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

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.

The Expression of Multiple Genes Can be Visualized Simultaneously Using Microarrays

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

The Expression of Multiple Genes Can be Visualized Simultaneously Using Microarrays

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