virtual Institute for Microbial Stress and Survival

summary:
We investigated the effects of low level oxygen on Desulfovibrio vulgaris Hildenborough using microarrays.
Keywords: time course, stress response

sample_organism: Nitratidesulfovibrio vulgaris str. Hildenborough

overall_design: Comparison of cells treated with 1000 ppm oxygen to untreated cells at 60,120,240 min.

first: Marcin

middle: P

last: Joachimiak

first: Aindrila

first: Jay


title: Total RNA from EPC derived microvescicles

status: Public on Jun 06 2007

city: Torino

first: Raffaele

last: Calogero

state: To

address: Via Nizza 52

department: Molecular Biotechnology Center

country: Italy

institute: University of Torino

summary:
mRNA present in EPC derived microvescicles were detected using a RNA quantity curve, in order to evaluate if these vescicles were shuttling a specific subset of mRNAs
Keywords: Microvescicles, endothelial progenitor cells, angiogenesis, NOS

overall_design: Respectively 0.25, 1 and 2.5 micrograms of total RNA extracted from two different preparation of EPC derived microvescicles were retrotranscribed, labelled and hybridized on the microarray.

first: Maria

middle: C

last: Deregibus

first: Vincenzo

last: Cantaluppi

first: Marco

last: Lo Iacono

first: Ciro

last: Tetta

first: Luigi

last: Biancone

first: Stefania

last: Bruno

first: Benedetta

last: Bussolati

first: Giovanni

last: Camussi


title: Trp Amino acid supplementation of Aspergillus flavus NRRL 3357 and Aspergillus parasiticus SRRC 143

city: Mississippi State

first: Jeff

last: Wilkinson

state: MS

department: Biochemistry and Molecular Biology

institute: Mississippi State University

summary:
Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. In order to better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined.  A. flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B1 and B2 biosynthesis, while A. parasiticus cultures had significantly increased B1 and G1 biosynthesis.  Microarray analysis of RNA extracted from fungi grown under these conditions revealed seventy seven genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB).  It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different.  These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.
Keywords: Aflatoxin, Aspergillus, flavus, Amnio Acids, Tryptophan

sample_organism:
Aspergillus parasiticus
Aspergillus flavus NRRL3357

overall_design:
Each experiment consisted of one 48 hour YES sample coupled to a 48 hour YES supplemented with 50 mM tryptophan sample for either A. flavus or A. parasiticus.  Each comparison was repeated with triplicate biological replicates and with duplicate dye-flip.   Briefly, 3 µg of total RNA was used to generate cDNA using Genispheres random and MPX dT primers with Supercript II, Dithiotreitol and 5X SuperScript II First Strand Buffer from Invitrogen (Invitrogen corp., Carlsbad, CA) as per Genisphere’s instructions.  The generated cDNA’s were purified using Qiagen MiniElute PCR Purification Kit (Qiagen Inc., Valencia, CA) as well as after terminal deoxynucleotidyl transferase tailing reaction and ligation to 3DNA capture sequence reactions.  cDNA hybridizations were performed using the 2X Formamide-Based Hybridization Buffer overnight at 49oC in Hybridization Cassette’s (ArrayIt, TeleChem International, Inc., Sunnyvale, CA).
Hybridized slides were scanned using either a ScanArray5000XL (GSI Lumonics, Packard Biochip, Packard BioScience Company, Billerica, MA) or a GenePix 4000B (Axon Instruments, Molecular Devices, Sunnyvale, CA), and the independent TIFF images from each channel were analyzed using TIGR Spotfinder software program.  After background correction and removal of flagged values, log base 2 expression ratios were median centered and linear transformed to obtain the log and linear values given in the data table.

first: Jiujiang

last: Yu

first: John

middle: M

last: Bland

first: William

last: Nierman

first: Deepak

last: Bhatnagar

middle: E

last: Cleveland


title: Human esophageal squamous cell carcinoma cell lines with downregulated PTTG1

city: Baltimore

first: Tetsuo

last: Ito

address: 1503 E Jefferson St Rm 106

department: Medicine

institute: Johns Hopkins University

summary:
We conducted a comprehensive genome-wide interrogation to identify gene targets regulated by PTTG1 using a bead-array platform.
The raw data and normalized logarithmic data were included in this subset.
Keywords: siRNA, esophageal cells

overall_design: HSA/c and KYSE140 cells were transfected with three siRNA oligos (NTC, P1, P2) separately or without oligos (LF+) at day 0 and harvested at day 3. Total RNAs were extracted by the RNeasy kit (Qiagen) from the eight samples. After the RNA quality measurement by the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), each RNA sample (100 ng) was amplified with the Illumina TotalPrep RNA Amplification kit (Ambion, Austin, TX), hybridized with the Illumina Human RefSeq8 version 2 BeadChip with 20,589 transcript probes comprised of optimized 50 mer-oligonucleotides (Illumina, San Diego, CA), washed, and stained with Cy3-streptavidin (GE Healthcare) as per the manufactures’ instructions. The arrays were scanned with the Illumina Beadarray Reader confocal scanner (Illumina) and the data were processed using the Illumina BeadStudio software (Illumina). The data were subjected to intensity-dependent normalization, and differentially expressed genes by PTTG1 downregulation were identified by the Significance Analysis of Microarrays (SAM) program.

first: Fumiaki

last: Sato

first: Stephen

last: Meltzer


title: Endogenous small RNAs from Chlamydomonas reinhardtii strain J3(mt-) vegetative cells

status: Public on Jun 04 2007

city: Norwich

first: Frank

last: Schwach

state: Norfolk

address: Colney Lane

country: United Kingdom

institute: Sainsbury Laboratory

type: Non-coding RNA profiling by high throughput sequencing

summary:
endogenous small RNAs from Chlamydomonas reinhardtii strain J3(mt-) vegetative cells
Keywords: High throughput 454 small RNA sequencing

overall_design: Size fractionated small RNA from total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.

first: Attila

last: Molnar

first: David

last: Studholme

first: Eva

last: Thuenemann

last: Baulcombe


title: Phenotypic and molecular characterisation of a novel Bt2 allele in maize

city: Beaucouze

first: sandrine

last: balzergue

address: Rue G. Morel

country: France

institute: INRA

summary:
At 35 DAP whole kernels (pericarp + endosperm + embryo) without glumes of green house grown ears of heterozygous (+/bt2-H2328), self-pollinated plants were visually divided into pools of phenotypically normal looking kernels (small indentation, slightly smaller than mutant kernels, genotype +/+ or +/bt2-H2328) and pools of phenotypically mutant kernels (plump, round kernels, slightly larger than normal kernels, genotype bt2-H2328/bt2-H2328). Pools consisted of 4 kernels. 3 different ears were used for a biological duplicate.
Keywords: gene knock out

overall_design: 4 arrays - maize

first: Sandrine

last: Balzergue

first: Peter

last: Rogowsky

first: Marie-Laure

middle: L

last: Martin-Magniette


title: Genetic dissection of NuA4-directed chromatin transactions throughout the S. cerevisiae genome

city: University Park

first: Melissa

last: Durant

state: PA

address: 452 N Frear

institute: Penn State University

summary:
Acetylation of histone tails has long been associated with gene activation.  Exactly how acetylation regulates gene expression is not fully known.  Acetylation events at specific sites or collections of sites on histones elicit distinct outcomes.  Here we examine the downstream consequences of histone acetylation by the histone H4 acetyltransferase NuA4 on a genomic scale.  Evidence is presented that Bdf1, which is known to bind to acetylated lysine H4 tails in vitro, binds to nucleosomes in vivo and that this binding is dependent upon Esa1, the catalytic subunit of NuA4.  Loss of NuA4 results in a coordinate depletion of Bdf1, the transcription complex assembly factor TFIID, and the H2A.Z assembly complex SWR-C at highly acetylated promoter regions.  This finding is consistent with known interactions between Bdf1 and TFIID and SWR-C.  Loss of Bdf1 results in little or no depletion of TFIID or SWR-C at these promoter regions, possibly due to substitution by the Bdf1 paralog Bdf2.  Consistent with this possibility, loss of Bdf1 results in accumulation of Bdf2 at sites normally bound by Bdf1. Together, the findings presented here strengthen the proposed cascade of events whereby nucleosome H4 acetylation by NuA4 at promoters target Bdf1, which then recruits TFIID and SWR-C to assemble the transcription machinery.
Keywords: chIP-chip, histone acetylation, transcription factor recruitment

overall_design: Experiment contains 22 total hybridizations.  Yeast enriched chIP DNA dual channel (Cy3/Cy5) cohybridization.  All experiments performed in duplicate (dye swap).  Dye swap data was normalized by centering to the average log2 ratio of the non-promoter regions.  Platform GPL1924: non-commercial nucleotide arrays. PCR amplified yeast (Saccharomyces cerevisiae) genomic DNA chip.  Printed on aminosaline glass slides by the Penn State University Microarray facility

first: B

middle: F

last: Pugh


title: Gene expression profiling in T-47D breast cancer cells following inducible expression of estrogen receptor beta

status: Public on Jun 01 2007

city: Provo

first: Chin-Yo

last: Lin

state: UT

address: 753 WIDB

department: Microbiology and Molecular Biology

institute: Brigham Young University

summary:
T-47D breast cancer cells were stably transfected with a tet-off inducible construct encoding estrogen receptor beta. Microarray experiments were carried out at multiple time points 1-30 hours following treatment with estradiol and under induction and non-induction conditions.
Keywords: Inducible expression of ERbeta

overall_design: Single biological samples were taking at each time point for each of the four treatment condition (uninduced-untreated, induced-untreated, uninduced-treated, and induced-treated.

first: Edison

last: Liu


title: In vivo genomic binding sites for the Drosophila CTCF Insulator

first: Steve

last: Russell

address: Downing Street

department: Department of Genetics

institute: University of Cambridge

summary:
Chromatin immunopurification with anti-CTCF was used to identify in vivo binding sites for CTCF in approximately 3% of the Drosophila genome using amplicon-based tiling arrays.  This includes the Bithorax-complex, a known site of CTCF activity, the Antp-Complex, the Achaete scute-Complex and the Adh region.  Reproducible binding identified new CTCF sites in the Adh region and the Bx-C.
Keywords: ChIP-chip

overall_design: Formaldehyde-crosslinked Chromatin was prepared from 4 independent biological replicates (Embryo1, Embryo2, Embryo5 and Embryo6) of 0-20hr wild-type Drosophila embryos (OregonR strain).  Each chromatin sample was immunopurified with rabbit Anti-CTCF and, in parallel with normal rabbit serum.  DNA purified from the experimental and control immunopurifications was amplified by LM-PCR. Four seperate aliquots (Technical replicates) of each PCR reaction were labeled and hybridised to independent tiling array slides, comparing experimental and control on each slide.  2 slides were dye swapped compared to the other two.  Each of the 4 technical replicates were normalised (VSN) and the average ratio of Experimental v control signals calculated.  The average ratio for each of the 4 biological replicates was assessed for statistical significance using CyberT to calculate an mean ratio and p-value.

first: Eimar

last: Holohan

first: Camilla

last: Kwong

first: Steven

first: Robert

last: White


title: Effect of growth in TSA or SCFA on gene expression in E. histolytica

status: Public on Jun 30 2007

city: Stanford

first: Gretchen

middle: Marie

last: Ehrenkaufer

address: Grant S-143, 300 Pasteur Dr.

department: Microbiology and Immunology

institute: Stanford Uninversity

summary:
In order to dtermine the role of histone actylation in gene expresssion, E. histolytica 200:NIH trophozoites were treated with the HDAC inhibitor Trichostatin A, or grown in short chain fatty acids, which have been shown to alter histone actylation patterns in Entamoeba species.
Keywords: Expression

overall_design: E. histolytica 200:NIH trophozoites grown in standard media (TYI or TYI-low glucose) were compared to trophozoites grown in TYI-LG + Trichostatin A (150 or 300 nM) for 16 or 72 hours, or in TYI-LG + short chain fatty acids (70 mM sodium acetate, 20 mM sodium propionate and 10 mM sodium butyrate). Differences in gene expression were assayed by hybridization to a full genome Affymetrix array.

first: Daniel

last: Eichinger

first: Upinder

last: Singh


title: comparison of gene expression in urogenital ridges of Wt1-/- and +/+ E11.5 mouse embryos

city: Jena

first: Christoph

last: Englert

address: Beutenbergstrasse 11

department: Molecular Genetics

institute: Leibniz-Institute for Age Research

summary:
The Wilms tumor protein Wt1 is a transcription factor known to play an important role in urogenital development. Mutations in the human Wt1 encoding gene (WT1) lead to several syndromes associated with defective renal and sexual development, namely WAGR-, Denys-Drash-, and Frasier-syndrome. During mammalian embryogenesis, urogenital development starts with formation of the urogenital ridges, consisting of the embryonic kidneys, called mesonephroi, and the gonads. In Wt1 knockout mice, these ridges start to form, degenerate, however, during further embryonic development . Although WT1 and the syndromes associated with its mutation are known for several years, it is still unclear which genes, regulated by Wt1, mediate the whole variety of Wt1's function in gonad and mesonephros development. To obtain a comprehensive view on Wt1-dependent gene activity in developing urogenital ridges, we employed microarray analysis. Comparison of gene expression in Wt1 wild-type and knockout mice, led to the identification of almost 150 genes with differences in expression levels higher than factor three.
Keywords: wild-type and knockout comparison

overall_design: urogenital ridges of wild-type and Wt1 knockout mouse embryos were isolated for RNA extraction and hybridization on Amersham CodeLink Mouse 20k microarrays. For both genotypes five embryos with 11-12 tail somites, a stage immediately before the beginning of sex differentiation, were used. Wild-type and knockout were measured three times.

first: Jürgen

last: Klattig


title: Effect of elevated CO2, warming, added rainfall and added nitrogen on Geranium dissectum growing in a natural grassland

first: Susan

middle: Nelle

last: Thayer

address: 260 Panama St.

department: Plant Biology

institute: Carnegie Institution of Washington

summary:
We used microarrays to discern patterns of gene expression in response to global climate change factors on leaf tissue of an annual dicot, Geranium dissectum, growing in a natural grassland.
Keywords: multifactorial global change treatments

sample_organism:
Arabidopsis thaliana
Geranium dissectum

overall_design: There are 5-8 biological replicates for each of the sixteen treatments - a total of 100 samples. The four global change factors at two levels are ambient or elevated CO2, ambient temperature or ambient + 1 degree C warming, natural rainfall (601.2 mm/yr) or natural rainfall supplemented with 50% of every storm added immediately following each storm with a 3 week season extension, ambient growth nitrogen or ambient supplemented with 7 g N m-2 y-1, supplied as Ca(NO3)2. There were 16 treatments applied in a nested design in the field.We hybridized the Geranium samples using cross-species hybridization to Arabidopsis cDNA arrays which had an Arabisopsis genomic DNA common reference in the Cy3 channel.

first: Samuel

middle: B

last: St. Clair

first: Shauna

last: Somerville

first: Christopher

last: Field