BIOLOGY 1A03 EXAM REVIEW

THEME 1: THE STRUCTURE OF THE CELL

T1M1 THE COMPOSITION AND STRUCTURE OF MEMBRANES
Cell: A membrane bound structure containing macromolecules

Nucleic acids: DNA/RNA hereditary material that codes for making proteins
Proteins: Structural elements (i.e. flagellum of bacteria) that performs metabolic
activities
Carbohydrates: Structural elements and sources of stored energy
o Cell wall + bacterial capsule

PHOSPHOLIPIDS AND FLUIDITY

The cell membrane separates the inside from the outside to protect the cell, dispose wastes
and take in specific compounds; it is composed primarily of phospholipids stacked as a lipid
bilayer

Phospholipids: Main amphipathic component of the cell membrane with a

hydrophobic core and hydrophilic head
o Glycerol linked to a phosphate + 2 fatty acid tails
Fatty acid tail = hydrophobic core
16-18 carbons in a single chain
Single bonds = saturated tail
Double bonds = unsaturated tail
Glycerol + phosphate = hydrophilic head
o Steroids (cholesterol) affect cell membrane fluidity and make up 50% of
molecules found in the bilayer membrane
4 ringed hydrocarbon structure

In addition to forming lipid bilayers, phospholipids can aggregate and form lipid micelles;
forms spontaneously

Important in absorbing fat soluble vitamins

Only one fatty acid tail forms cone shape and aggregates in the shape of a sphere

Phospholipids are fluid lateral movement is easy but transverse movement cant easily be
done without energy
FACTORS AFFECTING FLUIDITY
1. # of carbons in hydrocarbon chain
a. Longer chains = more tightly packed = less fluid
2. Unsaturated vs. saturated fatty acid tail
a. Unsaturated = double bonds in chain = kinks = less rigid = more fluid
b. Saturated = single bonds in chain = more rigid = less fluid
3. Temperature
a. Higher temperature = more fluid
b. Cold adapted organisms will be more unsaturated and have cholesterol that
allow it to maintain fluidity despite decreasing temperatures
4. Cholesterol
a. No cholesterol = too fluid
b. Cholesterol at high temperatures = packs membrane too close = less fluid
c. Cholesterol at low temperatures = prevents phospholipids from solidifying =
more fluid
TESTING FLUIDITY OF MEMBRANES
To test the fluidity of membranes, a genetic construct is made that causes embedded
proteins to fluoresce. After applying a laser to the membrane, some of the proteins are
photo-bleached to remove the fluorescence.
Over time, unbleached and bleached proteins intertwine laterally. This can be done to test
fluidity of various membranes.

Nuclear membrane = little recovery

o Due to lattice work of proteins it is less fluid
o Transport occurs via pores = more selective

ACTIVE AND PASSIVE TRANSPORT

Fluidity relates to permeability:

More fluid = high permeability

Less fluid = low permeability

Selective permeability: Ability for a cell to control the traffic of substances into/out of the cell
and organelles

What crosses?
o Small ions + molecules via diffusion
o Hydrophobic molecules

Fluid mosaic model: Membrane consists of proteins and carbs embedded in the phospholipid
bilayer

Isotonic environment: [extracellular] = [intracellular]; same osmolarity and therefore no net

movement (optimal form maintaining cell shape)
Hypotonic environment: [extracellular] < [intracellular]; water moves into the cell and
causes swelling
Hypertonic environment: [extracellular] > [intracellular]; water moves out of cell and causes
shrivelling
TYPES OF TRANSPORT
1. Passive/simple diffusion: Small ions and molecules move from areas of high
concentration to low concentration along a gradient
2. Facilitated diffusion: Diffusion with the help of transport proteins
a. Assists ions and hydrophilic molecules crossing the membrane
b. Most common
3. Active transport: Movement against the concentration gradient that involves proteins
fuelled by ATP on the intracellular side
a. Primary active transport:
i. Hydrolysis of ATP directly causes transmembrane protein to undergo a
conformational change to pump substances across a membrane
b. Secondary active transport:
i. Neighbouring transmembrane proteins use electrochemical gradients
established by primary active transport to pump their own solutes
ii. Example:
1. Sodium-potassium pump
a. Establishes concentration differences in Na+ and K+ on
either side of the cell by pumping 3 Na out and 2 K in
b. Extracellular environment always has more Na on the
outside and more K within the maintain the gradient
c. Na pump = primary active transport
T1M2 ORGANELLES AND ENERGY
For most of Earths history, cells were anaerobic and unicellular; the first photosynthetic cells
opened up doors for diversity. Interactions between unicellular organisms made them
codependent which helped them survive harsh environments.
Eukaryotes: Organisms whose cell contains a nucleus and membrane bound organelles

Contains an internal network of membranes

First appeared 2 billion years ago as unicellular organisms that contained organelles
and internal membrane systems with distinct structures and functions (now known as
mitochondria and chloroplasts)

Prokaryotes: Organisms without a nucleus and with little to no organelles

Lived on Earth longer than eukaryotes

Importance of organelles:
1. Compartmentalization of cellular functions
a. Increases efficiency

i. Specific enzymes, substrates and products are kept close and

concentrated
2. Increases membrane surface area
a. Increases potential metabolic capacity
3. Incompatible processes are kept separate
Carbohydrates are the most abundant ATP source formed by the polymerization of
monosaccharides

Monosaccharides: glucose, galactose, fructose

Disaccharides: sucrose, lactose, maltose, etc.
o Enzymes catalyze condensation reactions between the hydroxyl on C1 and C4
to form
Polysaccharides: Many monosaccharides linked together
o Many 5/6 carbon sugars form rings
o Starch: Storage in plants
o Glycogen: Storage in animals

ATP is composed of 3 phosphate groups + ribose + adenine. The three phosphates are
negatively changed which generates high potential energy.
EVOLUTION OF CHLOROPL ASTS AND MITOCHONDRIA
Chloroplasts: Organelles that manufacture sugars via photosynthesis

Structure: Double membrane exterior with inner thylakoid stacks

o Organized into grana stacks
o With thylakoid, pigments + enzymes undergo photosynthesis
Photosynthesis: 6CO2 + 6H2O C6H12O6 + 6O2

Mitochondria: Organelles that produce chemical energy via cellular respiration

Structure: Double membrane exterior

o Outer membrane surrounds the mitochondria
o Inner membrane separates the mitochondrial matrix and the intermembrane
space
Consists of cristae (folds in the inner membrane where most ATP is
synthesized)
Cellular respiration: C6H12O6 + 6O2 6CO2 + 6H2O

Lynn Morgulis: Proposed the idea of endosymbiosis

Endosymbiosis: Theory that eukaryotic cells originated as communities of interacting

prokaryotes that eventually joined together due to codependence
o Prokaryotes entered another cell and, over time, the host + guest developed
mutually beneficial interactions that lead to eukaryotes
Ancestral eukaryotes (produced little ATP) + mitochondria (produced a
lot of ATP but needed protection from a hostile environment) =
eukaryote engulfs mitochondria

Invaginations of the cell membrane led to compartmentalized genetic

information which led to more control over gene regulation and protein
processing
o Theory states that temporary relationships must become permanent and
heritable
Evidence:
o Living examples of acquires endosymbiotic relationship:
Eukaryotes will undergo photosynthesis after engulfing photosynthetic
bacteria
Green algae
Corals and dinoflagellates
Sea slug with photosynthetic algae
o Mitochondria and chloroplasts contain their own circular genome
Similar to bacteria
o

PHOTOSYNTHESIS AND CELLUL AR RESPIRATION

Photosynthesis: 6CO2 + 6H2O C6H12O6 + 6O2

Light energy is converted into chemical energy in the thylakoid membrane (ATP +
NADPH)
o Photo excites an e- obtains from H2O (water split into 2H+ and 1/2O2) which
passes through ETC 1 and 2 to produce NADPH and ATP
ATP produced from ATP synthase due to concentrations gradient in H+
Calvin cycle (stroma)
o Rubisco uses energy from NADPH to make a 6 carbon sugar from a 5 carbon
sugar using CO2
o Calvin cycle release two 3 carbon sugars

Cellular respiration: C6H12O6 + 6O2 6CO2 + 6H2O

Glycolysis (cytosol):
o Produces 2 pyruvates (3 carbon sugar), 2 ATP and 2NADH
Pyruvate is processed into acetyl coA in the mitochondrial matrix
o Releases 2 CO2
o Produces 2 NADH
Krebs cycle (mitochondria matrix)
o 6 NADH produced
o 2 FADH2 produced
o 2 ATP produced
o 4 CO2 produced
Electron transport chain (cristae)
o Proteins along the inner membrane allow e- to be transferred which generates
energy to pump H+ into the mitochondrial matrix
o High proton gradient = electrochemical gradient
ATP synthase uses the gradient to produce ATP
o 32 ATP molecules produced

T1M3 PROTEINS
Functions of proteins:

1. Transport and signalling

a. Cell membrane: Interaction with carbs
b. Cytoplasm: Allows for amplification of signals
2. Enzymes
a. Speed up reactions
3. Movement and structure
a. Movement: actin + myosin
4. Defense
Structural differences relate to function:

Soluble cellular proteins are globular in shape with a hydrophobic interior and
hydrophilic exterior that allows for solubility
Aquaporin
o Hydrophilic cores allow H2O to pass
o Hydrophobic exterior allows it to be embedded in the lipid bilayer
o Made up of 4 protein subunits (monomers) to form a tetrameric aquaporin
channel
Each subunit contains membrane spanning alpha helices that form a
central pore
Water molecules form H-bonds with the hydrophilic amino acid R
groups that line the core and displaces nearby H2O molecules ahead

Protein structures can be represented by either the space-filling diagram or the ribbon
diagram

Space filling diagram: Shows the relative size and location of atoms
Ribbon: Lines represent the backbone of the polymer

PROTEIN STRUCTURE
DNA RNA transcription translation into a functional protein
-

In prokaryotes, transcription is closely linked with translation

The nucleus is a double membrane bound domain that contains chromosomes which pack
and control DNA molecules. It has nuclear pore complexes that allow ribosomal subunits and
RNA out and allows building blocks of DNA/RNA + enzymes in.
Protein destination depends on what type of ribosome translated it

Free ribosome = proteins stay in the cytosol

o Example: enzymes in mitochondria, proteins that target organelles, histones
Bound ribosomes = proteins secreted or join cell membrane

Levels of protein structure:

1. Primary structure: Sequence information (amino acids in a polypeptide chain)
2. Second structure: Alpha helix + beta sheets
a. Alpha helix: Spiral formed by noncovalent bonds between carbonyl of carboxyl
groups and amide of amine groups
i. Variable side chains stick out from the helix
b. Beta sheet: Broad lines with arrow heads formed by H bonds between
carboxyl and amine groups of parallel strands
i. R groups extend above and below the beta sheets
3. Tertiary structure: Structures that occur due to interactions between the R groups
4. Quaternary structure: Association of different polypeptide subunits to form the fully
functional protein
Chaperones: Molecules that bind to hydrophobic regions of polypeptides to prevent incorrect
folding
Chaperonins: Large molecular complexes that form isolation chambers that proteins can fold
in without interference

AMINO ACID STRUCTURE

All proteins are derived from 20 amino acids
Structure:

Central carbon bonded to amine group + carboxyl group + H atom + one variable
side chain
o Bonds formed between two proteins (peptide bonds) occur between the
carboxyl and amine groups
R groups create distinct set of properties on different amino acids
o Interactions between side chains allow for the formation of stable proteins

THE ENDOMEMBRANE SYSTEM

Invaginations of the cell membrane formed the endomembrane system which allows for
compartmentalization
Glycosylation: The addition of a carbohydrate group to proteins as post-translational
modifications
Bound ribosomes synthesize proteins in the endoplasmic reticulum (site of protein
modification and glycosylation)

Signal recognition particle (SRP) binds to signal sequences in the amino-terminal end
of the growing polypeptide
o Halts translation
SRP bind to a SRP receptor (SRPR) on the ER membrane
o SRPR brings the ribosome to a transmembrane channel where SRP dissociates
o Protein synthesis resumes as the polypeptide chain is threaded through the
channel
Protein ends up in the lumen and is folded there
o Some proteins stay in the lumen
o Most protein travel via vesicles to other destinations
After the ER, proteins can go to the Golgi apparatus
o More glycosylation occurs
o At the Golgi, they may pinch off into more vesicles to travel further to the cell
membrane or to other organelles
o Proteins that leave have a tag that allows it to be packaged and transported
to the appropriate destinations

Cytoskeleton: A dense network of fibres that help maintain and change cell shape

Microtubules: Protein polymers that form long fibres

Functions as cellular roadways to transport vesicles

Kinesin + dynein: Motor proteins that walk and transports molecules

Requires energy from ATP

T1M4 NUCLEIC ACIDS

DNA in prokaryotes are found in the nucleoid region.

Plasmids: Small circular DNA molecules with one or two genes that replicate
independently from the core genome; they can be transferred from one bacteria to
another

Chromosomes: The organization of double stranded DNA molecules and its association with
proteins and RNA
Eukaryotes = long and linear
Prokaryotes = small, circular, supercoiled and helical
Fred Neufeld: Observed 2 strains of streptococcus pneumoniae (one that caused mice
death and one that didnt)

R strain = benign
S strain = virulent

Griffrith: Determined that genetic material from a cells environment can be incorporated
into the cell which changes cell behavior

Experimental set-up:
o Injected streptococcus pneumoniae R strain, S strain, heat-killed S strain, and
heat killed S strain + live R strain into mice in four separate trials
Result:
o The mice died in the second and fourth trials indicating that benign bacterial
cells somehow became virulent
o Transformation: The genetic alteration of a cell resulting from the direct
uptake and incorporation of exogenous genetic material from its surroundings

Oswald Avery, MacLeod and McCarty: Determined that nucleic acids carried hereditary
information

Experimental set-up:
o Using DNAase, RNAase, and protease, they selectively eliminated each type of
macromolecule (DNA, RNA, and proteins) by testing the molecules ability to
transform
Result:
o In the absence of protein + RNA, cells can still undergo transformation
o In the absence of DNA, cells are unable to undergo transformation

STRUCTURE OF DNA
Watson and Crick: Revealed the double helical structure of DNA based on x-ray
crystallography images of the molecule

Chargaff: Based on DNA properties and biochemical structures, hypothesized that G pairs
with C and A pairs with T

Chargaffs rules: DNA should have a 1:1 ratio of pyrimidines to purines

Rosalind Franklin: Used x-ray diffractions (crystallography) to aim x-rays at DNA to create
images that identified the helical nature of DNA

Calculations determined the dimensions of the molecule

X-shape orientation with black surrounding showed that the phosphate backbone was
spiraling

Nucleotides are basic nucleic subunits

3 components: phosphate group attached to 5 end of 4-carbon deoxyribose sugar +

nitrogenous base attached to 1 end
The 4 nucleotides are chemically distinguished by nitrogenous base
o Pyrimidines: Single ring
Cytosine, thymine
o Purines: Double ring
Guanine, adenine
o A binds to T (2 H bonds formed)
o C binds to G (3 H bonds formed)
Condensation reactions form phosphodiester bonds between 2 nucleotides
o The phosphate group on the 5 carbon binds with the hydroxyl group of a 3
carbon

STRUCTURE OF RNA
DNA vs. RNA
1. DNA has a deoxyribose which has an H atom at the 2 carbon
a. RNA has a ribose which has an OH atom at the 2 carbon
2. DNA thymine
a. RNA uracil
3. DNA = double stranded
a. RNA = single stranded
3 types of RNA: mRNA, tRNA and rRNA

mRNA: A genetic copy of genes the encodes for proteins

tRNA: Functional transport RNA that is never translated
rRNA: Functional ribosomal RNA that constitutes for a large proportional of total RNA
in the cell

DNA in eukaryotes: Found in the nucleus as linear chromosome molecules organized around
histone proteins to form chromatin
APPLIED LECTURES
FECAL ENEMA

Microbiome: The population of microorganisms or microbes in a particular environment

Microorganisms: Organisms not visible to the naked eye

The intestines are the largest microbiome reservoir in the body

o Bacteria allows for the fermentation of carbs + synthesis of vitamin B and K

There are 10x more bacterial cells on the human body compared to number of eukaryotic
cells; they make up 2-3% of our entire body weight
If bacteria localized to one area goes to another region, disorders/illnesses can occur
Dr. Ben Eiseman: Used fecal enema to treat disorders of the microbiome in the intestines

Had 4 patients with pseudomembranous colitis (resistant to antibiotics) caused by C.

difficile
o C. difficile usually not found in the colon because other bacteria keep it in
check
Pseudomembranous colitis typically occurs with immunocompromised people or
people whove had their good intestinal bacteria stripped
o Prolonged antibiotic use
o Weak immune system
o Exposure to C. difficile
Proposal: Using fecal enema to restore good bacteria
Results: C. difficile eliminated, intestinal flora restored

BACTERIA TO KNOW
1. Streptococcus salivarius
a. Normally found in the upper respiratory tract + oral cavity
b. Prevents plaque build up
2. Staphylococcus haemolyticus
a. Normally found on skin (reduces inflammation)
b. Pathogenic if it goes beyond the dermal layer
ENERGY RESERVES IN ANIMALS
CAMELS
Camel humps consists of accumulated fats that allow camels to survive 2 weeks with limited
food

Contains complex fat called tristearin (lots of potential energy)

o Also a source of H2O when metabolized (by-product)
110 H2O molecules produced

Camels last long without water because they have high water loss tolerance (30-40%) and
because they minimize water lost by condensing H2O in their nostrils, having dehydrated
feces and by producing water via lipid lysis for energy.
MIGRATORY BIRDS

Migratory birds can tap into fat resources by inducing lipolysis. During the spring and
summer, birds will bulk up because they can lose up to half their body weight while
migrating.
Dr. Grant McClellan: Studied the metabolic and respiratory processes induced by
challenging environments

Fat to the fire: the regulation of lipid oxidation with exercise and environmental stress
o Realized that there is an interaction between carbohydrates and lipid use
o High altitude + prolonged flight can induce lipolysis
Fat cell = adipocytes
Storage vesicles release fats as fatty acids and transport them though the circulatory
system to areas that require fatty acids as an energy source
Beta-oxidation: Lysing lipids to generate Acetyl coA
o Per molecule of fatty acid generates 3x more ATP than glucose

Dr. Graham Scott: Studied the physical adaptation based on the environment that allow
migratory birds to fly at higher attitudes despite hypoxic environments

Elevated performance: the unique physiology of birds that fly at high altitudes
o High altitude environments require more mechanical power to sustain flight
due to thinner air
O2 transport in birds support greater capacity for vigorous activities in
hypoxic environments
Unique features of high flying birds:
o Increased ventilation rate
o Larger lungs
o Greater amount of capillaries and alveoli
o Better binding in hemoglobin
o Mitochondria close to capillaries

LOCUSTS
Locusts are able to mobilize lipid stores during prolonged flights; carbs used first and lipids
are used after
Glucose vs. flight duration:

Blood
o
Blood
o

levels increase in glucose concentration for the first 15 minutes

Glycogen stores being utilized
levels decrease in glucose concentration after 15 minutes
Lipid levels in the blood increase after 15 minutes as fat stores are being used

KETOGENESIS AND KETONE ENERGY DRINKS

Dr. Kieran Clarke: Looked at using alternative fuel sources in humans and tried to
understand how to enforce fat metabolism in a natural way

Process of metabolizing fats:

o Hormones go to adipose cells
o Fatty acids metabolized
o Ketone body concentration increases

Ketone bodies contain 28% more recoverable energy that glucose; during stress hormones
increase fat metabolism to increase concentration of ketone bodies
Ketogenesis: The generation of ketones (occurs in the liver)

Ketones can be converted to acetyl coA

Ketone energy drinks:

Athletes went 2% further in exercise

Helps people rely less on carbs
Increases endurance and cognitive functions
Expensive and tastes bad

CYSTIC FIBROSIS
Cystic fibrosis is the most fatal genetic disease that mainly affects digestion and the lungs. It
is an autosomal recessive disease.

Symptoms
o Persistent cough + wheezing
o Thick mucous production
o Chest infections
o Salty tasting sweat
o Food cant be absorbed properly because pancreas cant secrete the proper
enzymes

Normally, airways are hydrated with relatively thin mucous layer. With cystic fibrosis, the
thick mucous builds us causing inflammation caused by bacterial infections.

Endoscopy reveals mucous build up, irritation and inflammation

Beneath the mucous layer is the airway surface liquid (water composite). Ciliated cell walls
help sweep away the mucous and pathogens in the airway surface liquid (the mucous traps
pathogens and dust). This is the manner by which the cells work together to maintain
healthy airways.

Airway surface liquid should have adequate volume to allow rhythmic ciliary beating
+ allows for mucocilary transport

Francis Collins and Lap Chee: Discovered the CFTR gene

CFTR is expressed on chromosome 7

Cystic fibrosis transmembrane conductance regulator gene
Codes for a protein that help transport chloride ions across the membrane
o Embedded in the apical part of epithelial cells
o Pumps Cl- out of the cell (transmembrane carrier)

Airway surface liquid height is mediated by a balance between Na + and Cl- transport in the
apical membrane of epithelial cells

In CFTR mutation, there is a hyper absorption of Na+

o Causes water to flow into the cell = reduced height of airway surface liquid
o Why?
Chloride channels arent pumping Cl- out of the cell and not
communicating with sodium ion channels
o Prevent cilia from beating in the mucous

CFTR is caused by the deletion of the amino acid phenylalanine at amino acid residue 508

Leads to improperly formed membrane

Mutation can affect:
o Number of CFTR protein channels
o Function of the CFTR protein channels

CFTR can be located via fluorescent tagging in which wild type CFTR are found embedded in
the membrane while mutant type CFTR are stuck in the ER and are unable to be trafficked
out properly.

THEME 2: FROM GENE TO PROTEIN

T2M1 TRANSCRIPTION
Genes: Sections of DNA molecules that contain info that is transcribed into an RNA copy;
genes code for specific proteins
Francis Crick: Defined the central dogma which states the process of copying and
interpreting genes into proteins

DNA information sequence of RNA sequence of amino acids

Transcription: DNA is used as a template to generate complimentary and antiparallel RNA

molecules
TRANSCRIPTION IN PROKARYOTES
1. RNA polymerase attaches to DNA promoter region
a. Promoter region: Region of DNA that initiates transcription
i. Located upstream
ii. In prokaryotes, a consensus sequence of TATAAT is located 10 pairs
upstream of the start site and serves as part of the promoter region
1. Additional consensus sequences include the TTGCCA sequence
which enhances the rate of transcription
2. RNA polymerase engages in open conformation and functions as a molecular
machine
a. Structural features unwind DNA to make a transcription bubble
b. Ribonucleotides enter and assemble in a complementary fashion
3. RNA polymerase continues to transcribe
a. Transcript threaded and the template strand and non-template strands moved
through different channels
b. DNA double helix restored after transcript is produced
4. Transcription stops at the termination sequence at the 3 end of the gene
a. Enables the release of the transcription complex
b. Rho-dependent termination sequence: Uses specific prokaryotic protein called
Rho factor which binds to DNA and uses ATP to move along RNA transcript
while unwinding it from DNA template
c. Rho-independent termination sequence: Consists of inverted nucleotide
sequences followed by 6 adenines which, when transcribed, fold back on
themselves to form a G-C rich hairpin loop which causes the polymerase to
pause
i. H bonds between the following A-U is week which causes the transcript
to separate
All prokaryotes use a single type of RNA polymerase
Transcription of prokaryotes require the association of RNA polymerase with sigma factors

Sigma factors: Proteins that facilitate binding to promoter regions

o Helps with identifying promoters and turning genes on/off
Initiation of transcription occurs when RNA polymerase binds to a sigma subunit to
create a holoenzyme capable of binding to and unwinding DNA for transcription

Transcription in prokaryotes is more precise than eukaryotes because in eukaryotic

transcription, the RNA polymerase proceeds further than it needs to and is cleaves off.
Consensus sequences: The most common nucleotides found at a specific DNA or RNA
location
TRANSCRIPTION IN EUAKRYOTES
Transcription requires general transcription factors to mediate the binding of RNA
polymerase to initiate transcription and allow for the binding to the promoter region

TFIID = largest general transcription factors in eukaryotes

Core promoter consensus sequence required to set up transcription initiation complex
o TATA box located 25 base pairs upstream
o 2 transcription factor subunits are required for the recognition of the TATA box

3 types of RNA polymerase:

1. RNA polymerase I = transcribes rRNA
2. RNA polymerase II = transcribes mRNA
3. RNA polymerase III = transcribes tRNA
POST-TRANSCRIPTIONAL MODIFICATIONS
Purpose:

To ensure mRNA stability

Allows for export of mRNA from the nucleus
Protects against ribonuclease enzymes that target phosphodiester bonds
Helps with ribosome attachment and translation initiation

5 cap: The attachment of modified methyl guanosine to mRNA through 5 to 5 triphosphate

linkages

The terminal 5 phosphate removed from mRNA by phosphatase

Guanosyl transferase attaches the modified methyl guanosine

3 poly A tail: A number of adenine nucleotides added to the 3 end of mRNA

150-200 adenines added

Polyadenylation: The process by which a polyadenylation signal sequence (AATAAA)
is transcribed
o mRNA is cleaved and a polymerase enzyme adds the adenine bases

In eukaryotes, RNA is processed by removing intron and splicing exons. RNA splicing occurs
at specific short nucleotide sequences at the end of introns

Catalyzed by spliceosomes (large molecular machines comprised of 5 small nuclear

ribonucleoproteins snRNPs)
o Spliceosomes start by base pairing with nucleotides at splice sites and
catalyzing a reaction that causes the branch site to attach the UG donor site
to form a phosphodiester bond (5 end)
Forms a loop
o Donor site then attacks the phosphodiester bonds at the AG acceptor site (3
end)
Forms new phosphodiester bonds between two exons
o Introns are released and exons bound together

TERMINATION OF TRANSCRIPTION
Transcription termination depends on the type of RNA polymerase used:
1. RNA polymerase I
a. Uses specific eukaryotic termination factors similar to prokaryotic rhodependent termination sequences
2. RNA polymerase II
a. Termination is coupled with mRNA maturation where 3 end is modified by
polyadenylation
3. RNA polymerase III
a. Termination sequence is transcribed and transcription is termination similarly
to prokaryotic rho-independent termination sequences
MRNA EXPORT
Nuclear pore complexes in the nuclear membrane act as gateways for molecules to move in
and out of; they are protein lined channel by which RNA processing occurs prior to exiting.
T2M2 THE GENETIC CODE

George Gamow: Suggested that 3 nucleotide bases coded for amino acids

Formed the RNA tie club

Determined that 1 or 2 base code could not code for all amino acids
o 1 = 4 amino acids
o 2 = 16 amino acids
o 3 = 64 amino acids (more than needed but it was a possibility)
Indicated a redundancy in code multiple unique triplets could code
for the same amino acids
o 4 = 256 amino acids
Less likely because it only increased redundancy

Marshall Nirenberg + Mattaei: Used a cell free system to decipher the 1st letter in code

Placed all the components needed for protein synthesis and determined what specific
amino acids were made from simple RNA sequences
o Only uracil = phenylaline residues only
o Uracil + cytosine = alternating serine and leucine
Confirmed that 3 nucleotides make up a codon
Showed that novel sequences of mRNA created distinct amino acids

RNA world hypothesis: The hypothesis that some type of ancestral RNA molecule was a
precursor for life. It is based on the vital role that RNA molecules play in converting genetic
information into proteins.
THE STANDARD CODE
Codons are written 5 to 3and can be simply determined by replacing U with T on the nontemplate strand (which is often called the coding strand for this reason)

Anticodons can be read from the template/non-coding strand

Open reading frame: The entire continuous sequence of a gene that begins with a start
codon and ends with a stop codon; it is the coding region of a gene and reflects the way a
nucleotide sequence is read

AUG = only start codon (methionine)

o Signals where protein synthesis should begin
There is a lot of redundancy in the genetic code (61/64 code for amino acids)
o 3 possible stop codons signal the end of mRNA translation

Reading frame: A way of dividing the sequence of nucleotides in a set of consecutive, nonoverlapping triplets

3 possible ways to read a nucleotide sequence (DNA has 6 reading strands in total)

Crick, Barnett, Brenner and Watts-Tobin: Conclusively determined that a codon had 3
nucleotides by adding single nucleotides to a sequence and observing the protein
synthesized

Adding 1 nucleotide = misread

Adding 2 nucleotides = misread

Adding 3 nucleotides = no change in reading frame
o Corresponded to the addition of a single amino acid
o Maintained the identity of most of the protein

T2M3 TRANSL ATION

After post-transcriptional modification occur and the mRNA is exported out of the nucleus,
tRNA transfers amino acids from a pool of cytoplasmically situated amino acids to the
polypeptide stand + ribosome.
Components of translation:
1.
2.
3.
4.
5.
6.
7.

mRNA
Initiation factors
Elongation factors
Release factors
Aminoacyl tRNA synthetases
tRNA
Ribosome

Structure of tRNA:

Single RNA strand 70-90 nucleotides long with a lot of complementarity

o Results in stretches of H bonds within the strand
4 helical segments with 3 characteristic loops
o In 2D, it appears to be clover shaped
The anticodon region has a specific nucleotide triplet written 3 to 5
The 3 end of tRNA has a protruding amino acid attachment site made of single
stranded CCA sequences
o The A is where the amino acid attaches to

Aminoacyl tRNA synthetases: Family of enzymes that carry out the activation of tRNA with
specific amino acids

Active site: Recognizes anticodon end of tRNA and the region of amino acid
attachment
When bound to the active site, catalyst of covalent attachments on the tRNA
molecule to its amino acid occurs
o tRNA gets charged
o Aminoacyl tRNA released from the enzyme

Wobble: Describes the flexibility for base pairing between the 3 rd codon and anticodon
PROCESS OF TRANSL ATION
Translation occurs in the cytosol of the cell
1. Initiation factors bind to 5 cap of mRNA
a. Other initiation factors bind to tRNA with methionine
2. Small ribosomal subunit recruited
3. Partially assembled ribosomal complex scans mRNA starting at the 5 end
a. mRNA is scanned until an AUG start codon is found
i. Prokaryotes: Translation initiation complex assembles at one or more
Shine Dalgarno sequences found a few bases upstream to the start
codon
4. Large ribosomal subunit recruited
5. Charged tRNA recruited
a. Initiation factors are released
b. Incoming charged tRNA delivered in association with GTP-bound elongation
factors
i. Once anticodon-codon pair made, GTP is hydrolyzed and the aminoacyl
end of tRNA is released from elongation factors

6. Transformational changes in rRNA occur after binding to charged tRNA allow for
peptidyl-transferase reaction
a. Transfers growing polypeptide chain onto A-site tRNA
7. Ribosome continues to translocate along mRNA
a. GTP bound elongation factors causes release of deacylated tRNA to move from
P site to E site
8. Once ribosome reaches stop codon, GTP bound release factors bind to A site
a. Catalyzes hydrolysis of bond between terminal amino acid and tRNA in P-site
9. More GTP hydrolysis enables dissociation of translation complex

ONE GENE ONE ENZYME THEORY

Beadle and Tatum: Worked with filamentous bread mold to establish the relationship
between genes and proteins

Neuspora can grow well on minimal medium therefore it must have enzymes
produced by a specific gene that converts substances into amino acids
Synthesis occurs in a series of steps in the metabolic pathway; transition between
steps require specific enzymes to form the subsequent intermediate compound

Precursor uses enzyme 1 ornithine uses enzyme 2 citrulline uses enzyme 3

arginine
Srb and Horowitz: Found that each gene contains the information needed to make an
enzyme

Performed genetic screening of radioactively treated neurospora to determine

whether there are specific genes that produced the enzyme needed in arginine
synthesis
Type

Mutant
Type

Arg1
Arg2
Arg3

Supplement
None
No growth
No growth
No growth

Ornithine
Growth
No growth
No growth

Citruline
Growth
Growth
No growth

Arginine
Growth
Growth
Growth

The one gene one enzyme theory was changed to one gene one polypeptide because genes
dictate the structure of all proteins produced by specific gene products
Exceptions:
1. Human proteome: Represents the full number of proteins that are expressed by our
genome
a. More than one protein can be produced by a single gene
2. Alternative splicing contribute to diverse numbers of mRNA transcripts

3. Post-translational modifications allow for production of diverse proteins

T2M4 THE COMPLEX PROTEOME
Human proteome: The full number of proteins expressed by all of the DNA genome

Alternative splicing and post translational modifications contribute to proteome

complexity
Composition of proteome can change in response to various factors
o Organisms developmental stage
o Response to internal/external signals

Compartmentalization of genetic information allows for intricate control in the regulation of

cellular processes.
Alternative splicing allows for one pre-mRNA to be spliced at different junctions to result in
many different mature mRNAs
Isoforms: Different types of mature mRNA from the same pre-mRNA transcript

Example:
o Insulin gene has 22 exons
In skeletal muscles, exon 11 is removed which translates to a higher
affinity version of the insulin receptor which uses glucose to meet high
energy needs
In liver cells, exon 11 is retained leading to a lower affinity for glucose

PROTEIN SIGNAL DETECTION AND ACTIVITY IN INSULIN

After eating a meal:

Stimulus: An increase in glucose levels

o Regulated by sensory responses in Beta islet cells of pancreas
Response: Pancreas modulates synthesis + secretion of insulin
o Insulin: Effector protein produced by pancreatic beta cells to decrease blood
glucose levels

The process from stimulus to cellular response is highly regulated and dependent on the
action of proteins and cell-cell communication (stimulus sensor effector response)
Insulin: Small protein

Translated polypeptide is 110 amino acids long

Functional protein is 51 amino acids long
o Dorothy Hodgkin: Used x-ray crystallography to determine insulin structure
2 amino acid chains:
Alpha = 21 amino acids
Beta = 30 amino acids
Post-translational modifications:
o Preproinsulin: Original 110 amino acid polypeptide
Contains N-terminal signal sequence which interacts with SRP to
facilitate translocation to the endoplasmic reticulum

o
o

Cleavage of signal sequence = proinsulin molecule

Proinsulin undergoes folding and formation of 3 sulfide bonds via chaperone
proteins
Folded proinsulin transported to the Golgi
More cleavage = mature insulin
Releases a C-chain of amino acids

Post-translational modification from preproinsulin to mature insulin allows for the N terminal
and C terminal amino acid residues in the A and B chains to bind to insulin receptors on
target cells
Insulin released from beta islet pancreatic cells binds to receptors expressed by target
tissues

Receptors: Proteins that receive and interpret information from signalling molecules
Insulin binds to receptor kinases
o Dimerization of receptor kinases lead to activation of cytoplasmic domains
which engage in phosphorylation of specific amino acids
Intracellular signal activates glucose transporter protein on the cell surface =
absorption of glucose into the cell

The process is initiated and maintained by positive feedback

A negative feedback loop can result in termination.
APPLIED LECTURES
RNA SPLICING AND SPINAL MUSCLE ATROPHY
RNA splicing of mRNA needs to occur. If not, the reading frame is thrown off, extra
unnecessary amino acids and produced and a truncated protein is created.
Survival of Motor Neuron (SMN): A protein involved in snRNPs assembly
Motor neuron diseases (MND): Diseases where nerve cells that control muscles are damaged

Muscles rely on simulation so the muscle cells weaken and die

Leads to classic symptoms of progressive muscle weakness
o ALS
o SMA

Spinal muscle atrophy: An inherited neuromuscular condition and number 1 genetic killed of
children under the age of 2 that affects ones ability to move and walk.

Diagonsis:
o Infant miss important physiological milestones
o Never crawls
o Cant hold head up
o Degenerative condition
o Muscle atrophy

SMA is an inherited autosomal recessive disorder carried in the SMN1 gene

Exon 7 is deleted
Protein is non-functional

There are multiple copies of the SMN gene SMN1 and SMN2 (they are spliced differently)

SMN1 = telomeric copy

SMN2 = centromeric copy

Different people have varied number of SMN2 copies

o The severity of SMA depends on # of SMN2 copies
More copies = later onset of the disease

When there is a mutation in the SMN2 gene, the same exon 7 is removed, creating the
truncated protein that SMN1 makes. In this case, as long as SMN1 remains okay, SMN1 can
make enough proteins to deal with the mutations in SMN2 (this does not occur vice-versa)
Treatment ideas:
1. Taking a copy of functional SMN1 gene, putting it into a vector and inserting it in the
nucleus
a. Single stranded antisense oligonucleotides (ASO) takes up into cell by
endocytic process where it enters the nucleus and binds to SMN2 pre-mRNA
b. Antisense recognizes intron region where nucleotide is defected and prevents
the silencer protein from binding to the intron so splicing can occur properly
ANTIBIOTIC RESISTANCE
30s (small ribosomal subunit)
1. Tetracyline: Blocks A site
a. Resistance: Protein flushes out tetracycline so that it cannot accumulate
enough internal concentration to block protein synthesis
2. Kasugamycin: Blocks initiator tRNA from forming a stable interaction with the start
codon
3. Aminoglycosides: Binds to small subunit and prevents mRNA from being threaded
though
a. Causes misreads in mRNA
50s (large ribosomal subunit)
1. Cloramphenicol: Blocks peptidyl transferase complex located in an RNA lined cleft
(i.e. it inhibits peptide bond formation)
a. Resistance:
i. RNA mutations of the rRNA by the PTC that disrupt the specificity of
antibiotic binding in the PTC (23S)
ii. Cfr methyltransferase methylation of the rRNA prevents antibiotic
binding to the 23S ribosomal rRNA
2. Macrolides: Blocks ribosomal exit tunnel
a. Resistance:
i. Mutation of 23S rRNA nucleotide at the tunnel entrance effects binding
of the antibiotic

ii. ErmC methyltransferase dimethylates 23S rRNA which confers

resistance
3. Erythromycin: Blocks translocation at the A site which prevent the addition of
incoming tRNA
a. Resistance:
i. Efflux: Antibiotic removed from the cell
ii. Small peptides that block erythromycin binding
iii. Chemical modification of the antibiotic
iv. Ribosomal mutations

THEME 3: RESPONDING TO THE ENVIRONMENT

T3M1 MODUL ATING TRANSCRIPTION
What is needed for prokaryotic growth?

Essential nutrients for growth and division

o Contains amino acids, vitamins, nucleotides and carbohydrates
o Favorable temperature

Gene regulation helps prokaryotes respond to the environment they need to adapt as the
environment changes
PROKARYOTIC GENE REGUL ATION
DNA is found in a bacterial nucleoid

Housekeeping genes: Genes required all the time for normal functions; constitutively
expressed
o Example:
Structural protein genes
RNA/DNA polymerases
Ribosomal protein genes
Actin gene
Regulated genes: Genes that can be turned on and off on an as needed basis; when
exposed to changing environments the cells alter expression pattern of these genes
o Allows for the production of important enzymes/proteins needed to cause
changes in growth/division

METABOLISING GLUCOSE AND LACTOSE IN PROKARYOTES

E. coli prefers to metabolize carbs but if they run out, gene expression mechanism allows
them to metabolise lactose instead

If glucose + lactose present in environment -> glucose metabolized first

Growth occurs at a steady rate until glucose consumed
No bacterial growth until lactose is able to be metabolized -> growth as lactose is
consumed

B-galactosidase: Enzyme that metabolizes lactose to produce glucose and galactose

Produced by turning on transcription of B-galactosidase gene (occurs when no

glucose present)

Francis Jacob + Jacques Monod: Investigated how the prokaryotic cell produces Bgalactosidase

Observation: Production of B-galactosidase enzyme dependent on the presence of

lactose in the environment
Experimental set up:
o Bacteria grown in lactose free medium = no B-galactosidase produced
o Lactose added to medium = amount of protein produced steadily increased
o Lactose removed from medium = production of enzyme stops
Result: Lactose in growth medium induces expression of B-galactosidase gene

LEVELS OF REGUL ATION

Gene expression is defined as when the functional product of a gene is made, modified and
activated. For protein coding genes, transcription, translation and protein modification must
be completed.
3 levels of regulation:

Transcriptional control
o Slowest -> must go through all steps for formation of a functional product
o Prevalent for more drastic environment changes
o Most efficient -> doesnt waste energy or resources to make protein
Cell only increases amount of enzyme when it is actually needed
Controls amount of mRNA produced in the cell
Activation of transcription requires that proteins bind to the promoter to
increase binding of RNA polymerase
o Cell can activate or inhibit transcription by binding proteins to promoter
Translational control
o Eukaryotes: Initiation of translation when binding of ribosome to 5 cap
o Prokaryotes: Ribosomes bind at the Shine-Dalgarno sequences
o Amount of protein produced affected by:
Rate of translation
Stability of mRNA
Post-translational control
o Fastest -> cell has a stockpile of inactive proteins
When cell receives a signal, all inactive proteins can be turned on
quickly
o
o

o
o

Primary sequence of polypeptide is not an active protein so control

mechanisms fold it into a 3D functional protein
Post-translational modification: Drives the assembly into complexes, binding
of substrates, unmasking of enzymatic domains

T3M2 PROKARYOTIC TRANSCRIPTIONAL REGUL ATION

B-galactosidase: Cytoplasmically situated, cleaves lactose into glucose and galactose

Lactose permease: Transport protein in bacterial cell membrane -> transports lactose into
the cell
Jacob and Monod: Discovered that bacterial genes were clustered into operons

Operons: Groups of functionally related genes organized into transcriptional units

Controlled by a single on/off switch called the operator: sequence of nucleotides near
the start of operon, regulated to allow or inhibit transcription
Also included the promoter

Polycistronic mRNA are punctuated with stop and start codons that signal where coding
sequences for each polypeptide starts and ends
The Lac operon uses positive and negative control -> lactose activates and glucose
represses

Highest levels of lac operon mRNA at point C (bacteria actively using lactose as
nutrient source)
Lowest levels at B
No lac operon expression at A (glucose being actively metabolized)

NEGATIVE REGUL ATION OF THE LAC OPERON

The repressor protein (made by the lacI gene) is constitutively expressed at low level and
binds to the lacO operator

Repressor protein: Tetrameric protein that binds to the lac operon to twist DNA into a
loop and prevent RNA polymerase from binding to the promoter

Negative transcriptional regulation can be allosterically inhibited when glucose is depleted

and lactose is present (lactose acts as the inducer molecule)

Lactose binds to repressor proteins near operator -> causes conformational changes
so that it cant bind

SUMMARY
Repressor protein (from lacI gene) binds to lacO operator to prevent RNA polymerase from
binding
Lactose (inducer molecule) acts as an allosteric inhibitor of repressor protein prevents
protein from binding and allows RNA polymerase to bind
POSITIVE REGUL ATION OF THE LAC OPERON

Positive regulation of the Lac Operon promotes the production of B-galactosidase and
lactose permease; a decrease in glucose levels in the environment causes an increase in
cAMP levels
Glucose levels contribute to cAMP levels:

High glucose levels = inhibition of the enzymes adenylyl cyclase which catalyzes
the production of cAMP from ATP
o Results in low cAMP levels

Low glucose levels = cells accumulate higher levels of cAMP due to increased
adenylyl cyclase activity

cAMP binds to CRP protein to form a CRP-cAMP complex which can bind to CRP-cAMP binding
sites on the operon the activator binding sites can be upstream, downstream or overlap the
promoter

Recruits RNA polymerase binding to the DNA promoter to allow for further activation
of B-galactosidase and permease

T3M3 EUKARYOTIC TRANSCRIPTIONAL REGUL ATION

Cellular differentiation describes cells with an almost unlimited ability to mature or
differentiate into any cell type; cells acquire specific fates dependent on signals sent and
which genes are turn on/off
Differentiated cells carry the same genome but have distinct functions
Regulation of gene leads to altered proteasomes
Transcriptional regulation in prokaryotes vs. eukaryotes:

Both:
o Proteins are involved in activating and repressing transcription
o RNA polymerase must bind to promoters to initiate transcription
Prokaryotes:
o Groups of related genes with similar functions clustered together in operons
transcribed by 1 promoter
Eukaryotes:
o Each gene is controlled by its own promoters and enhancers

METHODS OF ACTIVATING TRANSCRIPTION

Transcriptional factors: Proteins that bind to specific sequences in DNA that are able to
interact with the DNA double helix to control transcription of DNA to RNA
DNA is tightly wound around histone proteins to form a nucleosome (an octamer of 8 histone
proteins with DNA base pairs wrapped around it)
Tightly wound genes in chromatin are not accessible so chromatin must unravel for
transcription
Positively charged histone tails + negatively charged DNA phosphate = strong
interactions
1. Chromatin remodelling via histone modifications
a. Activator proteins recruit coactivator enzyme histone acetyltransferase
(HAT) which attaches acetyl groups to lysine amino acids on the positive tail
of the histone protein
i. Positive charge reduced on the tail interaction weakened =
loosening of heterochromatin allowing for transcription to occur
ii. ACETYLATION WEAKENS BONDS AND PROMOTES TRANSCRIPTION
2. Chromatin remodelling via methylation/acetylation of histone proteins
a. Modifications alter charges on the tail which alters histone binding to DNA

i. Opens space for transcriptional proteins

b. Degree of modifications to histone tails is part of the histone code that
determines whether transcription is activated or repressed
i. Methylation with 1 methyl group allows from transcription to occur
ii. Methylation with 3 methyl groups repressed transcription
3. Trans-acting factors
a. Trans acting factors: A DNA sequence that contrails a gene that encodes for a
protein that will be used in the regulation of another target gene; bind
specifically to DNA once its unwound
i. Basic helix loop helix
ii. Helix turn helix
iii. Zinc finger
iv. Leucine zipper
b. Most have alpha-helical domains to fit major grooves of DNA
c. Due to molecular interactions, when interactions are strong enough,
transcription factors assumes a conformation that allows for control of
transcription
i. Includes: Recruitment of other trans factors, RNA pol. + the activation
of transcription

4. Transcriptional activator proteins

a. Bind to specific enhancer sequences in regulator promoter region
b. Enhancer region: A short region of DNA that can be bound by activator
proteins to interact with the basal machinery at the promoter to enhance
transcription of a gene
i. Can be distance from the promoter
5. TATA box and transcriptional start sites
a. Form a part of the core promoter binding site required for the binding of
RNA pol. + associative trans factors
b. TATA box: A DNA sequence upstream from transcription start site that
indicates where a genetic sequence can be read and recoded
i. Recognized by a TATA-binding protein (TBP) subunit of transcription
factor TFIID
c. BRE region: DNA region close to TATA region which will be recognized and
bound to TFIIB general transcription factor that allows for protein-protein
interactions that help with assembly of transcription initiation complex
When general transcription factors bind to the promoter and transcriptional activator
proteins bind to enhancer, through the looping of DNA, the activator proteins, mediator

complex, RNA pol. and general transcription factors are brought into close proximity to allow
for transcription to proceed.
METHODS OF REPRESSING TRANSCRIPTION
Silencer regions: DNA sequences that can bind to transcriptional repressors to prevent
transcription by interfering with general transcription factor assembly and mediator activity
require for RNA pol. binding
1. Methylation near promoter of a gene
a. Characterized by chemical modifications of cytosine bases in DNA sequence
which occurs within a string of cytosine and guanine bases called CpG
islands
i. Frequently located in/near promoter sequencer
b. When island is heavily methylated, the shape of DNA binding sites for proteins
change which prevent binding not transcriptionally active
c. Methylation state is dynamic = it changes in response to environmental and
developmental cues
d. Promotes chromatin remodelling
i. Transcriptionally repressed methylated promoter can further promote
transcriptional inhibition
ii. Some proteins can only bind to methylated DNA
1. Histone deacetylases (HDAC) binds to methylated DNA and
promotes the removal of acetyl groups from neighboring
histones which allows nucleosomes to reassemble and leads to
masking of enhancer + promoter regions

T3M4 TURNING OFF THE SIGNAL AND RESPONDING TO THE ENVIRONMENT

Multi-level regulation allows cells to rapidly alter levels of active proteins in response to
internal/external signals

Points of regulation:

Transcription initiation
RNA processing
Overall stability of RNA molecules
Protein synthesis
Protein modification + transport
Protein degradation

MICROARRAY TECHNIQUES
Microarray techniques are used to look at the expression of thousands of genes at once;
helped visualize variations in gene expression; Based on base-pair interactions in which DNA
molecules in glass slides act as probes to detect gene expression

It is a tool to find differences in gene expression levels between normal and

cancerous cells
o Before: Cancer cells classified only on where they came from
o Now: With microarray, it is possible to differentiate between the patterns of
gene activity between the two types of cells
Isolated mRNA serves as templates for making complementary cDNA molecules to
the mRNA that are labelled with fluorescent nucleotides
o Uses a reverse transcriptase enzyme
DNA microarray chip contains:
o Large # of single stranded DNA fragments representing different genes
o cDNA from normal cells (green) and carcinoma cells (red)
They are tested for hybridization with the single stranded DNA
molecules in the microarray chips
Relative differences in fluorescence colour and intensity is measured at
each position
If gene is active they more labelled cDNA is able to hybridize
with the DNA on the microarray chip brighter spots
Relative gene activity is determined by measuring the intensity of fluorescence
o Brighter = more active gene
o No fluorescence = inactive

Probe: Single stranded segments of DNA/RNA that hybridizes with complementary targets
Oligonucleotides: Short fragments of nucleic acids
TURNING OFF GENE EXPRESSION @ A POST-TRANSCRIPTIONAL LEVEL
It is not sufficient enough to turn off gene expression by removing proteins from the cells if
mRNA is continually being transcribed; mRNA must be degraded to stop gene expression
Methods:
1. Length of poly-A tail
a. Regulation of mRNA stability controlled though the length of the poly A tail

2. miRNA and the RISC complex

a. The activation of RNA interfering machinery is activated by short, noncoding
regulator double stranded RNA molecules such as microRNA
i. Transcribed microRNA forms hairpin loops due to base-pairing in its
precursor transcript
ii. Hairpin loop is processed into smaller, single stranded mRNA
fragments to activate the RNA interference machinery
b. miRNA incorporated into RNA-induced silencing complex (RISC)
i. Single stranded miRNA contains complementary sequences to specific
target mRNA sequences that require regulation
1. microRNA in RISC complex binds in non-exact manner to target
mRNA
2. Proteins in the RISC complex inhibit translation
3. siRNA and the RISC complex
a. siRNA: Small interfering RNA that are transcribed + processed similarly to
miRNA and lead to mRNA degradation
i. siRNA strands are the exact complements of mRNA target
b. siRNA becomes associated with the RISC complex similarly to miRNA
c. siRNA + RISC complex induces cleavage of target mRNA
i. Destabilizes target mRNA and further contributes to posttranscriptional regulation of gene expression
TURNING OFF GENE EXPRESSION @ A POST-TRANSL ATIONAL LEVEL
Proteoasomes: Large protein complexes that can break peptide bonds and therefore degrade
unneeded or damaged proteins
Breaks long polypeptides into small fragments of a few amino acids
Ubiquitin proteins: Proteins that tag other proteins that require degrading via an enzymatic
cascade

They can turn off gene expression signal which results in degradation
Most ATP dependent allows for unfolding of proteins + subsequent cleavage
Facilitated by ubiquitin activation, conjugation to target substrate protein and ligation

APPLIED LECTURES
TREATING LACTOSE INTOLERANCE
Lactose: Disaccharide glucose + galactose

Energy source for most offspring mammals

Lactase (a B-galactosidase hydrolyzes lactose into its monosaccharides
o Present on the apical surface of absorptive intestinal enterocyte cells

Galactose undergoes biochemical reduction by pathways using 3 enzymes:

1. Galactokinase: Phosphorylates galactose
2. Gal-1-phosphate uridyltransferase: Turns galactose-1-phosphate into glucose-1phosphate after UDP-glucose is transferred onto the galactose-1-phosphate
a. Causes the UDP glucose to become UDP galactose
3. UDP-Gal epimerase: Turns UDP galactose into UDP glucose to be used by Gal-1phosphate uridyltransferase
Humans have evolved to have a mutation that have kept the lactase gene permanently on;
random mutations occurred in the MCM6 regulatory gene

Causes lactase non-persistence which allows humans to retain some lactase activity

Congenital lactase deficiency: A condition in which individuals are unable to digest lactose

Caused by LCT gene mutations on chromosome 2 which codes for the lactase
enzyme (single mutation which alters amino acids or leads to truncated polypeptides)
Results:
o Excessive lactose in the intestine attracts water molecules = prevents water
from being absorbed into the bloodstream
o High concentration of lactose in the gut
o Fermentation in the anaerobic environment
Leads to bloating, gas, cramps, nausea and diarrhea
Treatment options:
o Omitting lactose consumption in the diet
o Pre-treating milk with purified lactase

Galactosemia: A condition in which individuals are born without the enzymes needed for
galactose processing

Types:
o 1 Gal-1-phosphate uridyltransferase

o 2 Galactokinase
o 3 UDP-Gal epimerase
Caused by mutations in the GALT, GALE, and GLAK1 genes on chromosome 9
Results:
o Toxic accumulation of galactose
Organ and tissue damage
o Vomitting/diarrhea
o Jaudice
Treatment options: Omitting galactose consumption in diet

ELIE METCHNIKOFF AND PROBIOTICS

Metchnikoff developed the original modern hypothesis of the positive role played by certain
bacteria
Question: Can modifying the composition of the microbiome of the colon influence lactose
intolerance?

Microbial galactosidase in yogurt is present in active forms in the duodenum in

lactose intolerant individuals after eating yogurt
o Presence of the enzyme enhances lactose absorption in lactase-deficient
individuals
Enzyme retains its function b/c it is protected by the bacterial cell membrane + b/c of
the buffering capacity of the yogurt

Probiotics: Microorganisms that are associated with beneficial effects to humans when
ingested by modifying the microbiome of the colon
EPIGENETICS AND GENE REGUL ATION
Epigenetic processes are essential for normal development, cell differentiation and are
increasingly being recognizes as being involves in human diseases.
Epigenetic mechanism:
1.
2.
3.
4.

Modification of histone tails

DNA methylation
Chromatin remodelling
Packing of DNA around nucleosomes

Epigenetic mechanisms are affected by:

1.
2.
3.
4.
5.

Development
Environmental chemicals
Drugs/pharmaceuticals
Aging
Diet

Epigenomics: The study of epigenetic modifications across an individuals entire genome

(epigenetics = above the genome)
Cancer cells have abnormal epigenomes:

Lower levels of methylation

o Increases expression of genes that promote cell growth (leads to excessive
growth)
High levels of methylation
o Decreases expression of genes that are needed to keep cell growth in check,
repair DNA or initiate programmed cell death

AGOUTI GENE IN MICE

The Agouti gene encodes for a protein that signals melanocytes to switch from producing
black pigment to yellow Agouti mice with a yellow coat are also more predisposed to being
obese

Mice are shown to be genetically identical but the gene expression is different
Methylation changes in the CpG island around the promoter of the Agouti gene
affects expression
o Methylation keeps the Agouti gene off
Experiment:
o Maternal diet was modified with methyl groups to see how you can regulate
the expression of genes via epigenetic mechanisms
o Sources of methyl groups in the diet of the mother = non Agouti gene
offspring
o Even with a yellow mother, susceptibility to the disease could be altered by
feeding the mothers diets rich in methyl groups
Diet supplemented during pregnancy = offspring mostly brown and
healthy
No dietary supplements = offspring mostly yellow and unhealthy

IDENTICAL TWINS AND EPIGENETICS

Identical twins have the same genetic blueprint but can end up with different characteristics
due to epigenetics
When mapping chromosomal regions for DNA methylation it was revealed that there was
different methylation in twin pairs

Technique: Competitive hybridization with normal metaphase chromosomes

o 3-year-old twins show similar distribution of DNA methylation
o 50-year-old twins show abundant changes in the pattern of DNA methylation

THIS PHENOMENON IS CALLED EPIGENETIC DRIFT

Dr. Parminder Raina: Conducted the first world-wide study to measure the evolution of
epigenetic markers in a large cohort; related epigenetic markers to health related, social and
environmental measures using microarray approaches
Human epigenome project: A multinational project with the aim to identify, catalog and
interpret genome-wide DNA methylation patterns of all human genes in all major tissues

If successful, it can advance the field of pharmacogenetics by allowing researchers to

develop drugs capable of directly changing the way genes are expressed

THEME 4: DNA REPLICATION AND MITOSIS

T4M1 THE CELL CYCLE
Distinctions between prokaryotes + eukaryotes:

Eukaryotes:
o Larger DNA organized in linear chromosomes
o Highly condensed nucleus
o Chromosomes replicated to be separated into daughter cells
o More regulation
Prokaryotes:
o Bacterial nucleoid region

CELL DIVISION IN PROKARYOTES

Binary fission: The prokaryote form of asexual reproduction that allows identical genetic
material to be distributed amongst daughter cells

Initiated bacterial chromosome DNA is attached by proteins to the inside of the

plasma membrane
o DNA replication begins along an origin of replication
As chromosome replicates circularly
o Cell elongates until DNA attachment sites are at opposite pole
o Newly synthesized DNA remain anchored to the plasma membrane
When replication is complete, the cell constricts along the midpoint
o Synthesis of new cell membrane + cell wall
o Complete division into 2 daughter cells

CELL DIVISION IN EUKARYOTES

Humans have 23 distinct chromosome pairs from their mother and father
Walther Fleming: Developed the idea of mitosis stages based on morphometric
characteristics of chromosome changes by looking at stained developing salamander
embryos.
Phases:

Interphase preparations for cell division occurs (chromosome in the form of long,
thin chromatin fibres)
o S phase DNA synthesis
Replication of DNA -> Exact copies of each chromosome created to
form sister chromatids
o Gap growth phases

G1: Prepares cell for DNA synthesis

G2: Prepares cell for mitosis
During the transition from G2 to M phase, duplicated
chromosomes being to condense
G0: Phase in which cells are not actively dividing
M phase Mitosis + Cytokinesis
o Prophase
Each chromosome is visible as sister chromatids
Centrosomes radiate long microtubules called mitotic spindles
Moves to opposite poles of the cell
o Prometaphase
Nuclear envelope fragmentation
Microtubules attach to specialized regions on centromere
(kinetochores)
Kinetochores: Specialized protein structures that associate with
each sister chromatid on either side of the centromere
Polar microtubules interact with each other and push the poles away
from one another
o Metaphase
Alignment of chromosomes at the center of cell along metaphase plate
Kinetochore microtubules facilitate alignment
o Anaphase
Kinetochore microtubules shorten = sister chromatids pulled apart into
individual chromosomes toward opposite poles
Polar microtubules push against each other and elongate cell
End: 2 ends of cell have equivalent and complete sets of chromosomes
o Telophase:
Two new daughter nuclei form and nuclear envelope reforms
Chromosomes begin to de-condense
Spindle microtubules break down
Division of parent cell underway
o Cytokinesis:
Division of the cell
Animals:
Begins with formation of contractile ring made up of motor
proteins
Motor proteins contract bundles of actin gibers along the cell
midline to make a cleavage furrow which divides the cell
Plants:
Cell wall developed along cell plate region
Cell wall fuses with the original cell wall

DIFFERENTIATION
Differentiation: When stem cells turn into specialized cells
The adult body has stems cells but are not able to give rise to all cell types

Mammalian adult skeletal muscles = stable tissues with very little cell division

If it undergoes injury quiescent (non-dividing) satellite stem cells present in

the basement membrane of the muscle tissue are activated and divide to
being muscle regeneration
o Activation proliferation differentiation fusion of muscle precursor cells
called myoblasts which eventually become mature muscle cells called
mytofibers
Stem cells can reproduce indefinitely and undergo periods of quiescent
o

CELL CYCLE REGULATION

Tim Hunt: Measured protein level changes of cyclin protein of dividing sea urchin embryos

Added radioactively labelled amino acids to sea urchin eggs which would be
incorporated into newly synthesized proteins in embryos
Took samples of dividing embryos every 10 minutes and looked at changes using gel
electrophoresis
Results:
o Most protein bands became darker as cell division and embryonic
development progressed
o One band oscillated in intensity due to cyclic nature of protein
o He wasnt aware of protein function but suspected it was involved in playing a
regulatory role in cell cycle progression

Mitosis promoting facto consists of:

1. Cyclin protein
2. Cyclin-dependent kinases (CDK) protein
Kinases: Enzymes that activate and inactivate other proteins by phosphorylating key amino
acids on target proteins; triggers a multitude of changes that occur during the various cell
cycle events (phosphorylation of target proteins that promote cell division)
The activity of cyclin-dependent kinases is dependent on concentration of activating cyclin
protein
Cyclin-CDK regulation is important during 3 steps of the eukaryotic cell cycle:
1. G1/S cyclin-CDK complex needed for transition from G1 phase to S phase
a. Prepares cell for DNA replication
2. S cyclin-CDK complex helps initiates synthesis
3. M cyclin-CDK complex initiates mitosis
a. Facilitated by phosphorylation of key structural proteins needed for nuclear
membrane breakdown
b. Helps regulate the assembly of microtubules in mitotic spindle
Process:

Cyclins bind to and activate the CDK to create the cyclin-CDK complex to control
progression through the cell cycle
Active cyclin-CDK complex phosphorylates target proteins involved in promoting cell
division
As cycle progresses, the cyclin degrades, inactivating the complex

3 major checkpoint can pause cell division until preparation for the next step is complete
or damage is repaired
1. End of G1 DNA damage checkpoint
a. Only undamaged DNA is replicated
b. When damage occurs, specific protein kinases phosphorylates p53
i. P53: A protein that can inhibit cell cycle when turned on by turning on
genes to inhibit the cell cycle
1. Present at low levels in the nucleus (most exported + degraded)
ii. P53 blocks activity of G1-S cyclin-CDK complex
iii. Arrests cell cycle in G1 phase
2. End of G2 DNA replication checkpoint
a. Only cells with replicated DNA will enter mitosis
3. Before anaphase spindle assembly checkpoint
a. Mitosis only completed if all chromosomes are attached to a microtubule
b. Regulatory proteins associated with the spindly assembly monitor degree to
which sister chromatids are attached to microtubules of mitotic spindle at
kinetochore region
i. Unattached kinetochores = wait signalled recruitment of spindly
assembly checkpoint proteins
1. Activated by lack of tension in centromere area
ii. Once everything is set, separase breaks sister chromatid attachments
Genes that normally inhibit cell cycle progression is normally turned off (turned on only
when necessary)

T4M2 DNA REPLICATION

Models of DNA replication:
1. Semi conservative (correct): 1 new strand + 1 parent strand (2 new helices)
2. Conservative: Two complementary parent strands come back together in the end
3. Dispersive: All four strands combine in a mixture of new and old DNA
Watson and Crick: Proposed that DNA consists of a pair of complement template chains
arranged as a double helix

The inherent organization of base pairing is indicative of a copying mechanism for

genetic material
Believed that when a cell copies its DNA, each strand serves as template for the
ordering of new nucleotides following base pair rules into a new complementary
strand
o Semi conservative model = 1 new strand + 1 parent strand

Meselson and Stahl: Conclusively showed that DNA replicates semi-conservatively using
E. Coli

Experimental set-up:
o E. coli cultured on medium for many generations that contained nucleotide
precursors with radioactively labelled heavy isotope nitrogen (15N)
o Bacteria transferred to a medium with a light 14N isotope

Throughout the experiment, DNA samples extracted + centrifuged

Separated DNA based on differing densities of radioactively labelled
molecules
Results:
o With only 15N one distinct band observed
o One round in 14N single band with lower density (higher than 15N but lower
than 14N)
Meant that there must be a hybrid of 14N and 15N
o Continued growth + division in 14N medium 2 distinct bands (one in 14N
position and one in 15N position)
Consistent with semi-conservative model
o

In DNA replication

Template strand copied from 3 to 5 direction (daughter strand elongates from 5 to

3)
Each incoming complementary nucleotide H-bonds with nucleotide on template
strand
o 5 phosphate group interacts with 3 hydroxyl of existing polymer
Phosphodiester bonds form between growing daughter strand and incoming
nucleotides to form the DNA backbone

DNA REPLICATION PROCEDURE

1. Initiation
a. Unwinding of double helix occurs at the replication forks with DNA helicase
b. Single stranded binding proteins bid to and stabilizes parent strand
c. Topoisomerase 2 binds upstream of replication fork to minimize torsional
strain
d. RNA primase synthesizes RNA stretches of nucleotides (RNA primers) for DNA
pol. 3
i. Initiation requires the primer to base pair with template DNA b/c
enzymatic machinery that elongates daughter strand can only do so
from existing DNA/RNA
2. Elongation
a. DNA pol. 3 synthesizes a replicated DNA strand from RNA primers
b. Leading strand: Only one primer is required and DNA pol. continuous adds
nucleotides as replication progresses
i. Replicates the whole template strand
c. Lagging strand: DNA pol. 3 adds nucleotides to the 3 end of daughter strands
i. Results in segments of Okazaki fragments formed by separate primers
ii. Requires some post-replication processing:
3. Termination
a. DNA pol. 1 replaces RNA primer sequences with DNA nucleotides
b. DNA ligase joins 3 of a fragment to adjacent DNA nucleotide by catalyzing
formation of phosphodiester bonds
c. During replication, DNA pol. 3 is able to proofread each added nucleotide
relative to template strand
i. If incorrect nucleotide detected DNA pol. removes + correct
nucleotide added
ii. High degree of accuracy

Prokaryotes and eukaryotes have similar replication mechanisms but eukaryotes use a
different set of DNA pol.
TELOMERES
At the very end, RNA primers on the lagging strand cant be replaced with DNA nucleotides
because there is no 3 end available for nucleotide addition + phosphodiester bond
formation therefore DNA replication machinery cannot complete the 5 ends of daughter
strands of linear DNA
Telomeres: Regions on eukaryotic chromosome ends that protect the DNA from being eroded
by successive rounds of replication; specialized noncoding, repetitive nucleotide sequences

Acts as a buffer zone to protect coding genes

Eventually, telomeres become shorter as you age

Telomere shortening does not occur in gamete + stem cells:

Genome remains relatively unchanged

o Gametes cant contain missing information and embryonic stem cells must
replicate a large amount of times to produce organism
Telomerase: Enzyme that catalyzes the lengthening of telomeres in eukaryotic cells
o Reverse transcriptase
o Synthesizes DNA from RNA template
Enzyme is a ribonucleoprotein that contains an RNA template as part of
its complex which binds to the tail of the telomere and allows for the
enzyme to catalyze extensions
After telomerase extends DNA, DNA pol. + ligase goes back to complete daughter
strand replication

T4M3 APPLICATIONS OF DNA REPLICATION

POLYMERASE CHAIN REACTION
Kary Mullis: Developed the method of polymerase chain reaction (PCR) for the
amplification of DNA which allowed scientists to amplify millions of copies of DNA from small
starting samples
Process of PCR:

Sample of DNA placed in tube containing:

o Buffer solution, pair of single stranded DNA primers, free deoxyribonucleotides
(dNTPs), Taq pol.
Two primers for either DNA strands
They bind to specific regions on template DNA (starting points
for replication)
Taq polymerase used because it can withstand high temperatures
Tube placed in a thermocycler
o Undergoes phases of heating + cooling in programmed steps to facilitate
replication process

Cycles of PCR:
o Denaturation:
Double helix unwound
High temperatures separate strand by denaturing it
o Annealing
Solution is cooled
Primers anneal to complementary sequences of DNA template strand
o Extension:
Heat stable DNA pol. extends + polymerizes daughter strands using
dNTPs + primers
Extends in 5 to 3 direction

Each cycle = 2n number of copies

GEL ELECTROPHORESIS
Gel electrophoresis is a technique used to separate DNA fragments from other sources on
the basis of rate of movement through an agarose gel in an electric field; used to visualize
DNA molecules
Process:

Molecules loaded into well of porous gel

Due to negative charge of DNA/RNA (from phosphates), they are attracted to the
positively charged anode end of the gel
o Smaller molecule = faster = farther distance
o Larger molecule = slower = shorter distance
A standardized ladder is loaded into gel with DNA sample

Using dyes that intercalate with and stain DNA fragments, the DNA can be visualized as
bands under UV light
DNA SEQUENCING
DIDEOXY CHAIN TERMINATION METHOD
Frederick Sanger: Developed a DNA sequencing technique called the dideoxy chain
termination method
Limitation: Could only find sequences of small fragments of DNA
Components:

Denatured single stranded template DNA

Single stranded DNA primers which bound to DNA
Sufficient dNTPs
DNA polymerase
o Links adjacent deoxynucleotides by catalyzing covalent bonds between
5 phosphate and 3 hydroxyl

Sanger reasoned that ddNTPS that were missing the OH group on the 3 position
would not allow for further elongation for attachment of next nucleotide

Added labelled ddNTPs into sequencing reaction tubes (lead to a series of

interrupted daughter strands)
o Each ddNTPS labelled with different fluorescent dyes
The sequencing reaction contains millions of molecules of template DNA (b/c
insertion of ddNTPS was a random process which could interrupt daughter
strand anywhere)
After DNA synthesis + chain termination, the labelled strands loaded onto a
gel
o Continues until each DNA band emerges from the bottom of gel and
the dye is excited by a laser
o Fluorescence detector records amount of fluorescence emitted +
matches to a certain ddNTPs
Spectrogram trace generated each peak corresponds to nucleotides that
make up DNA sequence that is complementary to template strand and begins
after primer

Sanger would try to align the DNA sequences based off the pieces of tagged,
interrupted sections
SHOTGUN SEQUENCING
Gene Meyes and Jim Weber: Developed shotgun sequencing based on being able to break
the entire genome into different size pieces

Useful in large scale sequencing projects

Facilitates identification of entire genome of organisms
Phases
o Random sequencing of DNA in each fragment
o Identifying regions of overlap between generated fragments and inferring the
long, continuous sequence of nucleotides in the DNA molecule that makes up
each chromosome
o Annotating sequences

Contigs: Regions of overlapping DNA segments

Annotation software identify any protein coding stretches of DNA that are flanked by a
start/stop codons
Gene annotating: Identifying sequences of interest on DNA after sequencing it allows us to
gain a better understanding of nucleotide patterns on DNA

Step 1: Identifying the correct reading frame

o 6 possible reading frames for each DNA sequence
o Computer programs can scan sequences in both directions to identify reading
frames
Good reading frame = long stretches of codon with no stop codons
Step 2: Identification of patterns/sequence motifs in sequenced DNA molecules
o Identification of open reading frames in DNA
ORF consists of triplets of nucleotides that specify amino acids that
makes up protein
o Binding sites for transcription factors that regulate gene expression

DNA sequences that bind to transcription factors are often short

sequences present in multiple copies near a protein-coding gene
Some motifs can be identified from hypothetical RNA molecules from
sequenced DNA
Ex. RNA that makes up tRNA
Sequences can be inferred from a DNA molecule by looking for
nearby complementary sequences

In eukaryotes, 50% of their genome consists of repeated noncoding sequences which

functions by contributing to organism diversity.
T4M4 DNA MUTATIONS
Changes in genetic information of a cell can affect protein structure and function they can
be beneficial or have devastating consequences. Mutations are rare events and there is
variability to the likelihood that a new mutation will occur at a given nucleotide base pair in
a single round of replication across different organisms.

Mutations are responsible for genetic differences between organisms

Multicellular organisms = low mutation probability
Viruses = high mutation rate
o RNA viruses = highest mutation probability
o Discrepancy due to delicate nature of RNA backbone of RNA viruses and
retroviruses
More prone to damage
No proofreading capabilities in RNA genomes

Ways mutations could occur:

1. Environmental factors
2. Spontaneous mutation (most common)
3. DNA replication errors
In somatic cells:

Mutated cell will be progenitor of population of identical daughter cells

o Creates a patch of cells with new mutation
o The earlier in the developmental cascade, the larger the spread
Mutation in a G0 cell = largely negligible effect
Not heritable
Mutation only at specific cells in specific tissues

In germline:

Mutations are heritable

Every cell in developing embryo carries the mutation

Joshua and Esther Lederberg: Determined that mutations are random and not directed

Experimental set up:

o Plate 1: Bacteria grows in colonies on petri dish with non-selective
supplemented nutrients (agar)

Once colonies grew, the original plate was stamped onto cloth and the
stamped cloth was stamped onto a new selective plate containing antibiotic
penicillin in agar
Stamping: Referred to as replica plating preserves original
arrangement of colonies

Result:
o Only penicillin resistant bacteria grew
Lederberg predicted that few colonies carried mutation for resistance
Second experimental set up:
o Exposed suspected mutant colony in original plate to penicillin
Result:
o A pure culture of antibiotic resistant bacteria was made which meant that
there was a mutation for the resistance before exposure

Mutagens: Agents that increases the probability of mutations at specific regions along DNA
(includes chemicals and radiation)
CORRECTING DNA DAMAGE
1. Base excision repair
a. Uracil glycosylase enzyme is signaled by an incorporation of uracil in the DNA
i. Cleaves uracil from DNA backbone and leaves behind bare deoxyribose
sugar with no nitrogenous base
b. Lack of base detected by AP endonuclease
i. Cleaves backbone on either side of the area with no base
ii. DNA synthesis occurs at the gap facilitated by DNA pol. + ligase
2. Nucleotide excision repair
a. Similar to mismatch repair mechanism
b. Contrast: Mismatch corrects single nucleotide mismatch and nucleotide
excision removes + replaces more than one damaged nucleotide base
c. Damaged based signal specific enzymes to cleave DNA backbone flanking the
region
d. After removal, DNA synthesis fills excised gap with correctly match
nucleotides
3. Mismatch repair
a. Mismatching of single nucleotide pairs in replication corrected by proofreading
of DNA pol.
b. Mismatched nucleotide makes kinks in DNA that are recognized by proteins
that can scan DNA for error and damages
i. Allows for mismatch repair identification of mismatched DNA
sequence leads to single stranded cleavage of mismatched DNA
backbone by nuclease
1. Occurs some distance away from mismatched nucleotide region
2. After backbone cut, enzyme removes successive nucleotides
3. DNA pol. + ligase induces DNA synthesis to close the gab
MUTATIONS IN THE NUCLEOTIDE SEQUENCE
1. Point mutations: Single nucleotide pair changes in the DNA sequence
a. Small scale and can arise during DNA replication
b. Single nucleotide pair substitution most common type of point mutation

c. If it occurs in important protein coding sequences, it can have detrimental

effects:
i. Non-synonymous/missense mutations: Causes a single amino acid
substitution
ii. Synonymous/silent mutation: Point mutations that transforms one
codon into another that is translated into the same amino acid
iii. Nonsense mutations: Point mutations that changes the codon of an
amino acid into a stop codon
2. Insertions: Occur when one or more extra nucleotides are inserted into replicating
DNA
3. Deletions: Skipping or removal of one or more nucleotides during replication
4. Frameshift mutation: Insertions or deletions not in multiples of 3
a. Leads to improper grouping of nucleotides downstream of mutation site
b. Usually ends sooner or later in nonsense, premature termination
c. Almost certainly leads to non-functional proteins
CHROMOSOMAL MUTATIONS
Chromosomal mutations: Mutations that affect a larger region of DNA and leads to visible
changes in chromosomal structures
1. Deletions
a. Chromosomal fragment lost
b. Can lead to loss of entire genes from chromosomes
i. If centromere is lost, then often the entire chromosome can be lost in a
few cell divisions
c. If deletion occurs in diploid cell, the deletion may persist depending on
whether the other homolog can compensate to provide and code enough of
the gene product required
d. If deletion occurs in embryo it often leads to embryo death or fatal
abnormalities
2. Duplications
a. Chromosomes acquire small duplications during DNA replication
b. Usually causes little harm
c. Sometimes extra gene copies confer a specific advantage on affected
individuals
i. Can lead to new gene formed (duplication and divergence)
3. Reciprocal translocations
a. Portion of one chromosome attaches to a non-homologous chromosome
b. Often happens when breaks occur in the chromosome and translocation
happens before repair
c. Usually occurs in noncoding regions of DNA in larger genomes
d. Homologous chromosomes cant pair and move together during cell division
event
i. Individual might have gametes missing vital genes
APPLIED LECTURES
TELOMERES AND IMMORTALITY

For eukaryotic chromosomes, there is a problem at both ends once the end RNA primer is
degraded, it leaves behind a short region of nonreplicated DNA (nowhere to attach another
primer)

The single stranded DNA is cleaved off the end of the chromosome
o Results in shorter chromosomes with each replication
With each cell division, telomeres become short and shorter (chromosomes become
more unstable the shorter they get)
o In order to prevent cutting into coding regions, cell division stops
o When chromosomes are missing telomeres, they can fuse together and attach

Cell turnover: When older, differentiated cells die and need to be replaced through cell
division of adult stem cells
Tissue homeostasis: When generation of new cells occur at the same rate that cells are lost
Hayflick limit: The limited capacity for cells to divide unto which cells enter stage called
senescence
Telomerase has built in RNA primers that can elongate telomeres

Telomerase gene therapy in adults + old mice delays aging + increases longevity
without increasing cancer
o Increases lifespan by 20%
Telomerase inhibition can be used as cancer therapy

There is a correlation between shortening of telomeres and the aging of the cell

As we age, our stem cell pool is aging (telomeres shortening) therefore we cant
replace lost differentiated cells as readily resulting in failure to maintain homeostasis

MUTATIONS AND GENE EVOLUTION

Mutations promotes diversification of gene family members gene families can expand and
contract over evolutionary time scales

Species undergo divergent evolution when mutations occur

Usually, duplicates have so many mutations that are not functional called pseudogenes

Hemoglobin contains alpha-globin and beta globin

On chromosome 11, different version of the gene is expressed as human develop

from embryo to adult
o All 5 code for proteins that are not exactly the same but have the same
ultimate function
Multiple events lead to multiple copies of a gene family
o Exchange occurs across the homologous chromosome results in 2 different
chromosomes
One has a deletion
One has a duplicated segment

2 homologous chromosomes have 2 gene spots

When chromosomes have repeated sequences, they might misalign
When recombination occurs, the chromosome gets an extra copy of the
gene

When are different genes used?

Embryonic
o Epsilon-globin
Fetal form (4 weeks before birth)
o Gamma-globin
Adult
o Beta-globin
Alpha-globin is always expressed

Regulation:

2 transcriptional factors turn off gamma globin and turn beta version on (coregualted)
o KLF1 turns on beta globin and causes BCL11A
o BCL11A turns off gamma globin

Fetal hemoglobin has a different affinity for oxygen than adults which allow the fetal
hemoglobin to pull oxygen away from the mother
CRISPR: Technology used for disrupting gene function

Takes a target sequence of DNA and makes a small deletion

Protein complex + RNA molecules targets structure to specific region of genome
determined by sequence of guided RNA
Complex recognizes transcription factor genes, deletes it and fuses the DNA together
so that BCL transcription factor is not made

SICKLE CELL DISEASE

One amino acid change results in a change in the structure of the protein the tetramers
lump together in a fibre which changes the structure of the red blood cell

Sickle cells arent as flexible as regular cells and form blockades in the capillaries so
oxygen cant reach target sites
Short lifespan

Reduced red blood cells = anemia

Reduced oxygen supply = cell death, tissue degeneration, pain and death
There is a higher frequency of sickle cell disease in areas prevalent with malaria

Suggests that there is a selective advantage

o People with sickle cell allele survive better in presence of malaria
Sickle cell more easily engulfed by phagocytes
Plasmodium (parasite that causes malaria) cannot affect individuals with sickle cell
very well
o Infected mosquito bites human and inserts sporocyte which go to the liver
o Sprorocyte replicate in the red blood cell
o Plasmodium replicate in red blood cell
Doesnt replicate as well in sickle cell

Question: Can gene duplication provide a built-in back up gene? Can gamma globin be
turned back on?
Blood cells are much more resistant to genetic repair than genetic disruption
BETA-THALASSEMIA
Reduced or absent beta-globin caused by frameshift mutations (replacement of an adenine
by a uracil which changes lysine to a termination codon) which results in inefficient oxygen
carriage throughout the body

Beta-0 = no Hb-Beta protein produced

Beta+ = Reduced production of beta chains

THEME 5: THE PRINCIPLES OF INHERITANCE

T5M1 GENETIC VARIATION
Human genome: Contains the complete set of instruction that codes for life; the exons and
regulatory elements that code for proteins are only a small part of the genome

Single copy genes transcribed to make functional proteins

Repeated nucleotide sequences
o Tandem repeats: Repeats that can be several thousand nucleotides in length
and be present next to each other or in multiple identical or near identical
copies
o Simple sequence repeats: Repeats as short as two nucleotides repeated over
and over throughout the DNA sequence stretch

The different possible sequences spread throughout genomes lead to genetic variation
within and across organisms (not all variation have an observed affect)

Overall outcome of distribution of different sequences through the genome depends

on nature of change and where in the genome it occurs

Genome sequencing projects: Projects that try to map out each chromosome with high
resolution

Underlying DNA sequence determined

Provides insight into inherited diseases + genetic variability

DNA polymorphisms: One of two or more alternate forms (alleles) at a chromosomal region
(locus) that differs in either a single nucleotide base or have variable numbers of tandem
nucleotide repeats in a given population of individuals

Large number of DNA polymorphisms across genome of organisms (most in

noncoding regions)
Allows for assembly of high-density, genetic maps
Referred to as DNA markers
o Detectable using various techniques (microarray, PCR, southern blotting)
o Can show relatedness such as in DNA fingerprinting

DETECTING VARIATION
99.9% of human DNA sequences are the same genetic variation accounts for differences
between individuals
SNPs: Single nucleotide polymorphisms

Most common type of genetic variation (1 in every 350 base pairs)

o If found close to a coding gene, it can be used as a DNA marker for that
specific gene
o If close enough/linked to the gene of interest, every time the gene is passed
on from parent to child, the SNP is passed on too
Single nucleotide base change or substitution in DNA sequence (can be in coding or
noncoding region)
DNA microarray analysis widely used to detect SNP genotypes
o Oligonucleotides that match the common allele and all possible variant SNP
alleles attached to the chip to detect SNPs
o Millions of short, single stranded oligonucleotides of known sequence attached
to the chip to detect SNPs
o Fragments of single stranded fluorescently labelled DNA hybridized to the chip
Math fluorescent pattern with what SNP person has
Can see if homozygous or heterozygous for each SNP

Ability for researchers to probe for different SNPs allows variations in population of
individuals due to genome containing its own pattern of SNPs

Each individual has their own SNP profile

VARIABLE NUMBER TANDEM REPEATS

Differences in organisms observed can be due to variations in short sequences of DNA
repeated in tandem called variable number of tandem repeats VNTRs
Tandem repeats: Patterns of one or more nucleotides that are repeated that are directly
adjacent to each other and can be found in various lengths
Variable number of repeats can be identified using PCR + electrophoresis:

Tandem repeat site targeted and amplified

Sequence specific primers target flanking regions
Resulting fragments are separated and detected using gel electrophoresis

Process of identifying VNTRs largely used to assist in identifying individuals based on DNA
profiles

VNTR loci similar between closely related individuals

o Extremely unlikely to be the same in strangers
Different genotypes yield a unique pattern of bands
Detection of VNTR and other polymorphisms used in crime scene investigations and
genetic family relationships

VARIATION AND DISEASE

Most variations in the genome have no known affect b/c they are in noncoding regions
(silent variations). However, variations in protein coding or regulatory regions can be
harmful.
Genotype: Representation of a pair of alleles carried by a person
Phenotype: Cell or bodys interpretation of a genotype
SICKLE CELL ANEMIA:
Characteristic variations in gene sequence allele is passed down from parent to child
RBCs carrying variation of the hemoglobin protein have sickle shaped cells that behave
differently from normal biconcave round RBCs

Sickle shape is cellular phenotype of the disease

Oxygen not carries as efficiently and old sickled blood cells can block capillaries of
circulatory system
o Leads to pain (caused by damage of vital organs and tissues)
o Leads to anemia

Variation in the gene:

Gene for beta-globin is on chromosome 11

Normally:
o People have to HbA alleles that both code for the functional protein
Sickle cell patients:

People have HbS genotype caused by single nucleotide polymorphism in the

protein coding sequences of beta-hemoglobin gene
Amino acid substituted from glutamine to valine
Reduces oxygen binding capabilities
Biochemical change at the protein level leads to aggregation of betahemoglobin protein which creates rod like structures within RBCs
causing the sickle shape
If patient is heterozygous (HbA/HbS)
o Sickle cell trait is developed
o Some hemoglobin is sickled under certain condition but the rest is largely
normal
o No symptoms of sickle cell anemia exhibited
o

VARIATION IN POPUL ATIONS

No two human individuals have the exact same genome due to polymorphisms (DNA
fingerprinting makes it possible to conduct large scale population genetic analysis by looking
at variation across populations)
Variations in gene copy number can contribute to genetic difference between individuals:

Copy number variations (CNVs): When the number of copies of a particular gene
varies from one individual to the next
o Some occur in noncoding regions while others can be present as many
tandem copies of a coding region along a chromosome
o CNVs can be identified based on relative fluorescence intensities during
microarray analysis
More copies = higher fluorescent output
DNA duplications are usually found to be adjacent to each other on chromosomes
o Human AMY1 gene: Codes for amylase
Detectable number of copy number differences on chromosome when
comparing individuals with different ancestral diets
o Gene copy variations is a reflection of selective pressures
Selective advantage in high starch diets to have more amylase
production

SICKLE CELL DISEASE

Being heterozygous for sickle cell anemia in populations where malaria threat is endemic
and continuous is an advantage; heterozygous for sickle cell allele = resistant to malaria
Haplotype: A set of DNA variations or polymorphisms that tend to be inherited together
In areas with the highest prevalence of sickle cell anemia, there is 5 possible distinct betaglobin haplotypes that correlates with regional distribution of each distinct sickle cell anemia
single nucleotide polymorphism

Different nations have different haplotypes that are more or less prevalent
o Multiple haplotypes can be found in a nation
Haplotypes are broadly distributed

T5M2 MEIOSIS
Unlike asexual organisms, sexually reproducing organisms do not produce identical copies of
themselves

Chromosome passed from parent to offspring through gametes

o Gametes are the only cells not made by mitosis
Traits are inherited by offspring with lots of genetic variation
Two parents give rise to offspring with unique combinations of genes inherited from
both parent

In humans, males produce 4 haploid sperm cells per sex cell precursor while females
produce one egg with a large cytoplasmic volume and 3 non-gamete polar bodies which
serve to reduce chromosomal content of the egg.
The unique haploid gametes pass genetic information onto the next generation

If gametes were diploid, offspring would have double the chromosome number
needed

Meiosis leads to reduction in chromosome number followed by recombination of parental

homologous chromosomes to create a diploid zygote
CHROMOSOME VOCABULARY
1. Sister chromatids: A duplicated chromosome
2. Homologous chromosomes: The individual chromosomes that have been inherited
from each parent
3. Non-sister chromatids: Replicas of different chromosomes that are genetically similar
but not identical
a. Due to maternal and paternal nature of non-sister chromatids
At the end of meiosis: Daughter cells receive one set of unique chromosomes which makes
the cells genetically different from each other and the parent
PHASES OF MEIOSIS
Before meiosis 1 occurs, duplication of the chromosomes occurs during interphase.
Meiosis: Two consecutive cell divisions with no interphase between that results in the
production of 4 haploid daughter cells
MEIOSIS 1
1. Prophase I
a. Chromosomal condensation and synapsis of homologous chromosomes along
their length
i. Synapsis: Pairing of and physical connection of homologous
chromosomes
1. Gene for gene pairing
ii. All homologs pair during synapsis to allow for the formation of a
bivalent unit containing a pair of synapsed chromosomes

1. Forms a 4 stranded structure

2. Chromatids attach to different centromeres
b. Facilitated by synaptonemal complex
i. Construct that forms between homologous chromosomes and holds
them together during synapsis
c. Crossing over: As chromosomes condense, rearrangement of genetic
information occurs between homologs (specifically between non-sister
chromatids)
i. Occurs at the chiasma (x-shaped regions on paired chromosomes)
ii. Results in physical breakage and reunion between non sister
chromatids
1. Produces characteristic chiasmata
iii. Random process that can occur anywhere on the length of the paired
homologs
1. Allows for the production of recombinant chromatids with some
parental and some maternal segments of genetic information
2. Leads to genetic diversity
2. Metaphase I
a. Homologous chromosome bivalents become randomly arranged relative to
each other at metaphase plate by microtubules of spindle apparatus
i. Random arrangement = increased genetic diversity
3. Anaphase I
a. Protein that holds homologs together (synaptonemal complex) breaks down
i. Results in separation of homologs to opposite poles of cell
b. Sister chromatids DO NOT separate
4. Telophase I
a. Beginning of the phase: Each cell has a complete haploid set of duplicated
chromosomes
i. Each chromosome consists of a pair of sisters
ii. Called reduction cell division number of chromosomes reduced
b. Nuclear envelop reforms followed by cytokinesis
MEIOSIS 2
Meiosis II: Equational division parent cells at the start have the same number of gametes
that are produced by the end of meiosis II (haploid at the start and finish); similar to mitosis
except it is haploid instead of diploid
1. Prophase II
a. Two recombinant chromosomes condense
b. Nuclear envelope breaks down
c. Spindle apparatus forms
2. Metaphase II
a. Chromosomes align in the centre of the cell along metaphase plate
b. Sister chromatids are not genetically identical b/c of crossing over in prophase
I
3. Anaphase II
a. Sister chromatids are pulled apart
i. Protein that holds the sister chromatids together at centromere are
broken down
b. Chromatids moved to opposite poles of cell
4. Telophase II
a. Nuclear envelop reforms
b. Each cell has produced 2 daughter cell (4 in total)

c. Chromosomes de-condense
d. Cytokinesis follows
NONDISJUNCTION
Ideally, the meiotic spindles will distribute the appropriate number of chromosomes to
daughter cells without error but sometimes sister chromatids or homologous chromosomes
do no separate in meiosis I or II.

Results in some gametes having extra chromosomes while other are missing them
o Called nondisjunction

Non disjunction can lead to detrimental/lethal effect in generated offspring

MENDELIAN INHERITANCE
1800s: Concept of heredity was based on the blending hypothesis which stated that genetic
material from both parents mixed to produce visible traits in offspring
Gregor Mendel: A monk who identified and documented two basic principle of inheritance
based on traits observed in pea plants

Tracked traits that had two distinct and alternate forms

o Seed colour, seed shape, colour of pod, shape of pod, colour of flower, etc.
Only started with true-breeding plants (homozygous plants that carried two identical
alleles)
o Cross true breeding plants and then crossed the produced offspring to
determine mathematical patterns of future crosses and inheritance
Differentiated between traits as dominant or recessive

Dominant traits: Traits that appear in the offspring of a cross between true breeding or
homozygous parents
First generation cross:

Started with two contrasting, true breeding pea varieties and crossed the parents
o I.e. cross with a true breeding yellow seed pea with true breeding green seed
pea
Produced F1 of all yellow seed peas Yellow is therefore the dominant trait
o Considered the parental generation

Second generation cross:

If blended model was correct the F1 offspring would produce offspring with an
average between yellow and green colour
Real result: All f1 offspring had a yellow coloured seed
o If green trait was lost, then the F2 would also be all yellow
o BUT, when F1 seeds self-pollinated, they did not breed true
Dominant to recessive seed colour trait seen in 3:1 ratio

Hypothesis: The heritable factor for recessive traits do not disappear. Instead, it is masked
by the dominant allele

FIRST LAW: LAW OF SEGREGATION

First law of segregation: Stated that two alleles of a gene segregate into different games
during gamete formation in both parents; each offspring inherits one copy of a gene from
each parent

Based on assumption that alternative versions of genes accounted for variations in

inherited traits
If two alleles at a locus differ, the dominant allele determines the expressed trait
o If true breeding, the allele is found in all gametes
Explanation:
o Specific traits attributed to the separation of chromosomes during meiosis
Homologous chromosomes separate in anaphase I
After meiosis II: Copies of each allele in each chromosome separated
into one of four possible gametes in each parent

Punnett squares: Illustrates possible allele composition of an offspring from crossing

individuals of known traits

Predicts expected genotype and phenotype ratios of offspring

Test cross: Breeding a phenotypically dominant individual with a phenotypically
recessive individual to determine the zygosity of the former by analyzing proportions
of offspring phenotypes
o Determined the 3:1 phenotypic ratio F2 and 1:2:1 genotypic ratio this way

SECOND LAW: LAW OF INDEPENDENT ASSORTMENT

Law of independent assortment: States that when two or more characteristics are inherited,
individual hereditary factors assort independent during gamete production, giving different
traits an equal opportunity of occurring together
Monohybrid cross: A mating between two individuals with different alleles at one genetic
locus of interest

Following 2 traits at once allowed Mendel to identify the second law of inheritance

Process:
1. Crosses performed between plants that were homozygous for two traits
a. Strain that was yellow + wrinkled vs. strain that was green + smooth
2. Because of the dominant nature of the yellow and smooth traits, he found that the F1
offspring contained peas that were yellow and smooth
3. Allowed F1 plants to self-fertilize to observe genotypes of F2 generation
4. Observed a phenotype ratio of 9:3:3:1 in F2 offspring
a. The result of independent assortment is visualized in the four possible gamete
types that are observed for each parent in the F1 generation
i. Each parent will contribute to one gamete type to the produced F2
offspring
Note: The law of independent assortment only applies to genes that are located on different
chromosomes

Explanation:

Law of independent assortment largely dependent on the manner in which nonhomologous chromosomes align at the metaphase plate in meiosis I
o In one alignment, chromosomes with dominant allele of one gene segregates
to the same pole as the dominant allele of another
Another scenario: Dominant allele segregates to the same pole as
recessive

FROM GENE TO PROTEIN

Mendel studied the B gene which was later identified as SEB1
Importance of SEB1:

In the development of pea seed, sugar is converted to starch by an enzyme from

SEB1 gene
o In wrinkled peas, the gene is interrupted and does not allow for conversion of
starch from sugar
o In bb plants, the level of sucrose is nearly doubled of BB plants
Wrinkled bb young seeds contain more water because of high level
of sucrose, but as the seed matures, water is lost and the seed shrinks
to become wrinkled

Shows that principles of inheritance can give insight into inheritance of molecular variations
at a gene level
T5M3 SEX CHROMOSOMES AND LINKAGES
Geneticists look at family history to build a family tree/pedigree which allows for the visual
representation of segregation for the specific traits of interest. Pedigrees illustrate the
transmission of a trait from one generation to the next.

Males = squares
Females = circles
Affected = shaded in with a colour
Progeny of mating arranged horizontally with order of birth ordered left to right

Human have 23 pairs of chromosomes

Consists of 3 billion base pairs and over 20, 000 protein coding genes in addition to
genes coding for functional RNA

Alleles are linked on a single chromosome and must be able to become unlinked and
recombine in different combinations
THE INHERITANCE OF GENES ON THE X CHROMOSOME
The chromosomal basis for determining sex is unique; humans and other mammals have
two types of sex chromosomes (X and Y); females have two 2 X chromosomes and males
have 1 X and 1Y.

Most regions on X and Y chromosomes are largely non-homologous

o Almost none of the genes in the X chromosome have counterparts in Y
o Small regions at the tips of X and Y chromosomes allow for the chromosomes
to pair and segregate like homologous chromosomes during meiosis

Human Y chromosome:

78 genes
Codes for 25 proteins
o Half of these help determine sex

Human X chromosome:

1100 genes
Most genes have many functions unrelated to sex determination

Sex-linked genes: Genes on a sex chromosome; expression of phenotype depends on the

sex of the individual
Mendels principles of inheritance do not apply for genes on the X and Y chromosomes
X-LINKED INHERITANCE OF COLOUR BLINDNESS
The inheritance of alleles on the X chromosome can be monitored through the construction
of pedigrees; pedigrees follow grandparents to parents to children.
The recessive disorder of red/green colorblindness is inherited as an X linked trait that is
associated with an X linked gene on the X chromosome
Ishihara colour test: Common way to determine colorblindness

Utilizes a circle containing different coloured dots with an internal pattern of dots that
forms a number shape visible to people with normal vision

Women heterozygous for colour-blindness do not show the phenotype

They become carriers and can pass the allele to offspring

Only homozygous women are colour blind

Males with one colour-blind allele will be colour blind because they only have one locus for
the allele

Hemizygous: Only having one sex-linked allele (occurs in men) which disallow for the
rule of dominance and recessiveness

X-LINKED INHERITANCE OF HAEMOPHILIA

Haemophilia is a blood clotting disorder that is an X-linked recessive trait that results from a
mutation in a gene that encodes for a necessary protein required for blood clotting.

For a female to be affected, they would have to inherit the allele from the mother and
father

GENETIC LINKAGE
Genes can be physically attached or linked on a chromosome (colour-blindness and
haemophilia are associated genes)
Example: Homologous pair of x chromosomes in females

Chromosome has 155 million base pairs with 1100 genes

P arm = short arm
o Dystrophin gene: codes for protein needed for muscle cell development
Q arm = long arm
o HPRT1 gene: when mutated, can lead to severe and recurring cases of acute
arthritis
Genes positioned close together on the same chromosome are called linked genes
and tend to be inherited together (DO NOT SEGREGATE INDEPENDENTLY)

Linked genes are not always inherited together the linkage can be broken

In prophase of meiosis: all homologous chromosome pairs line up beside each other
and form chiasmata
o Linkages between neighboring genes on chromosomes can be broken
o Alleles of neighboring linked genes can be separated during recombination
events in meiosis I during gamete formation
Leads offspring that have genes (once linked) that independently are
inherited

RECOMBINATION OF ALLELES
During recombination, the position of genes do not change the relative association of
alleles changes
If linked genes are far enough apart, recombination chromatids that carry alternate allele
combinations are generated during cross over events

If linked genes are very close: No crossing over during meiosis I OR any crossovers
that do occur would not be in the region between the two genes
o Result: No recombination of alleles, little or no cross-over between the two
genes

Recombination between linked genes depend on distance between them

Closer = less recombination frequency

Farther = more recombination frequency

CONSTRUCTING LINKAGE MAPS

Linkage between the genes for haemophilia and colour-blindness would lead to the
prediction that the two genes would be inherited together sometimes this does not occur
(i.e. a child only has haemophilia or only colour-blindness)

Explained by the fact that the separation of these alleles must have occurred by
crossing over or recombination of alleles
o The frequency of these exception can tell us something about the distance
between the genes

The relative distance between genes on a chromosome allows researchers to make a linkage
map
Linkage map: Maps that show the distance between chromosomes as well as the order of
genes along the chromosome

Haemophilia and colour-blindness genes are about 12 map units (centimorgans)

apart on the X chromosome (about 12 million base pairs apart)
o Distance is large enough for crossing over to occur
Impractical for human applications

GENETIC MAPPING IN HUMANS

Instead of linkage maps, we can turn to SNPs and other markers that do not lie in the coding
regions of chromosomes to create high density maps

High density linkage maps: Linkages maps that identify genetic loci that are merely a
few thousand base pairs apart
o Principle is same as before
We are looking at the frequency of recombination to determine the
relative distance between two genetic loci (can be genes, markers or
both)

Process:

Gene is represented by two alternate alleles (mutant and non-mutant)

o Marker is similarly represented by two alleles (a G-C pair or an A-T pair)
In image b, Marker B is far from the disease gene
o Far enough away so that recombination will always occur in the interval
o G-C marker allele does not remain with the mutant allele and the A-T marker
does not remain with the G-C marker allele
In offspring, the G-C allele is found with the nonmutant alleles and the
A-T allele is found with the mutant allele
o Since we see all four combinations of alleles with the same frequency, 25% of
the time each, there is no association between the disease gene and Marker B
In image a, Marker A is close to the disease gene
o There is not an equal representation of the allele combinations
The mutant allele with the G-C allele and the nonmutant allele with the
A-T allele occur more frequently (49%)
Recombination between the disease gene and Marker A is rare
and the distance between the two loci is relatively small
o There is an association between the diseased gene and Marker A
Since we know the exact location of markers from human genome maps, the
approximate location of the diseased gene can be determined

GENOME WIDE ASSOCIATION STUDIES

Pedigree analysis limits studies to a few hundred or thousand individuals related through
ancestry
SNP-linkage maps and technology can identify SNPs in a quick and cheap manner to look at
tens of thousands of unrelated individuals to map the location of genes
Genome wide association study:

Looks across an entire SNP linkage map for association between phenotypes and SNP
o Example: Linking gene for increased height and a marker
Helps researchers identify genes that contribute to human characteristics

T5M4 OUR PERSONAL GENOME

Cellular proteasomes are driven by different transcriptional programs that direct different
cells to engage in specific functions in association with other cells or within tissues

Example:
o White blood cells can migrate in blood vessels and engage in monitoring for
infection, inflammation and pathogens
Very different from the role of red blood cells which carry oxygen to
tissues engaging in high metabolic activities

THE IMPORTANCE OF GENETIC VARIATION

Genetic variation: Refers to diversity in gene frequencies; can influence differentiation and
proliferation of different cell types

Variations largely contribute to genetic variability that can lead to positive or

negative effects on body
Information in genomes that codes for important functional proteins require high
fidelity of replication, transcription and translation
o Small alteration can alter the shape and function of the cell

Some genetic variations can contribute to diversity in a population:

Example: ABO blood typing system

Everyones RBCs bind and carry oxygen the same way

Transfusions of non-self blood of a different blood type = tragic consequences
o Showed that not all blood is the same
Blood type: Classification of blood based on presence or absence of inherited cell
surface proteins or enzymes
o ABO locus: 3 main alleles A, B and O
A and B code for specific glycosyltransferase enzymes
Catalyzes formation of A or B agglutinogens expressed on the
cell surface
O encodes for inactive glycosyltransferase
AB blood type has several polymorphisms that lead to formation of
slightly different transferases
o Alleles genetically inherited from parents

In the right environmental conditions, mutations can be beneficial

Example:

HIV virus invades T cells by interacting with 2 proteins on the cell surface (CD4
receptor and CCR5 co-receptor)
o When attached, the virus is engulfed by the cell and the infection begins

Leads to death of T cell and a compromised immune system

Mutations have developed to be beneficial by protecting against the disease
o Some people have mutations in the CCR5 gene = immunity to HIV
32 base pair deletion
Frame shift mutation that shifts the reading frame and creates an early
stop codon
Why is this mutation prevalent if beneficial effects seen only during infection?
o Mutation conferred resistance to mid-14th century outbreak of the bubonic
plague
Mutated individuals = more likely to survive
o Other theory: Selective pressure imposed by smallpox led to prevalence of
mutation

MICROBIOME VARIATIONS
There are 10x as many bacterial cells in the body compared to human cells; microbiome
diversity depends on history of exposure to different bacteria, exposure to antibiotics and
interactions with the environment
Researchers studying variations in microbiomes in the intestinal tract:

Examined DNA sequences from gut microbiomes of people in America, Japan and
France
Findings:
o Distinct mixtures of bacterial species associated with each region
o Different populations have distinct collection of genes
Could allow the production of different vitamins, enzymes and may
affect disease susceptibility

Variations in microbiomes do not correlate with ancestry but is actually associated with
recent dietary patterns

Suggests that microbiome can respond rapidly to diet and environment change

APPLIED LECTURES
NONDISJUNCTION AND DISEASE
Ideally, the meiotic spindle distributes chromosomes to daughter cells without error.
In the ovaries, females are limited to the number of eggs in the ovaries just after birth, egg
cells go into meiosis (some remain in meiosis I for the entire duration of the females
lifespan until the release of one egg each month)

Brief period of mitotic divisions occurs before the entry of all cells into meiosis
Some cells undergo apoptosis, others stay in prophase I and arrest
o Individual cells re-enter and complete meiosis after puberty

In the testes:

Brief period of mitosis and then entry into mitotic arrest

After puberty, male germ cells resume mitosis

At puberty, cells are stimulated to undergo meiosis

Nondisjunction: Failure of either of the two homologous chromosomes to pass to separate

cells during the first meiotic division or of the two chromatids of a chromosome to pass
separate cells during mitosis or second meiotic division

When occurring in mitosis leads to cell lineages with extra/missing chromosomes

o Seen in cancer cells
o Usually the somatic cell will just self-destruct

Aneuploid cells have an abnormal number of chromosomes; one may have too many copies
of a gene leading to too much gene product while the other may have too few copies leading
to not enough products

In sex cell development, can lead to in utero or abortive effects

Despite genetic abnormalities some fetuses survive to term and resulting infants display
developmental abnormalities:
1. Trisomy21 = down syndrome
2. XXY = phenotypes of female and male present
a. XXX or XXY they cant know if they have the mutation
Polyploidy: Fertilization of a diploid and haploid absolutely lethal
Monosomy: Absence of a chromosome in a gamete that combine with a normal gamete
(zygote only contain one copy) sometimes that missing chromosome can be compensated
2n-1 (2n is the haploid number and -1 = 45 chromosomes)

Trisomy: The presence of two of the same kind of a chromosome in one of the gamete
2n+1
DOWN SYNDROME
Down syndrome is causes by chromosomal alterations that results in 3 copies of
chromosome 21 due to nondisjunction in meiosis I

Predisposition in older women bearing children

o Eggs in meiotic arrest for so long that the machinery is not working
Affect 1/700 children
Symptoms:
o Developmental challenges
o Broad face, hands, nasal bridge, wide-set eyes
o Heart, immune and skeletal defects

Diagnosis of down syndrome can occur in utero by looking at biomarkers in the amniotic
fluid (looking for excess proteins from chromosome 21)

If a protein shows double the difference, it will be partitioned into its own separate
groups
In DS, over 150 proteins are expressed differently
Process:
o Characterize proteins present in amniotic fluid of both groups
o Compare relative differences
o Identify proteins that show greater than a 2-fold difference between the 2
groups
o Bioinformatics analysis infers the molecular interactions

KLINEFELTER SYNDROME
Klinefelter syndrome is the result of an extra X chromosome in a male, producing a XXY
individual (most common sex chromosome aneuploidy in humans)
Symptoms:

Partial breast development

Recessed/smaller testes
Relatively infertile
Tall, slightly feminized physique

TURNERS SYNDROME
Turners syndrome is the result of monosomy of the X chromosome to produce XO females

Symptoms:
o Webbing in neck
o Cardiac problems
o Unstructured reproductive structures
o Generally infertile

It is the oly known viable monosomy in humans but mostly result in spontaneous abortions

ANEUPLOIDY AND CANCER

Oncogenes: A gene that in certain circumstances can transform a cell into a tumor cell
Mitotic chromosome instability is a feature of most cancer cells; chromosome instability
leads to an imbalance in chromosome number which causes an activation of oncogenes and
inactivation of tumour suppressors

Causal relationship between aneuploidy and tumorigenesis

In cancer cells:

There is misregulation of cell cycle checkpoints

o Causes uncontrolled growth and division
Cells progress through the cell cycle unchecked and can form malignant tumours with
the potential ability to metastasize throughout the body

A weakened or overactive mitotic checkpoint facilitates chromosome instability

Weak checkpoint = chromatids may not be properly attached

Overactive checkpoint = separation happens too early and extra sister chromatid
stays

An anticancer drug isolated from the bark of the Pacific Yew Tree is now synthetically made
and can inhibit cell division and growth of cancer cells by binding to tubulin subunits and
stabilizing microtubules against depolymerisation

Cells remain in metaphase-anaphase which leads to apoptosis

GENOME WIDE ASSOCIATION STUDY

Linkage: The physical proximity along a chromosome that indicates the frequency of
recombination

Linked genes sit close together on a chromosome so that the two alleles on the one
chromosome are more likely to be inherited together

Association: Increased probability of seeing alleles of two genes or two markers together due
to linkage
Expansive maps of the genome are based on polymorphisms such as SNPs, repeating
regions, insertions and deletions

Some maps have base pairs only a few hundred base pairs apart
Linkage between a marker and a trait suggests the gene for the trait and marker are
close together

With genome wide association studies, linkages between a traits and millions of polymorphic
SNPs are observed across thousands of unrelated people
WELCOME TRUST CASE CONTROL CONSORTIUM
The Welcome Trust Case Control Consortium was the first large, genome-wide association
study to examine complex diseases using a SNP-chip

Chip enable scientists to scan an entire genome for SNPs in a single experiment

Process:

There are two distinct populations, different in a trait we are interested in

o For most SNPs, we wont see any association but for a few markers, there will
be a bias that suggests an association
Example a population may have an association where individuals are more likely to
have a C allele than a T allele as compared to a control group
o More likely to see C allele in affected individuals
o Equally likely to see C or T allele in controlled individuals
Odds ratio tells the likelihood that we will see a marker in one population more than
another

Using chi square analysis, a p value can be calculated

Manhattan plot: A graph in which each dot on the diagram represents a molecular marker

Location represented on the x axis

Probability value is on the y axis
Dots above the threshold are showing linkage to the disease allele and are
statistically significant
o Strongest associations occur at the top because they have the lowest
probability of occurring by change
o Lines show that several SNPs are lying next to each other that have an
association
Looking for peaks where they is a collection of dots to identify new candidate genes