Download doc BIOL 112 Course Summary 2013

BIOL-112 Condensed Notes
R. O’Loghlin
Bonding
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Covalent Bonds
o When an atom has an orbital with only one electron in it, it becomes reactive, wanting
to fill the space
o Strongest bonds
o Made up of 2 electrons, one from each atom
o The electrons are shared between the two atoms, not always equally, but shared
Hydrogen Bond
o Electrostatic interaction between partial negative charge of an atom and the partial
positive charge of another atom
o Both must be involved in polar covalent bonds
o Only occurs between Hydrogen and Oxygen or Nitrogen, or (more rare) Carbon and
Oxygen
Ionic Bond
o Occur between elements on the far left and far right of the periodic table
o Ionic bonds in biology aren’t as strong as the ones in chemistry due to the biological
ones usually being individual bonds such as those in the interior of proteins in the
absence of water
Polar molecules tend to be hydrophilic, and dissolve in water
Nonpolar molecules are hydrophobic, and are generally made up of c-c or c-h bonds
Hydrophobic interactions
o Water forces nonpolar molecules together, because doing so minimizes their disruptive
effects on the h-bonded water network
o Fairly weak
Van der Waal’s interaction
o Nonpolar molecules are also attracted to eachother via weak attractions caused by
transient dipoles (temporary)
o Strength increases with increasing molecular weight
In order of decreasing strength:
o Covalent bond (50-110 kcal/mol)
o H-bond and Ionic Bond (Both 3-7 kcal/mol)
o Hydrophobic interaction (1-2 kcal/mol)
o Van der Waals interaction (1 kcal/mol)
Acids & Bases
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Acids release/donate
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o Strong acids completely disassociate in solution
Bases accept
in solution, or you could say that they release
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ions in solution
o A strong base completely disassociates in solution
Buffers
o Make the overall solution resistant to pH change
o Can make a buffer out of a solution of a weak acid and its conjugate base, or a weak
base and its conjugate acid
o Law of Mass Action
 The rate of any given chemical reaction is proportional to the concentration of
the reactants
Functional Groups
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Carboxyl group
o Carboxylic acids
o –COOH (with double bond between C and one O)
o Acetic Acid
Amino group
o Amines
o –NH2
o Methylamine
Hydroxyl group
o Alcohols
o –OH
o Ethanol
Carbonyl Group
o –CO
o Aldehyde if the carbonyl group is at the end of the molecule
 Acetaldehyde
o Ketone if it is in the interior
 Acetone
o Phosphate group
 Organic phosphates
 -PO4
 ATP
o Sulfhydryl Group
 Thiols
 -SH
 Mercaptoethanol
Large Molecules
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Macromolecules are made the same way in all living things, and are present in all organisms in
roughly the same proportions
Proteins, nucleic acids, and lipids can form polymers of multiple molecules
o Reaction is called polymerization
o Condensation reaction releases a molecule of water for each bond formed
o Depolymerisation involved hydrolysis, consuming a water molecules to break a bond
Isomers
o Molecules that have the same chemical formula, but different atomic arrangements
o Structural isomers
 A group is attached to different carbon atoms
o Optical Isomers
 A group is attached in a different way to the same carbon atom
 Optical isomers are non-superimposable mirror images of eachother
 Occur whenever a carbon has four DIFFERENT atoms or groups attached to it
Sugars
o Carbohydrates (sugars) act as energy storage and building blocks for other molecules
o Serve a structural components
o Monosaccharides
 A single sugar such as glucose
o Disaccharides
 2 Sugars such as sucrose
o Polysaccharides
 Many sugars
o General carbohydrate formula is CH2O
o Glucose has two ring forms
 Alpha and Beta glucose
 Optical isomer caused by four different groups being bonded to carbons 2-5
DNA
o Contains genetic information
o Transcribed to RNA, which makes proteins
o Nucleotides have additional functions as signalling molecules and energy transducers
Phosphate groups
o Joined to the C5 hydroxyl of the ribose sugar
Nitrogenous Bases
o Purines
 Adenine
 Guanine
o Pyrimidines
 Thymine
 Cytosine
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 Uracil (only in RNA, replaces thymine)
o A purine always pairs up with a pyrimidine (chargaff’s law)
o Guanine-Cytosine bond is stronger than Adenine-Thymine due to having 3 binding sites
rather than 2
o Base pairs are stabilized by hydrogen bonds
Nucleotides
o Base connected to C1 of the Sugar
o Phosphate connected to C5
o Phosphate group of a nucleotide binds to the 3-prime hydroxyl group of the next
DNA is antiparallel
o One strand goes 3 prime to 5 prime, the other is opposite
o Polymerization always happens 3 prime to 5 prime
RNA
o Main function is to act as an intermediate between DNA and proteins
o Often forms 3d structures
o RNA evolved before cells did
Lipids
o Insoluble in water
o Aggregate away from water (Hydrophobic Interactions)
o Attracted to each other by Van der Waal’s forces
o Fats and oils are used for energy storage
o Phospholipids used in cell membranes
o Carotinoids used in capture of light energy in plants
Fatty Acids
o Long carbon chain with a lone hydroxyl at the end
o Saturated fatty acids carry the maximum amount of hydrogen atoms
 Straight, form part of animal fats
o Unsaturated fatty acids have at least one carbon-carbon double bond
 Causes kinks that prevent easy packing
 Caused by a cis-configuration around the double bond
 Part of plant oils
 Liquid at rom temp
Triglycerides
o 3 fatty acids bound to a glycerol molecule via ester linkages
Phospholipids
o Have two hydrophobic fatty acid tails
o One hydrophilic head
o All attached to a glycerol molecule
o Self-assemble into a bilayer due to H-bonding and hydrophobic interactions
o Hydrophobic tails stay inside the layer, the hydrophilic heads being on the outside
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In the winter, certain fish and plants increase the number of unsaturated fatty acids in
their membranes to keep them fluid
Proteins
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Proteins are polymers of amino acids
Range in size from a few amino acids to thousands
o Titin, the largest, is 33000 amino acids in length
Folding is crucial to the function of proteins
o Influenced by the sequence of amino acids
The alpha carbon in the amino acid is attached to an amino group, carboxyl group, and R group
o R-group determines identity of the amino acid
Peptide linkages are the covalent bonds between two amino acids
Every protein starts with an amino group (NH2), and ends with a carboxyl group (COOH)
The precise sequence of amino acids in a protein is the Primary Structure
Amino acids can be positively charged, neutral, or negatively charged depending on the R-group
o Some are polar, but do not carry a charge, such as Serine
 These are hydrophilic
 Occur with OH and carbonyl R-groups
o Some special cases
 Cysteine has an SH R-group
 Proline forms a ring between the alpha-carbon and the amino group
 The amine nitrogen is bound to two alkyl groups
A protein’s secondary structure consists of regular repeated patterns in different regions of
the polypeptide chain
o Alpha-helix
 H-bonds formed by the backbone are parallel to the axis of the helix
 Between amino and carboxyl groups
 Has a rigid structure
 Can insert into plasma membranes
 ONLY if the helix ONLY contains hydrophobic amino acid side chains
o Coiled coils
 Two alpha helices wrapped around each other
 Small stripe of hydrophobic amino acids occurring every 4th position
 One complete turn is 3.6 amino acids
 Fibrous structural proteins consist mainly of alpha helices arranged in coiled
coils
 Keratin
o Beta-pleated sheet
 Makes flat plates
 R-groups project up and down from the sheet
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The strands of the beta sheet can run in parallel or antiparallel direction
Can even come from different polypeptides
Occasionally can be inserted into the plasma membrane (Rare)
 Forms a barrel shaped object with R-groups pointing outside
Proline
o Alpha helix and Beta sheet breaker
 Appears at the end
o Doesn’t really fit in either structure because it makes a kink in the peptide
 Also because the N carries no H for bonding
Tertiary structure
o Tells how the short stretches of alpha helices and beta sheets fold together to make a
protein
o Determined by:
 Location of disulphide bridges
 Covalent bond between two cysteines
 Location of secondary structure
 Ionic interaction between positive and negative charges in the protein
 Hydrophobic aggregation of R groups stabilized by Van der Waals forces
 Most important factor of tertiary structure
 Tries to move interior away from water
Loss of a protein’s normal three-dimensional structure and function is called denaturation
o Caused by changes in temperature or pH
o Proteins will refold in a test tube
 Shows that proteins automatically fold
 All folding info is contained within the primary sequence
o Denatured proteins in cells do not typically refold because of other partially folded
proteins
Chaperones are specialized proteins that keep other proteins sequestered, providing optimal
folding conditions
Membranes
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Phospholipids self-assemble into the lipid bilayer
Cholesterol
o Hydrophobic, inserts into membranes causing them to become stiffer
 Caused by the rigid ring structure
It is energetically favorable for bilayers to seal/form an enclosed space
Lipid bilayer is fluid, so phospholipids can move around inside the membrane
o Lateral diffusion is most common
o Flexion
o Rotation
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Phospholipids and glycolipids are asymmetrically distributed along the membrane with respect
to inside/outside
Some proteins bind to specific phospholipids
Transmembrane proteins
o Have hydrophobic regions of amino acids that cross the membrane
o Alpha helices and beta pleated sheets (rare)
o Have specific orientations, with an inner part and outer part
Peripheral membrane proteins
o Lack hydrophobic regions, are not embedded in the bilayer
o Covalently attached to lipids, or bind noncovalently to other transmembrane proteins
 Used for signal transduction, molecule transport, energy generation, and cell
adhesion
Diffusion
o The passive mixing of substances resulting in transport along a concentration gradient
o Brownian motion – random movement of molecules due to thermal motions and
collisions
o Rate is effected by distance, temperature, size of molecule, and steepness of the
concentration gradient
 Mainly concentration gradient
o Hydrophobic molecules diffuse easily through cell membranes
o Gases and water cross freely
o Large polar molecules and ions cannot cross through diffusion
Osmosis
o Diffusion of water across a selectively permeable membrane
o Concentration gradient determined by concentration of dissolved solute in the water
o Cells shrink in hypertonic solutions
 Hypertonic – Inside cell has higher solute concentration than outside
o In a hypotonic solution, water will move into the cell, expanding it and possibly bursting
 Hypotonic – Inside cell has lower solute concentration than outside
Passive transport – Facilitated diffusion
o Two types of membrane proteins
 Channel and carrier
o Ion channels
 Most important channel protein
 Can be open or closed (gated)
 Specific for one type of ion
o Carrier proteins bind the substance to be transported
 Changes shape after binding to substrate
 Transition between the open out, bound, and open in is random and
reversible
 Can become saturated when all binding sites are occupied
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Rate of diffusion lowers when this happens
Active transport
o Requires expenditure of energy
o Substances are moved across the membrane against the concentration gradient
o Primary active transport
 Sodium potassium pump in animals
 3 NA ions out, 2 K ions in, uses 1 ATP
 Net change of -1, generates membrane potential
o Secondary Active Transport
 Use established gradients to move substances
 Symport
 When ions diffuse from outside to in, the energy generated allows the
symport protein to move the molecule or substance through
 Both molecules move in the same direction
 Antiport
 Uses the energy generated by an ion diffusing from inside to out to
move the molecules through
 The molecules move in opposite directions
 These are often paired with Primary Active Transporters
Cells
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Low end estimate of number of cells in the body – 10 trillion
Cells are small because most chemical reactions in cells require diffusion
High surface area to volume ratio is ideal for diffusion
Eggs (one cell) are large because most of the volume is storing food material
All organelles in eukaryotic cells are connected by vesicles that bud off of one compartment and
fuse to the next, moving molecules between organelles as needed
o Orientation of the membrane is preserved when this happens
The Nucleus
o Contains most of the cell’s DNA
o Ribosomes are assembled in the nucleolus
o DNA is replicated and transcribed into mRNA or rRNA by RNA polymerases
o rRNA is not translated, it directly folds into 3D structures
 4 rRNAs + about 80 proteins make up a ribosome
o In eukaryotes, ribosomes are found free in the cytoplasm, in mitochondria, bound to
the ER, and in chloroplasts
o Two lipid bilayers form the nuclear envelope
 It is perforates with nuclear pores which are selectively permeable to RNA and
some proteins
Endoplasmic Reticulum (ER)
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Network of interconnecting membranes distributed throughout the cytoplasm
Internal compartment, called the lumen, is a separate part of the cell with distinct
protein and ion composition
o The ER’s folding generates a huge surface area, larger than the plasma membrane
o Continuous with the outer nuclear envelope at certain sites
o Rough ER
 Has ribosomes attached
 They come from the cytosolic pool of ribosomes and are directed to the ER after
they have translated the first few amino acids containing a signal sequence that
directs them to the ER
 Oligosaccharides (3-12 subunits) are attached to the proteins
o Smooth ER
 Ribosome free segment
o All lipids are synthesized in the ER
o Enzymes in ER detoxify many substances by adding OH groups
Golgi apparatus
o Essentially the sorting and distribution center for the cell
o Receives proteins from ER and modifies them
o Adds oligosaccharides to membrane lipids
o Concentrates, packages, and sorts proteins before they’re sent to their destination
o Sugars added to lipids in the Golgi will end up on the outside of the cell membrane
Lysosomes and Endocytosis
o Lysosomes are cells that transport material from the Golgi apparatus and secrete it
out of the cell through exocytosis
 Have a low pH for breaking down and digesting any useful material before
excreting the waste
 Sites for breakdown of food and foreign material brought into the cell
 Phagocytosis
 Can eat their host cell if nutrient deprived
 Leaves in fall
o Endocytosis is when materials are taken into the cell by endosomes, budding inwards
into the cell
Mitochondria
o Outer plasma membrane and a highly folded inner membrane
o Cellular respiration occurs here
o Region inside the inner membrane contains many enzymes for respiration
Chloroplast
o Carries out photosynthesis
o Double membrane
Endosymbiont theory
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Mitochondria and chloroplasts are descendants of bacteria taken into a cell by
endocytosis
o Evidenced by double membrane
o They have their own genome
o Their own ribosomes as well, to translate their own proteins
Cytoskeleton
o Maintains cell shape and polarity
o Provides mechanisms for cell movement
o Acts as tracks for motor proteins that help move materials within cells
Actin filaments
o Long filament surrounding the cell
o Gives shape to cells
o Mediates cell shape changes, cell migration, and muscle contraction
Intermediate filaments
o Found only in multicellular organisms
o Rope-like assemblages in cells
o Give strength to tissues and nuclei
o Progeria is caused by defective nuclear intermediate filaments
o Form a coiled coil
Microtubules
o Largest of the cytoskeleton subunits
o Hollow cylinders made from tubulin
o Organize the cell
 Provide intracellular skeleton
 Determine cell polarity
 Function as tracks on which motor proteins can move vesicles and organelles
 Move chromosomes during Mitosis and Meiosis
 Make up cilia
Cell adhesion
o Cell binding to one another
o Leads to phagocytosis, DNA exchange, sperm-egg fusion
Cell Junctions
o Tight junctions
 Separate apical and basolateral membrane domains
 Important in the gut
o Adherens junctions
 Connect two adjacent cells
 Does so by connecting actin bundles
 Most ancient and important cell-cell junction
 Made of transmembrane cadherins
o Desmosomes
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 Provide mechanical strength (skin)
 Joins intermediate filaments in one cell to those of another
Gap junction
 Makes a small hole between two cells
 Made up of proteins called connexins
 Small molecules and water soluble ions can flow through
Focal adhesion
 Anchors actin filaments to the basal lamina
 Most important cell-matrix junction
 Made of transmembrane integrins. Several integrins from one cell bind to
extracellular matrix molecules
 Integrins – receptors that mediate attachment between a cell and the
tissues surrounding it
Basal Lamina – Thin mat of extracellular matrix underlying epithelia, surrounding
muscles etc
Energy
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Metabolism is divided into two types of activities
o Anabolic reactions
 Link simple molecules together to make complex ones
 Require energy
 Energy storing reactions
o Catabolic reactions
 Break down complex molecules into simpler ones
 Release energy
First Law of Thermodynamics
o During any conversion of energy, the total initial energy equals the total final energy
o Energy is neither created nor destroyed
Second Law of Thermodynamics
o Energy spontaneously disperses from being localized to becoming spread out if it is
not hindered from doing so (entropy increases)
o Energy conversions only happen if energy disperses in the universe
o Dispersing energy is the driving force for energy reactions
o Energy transformations always result in a state of higher probability (More
disordered)
Free energy (ΔG) = ΔH – TΔS
o If Delta G is negative, energy is released and the reaction can proceed
o If positive, extra energy will be required for the reaction to occur
o Four Types of Reaction
 Heat released, Disorder increased(ΔH<0 and Δ S>0)
 Always spontaneous
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 Most catabolic reactions
 Heat released, disorder decreases (ΔH<0 and ΔS <0)
 Only spontaneous below a certain temperature
 Protein folding
 Heat consumed, disorder increases(ΔH>0 and ΔS >0)
 Only spontaneous above a certain temperature
 NaCl dissolving
 Heat used, disorder decreases (ΔH>0 and ΔS < 0)
 Never spontaneous
 Most anabolic reactions
In principle, all reactions are reversible
o Adding reactant speeds up forward reaction
o Adding product speeds up reverse reaction
o Delta G = 0 when both rates equal each other, chemical equilibrium
Standard free energy (Delta G®) applies to 25 degrees Celsius and 1M concentrations of all
reactants and products
All living cells use ATP for capture, transfer, and storage of energy
o ATP is so useful as the energy currency because its ΔG° is intermediate between what
you gain in respiration and what you expend in anabolism
Direction of a reaction can be predicted is delta G is known, but not the rate
Enzymes and Glycolysis
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A Catalyst is any substance that speeds up a chemical reaction without itself being used up
o Most biological catalysts are called enzymes
Enzymes bind specific reactant molecules called substrates
Some enzymes require cofactors in order to function
o Anything that isn’t an amino acid that is required for enzymes to function properly
o Heme in hemoglobin
Enzymes work by lowering the activation energy required for the reaction to occur
Types of catalysis
o Orientation
 Enzyme’s active site forces the molecules into the right orientation so that the
reaction can occur
o Strain
 The optimal configuration for the substrate is one that induces strain on the
molecules
 Breaking covalent bonds
o Transfer of electrons
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Can transfer carboxyl or amino groups to the substrate, providing a charge for
the reaction to occur
 After the reaction, the provided group is returned to the enzyme
An inhibitor can bind to the active site of an enzyme, blocking the substrate from attaching,
effectively deactivating the enzyme
o Competitive inhibition
Noncompetitive inhibition is when inhibitor binds to a different site on the enzyme, causing
the conformation of the active site to change, no longer able to accept the substrate
o Allosteric inhibition
Allosteric regulation is more efficient than competitive inhibition because less inhibitor
molecules are required
Positive Allosteric regulation
o When the regulator attaches to the enzyme, then it becomes active
Cooperative allosteric transition
o Occurs with two or more subunits
o The more subunits, the more efficient the inhibitors will be at lowering enzyme activity
Glycolysis
o Catabolic pathways are long and complex in order to release energy slowly
o Glycolysis for the complete oxidation of a glucose molecule is -686kcal/mol (exergonic)
o Half the energy in glucose is collected in ATP, the rest is use to drive reactions
Redox reactions transfer electrons between molecules
o A gain of electrons is reduction
o Loss of electrons is oxidation
o Happen simultaneously
o Oxidation of organic molecules decreases number of CH bonds
o Methane is most reduced, Carbon Dioxide most oxidized
The Cofactor NAD is an essential electron carrier in redox reactions
o After oxidation, energy cannot be immediately stored in ATP
Glycolysis can be divided into two stages
o Investment of ATP to activate the sugar, followed by splitting C6 into 2x C3
o Oxidation of C3 giving NADD + H and ATP followed by recovery of initial ATP investment
Phosphate gets added to glucose
o Traps it in the cell because of negative charge
Hexokinase turns it into G6P, and so on through a chain of enzyme-assisted reactions
Energy harvesting reactions occur when the C3 compounds are oxidized, reducing NAD+
Substrate level phosphorylation occurs next, forming ATP
Final product of glycolysis is 2 pyruvate molecules
Sequential reactions
o If a reaction that you want to move in the forward direction is favoured in the
backwards direction, you can couple it with an extremely favorable reaction which uses
the products of the first reaction as its reactants, keeping the concentration down,
forcing the first reaction to speed up forwards
Citric Acid Cycle and Electron Transport Chain
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After glycolysis, the pyruvate is oxidized to acetyl CoA
o CO2 released
Glycolysis occurs in the cytoplasm, The Citric Acid Cycle in the mitochondrial matrix
o Electron transport chain in the inner mitochondrial membrane
Citric Acid Cycle
o Completely oxidized the 2-C acetyl group to 2 CO2 molecules with dehydrogenase
o Formation of ATP is the only step that isn’t oxidation
o Final product is 3 NADH, 1 FADH2, 1 ATP
 Per molecule of Acetyl CoA (2 per each glucose molecule)
o FAD is needed instead of NAD in the conversion of succinate to fumarate because the
redox potential of succinate is much stronger than that of NAD, but not as strong as
FAD, and the reactions tend to go towards the higher redox potential
Electron Transport Chain
o Uses NADH + H and FADH2 generated during sugar oxidation
o Flow of electrons in a series of redox reactions causes the active transport of protons
across the inner mitochondrial membrane, creating a proton concentration gradient
o NAD doesn’t pass the electrons directly to oxygen, instead going through a series of
complexes
 NADH can only be oxidized by NADH hydrogenase, which then passes it to the
rest of the chain
 This is done because energy needs to be released gradually so that it can be
captured and utilised
o Each NADH + H oxidized in the chain pushes 3 protons across the membrane, FADH
pushes 2
o Cyanide kills by binding to the active site of cytochrome c oxidase (Last stop on the
chain), blocking the final redox reaction
Pumping of protons in the ET chain followed by ATP synthesis is oxidative phosphorylation
ATP synthesis is reversible, though only occurs in bacteria growing without oxygen
Regulation
o Main control point in glycolysis is the kinase that adds a second phosphate
 Inhibited by high ATP concentration, activated by high [ADP]
o Main control point of the citric acid cycle is the first hydrogenase
 High [NAD+] activated, high [NADH + H] inhibits
o Electron transport chain is regulated by the H+ gradient
 Lower electrochemical gradient, faster electron transport
Fermentation
o Electron transport chain and citric acid cycle stop in the absence of oxygen
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Some cells continue glycolysis and produce limited amounts of ATP if fermentation
regenerated the NAD to keep it going
o Occurs in cytoplasm
o Reduces pyruvate instead of oxidizing it
 Pyruvate replaces oxygen as electron acceptor
o Only occurs in muscle cells of humans
o Results in 2 ATP per cycle
Cellular respiration produces 32 ATP per molecule of glucose
If inadequate food is available, glycogen stored in muscle and liver are used first
o Fats next, but the brain can only use glucose, so it must be synthesized
Photosynthesis
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Plants make all complex molecules on their own, except ammonium that they get from soil
Photosynthesis occurs in the chloroplasts
o Light energy captured in the thylakoid
Difference between NADH and NADPH
o Same properties and occur in plants and animals, but are made by separate pathways
and independently regulated
o NADP used for exclusively anabolic pathways
o NAD used exclusively for catabolic pathways
o NAD can be converted to NADP if needed
Capture of light energy
o Plants absorb light in the visible spectrum because it holds the right amount of energy
 IR/Microwaves provide vibrational heat (energy) only
 X-rays = damage
o Chlorophyll has alternating double bonds which result in delocalized electrons
o Plants have two predominant chlorophyll molecules: A and B
 Absorb blue and red wavelengths
o Other accessory pigments (eg carotenoids) absorb photons between red and blue and
then transfer a portion of that energy to chlorophyll
o When an electron absorbs light, it becomes excited and reaches a more energetic
state
 As it descends to its ground state, it releases that energy to a neighboring
chlorophyll molecule
 Continues along a chain of redox reactions
o These reactions occur within the light harvesting complex
 Made up of 100 chlorophylls plus carotenoids in the antenna
 Only one chlorophyll is attached to the electron acceptor
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Antenna is required because individual chlorophyll molecules are excited too
rarely
 Final step, in the middle of antenna system is a redox reaction
 Chlorophyll in the reaction center acts like a sink, its excited state has the
lowest energy
 Transfer of light energy into chemical energy occurs when the reaction center
chlorophyll gives up its excited electron to reduce the first member of the
electron transport chain
o The Light reactions
 Two different systems for transport of electrons in photosynthesis
 Cyclic and noncyclic electron transport
 Noncyclic electron transport produces NADPH + H, ATP, and Oxygen
 Water gives up its electrons to photosystem II, giving off protons and
oxygen
o Forms an electrochemical gradient, generating ATP
 Two photosystems are required because one quantum (photon) of
light does not have enough energy to transfer electrons from water to
NADP+ and make ATP
 Cyclic electron transport only produces ATP
 When the plant has enough NADPH and sugars stored, it just uses
photosystem I in a cyclic fashion to make ATP
 Ferredoxin transports the electrons back to the cytochrome complexes
and cycles through photosystem I
The formation of the proton gradient via the electron transport chain, followed by synthesis
of ATP is called phosphorylation
o Thylakoid region
Carbon Fixation
o Rubisco is the most abundant protein in the world
o Converts carbon dioxide to solid 3phosphoglycerate
o Rubisco is a carboxylase, adding CO2 to RuBP
o At low [CO2] and high [O2] it can also be an oxygenase, adding O2 to RuBP
 Photorespiration – Not good, uses ATP and NADPH, no beneficial function
Cells & the Cell Cycle
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Cell theory
o All organisms consist of cells
o Cells divide to produce new cells
o Higher organisms fuse their germ cells to produce a new organism
Chromosomes
o Single continuous strand of DNA
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Circular (Bacteria) or linear (most other organisms)
When the cell is preparing to divide, chromosomes condense by winding around
proteins called histones
 DNA winds around the proteins, packing tightly together to form the
chromosomes
o The number of chromosomes an organism possesses is a characteristic of its species
 Bacteria typically have one, humans have 46 (23 pairs)
o Eukaryotic chromosomes often come in identical pairs called homologs
Karyotype
o A way of organizing and identifying chromosomes
o Performed by flattening and staining a cell’s DNA as it is preparing to divide
o Chromosomes are paired up according to banding patterns
Before cell division can occur, each chromosome must be replicated to produce two copies of
each
o Chromatids are joined in the middle by a centromere (conglomeration of proteins)
o These paired up chromatids are called mitotic chromosomes
Segregating the replicated chromosomes during cell division is where most of the cell’s
resources are devoted
o Organisms need at least one of each chromosome, as they each carry essential genetic
material
o Organisms typically need EXACTLY one of each chromosome
 Down Syndrome results from an extra copy of chromosome 21
Steps in cell division
o S Phase
 Consists of the replication of the chromosomes (DNA)
o Mitosis (M phase)
 Process by which somatic cells make identical copies of themselves
o OR Meiosis
 Process by which germ cells make non-identical copies of themselves
o Cytokinesis (Optional stage)
 Dividing the cytoplasm and organelles between daughter cells
The cell cycle
o When cells aren’t dividing (Most of the time), and haven’t performed DNA synthesis (S
phase) yet, they’re in the Gap 1 (G1 phase) of the cell cycle
o There is a checkpoint in between the G1 and S phase that checks for external chemical
signals from other cells
 E.g. Cyclin E becomes active in response to hormone signals during mammalian
pregnancy, which results in proliferation of breast cells needed for lactation
o Followed by the S phase
 DNA synthesis
o Followed by the second Gap phase (G2), which has another checkpoint
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G2 checkpoint makes sure that all DNA has been replicated before allowing
the cell to divide
 This checkpoint can be disabled by treating a cell with caffeine
 Treating a cell with hydroxy urea will prevent DNA replication (Skips S phase)
 Treating a cell with both results in a cell with only one set of chromosomes
entering mitosis, where the daughter cells will be destroyed due to not
containing necessary genetic material
Proteins involved in regulation of the Cell Cycle
o Cdk4 and Cyclin D are important mitotic regulator proteins
o Cyclin D is synthesized in G1 when the cell is ready to replicate DNA
o Cyclin D binds to the active site of a Cdk4 molecule, activating it
 Sends signals to the cell telling it to enter the S phase, then breaks down,
restarting the process
o There are many other proteins involved in the cell cycle, these were just obvious ones
used as an example
Most cells of your body are not dividing, and have no plans to divide any time soon
o Cells that are not dividing are usually arrested in the G1 phase, waiting for external
chemical signals to tell the cell to divide
Cancer results from unregulated cell division
o If the G1-S checkpoint of a cell is defective, a cell can continuously divide in an
unregulated manner
o If Cyclin E is always active, or over abundant, a cell will repeatedly divide as if during
pregnancy
Mitosis and Meiosis
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First noticeable part of mitosis is the condensing of the chromatin to form the chromosomes
o Prophase
Microtubule organising centers (centrosomes) migrate to the poles of the nucleus
o Start forming dense fibers which will form the spindle
Prometaphase
o Nuclear envelope breaks down
o Tubules are now free to interact with the chromosomes
o Microtubules grow and shrink, hooking onto the chromosomes
 Some do not join with chromosomes, but become rigid and overlap, stabilizing
the cell
 Known as polar microtubules
 Kinetochores on the centromeres bind to the microtubules
o Chromosomes arrive on the metaphase plate
Metaphase
o All chromosomes have been captured by at least one microtubule from each
centrosome
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o The microtubules pull the chromosomes to align them along the metaphase plate
o Sister chromatids are bound to kinetochore microtubules on opposite spindles
Anaphase
o Centromeres holding the identical chromatids together separate
o The kinetochore microtubules begin shortening, pulling the sister chromatids to
opposite sides of the cell
o Makes sure that each daughter cell will receive one of each chromosome
Telophase
o Nuclear envelope reforms
o Chromosomes reach the poles
o Chromatin becomes diffuse
Cytokinesis
o In animals – Actin and myosin form a purse string that constricts and divides the cell
 Contracts much like in muscle cell
o In plants – Vesicles fuse to make cell membrane and cell plate, which becomes a new
cell wall
o Some cells don’t bother to divide their cytoplasm
 Muscle cells have many nuclei (Syncytial) because they undergo mitosis
without cytokinesis
Ploidy
o N = a set of chromosomes that includes exactly one of each homologue
o Multiples of N are named
 N = haploid
 2n = diploid
o In humans, somatic cells are diploid, gametes are haploid
Meiosis
o Meiosis I
 Early prophase I
 DNA begins condensing
 Centrosomes begin moving towards poles
 Mid prophase I
 Homologues start to pair up, forming tetrads
o Called synapsis – The pairing of homologous chromosomes
(2x2 homologs)
 Late prophase I
 Crossing over occurs between homologous chromosomes
 The point at which crossing over occurs is called chiasmata
 Genetic information is exchanged between them, forming recombinant
chromatids with genetic info from each parent
 Prometaphase I
 Nuclear envelop breaks down
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 Spindle fibers form
 Metaphase I
 Like metaphase in mitosis, except that pairs of homologous
chromosomes line up double file at metaphase plate, rather than in a
single file line
 Chromosomes lined up at the plate are held together by chiasmata
o This is why it is double file
 Anaphase I
 Kinetochore microtubules attach to entire homologues
 Centromeres don’t dissolve, junctions between the homologous
chromosomes do
 Homologous chromosomes move to ends of the cell
 In downs syndrome, this is the point at which both chromosome 21
homologs end up in one daughter cell
 Telophase I
 Regular cell division
 Meiosis II
 Acts just like mitosis, except that the four daughter cells formed each
contain a mixture of genetic material from each parent
Cells can arrest in meiosis for a very long time
o 40 Years in meiotic prophase I for some human female eggs
 When ovulation happens, the released egg completes meiosis
 The last eggs to ovulate have been arrested for 40-50 years
 This long wait in prophase I is what leads to older women being more
likely to have a child with Down Syndrome
Importance of ploidy
o It’s not about the absolute number of chromosomes that’s important, it’s the ratio of
homologues
 Odd Ploidys tend to be sterile because of problems in dividing up the
homologues during metaphase I (Hard to divide 3 sets into 2 cells)
 Even-n ploidy works fine for mitosis and meiosis
 4n Is similar to 2n, just tend to be bigger
Mendelian Genetics
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For a given character (colour, stature, facial features etc) offspring share traits with their parents
Types of variation
o Continuous variation – Seemingly infinite number of traits for a given character, falling
along a continuous spectrum.
 Height, skin colour, etc.
 Blended inheritance
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Discrete variation
 There are only two or a few traits for a given character
 E.g. fur colour in mice
 Mendel chose peas because they have several characters the show discrete
variation
Mendel used true breeding strains
o He crossed the two true breeding plants
 All the resulting peas were round
 Showed that round was dominant over wrinkled
o Then crossed this F1 generation, giving ¾ round, and ¼ wrinkled in the F2 generation
Mendel discovered that heredity exits in discrete units that travel through space and time
o The units are called genes
o Different gene “flavours” – alleles
o Having two of the same allele for a character – homozygous
o Having two different alleles for a character – heterozygous
The probability of two independent events is the product of the probability of each event
o Useful for predicting results of genetic crosses involving more than two characters
Genotype – The set of alleles that an organisms has
Phenotype – The displayed traits of an organism
Genotype determines phenotype, but phenotype may not determine genotype
Independent assortment
o Alleles of different genes segregate independently
Linkage and the Genetics of Continuous Variation
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Genes assort independently as long as they’re located on different chromosomes
o Gets more complex when they’re on the same one
Sex is determined by the sex chromosomes
o XX for female, XY for male
o Equal chances of either gender
Red green colour blindness
o Appears in ¼ of children of unaffected parents (recessive)
o Overwhelmingly appears in males (6%) vs females (<1%)
 Sex linked, X chromosomes
o Colourblind females will always have affected fathers
 Males have just one X chromosome, so to pass it on to daughter, they must be
colourblind
Sex-linked traits
o A character of trait determined by genes located on the sex chromosome(s)
o Do not exhibit independent assortment
Types of alleles
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o Wild type allele – predominant allele (>99% of population)
o Mutant allele – a change from the wild type, typically resulting from mutation
o Polymorphic allele - An allele that is present in less than 1% of the population
A linkage in trans – when alleles are linked on different homologous chromosomes
o In cis is when both are on the same homologous chromosome
Crossing over exchanges genetic material, causing alleles linked in cis to possibly become linked
in trans and vice versa
Recombination rate – Measure of distance
o Recombination occurs at random points on the chromosome
o Therefore the probability or rate of recombination occurring between two genes
depends on how far apart the genes are on the chromosome
Linkages and genetic mapping
o The more recombination, the further apart the genes are
o Distance measured in centiMorgans (cM) = 100*(# of recombinants/# total progeny)
o All genes linked together on the same chromosome are a linkage group
Heart attack risk is an example of a multigenic trait, as many genes can contribute to a higher
risk
Epistasis – when genes interact in a non-additive fashion
o E.g. when albino and agouti loci interact in mice
o Results from genes involved in different steps of the same process or pathway
Environmental contributions to phenotype
o Penetrance – percentage of individuals of a given genotype that show the phenotype at
all
o Expressivity – degree to which a phenotype is expressed
Factors that contribute to inheritance of a trait
o Number of genes
o Number of alleles in the population for each gene
o Environmental effects
o Semi-Dominance
 When the heterozygote is phenotypically distinguishable from either
homozygote
 E.g. RR=red, rr=white, Rr=pink
Discovery and Replication of DNA
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To purify a sample, you must first separate the components, and then Assay each component
Assays
o A way of measuring something
o Can measure a substance such as starch, or an abstract phenomenon like memory
How to purify DNA
o Grind up an organism
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o Extract lipids and proteins with an organic solvent
o Precipitate the DNA with ethanol
Transforming principle
o Discovered by Fredrick Griffith
o Experiment composed of Harmless R strain and dead but previously virulent S strain
mixed together to produce lethal S strain, showing that something in the S strain was
sufficient to transform the R strain
 That something was DNA
o Chemical components of one cell are capable of genetically transforming another cell
o Oswald Avery used Griffith’s assay to show that it was DNA that held this ability
o Hershey-Chase experiment determined that it is the DNA, not the protein component of
viruses which enters cells and directs assembly of new viruses
Hershey-Chase experiment
o Wanted to find out what bacteriophages injected into bacteria; protein or DNA
o Took a virus with a DNA core surrounded by a protein coat
o Radioactively labeled DNA with an isotope of Phosphorus and the protein with an
isotope of Sulphur
o Allowed the virus to infect bacteria, then but it in a blender
o Infected bacteria produced viruses that contained the phorphorus isotope, but almost
none of the sulphur isotope
o Determined that DNA must be the viral genetic material
Chargaff’s Rules
o A = T, and G = C (Proportion-wise)
o The ratio A+T/G+C varies with organism
Crystallography
o DNA consists of two strands, twisted around each other in a double helix shape
o Phosphates are probably on the outside of the helix
o The strands run antiparallel to each other
o Shape of DNA discovered by Watson & Crick
o The proposed bases formed H-bonds with each other such that A-T, and C-G only.
 Reverse compliments
Nucleotides
o Made up of a base, a sugar, and a phosphate group
o In humans, Adenine and Thymine make up ~31% of our total DNA each, while Guanine
and Cytosine are ~19% each
 In other organisms, it is closer to even distribution of each
The sequence of nucleotides is not constrained by the structure
o DNA can accommodate any sequence
o An arbitrary sequence can encode information
o The two strands of DNA encode the same information in complementary form,
suggesting a method of replication
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DNA follows the semiconservative method of replication
o Conservative Replication – each DNA strand makes a new strand, then the parent
strands reform together, and the daughter strands form together, preserving the
original pairing
o Semiconservative Replication – each DNA strand makes and remains annealed to a new
strand
o Dispersive Replication – DNA breaks apart and rejoins to produce four new strands that
each contain pieces from both parent strands
o Discovered by the Meselson and Stahl experiment
 Used radioactive isotopes of carbon to track the transmission of DNA strands
between generations of replication
To create DNA in a test tube you need:
o Triphosphate nucleotides
o DNA polymerase
o Template DNA with ragged ends
Steps of DNA Replication
o The enzyme helicase unwinds the DNA strands
o Primase makes short RNA primers, which act as a ragged end so the DNA polymerase III
can go to work
 Primer – Short pre-existing polynucleotide chain to which new nucleotides can
be added
o DNA polymerase 3 binds to the primer, and starts replication of the template strand
from there
 The points at which replication is initiated are called Origins of Replication
o As helicase unwinds DNA in each direction from the origin of replication, a bubble forms
with Replication Forks on either end where the old double stranded DNA is being split
to act as a template for the formation of two new strands
o One strand foes from 5’ to 3’, and is called the Leading Strand; the other strand goes
from 3’ to 5’ and is called the Lagging Strand
o On the lagging strand, since replication happens from 5’ 3’, DNA cannot be synthesized
continuously, it is synthesized discontinuously by putting down primers every few
hundred bases
o The short DNA fragments called Okazaki Fragments that form are later connected
covalently to form a continuous strand. This is because DNA synthesis can only occur in
one direction – 5’ to 3’, and the lagging strand runs in the opposite direction, requiring
discontinuous synthesis
o DNA polymerase 1 converts the RNA primers into replicated DNA
o DNA ligase connects the Okazaki fragments and the sections of DNA made from the
primers into a continuous strand of replicated DNA
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Also joins the end of the long strand of replicated DNA on the leading strand to
the newly synthesized segment (which used to be the primer), creating one
continuous chain
On circular chromosomes, there is one origin of replication, and opposite it there is a terminal
locus
o Replication complex moves around the parental strand, and then the two connected
loops of DNA are disconnected, making two circular chromosomes
Error correction
o Polymerase III proofreads the fragments before replicating
 Can correct mismatches and excision
 Mismatches are when there is an incorrect base in the sequence, and DNA
polymerase III removes it and takes the correct base from the cytoplasm
 Excision repair can take out a sequence of bases containing errors and replace it
with the correct sequence from the cytoplasm
o Exposure to UV light produces Thymine Dimers – excision repair helps to fix this by
removing them
The Genetic Code
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Alkaptonurea
o Recessive hereditary trait
o Deduced that it results from the absence of a specific enzyme
o Beadle and Tatum proposed that one gene codes for one enzyme
 Has been modified to a gene coding for a protein now
If an organism cannot convert one particular compound to another, it lacks an enzyme required
for the conversion
o Therefore mutations exist in the gene that codes for the enzyme
Central Dogma of Biology
o DNA -> RNA -> polypeptide
Transcription
o The process where DNA is transcribed into RNA
o The RNA polymerase makes an RNA polymer that is complementary to one gene at a
time
o The resulting mRNA strand starts slightly before open reading frame (ORF) and extends
slightly past the stop codon
o Knows when to start because of special sequences called promoters that the RNA
polymerase recognizes as the beginning of a gene
o The strand which is not transcribed but which has the same sequence as the mRNA is
called the sense strand
o The transcribed strand that has a sequence complementary to the mRNA is the
template strand (or antisense strand)
DNA sequence uniquely determines the amino acid sequence of a protein
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o Amino acid sequence of a protein does NOT uniquely determine the DNA sequence
o Redundancy in the code (multiple codons code for one amino acid)
Ribosomes have an amino acid attachment site that recognizes the sequences binding to it
There is at least one tRNA for each amino acid, and often more than one
o The tRNA for an amino acid contains an anticodon sequence that is the reverse
complement of the codon for that amino acid
o In addition to the different anticodons, each tRNA has unique sequences that cause it
to have a unique shape
Some tRNA molecules can be chemically modified, leaving the anticodon untouched, but
changing the amino acid to which it is bound, changing the amino acid sequence of the
polypeptide
Some tRNA molecules can undergo chemical mutagenesis, with a base of the anticodon being
replaced by another base, but leaving the amino acid untouched.
o Causes anticodon to bind to a different RNA sequence, but still adds the same amino
acid, just in a different location on the polypeptide
Because genetic code is arbitrary, shared by all organisms, and fixed by convention
o We must share a common ancestor with ALL living organisms, meaning that genetic
code is extremely old
Translation
o The conversion of mRNA to protein, done by ribosomes
o While synthetic RNA in a test tube translation mix will produce proteins from all three
different reading frames, but in a cell, the ribosome picks the right reading frame, as it
always starts with AUG, the start codon.
o The ribosome consists of two sub units (large complexes of rRNA and proteins)
 At the start, the small subunit finds the first AUG in the mRNA and binds it along
with the methionine tRNA
 The small subunit complex of tRNA and mRNA recruits the large ribosomal
subunit
o The large subunit has two tRNA binding sites, the A site and the P site
 When it is recruited, it binds so that the met-tRNA is in the P site, and then the
tRNA that corresponds to the next codon in line binds to the A site
o The carboxyl group of the methionine is transferred (with the help of the enzymatic
properties of the ribosome) from the 3’ hydroxyl of the met-tRNA to the free amino
group of the new amino-acid (eg sernine) in the A site (forming the first peptide bond)
o The ribosome then releases the met-tRNA and shifts one codon distance in the 3’
direction
o The tRNA that was in the A site is now in the P site, with all previous amino acids
o The process repeats until the ribosome reaches a stop codon (UAA, UAG, UGA)
 A protein called a release factor binds to the A site causing the last tRNA to
release the peptide chain and the ribosomal complex to disassemble
 The peptide then folds into a functional protein
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Wobble
o Recall there is a codon for every tRNA, but not a tRNA for every codon
o Some tRNAs can use more than one codon (some are able to recognize more than one
codon)
o Wobble pairing – the tRNA anticodon does not strictly obey base-pairing rules
o The 5’ residue of the anticodon (which corresponds to the 3’ residue of the codon) has
some wobble – it doesn’t always have to match up perfectly with the 3’ residue of the
codon
 E.g. CAU can also recognize GUG (instead of GUA) as it pairs up well enough
o Wobble pairing rules (anticodon 5’ position -> codon 3’ position)
 G - C or U
 C-G
 A-U
 U - A or G
 I (inosine) - A, U or C
 Covalently modified adenosine (A purine nucleoside that has adenine
bound to a ribose sugar by a glycosidic bond)
Mutations
o Point mutations – small changes in a single gene
 Silent mutations – do not affect the protein sequence because of the
degeneracy of the code (i.e. GGA and GGT both code for Glycine)
 Missense – changes one amino acid into another
 Nonsense mutations – change an amino acid into stop codon, thus truncating
the protein
 Frame shift mutation – can result from an insertion or deletion and changes
the reading frame from that point onwards
 Deletions or insertions that are multiples of three nucleotides do not
change the reading frame, but merely delete amino acids from or insert
amino acids into the peptide chain
o Chromosomal mutations – changes that affect a large portions of a chromosome, often
affecting many genes
 Deletions – remove a large piece of the chromosome including many genes
ABCDEF -> AB/EF
 Duplications - duplicate large chunks of the protein ABCDEF -> ABCD/CD/EF
 Inversion – when a piece of the DNA flips around, re-entering the chromosome
in the reverse orientation ABCDEFG -> AB/EDC/FG
 Translocation – results when a piece of DNA jumps from one chromosome to
another
 These can be reciprocal translocations where two chromosomes trade
places
o Causes
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Mistakes in replication, chemicals, radiation
Anything extraneous to the organism (e.g. chemicals) that cause changes are
referred to as mutagens
The effects of the changes depend on if it occurs in somatic tissue or germ line
tissue
 If somatic, then the change could kill the cell, make it sick or cancerous
o Although the mutation will be inherited by the mitotic progeny,
the change will not be transmitted to the progeny as somatic
cells do not make gametes
 If it’s a germ line mutation, which produces gametes, an egg or sperm
may end up with the mutated gene and transmit it to progeny
o Thus, the mutation (if not too deleterious) will become part of a
repertoire of alleles present in that population of organisms
o It is on these germ line mutations that natural selection acts to
produce new species
Viral and Bacterial Genetics
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Distinguished by bacteria by their small size
Dimitri Ivanovsky and the tobacco mosaic virus started a huge effort to identify and classify
viruses
Virons (virus particles) consist of
o A simple outer protein coat or capsid
o Nucleic acid inside
o Sometimes a lipid bilayer membrane around the protein coat
Virus classification
o Host specificity and pathology
o Genetic material (RNA or DNA)
o Size and shape
Viruses use a variety of genetic material
o Single stranded DNA
o Double stranded DNA
o Single stranded RNA
o Double stranded RNA
o Can be linear or circular
Viruses can undergo two different life cycles
o Lytic Cycle
 Virus injects genetic material into host cell
 Viral genome replicates separate from the host DNA
 Uses host cell to create new viral proteins
 Proteins and replicated genetic material reassemble into new viral cells
 Viral cells burst out of host cell, lysing the cell and destroying it
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 Bacteriophages that undergo the lytic cycle are called Virulent phages
o Lysogenic cycle
 Virus injects genetic material into host cell
 Genetic material (called Prophage) incorporates itself into host cell’s genome
 When host cell replicates, viral genome is replicated along with it
 Viral genome separates from host genome
 Can now either enter lytic cycle or restart lysogenic cycle
 Phages that undergo a lysogenic reproductive cycle are called Temperate
phages
Viruses replicate themselves by containing the information needed to produce the needed
enzymes used in replication
o “Early” genes code for the enzymes and proteins necessary to transcribe and translate
the sections of the genome that code for viral proteins and lysis proteins – “Late genes”
Retroviruses
o A special class of viruses that bend the rules of the central dogma
 E.g. HIV retrovirus
 Has envelope with glycoprotein, capsid protein, retrovirus RNA and
reverse transcriptase (a little protein that associates with the genome)
 Like influenza binds to proteins on the surface of the cell (binds to CD4,
a protein important to immune cells), HIV fuses to the cell and releases
its RNA
 Instead of making RNA copies of the genome, the reverse transcriptase
enzyme makes a DNA copy of the RNA viral genome which violates the
central dogma of going from DNA to RNA
 The DNA copy is then made double stranded, and is then inserted into
the host DNA (very much like a prophage)
 The viral genome is now in the cell genome, protected from anything
the immune cell may want to do, and can now make RNA copies of itself
through transcription, make more RNA, proteins etc.
 This makes it extremely hard to combat HIV, attacks immune system,
and is in the DNA and difficult to access
o Makes DNA from RNA
o The DNA of the retrovirus can be integrated into host chromosome and remain there,
inactive, for a long time before it starts procuring viral particles again
Bacterial Sex
o A means for bacteria to exchange genetic material
o Auxotrophic phenotypes (where a bacteria can only grow when certain substances are
added to their media) are the most useful to show this
o Specialized Transduction
 When a prophage excises itself from the chromosome, it may have accidentally
picked up a gene from the bacterial chromosome
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Generalized Transduction
 The phage could do a poor job of chopping up the bacterial DNA and instead of
packaging phage DNA into the virons, they package chunks of bacterial
chromosome
 When those virons infect another bacterium, the chunk of bacterial DNA get
injected into the infected bacterium, and can be incorporated into the
chromosome
 Since adjacent genes are more likely to be transferred together, generalized
transductions can also be used to map genes
o Transformation
 Bacteria sometimes take up naked DNA floating around in their environment
 The genomic DNA can then be integrated by recombination
 The non-genomic DNA plasmids can also be used to transform bacteria
 The plasmids don’t integrate, but are duplicated by DNA replication
machinery
 They carry and express only a few genes
 R-Factors are plasmids that carry antibiotic resistance genes
o Conjugation
 Plasmids - a linear or circular double-stranded DNA that is capable of replicating
independently of the chromosomal DNA that float around in a bacteria
 F Plasmid (in F+ E.coli) – can allow the F+ bacteria to form a Conjugation tube
with a F- bacteria and thus donate a strand of their F plasmid
 If the F-plasmid gets integrated into the chromosome
 Conjugation occurs more frequently
 The F DNA takes some of the chromosomal DNA with it during the
conjugation (strips off one layer to travel to the other cell, tries to bring
the rest of the DNA with it)
 The transferred DNA can be incorporated into the chromosome of the
F-cell by recombination
 These integrated F-strains are called Hfr (High frequency recombination)
 Upon conjugation, basically the entire chromosome acts like a big F-plasmid and
one strand threads its way into the F-cell
Gene mapping (with conjugation)
o By interrupting the conjugation process, you can see in what order the chromosomal
genes enter the F- and thereby map them
o The sooner the genes get transferred, the closer they are to the Hfr inserted plasmid in
the chromosome
Eukaryotes
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Tetraploid organisms are larger than their diploid counterparts
o Due to cells being larger
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Cell size can be used to estimate genome size
o Larger cells, generally larger genome
DNA is divided up linearly into:
o Transcription Promoter sequence -> Start codon -> Exon -> intron -> exon -> intron ……..
-> stop codon -> exon -> terminator of transcription
o What gets transcribed by the mRNA is initially all of the exons and introns
 Introns are segments of DNA that do not end up in the final mRNA product
 Introns are removed by snRNP molecules which splice the unnecessary
segments out of the mRNA chain, and then connect the exon segments
Splicing is a form of RNA processing
o Other forms
 5 prime G cap – Cap at the front of the primary RNA transcript
 Composed of 7-methylguanosine ( sequence of Gs)
 Polyadenylation – Addition of a polyA tail
 Long tail of adenine molecules bound to the end of the RNA transcript
Telomeres
o Non-coding DNA on the ends of chromosomes that act as a buffer
 Telomerase adds single stranded repeat sequences to the ends of the
chromosome
 The repeat is complementary to a guide RNA that is part of the telomerase
 The primer RNA binds to this single stranded repeat sequence, as do other
complementary buffer bases
o Most human cells do not express telomerase. As they replicate their DNA, the
chromosomes get shorter until a cell cycle checkpoint prevents further division
o Cancer cells often express telomerase
DNA Transposons
o Pieces of DNA that excise and then reinsert into the genome
o Composed of a segment of DNA called the Transposase gene, which has inverted
repeating sequences on either side of it
o Signals transposase to splice the segment from the strand, and inserts it elsewhere,
often in the middle of a gene-coding sequence, causing a change in the DNA sequence
o Transposons can be copied by the host cell’s DNA break repair
 If a transposon in one strand moves to another section, the new empty spot
where it used to be is across from its complementary sequence on the other
DNA strand, which is also a transposon
 Template-mediated repair occurs, which means that an enzyme adds base pairs
complementary to the other strand into the empty gap, creating another
transposon where the other one used to be
Retrotransposons
o Move through an RNA intermediate
o Require reverse transcriptase to do so
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Copy themselves to RNA, then back to DNA that may integrate with the genome
 Thus the reverse part of the name (RNA -> DNA)
o LTR Retrotransposons
 Have direct Long Terminal Repeats (LTRs)
o Non-LTR retrotransposons
 Don’t have LTRs
Pseudogenes
o mRNA from cellular genes can also be reverse transcribed and re-inserted into the
genome
o Known as processed pseudogenes (They lack introns)
Ribozymes
o RNA enzymes
o Can catalyze a variety of reactions just like proteins
RNA could be both genetic material and enzyme
o RNA polymerase ribozyme could replicate itself (Though no RNA polymerase ribozyme
has been found)
o Primitive organisms may have been entirely of RNA (and probably a lipid membrane)
Gene Regulation
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All cells in the body share the same alleles of the same genes
Cells appear different because they express different genes
Transcriptional regulation
o The main difference between cells in different tissues is that they transcribe different
subsets of genes
o The ability of RNA polymerase to begin transcription is controlled
o Promoter elements are required
o Accessory proteins that recognize regulatory DNA sequences are involved
Promoters include several elements
o GC box at beginning
o TATA box at the end
There are Three eukaryotic RNA polymerases
o Pol 1 – rRNA
o Pol 2 – mRNA
o Pol 3 – tRNA, snRNA
Enhancers and Silencers
o Act at a great distance (up to 20,000 bp from the promoter)
o Can be upstream or downstream of the promoter, and work in either orientation
o Short region of DNA that can be bound with proteins to enhance transcription levels of
genes
A cell will inactivate all but one of its X chromosomes, no matter how many there are
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o Inactive X chromosomes become Barr bodies
Euchromatin
o Lightly stained, less condensed, transcriptionally active
Heterochromatin
o Darkly stained, more condensed , transcriptionally inactive
Post-Transcriptional mechanisms of gene regulation
o Alternative splicing
 Can make different proteins depending on which exons are left and which are
spliced out
o During splicing, the order of exons in the mRNA is the same as their order in the
chromosomes
RNA stability
o Cells possess mRNAs in ways that affect their stability
o Stability of specific mRNA is a function of its unique 5 prime and 3 prime Untranslated
Region (UTR)
o Cells have specific mechanisms for recognizing and regulating specific mRNA
mRNA is like water in a reservoir
o The rate of inflow (transcription) and the rate of outflow (degradation) determine the
water (or mRNA) level
Translational control
o Affects the ability of mRNAs to be translated by the ribosome
o RNA interference
 RNA can be cleaved and form a complex with proteins, which regulate the
mRNA from genes complementary to the cleaved gene
Post-Translational control
o Affects the activity of proteins after they are translated
o E.g. addition or removal of phosphate groups from proteins causing
activation/deactivation
Why different regulatory mechanisms?
o Seems to be a balance between speed and efficiency
o Regulating at the level of transcription results in slow changes in expression, but is
efficient because RNA and proteins are not made when not needed
o Regulating at the level of protein is fast, but inefficient because the protein is made,
but not used or reused
Gene repression
o Repressor molecules can bind to DNA sequences, blocking transcription unless a certain
requirement is met
 E.g. when lactose is scarce in the body, a repressor will bind to the operator,
blocking transcription until there is enough lactose present.
 Lactose binds to the repressor, deactivating it, allowing transcription and
translation to continue
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Can occur in the opposite fashion
 When tryptophan is present in large quantities, a corepressor will
activate a repressor, blocking the segment of DNA that codes for
tryptophan until its levels drop enough that the co-repressor stops
binding to the repressor
Biotechnology
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Genetic engineering
o Changing the genotype of an organism by transferring genes from one species to
another
Selective breeding
o Used to domesticate animals and plants
o Only technology required is the ability to select and breed the next generation
o Can wait for useful mutations to arise by chance
o Can cross two related species
o Domestication of maize from teosinte
 Selection for plants with more, larger kernels created maize as we now know it
Gene cloning
o Moving a single gene from one organism to another
Cloning
o Used to make an organism express a new gene to that it has a new phenotype
o Also used to study genes in isolation
Problems encountered in trying to produce a gene within another organism
o Where to get the gene
o How to get it into another organism
o How to get it expressed
Viruses are often used as they have a mechanism of incorporating their genome into their
host cell
Restriction enzymes
o Cut DNA sequences at a certain point, creating two fragments
o Spliced fragments can be put together to form new sequences with the unwanted parts
removed
o OR the gene that you want inserted into a foreign genome can be inserted within the
gap created by the restriction enzyme
 DNA ligase moves phosphate groups around to stabilize the new addition to the
genome
Transformation is inefficient
o How do we know which bacteria have the plasmid?
We can fix this inefficiency by adding an antibiotic resistance gene to the plasmid, so that only
transformed bacteria will be resistant
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Bacteria will not always recognize foreign genes as genes, and may reject them
o Restriction enzymes can force the foreign gene into the genome, but it is not always
incorporated in the form/orientation/number that you want it to
Why clone?
o To understand what the gene encodes and how it works
Cystic fibrosis
o Characterized by respiratory infections, digestive problems, and sterility
o Autosomal recessive trait
o 1/2500 Caucasian newborns have it
If we could find the gene that is mutated, we can predict the protein it encodes
o Knowing the sequence of the protein might help us understand what it does
o Knowing what the protein does would help us understand what goes wrong when it is
missing, and help fix the problem
How to find a gene if you don’t know what protein it makes?
o Map it!
 First map the gene to a chromosome
 Then map the gene between known genes on the chromosome
o Problems
 There aren’t many phenotypes in humans that show simple Mendelian
inheritance for you to map against
 Can’t do controlled mating in humans
Restriction Fragment Length Polymorphisms (RFLPs)
o Polymorphisms in the DNA results in the presence or absence of a restriction site
o Polymorphisms segregate in a simple Mendelian fashion
o We can assay the presence or absence of many restriction sites
o RFLPs can be mapped just like any other allele
Polymorphisms
o 1/4096 chance that a random 6 base pairs area restriction site
o ~730,000 6-cutter sites per genome
o Roughly 4400 polymorphic restriction sites
Polymerase Chain Reaction (PCR)
o Amplifies a small stretch of DNA so you can study it
o DNA primers are used to stimulate DNA polymerase
 The primers are incorporated into the new strand, which causes a chain reaction
of replication, creating many copies of the target sequence
RFLPs are just like alleles
o An allele of a gene is a variation in the sequence of that gene just as an RFLP is a
variation in the sequence of a restriction site
o Both reside at a specific locus on the chromosome
o You can tell which allele of a gene is present by the phenotype produces
 You determine which RFLP is present by PCR/restriction digest
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Causes new band sizes in gel when undergoing electrophoresis because
of different restriction site locations
o RFLPs and alleles with phenotypes are two ways of identifying DNA variations, but they
are not mutually exclusive nor do they perfectly overlap
 Alleles with phenotype but no RFLP, and RFLP with no phenotype exist
Mapping only gets a group of genes, not a single gene
o You can find where the specific genes are by using Expressed Sequence Tags
 Look for cDNAs (Made from mRNAs) and find the genes that encode them
o Can also use bioinformatics
 Tell a computer what your sequence is, and wait for an answer
Cystic Fibrosis (revisited)
o Found to be caused by a mutation in the gene encoding a chloride channel that
regulates the flow of chloride across the plasma membrane of epithelial cells,
specifically into the lung
 When chloride isn’t regulated correctly, mucus becomes sticky and bacteria
are able to grow
Genetic screening
o Used to identify carriers of genetic disorders, so they can make informed family
planning decisions
Individuals with Cystic fibrosis are protected against typhoid fever, because the normal
chloride channel is a receptor for the bacteria that cause it
o Also may be protected against cholera, E.coli, and asthma