Transcription profiling of year-long 10 degree C reduction in water temperature on global gene expression in tail skeletal muscle from adult, male zebrafish
ABSTRACT: The effect of a year-long 10 reduction in water temperature on global gene expression in tail skeletal muscle from adult, male zebrafish was determined using a long oligonucleotide probe set (16,399 65mers from Compugen) spotted onto glass slides. Outbred male zebrafish were obtained from a commercial supplier (Liles Tropical Fish, FL) at 6 months of age. Fish were maintained at 28 until 10 months of age. (see Gerhard et al., Exp Gerontol 37,1055-68, 2002) For temperature reduction, water temperature was decreased by 2.5 per week for 4 weeks. Fish were maintained until 22 months of age. Fish were fed fish flakes (Wardely Corp. Secaucus, NJ) twice per day. Each feeding was a discrete event in which a small measured amount of granular food was sprinkled on the water surface. A second small aliquot was offered if the first has been eaten within a few minutes. The feeding stopped when food from the previous aliquot has not been eaten and feeding behavior has ceased. By providing a consecutive series of small aliquots until food is no longer accepted, every member of the tank has an opportunity to eat until satiety, yet the amount of uneaten food is minimized. At 22 months of age, fish were euthanized by decapitation. Total RNA was harvested from a pool of tail muscle samples from 5 fish. Two independent pools of 5 fish per pool were collected from fish maintained at 18 and 28. Flip-dye hybridizations were performed on each pool for a total of 4 hybridizations in this study. Experiments DAR011d0001 and DAR011d0002 are flip-dye hybridizations from Pool 1. Experiments DAR011d0003 and DAR011d004 are flip-dye hybridizations from Pool 2.
Project description:We isolated total RNA from staged embryos at 2 hour intervals over a 24 h period. Total RNA from 8 h embryos was used as a reference for the time course. This expression study used a >16,000 oligonucleotide (65-mers) probe set from Compugen to examine changes in gene expression in wild-type Danio rerio during development, emphasizing the period of segmentation, when the nervous system is forming. Embryos were provided by Scientific Hatcheries.
Project description:We investigate the relevance of RNA integrity in gene expression analysis as well as analysis methods to accommodate the possible effects of degradation using paired tumour and normal samples from colorectal cancer patients undergoing colonic resection.
Project description:Responses to social cues, such as pheromones, can be modified by genotype, physiology, or environmental context. Honey bee queens produce a pheromone (queen mandibular pheromone; QMP) which regulates many aspects of worker bee behavior and physiology. Forager honey bees are less responsive to QMP than young nurse bees engaged in brood care, suggesting that physiological changes associated with behavioral maturation may modulate response to this pheromone. Since cGMP is a major regulator of behavioral maturation in honey bee workers, we examined its role in modulating worker responses to QMP. Treatment with a cGMP analog, 8-Br-cGMP, resulted in significant reductions in both behavioral and physiological responses to QMP in young caged workers. Treatment significantly reduced attraction to QMP (the retinue response) and inhibited the QMP-mediated increase in vitellogenin levels in the fat bodies of worker bees. Genome-wide analysis of brain gene expression patterns demonstrated that cGMP has a larger effect on expression levels than QMP, and that QMP has specific effects in the presence of cGMP, suggesting that some responses to QMP may be dependent on an individual bees physiological state. Several functional gene categories were significantly differentially expressed, including genes involved in regulating GTPase activity, phototransduction, immunity, and carboxylic acid transmembrane transporter activity. Overall, our data suggest that cGMP-mediated processes play a large role in modulating responses to queen pheromone in honey bees, at the behavioral, physiological and molecular levels.
Project description:This experiment tests the gene expression of the RelA mutant (ORF03691/GSU2236) versus wild type G. sulfurreducens during log phase growth. Chemostat culture, Acetate-limiting electron donor, Fumarate electron acceptor.
Project description:1. Summary: SfrAB, the soluble ferric reductase of Geobacter sulfurreducens, is a two subunit complex that can catalyze the NADPH-dependent reduction of chelated Fe(III) and is encoded in a two gene cluster in which the sfrB gene (GSU_0510) is 126 bp upstream of the sfrA gene (GSU_0509)[Kaufmann and Lovley (2001) J. Bacteriol. 183:4468-4476]. In order to gain insight into the physiological function of SfrAB, a knockout mutant was constructed by replacing a 3.6 kb stretch of sequence encompassing the C-terminal 72% of the SfrB and the N-terminal 80% of the SfrA coding sequences with a kanamycin resistance cassette via homologous recombination. The SfrAB knockout mutant (Delta sfrAB::kan) was found to be initially unable to grow in media in which acetate served as the sole electron donor. However, following prolonged incubation (2-3 weeks) in acetate:fumarate medium, an acetate-adapted SfrAB-null strain with the ability to grow on fumarate with acetate serving as the sole electron donor was isolated. This acetate-adapted, SfrAB-null strain and wild type G. sulfurreducens were cultured in parallel in chemostats at an intermediate growth rate (0.05 hr-1) in acetate-limited freshwater fumarate medium (5 mM acetate:27.5 mM fumarate). After the chemostats reached steady state, cells were harvested by centrifugation and RNA was extracted. In order to identify genes that were involved in growth on acetate in the absence of SfrAB, this RNA was used to perform microarray analysis comparing gene expression in the acetate-adapted, SfrAB-null and wild type strains. 2. Growth condtions: Cells were cultured in chemostats at a dilution rate of 0.05 hr-1 in acetate-limited freshwater fumarate medium (5 mM acetate:27.5 mM fumarate) and harvested when the chemostats were at steady state. 3. Mutant designation: acetate-adapted SfrAB-null strain, genotype= Delta sfrAB::kan. (Note: there is an unadapted Delta sfrAB::kan strain which cannot grow on acetate and an adapted one which can. The acetate-adapted strain was used for the microarray analysis).
Project description:The LIM homeodomain transcription factor Lmx1a is a very potential inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyze the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other, previously generated expression arrays, and their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (drJ/drJ) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A overexpression cell system resulted in the identification of novel downstream targets of Lmx1A in mdDA neurons: Grb10, Rgs4 and Vmat2. RNA was isolated from MN9D cells. Each experimental sample consisted of a RNA pool derived from 3 separate 10-cm dishes containing Lmx1a overexpressing MN9D cells (transfected with pcDNA3.1(-)-Lmx1a). microarray analysis was performed in triplicate, each experimental sample was hybridized to the same reference pool of RNA derived from 9 10-cm dishes containing control MN9D cells (transfected with empty pcDNA3.1(-)). On each of three microarray samples, dye swap was performed to correct for dye effects.
Project description:The PI3K-PKB/c-akt-FOXO signalling network provides a major intracellular hub for regulation of cell proliferation, survival and stress resistance1. Here we report a novel function for FOXO transcription factors in regulating autophagy through modulation of intracellular glutamine levels. To identify novel transcriptional targets of this module we performed an unbiased microarray analysis after conditional activation of the key components PI3K, PKB, FOXO3 and FOXO4. Utilising this global pathway approach we identified glutamine synthetase (GS) as being transcriptionally regulated by PI3K-PKB-FOXO signalling. FOXO-mediated increase in GS expression specifically induced glutamine production independently of cell type, and this was evolutionary conserved. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes, which was dependent on GS activity. Increased GS activity resulted in increased autophagosome turnover as measured by LC3 lipidation, p62 degradation, and confocal imaging of LC3, p62, WIPI-1, ULK2 and Atg12. Inhibition of FOXO3-mediated autophagy resulted in increased apoptosis, suggesting that the induction of autophagy by FOXO3-mediated upregulation of GS is important for cellular survival. These findings reveal a novel signalling network that can directly modulate autophagy through regulation of glutamine metabolism. conditional activation of pkb and pi3k were followed in a timeseries. Each timepoint consists of 4 independent replicates, labeled with either cy3 or cy5 and put on array against time0.
Project description:Mating is a complex process that causes many behavioral and physiological changes, but the factors triggering these changes and the underlying molecular processes are not well characterized. Honey bee queens provide a convenient system for dissecting these factors (e.g., physical manipulation, insemination volume, insemination substance) via instrumental insemination. We examined the effects of carbon dioxide (CO2), a commonly used anesthetic in instrumental insemination that causes changes similar to those observed after mating, and physical manipulation, which presumably mimics the act of copulation, on the brain transcriptional changes in honey bee queens. We found significant gene overlap between our study and previous mating studies in honey bee queens and Drosophila. This suggests that molecular pathways regulating the mating process are conserved across different mating regimes of honey bees as well as across insect orders.