* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download The Power of Microarray Technology - People
Transposable element wikipedia , lookup
Public health genomics wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Epigenetics of depression wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Gene desert wikipedia , lookup
Minimal genome wikipedia , lookup
Gene nomenclature wikipedia , lookup
Ridge (biology) wikipedia , lookup
History of genetic engineering wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genome evolution wikipedia , lookup
Genomic imprinting wikipedia , lookup
Genome (book) wikipedia , lookup
Fetal origins hypothesis wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Microevolution wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Mir-92 microRNA precursor family wikipedia , lookup
Designer baby wikipedia , lookup
Gene expression programming wikipedia , lookup
The Power of Microarray Technology Ruth G. Alscher Gene Expression: Control Points Responses to Environmental Signals Glycolysis, Citric Acid Cycle, and Related Metabolic Processes Free Radicals Attack Cells: Survival Mechanisms? ROS Arise as a Result of Exposure to: • • • • • • • Ozone Sulfur dioxide High light Paraquat Extremes of temperature Salinity Drought Free radicals arise throughout the . cell when stress is imposed Wounding , Chilling Pathogens Ozone Cell Wall Pathogens Wounding , Chilling Ozone Cell Wall Mitochondrion Post-transcriptional Effects Drought , Salinity (ROS subcellul ar si tes unclear) Mitochondrion Pos t-tra nscriptiona l Effe cts Drought , Salinity Cytosol Cytosol Antioxidant genes Antioxidant genes Nucleus Nucleus Gene (ROS su bce llul ar si tes un cle ar) Ex pression Gene Expression Chloroplast Chloroplast Pos t-tra nscriptiona l Effe cts Post-transcriptional Paraquat , Effects High Light + Chilling Sulfur Dioxide Paraquat , High Light + Chilling Sulfur Dioxide , , Effects of Drought Stress on Gene Expression in Loblolly Pine Trees Virginia Tech: determining which genes are essential for resistance to stress Plant Biologists: Drs. Alscher and Chevone., Cecilia Vasquez CS: Drs. Heath and Ramakrishnan, Margaret Ellis, Logan Hanks Statistics: Dr Key, Xiao Yang. NC State (Forest Biotechnology): discovering new genes in loblolly pine. Investigating gene expression patterns in stressed loblolly pine Selected cDNAs are spotted on to a glass surface (can be up to 20,000 different sequences spotted on to one slide). cDNAs derived from mRNA populations obtained from treated or control tree are hybridized to the cDNAs on the slide. The Expression of Multiple Genes Can be Visualized Simultaneously Using Microarrays Relative Abundance Detection Detection Treatment 1 1 1 Control 1 2 2 3 3 3 3 2 2 Mix Spots: 1 2 (Sequences affixed to slide) 3 Hybridization 1 2 3 The Expression of Multiple Genes Can be Visualized Simultaneously Using Microarrays Iterative strategy for detection of genetic interactions using microarrays Detection of gene expression effects on microarrays 1 4 Genetic Regulatory Networks Test mutant phenotypes 3 Identify mutants Characterize 2 gene function Design of Microarrays • Clones on the drought-stress microarrays were replicated and randomly placed • Experiment involved 384 archived pine ESTs • Organized into 4 microtitre source plates after PCR • Pipetted into 8 sets of 4 microtitre plates each • Each set a different random arrangement of 384 ESTs • Printed type A microarrays from first 4 sets • Printed type B microarrays from second 4 sets • Each array has 4 randomly placed replicates of each EST • Each control versus stress comparison was done on 4 arrays — A and B; flip dyes; A and B • Total of 16 replicates of each EST in each comparison Who’s Who Computer Science Plant Biology Ruth Alscher Plant Stress Virginia Tech Boris Chevone Plant Stress Dawei Chen Molecular Biology Bioinformatics Ron Sederoff, North Ross Whetten Carolina Len van Zyl State Univ. Y-H.Sun Lenwood Heath (CS) Algorithms Virginia Tech Naren Ramakrishnan (CS) Data Mining Problem Solving Environments Craig Struble, Vincent Jouenne (CS) Image Analysis Forest Biotechnology Ina Hoeschele (DS) Statistical Genetics Keying Ye (STAT) Bayesian Statistics Statistics Virginia Tech Expresso People Ron Sederoff Lenny Heath Craig Struble Ruth Alscher Ross Whetten Keying Ye Boris Chevone Y-H .Sun Len van Zyl Dawei Chen Vincent Jouenne Naren Ramakrishnan Hypotheses • There is a group of genes whose expression confers resistance to drought stress. • Expression of this group of genes is lower under severe than under mild stress. • Individual members of gene families show distinct responses to drought stress. Selection of cDNAs for Arrays • 384 ESTs (xylem, shoot tip cDNAs of loblolly) were chosen on the basis of function and grouped into categories. • Major emphasis was on processes known to be stress responsive. • In cases where more than one EST had similar BLAST hits, all ESTs were used. Expresso: A Problem Solving Environment for Microarray Experiment Design and Analysis • Integration of design and procedures • Integration of image analysis tools and statistical analysis • Connections to web databases and sequence alignment tools • The software Aleph was used for inductive logic programming (ILP). Categories within Protective and Protected Processes Gene Expression Signal Transduction Protease-associated ROS and Stress Environmental Change Protective Processes Nucleus Cell Wall Related Trafficking Phenylpropanoid Pathway Development Protected Processes Secretion Cells Cytoskeleton Tissues Plant Growth Regulation Chloroplast Associated Metabolism Carbon Metabolism Respiration and Nucleic Acids Mitochondrion A Note about Categories Categories are not mutually exclusive; gene(s) may be assigned to more then one category. For example, heat shock proteins have been grouped under these different categories and subcategories – Abiotic stress – heat – Gene expression – post-translational processing – chaperones – Abiotic stress - chaperones Abiotic Biotic Stress Protective Processes Cell Wall Related “Isoflavone Reductases” Antioxidant Processes Phenylpropanoid Pathway Drought Dehydrins, Aquaporins Heat Non-Plant Heat shock proteins (Chaperones) Xenobiotics GSTs Chaperones NADPH/Ascorbate/ Glutathione Scavenging Pathway Sucrose Metabolism Cellulose Categories within “Protective Processes” Arabionogalactan proteins Cytosolic ascorbate peroxidase superoxide dismutase-Fe superoxide dismutase-Cu-Zn glutathione reductase Extensins and proline rich proteins Hemicellulose Pectins Xylose Other Cell Wall Proteins Lignin Biosynthesis isoflavone reductases phenylalanine ammonia-lyases S-adenosylmethionine decarboxylases glycine hydromethyltransferases 4-coumarate-CoA ligases CCoAOMTs cinnamyl-alcohol dehydrogenase Quality Control • Positive: LP-3, a loblolly gene known to respond positively to drought stress in loblloly pine, was included. • LP-3 was positive in the moist versus mild comparison, and unchanged in the moist versus severe comparison. • Negative: Four clones of human genes used as negative controls in the Arabidopsis Functional Genomics project were included. The clones did not respond. Spot and Clone Analysis • Image Analysis: gridding, spot identification, intensity and background calculation, normalization • Statistics: • Fold or ratio estimation • Combining replicates • Higher-level Analysis: • Clustering methods • Inductive logic programming (ILP) Current Status of Expresso • Completely automated and integrated – Statistical analysis – Data mining – Experiment capture in MEL • Current Work: Integrating – Image processing – Querying by semi-structured views – Expresso-assisted experiment composition Future Directions Next Generation Stress Chips 1. Time course, short and long term, to capture gene expression events underlying “emergency” and adaptive events following drought stress imposition. (Use all available ESTs for candidate stress resistance genes.) 2. Generate cDNA library from stressed seedlings. 3. Initiate modeling of kinetics of drought stress responses. Microarray Data Analysis How to use microarrays to learn more about the influence of drought stress on gene expression? Where the biologists need the computer scientists. A. Confounding factors in the raw data 1. Limitations in accuracy (technique) 2. Biological variation (individuals) B. How to apply corrections for these confounding factors to maximize the predictive power of the data. C. Modeling regulatory networks.