Genomics Training Genomics Training

A Cooperative Endeavor between USEPA Region 5

-and-USEPA Office of Research and Development

A Cooperative Endeavor between USEPA Region 5

-and-USEPA Office of Research and Development

Adam BialesAdam Biales

NERL Postdoctoral FellowNERL Postdoctoral Fellow

OverviewOverview

DNADNA RNARNA ProteinProtein PCRPCR

The Central DogmaThe Central Dogma

Central dogma of molecular Central dogma of molecular biology states that DNA carries biology states that DNA carries the genetic information which the genetic information which is transcribed to RNA and is transcribed to RNA and subsequently translated to subsequently translated to proteinprotein

DNA DNA RNA Protein RNA Protein

The Central DogmaThe Central Dogma

DNADNA RNA Protein RNA Protein

Overview of DNAOverview of DNA

DNA stores information – Blue printDNA stores information – Blue print– 1-5 billion base pairs in length1-5 billion base pairs in length– 2 meters in length2 meters in length– Very complexVery complex– Lots more DNA than genesLots more DNA than genes

Non-codingNon-coding– Repetitive (microsatellites, SINES, etc.) Repetitive (microsatellites, SINES, etc.) – IntronsIntrons– Structural – telomeres, centromeresStructural – telomeres, centromeres

Protein coding Protein coding ≈≈ 1% 1% – Complexity becoming clearComplexity becoming clear

1970 1970 ≈≈ 100 million genes 100 million genes 2002 2002 ≈ 30,000 genes ≈ 30,000 genes

Mitochondrial vs. NuclearMitochondrial vs. Nuclear

NucleosomeNucleosome

ChromosomesChromosomes

Overview of DNAOverview of DNA

DNA stores information – Blue printDNA stores information – Blue print– Very complexVery complex– Lots more DNA than genesLots more DNA than genes

Non-codingNon-coding– Repetitive (microsatellites, SINES, etc.) Repetitive (microsatellites, SINES, etc.) – IntronsIntrons– Structural – telomeres, centromeresStructural – telomeres, centromeres

Protein coding Protein coding ≈≈ 1% 1% – Complexity becoming clearComplexity becoming clear

1970 1970 ≈≈ 100 million genes 100 million genes 2002 2002 ≈ 30,000 genes ≈ 30,000 genes

Mitochondrial vs. NuclearMitochondrial vs. Nuclear

DNA structureDNA structure

DNA is a polymer of nucleotides DNA is a polymer of nucleotides – Purines (Adenine,Guanine)Purines (Adenine,Guanine)– Pyrimidines (Cytosine,Thymine)Pyrimidines (Cytosine,Thymine)

Two strands twist around each Two strands twist around each other (double helix) other (double helix) – Bases of the two strands face each Bases of the two strands face each

otherother– Forms base pairsForms base pairs– A pairs with T, G pairs with C A pairs with T, G pairs with C

NucleotideNucleotide

DNA structureDNA structure

DNA is a polymer of nucleotides DNA is a polymer of nucleotides – Purines (Adenine,Guanine)Purines (Adenine,Guanine)– Pyrimidines (Cytosine,Thymine)Pyrimidines (Cytosine,Thymine)

Two strands twist around each Two strands twist around each other (double helix) other (double helix) – Bases of the two strands face each Bases of the two strands face each

otherother– Forms base pairsForms base pairs– A pairs with T, G pairs with C A pairs with T, G pairs with C

The Central DogmaThe Central Dogma

DNA DNA RNARNA Protein Protein

Overview of RNAOverview of RNA

Different typesDifferent types– mRNA, tRNA, rRNAmRNA, tRNA, rRNA

Carries message from DNA to Carries message from DNA to ribosome – proteinribosome – protein

Often quick turn overOften quick turn over Highly regulatedHighly regulated All functions not understoodAll functions not understood

RNA structure RNA structure

– Single strandedSingle stranded– DNA and RNA have structural DNA and RNA have structural

differencesdifferencesPentoses (five-carbon sugar Pentoses (five-carbon sugar

component)component)– In DNA, the pentose is deoxyriboseIn DNA, the pentose is deoxyribose– In RNA, it is riboseIn RNA, it is ribose

Nitrogenous basesNitrogenous bases– thymine in DNA, uracil in RNAthymine in DNA, uracil in RNA

RNA StructureRNA Structure

The Central DogmaThe Central Dogma

DNA DNA RNA RNA ProteinProtein

Overview of ProteinsOverview of Proteins

Made from amino acids Made from amino acids strung togetherstrung together

Form regular 3-D structuresForm regular 3-D structures Structure dictates functionStructure dictates function Subunits add specificity to Subunits add specificity to

functionfunction Turned on and off by Turned on and off by

chemical modificationschemical modifications

Levels of Protein StructureLevels of Protein Structure

Overview of ProteinsOverview of Proteins

Made from amino acids Made from amino acids strung togetherstrung together

Form regular 3-D structuresForm regular 3-D structures Structure dictates functionStructure dictates function Subunits add specificity to Subunits add specificity to

functionfunction Turned on and off by Turned on and off by

chemical modificationschemical modifications

ProteinsProteinsProteinsProteins Proteins are used for a Proteins are used for a

variety of functions in the variety of functions in the cell:cell:– Structural supportStructural support– MetabolismMetabolism– MotionMotion– DefenseDefense– DNA replication, RNA synthesis, etc.DNA replication, RNA synthesis, etc.– And many more functionsAnd many more functions

DNA PolymeraseDNA Polymerase

PCRPCR

Polymerase Chain Polymerase Chain Reaction - Reaction - PCRPCR

Polymerase Chain Polymerase Chain Reaction - Reaction - PCRPCR

An An in vitroin vitro method for replicating method for replicating DNADNA

Makes billions of copies of a DNA Makes billions of copies of a DNA template in a few hourstemplate in a few hours

Requires only a tiny amount of Requires only a tiny amount of starting materialstarting material

Highly specific: primers Highly specific: primers determine the sequence to be determine the sequence to be amplifiedamplified

PCR reaction PCR reaction componentscomponentsPCR reaction PCR reaction componentscomponents

Template DNA strandTemplate DNA strand Nucleotides (dNTPS)Nucleotides (dNTPS) Heat stable DNA Polymerase – TaqHeat stable DNA Polymerase – Taq

– Same enzyme that replicates DNA in Same enzyme that replicates DNA in your cellsyour cells

– Isolated from hotsprings in Isolated from hotsprings in YellowstoneYellowstone

Primers (oligos) – 20ish bpPrimers (oligos) – 20ish bp

Primer specificityPrimer specificity4 nucleotides, 3.2 x 109 base pairs in the human genome…4 nucleotides, 3.2 x 109 base pairs in the human genome…An oligo of 10 bases in length = 4An oligo of 10 bases in length = 41010 (an exact sequence (an exact sequence match expected to occur ~ every match expected to occur ~ every 1 million bases1 million bases))

…expected to be represented in the genome 3,200 times

An oligo of 10 bases in length = 4An oligo of 10 bases in length = 41515 (an exact sequence (an exact sequence match expected to occur ~ every expected to occur ~ every 1 billion bases1 billion bases))

…expected to be represented in the genome 3 times

An oligo of 10 bases in length = 4An oligo of 10 bases in length = 420 20 (an exact sequence (an exact sequence match expected to occur ~ every match expected to occur ~ every 1 trillion bases1 trillion bases))

…expected to be represented in the genome…well you get the idea

PCR AmplificationPCR Amplification

CyclesCycles CopiesCopies

11 22

22 44

44 1616

1010 1,0241,024

1515 32,76832,768

2020 1,048,5761,048,576

2525 33,554,43233,554,432

3030 1,073,741,8241,073,741,824

Expression Based Technology Expression Based Technology

Expression Based TechnologyExpression Based Technology

Immediate responses on Immediate responses on cellular levelcellular level

Biological informationBiological information Can be specific for a given Can be specific for a given

stressorstressor RapidRapid

RegulationRegulation

Needs to be heavily regulatedNeeds to be heavily regulated Appropriate cell activation Appropriate cell activation If not regulated leads to If not regulated leads to

pathologypathology– Cancers Cancers

Cell-cycle Cell-cycle SignalingSignaling

Occurs on each level: Occurs on each level: – DNA, RNA, ProteinDNA, RNA, Protein

Gene StructureGene Structure

1000 - 250000 bp

Promoter

Cis element

RegulationRegulation

DNA Gene StructureDNA Gene Structure

1000 - 250000 bp

Cis element

Promoter GENE

Transcription/RNA processingTranscription/RNA processing

Intron Intron IntronExon ExonExon

AAAAAAN250

mRNA

5’ 3’

The Central DogmaThe Central Dogma

DNA RNA Protein

Reverse Transcriptase

cDNA

QPCRQPCRQPCRQPCR

Realtime quantitative PCRRealtime quantitative PCR– AdvantagesAdvantages

QuantitativeQuantitativeSensitiveSensitiveReproducibleReproducibleMinimal tissue requirementsMinimal tissue requirementsRapid resultsRapid results

““Real-Time” visualization of Real-Time” visualization of amplified productsamplified products

107 100

Dynamic range

Highly reproducible across entire range12 replicates each Ct < 0.50 cycles

Able to QA

MicroarraysMicroarrays

Monitor changes across 1000s of genes Monitor changes across 1000s of genes in 1 experimentin 1 experiment– Available for rainbow troutAvailable for rainbow trout– ZebrafishZebrafish– FHM – 2006FHM – 2006– DaphniaDaphnia

Signatures – 1 set of genes = 1 Signatures – 1 set of genes = 1 chemicalchemical

Systems biologySystems biology Information about mechanismInformation about mechanism

– Cluster analysisCluster analysis

Control (reference sample)Treatment (ie. effluent)

Isolate Tissue RNA Isolate Tissue RNART & label RT & label

1

12

12

3

1

12

12

3

Microarray

Message present Message present at equal levels in both treatmentat equal levels in both treatment

and controland control

Message present Message present only in treatmentonly in treatment

Message present Message present only in controlonly in control

Adapted from H. Hamadeh and C. Afshari, American Scientist 88:508-515

Lapointe, Jacques et al. (2004) Proc. Natl. Acad. Sci. USA 101, 811-816

Genes Expression Comparison between Genes Expression Comparison between High and Low Grade CancerHigh and Low Grade Cancer

ProteomicsProteomics

Similar to microarrays – global Similar to microarrays – global changeschanges

Protein data more biologically Protein data more biologically relevantrelevant– Higher biological levelHigher biological level– Less variableLess variable

2-D gel electrophoresis2-D gel electrophoresis– Non-directed, no assumptionsNon-directed, no assumptions– Can see modificationsCan see modifications

Study of protein populations of one cell versus another

2-D Gel Electrophoresis2-D Gel Electrophoresis

Control Treatment

Molecular/RNA/ProteinMolecular/RNA/ProteinChangesChanges

Cell death/Mitosis/Activation Cell death/Mitosis/Activation

HistopathologyHistopathology

Toxicity TestingToxicity Testing

Bio-assessment + CommunityBio-assessment + CommunityAssessmentAssessment

Population Decline, Adaptation Population Decline, Adaptation Population DNA Analyses Population DNA Analyses

BiologicalBiologicalEffect Effect

The Exposure-Effect HierarchyThe Exposure-Effect Hierarchy

BiologicalBiologicalLevelLevel

SubcellularSubcellular

CellularCellular

Tissue/OrganTissue/Organ

OrganismOrganism

PopulationPopulation

CommunityCommunity

Molecular/RNA/ProteinMolecular/RNA/ProteinChangesChanges

Cell death/Mitosis/Activation Cell death/Mitosis/Activation

HistopathologyHistopathology

Toxicity TestingToxicity Testing

Bio-assessment + CommunityBio-assessment + CommunityAssessmentAssessment

Population Decline, Adaptation Population Decline, Adaptation Population DNA Analyses Population DNA Analyses

BiologicalBiologicalEffect Effect

Current Molecular AssaysCurrent Molecular Assays

VitellogeninVitellogenin– Egg protein – only females in breeding Egg protein – only females in breeding

seasonseason– Males turn it on when exposed to Males turn it on when exposed to

estrogenestrogen– All kinds of fishAll kinds of fish

Fathead, trout, bass, gudgeon, shiners, Fathead, trout, bass, gudgeon, shiners, roach, flounder roach, flounder

– Can be used as an indicator to Can be used as an indicator to estrogen exposureestrogen exposure

EstrogensEstrogens

Estradiol, estrone, EEEstradiol, estrone, EE22 SourcesSources

– Farm runoffFarm runoff– ContraceptivesContraceptives

Environmental levels - EEEnvironmental levels - EE22

– Surface water Surface water 0.05 – 30.8 ng/L EE0.05 – 30.8 ng/L EE22

– Sewage effluents Sewage effluents 0.05 – 62.0 ng/L EE0.05 – 62.0 ng/L EE22

– LOEC Vg protein 0.1 ng/LLOEC Vg protein 0.1 ng/L– LOEC sex interchange 0.6 ng/LLOEC sex interchange 0.6 ng/L

Can have additive effects Can have additive effects Potent responses at low levelsPotent responses at low levels

– (4 ng/L EE(4 ng/L EE22))

Estrogenic EffectsEstrogenic Effects Male fish with ovatestesMale fish with ovatestes Decreased male fitnessDecreased male fitness Decreased size/physical abnormalitiesDecreased size/physical abnormalities Decreased reproductive success (.32 ng/L) Decreased reproductive success (.32 ng/L)

– NO EGGS PRODUCED at 3.5 ng/LNO EGGS PRODUCED at 3.5 ng/L Skewed sex ratiosSkewed sex ratios

– 0 males with secondary sex characters0 males with secondary sex characters– 3.5 ng/L3.5 ng/L

May have long-term effect on reproductive May have long-term effect on reproductive successsuccess– 50% reduction in reproductive success 29 days after 50% reduction in reproductive success 29 days after

exposure exposure – 5 months following treatment – decreased fertility5 months following treatment – decreased fertility

IssuesIssues Fish swimFish swim

– Not sitting by effluent for whole lifeNot sitting by effluent for whole life– Level of real world exposureLevel of real world exposure

Linkage between biological levels Linkage between biological levels not completely establishednot completely established

Why are there any fish at all?Why are there any fish at all? Needs field testingNeeds field testing MixturesMixtures Can we use gene expression as a Can we use gene expression as a

metric in bioassessmentmetric in bioassessment

ELAELA

Located in northwestern Ontario approximately 250 km east of Winnipeg and 50 km east-southeast of Kenora.

K. Kidd

ELAELA

Dose lake with 4-6 ng/L EE2 or not for 3 Dose lake with 4-6 ng/L EE2 or not for 3 continuous years (2001-03)continuous years (2001-03)

Measure Vg expression in wild fish, deployed fish, Measure Vg expression in wild fish, deployed fish, laboratory, embryo/larval exposureslaboratory, embryo/larval exposures

Water chemistryWater chemistry Sediment elutriate exposure in frySediment elutriate exposure in fry Other biological measurements – aggression, Other biological measurements – aggression,

mortalitymortality

ObjectiveObjective: To study the long term effects of : To study the long term effects of xenoestrogens on wild fish populationxenoestrogens on wild fish population

ELA resultsELA results Male FHM had elevated Vg expression Male FHM had elevated Vg expression

from 24 hours until October (last sample from 24 hours until October (last sample of year)of year)

Females had elevated Vg levels past end Females had elevated Vg levels past end of breeding seasonof breeding season

Male pearl dace also exhibited high Vg Male pearl dace also exhibited high Vg levelslevels

In fall of second year no age 0 fish foundIn fall of second year no age 0 fish found HistopathologyHistopathology

– liver hypertrohpyliver hypertrohpy– fibrotic sperm ductsfibrotic sperm ducts

Ohio RiverOhio River

Collaboration with Ohio River Valley Water Sanitation Commission Collaboration with Ohio River Valley Water Sanitation Commission ORSANCOORSANCO

Vg – Sewage Treatment Vg – Sewage Treatment PlantsPlants

Objective: Create Vg assay and identify the number of Objective: Create Vg assay and identify the number of species and geographic extent effected by estrogenic species and geographic extent effected by estrogenic compounds found in effluentscompounds found in effluents

3 sampling sites 3 sampling sites – Region 3 – Wheeling, ALCOSAN, ParkersburgRegion 3 – Wheeling, ALCOSAN, Parkersburg

Downstream – proximal Downstream – distal Upstream – reference

Multiple speciesMultiple species Multiple exposures – lab, deployment – high & low flowMultiple exposures – lab, deployment – high & low flow Chemical analysisChemical analysis Histopath analysisHistopath analysis Sex RatiosSex Ratios QuestionsQuestions

– Are male fish in reference sites producing Vg?Are male fish in reference sites producing Vg?– If fish aren’t stuck at effluent is there an effect?If fish aren’t stuck at effluent is there an effect?

Assessment of Ohio River Assessment of Ohio River Pools, 2004Pools, 2004

100908070605040302010

25%

Racine Mark15 Mark29 MyersNew C

25.5%

40%

26.4%41.4%

Per

cent

age

of s

ites

with

in a

poo

l with

fai

ling

IBI

scor

es

303d list

Ohio River - EDCOhio River - EDC Using probabilistic sampling to determine Using probabilistic sampling to determine

extent of exposure in a pool of the Ohio extent of exposure in a pool of the Ohio RiverRiver– Fish localized to a given pool - DamsFish localized to a given pool - Dams– 15 probabilistic sites15 probabilistic sites– Exposure differences – ecologyExposure differences – ecology

Multiple speciesMultiple species– Bottom feedersBottom feeders– Water columnWater column

– Sex ratiosSex ratios– Vg expressionVg expression– Result: does expression aid in identifying Result: does expression aid in identifying

causescauses

Forthcoming ProjectsForthcoming Projects

Several other projects targeting Several other projects targeting non-estrogenic compoundsnon-estrogenic compounds

AtrazineAtrazine– 2-D gels Gene expression markers 2-D gels Gene expression markers

5 different tissues5 different tissues

Androgen indicatorsAndrogen indicators Invertebrate sourcesInvertebrate sources MixturesMixtures Pulsed exposuresPulsed exposures

Expression Technology Expression Technology SummarySummary

Informative Informative Available - microarrays are online or Available - microarrays are online or

under developed for a number of aquatic under developed for a number of aquatic species species

SensitiveSensitive Targets changes early in exposureTargets changes early in exposure High through-putHigh through-put Making linkages to higher biological levelsMaking linkages to higher biological levels Assays are being developed for a number Assays are being developed for a number

of different species representing an array of different species representing an array of different ecological catagories (habitat, of different ecological catagories (habitat, feeding groups, etc.feeding groups, etc.