Lecture 1:

 Genes
o Coding regions encode for genes and gene products (not only proteins but also different
RNAs)
o Non-coding regions don’t code for anything (as name suggests)
o Other functional sequences
 Regulators; that regulate how protein folds, etc.
 Controversies in Behaviour genetics
o “Designer babies”
 Genetically manipulate genes to get specific babies
 Genotype to phenotype
o Variability of DNA sequences can result varying behaviours
 Even single nucleotide changes can be damaging
 How do genes determine physical features?
o Physical features are controlled by genes
 Genotype to phenotype
o What kind of DNA is present?
 There is mitochondrial DNA present as well
o RNA
 Any RNA transcripts in any given conditions are the transcriptome
 Transcripts can change under different conditions
o How much is available
 Based on time and location
o Protein
 Any protein in any given time and conditions
 Proteome
o Brain
 Connectome
 How all the neurons’ connections are being made
o Behaviors
 Set of behaviour that define certain species is a behavioural repertoire
 How do genes influence behaviours?
o Selective breeding
 Part of the pointing posture and herding is coming from set of genes (look at the
chart)
o Not complete picture without role of environment
 What role does the environment play?
o Any type of environment that can cause a change
 Example
 Stress; cells express specific set of gene that change the amount and types of
proteins
o Impact the emotions and behaviours
 The environment makes you stressed and changes the gene
expression patterns
 How does environment influence behavior?
 o Example
 If that aggression is met with aggressive counterattack, they become submissive in
return
 Bottom of social hierarchy
o Animal learns the aggression and submissive behaviour dynamic
 Social interactions fine tune the proteins and genes
(environment influence)
 Changes the protein functions or structure of the
brain
o Example
 Questioned for impaired intellectual ability when the features are identified
 Leaves adults with lowered expectations for intellectual capabilities; making
the environment less optimal for success
o Thus, is it influenced by genes or environment? (main question of
the course)
 Because of their physical appearance have a different environment with lower
opportunities
 Lower intellectual achievements
 Mixed-up Brothers of Bogota (twin studies for influence of environment)
o They had different characteristics even though they were identical twins because they were
raised in different environments
 Better intellectual vs physical stronger
 Different opportunities (different nutrition and etc.)
o Effecting behavioural and physical traits
 Origin of Behavioral genetics
o Darwin
 Passing more beneficial genetic information to offspring because of the better
survival
 Father of behavioural genetics
o Helped with biostatistics
 Nature vs nurture debate
o The debate hasn’t been resolved yet
o Operant conditioning
 Don’t touch a hot stove because know that it will burn you
o Examples
 Pigeons shake their head thinking it would get them food
 Superstitious behaviour
o Some behaviours are learned
 Nature vs nurture
o Dog training
 Establishing a conditional response
o Operant conditionings
 Mice learn to press the lever at certain times (when green) to get food
 Electrocuted when light is red
 Speaker played songs randomly
  Mice learned a superstitious behaviour that the song played will get them
shocked even if the light was green
o Think they will get shocked even if it was not connected to the food
o Behaviour imprinting
 Examples
 Ducklings following the mother even if the mother falls down the river
o Fixed action patterns
 Not learned, but know it instinctively
 Mating dance
o Innate behaviour
 Exist on their own
 Only some behaviours though
 Modern evolutionary synthesis
o Population genetics
 Frequency of genes
 Percent of population that has a specific gene or genes that will increase or
decrease if it is advantageous vs disadvantageous
o May disappear if detrimental or be apart of the species over
evolutionary time (many generations)
o ANOVA
 Changes in various populations
o Epigenetic landscape
 Looking at epigenetic modifications on proteins that bind to DNA and RNA
 Acetylation and methylations or deacetylation
o Example of histones (post translationally modified)
 DNA can stay compact or opens up for gene expression
 Controls when and where it can be expressed
 Model organisms
o Breed animals with specific defects or conditions and genetic sequences
 Example
 Knocking down or deleting the gene to look at the effects of that gene
o Study effects of environment and genes
o Lab conditions don’t mimic real life conditions (maybe close but never 1:1)
 Some variables can’t be controlled
 The basis of genetics: DNA
o Opening reading frame
 The DNA sequence that codes for something (gene product) between start and stop
codon
o Splice site between an intron and exon boundary
 Intron will be removed here
o mRNA process
 First the whole genome is transcribed (both exons and introns)
 Not an mRNA because the introns aren’t spliced off
 Then, the introns are removed by spicing and join the exons together
 After, it becomes mRNA and is translated later on
 o RNA polymerases carry out the transcription
 Making the primary transcript
o Ribosomes will read the mRNA sequence to make protein (translate the transcript)
 Genotype to phenotype
o Variation on DNA sequences sometimes cause variations in physical structures
 Could be very subtle so have no impact
 Variations are passed to offspring; making genetic predisposition or
susceptibility
o Example
 Some people are more prone to depression; predisposed to it
 Could be due to differential mRNA splicing or express gene differently once
depressed
o Causing altered brain patterning, synaptic dysfunction, impaired
connectivity = altered behaviour
 Genes and environment influence
 Encoding genetic information
o Each nucleotide is apart of one codon
 No overlapping
 Triplet of nucleotide
o Once ribosome reads it, each codon code for specific amino acids
 Each amino acid is only one codon
 Reading frame
 Genetic code
o Almost all proteins start with AUG (methionine) = start codon
 Every first amino acid synthesized is methionine for each protein (exceptions exists)
o Is the genetic sequence consistent or can it change over time?
 Genetic sequences change
 Due to mutations; change the nucleotide
 Changes in DNA sequences
o Point mutations are very common
 May or not have effect on protein and resulting behaviour
o Point mutations type
 Silent
 Change one nucleotide but still codes the same amino acid
o Why still the same amino acid?
 Because our genetic code is degenerate (redundancy in
genetic code)
 Several different codons code for the same amino
acids
 Nonsense
 Change in a nucleotide that results in stop codon (very serious)
o The protein will stop right there; will not translate the full length
protein
 Premature stopping (no full length protein)
 Missense
  Conservative (keeping the property of the amino acid)
o Single nucleotides change that results in not a detrimental effect
(most likely)
 Changing one basic amino acid for another basic amino acid
 Depends on where the amino acid is on the protein
(may even be beneficial)
 Non-conservative
o Changing single nucleotide change that chances are, will result in
damaging effects
 Not changing to another basic amnio acid
 In this example; changing from basic to polar amino
acid
o May cause catalytic product (most likely)
o Frameshift mutations
 Changes the reading frame (shift in reading frame = complete change of amino acid
sequence of protein)
 Anything b4 the mutation makes sense and anything after the mutations is
gibberish (doesn’t make sense)
o With 3 insertions, it restores the reading frame
 Makes sense again
 As long as it doesn’t change the protein structure
drastically due to the changing of amino acid
(folding process) or isn’t a stop codon
o With 3 deletions, you miss an amino acid but restore the reading
frame
 Frameshift mutation specifically are more serious if it happens towards the
beginning of the gene
o If it happens towards the end of the gene (misses the stop codon for
instance), it continues the gene translation until another stop codon
or ribosome falls off
o Deletion
 Break in the chromosome itself (genetic sequence)
 Cells will try to repair the damaged region, but the DNA region maybe lost
o Chunk of DNA missing in that chromosome
o Variation in metabolic mutation in cells or radiations can cause mutations
 Luckily, our cells can repair those mutations but sometimes do get thru
 Can also introduce mutagens to the cells or add modified genes (with certain
deletions) via experiment
 Ethics must be considered
 Depression and serotonin transporter (5-HTT)
o Serotonin transporter allows leftover serotonin to be taken back
o Study
 Showed that there was a promoter length polymorphism; different individuals have
different promoter length for the gene (short or long)
 Individuals that were prone to depression had longer promoter length of the
gene
 o Debunked (proven wrong); done with more sample size
 Original research article
o Found that there was a point mutation that changed AA isoleucine to valine at position 425 on
the protein
Lecture 2:

 Neurons
o Sometimes electrical signal is converted to chemical (vice versa)
 Electrochemical communication
o Collectively dendrites are called arborizations
 Dendrites are small projections from the cell body
o Axons conducts nerve inputs but also have vesicles and NT that transport neuronal
information
 Can be up to a meter or micrometer
 Within the brain (shorter connections) vs across the spinal cord
o Axon terminal where neurotransmitter is released and bring change to other neurons
 Neuronal structure
o Image
 Vesicles moving across the axon and localized to the axon terminal
 NT is synthesized in the cell body and send to the axon terminal
 When they are released into the cleft, they bind to target receptors in target
cells
o Image
 Generally, presynaptic are submitting info and post synaptic are receiving info
 In close proximity (not physically attached)
o Across the synaptic cleft
 Once the NT is released, it binds to the receptors of the post-synaptic cell
 In the synaptic cleft, a threshold of NT must be reached to activate the appropriate
receptors
 Sometimes, the wrong type of NT is released in small amount and causes a
small effect onto the post-synaptic cell (it happens even with regulation)
 Resting potential
o Diagram
 Charge gradient across the membrane
 Need channels, pumps and transporters to allow diffusion across the membrane
 Because we need to control the movement because the movement can
dissipate the concentration gradient and charge
o Once it goes in, it can easily come back out (without any regulation;
if all channels are opened)
 Also, many molecules have polarity (charge); making it harder to pass thru
 Several proteins in post synaptic cells have negative charge
 Carry negative charge = cultivating the negative charge of the cell
 Ion channels
o Not a free-flowing movement; controls and requires specific conditions
o Threshold level needs to be reached b4 voltage gated channels is opened
 o Right figure
 Sodium intake causes the voltage inside to increase (more positive)
 Reaching the threshold level and then opens the voltage gated channels
 Neuronal function
o Image
 Regular sodium channel lets in sodium ions which increases the charge gradient
allowing the opening of sodium voltage-sensitive channels to open
 Bringing in more Na ions which increases the intracellular charge (more
positive charge), opening the Ca voltage gated channels
 Ca ions cause the release of vesicles and fusion with NT
 Able to then release the cargo into the synaptic cleft
o Neuromodulators
 Depending on the signal; balances the neurotransmitter effect and resulting synaptic
communication
 One way to control the synaptic communication
 Neurotransmitter
o Bind to specific receptors on post synaptic cells continuously unless…
 (1) It diffuses across the concentration gradient because they are not abundant in
surrounding areas as opposed to the cleft
 (2) Receptor is degraded or broken down
 (3) NT is recycled back into the pre-synaptic cell
o Overactivation and over stimulation because of NT in the synaptic cleft, causes all the
receptors to be saturated (or broken down) and get internalized into the cell
 Cell will be desensitized to the NT
 One solution; break the neurotransmitter down into inactive components
(degradation) using an enzyme
o By themselves can’t bind to receptors
 Neurotransmitter reuptake
o Autoreceptors; regulate and monitor the concentration of NT
 If it gets to the right level, prevents further release of NT
 Inhibits fusion of NT with vesicles
o Receptors have a threshold to be reached to be activated based on concentration of NT
 When activated; send signals to components within the postsynaptic cell (bring about
change)
o Therefore; what NT, at what time and in what concentration is important
o If there are no receptors for the NT, there will be no response
 However, the abundance of that NT can promote receptor for that NT to be made
 Receptors are inserted into the cell surface allowing the NT binding and
activating the ion channels
 Synaptic Communication
o Sometimes, the transmission may not be activated because timing is not appropriate
 Synaptic receptors
o NT receptors also involved in transduction
 Convert the chemical signal into the electrical signal
o The functional change in the post synaptic cell
  Simple vs complicated (opening ion channels vs changing gene expressions)
o Ionotropic receptors and metabotropic receptors
 Ionotropic are short activation generally
 Metabotropic take longer to activate but activate for a longer time
 Ionotropic receptors
o Immediate effect generally
o 2 main domains
 Extracellular domain
 Membrane spanning domain
 Cytoplasmic domains (almost all have a 3rd domain)
 Connected to other signaling proteins
o By connecting into a protein complex
o Figure
 Glutamate example
 As glutamate binds to the receptors, there is a 21 degrees shift in the protein
that opens the ion channel to allow ions to pass thru
o Cytoplasm charge changes depending on the ion passage
 Changes the local current across the membrane
 Basically, changing chemical to electrical signal
o Left figure
 The right ligand is present and binds to ligand binding domain of the receptor
 Ion channel opens, allowing Na ions to pass thru
 As Na ions come in, it increases depolarization (more positive than b4)
o Easier to activate for AP because more positive = closer to threshold
level of AP
o Right figure
 As Cl ions come in, it causes hyperpolarization inside the cell (more negative than
b4)
 Generally, inhibits the signal because you are negative
o Move away from the threshold for AP
 Measuring electrical potential
o The probe (electrode) doesn’t damage the membrane as it closes the ion channels by forming
a seal (no leakage)
 Measuring using voltmeter
 Needs to be sensitive = involved in small changes (mV)
 Metabotropic receptors
o Generally, don’t conduct current
 Don’t conduct ions fast; slow activation
o How it works
 Once the right NT (ligand) binds to the receptor, it causes a conformational change in
the receptors
 Causing a signalling cascade involving G-proteins and second messengers
 The G-proteins activate other enzymes that is in the membrane or that it’s attached in
a complex
  In the example, it activates the adenylyl cyclase which synthesizes cAMP
from ATP (strong second messenger)
o Second messenger are molecules inside the cell
 Take the signal from the first messenger and promote a
useful signal; activate other enzymes/protein
 In the example, the cAMP activates the cAMP-dependent kinase
 The kinase then activates the right ion channel by phosphorylating the channel
 Conformational change that opens the channel and lets in ions
o In some cases, the phosphorylation inactivates the ion channels and
receptors
 Complex
o Effects cell function over a long period of time
 Slower response
 Synaptic integration
o Diagram
 No summation; first signal doesn’t reach threshold because the second signal didn’t
come in time (spatially or temporally)
 No addition in space vs time
 All or nothing principle
 Fire at threshold but don’t fire if not at threshold
o Only 2 options
 (d)
 Inhibitory comes in first = hyperpolarization
 Excitatory signal comes in next = still hyperpolarized so no impulse firing
even if its excitatory
o Summation by space
 Genetic influence on neuronal functions and behaviors
o When looking at neuronal receptors and NT, a lot of effects are ambiguous and unclear
 Not only serotonin effects depression but also norepinephrine
o A lot of neurons combined effect behaviours
 Not complete picture is known
 Neuropharmacology
o Brain and nervous system can be dependent on the drugs
 Dependence formed = hard to come off of it
 Agonists and antagonists (must look at overall physiological response)
o Agonists; methods
 (1) Activates postsynaptic receptors
 Represents the ligand the receptors bind to usually
o Bind to the binding sites of the target receptors only because if it
activates other receptors = more complicated and negative
consequences maybe (not desirable)
 Side effects
 (2) Inhibiting breakdown of NT
 (3) Block NT uptake
 (4) Block autoreceptors
  Acts as antagonist on a cellular level for that specific receptor but overall,
acting as agonist (increasing neurotransmission)
o Antagonist; methods
 (1) Block post-synaptic receptor
 Activate another channel that phosphorylates the receptors to stop it from
activating
 Or adding a molecule that binds to the receptor that can’t be taken up by the
cell
o Reduces overall transmission because there is no change happening
within the cell
 (2) Slow release of NTs
 Slow down the vesicle fusion
 (3) Stimulate autoreceptors
 Again, it can act as an agonist for a specific receptor but overall, it can act as
antagonist (decreasing neurotransmission)
 SSRI
o SSRI
 Inhibits the serotonin reuptake back into the pre-synaptic cell
o Not an instantaneous effect but gradual
 There is gradual structural and functional changes in the NT as well as the receiving
cell
o Drug may not work on u or adjustment of dosage may not work either
 Causing severe side effects
 Trial and error with other drugs and right dosages
o Diagram
 Problem
 Overactive serotonin system
o Due to either overactive receptors or NT
 Giving SSRI drug (as antidepressants) at first causes worsening symptoms in the
beginning of medication
 Normal reaction of neurons reacting to the drug
 Eventually reach the borderline (threshold) where serotonin activation is just right
(reduced the activation of serotonin)
 New baseline reached that works
o Happens overtime (balances the amount of NT and receptors); don’t
know the exact concentration and balance
 If instantaneous and not overtime, it may backfire and cause
very bad effects
 Antipsychotics
o Off label use
 Using a drug that isn’t what it was intended or used for usually
 Viagra supposed to be for heart medication
o Dopamine receptor antagonists; inhibit dopamine from coming in
o Seroquel = severe usage for insomnia (may put to sleep for days)
 Benzodiazepines
 o Not commonly used anymore (unless necessary) because they are highly dependent
(withdrawal symptoms)
 Damage neurons in the long run
 Deactivate neurons entirely at times
o GBA receptor agonist
 Hyperpolarizes the post synaptic cell
 Not able to fire as much (more away from the threshold for firing)
o The drugs work very quick
 However, quitting benzodiazepine = major side effect is insomnia
 Pharmacogenomics
o DNA profiling (of people with the same condition)
 Divide into control and treatment groups
 Based on the measurement or symptoms
o Have good responders, bad responders and no responders
 Able to look at gene variation between the responders
 For personalized medicine
 Goals of HGP
o Stored in databases for accessibility around the world
o Using AI analysis for gene sequencing
o Made available to private sector
 Profit reason
 Private sector can put in the money and develop more data for development of drugs
that work for specific set of genes
 HGP
o Based on only the samples they had available (not representative of the entire world)
 Now updated to accommodate for ethnicities and environment as well as different
populations
o Clone-by-clone sequencing (takes a long time)
 How it works
 (1) Take a piece of DNA and generate large number of the fragments of the
DNA from the genome that you want to see
o By breaking or cutting it down
 (2) Map the fragments onto the chromosomes
o Now, know where the fragments came from but don’t know what the
sequence of the fragment is
 However, know which clone they came from
 Why called clone-by-clone sequencing
 (3) Then, insert those fragmental clones into BACs (BAC = bacterial
artificial chromosome)
o BACs are used because it can accept large pieces of DNA
 Can have large pieces of DNA cloned into a BAC
 (4) Grow the BACs with the target sequence within the bacteria to get
multiple copies (lots of identical copy)
 (5) Then, you purify that BAC DNA
 o Done because initially the DNA sequencing reaction and reading of
the DNA sequence only worked up to 150-200 nucleotides (can’t do
the whole thing at once)
 Only want the DNA (no contaminants)
 (6) After, fragment it into smaller pieces
o It can only sequence 1000 bp at a time (on very favourable
conditions)
 Sequencing millions of BP will take forever
 Thus, must divide it into smaller fragments
o The fragments must be less than 2 kilobases
 Because there will be overlap between the fragments
(complementary parts of strands)
 Then the sequences can be merged and joined
computationally
o Able to get the longer strands as long as you
have overlapping regions (where you know
where pieces fit)
 (7) After, you take those DNA fragments of interest and clone them into a
different BAC (sequencing vector)
o Subclone
 Clone of a clone
o With specialised DNA sequences that make it easier to get the
sequence of the fragment we like to clone
 (8) Then every single clone is sequenced
o Get several of thousands of fragments that have overlapping regions
between them
 (9) Assemble them back (align them up)
o Tricky because the fragments must be put back together like a puzzle
 Difficult in those times
 Look for overlapping fragments (there is similarity
at their ends) and complementary base pairing
o Lots of computation power and analysis
required
 Takes a long time
o With the combined sequences, get the full million base pairs
o Shotgun sequencing (new technique; fairly fast)
 Do not clone
 Take the DNA that u want to sequence (human DNA) and directly break it
down into smaller fragments
o No BAC required and isolating it as well as no purification
 From million BP to 1600 BP
 How it works
 (1) Isolate the DNA and break it into many fragments
 (2) Now have thousands of fragments that are overlapping in certain regions,
which you will clone them into sequencing vectors
o Sequence it
  (3) Once you get the sequence back (with millions of fragments), you will
assemble them back like puzzles
o Now use computerization power
 Major findings
o Same number of genes as mouse (surprising finding)
 Because most of genes don’t encode for gene products or protein
o 1350 bp is average gene
 Not very big
o Repetitive DNA
 LINES, SINES and transposons are evolutionary relics that got inserted into our
genome long time ago
 Non-coding DNA sequence
o Satellite sequences (in tandem; one repeated after another)
o siRNA (small interfering RNA)
 Used to control how an mRNA is translated into a protein
o miRNA (micro RNA)
o snRNA (small nuclear RNA)
o Sequences involved in 3D interactions
 The folding of DNA
 The binding to proteins
 Identifying genes in the human genome
o (A) Compare genomic DNA sequences to mRNA
 If an mRNA being made, it is likely part of the gene
 Have to be careful because
 Only subset of genes is expressed under specific conditions
o Must look at different cell types with controls and treatment groups,
under multiple conditions (time consuming)
o (B) Look for specific nucleotide sequences
 Sequences indicate the presence of an ORF
 ORF = DNA sequence from start to stop codon that encodes for a gene
product (RNA or protein)
 Promoter sequences are generally conserved (don’t deviate too much)
 Polyadenylation signal sequences (at 3’ end of mRNA)
 Splicing sites
 Specific nucleotide sequences that are recognized by splicing sites
o There are donor and acceptor sites; predict where the splicing events
occurred
o (C) Compare genomic sequences from other species
 Lots of conserved sequences = functionally important (serve important functions)
 Highly conserved
 Human genetic variations
o What kind of variations can we see?
 Genetic variations
o Polymorphisms is slightly difference in nucleotide sequences or slightly different order of
nucleotides sequences
  SNPs
 Most common type
 Happens in protein coding and non-coding sequences
 CNPs
 Multiple copies of gene
 Structural variations (in the chromosome)
 Largest segments of the genome can change
o If protein coding regions are affected; potential in changing different
phenotypes
o Image
 Deletion
 If the gene is deleted without any backup or redundancy and its important for
the cell’s function, the cell will die
 Inversion = expecting no changes
 See segment of genome is inverted so gene order has changed
o Still making the same protein so not expecting a change
 But what if the inversion lead to different promoter for
different proteins?
 Protein with the different promoters will not be
made
 Another scenario is that maybe multiple copies of
the gene is made because the different promoter
encode different amount of protein depending on the
gene (which is not required) = effect on the cell
 Insertion
 Depends on what the surrounding sequences, it may/may not have an effect
 SNPs
o Depending on the genotype (SNPs differ), the people respond to drugs differently
 Data can be used for drug testing
 GWAS
o Must be in the same ethnic background; different SNPs in different populations
 Right controls
o Association (not causing agent but correlational)
 Know what we look for
 Don’t have to sequence the entire genome
o Goal
 Don’t have to know what the gene does but know where the gene is
 Starting point that can be further analysed
Lecture 3:

 GWAS
o (4) Replicate to make sure the results are accurate and true
 SNP genotyping
o Tag SNPs
 Hard to analyze every single SNP (too much work)
  Therefore, use tag SNPs; representative SNPs for each haplotype
 Are like points of interest
 Tells you there is a SNP and indicates what’s around that area
o Image
 Different individuals
 These individuals have the 4 SNPs that are inherited together = first
haplotype block & SNPs 5-7 are inherited together = second haplotype block
o Can see that SNP3 and SNP5 are the most common one which
becomes the Tag SNPs
 Indicates there is a change
o DNA chip can cover millions of different SNPs
 Image (GeneChip)
 Glass slide that contains millions of chips within a grid to which you know
the coordinates to and is read by the machine
o Since DNA is very small, can have millions of fragments within the
slide
o How it works?
 Image
 Sample DNA from patients and non-patients
 Then hybridize the samples; make DNA single stranded and add to the chip
o If there is a probe complementary (complementary sequence) to the
strand, it will bind (helps identify which SNP is present)
 Know what is present where at all times
o If not, then it will wash off
 Can see what SNP shows up more frequently in patients than non patients
o Then allows you to draw associations behind the disease
 SNP Genotyping Techniques
o A typical genotyping protocol
 3 major steps (with multiple techniques)
 (1) Target fragment amplification (amplify the target fragment)
o PCR
o Strand displacement amplification (variation of PCR)
 As the polymerase synthesizes new DNA, it displaces one of
the strands and synthesizes the new strand in that spot
 (2) Allelic discrimination reaction (to differentiate between the alleles and
identify which SNP is present)
o Many ways
 Look at structure specific cleavage via restriction enzymes
that digest the product
 Carry out ligation or hybridization
 Used to be common
o But sequencing is much faster
 (3) Allele specific product identification
o Identify the product that the specific allele came from
 Many ways
  Looking at fluorescence intensity using FRET
o Allows us to see how close the molecules
are
 For large scale SNP analysis (large samples)
o Microarray is used (our focus)
 Can look at millions of SNPs at the
same time
 Example: Hybridization
o How it works
 Make the target single stranded and mix it with the sample (also single stranded)
 If the sequence is complementary, they will base pair via hydrogen bonds
o AT has 2 hydrogen bonds while GC has 3 hydrogen bonds
 Increasing temperature will break the bonds
 100% base paired = harder to break down the bonds
 Vs a mismatched base pair
o Image
 Wild-type gene
 Under normal conditions, it has an identified SNP site (A and T)
 A specific probe is fluorescently labeled and complementary to the target
SNP
o It will base pair
 As the temperature increases, the DNA will denature
 However, the double stranded molecule becomes
single stranded at a higher temperature (100% base
paired)
o The probe is designed that there is no
fluorescence when DNA is double stranded
 Mutant gene
 Where there is a SNP, instead of a T, there is a C (in this case)
o Therefore, the probe will not base pair with DNA
 As a result, the molecule is slightly weaker
 Now, as temperature increases it dissociates but it
will dissociate under lower temperatures because its
not 100% complementary
o It fluorescent; telling you that the fragment
has a SNP
 SNP Genotyping
o Image (chip; a mix of techniques mixed together)
 Can have arithmetic probes on the chip
 Either has a millions of probes with known SNPs on it or a representation of
the genome onto the chip
o Again, based on the fluorescence, can determine if there is a SNP or
not
 How it works
  Each chip will have millions of probes on each array to which the DNA with
the SNP will attach
o The sequence and location are known (know exactly what probe is
present on the grid)
 Then, DNA fragment is labelled (fluorescence) which can be detected by the
chip reader
o Different labels = different colour of fluorescence
 Can’t use yellow because red and green overlap to make
yellow
 After, you add the labelled DNA fragments to the chip
o Give it time to form base pairs with the probe (on the chip)
 Then wash off excess and anything they didn’t bind
 Chip reader will detect the fluorescence and indicate where it is
o Based on the fluorescence, can figure out where it is located on the
grid and know whether it’s a SNP or not because of hybridization
 The grid already has all positions mapped out (know exactly
where the SNP is)
 Could combine with temperature technique but its not commonly used
 Affymetrix GeneChip Mapping Assay (want to analyze a particular region of genomic region)
o The particular DNA fragment is surrounded by 2 restriction sites
 Cut by the enzyme Xba (3 cuts) thru the restriction digestion process (RE digestion)
 Get 1 red fragment and 2 green fragments
o Have to know where the restriction enzymes are based on prior
sequencing analysis or restriction enzyme analysis
o Then, adaptors/barcode are attached to the fragments
 Has known DNA sequences that you ligate to the fragment
 Adaptors provide stability to small fragments of DNA
o Without it, it will digest further
o After, carry out PCR; amplification
 Designed primers for the adaptors
 Amplify whatever fragments that have the adaptors attached to them
o Universal primers will bind with all types of fragments with base
pairs
 Get lots of copies of the fragments
o Next, carry out complexity reduction
 Whereby, you purify the fragment with only the Xba (the one you are interested in)
 Get rid of fragments that you don’t need and save the fragments that we need
o That’s why specific primers are used to amplify the specific regions
o Fragment it, and label it is using fluorescence or some other label
o Put it into the microarray chip
 Make sure it is single stranded (both probes and DNA fragments) since PCR makes
double stranded DNA products
 Easiest way to do so is raise the temperature to enable the DNA to break
apart and then add it
 o Fragments will bind to the probe that is complementary (it will bind to wherever the target
SNPs are)
 If it hybridizes, it will stay onto the chip and anything that doesn’t bind will be
washed off with a buffer solution
o Lastly, scan + record the fluorescence and analyze further to see if it’s a SNP or not
 Know where exactly they are and exactly what sequence its binding to
 Just from the grid coordinates
 Stat analysis
o Need to test for association of the genome with the SNP with a particular disease
 Calculate the odds ratio
 Measure of an effective allele on risk of developing a disease
o Image
 Calculating the odds of getting a C
 Case C divided by Case A as well as Control C over Control A
o How many C over A under control and case conditions; ratio given
o Image (Manhattan plots)
 Plot all the scatterplots
 There is a threshold; log value of the p-values
o Anything above the threshold is likely associated or involved with
the disease
 SNPs are given numbers to give the exact location of the
SNP (on chromosome)
 Early Bird or Night Owl
o SNP of A vs SNP of B
 Effect on when you wake up
o Table 2
 OR ratio of 1.19 means there is 19% more chance of being a morning person if you
have a T allele for that gene
 Other way around if its less than 1.0 ratio
o Environment can change the circadian rhythm (forcing it to change)
o Table 4
 All phenotype is less than 1 except for sleepwalking
 Clinical applications
o (1) First, identify variants that lead to susceptibility of that disease
 What SNP is associated?
 Inheritance of genetic traits
o Quantitative traits
 Occur over a bell curve (usually)
 PKU
o Disease or disorder where the enzyme phenylalanine hydroxylase (PAH) that breaks down
phenylalanine is deficient
o Image (b)
 If defective, will instead produce excess phenyl pyruvic acid
 Causes neurotoxic effects
o Can be treated by controlling the environment
  Diet that supplements tyrosine and reduce phenylalanine (no buildup of phenyl
pyruvic acid)
 Dominant and recessive alleles
o All somatic cells have 2 copies of the allele
o Capital = dominant & lowercase = recessive
 Genetic linkage and recombination
o Sometimes, some traits are inherited together (linked together)
 Example; red hair and lighter skin tone is associated together most times
o Can also have genetic recombination (also called crossing over of chromosome)
 When fertilization has taken place and cells are dividing & gametes are forming;
there could be genetic recombination
o Because sometimes genes are too close together, there is no space for recombination
 Therefore, the further apart the genes are, the higher the probability of there being a
recombination in that region
 Image (bottom)
o Cross over event occurring
 Now have a new phenotype that didn’t exist b4
 Contributes to genetic diversity
o Can map the gens on the chromosomes using genetic linkage and recombination
 Very time consuming
 Know where exactly the genes are without knowing the DNA sequence
o Old method prior to being able to perform DNA sequencing in large
amounts
o Linkage analysis
 Example; may know some known genetic markers for the disease
 You can analyze the nearby regions to see what the position of those genes
are on the chromosome and relate them to each other
o See whether or not certain genes are inherited together in that
condition
o Genetic markers
 Microsatellites
 In different individuals, can be repeated different number of times
 DNA fingerprinting
o How its done
 (1) Take a sample of DNA and carry out restriction fragment length polymorphism
(RFLP)
 Image
o Individual A has an Eco R1 (restriction guided site) on either end of
the gene which is also conserved in individual B
 What’s different is how many repeated sequences they have
in between those sites
 Individual A has 40 repeats and individuals B has 70
repeats
o Then you carry out analysis and take the DNA to amplify that
specific region via PCR
 o After, run the sample onto the gel electrophoresis
 The smaller fragment (40 repeats) and larger fragment (70
repeat)
 Thus, can be distinguished between the 2 individuals
o The M on the gel is the molecular weight
model that lets you know the size of the
samples
o Normally, there are 13 specific VNTR clusters (loci) that are analyzed
 Chances of individuals having identical VNTR within those loci is rather low
 Almost non-existent
o Why genetic test results are 99.9% chance of being correct
o If the VNTRs are not similar = not related to their parents
o Looking for different number of repeats that changed the restriction fragment length that we
get
 The more repeats you have the bigger the fragment length
 More complicated
o Need to know the restriction site near the loci you want
 PCR is much easier because you can design specific primers
that amplify specific loci or specific VNTRs
 Based on the product size can be differentiated
because you can figure out the genotype of the
individuals
o Match them to the individuals to figure out
criminals or paternities
 Microsatellite analysis
o Depending on the PCR product length
 Decide how many number of the repeats
o Microsatellites are repeats of short sequences (2 to 5bp)
 That are repeated right after each other
o How do we analyse the repeats thru PCR?
 Have to have the right set of primers; designed to be outside of the region I want to
analyse
 Make 2 sets of primer; forward and reverse (short sequences (18-25
nucleotides)) that bind to the target DNA sequence
o Have to know b4hand where the primers should be (need info about
genome)
 DNA synthesis is from 5’ to 3’
 Therefore, under the right PCR conditions, it will make lots of copies
(amplify) of the region between the primers
o Number of repeats determine the size of the PCR
o Example
 Can then analyze the fragments with different individuals by looking at the fragment
sizes
 Figuring out how many repeats there might be
 Huntington disease
 o Huntingtin protein is naturally produced and under controlled by the appropriate repeats
 Genetic marker = more than 37 CAG repeats
 3 repeats will not affect the reading frame
o Too many polyglutamine = toxic to cells
 Depending on the number of repeats make more glutamine that join to make
polyglutamine
o Onset = age when symptoms arise
 Image (left)
 Under 35 CAG repeats, normal nonmutated protein is produced
 More than 37 repeats, end up with polyglutamine that affects the function of
protein
o Toxic to neuron = degenerate
 Image (right)
 The more repeats, the sooner the disease will manifest
o G8 marker
 Tend to find this marker in individuals that will develop the disease
 Image
 Huntingtin disease is passed on via genes
o Overtime, a lot more individuals will have the disease (subsequent
generations)
 Overtime, the number of repeats can start to change
o HD allele
 The reason why its 67% is because one of the offspring dies
 2/3 instead of 3/4
 Human language and FOXP2
o Why is being a TF significant?
 Regulates expression of other genes
 When mutated, affect many gene expressions
o Once made into a protein, will affect other proteins
o If mutated, develop orofacial and speech dyspraxia (motor disorder; can’t move mouth
properly)
o Are those 2 amino acids enough to stop speech production?
 Highly unlikely but not certain
 Gene conversion
o Non-reciprocal
 The donor will not change but only the acceptor (recipient) chromosome will change
o When crossing over occurs and there is an error, the chromosome tries to match it but it uses
the other chromosome as a template sometimes
 Results in both chromosomes having the same sequences
 Chromosomal translocation
o Balanced translocation
 Fairly large pieces of DNA exchanged equally
 Both chromosomes are affected
o Unbalanced translocation
o Image
  P fragment is the small fragment
 Q fragment is the large fragment
 Used as a coordinating system
o 7p 12-14
 Bands must be dyed to be analyzed (can’t be seen without staining)
 If dots = sub bands (more specific)
 Neurofibromatosis 1 - symptoms
o Skeletal dysplasia
 Bones start to bend
 Neurofibromatosis 1 – signaling pathway
o NF1 is a tumor suppressor
 Under normal conditions, will supress tumor development
 By inhibiting the Ras protein via hydrolysis of the GTPase
o GTPase meaning it hydrolyzes GTP
 When GTP is bound to Ras, it is active
 When hydrolyzed, it becomes GDP and Ras is now
inactive
o If mutated NF1 gene, then the Ras is active
 Promote more cell growth and proliferation pathway when not needed
 Major changes in cells
o Eventually develop cancers
 Genetic imprinting
o Where the origin of the allele matters; from mother or father
o Figure (right)
 Maternal vs paternal inheritance
 Look at the source of the allele
o It has to do with methylation and other epigenetic modifications onto
the genes
o Angelman syndrome
 Prone to seizures
 Ataxia
 Can’t move very well
o Prader-Willi syndrome
 Inheritance of mitochondrial DNA
o mtDNA is passed from the mother
 When fertilization takes place, the sperm’s tail doesn’t make it in (where
mitochondria is abundant) whereas the sperm’s head that makes it in has little
mitochondria
o Image (ancestral)
 mtDNA comes down the female (moms)
 Can trace back to early on the evolution
o LHON
 Affects the genes of complex 1 of the oxidative phosphorylation chain
 Image
 If mother is affected, the offspring is affected
 o mtDNA is inherited from mother
 If a male is affected, the offspring is not affected because it doesn’t inherit
mtDNA from the father
o Image
 With little mutant mitochondria (20% maybe), show mild to no symptoms like the
mother
 The more mutant mitochondria, the more severe symptoms
Lecture 4:

 QTL
o On their own, the genes affecting a trait can be subtle but when combined together, it tends to
have additive effects
o Scenario 1 (2 genes)
 No clear dominance
 If there was dominance, there will be qualitative not quantitative (common)
 See a normal distribution
o Scenario 2 (3 genes)
 The more genes involved = the more closer to the normal distribution
 Infinite number of loci model
o With more genes = more like a bell curve
 Studying QTLs
o Fairly long process
 When looking for traits
 Linkage studies (family history)
o Genetic markers can allow the indication of region of chromosome where it might cause the
disease, possibly
o Generally, markers are inherited together because they are very close to each other
 Association studies
o Only correlations
 Not giving the cause of the trait
 Linkage vs association studies
o Linkage studies are good for rare and dominant traits
 Association studies are more useful for common traits
 QTL mapping
o Genetic markers can just simply be a SNP
o Requirements
 Group of genetically related individuals that differ only genetically for that trait in
question
 Able to differentiate between having the traits vs not having the traits
o Procedure
 Measure the trait of interest
 Carry out analysis studies to figure out the association or linkage between the marker
and trait
 QTL mapping in lab animals
o In lab using animals, can create specific genetic lines
  Have individuals that differ in that particular traits and analyze their genome
 Image
o The lines are created by divergent artificial selection
 Started out together but start to carry out individual
experiment
 Diverge more and more in relation to the particular
expression or trait
o Line R = shows trait strongly
o Line B = doesn’t show it strongly or doesn’t
have it
o How it works?
 Cross the parental strains to get the heterozygous F1 individuals (one copy from
mother and another copy from father)
 Cross F1 generation to get F2 generation
 Now have different fractions of the genome of R and B parental lines
o Different crossing over and recombining allows different fractions
 Within the F2 line, can identify markers
 In this case, looking for 30 cM apart (30% being the cutoff)
o Meaning, there is 30% chance of the markers being separated during
recombination
o There is no point in using genetic markers that are the same because there is no point
 Genetic markers should allow us to differentiate between the strains
 Image
o All the dashes are different markers
 The strains have differences in the marker positions (could
be just SNPs)
o Time consuming because have to carry out stats analysis for each makers and their specific
genotypes
o Graphs
 Marker 1
 Very wide distribution related to the phenotype
o Be careful, there is are variations within the markers and phenotypes
 More likely to be associated with that trait because there are a lot more
difference in the markers
o See major differences in the phenotype
 Whereas with marker 2, see little change in phenotype
between the genotypes
o Grach c
 Determine the threshold
 Anything above the threshold is likely to be associated with the trait
o Having that marker is associated with the phenotype
 Limitations
o Inbred populations
 What if the crossing over at points that they don’t normally? What if the
chromosomes aren’t segregating as they should?
 o Not a large effect
 May see as statistical noise but still is real
 But its too small of a difference within populations
o Resolution is poor
 Gives a lot of genes to look at
 Within the 20 cM, there are hundreds of genes possibly
o Must then analyse, gene by gene
 What about regulator sequences affecting other sequences that’s not in the same area?
 Distribution of quantitative traits
o Figure (right)
 Liability threshold
 Show symptoms when above the threshold
o Don’t show symptoms when below the threshold
 Liability threshold (when clinical diagnosis happens)
o The threshold depends on the previous data
 If the threshold is not reached (below threshold), patients will not be diagnosed with
the condition
 Don’t meet the diagnostic criteria
o Image (right)
 May say we have more diagnosis of autism now than ever b4
 Its because there is better diagnosis of autism
o Therefore, since criteria for the disease can change so does the
liability threshold
 Updated criteria for disorders are done every 2 years
o Symptoms of the multiple QTL-influenced disorder reflect only a subgroup of individuals in
the extreme upper end
 Because if you aren’t showing symptoms, you will not go to the doctor
 Therefore, the graph will show subpopulation (not true population) for severe
end of the disorder
o As you get closer to the liability threshold, eventually see more and
more individuals seeking treatment
o Within simple severity, see normal distribution (see a normal distribution within a normal
distribution)
 Individuals seeking treatment vs not seeking treatment vs can’t seek treatment
 At the very end, it drops off because there just isn’t a lot of patients in that
extreme end
 QTLs contributing to a disorder may overlap
o QTLs contributing to different conditions may overlap and share between the traits
o Image
 After passing the second threshold, you are more likely to have schizophrenia than
personality disorder
 Narrow range but the two are linked (overlap)
o Personality disorder is a less severe form of schizophrenia
 Gene-Environment interaction
o Left image
  Higher probability with high stress in developing schizophrenia symptoms
 Environment is playing a role
o Right image (disproven)
 Homozygous for short allele with the stress events = lots more symptoms manifest
Lecture 5:

 Genes and Environment

o Genes + environment and lifestyle = phenotype
 Evolution of traits
o Adaptive changes
 To best suit their environment to increase chances of survival and reproduction
 More favorable features = adaptive change
o Change over evolutionary time; don’t stay stagnant (over several
years)
o Evolution occurs by changing frequency of favorable alleles within a population (not
individuals)
 Beneficial alleles in specific environmental conditions (becomes more common)
o Darwin
 A lot more individuals are born, not all will survive
 If those that do survive, not all reproduce
 Darwin’s finches
o The finches within those islands, lived in different areas/ habitats, resulting in different traits
 Specifically, the beak sizes and shape were different
 Adapted to their different diets (evolved to match)
o A whole spectrum of beak shape and size
o Adaptive radiation
 Diagram (hypothesis)
 Ancestral finch (from SA mainland) that was isolated for long time and
developed into many different finches
o Beak shape changed due to diet
 Evolutionary tree example
 Each branch point indicates adaptive radiation
o But all come from a single ancestral specie
o Example
 Environment change; prone to drought = reduces the number of insects on the ground
 Therefore, individuals that have better suited traits such as flying and faster
agility will have a higher chance of survival and eventually higher chance of
reproduction
 Drought conditions continue on and eventually, the environmental changes
results in more and more insect in trees (rather than the ground)
o Insects will evolve as well (evolution looks at population rather than
individuals)
 Overtime, it will cause speciation and the new specie will
move to a new habitat (go from completely ground dwelling
to tree dwelling)
  In turn, the finches will evolve in response to this
change and evolve to favour finches that are able to
go onto trees rather than ground
 Adaptation examples
o Camouflage (adaptation to surroundings)
 Stick insect (adapting)
o Changes to social behaviours
 Wolve packs and social hierarchies
 Adapting to social behaviours
o Hibernation
o Mimicry
 Model is Monarch butterfly and the mimic, Viceroy butterfly copies the Monarch
butterfly
 Develops similar characteristics in order to mimic the Monarch butterfly
since they are toxic and unpleasant for predators
o Predators will be confused and thus, lead to them being able to
survive (even if they don’t have the toxic trait)
 Predators will still avoid them as they can’t be distinguished
 Natural selection and sexual selection (more about reproduction)
o SS (2 categories)
 Intrasexual selection
 Example
o Large antlers vs small antlers
 Antlers with large antlers have physical advantage during
altercations
 May induce non-violent intimidation
 Intersexual selection
 Example
o Peacock; more colourful the feathers = the more sexually desirable
they are
o Table
 Features
 Superficial = features that may not be beneficial for survival but necessary
for reproduction in individuals
 Utilitarian = features are all for survival
 Evolution of traits
o Changes at genetic level leads to evolution (essentially)
o Question?
 Eventually, the normal distribution will shift in order to accommodate for the extreme
end of genotypes as they are more adaptable (there will more individuals with those
genotype and alleles = allele frequency increases)
 Depending on environments, may return back to normal distribution as b4 if
the environment is consistent
 Inclusive fitness
o Inclusive fitness
  Genetically related = doesn’t mean from the same family (siblings or parents) but
rather a part of the population of species
 Inclusive fitness increases the likelihood of reproduction of individuals and increases
likelihood of reproduction of related individuals
 One individual may help in ways that increase the chance of reproduction of
another related individual
o Warning call (example)
 Some animals are preyed by a predator, a warning call by an
individual of the same specie will save a whole bunch of
related individuals from the same specie
 The individual issuing the warning call may suffer at
the hands of the predator (not survive) but it
increases the overall survival of the entire population
 Hypothesis (selfish genes)
 There are genes controlling and promoting their own survival
 Video
o California ground squirrel warning call
 Taking a survival risk (predator hears the call) on their own for the betterment of the
other related individuals in the population
 Predator may attack the warner squirrel first
o Battle at Kruger
 Pack of lions catch (kidnaps) the calf from buffalos
 Herd of buffalos are coming to take back the calf from the lions (rushing and
separating them from the calf together)
o Risking their own safety and lives for the offspring
 Successfully does get the calf back
 Saving genetically related individuals while risking
your own lives (inclusive fitness)
 Genetic diversity
o Genetic diversity arises from allelic variation, which can be acted on by natural selection
 Critical for survival of species
 The more genetic diversity a species has, chances are they are going to have
more physical and behavioural diversity in traits
o The individuals within the population that have more of these
features will have a better ability to adapt to environmental changes
 Low genetic diversity is common in very small populations (with inbreeding)
 Major problem in conservation of endangered species (inbreeding of
individuals will occur)
o Problem is a lot of deleterious effects tend to show up because a lot
of the individuals start to become homozygous
 Adaptive radiation
o For speciation, sufficient allelic diversity is required because if everyone has the same allele,
what is there to select for?
 For selection to work, need allele diversity
 Factors influencing genetic diversity
o Inbreeding
  Over many generations, more individuals will become homozygous for different
traits and less heterozygous individuals
 Gets to a point where there is reduction of viability in offsprings
o Concept is called inbreeding depression
o Gene flow
 Can happen in one direction or other
 Introducing new alleles within the population; that increases genetic diversity
o Mutations can change genetic diversity
 Could be spontaneous mutation that occur overtime or induce mutations (radiation
damage)
 Human evolution
o Humans have the unique ability to change their own environmental pressures
 If feeling too hot, you can turn on the AC
 Change the environment regardless of the surroundings
o Why might detrimental alleles be so prevalent?
 A lot of the alleles don’t exhibit lethal effects until after the individual has reproduced
 When individuals have reproduced and passed on their genetic information,
that’s when they develop these detrimental effects
 Medications can help treat complications associated with negative diseases
 Environmental effects on genetics and behaviour
o Prenatal environmental effects; agents that effect phenotype b4 a child is born
 Nutrition of the mother
 Hormones the fetus is exposed to
 Exposure to drugs and chemicals during pregnancy
 Psychological stress on the mother
o Postnatal environmental effects; agents or processes that effect phenotype after birth
 Parenting styles (abusive vs nurturing)
 Birth order; treating children differently (older child vs younger child)
 May affect behaviour of the other child
 Prenatal environmental effects
o Neural Darwinism
 Neural pathways and neurons that are inactive will eventually be removed
 Via apoptosis; genetically controlled cell death
o Figure
 (A)
 Multiple connections to a common site
o Making neural connections and pathways
 (B)
 This cell sends a lot of signals to another cell
o Now, the feedback from the postsynaptic cell will make sure that
apoptosis doesn’t occur
 As simple as sending an anti-apoptotic signal to the
presynaptic cell
 (C)
 Since there is no feedback signal from the postsynaptic cell, these are
inactive neurons and will be removed via apoptosis
 o Not a part of the neural circuitry
 (D)
 Based on what synapses are strengthened and active, it can cause specific
circuit to be kept
o Environmental effects can basically rewire the brain more or less
 Depending on what circuits u strengthen and which circuits
are weaker or not responding
 Exogenous factors (effects that are external from the body)
o FAS
 Key symptoms
 Smooth philtrum
o No groove between upper lip and nose
 Indicates FAS
 Low physical growth
o Height is shorter and weight less than normal
 MRI image (child brain)
 There are major structural changes in brain development when exposed to to
alcohol
o Brain surface is smoother (less surface area due less ridges and
grooves)
o Head is smaller
o Fewer folds in the brain
 Leads to behavioural and psychiatric issues
 Endogenous hormones
o Endogenous hormones can control the development of external genital organs
 Depending on which hormones are present
o In humans, the developing fetus is bipotential
 Undifferentiated gonads can grow into testes or ovaries depending on hormonal
signals
 Estrogen, testosterone and other hormones
o Depending on the mix of hormones
 The undifferentiated gonads can develop into testes or
ovaries
o If the Y chromosome is present
 Testes develop in abdomen and starts to secrete testosterone and other androgens
 Resulting in male genitalia and other male features
o If the Y chromosome is absent
 No testosterone is produced and instead of testes, ovaries start to develop in abdomen
 Resulting in female genitalia from the same tissue
o CAH
 Autosomal recessive condition
 Adrenal glands secrete abnormal high concentrations of hormones (cortisol) during
fetal development
 Start to have testosterone-like effects on sex-specific development
  Effects in XY individuals are not dramatic because they are already developing
testosterone
 In XX, develop intermediate genitalia between male and female (incomplete male
genitalia)
 Because you are getting testosterone-like effects from the other hormones
o But still have fully functional ovaries and other reproductive organs
 Still can reproduce
o AIS
 Testosterone is there but the receptor can’t respond to the hormone (can’t bind to it
due mutations)
 In the case of XY individuals, even though testosterone is there, its won’t
respond to it
o Genetically, they will be XY but not develop any male sex specific
features
 Complete AIS (more severe form)
 Develop features that are the same as XX females although, they are XY
individuals
 Brain development
o Sexual dimorphic = can have male or female features
o MPOA
 Diagram
 Located adjacent to hypothalamus
 Its activity is linked to male typical behaviours (aggression)
 MPOA region is larger in males than in females
 The presence of testosterone will inhibit apoptosis in that region = more cells
will develop (neural Darwinism)
 Damaged areas of MPOA = no masculine behaviour
 Tells you that MPOA region is more involved with masculine behaviour
 Nature vs Nurture
o In qualitative traits; phenotype is mostly based on genotype and little to no effect by
environment
o In quantitative traits, phenotype is determined by both environmental effects and genotype
 Generally polygenic traits; traits that are affected by multiple genes
 Phenotypic variation
o When looking variations, we are looking at populations (not individuals)
 Quantitative genetics
o Can also answer which variation in a population is caused by environmental effects?
o The probability of genetic differences that will be passed onto the next generation is the
measure of heritability
 The higher the heritability = the higher the chance of passing it onto the next
generation
 Calculating variance
o Diagram
 Normal distribution in both ages of boys in terms of height
 The mean is 135 cm and 175 cm
 o Calculated by sum of all data points, divided by number of data
points
o Variance
 Tells us how much variability is present in a group
 Looks at the spread of numbers
o The larger the variance value, the bigger the spread of the curve (the
more variance exists in a population)
 Calculated by (data point minus mean) and then squared,
 After, divided by number of data points minus 1
 Diagram
 The smaller the variance, the more narrow the normal distribution
o 0.25; peak is sharp and not well spread
o 1 and 4; the numbers are more spread out
 Phenotypic variance
o Generally looking at a single trait at a time
 Don’t combine unless there is a link
o Basically, phenotypic variance is sum of…
 Variance in genetics (VG), variances in environment (VE) and what effects the
environment has on the genes (VGE)
 Heritability
o It’s the portion of phenotypic variation in a population that is the result of genetic variation
 Does not take into consideration or indicate any genetic effects on a trait at an
individual level
 Looking at populations
o Heritability is statistical measure for a specific population in a study
 Components of genetic variance
o Additive genetic variance
 Variation or variance in a mean phenotype due to inheritance of a particular allele
 A particular allele that contributes to the variation (essentially)
 Alleles tend to contribute a fixed value to the quantitative trait (what’s an allele’s
relative effect on a phenotype?)
 Example;
o 10 different alleles; each allele contributes a certain percentage
 Overall; added up = additive genetic variance
o Dominance genetic variance
 Example
 Parents have white flowers and offspring has pink flowers
o The heterozygous tend to show some level of dominance in the
phenotype
 It’s the interaction between alleles at each locus
 Red and white interact to give us pink
 Intragenic interaction
 Interactions between different alleles or different variance of a single gene
o How the variation in a specific trait come together to give the
variance?
 o Epistatic genetic variance
 Intergenic interaction
 Interaction between different genes at different loci (controlling how you get
variance)
o Gene to gene interaction
 Example; how different genes interact to give a phenotype?
 A gene that controls the expression of another gene
 Heritability
o Within heritability, there is broad sense and narrow sense heritability
 Broad sense heritability
 All the genetic effects or variation
o Ratio of total genetic variance to total phenotypic variance in a
population
 Effect of all gene factors on the phenotype
 Narrow sense heritability (more focused)
 How the additive variance affects the phenotypic variance
 Estimation of heritability
o Three methods:
 Estimation of variance components
 What contributes to the variance?
o Easily done in lab animals
 Can control genetics and environment
 Difficult to do in natural populations
 Mid-parent offspring regression
 Comparing phenotype of offspring to the parent
o Need average phenotypic value of parents and offspring (or multiple
offspring)
 Degree of genetic relatedness
 How related are they genetically?
 Twin studies used because you can see the effect of various components
o Using lab animals
 If you can control one component and make it a 0, it will allow you to estimate the
other variance
 Either make the offspring all identical or make them grow in identical
environments
 Example; using inbred lines (VG = 0)
 (1) Phenotype is entirely dependent on the environment
o Controlled genetic variation
 (2) Then you replicate the same experiment but with outbred population (has
a lot of genetic variation or diversity in the population)
o But you perform it with the same environment (as well as the same
environmental conditions)
 (3) Since the VE is determined from the inbred line, you can rearrange the
equation to find out the effect of variance in the genetics
 o Subtract the environmental variation from phenotype = variation in
genetics
 (4) From there can now, calculate the broad sense heritability
 Not possible to make genetically identical individuals in all species
 Some species can’t be cloned easily or made into inbred lines
o Mid-parent offspring regression
 Taking averages of the offspring and comparing them to averages of the parents
 Offspring tend to resemble their parents but don’t look identical because
there is genetic variation
 Diagram
 Once you have the data points, you can draw the line of regression
o Line of best fit that best fits the data points
 Covariance / variance = narrow sense heritability
 Figure (height)
 If heritability is 0, regression line is more or less a horizontal line
o Indicates the phenotype of offspring is not associated with phenotype
of the parents
 If heritability is 1, shows that mid-parent phenotype is a strong predictor of
the offspring phenotype
 Twin studies - Falconer model
o Chances are the environment will be similar and shared
 Not always the case
o Regression in monozygotic twins (rmz) and dizygotic twins (rdz)
 Broad sense heritability can be calculated by 2 (rmz - rdz)
 Twin studies – Concordance rate
o Looking at twins not genetically unrelated individuals
o If one twin has a condition, what are the chance of the other twin having the condition
o Example
 If concordance rate for schizophrenia in MZ is 0.48 and if one twin is diagnosed, the
other twin has 48% chance of developing the same disorder
o Table
 Compare the concordance rate in monozygotic twins to that of dizygotic twins
 Blood type – difference of .34 vs eye color – difference of .29
o The more the difference = the greater the heritability
 Therefore, blood type is more heritable
 Handness (.79 and .77)
o Heritability is low

Lecture 6:

 Model organism
o Can keep the genetics the same and study the environmental effects (vice versa)
o 2 main questions to think about
 How relevant are the findings to laboratory animals to natural conditions?
 The animals are grown in labs and never see the real/wild environment
 How relevant are such findings to humans when it comes to treating diseases?
  Not everything is related to humans
o For molecular genetics, Arabidopsis is a common lab animal used
 Characteristics
o Grow and reproduce quickly
 Importance; easier to study (don’t want to spend decades to wait for the next
generations to see effects overtime)
o Easy to maintain
o Their biology has been well established
 No need for deep biological analysis
 If you don’t know, then you will not understand why certain things are
occurring in the animal
o Basic biological processes are identical or closely resembles other organisms
 Glycolysis
 Happens in every single living organism
o Based on the available genome sequence, can do genetic analysis
 Genome databases
o Figure (information you can get)
 What are the known genes?
 What is their function?
 What are the different strains are available?
 Any disease models?
 Can do QTL analysis?
 Impact
o Yeast
 First understanding of cell cycle and how cancer is caused by defects in the cell cycle
o Drosophila
 First discovered apoptosis in this organism
o Rats
 Finding carcinogenic properties in drugs (if it causes cancer)
o Mice (easier to genetically engineer)
 Modifying model organisms
o Can modify to study the effects of genes on various aspects of development, growth and life
cycle
 By mutating a gene and see the effects
o Diagram
 Remove a gear
 Equivalent to knockout of a gene = deletion of gene
o See the importance of that gene in a specific pathway and process
 Insert a worn out gear
 Equivalent to simple mutations that changes the function of proteins or
knockdown
 Contain 2 broken gear
 Mutate two genes
o Can also mutate regulatory sequences, promoter, enhancer and etc.
 See the function it serves in an organism
 Mouse as a model organism
o Can have successful generations every 2 months
 Produce a relatively large pool of offspring to work with
o Commonly used mice are albino mouse (white) or agouti mouse (brown fur)
 Can use different strain or a mix depending on the experiment
 Generating transgenic mice
o (1) Injecting foreign DNA into single cell embryos
 Where DNA is randomly integrated into the genome (less targeted)
 Generally multiple copies of randomly integrated DNA
o (2) Using homologous recombination at specific genetic loci
 More targeted
 Gene knockout = deleting or knocking out the function of that gene
 DNA microinjection into single cell embryos
o Super-ovulating (induce ovulation by feeding them specific hormones) females are used to
mate together with a stud male to produce large number of eggs
 After fertilization, the eggs can be isolated
o One thing to be careful about...
 You must remove the fertilized egg at a stage where the cell has not started to divide
yet (haven’t formed an embryo yet = single cell embryo stage)
 How do we know that they haven’t fused yet and performed cell division?
o Time it after fertilization
 Know the timing of when the eggs are developing into an
embryo and how long it takes to for it to start to divide
 Thus, can isolate prior to the cell division stage
o Fairly quick after fertilization
o After isolation, microinject the experimental foreign DNA into the male pronucleus (sperm
nucleus before it fuses with a female egg)
 The nuclei of female and male are there but the genetic material haven’t been mixed
yet
 Male pronucleus is easier to inject rather than female pronucleus (deeper into
the egg itself)
o Diagram
 A section pipette holds the embryo in place and a very sharp
needle injects the DNA by going into the nucleus
 Needle doesn’t damage the egg or embryo
o How does the integration work?
 When you inject multiple copies of DNA into the nuclei, they get integrated into
random sites within the genome
 Diagram
o a
 Each arrow is a piece of DNA
o b
 After the injection, the DNA copies are integrated as head-
to-tail concatemers (multiple copies of DNA or the same
gene are connected in the same direction)
  That’s just what a cell nucleus does when a foreign
DNA is introduced because cell will see it as
damaged so, it tries to join it together and tries to fix
it
o Concatemers will be integrated into the
genome using homologous recombination
 Can do the modification in somatic cells as well but it won’t get transferred onto the
next generation
o Next step, take 20-30 eggs to implant them into the oviduct of a pseudopregnant female
 Pseudopregnant
 Female recently mated to vasectomized male, so female thinks it is pregnant
and so produce the right hormones, but we are just implanting eggs into its
oviduct
o Not actually pregnant thru mating but thru implantation
 Many eggs are implanted because not all eggs will fertilize and develop
 Only some eggs grow into embryos
 Diagram
 Injected the eggs in the pseudopregnant female
 Given 19 days, produce first batch of pups
o Some of them may have transgene (indicated by the blue coloration)
 After 3 weeks, can see foreign DNA is integrated in random sites
o As embryo grow, bind the DNA into various regions
 Analysis
o Use kits and machines to analyse the
 For the ears, can clip them for DNA sample
 May be required (not necessary)
 The tails (less harmful) for sampling (can regrow)
 Run PCR tests
 PCR and gel electrophoresis (to identify)
o Mice 1 is a control but not the transgene
o Mice 2 and 4 actually carry the transgene (foreign DNA)
 Only these mice can pass it down to the next generation
 Backcross (making stable genetic line)
o Take the non-transgenic mouse and cross them with the transgenic
mouse
 Get G1 pups (see if the next generation has the transgene via
PCR test)
 M4’s offspring doesn’t have the transgene (means
M4 has the transgene integrated into the somatic
cells)
 M2 offspring does have the transgene
o Can carry out mating over multiple
generations
 Integrated into the germline
 Using transgenic mice
 o Reporter gene
 Make a gene fusion with the gene fluorescent along with the gene of interest
o Based on the fluorescence patterns, can know where the gene of interest is being expressed
and replicated
o Intensity of fluorescent = more expression of the gene in the area
 Use of reporter genes
o Can use drugs to see if it changes gene expression
 Diagram
 With the enzyme (LazZ reporter gene), get blue fluorescence
o Gene is being expressed in the nervous system; brain, and spinal
cord
o Can regulate when genes are expressed via drugs and environmental conditions
 But must know the regulatory sequence that respond to that drug
 Example
 WT
o No fluorescence
 Ubiquitin with attached red fluorescent protein and GFP (2 reporter gene)
o With the drug addition, it converts red to green fluorescent
 Via the fluorescent change, can know the expression pattern
 Cell type specific overexpression
o Example
 Use specific promoter to see if gene is expressed in the heart cells
o The construct with the promoter and reporter gene must be artificially engineered
o Figure
 Expression in eyes, spinal cord and brain
 Confirmed PAX6 involvement
 Generating transgenic mice
o Homologous recombination is more targeted
 Add DNA at specific places
 Using homologous recombination
o Image
 Target vector with neomycin resistance (neor) and HSV-IK
 2 ways it can be integrated
o Random integration
 Once the vector is added to the embryo, it randomly
integrated
 Gene is integrated into random spots in the genome
 It will form ring looking constructs
 Can’t control where its integrated
o Gene targeting (control where its integrated)
 Use homologous arms to target any area on the mouse
genome
 Add homologous arms that are similar or identical to
the region of target
 o Homologous arms (genes similar to the area you want to insert) are added into the targeting
vector
 Why are homologous arms are required?
 Homology regions will match with the target chromosome regions, and
homologous recombination occurs
o The vector sequence is inserted
 Again, need to know where the gene of interest is located to
allow generation of homologous arms
o Typically, the targeted gene is inactivated by inserting selective marker (resistance to
antibiotics)
 Grow the cell in the presence of an antibiotic called G418
 If they have the resistance gene = will survive
o Selectable marker
 Will allow us to select for cells that have the foreign construct integrated into their
genome because that will allow them to survive in the presence of the drug
 Anything that does not have the transgene, they will die
 Embryonic stem (ES) cells
o Image
 Take an super ovulating agouti female (brown) mated with stud agouti mouse
 Wait three days
 Get/isolate a blastocyst
o Very early embryo, that has undergone a few cell division
 Culture the blastocyst into feeder cells (give a matrix to grow upon)
 Get an ICM (stem cells)
 Take the ICM and subculture them
 Within them, some will develop to become epithelial cells and some remain
as stem cells
 The embryonic stem cells are expanded
 ES cells
o Image
 ES cells growing on a fibroblast
 Fibroblasts provide a matrix to grow on
 Transfecting ES cells
o Now that you have the ES cells, how do we add the foreign DNA?
 Many ways to transfect
 Electroporation
o Using electrical currents to transfect the cells
 Via a machine that applies electrical current to allow the
foreign DNA to go into the ES cell
 Lipofection
o Making lipid nanoparticles that contain the foreign DNA
 Transgene in the lipid droplet
 Lipofection allows the droplet to fuse that with the
host cell
o DNA is inside the cell, it goes into the
nucleus and is integrated
 o Image
 Make liposomes (lipid vesicles) that have an aqueous cavity
and form DNA lipid complex to add into the cells
 The cells will carry out endocytosis and takes it inside
 It converts into endosome and DNA will eventually be
released
 As the cell divides, it gets integrated into the nucleus
 Micro injection
o Micro inject DNA into the nucleus directly
 Viral transfection
o Take a virus that’s modified to have the transgene in the genome and
infect the cells
 It releases its genome inside the nucleus and its integrated
into host genome
o Image (in this case)
 After transfection; grow the cells in the presence of G418
 Any cells that have the neomycin resistance will grow while any who don’t,
will not survive
o Transfection is not 100% efficient; must get rid of cells that haven’t
undergone transfection
 Positive selection
 To identify the clonal ES cells containing the gene disruption
 Southern blotting (not used as often; 2 days for results)
o Probe that binds to the target transgene and carries out southern
 PCR (More used; several hours for results)
o Using specific primers
 Using homologous recombination
o Problem
 (1) The construct is being inserted randomly (some do accurately but not often)
 (2) If the DNA gets randomly inserted or targeted insertion, either way; the cells will
survive and not be able to tell where it was integrated
 Until further analysis
o Solution
 Negative selection
 Inhibiting the growth when right conditions are met
 Example
o HSV-tk
 If marker stays intact, the cell will not grow
 With negative and positive markers, can get the right cells where DNA was inerted at
the right spots
 Insertion or deletion of a gene
o Gene knock in
 Pronuclear injection is more random
 Knock in is more consistent and accurate
o Gene knock out
  Conventional
 Conditional
 Delete the gene at specific time or specific cell type
o Under the given conditions
o Conditional knockout
 Better define gene function
 Under which physiology and behavioural conditions
 Conditional knockout
o Cre-LoxP recombinase system (biologically engineered)
 Involves 2 componenes
 Cre
o Endonuclease (like scissors)
 Digestion and recombination
o The DNA between the LoxP site is cut and recombined
 Innate preference test
o Kills certain cells in the olfactory system
 Wild type
 With fox smell, is afraid of it and distances itself
o Freezes in a corner
 Experimental type
 Can’t smell
o Roams around carefree
 Fearless mouse
o Doesn’t escape from the cat
 Since it can’t smell the cats smell
 From gene to behaviour
 Neural circuits
o Billions of neurons = a functional unit
 Leading to trillions of synaptic connections
o Vertebrates
 Groups of neurons of the same type
 Performing the same functions
o Studying the cell types and connectivity = determining how the circuit is wired
o Diagram (simple neuronal circuit
 Connection between the layers and across the cortex
 Pyramidal neurons and inhibitory interneurons
 Studying the effects of gene in neurons
o (1) Have to identify the neurons with expression of the gene of interest
o (2) Measure the activity
o (3) Look at the connections they form
o (4) Modulate the activity to understand the effects
 Identifying neurons expressing gene of interest
o Why use preserved tissues?
 Not all mRNA is not present in the tissues
 Under specific conditions, it may exist for minutes or days
  Preserved tissues
 Allows the mRNA to stay there
o Its not alive so it wont move
 Method
o Formaldehyde usage to permanently stop the mRNA in place
o Probe
 Make it such that it can be labelled and detect where the probe is binding to
 Generation of riboprobes
o Diagram
 2 polymerase binding sites
 Polymerase read the sequence and make sense as well as antisense probes
o Use both strands to make the gene
 PCR
 Target gene
 Design primers such that it has binding sites for the polymerase + more
sequence that can be identified by the polymerase
 DNA molecules is multiplied and can carry out transcription
o Able to developed antisense and sense strands
 In situ hybridization
o Fluorescent tags is more useful because you can make multiple probes with different
florescent to detect many mRNA
 More expensive
o Colorimetric
 A molecule on the probe with an antigen
 It will be detected by the antibody
o It will be
o Fluorometric
 Antibody against antigen
 Antibody will recognize wherever there is DIG molecule
 Problem
 Not as specific but fairly good
 Genepaint
 Genes to neuronal phenotype
 Transgenic approaches using promoters/enhancers
o Why do we want to isolate?
 If we want to understand how the gene is being regulated and transcribed
 Enhancers can be fairly away from gene of interest
 Promoters can be close to the gene
 Then test the gene expression
o (3) to get the foreign construct into the genome
 Diagram
 Promoter comes from gene of interest and GFP molecule
o Limitations
 Far from gene
 Separated from noncoding sequences
  Sometimes relevant genetic elements are not included into the construct
 Random integration can occur
 What if it integrates into an area that is not expressed?
o No expression = no studying
 Transgenic approaches using BACs
o BAC = for large pecies of DNA
o Same problem with random integration
 Unless used homologous arms and integrations
o How its done
 Identify the clone that has the target gene
 Clone several large fragments into different BACs
o Have to look for the BAC with the gene of interest or geen region
 Have to screen
 Then, add a transgene
 Then implant them into the embryos
 Founder animals and so forth
o Stable transgenic line
o Table
 Clone a reporter protein into the BAC
 Allow the identification of neurons where the gene is expressed
o Fluorescent signalling
 Add a TAG
 Make a fusion protein with the TAG
 Use GFP tagged to a ribosomal protein
 Part of the 16 subunit
o Fluorescently label the ribosomal protein as well
 Look at translation profile
 Diagram
o Antibody is attached
 Have a gene modifier
 Carry gene knock out and gene knock in
 Knock-in approaches
o Diagram
 Add the transgene right next to the promoter
 Make a fusion protein with gene of interest and GFP protein
 Recombinase0mediated approaches
o Instead, can use CRE to express a gene
 Diagram
 Cell specific rembinase where CRE gene will be expressed
o If you want to express wherever the
 Make a flus mouse with a transcriptional stop signal; next to the transgene
gene
 Phenotypic analysis – general health
o Find a work around the limitation
o Table
  Different criteria = score
 Depending on score and comparison to control
o Know the effect
 Social behaviours
o Barbering
 Grooming
 Problem
o Barbering can be extreme in some mutants
 If genes are involved in social behaviours
o Social approach
 2 compartment; one with mouse, one with object and one with nothing
 Doors which the mouse can go at any time
 Can measure time spend in each chamber
o First familiarize the surrounding then move to play with new object
 Diagram (result)
 Each line is a trace picked up by software based on camera targeting
o Social mouse
 Playing with mouse and spending time with other mouse
 Most time spend with other mouse than with object
but little time spent alone
o Baseline of control estbalsihed
 Perform the same test for mutants
o Ultrasonic vocalizations
 Example
 Male smell the urine and call out for the female mice
o Testing olfactory system
 Mouse sounds like birds
o Ultrasonic (cant hear otherwise)
 Slowed down 16 times
 Measuring their chirps
o Nest building (parenting)
 At first little to no nest build; score of 1
o Pup care
 Feeding or lactating for the pups
 In extreme hunger, can eat the pups
 Test
 Scatter the pups
o The mom with clump them together
o Aggression
 dd
Lecture 8:

 learning and memory

o T-maze
 One with a reward sand one side with no reward
 Barnes maze
o Have an aversion to noisy and bright environments
o Extremely motivated to find escape tunnels
 Underneath a hole; there is an escape box
 Able to find spatial cues to navigate thru the hole
o Within 1 to 2 trails
 Find the hole based on landmarks
o The more training the easier for to them to find
 Aversive learning
o Contextual fear conditioning
 Diagram
 In a forest; see a shadow that resembles a snake and so think of it as a snake
o Be scared
 Snake in a zoo; context is different
o Not scared
 Know it wont attack u
 Depending on context; remember an unpleasant stimulus in a context
 Diagram
o See mouse will freeze in that context; based being on the same room
o Compared to a different room
 No freezing
 No linking
 Passive avoidance test
 Taste aversion test
Lecture 9:

 Emotional behaviors – fear related

o Conditioned fear response
 Example; shock response that will be conditioned depending on context
 Cured fear conditioning
 Ring a bell or light is shined b4 a fear response
o Associated with the shock
o Unconditioned fear response
 Mouse don’t like open and bright spaces; like to hide
 Approach-avoidance behaviour
o See what anxiety or fear response they have
 Open field test
 Box with animal and animal try to stay in the
outskirts (more dark)
 Elevated plus maze
 Uncovered and covered arms
o Avoid exposed arms of the maze
 Marble burying
 Try to bury unfamiliar objects if anxious
 Shock probe burying
  If close to shock probe; get shocked
o Burys it
 Tests
o How many times they poke their head out to lookout?
o Elevated zero maze
 Tend to go where there is less light
 Curious animal tho; still look in light area
o Time spend in dark vs light areas
 Open field test
o Balance between being scared (edges) and exploring new environments (middle and roaming)
 If high anxiety; more avoidance of open area and less exploration
o More anxiety = stick to edges
o Graphs
 Measuring the distance the animal have traveled
 Time spend in each zone
 In open field; the transgenic mouse covers more distance (more active0
o Where does it explore more time?
o Conclusion
 Assuming they spend time all over the zones
 Have less anxiety response
 Elevated plus maze
o Placed in the middle (setup)
 Higher anxiety; spend more time in the covered arms
o Left graph
 A lot more time spend in closed arms vs open arms (control animals)
o Right graph (how many times do they enter the arms?)
 More entries in the closed arms
 Innate natural behaviours
 Actual data
o Vehicle
 Drug can be dissolved in saline solution (be careful)
o Data shows….
 First graph
 NpY is lowering the anxiety in control
o Not happening in knockout model because the receptor isn’t there to
allow the drug in
 Second graph
 NpY is having an anxiolytic action
o Increase in dosage doesn’t always lead to desirable effect (a limit)
 Third graph
 NpY not having any effect on # of total entries
o Only effect in open area and time spend
 Tells you that presence of NpY allows more time spend but doesn’t change
the fact that they still choose to explore
 Light-dark exploration test
 o Graph
 Knockouts are much more anxious
 No time spend in light area boxes
 Actual data
o Homozygous; heterozygous + homozygous knockouts
o GABA is inhibitory = makes them calm down
o Data shows…
 First graph
 With knockout; not much transition
 With one allele knockout; not significant effect
o Statistically not significant
 Maybe still biologically significant
 Second graph
 Knockout spends less time in light areas
 Heterozygous knockout
o Spends more time in light areas
 Not statistically significant
 Repeat experiment with more test subjects to address
 Third graph
 Knockout; goes much faster in the dark
o Prefer to be in the dark
 Marble burying test
o Fpt
 Wild type has more anxiety
 More burying
o Gene KO and anti-anxiety drugs
 Have an effect; lowering the anxiety
 Chlordiazepoxide has a slightly less marbles buried = more anxiety calming
 Forced swim test
o Testing depression related behaviour
 If show more depression like behaviour = they will just float (not escaping)
o Placed in warm water (not unpleasant)
 Depression related
o With SSRIs
 Less time being passive when having the drug
 But different in terms of climbing and swimming between the drugs
o With fluoxetine
 More effective in swimming
o With desipramine
 More effective in climbing
 Meaning; they are affecting different pathways
 Both drugs still have an effect in passive
o Still effect depression
 Tail suspension test
o Depression like behaviour = more immotile behaviours
  Video
 Physical activities (running in treadmill) alleviate some depression like
behaviours
 Actual data
o With SSRIs
 Have less immobility
 Less spend being immobile
 As age goes up; depression related behaviour increases (significant)
 Desipramine works better in younger mice
 Schizophrenia related
o Sensorimotor gating test
 Play a loud sound and measure their startle response
 Pre-pulse = start with a light sound followed by the loud sound
o Sensorimotor gating
o Image
 Startle stimulus trail
 Startle
o How much or far they jump
 Pre-pulse inhibition
 Start with a sound that is higher than the background noise and followed by a
loud sound (with 100ms)
o See amplitude of startle is much lower
 Pre-pulse inhibition
o Pre-pulse inhibition will not work in schizophrenia patients
 Actual data
o Left graph
 Much stronger pre-pulse inhibition
 Drug works
o Right graph
 Pre-pulse inhibition is increased
 Drug works
 With 10 mg and 20 mg
 Higher dosage doesn’t equal a higher effect
o But still different than control
 Improved sensorimotor gating
 Reward seeking behaviour
o Context dependent test
 Image
 Checkered pattern room is where heroin is injected
 Circle pattern room is where saline is injected
o Spend more time in checkered room
 Where the pleasant drug is injected
 If we injected an unpleasant drug
o It will spend more time in the opposite room in which the unpleasant
drug was administered
 Self-administration of drug
o How many times do they self-administer?
 Drug vs saline
o Graph
 Modafinil is derivative to amphetamine (used to make you awake)
 Graph shows…
 Amphetamine is more addictive; keep on pressing the lever
 Faily high dosage of modafinil is required to see high amounts of self-
administration
o At low dosages is likely not very addictive
o Even in high dosages; it starts to plateau
 Actual data
o Cued behaviour test
 Come back next week (analysis)
 Balance beam test
o Shelter = dark box
 Record how much time it takes to cross the beam and the # of paw slips (slipping
when running)
 Can explain their motor functions; if they have impairment in motor
functions
 Rotarod test
o Rod can be controlled; speed and how far
 Record how much they slip off?
o See improvement as trails increase if there is no deficits in motor functions
o Graph
 Transgenic mouse
 Huntingtin disease has a later onset
o Older mice stay less time at the rod
 Effects the motor functions
o Age itself is affecting time on rod
 Footprint analysis
o Graph F
 As they age, transgenic mouse’s stride length goes down
 Cause huntingtin disease shows up more and more with age
o Effects motor function
 Grip strength
o Record the amount of force on their paws
 Graph H
 After 6 weeks, there is a difference
o Grip strength goes down
 Deficit in motor function
 Visual acuity test
o Mouse will perceive the height difference due to the glass placed across it
 If there is a deficit in visual acuity; they will go straight and not stop at the edge
 Auditory acuity test
 o Sensor will record the jump; startle response recorded
 No issues with auditory acuity; they will jump or their amplitude will be higher
 Olfactory acuity
 Pain sensitivity
o Graph
 With increasing dosage of morphine (decreasing their pain sensitivity)
 Can tolerate pain longer
 Not linear response
 5 mg will work as an appropriate dosage (higher dosage = no difference from
5 mg)
Lecture 9:

 Conditional ablation of Beta-actin

o Making sure of beta-actin is knocked out
 DAPI stain (ds-DNA)
 Tells where cell bodies are
 No beta-actin seen
 No change in gamma-actin or alpha actin (smooth muscle actin; present around blood
vessels)
 Characterization
o Graph B
 Clear different
 KO mice are smaller than control mice
o Graph C
 KOs are eating more; more caloric intake
o Graph D
 Neurological dysfunction involved
 Paws are clasped
o Graph E
 No physical changes in gross structure of the brain
 Doesn’t mean no changes at all
o Graph A
 Looking at cerebellum organization
 Abnormalities seen
o Graph B
 Looking at hippocampus
 Abnormalities found
o Table A and graph B
 In KOs mothers are not bunching the pups together (hurdle) nor is it making the nest
properly
 If pups in foster mother; normal
o Rotarod analysis
 Graph A
 No changes in motor functions
  Image B
 KOs are more active; running around all over the place
 Graph C
 KOs travel at greater distances in higher speeds; cover more distances
o KOs are more hyperactive
o Graph F
 No major changes; until final day of learning
 Long term memory deficit
o Graph G, H and I
 Learning and memory deficit
o Image A and Table D
 Again; there is actin ablation
 Neurons organize in the same way morphologically even if actin is not present
Lecture 10:

 IQ score
o The standardized behavioural test is usually self reported
 People will choose to cheat on such test
 Have to account for this variability
o Graph
 Cognitive enhancement
 Don’t know if this cognitive enhancement is part of the IQ score
o Not a lot of people are within that range
 Different things are tested
 Language ability, mathematical ability
 Intelligence
o Intelligence
 Means to learn from the information
o Cognitive abilities
 Can be measured to get a hint on their intelligence
o Hierarchical organization chart
 However IQ tests aren’t accurate
 Someone who is better at taking test will do better than someone who isn’t
 To measure g-factor, have to measure the various abilities that contribute to it
 There is also sub traits
o Figure
 Some are contributing to g-factor more than others
 Perpetual speed and mathematical ability > verbal comprehension
o Some tests are designed to give more weight to certain important ability
 Intelligence – heritability
o 50% of intelligence can be explained by genetics
 Environmental factors influence (twin studies)
o Rats were breeded for intelligence (if they can solve the maze)
 Graph
 There is difference between Maze dull and maze bright rats’ group
 o There is certainly major genetic component (not just one gene)
 Multiple genes are contributing to the intelligence
o Another experiment to test environmental factors
 Impoverished (nothing to play for and no change in toy position = poor environment)
vs enriched environment
 Graph
o Under normal conditions; maze -dull made more errors (expected)
o Under enriched environment; maze dull made less errors
 Meaning environment is responsible
o Under impoverished environment; no difference
 Maze bright rats lost their advantage due to the poor
environmental conditions
 Conclusion; both genetics and environment play a role
 NR1 knockout (subunit for AMDA receptor; involved in learning and memory)
o Knocking out the NR1 receptors in the hippocampus
 Result; decline in learning and memory (expectation)
o Experiment to confirm
 KO mouse had higher latency (worse performance)
 Why comparing it to such controls?
o Bc, to get to the CA1-KO; u added Cre and flox modifications
 Thus, if they had no impact as well (they are essentially the
controls)
 NMDA receptor overexpression
o Transgenic mice with overexpression = increased performance due to enhancement in
learning and memory
 Overexpression of NMDA receptors = better performance
o Contextual fear conditioning test
 Seeing how much of the information they retain after an hour
 Transgenic perform better (retain information better) even after 10 days
 Genes underlying human intelligence
o BDNF
 LTP is how memory is formed while LTD is how memory is forgotten
 Circuit is kept vs removed
o Want to see increase in LTP and decrease in LTD
 BDNF polymorphism (single ammino acid substitution)
 Memory and cognition suffers
o COMT
 Substitution = dopamine increased
 Better working memory
 Personality
o Tend to think and act in the same way in each situations
o Self reported questionnaires = how accurate are they?
 Some want to produce fabricated results
o FFM
 OCEAN acronym
  Resuls in a sample in personality portfolio
o Comparison between other people’s score
 Where u fall in the normal distribution scale
 Genes underlying human personality
o DRD4
 VNTR length polymorphism
 Short vs long allele (clear difference and their effect on personality)
o Long allele have increased novelty seeking behaviours
 Again self-reported scores
 Can explain addiction
o Graph
 Long allele is associated is seeking new things
o Chart
 People with long alleles tend to crave (like cigarettes)
 Reward dependence impacts
o Exposed to lit cigarette = craving goes up
 People with shor alleles don’t have changes to craving when seeing a lit cigarette
o Vervet monkey
 Graph
 Lower latency = more eager to look for new things
o Increase in novelty behaviour
 Different from human results
 We don’t know why tho
o Niimi et al.
 Golden retrievers = more common DRD4 S alleles
 Shiba = more common DRD4 L alleles
 Clear difference in personality
o Dulawa et al.
 KO the DRD4 receptor
 Place a cup in the middle
o See how much time they spent in the center where the object is
 Graph
 (a)
o Wild type
 Interact a lot with the cup in the
middle
o KO
 Spend not as much in the center as
wildtype
 (b)
o Not much change in the # of times they went
to the middle
 Sexual behaviour
o A normal distribution between hypersexual and asexual
o MPOA
  Dopamine agonist = increase in dopamine synaptic activity = increase in sexual drive
 Genetic influences on sexual behaviour
o Garcia et al. data
 Individuals with 7+ repeats = cheating on their partiners
 Graph
o Clear difference in the reported graph based on their genotype
 Ethics question?
 May be available and common knowledge in dating apps/profiles
 Intellectual deficits
o Intellectual deficits can be caused by genetic changes and environmental aberrations
o All the sub traits have many genes involved; very complex to measure intelligence
 Intellectual disability
o For diagnosis
 Anything below 70 IQ = intellectual disability
 Within them, there are further subdivisions
o Table
 Mild retardation
 50-70 IQ
 Moderate retardation
 35-49 IQ
o Sheltered workshops
 Only do what they are told
 Severe
 20-34 IQ
o Generally, can’t work and support themselves
 Profound
 Below 20 IQ
o Genetic and environmental causes
 28% is due to their inheritance patterns but 50% were idiopathic (their genetics and
environment is normal)
o Teratogen
 Viruses that the mother has can affect the offspring
 Syphilis for example
o Table
 There both pre and post natal effects
 Trisomy
o Down syndrome
 Rate of down syndrome is increasing
 Having kids later (increasing the chance)
 3 copies of chromosome 21 instead of 2 copies
 Tend to develop schizophrenia later in their lives
 Chromosomal non-disjunction
 Can occur in meiosis I and II
 Resulting in trisomy or monosomy
o In monosomy; tend to not live
  Images
 Table
 As age of pregnancy increases, the chance of Down syndrome children
increases
o Patau syndrome
 Rare case of being alive; severe mental retardation is evident
 Single gene mutation
o Fragile X syndrome
 Premutation
 Mutation is there but doesn’t have the full effect
o Unable to diagnosis the syndrome; can still function normally
 Changes in how the brain is structured
 More prevalent in X chromosome inactivation
 Image
o Shaded area has the repeats
o By random chance, the chromosome without the repeats are silenced
 The repeats area is still active = fragile X phenotype
o In males
 There is no copy of X chromosome to compensate for it
o Rett syndrome
 Any gene influenced by the MeCP2 gene can be affected
 Why is this the case?
 ?
 Symptoms
 Holding their breaths
 Tend to place hand near the mouth (image)
 Prone to seizures
o PKU
 Microdeletion
o DiGeorge syndrome (deletion in chromosome 22’s short arm)
 Cleft palate (used for diagnosis)
 The palate of the infant doesn’t close properly (normally)
 Due to microdeletion in small region of chromosome 22
o Prader-Willi and Angelman Syndrome
 Learning disabilities/disorders
o Low IQ doesn’t mean learning disabilities
 Not always
o Dyslexia
 Jumping letters
 Letters change positions
 Learning disabilities
o Heritability of dyslexia is relatively high
 Genetics of dyslexia
o ROBO1 (stronger candidate gene)
  Its protein is implicated in guidance of axons that cross between brain hemispheres
and guidance of dendritic connections
 Remember a lot of the them have many genes involved and there is
environmental effects
Lecture 11:

 Dementia
o Mostly age related; prevalence increases with age
o More prevalence in women than man
o AD
 Most common
o Vascular dementia
 The second most prevalent
 AD
o Symptoms
 Forgetting and substituting words (not in the right context)
 Motor deficiencies
 Muscle rigidity
 Image
 Overall structures are changed
o Hippocampus is major change
 As you age, some shrinkage is normal
 However, in people with mild cognitive impairment,
hippocampus is even smaller
 In AD patients, hippocampus is very evry small
 AD
o Amyloid plaques
 APP protein is processed by amyloid beta protein
o Neurofibrillary tangles
 Tau associates with the microtubules
 Image
o Accumulates in the cell; see tangles in the neuron
o Actual tissue image
 See little plaque and no tangles in normal patients
o Neurodegeneration
 Normal brain vs AD patient brain
 Hippocampus shrinks severely
 Ventricles with CSF are enlarged
 Tau
 Low amounts of tau and amyloid plaques in normal patients
 High amounts of tau and amyloid plaques
o But may not show symptoms
 Why?
 Diagnostic criteria; some have symptoms but don’t
reach the liability threshold and so are not diagnosed
  Progression of AD
 Takes many years to manifest
 May live after severe changes for couple of years but will have to soon
monitor
 Symptoms first to manifest
o Memory loss
o Types of AD
 FAD
 Early onset
o See symptoms b4 age 65
 Cause could be mutations to gene encoding for…
o Presenilin 1 and 2
 Up to 70% of individuals
o Amyloid beta precursor protein
 Up 10 to 15% of individuals
 Sporadic AD
 Lat onset
 Linked to genetic variations in APOE
o APoE 4 allele polymorphism (Cys130Arg; at nucleotide position fo
130, there is a substitution from cystine to arginine)
 At least one of the defective allele is present in 40-65% of
AD patients
 Doesn’t fully explain the cause of the disease
 Vascular Dementia
o Blood clot that disrupts blood to the brain
 Neurons are starved of nutrients and oxygen
o Symptom progression
 Severeity of stroke
 Different people are affected differently
 Also depends on how fast you can get treatment
o Down image
 Blood clot
 Whatever brain area that that vessel projects to, will be affected
o Right image
 After blood clot (broken down), brain areas is saved
 However, the symptoms may still persist
 Parkinson disease
o 3rd common type of dementia after AF
o Brain section image
 SN region of the brain (called bc there is a lot of melatonin in that region)
 In PD patients, shrinkage occurs and see Lewy bodies (protein aggregates
present in the cell)
o Gets toxic to the cells
 Affects protein trafficking and transportation
o A image
  See neurodegeneration of SN in PD patients
 Lewy body and frontotemporal dementia
o Lewey body dementia
 Lewy bodies occurring not only in SN region but everywhere
o Frontotemporal dementia
 What can cause the accumulation of tau protein in term of mutation to MAPT gene?
 Mutation that causes Tau protein structure to change
 Mutations that cause Tau protein to be unable to be broken down
o Higher unlikely that there is a disruption in protein degradation
 Bc, if degradation pathway is affected, other proteins will be
affected (but don’t; so we can rule it out)
 Mutations that causes highly expression of Tau proteins via transcriptional
changes
o Half life of mRNA increases
 Identifying genetic variants by risk allele frequency and strength of genetic effect (odds ratio)
o Chart
 Can have alleles that are rare in populations but have a strong affect
 Follows mendelian inheritance
 Can have rare alleles that have small effect
 Hard to identify
 Can have common alleles that range with small and high effects
 Some common alleles have a higher effect on the genes
 Common alleles with high effects are easier to detect in a population
 Followed by common alleles with low effects
 Schizophrenia
o Positive symptoms (doesn’t mean as being good; don’t normally see these symptoms in
normal people)
o Negative symptoms (present in normal people but disrupted in schizophrenia patients)
 High muscle rigidity
 Lack of some emotions
 Lack of social interaction
o Severe symptoms can be really bad
o Prevalence
 Prevalence of 1% in entire population is misleading
 Social stigma exist; people may not want to be diagnosed
 Figure
 Can pose major economical impact
o Working individuals will need to be hospitalized
o Symptoms
 Hallucinations (auditory, visual)
 Delusion
 Thinks they maybe Jesus
 DA hypothesis of schizophrenia
 NFG1
o Some change in NRG1 gene = higher chance of developing the disease
 o John Nash
 Had schizophrenia with the polymorphism of NRG1 gene
 Autism
o To be diagnosed; must be present b4 age of 3
o Key traits
 Deficits in social interactions
 Resist cuddling
o High skin sensitivity
o For interventions to work; must be diagnosed very early
 Autism – Symptoms
o Self injury
o GI disorders
o Immune dysfunction
o Language changes
 ASD
o PDD-NOS
 Diagnosed if outside the criteria of classic autism and Asperger syndrome
 ASD prevalence
o Increase in prevalence
 ASD - environmental factors
o There is increasing prenatal environmental factors that contribute to ASD
 There are certainly some environmental factors that increase prevalence
o Vaccine hypothesis
 As a result, previous childhood diseases are not under controlled
 ASD – heritability
o Is it due to rare mutations or rare combo of alleles?
 Don’t know yet
 Autism – genetic components
o Families with ancestor
 intermarital marriages
o Saw major deletion in chromosome 3q
o Synaptic cell-adhesion pathways play a role in autism
 Why?
 A lot of the genes affected are involved in cell to cell commincation
o How things move in cells
o Neural activity
 Mouse models of autism – behavioral phenotyping
o BTBR T+tf/J strain shows the 3 key traits (that you see in humans)
o Graphs
 When exposed to B6 female urine…
 B6 subjects
o Respond very quickly
 BTBR subjects
o Don’t respond
 When exposed to BTBR female urine
  BTBR subjects only respond and then don’t respond
 B6 subjects
o Same response as b4
o B6 and FVB/Ant strain
 High social ability
 Close together
o Touching nose
 Less self-grooming
o BTBR strain have social deficits
 Repetitive behaviours
 High self-grooming
 Not much deficit in exploration (even more exploration than B6)
o Looking at social interactions
 Interact more with other mouse, object or by itself?
 Graph b
o BTBR
 Spend more time by themselves
 Don’t spend much time with other mouse
 Spend less time with novel objects
o Looking at repetitive self-grooming
 Much higher self-grooming in BTBR strain
 Decreases with age (normal)
o