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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
 Central
dogma of molecular
biology states that DNA carries
the genetic information which
is transcribed to RNA and
subsequently translated to
protein
DNA
RNA
Protein
The Central Dogma
DNA
RNA
Protein
Overview of DNA
DNA stores information – Blue print
–
–
–
–
1-5 billion base pairs in length
2 meters in length
Very complex
Lots more DNA than genes
 Non-coding
– Repetitive (microsatellites, SINES, etc.)
– Introns
– Structural – telomeres, centromeres
 Protein
coding ≈ 1%
– Complexity becoming clear
 1970 ≈ 100 million genes
 2002 ≈ 30,000 genes
Mitochondrial vs. Nuclear
Nucleosome
Chromosomes
Overview of DNA
DNA stores information – Blue print
– Very complex
– Lots more DNA than genes
 Non-coding
– Repetitive (microsatellites, SINES, etc.)
– Introns
– Structural – telomeres, centromeres
 Protein
coding ≈ 1%
– Complexity becoming clear
 1970 ≈ 100 million genes
 2002 ≈ 30,000 genes
Mitochondrial vs. Nuclear
DNA structure

DNA is a polymer of nucleotides
– Purines (Adenine,Guanine)
– Pyrimidines (Cytosine,Thymine)

Two strands twist around each
other (double helix)
– Bases of the two strands face each
other
– Forms base pairs
– A pairs with T, G pairs with C
Nucleotide
DNA structure

DNA is a polymer of nucleotides
– Purines (Adenine,Guanine)
– Pyrimidines (Cytosine,Thymine)

Two strands twist around each
other (double helix)
– Bases of the two strands face each
other
– Forms base pairs
– A pairs with T, G pairs with C
The Central Dogma
DNA
RNA
Protein
Overview of RNA
 Different
types
– mRNA, tRNA, rRNA
 Carries
message from DNA to
ribosome – protein
 Often quick turn over
 Highly regulated
 All functions not understood
RNA structure
– Single stranded
– DNA and RNA have structural
differences
 Pentoses
(five-carbon sugar
component)
– In DNA, the pentose is deoxyribose
– In RNA, it is ribose
 Nitrogenous
bases
– thymine in DNA, uracil in RNA
RNA Structure
The Central Dogma
DNA
RNA
Protein
Overview of Proteins
 Made
from amino acids
strung together
 Form regular 3-D structures
 Structure dictates function
 Subunits add specificity to
function
 Turned on and off by
chemical modifications
Levels of Protein Structure
Overview of Proteins
 Made
from amino acids
strung together
 Form regular 3-D structures
 Structure dictates function
 Subunits add specificity to
function
 Turned on and off by
chemical modifications
Proteins
 Proteins
are used for a
variety of functions in
the cell:
–
–
–
–
–
Structural support
Metabolism
Motion
Defense
DNA replication, RNA
synthesis, etc.
– And many more functions
DNA Polymerase
PCR
Polymerase Chain
Reaction - PCR
 An
in vitro method for
replicating DNA
 Makes billions of copies of a
DNA template in a few hours
 Requires only a tiny amount of
starting material
 Highly specific: primers
determine the sequence to be
amplified
PCR reaction
components
Template DNA strand
 Nucleotides (dNTPS)
 Heat stable DNA Polymerase –
Taq

– Same enzyme that replicates DNA in
your cells
– Isolated from hotsprings in
Yellowstone

Primers (oligos) – 20ish bp
Primer specificity
4 nucleotides, 3.2 x 109 base pairs in the
human genome…
An oligo of 10 bases in length = 410 (an exact sequence
match expected to occur ~ every 1 million bases)
…expected to be represented in the genome 3,200 times
An oligo of 10 bases in length = 415 (an exact sequence
match expected to occur ~ every 1 billion bases)
…expected to be represented in the genome 3 times
An oligo of 10 bases in length = 420 (an exact sequence
match expected to occur ~ every 1 trillion bases)
…expected to be represented in the
genome…well you get the idea
PCR Amplification
Cycles
Copies
1
2
2
4
4
16
10
1,024
15
32,768
20
1,048,576
25
33,554,432
30
1,073,741,824
Expression Based Technology
Expression Based Technology
 Immediate
responses on
cellular level
 Biological information
 Can be specific for a given
stressor
 Rapid
Regulation
 Needs
to be heavily regulated
 Appropriate cell activation
 If not regulated leads to
pathology
– Cancers
 Cell-cycle
 Signaling
 Occurs
on each level:
– DNA, RNA, Protein
Gene Structure
Promoter
Cis element
1000 - 250000 bp
Regulation
DNA Gene Structure
Promoter
Cis element
1000 - 250000 bp
GENE
Transcription/RNA processing
Intron
Exon
Intron
Exon
5’
Intron
Exon
3’
mRNA
AAAAAAN250
The Central Dogma
Reverse Transcriptase
cDNA
DNA
RNA
Protein
QPCR
 Realtime
quantitative PCR
– Advantages
 Quantitative
 Sensitive
 Reproducible
 Minimal
tissue requirements
 Rapid results
“Real-Time” visualization of
amplified products
Dynamic range
107
Able to QA
100
Highly reproducible across entire range
12 replicates each DCt < 0.50 cycles
Microarrays

Monitor changes across 1000s of genes
in 1 experiment
–
–
–
–



Available for rainbow trout
Zebrafish
FHM – 2006
Daphnia
Signatures – 1 set of genes = 1
chemical
Systems biology
Information about mechanism
– Cluster analysis
Control (reference sample)
Treatment (ie. effluent)
Isolate Tissue RNA
RT & label
Isolate Tissue RNA
RT & label
1
1
1
3
2
1
2
2
3
1
2
Microarray
1
Message present
only in treatment
Message present
only in control
Message present
at equal levels in both treatment
and control
Adapted from H. Hamadeh and C. Afshari, American Scientist 88:508-515
Genes Expression Comparison between
High and Low Grade Cancer
Lapointe, Jacques et al. (2004) Proc. Natl. Acad. Sci. USA 101, 811-816
Proteomics
Study of protein populations of
one cell versus another



Similar to microarrays –
global changes
Protein data more biologically
relevant
– Higher biological level
– Less variable
2-D gel electrophoresis
– Non-directed, no
assumptions
– Can see modifications
2-D Gel Electrophoresis
Control
Treatment
The Exposure-Effect Hierarchy
Biological
Level
Community
Biological
Effect
Bio-assessment + Community
Assessment
Population
Population Decline, Adaptation
Population DNA Analyses
Organism
Toxicity Testing
Tissue/Organ
Histopathology
Cellular
Cell death/Mitosis/Activation
Subcellular
Molecular/RNA/Protein
Changes
Current Molecular Assays

Vitellogenin
– Egg protein – only females in breeding
season
– Males turn it on when exposed to
estrogen
– All kinds of fish
 Fathead,
trout, bass, gudgeon, shiners,
roach, flounder
– Can be used as an indicator to
estrogen exposure
Estrogens


Estradiol, estrone, EE2
Sources
– Farm runoff
– Contraceptives

Environmental levels - EE2
–
–
–
–


Surface water 0.05 – 30.8 ng/L EE2
Sewage effluents 0.05 – 62.0 ng/L EE2
LOEC Vg protein 0.1 ng/L
LOEC sex interchange 0.6 ng/L
Can have additive effects
Potent responses at low levels
– (4 ng/L EE2)
Estrogenic Effects




Male fish with ovatestes
Decreased male fitness
Decreased size/physical abnormalities
Decreased reproductive success (.32 ng/L)
– NO EGGS PRODUCED at 3.5 ng/L

Skewed sex ratios
– 0 males with secondary sex characters
– 3.5 ng/L

May have long-term effect on reproductive
success
– 50% reduction in reproductive success 29 days after
exposure
– 5 months following treatment – decreased fertility
Issues

Fish swim
– Not sitting by effluent for whole life
– Level of real world exposure
Linkage between biological levels
not completely established
 Why are there any fish at all?
 Needs field testing
 Mixtures
 Can we use gene expression as a
metric in bioassessment

ELA
Located in northwestern Ontario approximately 250 km east of Winnipeg
and 50 km east-southeast of Kenora.
K. Kidd
ELA
Objective: To study the long term effects of
xenoestrogens on wild fish population





Dose lake with 4-6 ng/L EE2 or not for 3
continuous years (2001-03)
Measure Vg expression in wild fish, deployed fish,
laboratory, embryo/larval exposures
Water chemistry
Sediment elutriate exposure in fry
Other biological measurements – aggression,
mortality
ELA results





Male FHM had elevated Vg expression
from 24 hours until October (last sample
of year)
Females had elevated Vg levels past end
of breeding season
Male pearl dace also exhibited high Vg
levels
In fall of second year no age 0 fish found
Histopathology
– liver hypertrohpy
– fibrotic sperm ducts
Ohio River
Collaboration with Ohio River Valley Water Sanitation Commission
ORSANCO
Vg – Sewage Treatment
Plants


Objective: Create Vg assay and identify the number of
species and geographic extent effected by estrogenic
compounds found in effluents
3 sampling sites
– Region 3 – Wheeling, ALCOSAN, Parkersburg








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Downstream – proximal
Downstream – distal
Upstream – reference
Multiple species
Multiple exposures – lab, deployment – high & low flow
Chemical analysis
Histopath analysis
Sex Ratios
Questions
– Are male fish in reference sites producing Vg?
– If fish aren’t stuck at effluent is there an effect?
Percentage of sites within
a pool with failing IBI scores
Assessment of Ohio River
Pools, 2004
100
90
80
70
60
50
40
30
20
10
303d list
40%
41.4%
25.5%
26.4%
25%
New C
Racine
Mark15
Mark29
Myers
Ohio River - EDC

Using probabilistic sampling to determine
extent of exposure in a pool of the Ohio
River
– Fish localized to a given pool - Dams
– 15 probabilistic sites
– Exposure differences – ecology

Multiple species
– Bottom feeders
– Water column
– Sex ratios
– Vg expression
– Result: does expression aid in identifying
causes
Forthcoming Projects
Several other projects targeting
non-estrogenic compounds
 Atrazine

– 2-D gels
5
Gene expression markers
different tissues
Androgen indicators
 Invertebrate sources
 Mixtures
 Pulsed exposures

Expression Technology
Summary







Informative
Available - microarrays are online or
under developed for a number of aquatic
species
Sensitive
Targets changes early in exposure
High through-put
Making linkages to higher biological levels
Assays are being developed for a number
of different species representing an array
of different ecological catagories (habitat,
feeding groups, etc.