Holy Bible
Your guide to A lvl bio mugging
Biomolecules
Cellulose: β-1,4-glycosidic bond (structure → property)
Straight chains of β glucose run parallel to each other with numerous inter-chain
and intra-chain hydrogen bonds between hydroxyl groups, forming microfibrils, thus
insoluble as there are not many OH groups available for hydrogen bonding with
water
- Highly rigid
- Microfibrils are arranged in larger bundles to form macrofibrils. Macrofibrils of
successive layers are interwoven and embedded in gel-like matrix thus having
high tensile strength
- Ideal for formation of cell wall
Describing bond: β-1,4 glycosidic bond is formed between the hydroxyl group of carbon1 and OH group of carbon-4, in a condensation reaction where 1 water molecule is
removed. Enzyme is needed to catalyse reaction.
-
Synthesis of cellulose: It is synthesised in cell surface membrane. Cell wall is outside
membrane. Cellulose synthase enzymes are embedded on cell surface membrane thus
allowing alignment of microfibrils outside cell. If cellulose is synthesis in cell, it is too
large to exit cell.
Reducing sugar test: Add 2cm3 Benedict’s soln, H2O, place in water bath for
2min. Red=reducing sugar
Starch: α-1,4-glycosidic bond & α-1,6-glycosidic bond. Thousands of α-glucose residue
- Consist of amylose and amylopectin
Amylose: α-1,4-glycosidic bond. Gives blue-black colour under iodine
o Coils into helical compact structure stabilized by hydrogen
bonds. Unbranched
Amylopectin: α-1,6-glycosidic bond. Gives red-violet colour under iodine
o
-
Coils into helical compact structure, allowing many glucose molecules to
be stored in small volume within cell
Large molecule, not many free OH group to form H bond with water, hence insoluble
Glycogen: α-1.4-glycosidic & α-1,6-glycosidic bond. More extensive branching compared to
amylopectin leading to more compact helical structure stabilized by H bonds.
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Each branch on glycogen ends with a non-reducing sugar unit. This makes it
unreactive and ensures it is only broken down when needed as a specific enzyme
is needed to act on it
Triglyceride: 3 hydrophobic fatty acids & 1 glycogen molecule.
-
Long hydrocarbon chains, thus more C-H bonds to release more ATP
upon hydrolysis and oxidation, yielding 2x metabolic energy than
carbohydrates
- Higher calorific value than carbohydrates, because they are less dense as they
are more compact and unhydrated due to hydrophobic, saturated fatty acid chain
- Can be stored in large amounts without exerting osmotic effect. Molecules are
large and non-polar due to presence of hydrocarbon tails thus insoluble in water.
They cannot diffuse out of the cell
- Soluble inorganic solvent as hydrophobic interactions can be formed between
fatty acid chain and solvent molecule
Ethanol emulsion test: Add 2cm3 ethanol, shake vigorously, add 2cm3 water, emulsion
(white) = lipid
Formed via 3 ester bonds (condensation)
Phospholipid: 1 glycerol, 2 hydrophobic carbon chains, 1 hydrophilic phosphate
group(amphipathic)
-
Formed from condensation rxn of 2 ester bonds and 1 phosphoester bond
Forms phospholipid bilayer. Associated with oligosaccharides to form glycolipids
for cell-cell recognition
- Needed to form acetylcholine. Micelles used to transport fat from stomach to liver
- Hydrophilic head faces aq medium, the hydrophobic chains face each other in
the middle of the bilayer to form hydrophobic core
<insert image to make sense of whatever you’re writing>
Cholesterol: Carbon skeleton of four fused carbon rings. Insoluble in water as they are nonpolar
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Regulate membrane fluidity. Synthesise sex hormones by acting as precursor
*Proteins: Start with NH3, end with COOH
Peptide bond: Formed between COOH of 1 amino acid and NH3 of another amino acid by
condensation reaction.
Biuret test: Add 2cm3 H2O, biuret soln. Purple = protein, ↑ intensity = ↑ protein
No. of pp combi = nr, n = types of aa, r = no. of aa in pp chain
Protein structure:
-
Primary structure: Specific no, and sequence of aa joined by peptide bond in
pp chain.
Secondary structure: Repeated folding & coiling of primary structure. H bonds
formed between peptide bonds. H bonds formed between N-H in a peptide bond of
an aa and C=O of another aa.
o α-helix: unbranched polypeptide chain tightly coiled to spiral, where each
turn of helix consists of 3.6 amino acids. Held by intra-chain H bonding
between N-H and C=O 4 amino acids away in peptide bond. Highly rigid but
flexible
o β-pleated sheet: adjacent extended regions of a single polypeptide chain
arranged in parallel/antiparallel manner. Held via intra-chain H bonding. NH group in peptide bond of a region in 1 chain and C=O group in peptide
bond
-
-
of adjacent region. Numerous straight chains make it rigid → high tensile
strength.
Tertiary structure: Specific, unique 3D conformation, maintained by Hydrogen, ionic,
disulphide bonds and hydrophobic interactions (H2ID) between R groups on a single
pp chain
Quaternary structure: >1 pp is held together by H2ID between R groups of
different polypeptide chain
Collagen:
-
Each molecule is tropocollagen consisting of 3pp held by H bonds to form triple
helix, making it relatively rigid.
- Triple helices lie parallel in stagger pattern to form fibrils, with covalent bonds
between neighbouring chains. Fibrils unite to form fibres → high tensile
strength
- Hydrophobic aa are found at exterior surface allowing it to be insoluble
(use these 3 features to explain fibrous proteins)
-
Proline & hydroproline are bulky & relatively inflexible
Every 3rd aa is glycine as only glycine is small enough to fit into centre. This makes it
compact → high tensile strength
- No tertiary structure. Unfolded, parallel polypeptide chains
- Only H bods, no H2ID
- Structural and support
function Haemoglobin:
-
-
4 polypeptide chains, each chain repeatedly coiled to form α-helix. Further folding
of α-helices to form compact spherical shape (tertiary), allowing many haemoglobin
molecules to be dissolve into cytoplasm of RBC for max O2 absorption
4 subunits, each containing 1 haem grp
Soluble, hydrophobic aa in interior and hydrophilic aa at exterior
Involved in metabolic rxn → transport O2 in blood
- Haem grp held in hydrophobic pocket → allow reversible O2 binding
Disulfide bond: formed when 2 SH grps are oxidised. Strong covalent bonds that
maintain globular structure by increasing resistance to high temp. Only extreme pH/high
temp can break this bond.
*Significance of R groups to protein structure:
-
R groups are of diff nature: polar/non-polar, acidic/basic
Interactions between R groups can form H2ID
Giving rise to unique 3D conformation which determines tertiary and
quaternary structure respectively
*change in primary structure: Affects:
-
Number & sequence of aa
How protein folds due to diff R grps and bonds formed, changing 3D structure
R groups of aa can form H2ID at diff locations within pp chain
Denaturation: Loss of specific 3D conformation by breaking bonds maintaining protein
structure causing protein to lose biological function (give eg if more marks)
Factors affecting protein structure:
1. pH: Changes in pH disrupts ionic & H bonds between R groups of aa, distorting
specific 3D structure of enzyme. If too acidic, acidic R group COO- accepts H+ ions to
form COOH. If too basic, basic R group NH2 donates H+ to form NH3+. As R groups
are no longer charged, ionic bonds are disrupted leading to denaturation.
2. Temperature: Increases kinetic energy, disrupting weak H2I bond eg. Egg
white becomes insoluble upon heating
3. Heavy metals: positively charged thus form ionic bonds with acidic R groups
disrupting ionic bonds in protein. With less negative charges on protein, solubility
is reduced.
4. Reducing/oxidising agent: Disrupt disulfide bonds → lose 3D conformation
5. Organic solvent: disrupt H bonding and H interaction
G-protein linked receptor: Transmembrane protein for cell signalling
-
Associated with a G protein which binds to GDP/GTP
Single pp chain with 7 hydrophobic transmembrane α-helices → allow protein to be
embedded @ hydrophobic core
Extracellular NH2 and specific loop between helices: extracellular ligand binding site
Intracellular COOH and specific loop between helices → intracellular G protein
binding site
Structure of G-protein: Has 3 subunits: Gα, Gβ and Gγ
Cell Biology
Nucleus: Double membrane nuclear envelope. Nuclear pores allow mRNA synthesised in
nucleoplasm to move to cytoplasm. Heterochromatin (highly condensed chromatin) is not
transcribed due to compact nature. Euchromatin (less condensed chromatin) is frequently
transcribed. Nucleolus contains DNA for rRnA synthesis.
Function: Contain hereditary material of organism in the form of DNA. DNA
replication occurs here. Nucleolus is site of rRNA synthesis. Ribosomal proteins
imported from cytoplasm assembles with rRNA in nucleus to form large and small
ribosomal subunit. These subunits move to cytoplasm to associate into ribosomes.
*Ribosome: Synthesises protein. Transport vesicles containing protein bud off from Rough
ER and move to cis face of Golgi Apparatus, fusing with it. It is ten chemically modified then
concentrated in Golgi Apparatus. Secretory vesicle buds off trans face of GA, moves along
cytoskeleton to cell surface membrane. Membrane of secretory vesicle fuses with cell
surface membrane, releasing contents by exocytosis.
→ Non-membrane bound spherical organelles of large and small subunit. Each unit is made
of rRNA and ribosomal proteins
Rough ER: 3D network of interconnecting flattened membrane bound sacs. Lined with thin
single membrane continuous with outer nuclear membrane.
Function: Folds polypeptides into functional proteins. Transport of proteins as
transport vesicles bud off from rER and are transported to GA. Carbohydrate chains
are added to proteins by glycosylation to form glycoproteins.
Smooth ER: 3D network of interconnecting membrane bound sacs called cisternae. More
tubular than rER, it is lined with thin single membrane.
Functions:
o
Synthesise lipids – enzymes in sER are needed to synthesise cholesterol
Detoxification – enzymes add hydroxyl grps to make toxic substances
soluble for expulsion out of the body
o Carbohydrate metabolism – enzyme for breakdown of glycogen to
glucose found in sER lumen
o Calcium storage – pumped & stored in sER. Release of Ca2+ triggers muscle
contraction
Golgi body: Role in chemical modification of proteins & lipids from rER and sER.
Glycosylation involves addition of short carbohydrate chain to form glycolipids and proteins.
Proteins formed in rER are temporarily stored in GA then packaged into secretory vesicles.
o
-
Formed lysosome. Vesicles containing hydrolytic enzymes bud off GA to
form lysosome
Trans-face secretory vesicles: used for transport of contents out of cell by exocytosis.
Structure: single membrane sacs
Lysosome: Contain hydrolytic enzymes eg. Protease. Single membrane bound.
Function: Digestion of foreign particles. Membrane of lysosome fuses with membrane
of phagocytic vesicle. Hydrolytic enzymes in lysosome digest contents to soluble
products, which diffuse to cytoplasm for cell use. Undigested materials are released
by exocytosis.
Autolysis: self-destruction of cell by release of hydrolytic enzyme eg. When tadpoles
mature into frogs, autolysis occurs and tail shortens
Mitochondrion: Cylinderical/rod-shaped. Bound by double membrane. Outer membrane is
smooth continuous boundary and inner membrane is folded extensively to cristae.
Cristae: ↑ SA for proteins and enzymes to be embedded in inner membrane eg.
Stalked particle with ATP synthase and electron carriers
Mitochondrial matrix: contains 70s ribosomes/circular DNA/glycogen granules/Krebs
cycle enzymes
Function: site of cellular respiration where glucose is metabolised to
synthesise ATP. ATP is needed for cellular processes such as
macromolecule synthesis
Chloroplast: Cylindrical. Bound by double membrane. Outer membrane – smooth,
continuous. Inner membrane gives rise to thylakoids and lamellae. Thylakoids stacked to
form grana. Extensive thylakoid folding ↑ SA for electron carriers, stalked particles
containing ATP synthase to be embedded in its membrane.
Stroma contains circular DNA, 70s ribosomes, Calvin cycle enzymes, starch grains
Function: site of photosynthesis, where light energy is used to synthesise organic
compounds like glucose from CO2 and H2O.
*Both mitochondrion and chloroplast are about 1-10µm in size and divide by binary fission.
Endosymbiont Theory: Mitochondria and chloroplasts’ outer membranes have structural and
chemical similarities. 70s ribosomes are found there, both divide by binary fission.
Centrioles: Pair of rod-like structures, positioned at right angles to each other. Composed of
9 sets of triplet microtubules arranged in a ring, found next to nucleus.
Function: During cell division in animal cells, centrioles replicate & migrate to
opposite poles of cell. Organise microtubules to spindle fibres for chromosomal
movement during cell division.
Cytoskeleton: Consists of microtubules, microfilaments.
Microtubules: Made up of helically arranged globular tubulin
- Designed to resist compression
- Lengthen and shorten by polymerisation/depolymerisation of
tubulin Function: Facilitate chromosomal movement during cell division
Microfilament: Made of solid rods of globular actin
- Designed to resist tension
- Thinnest class of cytoskeleton fibres
Function: Cleavage furrow during cell division. Ring of actin-myosin contracts to form
cleavage furrow. Maintain & change cell shape by disassembling/assembling
microfilament.
Cilia & Flagella: Ring of nine microtubule doublets surrounding central pair of microtubules.
Both have a basal body at base
Function: Allow locomotion of unicellular organisms
Vacuole: Fluid filled sacs, single membrane bound
Function:
o
o
o
o
Storage of pigmented colourings
Storage of defensive compounds
Storage of hydrolytic enzymes
Deposition site for metabolic waste products
Cell wall: Made of cellulose microfibrils, have pectin/cellulose/hemicellulose
Function: Provide mechanical strength and support. Maintain cell shape due to high
tensile strength. Orientation of microfibrils limit cell growth.
Analysis of cell membrane: Cell frozen and fractured with knife, split along hydrophobic
interior, revealing fluid mosaic structure
Fluid: phospholipids and protein move freely within bilayer. Phospholipids move laterally
within a layer while embedded proteins move slowly. Some have restricted movement as
they are attached to cytoskeleton.
Mosaic: Protein is scattered and embedded among phospholipids
Proteins: External extrinsic proteins are attached to fibres of extracellular matrix, keeping cell
in place. Internal extrinsic proteins are attached to cytoskeleton. They maintain cell shape &
mobility. Easily removed from membrane.
Intrinsic proteins: Embedded in hydrophobic core. Contain both hydrophobic & hydrophilic
regions. Hydrophobic regions form hydrophobic interactions with hydrophobic fatty acid tails.
Hydrophilic regions form H bonds with aq medium and hydrophilic phosphate heads. Can be
removed without disrupting membrane.
Protein functions:
-
Channel proteins – hydrophilic pores for movement of charged/polar molecules (eg.
Cl channel). Aid in facilitated diffusion.
Carrier proteins – Aid in facilitated diffusion/active transport as they have specific
binding sites and undergo conformation change when solute binds to it (eg.
glucose transporter)
Enzymes – Stalked particles containing ATP synthase. Carry out sequential steps
in metabolic pathway.
- Signal transduction – Receptor proteins have sites of attachments for
signal molecules to bind (eg. Insulin receptor)
Glycoproteins & glycolipids: Found on outer surface of membrane
-
Function:
o Cell – cell recognition
o Cell – cell adhesion
Cholesterol: Found in membrane of animal cells. Amphipathic, interspersed among bilayer.
Function:
o
o
Regulate fluidity. At high temp, prevents phospholipids from moving too
far apart by forming hydrophobic interactions with fatty chains. At low
temp, prevent phospholipids from packing too close, preventing cell
freeze, membranes remain functional as they are fluid.
Maintain mechanical stability of membrane.
Factors affecting membrane fluidity:
-
Temp
o Low temp – molecules have low KE, less fluid, entering gel-like state.
o High temp – molecules have high KE. Proteins are denatured as weak H
bonds and interactions disrupted, causing loss of specific 3D config., loss of
tertiary structure, resulting in gaps formed in the spot the protein was once
in, increasing permeability.
Importance:
o
o
o
Cell elongation & division
Formation of vesicles for exo/endocytosis
Growth and self-healing of membrane
Function of cell membranes:
-
Boundary: separate cell contents from external environment. Allow control of
internal environment for optimum function.
Compartmentalisation: Prevent indiscriminate mixing. Allow for specialised
cell function by concentrating specific substances.
Partially permeable: Hydrophobic tails repel hydrophilic molecules like
charged/polar molecules. Allow passage of hydrophobic molecules (eg CO2) or
small polar molecules like H2O
Signal transduction: Receptor proteins have sites of attachments for signal molecules to bind
to for cell signalling.
Types of transport:
-
Passive transport: Movement of substances down conc. gradient w/o ATP
Diffusion: Net movement of mols down conc. gradient until dynamic eqm reached.
Factors in diffusion: Size of mols, conc. gradient, distance, surface area
Facilitated diffusion: Net movement of mols down conc. gradient with help of
specific transport proteins till dynamic eqm.
Osmosis: Net movement of water mols down water potential across
partially permeable membrane until dynamic eqm.
-
-
-
-
Active transport: Movement of substances against concentration gradient using
ATP. Highly selective & unidirectional, involving specific transmembrane carrier
protein
Bulk transport: Transport large mols using ATP
Endocytosis: Intake of substances by invagination of cell surface membrane or
extension of pseudopodia. Small area of cell surface membrane pinches off to form
a vesicle.
Phagocytosis: Uptake of solid, large materials. Specific process: cell extends
pseudopodia to engulf bacteria, forming phagocytic vesicle membrane. Hydrolytic
enzymes digest contents to soluble products, diffusing to cytoplasm for cell
usage. Membrane of vesicle fuses with cell surface membrane, releasing
undigested material by exocytosis.
Pinocytosis: Uptake of droplets of extracellular fluid. Unspecific as all solutes
are taken in. Carried out by invagination of cell surface membrane.
Receptor-mediated endocytosis: Specific as specific substances (ligands) bind to
proteins with specific receptor sites. The receptor proteins cluster in coated pits
which forms a vesicle containing ligand by invagination of cell surface
membrane. After release of ingested material, emptied receptors are recycled to
cell surface membrane.
Enzymes
-
Biological catalyst speeding rate of metabolic rxns while being chemically unchanged
Highly specific to substrates – Active sites specific and complementary to
substrate by SSCO(shape size charge orientation)
Effective in small amts due to high turn-over rates
Require cofactors for functions → activate the enzyme
-
o Cofactors: inorganic ion
o Coenzymes: bind loosely to enzyme
o Prosthetic grp: organic molecules tightly bound to enzymes
Active site: held by 3 to 12 aa from different parts of single pp
E – S complex:
1. Substrate enter active site, enzyme change shape such that active site
enfolds substrate. Held by weak interactions
2. Lower EA and speed up exn by providing favourable microenvironment
3. Convert substrate to product. Release products
4. Active sites available again
Lock & key: Enzyme fits precisely with substrate. Active site perfectly complementary by
SSCO. Probable for enzymes that only act for one type of substrate.
Induced-fit: Enzymes work in more flexible manner. Active site not perfectly complementary
by SSCO. Upon forming some bonds with substrate, enzyme changes shape to lead to more
precise fit eg. Lipases
EA: ↑temp, reactants↑KE, colliding ↑frequency and force. Thermal agitation of atoms in mols
contort reactants, making bonds likely to break. When they absorb enough energy for bonds to
break, they enter transition state.
Measure rate of rxn: eg. measure product – catalase
1. Use syringe, add 5.0cm3 5% H2O2 to boiling tube
2. Use syringe, add 1.0cm3 2% catalase to small vial
3. Place small vial held by string containing boiling tube containing H2O2 & seal
with rubber bung
4. Place boiling tube in water bath and equilibrate for 5min
5. Connect delivery tube to deliver enzyme to H2O2 and start timing
6. Measure V(O2) per min for 10min
Michaelis constant (Km): [substrate] at ½
Vmax
Factors affecting enzyme activity:
[substrate] – linear, proportional ↑in rate or rxn. As [substrate] increases, increase in
rate slows down, before plateauing off and max rate reached.
Explain: ↑[substrate] → ↑frequency of effective collision → ↑ E-S complex formed per unit
time → proportional ↑in rate of rxn. [substrate] is limiting. At high conc, all active sites of
enzymes are occupied → max E-S complex formed per unit time. [enzyme] is limiting.
- [enzyme] – Linear ↑as [enzyme] ↑. As it keeps increasing, increase in rate slows down,
plateauing off eventually and max rate reached
Explain: At low [enzyme] → ↑in [enzyme] → ↑no. of active sites → ↑in frequency of eff.
collisions →↑E-S complex formed per unit time → ↑in rate of product formation →
[enzyme] limiting
High [enzyme] → no further ↑in E-S complex formed per unit time as substrate is not enough to
occupy all active sites. Other factors limiting.
- Temperature: ↑temp → ↑KE of enzyme & substrate → affect stability of protein
structure. At low temp, rate is slow. Rate doubles for every 10% increase. At optimum
temp, rate is at max. At high temp, there is drastic fall in rate and it falls to zero.
Explain: low KE → move slowly → few eff. collisions → low rate of E-S complex formed at low temp.
As it ↑to optimum temp, KE ↑, molecules move faster. ↑eff. collisions → ↑in rate of E-S
complex formed. Substrate has higher chance to overcome EA barrier and form
products. At optimum temp, max E-S complex formed. >optimum temp, thermal
agitation disrupts weaker bonds (H2I) → distorts specific 3D conformation of enzyme
-
→ active site distorted and not complementary to substrate → no E-S substrate
formed. Enzyme denatured → ↑% of enzymes denatures, thus fall to zero.
- pH curve: Bell-shaped, symmetrical about optimum pH. As pH varies slightly,
rate falls drastically
Explain:
o Optimum pH – all ionic, H bonds between R groups intact. Max E-S
complex formed per unit time.
o Slight pH change – change affected on basic and acidic R groups of aa at
active site, ↓ binding ability of substrate to active site, rate of E-S complex
formed ↓ due to weakened bonds.
o Drastic pH change – disrupt ionic bonds between ionic and basic R
groups and H bonds between polar R groups. 3D conformation distorted,
specific active site distorted, no longer complementary, ↓ E-S complex
formed, denatured.
At low pH, there are still a small number of functional enzymes carrying our rxn.
Reversible inhibition: When inhibitor & enzyme are held by weak bonds (eg H bond)
Competitive inhibition: Structurally similar by SSCO, bind to active site and compete with
substrate for active site. ↓ no. of active sites for substrate to bind and form E-S complex.
-
Km ↑, Vmax can be eventually reached
-
At low conc. of substrate, more likely to form E-I complex, ↓ no. of active sites, ↓ E-S
complex
High conc. of substrate, ↑E-S complex. Inhibitor has no effect on Vmax
-
Non-competitive inhibition: Structurally unsimilar to substrate. Bind at site away from active
site. Alters specific 3D conformation → active site distorted hence uncomplimentary to substrate
- Km unchanged, Vmax lowered
Irreversible inhibition: Permanent binding & dmg to enzyme. Strong covalent bonds
between enzyme & inhibitor
Cooperativity: Binding of one substrate locks all subunits in active conformation.
Allosteric activation: Activator/inhibitor binds to allosteric site. Activator stabilises shape of
enzyme with functional active site.
Phosphofructokinase is product of respiration. Feedback inhibition occurs where excess
phosphofructokinase inhibit enzymatic (Krebs cycle) activity.
How does enzyme speed up reaction: Provide alternative pathway of lower EA. By allowing
close proximity of reactants of reaction due to E-S complex formation, ensuring correct
orientation of reactants for effective collisions, destabilising bonds of reactants to facilitate
formation of transition state.
DNA genomics
DNA (deoxyribonucleic acid)/RNA (ribonucleic acid)
Nucleotides joined by condensation rxn, phosphodiester bonds. Nitrogenous base bonded to
1’C of pentose, phosphate to 5’C of pentose.
Nitrogenous bases: A=T, C≡G. A&G – purines. One 6 membered ring to 1 5 membered ring. 5’ end of
polynucleotide: Phosphate grp to 5’C of sugar. 3’ end: OH group on 3’ of sugar
DNA structure: Antiparallel. 1 strand runs from 3’ to 5’ and the other from 5’ to 3’. Pentose
sugars are on 1 strand inverted wrt other strand. Since DNA polymerase can only work from
5’ to 3’, presence of lagging & leading strand shows they are antiparallel.
-
Full turn every 3.4nm to base pairs in each turn
Soluble. Hydrophilic sugar-phosphate backbone outside helix. Hydrophobic
bases paired inside helix. Base pair stacking leads to hydrophobic interactions.
- Polynucleotides held together by H bonds between bases, making it rigid and of
high tensile strength.
Function: DNA is the genetic material organisms inherit from parents. Each DNA
molecule contains numerous genes, which are units of inheritance, storing coded
instructions for synthesis of RNA/protein.
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Replication of DNA at cell division. Weak H bonds allow double stranded DNA to be
easily broken to 2 single stranded DNA, to serve as templates for semi-conservative
replication.
RNA: Single polynucleotide chain. Less stable than DNA as it is more prone to hydrolysis by
intercellular enzymes. Can develop secondary structures by complementary base pairing
within RNA molecule.
mRNA: Synthesised by transcription in nucleus, used as template to synthesise protein.
rRNA: Synthesised in nucleolus. rRNA and ribosomal proteins assembled into small and
large ribosomal subunit, then transported to cytoplasm and associate to form ribosomes.
Site of protein synthesis: rRNA of large subunit has peptidyl transferase which catalyses
formation of peptide bond between aa to form pp.
tRNA: Single stranded molecule. Anticodon present on tRNA which forms H bonds with
codon on mRNA by complementary base pairing. 3’ acceptor stem serves as site for aa
attachment. Clover-leaf structure of tRNA further folds to 3D structure so that tRNA can fit
into ribosome during translation. tRNA transfers correct aa to ribosome for translation.
Semi-conservative replication (SCR): Double helix parental DNA replicate and each
daughter DNA has a parental strand and newly synthesised strand. Two strands of parental
molecule separate by breaking H bonds between complementary bases, each strand acts
as template for synthesis of new strand.
Conservative replication: Parental molecule is intact and resulting daughter mol formed from
2 newly synthesised DNA strands. Both strands of parental mol act as templates for
synthesis of entirely new DNA mol. Two parental strands reassociate to restore parental mol.
Dispersive replication: Each daughter strand has a mixture of old and newly synthesised
DNA. Parental DNA molecule breaks up to short segments, acting as template for DNA
synthesis. Segments are joined together, resulting in both old and new DNA interspersed
along each strand in both daughter DNA mols.
Expt to prove SCR: Bacteria used as they reproduce quickly so many generations can be
studied in short time, easy to culture in large quantities.
1. E-coli containing NH4Cl with 15N grown in medium for many generations
2. Bacteria transferred to 14N medium with 15N medium and allowed to grow. Two
or more generation samples after transfer collected.
3. DNA extracted and put to C5Cl2 soln and spun at 40,000g at centrifuge.
4. C5Cl2 sink to bottom of test tubes, forming density gradient.
5. Tubes observed under UV rays. DNA appear as fine bands in different heights
based on density.
Results: By SCR, 1st generation has intermediate density. ½ of 2nd generation DNA is 15N14N
and the other half is 14N14N.
Explain: Original parent mol consisting both 15N strands separate, acting as template for
G1 synthesis. In G1, all DNA mols have 1 original parental 15N strand and a newly
synthesised
14N strand, this intermediate density. In G2, both strands in G1 separate, each 14N and 15N
stand act as templates for synthesis of new complementary strand.
Replication: Occurs at S phase of interphase.
1. Portion of double helix unwound and unzipped at origin of replication by
DNA helicase and H bonds between parental strands broken.
2. Each strand bound and stabilised by single-stranded binding proteins,
preventing them from rewinding behind replication fork.
3. DNA topoisomerase introduces break in single strand, allowing it to rotate around
the break and reseals strand, eliminating positive supercoil.
4. Each parental strand acts as a template for synthesis of new daughter strand.
5. Primase synthesises RNA primers in 5’ to 3’ direction by adding ribonucleotides
via complementary base-pairing using parental strand as template.
6. DNA polymerase adds deoxyribonucleotides to 3’ OH end of RNA primer by
complementary base-pairing with parental strand and catalysing formation
of phosphodiester bond between nucleotides.
7. Adenine pairs with thymine with 2H bonds, guanine pairs with cytosine with 3
H bonds.
8. Leading strand synthesised continuously from 5’ to 3’ direction. Lagging strand
synthesised discontinuously via series of Okazaki fragments away from replication
form, catalysed by DNA polymerase. Each Okazaki fragment is primed separately.
Primase catalyses synthesis of RNA primers, which are excised and replaced with
deoxyribonucleotides by another DNA polymerase III. DNA ligase catalyse
formation of phosphodiester bond between 2 Okazaki fragments.
9. Product of SCR is daughter DNA formed form 1 parental mol. Each daughter
mol contain 1 strand from parental mol and newly synthesised strand.
End-replication problem: DNA polymerase require free 3’ OH group to add
deoxyribonucleotides to. RNA primers at 5’ end are excised but cannot be replaced due to
absence of 3’ OH end. Newly synthesised strand has shorter 5’ end due to removed primers,
while parental template strand at 3’ end is longer.
DNA molecule stability: Nitrogenous bases held by H bonds. Hydrophobic interactions
between stacked bases stabilise structure of double helix. Nucleotides are held by strong
covalent phosphodiester bonds, which are not easily broken, maintaining integrity of DNA
base sequence.
DNA replication: Essential for accurate replication so daughter nuclei have identical DNA
copies as parent nucleus. Double helical structure allows SCR to occur (Explain SCR). DNA
polymerase ensures accuracy by pre-synthetic error control/proofreading.
Transcription: Synthesis of RNA molecule using DNA strand as template
Prokaryotes
-
Sigma factor of RNA polymerase to
-35 and -10 sequence of promoter (10 = Pribnow Box)
- Sigma factor released from core
enzyme
- RNA polymerase unwinds and
separates 2 strands of DNA by
breaking H bonds between bases
- Template strand available for CBP
with ribonucleotide
Eukaryotes
-
-
TATA binding protein recognises
and binds to TATA box of promoter
General transcription factors and
RNA polymerase recruited to form
transcription initiation complex
RNA polymerase unwinds and
separates 2 strands of DNA by
breaking H bonds, template
available for CBP with
ribonucleotide
-
-
-
RNA polymerase adds
ribonucleotides to free 3’ OH end of
growing RNA chain, synthesising
new RNA strand in 5’ to 3’ direction
via complementary base pairing
with template strand. This is done
by catalysing formation of
phosphodiester bond between 3’
OH end of RNA and 5’ phosphate
grp of NTP. Energy needed
originates from 2 phosphate grps
removed as NTP joins growing end
Termination occurs after terminating
sequence is transcribed
Short RNA-DNA hybrid separated,
releasing newly synthesised RNA
polymerase & RNA transcript
No further modification
-
-
Termination after polyadenylation
signal sequence is transcribed
Codes for AAUAAA in pre-mRNA
At 10-35 NTP downstream, premRNA is cleaved, releasing premRNA and RNA polymerase
Post-transcriptional modification,
releasing mature mRNA
Post-transcriptional modification: occur @ nucleus
(Not in prokaryotes due to lack of nucleus & translation occurs simultaneously with
transcription)
1. 5’ methylguanosine nucleoside triphosphate: added to 1st nucleotide by 5’-5’
triphosphate linkage. Catalysed by guanylyl transferase.
Function: Facilitate export of mature mRNA from nucleus to cytoplasm. Protect mRNA from
5’ exonucleases, conferring stability in mRNA. Facilitate in binding of ribosomes to
mRNA.
2. RNA splicing: Introns are non-coding DNA. Splicing occurs to remove introns and ligate
exons to form continuous coding sequence. Alternative splicing gives rise to alternative RNA
arrangements and different pp chains can be made from same gene.
↑ no. of proteins without ↑ genome size.
How: specific small nuclear ribonucleoproteins (snRNP) located in nucleus recognise and
bind to 5’ & 3’ splice site. Additional proteins interact with snRNPs to form
spliceosome. As exons are brought closer together, intron loops. Spliceosome excise
introns and join exons that flanked intron, releasing introns in lariat structure.
3. 3’ poly-A tail: 200 adenine residues added to 3’ end of pre-mRNA. Catalysed by
poly- A-polymerase. Slows degradation by 3’ exonuclease.
Genetic code: Gene-specific nucleotide sequence of DNA coding for functional product
-
-
Codon: 3 consecutive nucleotide bases.
Non-overlapping: ribosomes can read codons one after another with no space
between codons. Thus ribosome joins aa in correct seq as coded by codon
sequence to form polypeptides’ primary structure.
Degenerate: >1 codon can code for same aa
Triplet: 3 consecutive nucleotide bases code for 1 aa → specific codon sequence
determine specific aa sequence
Start codon: AUG. Stop codons: UGA/UAA/UAG
Translation: synthesis of protein using mRNA as template
Accurate translation requires: correct match between tRNA and aa during aa
activation/correct match between tRNA anticodon and aa codon (CBP)
Amino acid activation: Each aa is joined to correct tRNA by aminoacyl-tRNA synthetase.
-
Active site of aminoacyl-tRNA synthase recognises and binds to specific pair of
aa and tRNA
Synthase catalyses covalent attachment of aa to 3’ acceptor stem of
corresponding tRNA
Each tRNA has specific anticodon which binds to codon on mRMA
by complementary base pairing
Resulting aminoacyl-tRNA/activated aa is released from enzyme and delivers aa to
growing pp chain on ribosome. This process is genetically controlled and takes
place in cytoplasm
Translation steps:
1. Small ribosomal subunit binds to 5’ end of mRNA and specific initiator tRNA which
carries aa methionine, with aid of initiation factors (note: in prokaryotes, initiator
tRBA carries N-formyl methionine)
2. Small ribosomal subunit scans downstream along mRNA until it reaches AUG,
start codon, establishing start of proofreading frame for mRNA
3. H bonds formed between complementary bases of anticodon on tRNA and
start codon on mRNA
4. Large ribosomal subunit attaches and binds to mRNA forming translation
initiation complex
5. At completion of initiation process, initiator tRNA sits in P site, and vacant A is
ready for next aminoacyl-tRNA
6. Anticodon of incoming aminoacyl-tRNA CBPs with mRNA codon in A site
7. Peptidyl transferase @ rRNA of large ribosomal subunit catalyses formation of
peptide bond between new aa in A site and COOH end of growing pp in P site,
transferring pp to A site
8. Ribosome translocates by advancing a codon along mRNA. Empty tRNA in P
site moves to E site to be released
9. Elongation continues until stop codon in mRNA reaches A site of ribosome
10. UAG/UAA/UGA binds directly with release factor, causing addition of 1
water molecule, hydrolysing pp chain
Polyribosomes allow cell to make many copies of pp directly
Control of genome:
Genome: complete genetic sequence on one set of chromosomes. Including genes
& non-coding sequence
Spatial control: specific sites.
Temporal control: produced at certain time
Constitutive/housekeeping genes: constant levels of expression
Sizes of DNA structures/how DNA is packaged in cells:
-
Nucleosome: Double stranded DNA wrapped around core of histone proteins,
joined by linker DNA, giving rise to beads-on-string structure. Histones have high
levels of
+ve lysine & arginine residues
↓30nm solenoid/chromatin fibre: folding of these 30nm fibres to loops attached to
scaffolding proteins
- ↓300nm looped domains: 700nm fibres when looped domains are further folded Gene
density: Number of genes in specific no. of base pairs. Eukaryotes have ↓density due to large amt
of non-coding sequence
-
Non-coding DNA: DNA sequences that don’t code for any functional product (UTR, control
element, introns, etc)
<insert drawing>
Transcriptional control:
Core promoter region: has TATA box and transcriptional start site. Found upstream of coding
sequence. TATA binding protein recognises & binds to TATA box and recruits other transcription
factors and RNA polymerase to initiate transcription. General transcription factors bind at core
promoter to initiate transcription by facilitating binding of RNA polymerase → basal rate of
transcription
Enhancer: Activators recognise & bind to enhancer. As a result of DNA looping, transcriptional
initiation complex stabilised through protein-protein interactions between activator protein,
general transcription factors & RNA polymerase assembling & position transcription initiation
complex @ promoter → ↑ rate of transcription
Silencer: Repressor recognise & bind to silencer near promoters/overlap them.
↓transcription by
-
Preventing activator from binding to enhancer
Recruiting enzyme to ↑ condensation of chromatin thus inaccessible to RNA
polymerase
- Disrupt formation of transcription initiation complex by recruiting proteins bound to
general TIF, destabilising transcription initiation complex
- Impeded movement of RNA polymerase downstream
Centromere: DNA in nature, holds 2 sister chromatids together. Play a role in chromosomal
movement (give eg. of phase), facilitate binding of microtubules of spindle fibres to
centromere via kinetochore
Telomeres: Ends of linear eukaryotic chromosome consisting of short tandem repeats. DNA
in nature, does not contain gene.
Function:
-
Prevent ends of linear DNA from being degraded by exonucleases.
Delay degradation of genes near telomeres due to shortening of DNA
during replication (due to end replication problem)
- Prevent ends of chromosomes from fusing with each other
- Serves as counting mechanism for no. of cell divisions a cell has
undergone, preventing uncontrolled cell division (reach Hayflick limit)
- 3’ single stranded overhang folds back and forms T-loop, hiding overhang.
Overhang hybridises with earlier repeat of some complementary sequence at opp.
strand.
Telomere capping proteins bind at T-loop to maintain stability of structure
Telomerase: A ribonucleoprotein. Maintains telomere length
-
Functions as reverse transcriptase, synthesising double stranded DNA from
single stranded RNA template
-
3’ overhang extended using telomerase RNA as template. DNA NTP
complementary to RNA telomerase is added
Telomerase translocates towards 3’ end of newly added sequence for
further extension
Extended 3’ end used as template to add primers and RNA polymerase
synthesise strand complementary for 3’ overhang
Control of gene expression: Although genes in al nuclei are identical, cell differs
morphologically & functionally due to differential gene expression. Only genes necessary for
specific functions is transcribed, allowing for cell differentiation.
Chromatin structure:
1. Histone methylation:
- DNA methyl transferases catalyse transfer of CH3 to carbon 5 of cytosine ring
- Methylated histones results in proteins recognising and binding to it, leading to
more compact chromatin due to protein-protein interactions. RNA polymerase
cannot access compact DNA
- Methylated DNA attracts other proteins which in turn recruit histone
deacetylation enzymes
2. Histone acetylation:
- Addition of acetyl groups to +ve lysine and arginine residues in tails of
histone proteins, neutralising protein
- Does not bind to -ve charge DNA, ↓interaction between histone protein and DNA,
making it less compact and more accessible to transcription machinery
- Catalysed by histone acetyltransferases
3. Histone deacetylation:
- Histone deacetylase catalyses removal of acetyl grp, inhibiting transcription
Post-transcriptional control:
-
-
Alternative splicing, RNA processing
RNA transport. Eukaryotic mRNA must be transported from nucleus to
cytoplasm due to nuclear membrane preventing simultaneous transcription and
translation
Unprocessed RNA is degraded in nucleus and gene not expressed
Translational control:
Phosphorylation: Inactivate translation initiation factors needed for transcription initiation
complex
Translational repressors: These regulatory proteins bind at 5’ UTR preventing initiation.
Cytoplasmic polyadenylation: Allows temporal control, Cytoplasmic polymerase catalyses
cytoplasmic polyadenylation of 3’ end, activating mRNA translation
Localisation of mRNA: Allow spatial control
Length of poly-a-tail:affect half life of mrna
Post-translational control:
Biochemical modification: Attaching other biochemical functional grps like lipids to activate
protein
Structural modification: Remove aa from protein to produce mature protein
Protein degradation: Proteasomes degrade excess proteins. Ubiquitin covalently attach to
unwanted protein and inject it to core of proteasome and release ubiquitin. Inside, it is
degraded to aa which are recycled back to cytoplasm.
Polymerase chain reaction
Purpose: Amplify specific segment of DNA, synthesising large amts of DNA from minute
starting amt.
Reaction mixture: Taq polymerase (thermostable), single stranded DNA primers. 2 different
DNA primers complementary to flanking sequence of target DNA sequence to be amplified
needed. DNTPs.
Advantage of Taq polymerase: During DNA denaturation, temp ↑ to 95°C. Taq polymerase not
denatured at high temp. Presence of strong disulfide bonds holding tertiary structure make it
thermostable. Thus it can be reused in repeated cycles of PCR, reducing time and cost of carrying
out PCR.
PCR process:
1. Denaturation of double stranded DNA to single stranded DNA at 95°C by breaking
H bonds between bases
2. Annealing of primers via H bonding to flanking sequence of target DNA at 50-60°C.
Binds due to CBP to single stranded DNA. Added in high concentration compared
to sample to prevent reassociation to single strand
3. Taq polymerase adds NTP to 3’ OH end of primers using DNA as template at
72°C Advantages:
-
Sensitivity: Large amts of DNA produced from minute starting amount of materials
Specificity: Specific DNA sequence amplified by using specific primers
Speed & accuracy: Large amts of DNa produced in short time with relatively high
acc of replication
Limitations:
-
Target DNA sequence to be amplified limited to 3kb. Efficiency of amplify ∝1/length
-
Exogenous DNA amplified contaminate PCR tubes
Knowledge of DNA needed to synthesise flanking nucleotide primers
Taq polymerase does not perform proofreading thus mistakes in complementarity
of NTPs may be amplified
- Non-target DNA sequence may be amplified alongside target DNA sequence
Gel electrophoresis: Separating nucleic acids based on size/electrical charge/other
properties by passing them through gel acting as molecular sieve in electric field.
Nucleic acid hybridisation: CBP of nucleic acid to another nucleic acid to form double
stranded molecule. Carried out using nucleic acid probe to locate gene of interest.
Southern blotting: Transfer of separated DNA fragments onto membrane for hybridisation.
Agarose gel electrophoresis: Separate DNA by size
1. Agarose gel prepared by heating agar powder with buffer soln to dissolve
it. Concentration of gel altered to adjust resolution (porous) of gel
2. Agarose gel poured to gel tray and cooled. Comb added at one end of gel to
create walls for DNA loading
3. When it has cooled and hardened, comb removed to reveal walls, then placed
in electrophoresis chamber filled with buffer soln
4. Wells placed at negative electrode and loaded with DNA samples. Loading
dye added to give indication of progress of electrophoresis
o Glycerol added to loading dye as it is dense & allow DNA to sink into
wells Kb(kilobase) ladder loaded to estimate DNA size
5. DC turned on. When DNA run to 2/3 gel length, current stopped to prevent
overrun where samples move to buffer soln.
6. Ethidium bromide added in gel before cooling. When viewed under UV, DNA
bonds fluorescence
7.
Southern blotting (remove the other unneeded genes) + nucleic acid hybridisation:
1. DNA samples cut with restriction enzymes
2. Gel electrophoresis
3. Double stranded DNA denatured to single strands via NaOH as H bonds
between complementary bases are broken
4. Bands on gel transferred to nitrocellulose membrane via capillary action
5. Gel placed in basin of buffer with NaOH. Piece of the nitrocellulose membrane
placed directly above gel. Large amts of paper towels (dry) and heavy weight
placed on top for capillary action, transferring DNA bands to nitrocellulose
membrane corresponding to those on gel
6. Membrane subjected to UV light/high temp to permanently cross-link single
stranded DNA to membrane
7. Membrane containing DNA incubated in labelled DNA probe with nucleotides
containing radioactive 32P. Probe binds to complementary DNA fragment of
interest by H bonds
8. When exposed to x-ray film, radioactive isotope exposes film. Band shows up as
dark band/DNA probe used as luminescence molecule, band can be captured on
polaroid photographs
Outline how nucleic acid can be used to identify troponin: Extract DNA from cells, add
restriction enzymes which cut sequence flanking troponin DNA, perform gel electrophoresis
to separate DNA by size. Gel treated with NaOH to denature double stranded DNA to single
stranded DNA. Radioactive single stranded DNA bind to troponin DNA via complementary
base pairing by forming bonds. Carry out autoradiography by placing x-ray on membrane.
Band containing gene show up as dark band on autoradiogram.
Cell division
Interphase: Cell growth & synthesis of cell materials
1.
-
G1 phase:
Intensive synthesis of organelles
Nucleolus actively synthesis rRNA
Synthesis of proteins
↑cytoplasmic volume
2. S phase:
- DNA SCR, doubled DNA content
- Histone proteins synthesised
- Each chromatin fibre is made of 2 DNA molecules
3. G2 phase:
- Intensive synthesis of organelles
- Replication of centrosome. Each is made of a pair of centrioles
- Synthesis of ATP &
protein Chromosome:
-
Unduplicated: 1 DNA molecule
Duplicated: Double arm structure as each molecule undergoes SCR during
synthesis phase of interphase, giving rise to 2 DNA molecules which condense
during prophase to form 2 identical sister chromatids joined at centromere
Centromere: Satellite DNA bound to kinetochore proteins for attachment of spindle fibres.
Hold 2 sister chromatids together & involved in chromosomal movement @ cell division.
*Homologous chromosomes:
- Same length
- Same centromere position
- Same genes at same loci
- One homolog derived from maternal parent, the other from paternal
- Homologous chromosomes pair to for bivalents @ prophase I
- Each homolog has double structure with 2 identical sister chromatids
- Different alleles
*Difference in chromosomal behaviour @ meiosis vs mitosis:
-
Homologous chromosomes form bivalents in meiosis @ prophase I but
homologous chromosomes do not pair up in mitosis @ prophase
Crossing over occurs at meiosis prophase I but it does not occur in prophase
of mitosis
Homologous pairs of chromosomes line up at equator during metaphase I of
meiosis but chromosomes line up single at mitosis metaphase
Homologous chromosomes separate in anaphase I of meiosis but sister
chromatids separate in mitosis anaphase
Mitosis: Division of parental nucleus once to form 2 genetically identical daughter nuclei with
same no. and type of chromosome as parent
Meiosis: parent nucleus divide twice to produce 4 genetically non-identical haploid daughter
nuclei. Vital for sexual reproduction and take place in reproductive organs
Checkpoints of cell cycle:
1. G1: Adequate cell size & sufficient nutrients, growth factors
2. G2: Adequate cell size & SCR completed successfully
3. Metaphase: All chromosomes attached to spindle fibres
Mitosis VS Meiosis
Prophase
Prophase I
-
Mitosis
Chromatin fibre condense
to chromosomes
Each duplicated
chromosome appears as 2
identical sister chromatids
-
Meiosis
Chromatin fibre condense to
chromosomes
Each duplicated chromosome
appears as 2 identical sister
chromatids joined at
joined at centromeres
Centrosome organise
microtubules to spindle
fibres and radial array to
asters
- Centrosome migrate to
opposite poles of cell
- Nuclear envelope
fragments. Nucleolus
disperse and seem to
disappear
-
Metaphase
Metaphase
I
-
-
-
Anaphase
Anaphase I
-
-
-
Telophase
Telophase I
-
-
centromeres
Homologous chromosomes
pair to form bivalents
- Crossing-over occurs
between non-sister
chromatids of homologous
chromosomes → chiasmata
- Centrosome organise
microtubules to spindle fibres
& radial array to asters &
migrate to opposite poles of
cell
- Nuclear envelope fragments.
Nucleolus disperse and seem
to disappear
-
Microtubules attach to
kinetochore to form
kinetochore microtubules
Centromeres of
chromosomes are aligned
along metaphase plate
Non-kinetochore
microtubules interact with
those on opposite poles of
cell
-
Centromere separates & 2
sister chromatids split,
becoming 2 daughter
chromosomes
Daughter chromosomes
migrate to opposite poles of
cell, with centromere
leading the way as
kinetochore microtubules
shorten
Non-kinetochore
microtubules lengthen,
leading to cell elongation
Nuclear envelope reforms
to 2 nuclei
Nucleolus reappears
Chromosome becomes less
condensed to from
chromatin
Microtubules disperse by
depolymerising
-
-
-
-
-
-
-
-
Bivalents align themselves at
metaphase plate
Chromosome arrangement is
independent of arrangement
of other bivalents
Non-kinetochore microtubule
interact with those from
opposite poles of cell
Both chromatids of 1 homolog
are attached to kinetochore
microtubules
Each homolog of bivalent
separates
Homologous chromosomes
migrate to opposite poles of
cell, with centromere leading
the way as kinetochore
microtubules shorten
Non-kinetochore microtubules
length, leading to cell
elongation
Nuclear envelope reforms to
2 nuclei
Each nuclei has haploid set of
chromosomes (reductional
division)
Nucleolus reappears
Chromosomes become less
condensed to form chromatin
Microtubules disperse by
depolymerising
-
Cytokinesis @ animal
Cell surface membrane invaginate
towards metaphase plate
Ring of actin contracts by interacting
with myosin
Cleavage furrow deepens →
parental cell pinched to 2
Cytokinesis @ plant
Golgi vesicles move along
microtubules towards metaphase
plate and fuse to form cell plate
- Cell plate enlarges with ↑fusing,
until surrounding membrane fuses
with cell surface membrane
-
What happens to chromosome between end of anaphase & start of next mitosis?
-
Chromosomes decondense to chromatin during telophase
Transcription takes place to produce mRNA and synthesise histone proteins
Replication takes place to produce new DNA. SRC occurs where parental DNA
unwinds and unzips and each strand is used as template to form new strand by
complementary base pairing to form 2 genetically identical daughter DNA
molecules, with 1 strand from parent and another newly synthesised
Mitosis importance: Produces 2 genetically identical cells in terms of chromosome number and
structure as parent, enabling ↑in no. of cells for organism growth/replace lost cells during tissue
repair/asexual reproduction
Meiosis:
-
Produce haploid gametes to allow fusion of gametes from different parents during
fertilisation
- Ensure maintenance of chromosomal number in offspring and prevent doubling
of chromosomal numbers during fertilisation
- Generates genetic variation in offspring through crossing-over between nonsister chromatids of homologous chromosome/independent assortment of
homologous chromosomes
Crossing over: Occurs between non-sister chromatids of homologous chromosomes
resulting in chiasmata during prophase I. Results in exchange of corresponding DNA
segment of Chromatids, separating alleles of linked genes and creating new allelic
combinations in chromatid
Involves:
1. Breakage of corresponding DNA from each non-sister chromatid
2. Exchange
3. Rejoining of DNA segment to the other chromatid → chiasmata
Random fertilisation: Genetic material from 2 different individuals is combined with random
fusion of gametes. Possible combinations = 2diploid no.
Nondisjunction: When pairs of homologous chromosomes fail to separate @ anaphase I of
meiosis/sister chromatids fail to separate @ anaphase II → aberrant gametes → when fused with
normal gamete, zygote has abnormal no. of chromosomes → aneuploidy
Mutation and Cancer
Mutation: Change in nucleotide sequence of DNA/chromosome structure/no. of
chromosomes
Germline mutation: Occur @ gamete cells, passed on to next generation
Gene mutation: Change in nucleotide seq → change in mRNA seq → change in aa seq → change
in primary structure → change in 3D conformation → change in protein
Types of gene mutations:
1. Substitution: A type of point mutation → Usually silent mutation (effect)
2. Addition/deletion: Involve 1 or more nucleotide pairs → usually missense/nonsense
mutation
Frameshift mutation:
-
When nucleotides inserted/removed are not in multiples of 3, frameshift
mutation occurs
- All nucleotides downstream improperly grouped to codons, resulting in
extensive change in aa seq
- Leading to nonsense mutation, causing premature termination of
translation Silent mutation:
-
Base pair substitution result in mRNA codon changing from
to
, coding
for same aa
due to
degeneracy of genetic code
- Gene mutation has no change in aa seq → no change in specific 3D structure →
binding site remains the same
Sickle-cell anaemia: Autosomal recessive. Substitution of single nucleotide from CTT to CAT in DNA
template of chromosome II → glutamate to valine
Effect: Tend to polymerise to long chains, distorting membrane of RBC giving it a distinct sickle
shape
-
↓O2 carrying ability of RBC, sickle shaped cells have ↓ lifespan
- Sickle cells may clog small blood vessels → organ damage
Chromosomal aberration: Deletion – cri du chat caused by specific chromosomal deletion of
chromosome 5
Translocation: Chronic myelogenous leukaemia → extra long chromosome 9 and short 22
Nondisjunction:
- If occurs @ anaphase I, all gametes are aberrant
- Anaphase II: ½ only. If aberrant fuse with normal, zygote is
2±1 Examples:
1. Down syndrome: 3 chromosome 21. Autosomal chromosomal aberration
2. Turner syndrome: Only 1X chromosome. Phenotypically female. Sterile due to
non- mature sex organ
Cell cycle regulation
Proto-oncogene: Gain in function mutation to oncogenes, lead to ↑amount of protein
product
-
Eg. ras oncogene → ras protein permanently activated → trigger kinase cascade
without growth factor → uncontrolled cell division
Types of proto-oncogene: Growth factor, growth factor receptor, signal transducer,
transcription factor, programmed cell death regulator
Why dominant: only one allele needs to be mutated to have same effect as
dominant allele. Dominant allele produces sufficient mutated protein to mask
recessive allele
Tumour suppressor gene: Loss in function mutation, no protein product → uncontrolled cell
division
-
Recessive allele as 2 alleles need to be mutated to have effect as insufficient
mutated protein produced to mask function of functional protein eg. p53
protein
- Types: proteins promoting apoptosis, enzymes that repair DNA, receptors for
hormones inhibiting cell proliferation, intracellular proteins @ cell cycle
checkpoint
Cancer cells: No Density dependent inhibition, No anchorage dependence
Benign tumour: Mass of abnormal cells that remain at original location
Malignant tumour: Invade surrounding tissue & can metastasise
Genetic factor: Inherited mutated oncogenes/TSG found in gametes of parent
Eg. colon cancer runs in family
Biological factor:
-
Virus: Alter DNA sequence of host → oncogene eg. cervical cancer
- Fungus: Mycotoxins cause liver cancer
- Genetic: Colon
cancer Chemical factor:
- Ethidium bromide: Cause chemical change in bases leading to incorrect BP
Ionising radiation: ∝radiation: form chemically active ions capable of breaking and damaging DNA
Cancer: Multi-step process requiring several accumulated mutations
-
-
Proto-oncogene mutation
TSG mutation
Accumulation of these mutations remove natural limit on no. of cell divisions →
uncontrolled cell div. forming malignant tumour → cancer due to loss of anchorage
dependence/density dependence inhibition + chromosomal aberrations & other
events
Tumour angiogenesis (growth of blood vessels to tumour) allow cells to
enter circulatory system
Invasion of cancer cells to surrounding tissues
Metastasis of cancer cells to distant sites → secondary tumours
How proto-oncogenes are mutated: Amplification of proto-oncogene →↑no. of copies of
proto-oncogene ↑ amt of protein expressed
Movement of transposable elements place a more active promoter near proto-oncogene,
↑expression
How dysregulation of checkpoints lead to cancer: There are 3 checkpoints
(G1/G2/metaphase). Cell cycle only proceeds to next stage when all checkpoints are passed
When dysregulation of checkpoints occur, cell that don’t meet criteria are allowed to divide &
multiply, accumulating mutations and becoming cancerous/chromosomal aberration
-
Give eg. of checkpoint, if M dysregulated, not all chromosomes attached to spindle fibre,
before proceeding to anaphase. Daughter cell may be aneuploid → cancer
Stem cells
Unspecialised cells that divide during a single division to one identical daughter cell and one
more specialised daughter cell which can undergo further differentiation
Features:
-
Capable of dividing & renewing themselves for long periods. Stem cells contain
telomerases which extend telomeres at chromosomal ends. Other cells have no
telomerase and are subjected to end-replication problem, where after every round of
replication, new daughter 5’ DNA strand is shorter. Once telomeres shorten to
critical length, it signals cells to stop cell division or initiate apoptosis
Potency → specialised = unipotent
Totipotent: zygotic stem cells can differentiate into any cell type to form whole organisms,
thus they are also pluripotent and multipotent
Pluripotent: Embryonic stem cells are unspecialised and can differentiate into almost any cell
type to form any organ/cell.(cannot form placenta or umbilical cord) They give rise to
ecto/meso/endoderm, giving rise to specialised cell types that make up heart, skin, lungs etc.
Differentiation is due to differential switching of genes
Multipotent: Differentiate to limited range of cell type, usually a closely related family of cells.
Maintain steady state of function of cell by generating replacements for damaged cells.
Blood stem cells differentiate to myeloid & lymphoid progenitor cell then further differentiate
to RBC/platelets and T/B lymphocyte respectively
Embryonic stem cell treatment VS altering adult cells (ISPC):
-
Social perspective of ISPC: Patient will not mount immune response to his own cells,
↓risk of transplant rejection, ↓need for lifelong immunosuppression drugs
Ethical perspective of ISPC: Using adult cells do not destroy embryos. Destruction of
embryos is morally unaccepted as it is considered a life.
Easier to create: It can be performed in any moderately equipped molecular bio
lab and does not require hard to obtain materials (eg. eggs)
Inheritance
Mendel’s Law of Segregation: Each characteristic is determined by a pair of alleles during
meiosis, they segregate from each other such that each gamete receives 1 of the alleles
Second Law: Alleles of one homologous chromosome pair segregate independently during
anaphase I. Alleles of genes on different chromosomes assort independently of one another
to different gametes. During metaphase I, pairs of homologous chromosomes align along
metaphase plate independent of other bivalents
Test cross: Organism with dominant phenotype of unknown genotype is crossed with
homozygous recessive to determine its genotype (state results → Aa x aa → all dominant)
Ratio of all crosses:
-
Aa x Aa – 3:1
Aa x aa – 1:1
AA x aa – All dominant
AaBb x AaBb – 9:3:3:1
AaBb x aabb – 1:1:1:1
Epistasis – All add up to 16
o Dominant – 12:3:1
o Recessive – 9:3:4
o Complementary – 9:7
- Codominance – 1:2:1
Genes on same chromosome = Linked genes – no independent assortment
*Mendel’s ratio are only valid without crossing over
Linked genes with crossing over: During prophase I of meiosis, homologous chromosomes
pair to form bivalents. Crossing over occurs between non-sister chromatids of homologous
chromosomes leading to chiasmata. Correspond DNA between non-identical sister
chromatids are broken, exchanged and rejoined, giving rise to recombinant gametes
Phenotypic ratio not observed as genes are linked. However, crossing over between
homologous chromosomes may occur @ prophase I producing recombinant gametes. As
crossing over may/may not occur, recombinant phenotypes occur less frequently than
parental phenotypes, ratio of parental : recombinant phenotypes are higher than that for
unlinked genes
Recombinant frequency: No. of recombinant/total no. of organism x 100
The further apart the 2 genes, ↑recombinant frequency, ↑% crossing over. If expected ratio is
seen, there is no linkage
Incomplete dominance: Heterozygote exhibits intermediate phenotype: An allele does not
show complete dominance over another allele (eg. pink colour as a result of red x white)
Reason: Dominant allele produces insufficient enzyme to synthesise enough red pigment.
Allele CW codes for non-functional enzyme, thus heterozygotes are pink (intermediate
phenotype)
Codominance: Both alleles of gene fully expressed. Heterozygote expresses both
phenotypes
Reason: Both alleles produce functional gene products while appear in heterozygote
Sex determination: Females – homogametic sex (XX), males – heterogametic sex (XY)
Sex-linked genes: Genes @ sex chromosomes
Y-linked genes are transmitted to all sons and never to
females X-linked gene has >1000 gene while Y has only 78
Reciprocal crosses: Determine if node of inheritance is sex-linked
(node of inheritance – dominant/recessive of autosomal/sex-linked)
If a gene is sex-linked, different phenotype distribution on different sexes. In one cross,
female with disease x healthy male. In another, male with disease x healthy female
Haemophilia: Deficiency of clotting factors for blood – excessive bleeding
X-linked gene recessive people with severe haemophilia treated by injection of concentrates
of bleeding factor
Haemophilic female (XnXn) x normal male (XNY) – 1 haemo-male, 1 carrier
female Haemomale (XnY) x normal female (XNXN) – 1 carrier female, 1 normal
male
Red-green colour blindness: Sex-linked recessive allele, X
Normal female (XNXN) x colour blind male (XnY) – 1 carrier female, 1 normal male
Female carrier (XNXn) x normal male (XNY) – 2 normal female : 1 normal male : 1 colour
blind male
Epistasis: Type of gene interaction where phenotypic gene expression of 1 gene alters
phenotypic effects of another independently inherited gene. Gene that masks expression of
another gene is epistatic to that gene. Gene A and B code for enzymes involved in metabolic
pathway in synthesis of purple pigment. Enzyme A catalyses rxn to form colourless product,
which becomes substrate for B to form purple pigment.
Recessive epistasis: Homozygous recessive A is epistatic over
Complementary loci epistasis: Homozygous recessive A/B is epistatic over the other
dominant gene
Dominant epistasis: Presence of dominant allele @ A mask phenotypic effect of B. New
phenotypes produced
Chi-square test: Types of cellular response: Regulate gene expression/activity of existing
proteins
Degree of freedom = no. of phenotypic category–1
Explain value: At 0.05 level of significance, calculated chi-square test value of 1.60 < critical
value of 3.841. Difference between O and E is not significant and due to chance. Probability
lies between 0.5 and 1. Thus is supports the hypothesis that
Pedigree chart: Diagram of family tree over many generations showing absence/presence of
particular trait in all members
Autosomal dominant: Appears in all generation. Every affected child has affected parents,
thus dominant
-
Affected male x normal female passed gene disease to son thus not sex-linked.
Equal no. of male & female affected
- Eg. widow’s peak
Autosomal recessive: Rare trait (not all generations). An affected child can have unaffected
parents as both are carriers (eg. sickle cell anaemia)
X chromosome sex-linked dominant:
- Affected male pass trait to all daughters
- Affected male x normal female does not pass trait to
sons X chromosome sex-linked recessive:
- Males affected > females
- All sons of affected female are affected
- Phenotypically normal parents produce affected sons
Continuous variation: phenotype controlled by many genes. Phenotypic expression affected
by environment factors. Additive effects of each gene give rise to phenotype range.
Characteristics exhibit complete range from one extreme phenotype to the other without
break eg. height
Discontinuous variation: 2 or more distinct, non-overlapping classes with clear-cut
differences & no intermediate between them. Controlled by 1/2 major genes, relatively
unaffected by environment conditions eg. blood group
Organism eg: Honey bee. Drones arise from unfertilised eggs. Females arise from fertilised egg.
Although queens and workers have same amt of genetic material, they are phenotypically
different due to diet of larvae @ development. Royal jelly stimulates protein formation and
maturation of female sex organ → queen bee
Cell signalling
Why can’t interferon act directly on the nucleus?
-
It is a large and hydrophilic molecule. It is unable to pass through hydrophobic
core of phospholipid bilayer which makes up cell surface membrane
- There are no transmembrane proteins to transport interferon across
membrane Explain how structure of TGFBR allows it to carry out its function as a
receptor:
-
TGFBR has extracellular binding site complementary to ligand
Intracellular tails allow dimerization of receptors
It is a transmembrane protein embedded on cell surface allowing it to receive
signal and transmit to cell interior
Types of cellular response: Regulate gene expression/activity of existing proteins
Termination: Ligand detaches/dephosphorylation by phosphatase/degradation of
2nd messenger
Signal amplification: Multi-step process. Enzymes (if not enzyme, X signal amplification)
must remain active for some time
Effect of glucagon on GPLR:
-
Binding of glucagon to extracellular side of GPLR activates receptor and changes
its conformation
- Cytoplasmic side of GPLR binds to an inactive G protein, making it exchange
bound GDP to GTP
- G protein is activated and dissociates from receptor. Activated G protein binds and
activates adenylyl cyclase which catalyses conversion of large number of ATP to
cAMP. During signal transduction, cAMP binds and activates protein kinase A.
PKA then initiates sequential phosphorylation and activation of glycogen
phosphorylase kinase, resulting in phosphorylation cascade, amplifying signal
- This results in cellular response of increased rate of glycogenolysis, glycogen →
glucose
- Termination:
o Glucagon released from receptor
o Phosphodiesterase converts cAMP to AMP
o GTPase activity intrinsic to G protein hydrolyses bound GTP to
GDP Effect of insulin on receptor tyrosine kinase (RTK)
-
During ligand-receptor interaction, binding of insulin to extracellular binding sites of
RTK causes 2 RTK proteins to form a dimer. Dimerisation activates tyrosine
kinase function found in extracellular tails of RTK and autophosphorylation of
tyrosine residues of other RTK protein occurs
- During signal transduction, activated RTK protein triggers assembly of relay
proteins on receptor tails, activating them. Each activated protein initiates
sequential phosphorylation and activation of protein kinase, resulting in
phosphorylation cascade, amplifying signal
- Vesicles embedded with glucose transporters move to cell surface membrane
and fuse with it, inserting transporters to cell surface membrane, resulting in
glucose uptake into muscle cells
- Large number of glycogen synthase activated, catalysing glycogenesis
- Termination:
o Phosphatase inactivate protein kinase by dephosphorylation
o Insulin released from receptors, tyrosine residues dephosphorylated
by phosphatases, dimer disassociates back to individual RTK proteins
How signal amplification occurs:
-
When one activated GPLR can activate many G protein molecules/activated
adenylyl cyclase catalyses large conversion of ATP to cAMP, activated protein
kinase A catalyses phosphorylation and activation of many glycogen phosphorylase
kinase (note: all are enzymes)
- This is because enzymes remain chemically unchanged throughout reaction and
can be reused
Advantages of signal pathway:
-
Signal amplification allows one signal molecule to trigger large cellular response
One signal molecule can activate man transduction pathways to trigger
numerous cellular reactions simultaneously
Binding of signal molecule to receptor at cell surface membrane results in
activation of gene transcription in nucleus
Specificity in ligand-receptor interaction allow signal molecule to elicit responses
in specific target cell
Ability of signal molecule to activate many different cells simultaneously allow
for regulation and control of response
Respiration
1. Glycolysis: Oxidation of glucose to 2 pyruvate @ cytoplasm without
O2 Products: 2 reduced NAD + 2 ATP + 2 pyruvate
2. Link rxn: Oxidation of pyruvate to acetyl-coA @ mitochondrial
matrix Products: 2 CO2 + 2 ATP + 2 acetyl-coA
3. Krebs cycle: Further oxidation of acetylcoA via series of rxns @ mitochondrial
matrix Products: 4 CO2 + 6 NADH + 2 FADH per glucose molecule (per cycle → 1/ 2)
4. Oxidative phosphorylation: @ inner mitochondrial membrane/membrane of
eukaryotes
Products:
o 1 NADH – 2.5 ATP
o 1 FADH – 1.5 ATP
Glycolysis:
-
Occurs in cytoplasm and does not need O2. Involves breakdown of glucose to
2 pyruvate
- Divided into energy investment & energy payoff phase. In investment phase, 2ATP
is used per glucose. Activation of glucose with phosphorylation of glucose using ATP
to glucose-6-phosphate, catalysed by hexokinase. Glucose-6-phosphate is then
isomerised to fructose-6-phosphate by isomerase
- Phosphorylation of fructose-6-phosphate using ATP produce fructose-1,6bisphosphate, catalysed by phosphofructokinase. Cleavage of fructose1,6- bisphosphate to 2 glyceraldehyde-3-phosphate
- At energy payoff phase, 4ATP produced per glucose via substratelevel phosphorylation
- Each GALP converted to pyruvate via multiple steps where 2ATP produced by
substrate lvl phosphorylation and protons+electrons released via
dehydrogenation transferred to 1 oxidised NAD to form NADH
- 2 pyruvate, 4ATP, 2NADH formed per glucose molecule (net ATP = 2)
Phosphofructokinase: low activity of ATP promotes PFK activity, increasing rate of
glycolysis. PFK inhibited by high ATP lvls due to binding of ATP to allosteric site of
PFK, resulting in distortion of active site, slowing glycolysis
Link rnx:
1. Decarboxylation: Carboxyl grp of pyruvate removed. CO2 released
2. Dehydrogenation: remaining 2 C molecule undergo oxidation via dehydrogenation
by transferring protons and electrons to NAD, converting it to NADH. Acetate
formed
3. Co-enzyme A added to form
acetylcoA Outline Krebs cycle [8]
-
Occurs in mitochondrial matrix
Products of Krebs cycle: 4 CO2, 6 NADH, 2 FADH, 2 ATP
-
AcetylcoA attached to oxaloacetate to form citrate, which is gradually reconverted
to oxaloacetate making it a cycle
- At 2 stages in Krebs cycle, oxidative decarboxylation occurs, removing carbon from
intermediate compounds. 2 CO2 formed per cycle which diffuses out of mitochondrion
to cell
- 1 molecule of ATP formed per cycle by S.L.P where the inorganic phosphate
is derived from guanosine triphosphate
- Intermediate compounds undergo dehydrogenation by transferring protons
and electrons to NAD and FAD, forming NADH and FADH
- These coenzymes subsequently transfer high energy protons and electrons to
ETC for ATP synthesis
- Since 2 molecules of acetylcoA formed per glucose at link rnx, Krebs cycle runs
twice to completely utilise them
Role of NAD in Krebs cycle:
-
Coenzyme which removes proton and electrons from Krebs cycle
Transfer high energy proton and electron to ETC
Which is embedded in inner mitochondrial membrane where
oxidative phosphorylation occurs
Oxidative phosphorylation:
How proton gradient is formed:
-
NADH and FADH transport high energy proton and electron to ETC
Electrons passed along ETC from 1 electron carrier to next, each with energy lvl
lower than the preceding one. Impermeable nature of inner membrane to protons
allow proton accumulation
- Energy released from electron flow used to actively pump protons from
mitochondrial matrix to intermembrane space by conformational change of proteins
in ETC
Why is O2 needed?
-
Final proton and electron acceptor with regeneration of NAD and FAD
At end of ETC with formation of H2O, catalysed by cytochrome oxidase,
maintaining electron flow along ETC
How proton gradient forms ATP: High energy electrons passed along ETC form 1 electron
carrier to the next of lower energy lvl. Energy from electron flow used to pump protons
actively from matrix to intermembrane space by conformation change of proteins in ETC,
generating electrochemical proton gradient across inner mitochondrial membrane. Protons
diffuse across gradient back to mitochondrial space through ATP synthase, synthesising
ATP from ADP and Pi
Alcoholic fermentation:
1. CO2 released from pyruvate → acetaldehyde (catalysed by decarboxylase)
2. Ethanal/acetaldehyde → ethanol (catalysed by alcohol dehydrogenase). NADH
oxidised, regenerating oxidised NAD → allow glycolysis
Lactate fermentation: Pyruvate → lactate (catalysed by lactate dehydrogenase)
Reconverted to pyruvate in liver when O2 supply restored
Respirometer – How it is used to measure O2 uptake: soda lime absorbs CO2 give out,
change in lvl of fluid/time indicates O2 taken in. Boiled seeds left in setup serve as negative
control
How it can be modified to measure RQ: remove soda lime, change in fluid indicates net
difference between O2 absorbed and CO2 released. Difference between this value and value
when soda lime is present indicates V(CO2). RQ = volume of CO2/volume of O2
Significance of RQ values: Different RQ values used to identify substrate. When glucose is
respiratory substrate, exactly same volume of CO2 and O2 released and produced
respectively, RQ = 1. If other substrates used, RQ<1 (protein = 0.8. fat = 0.7)
Why does glycolysis rate increase significantly when yeast cells switch from aerobic to
anaerobic: aerobic produces 36 ATP/glucose while anaerobic produces 2ATP/glucose. To
produce same amt of ATP, more glucose broken down during glycolysis
Prokaryotic genome
Prokaryote vs eukaryote chromosome: In prokaryotes, formation of loop domains anchored
by DNA binding proteins, followed by negative supercoiling, whereas in eukaryotes, DNA
wrapped around histones formed nucleosomes joined by linker DNA forming beads-on-string
structure. Nucleosomes and linker DNA is further folded to form solenoid structure which is
further coiled to looped domains anchored and coiled by scaffolding proteins. Loop domains
are further coiled and condensed to form metaphase chromosome
-
-
In eukaryotes, double stranded linear chromosomes whereas in prokaryotes,
double stranded circular chromosomes
In prokaryotes, single origin of replication within each chromosome, while
in eukaryotes multiple origin of replication
In prokaryotes, no telomeres and centromeres present whereas in eukaryotes
presence of 1 centromere within telomeres and 1 telomere attached at each end
of chromosome
Eukaryotes have 1011 base pairs while prokaryotes such as E. coli have 106
Prokaryotes are haploid but eukaryotes are diploid
Prokaryotes have high gene density but eukaryotes have low gene density
Prokaryotes do not have introns but eukaryotes have introns
Explain role of F plasmid: F factor contains several genes, most required to produce sex pili.
Genes within F factor code for proteins promoting transfer and replication of one strand of F
plasmid DNA by rolling circle mechanism. F plasmid allows bacteria to mate via conjugation
where there is DNA transfer from donor to recipient and through the process facilitate
genetic recombination leading to genetic variation. F plasmid contains own origin of
replication, allowing replication independent of bacteria chromosome and origin of transfer,
where a single strand is nicked and transferred to recipient cell
DNA transfer:
Transduction:
Specialised transduction: Bacteria genes adjacent to prophage site are transferred from
one cell to another by a temperate bacteriophage. Error during induction of lytic cycle result
in prophage incorrectly excised and excised phage DNA incorporated some bacteria DNA/
Phage can then attach to another bacteria and inject phage DNA containing bacteria DNA
from first cell. Bacteria DNA is then incorporated in recipient cell by homologous
recombination
Generalised transduction: Bacteria genes are randomly transferred from one bacteria cell to
another by a bacteriophage. A small piece of bacteria DNA is accidentally packaged within
phage capsid in place of phage DNA before release of completed phage from host cell.
Such a virus may not be able to replicate as it lacks some/all of its genes. However, after
release from lysed host, it can attach to another bacteria and inject the piece of bacteria
DNA from first cell into recipient cell. It can then be incorporated by homologous
recombination.
How plasmids differ from bacteria chromosome:
1. Plasmid is fewer in base pairs and hence has fewer genes than chromosome
2. Gene coding for antibiotic resistance is found in plasmid but gene coding for
enzymes for metabolism is at chromosome – chromosome contains essential
DNA while plasmids contain useful DNA (good to have but not needed for basic
cell survival)
Transformation:
Naked foreign DNA released from lysed bacteria into surrounding environment is taken up
by another suitable recipient bacteria using specific cell surface proteins. Foreign DNA is
incorporated by homologous recombination
Conjugation:
Sex pilus of F+ bacteria cell attach to F- bacteria. Pilus retracts and temporary cytoplasmic
mating bridge is formed. 1 strand of F plasmid DNA is nicked at origin of transfer, and DNA
strand travels through cytoplasmic bridge from donor to recipient cell with the help of protein.
Synthesis of complementary strand begins in both cells by DNA polymerase at 3’ free OH
end. Each parental strand acts as template for synthesis of 2nd strand of DNA by
complementary base pairing, synthesising double-stranded DNA in recipient and donor cell.
In recipient cell, 2 ends of each F factor DNA strand is joined together to form circular
molecule
Benefits of conjugation: Plasmid contain antibiotic resistance genes, conferring bacteria
selective advantage in environment with antibiotic. Plasmid may contain xenobiotic
resistance gene, conferring bacteria selective advantage in environment with foreign
chemical. Plasmid may contain gene encoding enzyme to metabolise new metabolite,
conferring bacteria selective advantage in environment with metabolite
Control of prokaryotic genome:
Operon – cluster of genes encoding enzymes of same metabolic pathway under
transcriptional control of 1 promoter
Difference between euk & prok genes:
-
In eukaryote, each gene is controlled by 1 promoter while in prokaryote, cluster
of genes controlled by 1 promoter
- In eukaryote, expression controlled by silencer while in prokaryote,
expression controlled by operator
- In eukaryote, introns present while introns absent in
prokaryotes Lac operon: Structural features
-
Consists of promoter, operator and cluster of genes coding enzymes to
metabolise lactose
- Regulatory sequences in lac operon: one CAP site recognised by CAP protein,
promoter found upstream of structural genes and where RNA polymerase binds to
to initiate transcription of lac genes, operator which is beside promoter and
structural genes where lac repressor binds to, terminator which is downstream of
structural genes and signals end of transcription and structural genes coding
enzymes for lac metabolism
- Its structural genes
o lac Z: codes for beta-galactosidase, which hydrolyse lactose to glucose
and galactose and convert a small % of lactose to allolactose
o lac Y: Codes for lactose permease which is a membrane protein to
transport lactose in cell
o lac A gene coding for galactosidase transacetylase whose role is
unknown Why operons are necessary in bacteria:
1. operon arranges genes coding for enzyme in same metabolic pathway together
for more efficient control of gene expression
2. 2. Since bacteria do not have nuclear envelope, transcription and translation occur
simultaneously. Thus regulation of gene expression usually occurs @
transcriptional level
3. So that bacteria only produce enzyme when necessary and do not waste
energy, conferring selective advantage
4. Operons allow bacteria to use variety of sugar like lactose/glucose
Explain how lactose switch on lac operon: Lac operon is an inducible operon. Without
lactose and hence allolactose, lac repressor bind to promoter and block RNA polymerase
from transcribing structural genes inhibiting transcription. However binding of repressor to
operator is weak and basal transcription occurs, producing small amount of β-galactosidase,
lactose permease and transacetylase. In lactose presence, small amount of lactose is
transported to cytoplasm by lactose permease, β-galactosidase convert lactose to
allolactose, which bind to repressor causing repressor to have conformational change,
preventing it from binding to operator. RNA polymerase can then transcribe structural genes
High glucose, NO lactose: cAMP is low and CAP is inactive and does not bind to CAP site of
lac operon. When there is no lactose, repressor is active and bind to repressor
No glucose, high lactose: cAMP is high, binding to CAP and activating it causing CAP to
bind to CAP site. Allolactose bind to repressor causing conformational change and inactive
repressor cannot bind to operator. RNA polymerase can bind to promoter and transcribe
structural genes
Trp operon: repressible operon. Trp repressor is synthesised in inactive form which has little
affinity for operator. Only if tryptophan bind to trp repressor does repressor change to active
form that bind to operator inhibiting transcription of structural genes (end-product inhibition)
Binary fission:
-
Before cell divides, semi-conservative replication of parental DNA begins at origin
of replication to give rise to 2 origin
Each origin moves rapidly toward opposite end of cell and adhere to cell membrane
When chromosome is replicating, cell elongates, separating the 2
identical chromosome copies
When replication is complete and cell reaches twice of initial size, cell membrane
invaginates and deposit new cell wall material, dividing parent cell to two
genetically identical cells. Each cell inherits a parental strand of DNA
Inducible vs repressible operon:
Similarity: Both have promoter, operator and cluster of genes coding enzymes of metabolic
pathway. Transcription of structural gene produce polycistronic mRNA
Differences: eg. lac vs trp operon
Usual state of operon
Effect if effector
molecule
Event when lac/trp is
present
Lac operon
Trp operon
Not expressed
Expressed
Transcription turned on when
Transcription turned off when
inducer allolactose binds to
corepressor or tryptophan
repressor
binds to repressor
Allolactose bind to repressor,
With tryptophan produced,
changing conformation,
tryptophan bind to repressor,
repressor no longer bind to
changing conformation,
operator, transcription of gene repressor bind to operator,
no longer inhibited
transcription inhibited
Explain how single mRNA produces multiple enzyme:
-
Polycistronic mRNA formed from transcription of structural gene which
contain coding sequence for more than 1 pp
Polycistronic mRNA has 3 start/stop codon, signals where coding sequence of
each pp begin and end thus forming 3 different enzymes
Translational control:
Translational repressor:
1. Recognise mRNA sequences, bind near ribosome binding site and block
ribosome from initiating translation
2. Synthesise antisense mRNA. mRNA forms duplex with antisense mRNA,
translation blocked as ribosome cannot access nucleotides in mRNA
Infectious diseases
Types of white blood cell:
1. Granulocytes:
o Neutrophil, eosinophil, basophil: polymorphonuclear and contain granule
in cytoplasm. Involved in innate defence during innate immunity. Engulf
pathogens by phagocytosis.
▪ Neutrophils: most numerous WBC, kill microorganism y
releasing antibacterial protein
▪ Eosinophil/basophil defend against parasites
2. Antigen-presenting cell: Macrophage & dendritic cell
o Engulf pathogen by phagocytosis, digest antigen as peptide and present
them as antigen fragments to T lymphocytes, activating them
o Monocyte reside in blood and migrate to tissue to differentiate
into macrophage
3. Lymphocytes: antigen specific – B & T lymphocyte, non-specific – natural killer cell
o B VS T lymphocytes:
B lymphocyte
T lymphocyte
Location of
Bone marrow
Thymus
maturation
Type of immune
Humoral
Cell mediated
response
Antigen binding
Bind to surface
Only bind to antigen
antigen directly &
presented on infected cells
present antigen
Substance secreted
Antibody
Cytokines/cytotoxic protein
Cell types formed
Memory B/plasma
Helper/cytotoxic/memory T
cell
cell
Lifespan
Short
Long
Function
Defend against
Defend against
extracellular
intracellular pathogen eg.
pathogen @ blood &
fungi
lymph eg. virus
Innate immunity: Barrier & chemical defence, cellular innate defence
1. Barrier defence:
a. Physical barriers: protective covering – epithelial tissues forming skin and
mucous membrane have tight junction, blocking pathogen entry. Mucuscovered epithelial tissue – mucus enhances defences by trapping
pathogens for easy removal eg. trachea, ciliated cells transport mucus and
pathogen to stomach and killed by HCl
b. Chemical barrier – HCl @ stomach, antibacterial enzyme (lysosome) @
tears, saliva
2. Cellular innate defence: pathogens breach epithelial barrier, causing phagocytes to engulf
pathogens by phagocytosis. Macrophage release cytokines & chemokines and
↑permeability of blood vessels and recruit cells like neutrophils to infected tissue.
Chemokines act as chemoattractants to attract phagocytes to infected tissues.
Cytokines act as signal molecule to enhance immune response. This is known as
inflammation, characterised by heat, pain, redness and swelling
Discuss role of phagocytes (NOT T cell) in resisting infection:
Phagocytes like macrophage residing in tissues engulf bacteria by phagocytosis via extension of
pseudopodia. As cell surface membrane pinches off, phagocytic vesicle is formed. Lysosome
membrane fuse with phagocytic vesicle membrane; hydrolytic enzyme digest cell. Macrophage
release chemo & cytokine which ↑permeability of blood vessel to recruit more phagocytes to
infected tissue. This is known as inflammation, characterised by heat, pain, redness and swelling
Immune responses: cell mediated (T cell) VS humoral (B cell antibodies)
Cell mediated response: A Cow Die At Moon
Antigen recognition
Clonal selection and expansion
Differentiate
Antigen eliminate
Memory
A – naïve T lymph circulate blood and lymph, possessing repertoire of antigen receptor.
Each cell receptor is specific for 1 particular antigen. T cell receptor bind to antigen
presented by MHC complex on macrophage/dendritic cell
C – naïve T lymph activated to proliferate and produce many identical progeny by clonal
expansion
D – progeny differentiate to effector cells with different function. Effector cells have same
antigen specificity. CD4+ → helper T/memory T cell. CD8+ → cytotoxic T cell. Helper T release
cytokine to help differentiation of cytotoxic T cell
A – recognise & bind to class I MHC-antigen complex on infected cell. Release perforin,
forming pores @ membrane. Granzymes break down proteins, initiate apoptosis
M – most effector cells die. However, activated memory cells persist and provide lasting
protective immunity & mediate rapid, effective secondary response
Explain how 2nd infection response differ from 1st:
-
Secondary response is more rapid & effective and pathogen removed faster
Memory lymph produced @ first infection
-
Reactivated more quickly than naïve lymph and there are many more cells
specific for pathogen
- Faster production of antibody/helper T/plasma cell/cytotoxic T
cell Explain role of cytokines:
- Inhibit viral replication and induce expression of MHC molecule
- Help in class switching in activated B lymph to produce antibody of different class
- Help activate CD8+ T lymph to cytotoxic T cell for cell-mediated immunity
- Help B lymph differentiate to plasma cell for humoral
immunity Humoral response:
A – Naïve B cell circulate blood & lymph, possess repertoire of antigen receptor. Each cell
receptor is specific to a specific antigen. B cell receptor bind to intact antigen which is taken
up via endocytosis, hydrolysed to short peptides and presented on MHC molecule. T cell
receptor of activated t cell bind to antigen-MHC complex, activating B cell. Cytokines
secreted by helper T cell to activate B cell
C – Activated B cell proliferate & produce many identical progeny via clonal expansion
D – Progeny differentiate to plasma and memory B cell. Plasma secrete antibodies which
recognise antigen that activated B cell. Antibodies secreted by exocytosis
A – Neutralisation of toxin: Antibody bind to toxin, preventing them from interacting with host
cell. It is then phagocytosed and degraded by macrophages. Opsonisation – antibody bind
to antigens on bacteria cell. Antibody bind to receptors expressed on macrophage
facilitating phagocytosis
M – memory cells persist and can be reactivated more quickly than naïve lymph, provide
lasting immunity and mediate more rapid and effective secondary response
Antibody: IgG (most numerous), IgE, IgD, IgM, IgA
Explain molecular structure of antibody with IgG as example:
-
Large quaternary protein of 4 polypeptide chain
Consist of 2 heavy and 2 light chain linked by disulfide bond
Heavy and light have constant and variable region
VH and VL are at amino terminus, forming antigen binding site which determine
antigen specificity of antibody
- CH and CL are at carboxyl terminus and determine function (class) of antibody
- As antibody has 2 identical variable region, there are 2 identical antigen binding site
- Flexible stretch joining Fab and Fc region is known as hinge region
- Allow flexibility of antibody to bind to multiple antigen → Increase total binding
strength
- Specialised Fc receptor expressed on surface membrane of neutrophil/macrophage
bind to Fc portions of IgG antibody, facilitating phagocytosis of pathogen coated
with antibody
Explain why antibiotics are ineffective against viruses:
- Antibiotics inhibit cell structure/metabolism but virus lack metabolism or cell
structure Predict 1 component of virus of importance to produce vaccine:
-
Viral glycoprotein. It allows B cell to recognise and bind to it, activating B cell
for humoral response. Clonal expansion & selection to produce memory cell
Explain why phagocytes act only on bacteria: Bacteria’s antigens are foreign while human
cells have self antigens, self antigens are encoded by genes in the body, non-self antigens
will trigger phagocytosis by APCs and phagocytes bind to antibodies complexed with nonself antigens
Explain how genetic recombination produce repertoire of antibody molecules:
1. Somatic recombination:
- Variable region of antibody are coded by gene segments rearranged be somatic
recombination during development of naïve B cell. It begins with rearrangement
of the heavy chain then light
- DA first joined to JH segment, followed by joining of DJH with VH to form complete exon
for heavy chain
- Light chain is coded by VL and JL. Joining of the two form variable sequence by RNA
splicing after transcription.
- VJL is joined to CL by RNA splicing after transcription forming complete mRNA of light
chain
- Translation of light and heavy chain mRNA forms light and heavy chain polypeptide
- There are many different copies of V, D, J gene segments in germline DNA at
gene loci of antibody. Random selection of one of each type of gene segment
forms different combination of gene segments, thus combinatorial diversity.
Different combinations of light & heavy variable region that pair to form antigen
binding site further generating combinatorial diversity
Class switching: Occurs in activated B cell during clonal expansion, where some rearranged
VDJH is linked to different heavy chain constant region. It is then translated to form antibody
of different class with same specificity but different function. It is regulated by helper T cell
Somatic hypermutation: occur @ activated B cell during clonal expansion. Introduces point
mutation in rearranged variable region gene, resulting in change of one to a few aa,
producing closely related B cell progeny with B cell receptors lightly differing in antigen
specificity and affinity
Natural VS Artificial immunity:
Source
Example
Natural immunity
Inherited naturally
Breastfeeding/from mother to
child via placenta
Immunity is result of previous
infection that produce memory
cell which can be reactivated
when a person is infected with
pathogen and produce own
antibody
Artificial immunity
Acquired deliberately by exposure to
antigen/antibody by non-natural
means
Injection of antibody from another
person
Result of immunisation where immune
response is induced without them
having symptoms
Explain how vaccination gives immunity:
-
Vaccination is intentional provision of killed pathogen/antigen to body
Inducing specific adaptive immune response that protects individual later on due to
production of memory cell. During primary response, particular antigen is
recognised by specific B cell. Activated B cell undergoes clonal expansion by
dividing and differentiating to memory and plasma cell. During secondary response,
memory cell quickly activate by recognising surface antigen of pathogen, rapidly
undergo clonal expansion and differentiate to plasma cell which produce antibody,
neutralising virulent pathogen
Penicillin: Inhibit transpeptidase, alter active site shape, peptidoglycan chain cannot link
up, cell wall breaks down and bacteria swells then lyses
Herd immunity: When proportion of people immune to pathogen is so high that pathogen
cannot find host to infect and cannot survive in population
Ideal vaccine: cheap/easily stored in high temperature/do not need booster, long lived
immunity/no side effect
Discuss risks & pros of vaccination:
Pros
Protect individual against future
potential diseases that can pose
long term health problems
- Protect not only individuals but
entire communities by herd
immunity
- Protect future generations as
immunisation has eliminated many
diseases eg. smallpox
- Cost of vaccination < healthcare
cost of treating diseases
-
-
Cons
Can cause side effects. However,
they are usually mild eg. redness
Risk of reversion to virulence when
live inactivated pathogens are used
in vaccines
How HIV cause disease:
-
HIV recognise CD4 on T helper cells and infect them, causing gp120 to be
presented on cell surface membrane of infected T helper cell, causing them to be
destroyed by cytotoxic T cell. Loss of helper T cell cause immune system to fail, thus
unable to mount immune response
- Person susceptible to opportunistic infections. AIDs result. Cancer may result if HIV
integrates to middle of tumour suppressor gene to switch it off. Inhibition of host cell gene
→ altered cell function, depletion of cellular materials needed for cell function
Why TB is not eradicated yet:
-
TB needs long term antibody usage. Incomplete antibody treatment leads to
resistant TB
Bacteria undergo random mutation to form antibiotic resistant gene
Bacteria multiplying in macrophage cannot be detected by antibiotic
Easy spreading via airborne droplet. Some people not vaccinated. 3rd world country
has no vaccination
Viruses
Are viruses living beings?
Agree
Organisation
Metabolism
Growth
Homeostasis
Respond to stimuli
Reproduce
Direct host cell’s
metabolism
Via host cell machinery
Disagree
Not composed of cells
Does not have it’s own
Does not grow
No
Not in virion state
Not independently
T4 phage (lytic cycle)
1.
2.
3.
4.
5.
6.
7.
T4 uses tail fibres to bind to specific receptor sites
Sheath of tail contract, injecting phage DNA into cell
Enzyme that degrade host cell DNA coded for
Phage DNA direct replication of viral phage DNA and synthesis of phage protein
Phage components assembled with help of non-capsid protein
Phage DNA is packaged within capsid
Phage directs synthesis of lysozyme that damages cell wall, causing fluid to enter.
Bacteria swells and lyses
λ phage (lysogenic cycle)
1. λ phage uses tail fibres to bind to specific receptor sites
2. λ phage makes use of specific pores to inject phage DNA into cell. Once within
cell, phage DNA circularizes.
3. λ phage DNA codes for integrase, which cuts chromosomal DNA of host and
insert phage DNA to host DNA, forming prophage
4. Prophage DNA codes for gene preventing transcription of host DNA. Phage
genome is dormant
5. Every time cell replicates it replicates phage DNA, thus allowing phage to
propagate without killing its host cell
6. Environmental signals like radiation induce phage to transit from lysogenic to
lytic cycle
7. Prophage is excised and phage enters lytic cycle, via synthesis of viral
components, assembly and release of new phage
Influenza:
1. Haemagglutinin glycoprotein of viral envelope recognise and bind to specific
sialic acid receptor on cell surface membrane of epithelial cells on respiratory
tract, promoting viral entry to cell
2. Virus enters host cell via endocytosis, forming endosome. Viral envelope fuses
with endosome membrane, exposing capsid to digestion by cellular enzyme
releasing RNA molecules, viral protein and enzymes to cytoplasm
3. Viral genome functions as template for synthesis of complementary RNA strands
by viral RNA dependent RNA polymerase. Positive sense RNA functions as mRNA,
which is translated to capsid proteins in cytosol and viral glycoproteins (modified in
RER and GA). Vesicles embedded with viral glycoprotein migrate toward and fuse
with cell surface membrane, hence viral glycoprotein is embedded on cell surface
membrane. Positive sense mRNA also serves as template for replication of new
copies of viral negative sense RNA genome
4. Capsid protein encloses viral genome and viral protein. Capsid then assembles
with viral glycoproteins during budding
5. Each new virus buds from cell, surrounded by host cell surface membrane
studded with viral glycoproteins
HIV:
1. Gp120 glycoprotein on viral envelope recognise and bind to CD4+ specific receptor
molecule on cell surface membrane of T helper cell
2. Virus envelope fuses with cell surface membrane. Capsid proteins are degraded by
cellular enzymes of host, releasing viral DNA and reverse transcriptase to
cytoplasm
3. Reverse transcriptase catalyses synthesis of single DNA strand complementary
to RNA strand
4. Viral RNA is degraded and reverse transcriptase catalyses synthesis of 2nd DNA
strand complementary to 1st. newly synthesised double stranded viral DNA enters
cell nucleus and integrates as provirus into host cell DNA via integrase
5. Proviral genes are transcribed to RNA molecules by RNa polymerase, which
functions as viral genomes for next generation/mRNAs, which are translated to
form viral glycoprotein and capsid proteins. Vesicles embedded with viral
glycoprotein migrate towards and fuse with cell surface membrane. Viral
glycoproteins become embedded on cell surface membrane
6. Capsid proteins enclose viral genome and protein. Capsid assembles with
viral glycoproteins during budding
7. Each new virus buds from cell, surrounded by host cell surface membrane
embedded with vial glycoproteins. Protease catalyse cleavage of polyprotein
to functional proteins
Describe how HIV acquires outer envelope
-
Viral surface glycoprotein (gp120 and gp41) are synthesised by host cell
ribosomes attached to RER
- Vesicles with viral glycoproteins embedded in vesicle membrane migrate
towards and fuse with cell surface membrane
- Host cell surface membrane studded with viral glycoprotein encloses viral RNA
and capsid and buds from cell
Explain how HIV inhibitors work in treating HIV infection:
-
Inhibitor block active site of protease. Viral glycoprotein cannot bind to active site
and will not be hydrolysed. (*link to end goal) – virus will not be functional
Describe how influenza enters cell: Haemagglutinin recognises and binds to sialic acid
receptors on epithelial cell surface membrane of respiratory tract, facilitating entry into cell.
Virus enter by endocytosis forming endosome. Endosomal membrane fuses with viral
envelope, exposing capsid to digestion by enzymes (host) releasing viral DNA
Explaining how inhibiting neuraminidase prevents influenza:
-
New virus cannot bud off infected cell, hence other cells will not be infected
Terminal sialic acid cannot be hydrolysed from newly formed viral glycoprotein
and host-cell membrane glycoproteins
Compare lytic and lysogenic cycle:
Lytic
Phage DNA is not integrated and remains
in cytoplasm
Phage directs synthesis of phage DNA and
proteins
Cell (host) lyses to release bacteriophages
Lysogenic
Phage DNA is integrated in bacteria DNA
as prophage
Phage DNA is replicated together with
bacteria DNA during binary fission
Host cell remains intact unless phage is
induced to transit to lytic cycle
Suggest advantages of lysogenic cycle:
- λ phage can propagate without killing host cell
- large amount of phage DNA is replicated via binary
fission Antigenic shift (only influenza):
How have the new combination of RNA segments in influenza arisen?
-
-
Animal and human harbouring classical swine, North American avian and
human H3N2 influenza virus live in close proximity to each other and coinfect a
host
Segments form North American avian strain, classical swine strain and human
H3N2 strain were randomly assembled into new virion by genetic recombination
leading to antigenic shift
Antigenic drift: substitution mutation in RNA, leading to antigenic drift where viral RNA
dependent RNA polymerase lack proofreading ability, resulting in minor change to specific
3D configuration and enhanced binding of glycoprotein to host cell receptor
Suggest advantage for phage with lytic and lysogenic cycle:
- Under unfavourable conditions, phage can remain dormant in host cell
- Phage genome replicated each time host cell replicate and cell is not killed
- Allowing large number of virions to be produced when induced to lytic cell
- Survival of bacteriophage ensured since host
survives Outline structure & function of viral nucleic acid:
-
In the form of DNA or RNA, either single or double stranded
If single stranded RNA, can be positive or negative sense
There can be 1 copy of genome (influenza) or more (2 in HIV)
Genetic information can be obtained in 1 molecule (HIV) or segmented to more
than 1 molecule (influenza)
- Code for synthesis of viral components for assembly and synthesis of
offspring Explain why it is necessary for viral RNA to enter nucleus:
-
Influenza lacks ribonucleotide for synthesis of viral RNA which is found largely
in hose cell nucleus
- Viral RNA hence enters host cell nucleus so that viral genome can function as
template for synthesis of complementary RNA strands by viral RNA dependent
RNA polymerase
- Newly synthesised positive sense viral RNA can undergo post
transcriptional modification, adding 5’ cap and 3’ poly-A tail
Outline how new influenza strains arise
-
Existing viruses can spread from 1 host to another, causing multiple strains
to coinfect single host
- Antigenic shift occur which is genetic recombination of viral genomes leading to new
RNA segment combinations during RNA assembly where new viruses are
assembled in host cell
- Drift: High mutation rate of viral RNA genome of existing strains due to errors
in replication not being corrected due to lack of proofreading ability of viral
RNA dependent RNA polymerase
- These mutates the haemagglutinin and neuraminidase genes, changing 3D
conformation of glycoproteins. They are unrecognisable to pre-existing host
immunity, thus cannot be inherited by antibodies for previous strains
Suggest how virus cause disease other than cancer:
HIV causes gp120 fragments to be exhibited on infected helper T cell’s cell
surface membrane causing these cells to be killed by other helper T cells
- Loss of helper T cells cause immune system to weaken, leading to
opportunistic infections causing disease
- Influenza cause viral antigen to be exhibited on cell surface membrane of infected
epithelial cell on respiratory tract, causing death of epithelial cell by necrosis. Loss
of epithelial cell makes one more susceptible to pneumonia
- Depletion of host cell’s cellular materials
- Trigger release of hydrolytic enzymes from host cell lysosomes
- Inhibition of host cell gene leads to altered cell
functions Suggest how HIV cause cancer:
-
-
HIV randomly integrate genetic material to chromosomal DNA of infected host,
causing gain in function mutation of proto-oncogene leading to uncontrolled
cell division
-
Loss in function mutation of tumour suppressor genes leading to uncontrolled
cell division
They can produce proteins inactivating p53 gene
Photosynthesis
Carotenoids: Carotenes & xanthophylls – absorb strongly in blue-violet light spectrum,
protect chlorophylls from excess light and oxygen produced during photosynthesis
Light dependent rxn @ grana – non-cyclic phosphorylation
-
-
-
Light of particular wavelength strike accessory pigment molecule in light
harvesting complex of PSII and PSI
This energy is relayed to neighbouring accessory pigment molecule and
accumulates and reaches one of the 2 P680 chlorophyll a molecules at reaction
centre of PSII. The same occurs for P700 chlorophyll a molecules at PSI
This excites one of the P680 electrons and P700 electron to higher energy state,
which subsequently gets emitted and captured by primary electron acceptor
within each PS.
A positive hole is left behind in each P680 and P700 chlorophyll a molecule
Photolysis of H2O forms oxygen, electrons and protons
Electrons from photolysis of H2O are used to fill up positive hole in reaction centre
of PSII and return P680+ to ground state
The photoexcited electron emitted by P680 in PSII previously passes from
primary electron acceptor of PSII to P700+ in PSI to fill positive hole in P700+
This occurs via ETC made up of electron carriers, each with a lower energy
level than the previous one
Energy from the transfer is used to actively pump H+ from stroma to thylakoid space
This generates electrochemical proton gradient for ATP synthesis. Protons diffuse
through stalked particle containing ATP synthase which catalyse synthesis of ATP
from ADP and Pi (chemiosmosis). Electrons and protons are passed down 2nd
ETC from primary electron acceptor to ferredoxin. NADP reductase catalyse
electron transfer from ferredoxin to NADP oxidised to form NADPH
Cyclic phosphorylation:
-
-
-
PSI is now a donor and acceptor of electrons. Excited electrons in primary electron
acceptor of PSI pass to ferredoxin and back to cytochrome complex in ETC.
Electron returns to PSI reaction centre eventually
PS involved: PSI & PSII – cyclic VS non-cyclic (pathway – linear vs cyclic)
Cyclic photophosphorylation does not involve photolysis of water but non-cyclic
photophosphorylation involves photolysis of water to form oxygen, protons and
electrons
Cyclic photophosphorylation yields only ATP but non-cyclic
photophosphorylation yield SO2, ATP and NADPH
Energy released from cycle of electrons allow protons to be actively pumped
from stroma to thylakoid space generating electrochemical proton gradient
across thylakoid membrane
Light-independent rxn @ stroma (Calvin cycle)
-
Rubisco catalyse fixation of CO2 by ribulose bisphosphate, giving unstable 6C
intermediate which breaks down to 2 molecules of glycerate-3 phosphate
(GP)
-
-
Reducing power of NADPH and energy from hydrolysis of ATP converts GP to
glyceraldehyde-3-phosphate which is the 1st carbohydrate formed in photosynthesis.
About 1/6 of GALP is needed to synthesis glucose, other carbohydrate and glycerol
About 5/6 GALP is used to regenerate RuBP consumed in 1st reaction, requiring
energy from hydrolysis of ATP
1 GALP requires 3CO2/9ATP/6NADPH
Explain effect of light and CO2 and limiting factors of photosynthesis
-
-
Limiting factor is one that determines rate of reaction and is in the shortest supply
CO2 is major limiting factor as concentration in atmosphere is low
CO2 is required in CO2 fixation in Calvin cycle and its increased concentration
increases rate of photosynthesis
Light intensity is an important limiting factor in light dependent stage to
excite chlorophyll molecules for photophosphorylation to occur
The higher the light intensity, more light energy is absorbed by chlorophyll leading to
higher rate of electron emitted from special chlorophyll a molecule in reaction centre
of PSII and PSI. However, it is seldom the limiting factor during daylight hours
Wavelength of light is also a limiting factor as rate of photosynthesis is highest at
red and blue-violet region and lowest at green region
Guard cells are the only cells found on lower epidermis of leaf that can
photosynthesise and the synthesis of sugars increase solute potential within
guard cells, causing H2O to enter and subsequently opening stomata, allowing
gaseous exchange
Suggest why plant cells with chloroplast also have mitochondria:
-
Mitochondria are sites of cellular respiration for ATP synthesis
ATP synthesised by mitochondria is released to cytosol for processes such as
active transport. ATP produced by chloroplast is used for CO2 fixation @ stroma
Mitochondria VS chloroplast
Similarity: Both have >1 membrane, inner membrane have large surface area
Difference:
-
Photosynthetic pigment @ chloroplast VS absence @ mitochondria
Inner membrane extensively folded to thylakoids (chloroplast) VS inner
membrane extensively folded to cristae (mitochondria)
Chloroplasts have 3 membranes while mitochondria have 2
membranes Suggest role of O2 @ photo and oxidative phosphorylation:
-
-
In OP, O2 is required as final electron and proton acceptor and combines with
electron and proton to form H2O, catalysed by cytochrome oxidase to
regenerate NAD and FAD
In PP, O2 is a by-product of photolysis of H2O to give protons, electrons and O2 which
electrons are used to fill positive hole in P680+ of PSII
Explain the term photoactivation of chlorophyll:
-
Photo: light strikes accessory pigment molecule in light harvesting complex
and energy is relayed to neighbouring accessory molecule
Activation: Until it accumulates and reaches a chlorophyll molecule in reaction
centre, where its electrons get excited to higher energy level and get emitted
Explain role of membranes @ chloroplast
-
-
-
-
Chloroplast is bound by double membrane. Outer is smooth, continuous bounding
and inner membrane is extensively folded to thylakoid & lamellae. Stacks of grana
are joined by intergranal lamellae
Thylakoids are stacked to grana, which serves as boundary between thylakoid
space and stroma
Extensive folding of thylakoid increase surface area for proteins, enzymes and
pigments to be embedded in close proximity in thylakoid membrane such as:
electron carriers, stalked particles containing ATP synthase and chlorophyll
Thylakoid membrane is impermeable to H+ ions, allowing electrochemical proton
gradient to be formed between stroma and thylakoid space
Chloroplast membrane allows compartmentalisation and prevent indiscriminate
mixing between cytoplasm and stroma – allows specialisation of chloroplast
function by concentrating specific enzymes (eg. rubisco @ stroma)
Enzymes for Calvin cycle are kept in optimal condition and concentration for
CO2 fixation
Outline role of NAD and NADP:
-
-
-
Both NAD+ and NADP+ are coenzymes of dehydrogenase and electron acceptors. In
reduced form, NADH and NADPH respectively store electrons and protons which
are energised temporarily and transfer them to ETC
NAD found in mitochondrial matrix and cytoplasm of cells is reduced to NADH
during glycolysis/link rxn and Krebs’ cycle by dehydrogenation reactions
NADH donates electrons to ETC embedded in inner mitochondrial membrane
during oxidative phosphorylation
Transfer of electrons through ETC down energy gradient results in
electrochemical proton gradient and ATP synthesis
Each NADH forms 2 SA TP. Regeneration of NAD ensures glycolysis/link
rxn/Krebs cycle can continue to proceed to form ATP
NADP found @ stroma of chloroplast is final electron and proton acceptor of ETC
embedded at thylakoid membrane during non-cyclic photophosphorylation.
NADPH is needed at Calvin cycle and produced at light dependent rxn
NADPH reduces GP to GALP. Some GALP will form other sugars and
eventually starch/protein/fat. By donating H+ to GP, NADP is regenerated and
continues to accept electrons and protons from light dependent rxn to form
biomolecules
Calvin cycle VS Krebs cycle
Calvin cycle
Krebs cycle
Site
Stroma of chloroplast
Mitochondrial matrix
Coenzyme
NADPH
NADH & FADH
Oxaloacetate
Regeneration RuBP
of
starting
material
Products
ATP/NADPH
4CO2/6NADH/2ATP
formed
GP forms GALP
2FADH
Role of CO2
CO2 fixation
Oxidative decarboxylation
Energy from ATP hydrolyses Synthesised via substrate level
Role of ATP
reduced GP to GALP and phosphorylation whereby enzyme
regenerate RuBP
transfer phosphate group from
substrate to ADP
Biological evolution
Explain why evolution of different species supports Darwin’s theory of natural selection:
-
Different selection pressures on different islands
Geographical isolation interrupting gene flow
Descent with modification from ancestral birds to form the different
species Explain ways islands favour formation of new species:
-
Variation in <trait> due to different alleles in a population of <organisms>
Identify selection pressure (unequal) – different environmental conditions (eg.
predator)
Survival of the fittest – well adapted survive till maturity and reproduce to form viable
fertile offspring
Like produce like (pass down beneficial alleles)
Identify isolation mechanism (eg. geographical – prevent gene flow due to
no interbreeding)
Over time (long period), genetic difference increase – speciation (eg.
allopatric speciation)
Explain how new species arise:
-
Well adapted individuals have selective advantage and survive and pass
on beneficial allele to offspring, causing change in allele frequency
Presence of geographical barrier prevents interbreeding, preventing gene
flow between populations
Populations accumulate genetic differences leading to allopatric/sympatric speciation
When two populations can no longer interbreed, new species
arise Explain why islands have unique species:
-
Geographic isolation disrupts gene flow
Different niches lead to different selection pressures and different mutations as
a result – change in allele frequency
Founder effect: colonisation of island by small group of founding individuals cause
resultant population on island to have gene pool different from original population,
forming new species
Using 2 named examples explain how environment forces act as force of natural selection
1. Peppered moth:
o There is existing heritable variation for a particular trait (colour of moth)
o There are 2 types of moths – lighter form and melanic (darker) form
o Before 1848, light moths have selective advantage as they camouflage
against light coloured tree barks. They were selected for, survive to
maturity and reproduce to form viable fertile offspring, passing beneficial
allele.
Peppered moth was found in high frequency
o With industrial revolution, barks were covered with soot and became darker
o Lighter moths selected against and were easy prey, declining in number
o Melanic moths camouflaged well and proliferated
o Hence there was differential survival and reproductive success
associated with a trait leading to change in allele frequency
2. Galapagos finches:
o There is inheritable variation for size & depth of beaks
o Finches with large powerful beaks selected for
o Type of beaks selected for depends on availability of food source due
to different habitat
o Finches more adapted survive to maturity, reproduce to form viable
offspring and pass alleles to next generation
Selection types:
-
Directional selection: Acts against 1 tail of curve (eg artificial selection of
removing low milk producing cows)
Disruptive selection: Produce individuals with traits at 2 extremes
Stabilising selection: Intermediate phenotype survive (eg weight of human
babies) Adaptive radiation VS divergent evolution: Very fast / takes a long time
Discuss sources of variation for natural selection:
-
-
-
Genetic variation inherited if mutation occurs @ gametes
Crossing over of genes between non-sister chromatids of homologous
chromosomes forms new combination of alleles
Independent assortment of homologous chromosomes leads to new
allele combination in gametes
Random fusion of male and female gametes at fertilisation
Gene mutation is change in DNA sequence (eg. deletion) giving rise to new alleles
and cause change in aa sequence and protein structure leading to new phenotype
(eg. sickle cell anaemia)
Chromosomal mutation is change in number or structure of chromosome (eg.
inversion giving rise to new genotypes due to reshuffling of allele of
chromosome
Change in chromosome number (eg.
aneuploidy/polyploidy) How is genetic variation preserved?
-
-
-
Diploidy: genetic variation hidden via recessive alleles in heterozygotes – maintain
huge allele pool that may not be favourable at present but bring new benefits when
environment changes
Balanced polymorphism: heterozygote advantage – heterozygotes selected for
compared to homozygotes (eg. sickle cell anaemia – heterozygotes not
susceptible to malaria)
Frequency dependent inhibition: survival and reproduction of 1 morph decrease
if phenotypic form becomes too common eg. scale eating fish
Neutral mutations: do not confer selective advantage or disadvantage
Increase/decrease due to genetic drift
How embryological/molecular/anatomical homology supports Darwin’s Theory
-
-
-
-
Homology: similarity between different species due to common ancestry.
Comparison of homology shows descent with modification as ancestral homology is
modified in offspring through natural selection
Anatomical: comparing anatomical structures within group of organisms show
they are based on common prototype derived from common ancestor. Since
organisms are modified from ancestors, they retain underlying features and
modify them for different functions due to natural selection
Morphological: track modification from ancestor through time from one species to
the next, supporting Darwin’s theory eg. pentadactyl limb is a morphological feature
Embryological homology: the more closely related species are, the longer
embryological development remains similar. Embryological homology shows
historical continuity as there are remnants from ancestors persisting in embryos
of offspring eg. throat pouches
Molecular: all forms of life use DNA and RNA and genetic code is universal. Thus, it
is likely all species descended from common ancestors using this code. Two
species show high homology by comparing DNA sequence
Suggest advantages of using mitochondrial DNA:
-
Direct phylogeny via maternal lineage can be deduced as sequence is
not confounded by genetic recombination
Mitochondrial DNA mutates @ higher rate than nuclear DNA, giving more
magnified view of diversity
Mitochondrial DNA mutates at consistent rate, so time taken for species
divergence can be found
Accumulation of neutral mutations is in mitochondrial DNA
Describe advantage of using molecular data to classify
organisms:
-
Evolutionary change between species compared via similarity/difference
in nucleotide though they differ by morphology
It allows us to classify closely related organisms more accurately as they
usually share very similar base sequences
Degree of divergence between species can be quantitatively measured by
comparing sequences
Allows us to classify extinct species as long as DNA is
available Explain why molecular evidence > morphological for
phylogeny
-
Unambiguous & objective
Quantifiable & open to statistical analysis
Morphological evidence can be due to convergence
Molecular evidence can detect neutral
mutation Define species [2]
-
Group of populations where members can interbreed in nature to form viable,
fertile offspring
Classification VS phylogeny
-
-
Classification: organisms of species via particular traits; may not account for
evolutionary relationship between species. Phenotypic classification is grouping
species to individual phenotypic classes based on morphology. Hierarchical
classification is grouping of organisms to levels of increasing inclusive categories.
A category in any rank unites levels below it based on shared characteristics
Phylogeny: organisation of species via particular traits accounting for
evolutionary relationships
Phylogenetic trees are used to depict hypotheses about evolutionary
relationships and each branch point represents divergence of 2 species from
common ancestor
Hierarchical classification:
-
Domain
Kingdo
m
Phylum
Class
Order
Family
Genus – 1st part of name – underline!
Species – 2nd part of name – underline!
Species concept definitions:
-
Biological: populations whose members can interbreed in nature to form viable,
fertile offspring. Members of species defined in terms of reproductive capability
o Limitation: does not apply to asexual/extinct organisms
-
-
Ecological: based on niche – how members of each species interact with
the environment eg. finches are different species due to what they feed on
o Limitation: hard to identify
Morphological: Based on structural features
o Limitation: subjective
Phylogenetic: species is defined as small group of individuals with common ancestor
o Limitation: degree of difference to indicate different species is subjective
Genetic: group of genetically compatible interbreeding natural populations
genetically isolated from other groups
o Limitation: rely on human judgement how much difference indicate
difference species
Isolation mechanisms:
-
Geographical isolation – lead to allopatric speciation
Physiological & behavioural isolation – lead to sympatric speciation
o Physiological: mating is attempted, but physiological difference impedes it
o Behavioural: courtship rituals attracting mates/other behaviour unique to
a species are effective reproductive barriers
Bottleneck effect: sudden environmental change leading to drastic population fall. Certain
alleles may be overrepresented among survivors, others may be underrepresented and
some may be absent altogether. Genetic drift occurs, causing small population to change in
allele frequency
Suggest how viruses evolve:
-
Viruses are polyphyletic in origin – no common ancestor they coevolve with host
Viruses may have evolved from escaped genes, DNA/RNA from larger organisms
Viruses may have been small cells that parasitized larger cells
Variation in traits between viruses within a population exists and some viruses
totally depend on parasitism
These viruses have selective advantage as they are smaller and infect new
cells easily
Over time, may genes not needed by viruses as they ca depend on host for survival
Like produce like and subsequent viral generations do not carry
genes Describe how classification differs from phylogeny
as DNA, RNA, etc
Organisms may be wrongly classified such Rarely classified wrongly as cases of
as when they are unrelated but look similar convergent evolution are placed on different
due to convergent evolution
branches
Climate change
Explain how greenhouse gases cause global warming:
- Greenhouse gases accumulate and remain in atmosphere
- Layer of gases allow incoming solar radiation to pass through to earth’s
surface where it is converted to heat
- Heat cannot all pass through layer of gases and some are reflected back to
earth causing the surface to become warmer
Describe processes where carbon is added and lost to atmosphere:
- Carbon sequestration(removal): Photosynthesis – loss of CO2 carried out by green
plants
- Algae and phytoplankton carry out photosynthesis by absorbing dissolved CO2
- Photosynthesis is synthesis of organic compounds using CO2, light and water.
Oxygen is released in the process
Addition of CO2: respiration – breakdown of organic molecule to produce energy in the form
of ATP and CO2 and H2O
CO2 is released via cellular respiration of plants and animals
Decomposition: dead organisms are decomposed by microorganisms and carbon in their
bodies return to atmosphere as CO2
Combustion: CO2 is released to atmosphere by human activity involving combustion of fossil
fuels like oil
Volcanic action also releases CO2
Weathered limestone on seabed exposed to air releases CO2
How has human activity changed balance of CO2 and what activities altered it?
- Increased CO2 concentration in atmosphere due to decreased capacity of carbon
sinks and increased emission
Decreased carbon sink: Deforestation/increased water temperature – Gas decreases in
solubility and sea had decreased carbon sink effect
Increased emission: Increased industrialisation/car usage or meat consumption
Suggest effects changes have on biosphere:
-
Biosphere=all ecosystems (land and ocean) and living organisms
Shrinking freshwater supply-increased saltwater intrusion destroying many crops
Increased extreme weather events like heat waves damaging crop and livestock
-
Melting of frozen organic matter releasing ancient viruses and bacteria
infecting organisms
Increased sea levels leading to more storm surges destroying
mangrove ecosystem/coral reefs
Melting of polar ice caps destroying habitats
Death of coral reef due to ocean acidification
Migration of organisms to colder region-invasive species upset ecosystem
Explain reasons for concern for long term survival of arctic species:
- Oceans may cease to act as carbon sink
- Atmospheric co2 concentration may rise more rapidly
- Atmospheric co2 concentration is at all time high and temperature is at all time high
- Increased sea temperature decreases availability of water
- Significant change in habitat hence populations may be unable to adapt
Suggest why corals without zooxanthellae live at greater depth:
- No reliance on light as there is no zooxanthellae to photosynthesise
- The deeper the water, the less light there is
- Different feeding methods/deeper water may be nutrient rich
Suggest benefits to zooxanthellae for being with corals:
- Physical support for reefs located in shallow waters to obtain light
- Protection from predation/too much UV radiation as coral make compounds acting
as sunscreens
- Prey caught by coral serve as nutrients for algae growth
- CO2 from respiration of corals used as raw material for photosynthesis by
zooxanthellae
Suggest why loss of zooxanthellae cause coral to die:
- Decreased food source in the form of sugar decreasing chemical energy source
- Loss of protective algae layer from sunlight
- Loss of inorganic ions for deposition of exoskeleton that algae obtain from sea
Explain why seas with corals are susceptible to increased temperature:
- Shallow waters heat up quicker
Suggest how global warming increases coral bleaching rate caused by bacteria:
- Increased bacteria multiplication as bacterial infectivity increases with
increased temperature, stress causes coral to expel zooxanthellae
Explain why loss of corals is considered reduced biodiversity on many levels:
- Genetic biodiversity-loss of genome if species become extinct
- Loss of genetic alleles within species
Species biodiversity: loss of coral species and species reliant on coral
Ecosystem biodiversity: loss of autotrophs, loss of habitat for other species, matter recycling
altered
Suggest why polar bears are endangered:
- Decreased ice sheet extent-decreased seals/prey + Increased competition for
food, loss of breeding sites
- Increased distance to find food, more diseases spreading due to crowding
- Due to human activity-killing
Explain causes for shrinking freshwater supply:
- Unprecedented rate of glacial ice melting-no water at summer
- Increased evaporation leading to drought in arid areas – no freshwater
- Increased surface runoff due to flooding, contaminating water
- Increased saltwater intrusion in freshwater areas
Explain importance of mangrove:
- Breeding /wildlife habitat for commercial fish or migratory species
- Increased water quality at coasts as mangrove roots slow water flow
facilitating sediment deposition
- Supply nutrients to coral reef, natural sunscreen for coral reefs
Diseases: Describe how malaria is transmitted:
- Anopheles (female) takes blood from infected person then feeds on healthy person
- Plasmodium transmitted in mosquito saliva/ blood transfusion/ across placenta
Explain why malaria is concentrated but TB is everywhere:
- Anopheles vector survive within tropics; Plasmodium needs to survive and
reproduce within anopheles
- Concentrated as mosquito control programmes non-existent in LDCS/
mosquito resistant to DDT
- Eradicated in some countries due to pest control
- TB transmitted by droplets that are airborne and do not need vectors
Explain why blood is source of protein:
- Contains haemoglobin/plasma proteins/ antibody/ enzyme
Describe difference in anopheles and malaria distribution:
- Not all countries with anopheles have malaria, only in subtropical and tropical areas
- Plasmodium not present in all areas, some countries eradicated the disease
- Vaccination
Suggest why anopheles gambiae is responsible for most plasmodium transmission:
- It proliferates in large population of humans-many victims to feed on
- Plasmodium is able to survive at where anopheles is at
- Anopheles more adapted to complete life cycle
- More resistant to DDT
- Feed mainly on human blood and require frequent blood meals
- It is a good host for plasmodium
Describe life cycle of mosquito:
- Eggs laid on surface of stagnant water dry out subsequently
-
Larva hatch from eggs upon being submerged in water again, moults over 5 days
Pupa stage follows for 5 days
Emergence of adult female which feed blood to produce egg
Explain why dengue range is same as Aedes:
- Dengue evolved and is adapted to its vector Aedes Aegypti
- Insect physiology greatly affected by temperature range hence mosquito thrive
where virus thrives
How has Aedes mosquito range followed human expansion:
- Aedes adapted to human habitats as human activity provide viable habitats for
Aedes to thrive
- In colder habitats, larger cities provide warmer habitat
Explain how climate change affect spread of dengue beyond tropics:
- Increased global temperature sees more favourable physiological condition
for mosquito and dengue
- Current range limit restricted by 10 degrees temperature barrier, climate change
may speed up increase in global temperature
How dengue is transmitted: female Aedes aegypti first feed on infected human blood with
DENV. DENV transferred to mosquito and undergo extrinsic incubation period where DENV
replicates and disseminates in mosquito until it reaches salivary gland. It then feeds on
healthy human blood, transferring DENV to him hence dengue
Explain how temperature affect insect metabolism:
- As temperature increases, developmental time from larvae to adult decreases
- Metabolic processes are enzyme mediated, increased temperature
increases metabolism leading to growth
- Mosquito migrate poleward, increasing spread
Immunity:
Describe how innate immune system responds to DENV:
- Macrophage recognise receptor on microbe, leading to receptor
mediated phagocytosis
- Phagocyte activated-secrete cytokine which recruit circulating phagocytes
- Inflammation occurs; recruited phagocytes assist in eliminating microbe
Explain how innate immunity is ineffective against DENV:
- Macrophages are target cells for DENV-phagocytosis allow entry of virus into cell
- Virus able to escape lysosomal degradation and replicate within cell,
increasing DENV number
Explain how antibody structure allow successful recognition and binding of DENV:
- Consist of 2 antigen binding sites, each composing of Vh and VL precisely folded to
give specific 3-dimensional conformation complementary to DENV antigen, allowing
formation of weak bond with DENV
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Explain how mosquito has evolved to resist deltamethrin:
- Mutation leads to genetic variation-some mosquito susceptible, some resistant
- Deltamethrin acts as selection pressure-resistant mosquito have selective advantage
- Differential survival and reproductive rate
- Allele coding for resistance passed to offspring- increased allelic frequency
of resistance allele
Define ecosystem: community of organisms interacting with one another and the
environment they live in (eg. Pond/lake or tropical rainforest)
Suggest difference between corals and plants:
- Corals are not photosynthetic
- They do not have chloroplast and cell wall or vacuole
Outline effect of temperature rise on Arctic ecosystem:
- Reduced range for arctic species like Pika/polar bear
- Melting of permafrost/ increased detritus decomposition previously on
trapped permafrost
- Increased sea levels, loss of ice habitat, climate patterns changing
- Extreme weather events like storms
- Migration of species to arctic habitats altering food chains
- Marine species sensitive to temperature changes die off