adam biales nerl postdoctoral fellow
DESCRIPTION
Genomics Training. A Cooperative Endeavor between USEPA Region 5 - and- USEPA Office of Research and Development. Adam Biales NERL Postdoctoral Fellow. Overview. DNA RNA Protein PCR. The Central Dogma. - PowerPoint PPT PresentationTRANSCRIPT
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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
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OverviewOverview
DNADNA RNARNA ProteinProtein PCRPCR
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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
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The Central DogmaThe Central Dogma
DNADNA RNA Protein RNA Protein
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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
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NucleosomeNucleosome
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ChromosomesChromosomes
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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
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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
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NucleotideNucleotide
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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
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The Central DogmaThe Central Dogma
DNA DNA RNARNA Protein Protein
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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
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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
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RNA StructureRNA Structure
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The Central DogmaThe Central Dogma
DNA DNA RNA RNA ProteinProtein
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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
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Levels of Protein StructureLevels of Protein Structure
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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
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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
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DNA PolymeraseDNA Polymerase
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PCRPCR
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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
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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
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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
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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
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Expression Based Technology Expression Based Technology
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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
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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
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Gene StructureGene Structure
1000 - 250000 bp
Promoter
Cis element
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RegulationRegulation
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DNA Gene StructureDNA Gene Structure
1000 - 250000 bp
Cis element
Promoter GENE
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Transcription/RNA processingTranscription/RNA processing
Intron Intron IntronExon ExonExon
AAAAAAN250
mRNA
5’ 3’
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The Central DogmaThe Central Dogma
DNA RNA Protein
Reverse Transcriptase
cDNA
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QPCRQPCRQPCRQPCR
Realtime quantitative PCRRealtime quantitative PCR– AdvantagesAdvantages
QuantitativeQuantitativeSensitiveSensitiveReproducibleReproducibleMinimal tissue requirementsMinimal tissue requirementsRapid resultsRapid results
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““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
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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
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Control (reference sample)Treatment (ie. effluent)
Isolate Tissue RNA Isolate Tissue RNART & label RT & label
1
12
12
3
1
12
12
3
Microarray
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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
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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
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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
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2-D Gel Electrophoresis2-D Gel Electrophoresis
Control Treatment
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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
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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
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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))
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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
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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
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ELAELA
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Located in northwestern Ontario approximately 250 km east of Winnipeg and 50 km east-southeast of Kenora.
K. Kidd
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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
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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
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Ohio RiverOhio River
Collaboration with Ohio River Valley Water Sanitation Commission Collaboration with Ohio River Valley Water Sanitation Commission ORSANCOORSANCO
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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?
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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
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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
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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
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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.