title: Halobacterium NRC-1_oxygen_response_timecourse_set3

status: Public on Jul 31 2007

first: Amy

last: Schmid

institute: Institute for Systemsbiology

summary:
In order to ensure the reproducibility of the transcriptional response of Halobacterium NRC-1 to oxic/anoxic transitions, we repeated global mRNA measurements for the oxygen time series data in GSE5924, except that cultures were equilibrated to low oxygen for 24 hours prior to the start of the experiment rather than 12 hours, and time course sampling continued to 12 hours post-shift to high oxygen rather than stopping at 6 hours.  The results of these data suggest that there is good reproducibility between datasets, and that Halobacterium responds robustly to oxic/anoxic transitions.
Keywords: time course

overall_design: This experiment was a replicate of Oxygen time series 1, except that cells were equilibrated at low oxygen for 24 hours prior to the experiment start.  Also, time course sampling was more frequent after the shift to high oxygen and sampling was continued beyond 6 hours to 12 hours.  Halobacterium sp. NRC-1 (ATCC700922) was routinely grown in complex medium (CM; 250g/L NaCl, 20g/L MgSO4.7H2O, 3g/L sodium citrate, 2g/L KCl, 10g/L peptone) at 37ºC under full-spectrum white light.  For turbidostat experiments, starter cultures of NRC-1 were inoculated into 2L of CM in a 3.0 L vessel (5-10% inoculum) and grown to mid-logarithmic phase (OD600 ~ 0.5) in batch mode in a BioFlo100 modular bench top fermentor (New Brunswick Scientific, Edison, NJ) at 300 rpm, pH 7.0.  Prior to each experiment, an oxygen sensor (model InPro 6000, Mettler Toledo, Columbus, OH) was calibrated to 100% oxygen at 1200rpm and sparging with 3.2 VVM of air. These conditions were approximately equivalent to oxygen saturation in CM medium, which is 1.6 mg/L (~5uM).   Once the culture reached mid-logarithmic phase, the oxygen level was rapidly decreased to ~0-0.5% within 5-10 minutes (achieved by cutting off airflow and decreasing agitation to 250 rpm), and allowed to incubate under anoxic conditions for 24 h prior to the start of sampling. The oxygen concentration in the culture was then rapidly increased to approximately 85-100% (achieved by agitating at 1100 rpm and sparging with air at 3.2 VVM) and sampling commenced and continued at the times indicated in the Sample records.  The culture was subsequently maintained in high oxygen conditions for 12hr.  During these perturbations, all other parameters were kept constant (pH 7.2-7.3, 37ºC, ambient light, O.D.600~0.5-6.5). Each culture sample taken at the times indicated in the Sample records was split in half, one half being used for RNA extraction and the other for ATP measurement.

first: David

last: Reiss

first: Amardeep

last: Kaur

first: Nichole

last: King

first: Phu

last: Van

first: Laura

last: Hohmann


title: Halobacterium NRC-1 oxygen, light and pertubations

status: Public on Jan 03 2008

city: Seattle

first: Marc

last: Facciotti

state: WA

address: 1441 North 34th Street

country: USA

institute: Institute for Systems Biology

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

overall_design: Refer to individual Series


title: Halobacterium NRC-1 Growth curve and GTF perturbation

status: Public on Feb 23 2007

summary:
Total RNA was collected at 6 or 8 points throughout the growth curve of Halobacterium NRC1 for wild-type, non-native expression of general transcription factors and knock-out strains of general transcription factors.  This RNA was hybridized against our standard reference RNA (wild-type OD600 0.5-0.6).
Keywords: growth genetic modification

overall_design:
Total RNA was collected at 3 or 4 points throughout the growth-curve for each strain.  Biological duplicates were grown and collected from for each strain making a total of 6 or 8 observations throughout the growth curve.

A total of 161 biological samples make up this experiment.

Strains include (wild-type NRC-1, delta-ura3 knockout control strain, pMTFcmyc plasmid-only control strain, TFBa,b,c,d and e knockout strains, TFBb, c, and d knock-out strains, cmycTFBa, b, c, d, e, f and g non-native expression strains, and cmycTBPa, b, c, d, e, f non-native expression strains).

Samples were hybridized against in-house spotted oligo arrays.  Each sample is represented on two arrays as dye swaps.

middle: T

first: Min

last: Pan

first: Madhavi

last: Vuthoori

middle: J

first: Richard

last: Bonneau

first: Paul

last: Shannon

first: Alok

last: Srivastava

first: Samuel

last: Donahoe

first: Leroy

last: Hood

first: Nitin

middle: S

last: Baliga


title: RNA expression data from Halobacterium NRC-1 in response to oxic/anoxic transitions, verification experiment

middle: K

summary:
In order to ensure the reproducibility of the transcriptional response of Halobacterium NRC-1 to oxic/anoxic transitions, we repeated global mRNA measurements for the oxygen time series data in GSE5924, except that cultures were equilibrated to high oxygen for 12 hours prior to the start of the experiment rather than low oxygen.  The results of these data suggest that there is good (~60%) reproducibility between datasets, and that Halobacterium responds robustly to oxic/anoxic transitions.
Keywords: time series

overall_design: This experiment was a replicate of Oxygen time series 1, except that cells were equilibrated at high oxygen for 12 hours prior to the experiment start.  Halobacterium sp. NRC-1 (ATCC700922) was routinely grown in complex medium (CM; 250g/L NaCl, 20g/L MgSO4.7H2O, 3g/L sodium citrate, 2g/L KCl, 10g/L peptone) at 37ºC under full-spectrum white light.  For turbidostat experiments, starter cultures of NRC-1 were inoculated into 2L of CM in a 3.0 L vessel (5-10% inoculum) and grown to mid-logarithmic phase (OD600 ~ 0.5) in batch mode in a BioFlo100 modular bench top fermentor (New Brunswick Scientific, Edison, NJ) at 300 – 500 rpm, pH 7.0.  Prior to each experiment, an oxygen sensor (model InPro 6000, Mettler Toledo, Columbus, OH) was calibrated to 100% oxygen at 1200rpm and sparging with 3.2 VVM of air. These conditions were approximately equivalent to oxygen saturation in CM medium, which is 1.6 mg/L (~5uM).   Once the culture reached mid-logarithmic phase, the oxygen level was rapidly increased to ~90-100% within 10 minutes (achieved by on airflow to 3.2VVM and increasing agitation to 1200rpm), and allowed to equilibrate for 12 h prior to the start of sampling. The flow rate was 2.5-5mL/min during high oxygen conditions.  The oxygen concentration in the culture was then rapidly decreased to approximately 0-10% (achieved by agitating at 250 rpm and turning off sparging) and sampling commenced and continued at the times indicated in the Sample records.  The culture was subsequently maintained in low oxygen conditions for 6hr.  During these perturbations, all other parameters were kept constant (pH 7.2-7.3, 37ºC, ambient light, O.D.600~0.5-6.5). Each culture sample taken at the times indicated in the Sample records was split in half, one half being used for RNA extraction and the other for protein preparation.


title: EMT initiation in MCF10A cells subjected to ionizing radiation and treatment with transforming growth factor beta-1.

status: Public on Jun 25 2007

city: Berkeley

first: Mary

middle: Helen

last: Barcellos-Hoff

state: CA

address: 1 Cyclotron Rd MS977R225A

department: Life Sciences Division

institute: Lawrence Berkeley National Laboratory

summary:
Transforming growth factor beta-1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A, HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture, or treated with a low concentration of TGFbeta (0.4 ng/ml), or double-treated. All double-treated (IR+TGFbeta) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin, beta-catenin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin and vimentin. Furthermore, double-treatment increased cell motility, promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, even though IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.
Keywords: response to ionizing radiation, response to transforming growth factor-beta, nonmalignant human mammary epithelial MCF10A cells, epithelial to mesenchymal transition

overall_design: Nonmalignant human mammary epithelial MCF10A cells (passages 106 and 108) were seeded at cloning density in 35mm dishes (10^5 cells/dish). Cell culture medium consisted of 3ml/dish of MGEM serum free medium (Cambrex Inc.), supplemented or not with 400pg/ml recombinant Transforming Growth Factor-beta. Cells were irradiated or not 5h post plating using 160 KV X-ray with a total dose of 2Gy. Sham, IR-treated, TGFbeta-treated and double-treated (IR+TGFbeta) MCF10A cells were harvested 8 days post-IR. Briefly, cells were washed with PBS, denatured in Trizol, scraped off the dish and subjected to chloroform extraction. After centrifugation, the upper phase was precipitated with an equal volume of isopropanol. RNA precipitates were resuspended in RNase free water and further purified on RNeasy columns (Qiagen, Germany). RNA quality was assessed on an Agilent Bio-Analyzer. The dataset analyzed by microarray included biological duplicates for each treatment in two independent experiments and three sham treated samples. Microarray data were generated at the Lawrence Berkeley National Laboratory Molecular Profiling Laboratory (http://hta.lbl.gov) using a high-throughput, automated GeneChip system (Affymetrix). Briefly, target preparation, HT_HG-U133A array plate hybridization setup, washing and staining were performed on an Affymetrix robotic system (GCAS) using version 2.1 protocols. Scanning (protocol version 2.2.09) was performed on a CCD-based high throughput scanner (Affymetrix). Samples were analyzed and clustered with the (UNO) One Color GenetrafficTM software version 3.2-12 (Iobion Informatics LLC, Stratagene, La Jolla, CA). Genes whose expression was specifically altered by treatment were defined as those in which dye ratio was more than 1.75-fold (|mean log2ratio|>0.8) from baseline in at least three out of the four treated samples compared to the three sham samples. Significance analysis tests (p<0.05) were performed using Excel between sham samples and either IR, TGFbeta or TGFbeta+IR samples.


title: RNA expression data from Halobacterium NRC-1 in response to oxic/anoxic transitions, initial experiment

summary:
To gain a comprehensive systems-level understanding of cellular phenotypes, it is critical to characterize the relationship between the dynamic transcriptome and proteome during environmental perturbations. Previous comparisons have shown a lack of correlation between mRNA and protein level measurements suggesting a predominant role for post-transcriptional regulation in mediating cellular environmental responses.  To investigate the extent of post-transcriptional regulation, we have analyzed transcriptome and proteome level changes over a 13-hour 28-point time course during transitions between oxic and anoxic physiologies of Halobacterium. Integrated computational analyses of these data show that temporally shifting mRNA and protein profiles relative to one another significantly increases the mRNA/protein correlation.  Although time lags for unrelated genes vary widely, we observe similar temporal lags between the transcription and translation of functionally related genes.  In contrast, no significant temporal separation was observed within the transcript profiles. Taken together, these data suggest that while there is indeed a direct correlation between many corresponding changes at mRNA and protein levels, translational delay may be the predominant mechanism for the temporal regulation of protein abundance during physiological oxic/anoxic transitions in Halobacterium. The approach and algorithms delineated in this study provide a framework for incorporating the temporal dimension of information processing across many different layers of gene regulation.
Keywords: time course

overall_design: Halobacterium sp. NRC-1 (ATCC700922) was routinely grown in complex medium (CM; 250g/L NaCl, 20g/L MgSO4.7H2O, 3g/L sodium citrate, 2g/L KCl, 10g/L peptone) at 37ºC under full-spectrum white light.  For turbidostat experiments, starter cultures of NRC-1 were inoculated into 2L of CM in a 3.0 L vessel (5-10% inoculum) and grown to mid-logarithmic phase (OD600 ~ 0.5) in batch mode in a BioFlo100 modular bench top fermentor (New Brunswick Scientific, Edison, NJ) at 300 – 500 rpm, pH 7.0.  Prior to each experiment, an oxygen sensor (model InPro 6000, Mettler Toledo, Columbus, OH) was calibrated to 100% oxygen at 1200rpm and sparging with 3.2 VVM of air. These conditions were approximately equivalent to oxygen saturation in CM medium, which is 1.6 mg/L (~5uM).   Once the culture reached mid-logarithmic phase, the oxygen level was rapidly decreased to 0.8-4.8% within 10 minutes (achieved by turning off airflow and reducing agitation to 250 rpm), and allowed to equilibrate for 12 h prior to the start of sampling. The flow rate was zero during low oxygen conditions.  The oxygen concentration in the culture was then rapidly increased to approximately 90-95% (achieved by agitating at 1200 rpm and sparging the medium with 3.2 VVM of air) and sampling commenced and continued at the times indicated in the Sample records.  The culture was subsequently kept in active growth at a flow rate of approximately 2.25 ml/min (specific growth rate ~0.1/hr) for 6hr.  Cultures were then rapidly shifted (within 10 minutes) back to low oxygen for 6hr, and then finally back to high oxygen for 30 min.  During these perturbations, all other parameters were kept constant (pH 7.2-7.3, 37ºC, ambient light, O.D.600~0.5-6.5). Each culture sample was split in half, one half being used for RNA extraction and the other for protein preparation.  Samples were removed from the turbidostat according to the times indicated in the titles of the Sample records.


title: Androgen-starved LNCaP cells treated with celastrol and androgen, androgen alone, or vehicle alone for 24h

status: Public on Sep 01 2006

city: CAMBRIDGE

first: Haley

last: Hieronymus

state: MA

address: 7 Cambridge Center

institute: Broad Institute of Harvard and MIT

summary:
celastrol is a natural product that affects LNCaP androgen-signalling by 24h
We used microarrays to detail the androgen-responsive program of gene expression affected by celastrol treatment at 24h
Keywords: drug treatment

overall_design: LNCaP cells were grown to 50% confluency and deprived of androgen, and subsequently treated with celastrol plus androgen, androgen alone, or vehicle alone for 24h prior to direct Trizol lysis and RNA isolation

first: Todd

middle: R

last: Golub


title: LNCaP cells treated with gedunin or vehicle for 6h

summary:
Gedunin is a natural product that affects LNCaP gene expression by 6h
We used microarrays to detail the global programme of gene expression affected by gedunin treatment at 6h
Keywords: drug treatment

overall_design: LNCaP cells were grown to 50% confluency and treated with gedunin for 6h prior to direct Trizol lysis and RNA isolation


title: Halobacterium NRC-1 in response to oxic/anoxic transitions (initial and verification experiments)

first: Amy

last: Schmid


title: LNCaP cells treated with celastrol or vehicle for 6h

summary:
Celastrol is a natural product that affects LNCaP gene expression by 6h
We used microarrays to detail the global programme of gene expression affected by celastrol treatment at 6h
Keywords: drug treatment

overall_design: LNCaP cells were grown to 50% confluency and treated with celastrol for 6h prior to direct Trizol lysis and RNA isolation


title: Halobacterium NRC-1, ZnSO4 Stress Response series 2

status: Public on May 31 2006

summary:
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.
Keywords: stress response, dose response

overall_design: 4 samples were analyzed (0mM, 0.005, 0.01 and 0.015mM Zn). Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.


title: Halobacterium NRC-1, CoSO4 Stress Response series 2

summary:
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.
Keywords: stress response, dose responseH

overall_design: 4 samples were analyzed. Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control

first: Baliga

last: Nitin

first: Pan

last: Min

first: Kaur

last: Amardeep

first: Meislin

last: Megan

first: Facciotti

last: Marc

first: El-Geweley

last: Raffat


title: Halobacterium NRC-1, NiSO4 Stress Response series 2

first: Marc

last: Facciotti

overall_design: 4 samples were analyzed. Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.

first: Min

last: Pan

first: Amardeep

last: Kaur

first: Megan

last: Meislin

first: Raffat

last: El-Geweley


title: Halobacterium NRC-1, NiSO4.6H2O Stress Response

first: Michael

middle: Hiroshi

last: Johnson

department: Computational Biology

overall_design: 4 samples were analyzed.  Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.

first: Rafaat


title: Halobacterium NRC-1, FeSO4.7H2O Time Series

summary:
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.
Keywords: time series

overall_design: 11 samples were analyzed.  Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.

first: Nitin

last: Baliga


title: Halobacterium NRC-1, CoSO4.7H2O Stress Response


title: Halobacterium NRC-1, ZnSO4.7H2O Stress Response

overall_design: 3 samples were analyzed.  Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.


title: B. thetaiotaomicron before and after glucose addition to tryptone media

city: St. Louis

first: Magnus

last: Bjursell

state: MO

address: 4444 Forest Park Ave

department: Molecular Biology and Pharmacology

institute: Washington University School of Medicine

summary:
B. thetaiotaomicron was grown in a batch culture fermenter containing tryptone media. Glucose (final concentration 0.2%) was added at OD600 = 0.3. Samples were taken immediately before and 60 minutes after addition.
Keywords: time course

platform_organism:
Bacteroides thetaiotaomicron
Bifidobacterium longum

first: Eric

middle: C

last: Martens

first: Jeffrey

middle: I

last: Gordon


title: Halobacterium NRC-1, ura3 Knockout, vng1179 Knockout, Cu Stress Response

summary:
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.
Keywords: stress response, dose response, gene knockout

overall_design: 8 samples were analyzed.  Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.


title: Halobacterium NRC-1, CuSO4.5H2O Stress Response


title: Halobacterium NRC-1, EMS Stress Response

summary:
100mM EMS was added to mid-log phase Halobacterium NRC-1 cultures. After constant stress of EMS for 30 minutes, cultures were spun down and pellets were re-suspended in same volume of GN101 media. Cultures were then harvested by centrifugation and pellets were snap frozen on a dry-ice ethanol bath. Samples for RNA preparation were collected during recovery time points at 0, 10, 20, 30, 40, 60 and 120 minutes.
Keywords: stress response, dose response

overall_design: 16 samples (8 samples from EMS perturbed and 8 non-perturbed controls) were analyzed on replicate arrays (dye-flips) all against the same standard reference sample.


title: An integrated systems approach for understanding cellular responses to gamma radiation.

status: Public on Aug 18 2006

address: 1441 North 34th St.

summary:
Cellular response to stress entails complex mRNA and protein abundance changes which translate into physiological adjustments for maintaining homeostasis as well as to repair and minimize damage to cellular components.  We have characterized the response of the halophilic archaeon Halobacterium salinarum NRC-1 to 60Co ionizing gamma radiation in an effort to understand correlation between genetic information processing and physiological changes.  The physiological response model we have constructed is based on integrated analysis of temporal changes in global mRNA and protein abundance along with protein-DNA interactions and evolutionarily conserved functional associations.  This systems view reveals cooperation amongst several cellular processes including DNA repair, increased protein turnover, apparent shifts in metabolism to favor nucleotide biosynthesis and an overall effort to repair oxidative damage.  Further, we demonstrate the importance of time dimension while correlating mRNA and protein levels and suggest that steady state comparisons may be misleading while assessing dynamics of genetic information processing across transcription and translation.
Keywords: Time course after gamma irradiation

overall_design: The wild type strain of Halobacterium salinarium strain NRC-1 was used for the gamma radiation experiments.  Culturing of all strains was done in a liquid Complete Medium (CM; at 42 ºC with shaking at 220 rpm. Cell pellets from two 180mL cultures (control and experimental) of Halobacterium NRC-1 (OD600nm= 0.4) were resuspended in 1/20 volume in a basal salt solution (CM without peptone) and exposed to 2500 Gy of  gamma-ray at 22oC  using a 26,000-curie (9.6E14 Bq) 60Co gamma source at Univ. of Maryland College Park Gamma Test Facility at a dose rate of 62.01 Gy/min. Irradiated and control cultures were resuspended in the original volume of CM, split into 20mL aliquots in baffled flasks and incubated at 42oC  and 220rpm shaking. Time course samples were placed on ice, pelleted (5000 x g, 4oC, 5 min) and flash-frozen in a dry ice/ethanol bath after decanting the supernatant. RNA extractions were performed using the Stratagene Absolute RNA kit and RNA quality checked with the Agilent Bioanalyzer and with PCR.  Microarrays were fabricated at the Institute for Systems Biology Microarray Facility.  The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1.   Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip).  Raw data was processed and converted into log10 ratios with lambda values determined by a maximum likelihood method.

first: Kenia

last: Whitehead

first: Adrienne

last: Kish

first: David

last: Reiss

first: Nichole

last: King

first: Laura

last: Hohmann

first: Jocelyne

last: DiRuggiero


title: Halobacterium NRC-1, ura3 Knockout, Metal Stress Response

overall_design: 14 samples were analyzed.  Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.

first: Marc


title: Halobacterium NRC-1, 2,2 Dipyridyl Stress Response

first: Min


title: Halobacterium NRC-1, FeSO4.7H2O Stress Response

summary:
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux
of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms.
Keywords: stress response, dose response

first: Amardeep


title: Halobacterium NRC-1, MnSO4.H2O Stress Response

status: Public on May 26 2006

first: Megan


title: Halobacterium NRC-1, ura3 Knockout, sirR Knockout, MnSO4.H2O Stress Response


title: xray induced expression profile change in sensitive vs resistant embryos

status: Public on Jun 29 2004

city: Gainesville

first: Lei

last: Zhou

state: FL

department: Molecular Genetics and Microbiology

institute: University of Florida

summary:
stage 9-11 embryos are sensitive to x-ray induced cell death, while stage 13-15 embryos are highly resistant.  The gene expression changes induced by x-ray in sensitve vs resistant embryos appear to be qualitatively different.
Keywords: repeat sample

first: Pamela

middle: M

last: Carroll


title: Halobacterium NRC-1 response to sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)

first: Michael

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

overall_design: Refer to individual Series