Download Genomics and Behavior “Central Dogma” Outline

Genomics and Behavior
“Central Dogma”
“gene expression”
DNA
RNA
Protein
Behavior
Outline
• Transcription
• Analyzing genes and gene expression
– Early life experience and serotonin transporter
• Manipulating gene expression
– Pair bonding behavior
• Epigenetics
– Maternal behavior
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Protein Synthesis
DNA
Transcription
nucleus
ribosome
mRNA
cytoplasm
Translation
protein
Transcription (Gene expression)
gene
• DNA double helix is
unwound
• A strand of RNA is
produced from the
complementary DNA
strand by RNA
polymerase
ATG AAC TCC
TAC TTG AGG
AUG AAC UC C
TAC TTG AGG
Translation
• mRNA migrates out of the
nucleus to the ribosome
• Protein synthesis takes
place based on the
genetic code
• A three base codon
codes for an amino acid
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Mutation can Change Protein
Structure
• A mutation in DNA can
change physiology or
behavior by changing
properties in the resulting
protein
• Siamese cats have a
mutation that makes the
tyrosinase enzyme heat
sensitive
J. Hered. 21: 309-318, 1930.
Gene Regulation
• Although mutations causing changes in
protein can have important effects on
behavior, most mutations are either silent
or harmful
• Usually it is a difference in gene
expression that influences a behavior
Transcription
• The production of messenger RNA
(mRNA) from a sequence of DNA (gene)
– Gene expression
– “Turning on the gene”
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Important DNA Sequences
• Promoter
– Indicates where transcription should begin
• Coding region
– Sequence that codes for a protein
• Terminator
– Indicates where transcription should stop
promoter
terminator
coding region
Transcription machinery
• RNA Polymerase
– Transcribes DNA into RNA
• Transcription factors
– Help RNA polymerase bind to DNA
– There are hundreds of different transcription factors
– Steroid hormone receptors
Transcription factors
RNA polymerase
Initiation of Transcription
• Transcription factors bind to promoter region
Transcription factors
promoter
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Initiation of Transcription
• Transcription factors allow RNA polymerase to
bind to promoter region
RNA polymerase
promoter
Transcription
• RNA polymerase begins to move down the
strand of DNA and transcribe it into RNA. It
unwinds the DNA as it moves down the strand.
RNA
RNA polymerase
promoter
Transcription
RNA
RNA polymerase
promoter
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Termination of Transcription
• When the RNA polymerase reaches the
terminator sequence, it stops transcribing
RNA
RNA polymerase
terminator
Transcriptional Regulation
• Promoter sequence
• Expression of
transcription factors
• Splicing
Last point on transcription
• Gene regulation doesn’t mean much
unless a change in protein occurs
• Translating the genetic code of RNA into a
string of amino acids
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The entire mRNA molecule is not
translated
Start codon
AUG
coding region
Stop codon
UGA
UAA
UAG
Somatostatin receptor: AY585720
5’ untranslated
coding region
3’ untranslated
Start codon
aca gac aca cac gtg acg aag atg aac tcc tcc tcg…..
Stop codon
…..aag acc ttc aca tcc tgc tga gag cct cct gct cct
Untranslated mRNA
• 5’ and 3’ untranslated mRNA can affect
stability of the mRNA molecule and also
how it is translated
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Coding vs. Noncoding region
• Across species, usually it is the coding
region of a gene that is more strongly
conserved than the noncoding region
• Most mutations affecting the function of a
protein are harmful, so they tend to be
selected against
Measuring Gene Expression
PCR and Microarrays
Using a sequenced genome for
behavioral research
• Most methods for measuring gene
expression require knowing the sequence
in advance
• It’s much easier to go to a database to
retrieve the sequence that have to
sequence it yourself
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Sequenced Genomes
• Mammals: Human, mouse, rat, cow, pig
• Birds: Chicken, zebra finch
• Fish: Zebrafish, Fugu (pufferfish), tilapia
Polymerase Chain Reaction (PCR)
• Generally credited
to Kary Mullis
– 1993 Nobel Prize
• One of the most
widely used
techniques in
molecular biology
PCR Steps
• Melting
– Heat up sample so that double stranded DNA
unwinds
• Annealing
– Cool down the sample so that gene specific
primers anneal to gene of interest
• Extension
– Heat up sample so that DNA polymerase
synthesizes new strand of DNA
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Materials for PCR
•
•
•
•
Taq polymerase
buffers
dNTP: raw materials
Specific primers: short sequences of DNA
that flank the region of interest
• Template: a source of DNA
– Can be genomic or cDNA
PCR animation
• Run PCR.exe file
Gel Electrophoresis
• Run gel animation
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PCR Applications
• Amplifying specific sequences of DNA
used for
– Detecting genetic differences (mutations,
deletions, etc.)
– Genetic fingerprinting and paternity testing
– DNA sequencing
– Manipulation of DNA (recombination)
Comparative Genomics: 5HTT
• Individual variation in the length of the
serotonin transporter promoter observed in
rhesus monkeys
Serotonin Transporter
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Activity
• The rhesus monkey
short allele has
reduced
transcriptional
activity compared
to the rhesus
monkey long allele
Serotonin function
• Researchers examined a serotonin
metabolite (5HIAA) in cerebral-spinal fluid
(CSF)
• Examined monkeys raised in two different
environments
– Peer-reared (nursery)
– Parent-reared (mother)
Serotonin metabolites
• In monkeys raised
in nursery,
heterozygotes had
reduced 5HIAA
• In monkeys raised
by mothers, there
was no effect of
genotype
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Serotonin and Behavior
• Individuals with the lowest CSF 5-HIAA
levels are more likely to show aggressive
behavior and engage in risk taking
behavior (making extreme jumps, also
drinking excessive amounts of alcohol)
• What’s missing from the story???
Real-time PCR
• A method for measuring the relative amount of
gene expression in tissue samples
• Extremely sensitive, often referred to as
quantitative PCR or qPCR
• All methods depend on increasing fluorescent
signal as target gene is amplified
Why is it called real time?
• After each cycle, the instrument measures
the amount of fluorescence in the reaction
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Cycle Threshold (Ct)
• Ct: the number of
cycles needed to
reach a certain
level of
fluorescence
• The more copies of
an mRNA present,
the lower the Ct
Cycle thresholds
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Cycle threshold
16
14
12
10
8
6
4
2
0
0
00
0,
10
00
,0
10
0
10
0
00
1,
10
1
• Every ten-fold
increase in mRNA
should lead to a 3.33
increase in cycle
threshold
• A standard curve can
be used to determine
how many copies of
mRNA are present in
a sample
Copies
Hormone Receptors in Cichlid Fish
• Cichlid fish
Astatotilapia can
have two
phenotypes
• Dominant males are
more aggressive
than subordinate
males
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Measuring hormone receptor
mRNA
• Dominant males had
more androgen
receptor mRNA in
forebrain
• Suggested that
dominant males may
be more sensitive to
androgens
Burmeister et al. 2007, Horm. Behav: 51 164-170.
Real time PCR +s and -s
• Advantages
– Relatively easy
– Very sensitive
– Gold standard of measuring gene expression
quantitatively
– Often used to confirm microarray results
• Disadvantages
– Usually poor spatial resolution
– Gene expression does not always reflect protein
expression
Microarrays
• Allow for the measurement of tens of
thousands of genes simultaneously
• Generally requires that the species’
genome sequenced
– Sometimes can use a closely related species
– Human, mouse, rat, zebrafish commercially
available
– Zebra finch, honey bee, and cichlid fish also
exist
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Honey Bee Behavior
• Younger bees stay
inside the hive and
take care of brood
(nurses)
• Older bees forage
for nectar and
pollen outside the
hive
Age vs. Behavior Confound
• In a normal colony age is confounded with
behavior
• To address this confound, researchers
created colonies in which all the bees
were the same age
– In this environment, some young bees will
become foragers
Behavioral Genomics
• Abbreviations
– Young nurse (YN)
– Young forager (YF)
– Old nurse (ON)
– Old foragers (OF)
• Yellow = increased gene
expression
• Blue = decreased gene
expression
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Different Behavior, Different Genes
When bees switch from nursing to foraging
These genes turn off
These genes turn on
Microarrays +s and -s
• Advantages
– Can generate a mind blowing amount of data
– Can identify gene networks
– Can help identify unexpected new directions for
research
• Disadvantages
– Frequently yield false positives and false negatives
– Large datasets can be overwhelming, sometimes
hard to make sense of it all
– Expensive
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RNAseq
• Instead of calculating relative expression
(group A has greater expression than
group B), count the number of transcripts
• Can identify splice variants
– Some genes like brain derived neurotrophic
factor have many splice variants
• Can identify single nucleotide
polymorphisms and other mutations
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Analysis
• Sequencing reads need to be assembled
to that the number of transcripts can be
quantified
– Usually done with a reference genome
– Methods are available to assemble transcripts
“de novo” using closely related species
– Want to know more? Take PSC 217 winter
quarter
Dealing with data
• Both microarray and next generation
sequencing methods produce massive
amounts of data
• Bottleneck is almost always at the analysis
level, as sophisticated statistical
approaches are needed to analyze
datasets
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Manipulating Gene
Expression
Knockout mice
• Breeding of knock-out mice is becoming
routine. Many universities have facilities
that will create lines of mice for a fee
• Key point: traditional knockout mice lose
function of the gene of interest through
development and as adults
Transgenic Mice: “Knock-ins”
• Similar procedure as creating knock-out mice.
• Introduce a construct containing the gene of
interest to mouse embryonic stem cells
• The construct should include a strong promoter,
to ensure the gene will be expressed. This
technique can also be used to determine if a
given promoter is, in fact, strong
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Vasopressin
• Vasopressin
• Behavioral effects
– Aggression
– Parental behavior
– Pair bonding/affiliation
• Vasopressin receptors
– V1a
– V1b
Prairie vole (Microtus ochrogaster)
Vasopressin Receptor 1a (V1a) Gene
• Prairie vole (Microtus ochrogaster) and meadow
vole (M. montanus) genes are 99% identical
• Biggest difference lies in the promoter region
(more extensive in M. ocrhogaster)
Does the Prairie Vole Promoter
Affect Behavior?
Regulation of the transcription of the V1a
RNA can influence the amount of V1a
protein
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Prairie Vole V1A Promoter in Mice
• Transgenic mice
expressing the prairie
vole V1a gene show
patterns of receptor
expression like prairie
voles
– White: wt mice
– Striped: transgenics
– Black: voles
Behavioral Effect of Prairie Vole
Transgene
• Increased expression of the receptor alone had
no effect on affiliative behavior
• Injection of vasopressin (AVP) in transgenic
mice increased sniffing and grooming of a
female
Complicating
Issues
• The prairie vole
promoter gene is
present in many
species of voles
that are not
monogamous
Fink et al. 2007, PNAS 103:10956
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Partner Preference vs. Monogamy
• Partner preference behavior has been examined
in only a few species of voles
• It may be that many non-monogamous species
show some form of partner preference that is
affected by vasopressin
• The effects of the vasopressin promoter may
depend on the expression of other genes
Epigenetics
Glossary
• Epigenetic: modifications to genes that do
not involve changing the DNA sequence
itself and can be transmitted across
generations
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Levels of Organization
coarse
• Chromosome
–Histone
• Gene
–DNA
fine
Packaging of DNA
DNA Must be Unpackaged Before
it is Read
• Unwrapping DNA from around histones
allow polymerases to read DNA for either
transcription or replication
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Methylated DNA
Shipping crate
Non-Methylated DNA
Box closed with tape
Methylation
A specific kind of chemical
change that can affect how easily
DNA is unpacked from the
histone
DNA Methylation
• Methylated DNA
has a small methyl
group attached to
some of its bases
• Usually methyl
groups attach to
cytosine (C) only
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Methylated DNA
Non-Methylated DNA
Maternal Care, Methylation, and
“Anxiety”
• Previous studies showed that
pups raised by high licking and
grooming dams showed
– Increased exploratory behavior
– Reduced physiological responses to
stress (namely corticosterone
secretion)
Methylation of Glucocorticoid
Receptor Promoter
• Cross-fostering
experiment showed
that rat pupss raised
by low
licking/grooming
dams had increased
methylation of the
glucocorticoid
receptor promoter
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Effect of Methylation on Stress
Response
• Experimentally
blocking
methylation with a
drug blocks the
effect of low
licking/grooming on
corticosterone
secretion during
stress
Methylation of Estrogen Receptor
• Low parental care rats
have low estrogen
receptor expression
• Estrogen receptor
promotes parental care
• Maternal behavior
reduces methylation of
estrogen receptor
Cross Generational Effects
• Pups raised by low
licking/grooming
dams grow up to
lick their own pups
at a low rate
• Behavioral patterns
are transmitted
across generations
Cross fostered
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Question
• Is this inheritance of
an acquired
characteristic?
• Some people think
so, others are not
convinced
• What do you think?
“The Modern Synthesis”
• Refers to the integration of ideas on
genetics with theories of natural and
sexual selection
– Hardy-Weinberg Theorem
– Genetical Theory of Natural Selection
• Continuous variation has same method of
inheritance (quantitative) as discontinuous
variation (discrete)
“The Modern Synthesis”
• Populations contain genetic variation that
arises randomly (mutation, recombination)
• Evolution as changes in gene frequency
• Most adaptive genetic variants have small
effects on phenotype (evolution is slow)
• Acquired traits are not inherited
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• Until Ronald Fisher’s “Genetical Theory of
Natural Selection”, many evolutionary biologists
held views on inheritance that are generally not
accepted today. One prominent theory was the
“inheritance of acquired characteristics”, in
which traits acquired by parents could be
passed on to successive generations. Although
usually attributed to Jean Baptiste Lamarck, it
was a commonly accepted method of
inheritance in the late 19th and early 20th
centuries. The rise of the “modern synthesis” of
evolution rejects this mode of inheritance, but
recent findings in epigenetics raise some
interesting issues. Do recent findings showing
behavioral modification of DNA methylation
constitute a challenge to the “modern synthesis”
or do these results conform with existing ideas
from the modern synthesis?
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