ROS Arise as a Result of Exposure to:
• Ozone
• Sulfur dioxide
• High light
• Paraquat
• Extremes of temperature
• Salinity
• Drought
Mitochondrion
Chloroplast
Nucleus
Cytosol
Cell Wall
WoundingChilling Ozone
Drought,Salinity
ExpressionGene
Antioxidant genes
Post-transcriptionalEffects
ParaquatHigh Light + Chilling
Sulfur Dioxide
,,
subcellularROS
sitesunclear
(
)
,Pathogens
Post-transcriptionalEffects
Mitochondrion
Chloroplast
Nucleus
Cytosol
Cell Wall
WoundingChilling Ozone
Drought,Salinity
ExpressionGene
Antioxidant genes
Post-transcriptionalEffects
ParaquatHigh Light + Chilling
Sulfur Dioxide
,,
subcellularROS
sitesunclear
(
)
,Pathogens
Post-transcriptionalEffects
.Free radicals arise throughout the cell when stress is imposed
-
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.
Ying-Hsuan Sun, Drs. Sederoff and Whetten:
Effects of Drought Stress on Gene Expression in Loblolly Pine Trees
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.
Spots:(Sequences affixed to slide)
1 2 3
11
2
21
3
1 2
2333
Treatment Control
Mix
1 2 3
Excitatio
n
Em
issi
on
Detection
Relative AbundanceDetection
Hybridization
Detection of gene expression effects on microarrays
Characterize gene function
Test mutant phenotypes
Genetic Regulatory Networks
Identify mutants
1
2
3
4
Iterative strategy for detection of genetic interactions using 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
Design of Microarrays
Who’s Who
Ruth Alscher Plant Stress
Boris Chevone Plant Stress
Ron Sederoff, Ross WhettenLen van ZylY-H.SunForest Biotechnology
Plant BiologyComputer Science
Lenwood Heath (CS)Algorithms
Naren Ramakrishnan (CS)Data Mining
Problem Solving Environments
Craig Struble,Vincent Jouenne (CS)
Image Analysis
Statistics
Ina Hoeschele (DS)Statistical Genetics
Keying Ye (STAT)Bayesian Statistics
Virginia Tech
North Carolina State Univ.
Virginia Tech
Virginia Tech
Dawei Chen
Molecular Biology
Bioinformatics
Expresso People
Ross WhettenBoris Chevone
Ron Sederoff
Y-H .Sun Dawei Chen
Lenny Heath
Ruth Alscher
Vincent Jouenne
Naren Ramakrishnan
Keying Ye
Len van Zyl
Craig Struble
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.
• 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).
Expresso: A Problem Solving Environment for Microarray
Experiment Design and Analysis
Categories within Protective and Protected Processes
Plant Growth Regulation
Environmental
Change
GeneExpression
SignalTransduction
ProtectiveProcesses
ProtectedProcesses
ROS and Stress
Cell Wall Related
PhenylpropanoidPathway
Development
Metabolism
Chloroplast Associated
Carbon Metabolism
Respiration and Nucleic Acids
Mitochondrion
Cells
Tissues
Cytoskeleton
Secretion
Trafficking
Nucleus
Protease-associated
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
ProtectiveProcesses
Stress
Cell Wall Related
PhenylpropanoidPathway
AbioticBiotic
Antioxidant Processes
Drought
HeatNon-Plant
Xenobiotics
NADPH/Ascorbate/GlutathioneScavenging Pathway
Cytosolicascorbateperoxidase
Dehydrins, Aquaporins
Heat shock proteins(Chaperones)
superoxidedismutase-Fe
superoxidedismutase-Cu-Zn
glutathionereductase
Sucrose Metabolism
Cellulose
Arabionogalactan proteins
Hemicellulose
Pectins
Xylose
Other Cell Wall Proteins
isoflavone reductases
phenylalanine ammonia-lyases
S-adenosylmethionine decarboxylases
glycine hydromethyltransferases
Lignin Biosynthesis CCoAOMTs
4-coumarate-CoAligases
cinnamyl-alcoholdehydrogenase
Chaperones“IsoflavoneReductases”
GSTs
Extensins and proline rich proteinsCategorieswithin
“Protective Processes”
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.
• 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)
Spot and Clone Analysis
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 DirectionsNext 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.
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.
Microarray Data Analysis