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.
Transcription
• The production of messenger RNA
(mRNA) from a sequence of DNA (gene)
– Gene expression
– “Turning on the gene”
Important DNA Sequences
• Promoter
– Indicates where transcription should begin
• Coding region
– Sequence that codes for a protein
• Terminator
– Indicates where transcription should stop
promoter
coding region
terminator
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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
Initiation of Transcription
• Transcription factors allow RNA polymerase to
bind to promoter region
RNA polymerase
promoter
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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
Termination of Transcription
• When the RNA polymerase reaches the
terminator sequence, it stops transcribing
RNA
RNA polymerase
terminator
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Transcriptional Regulation
• Promoter sequence
• Expression of
transcription factors
• Splicing
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 Next Generation
Sequencing
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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
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
New Frontiers: Mass Sequencing
• New sequencing techniques allow for the
ability to sequence all of the mRNAs in a
single sample
– Illumina
– 454 Sequencing (Roche)
• Can detect mutations in sequences
• This technology is making microarrays
obsolete
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RNA Seq
•
•
•
•
Extract and isolate mRNAs (poly A RNA)
Reverse transcribe and fragment mRNAs
Add sequencing adaptors
Amplification of each sequence creates
clusters
• Each sequence read base by base
• Align fragments and quantify
RNAseq strategy
• Analysis strategy based on counting
number of transcripts in experimental
gorups
• Can identify splice variants
– In come cases, part of a gene will not be
included in the mRNA
• Can identify single nucleotide
polymorphisms and other mutations
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Analysis
• Sequencing reads need to be assembled
so that the number of transcripts can be
quantified
– Typically done with a reference genome
– Methods are available to assemble transcripts
“de novo” using closely related species
• Unless you can analyze data yourself, now
it is cheaper to do the sequencing than to
do the analysis
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Manipulating Gene
Expression
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)
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Does the Prairie Vole Promoter
Affect Behavior?
Regulation of the transcription of the V1a
RNA can influence the amount of V1a
protein
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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