The Cell & Molecules of Life (9744) Carbohydrates 2018

Carbohydrates

Describe the structure and properties of α glucose and β glucose monomers and the formation and the breakage of a glycosidic bond.
Carbohydrates: Cn (H2O)m
Monosaccharides: (CH2O)n Features

α glucose
Disaccharides: Cn(H2O)n-1
1. Have a free carbonyl (C=O) group hence are all reducing sugars. (Property of glucose)
2. Small size and have multiple hydroxyl (OH) groups and can form hydrogen bonds with water
hence readily soluble in water (Property of glucose) and easily transported in animal and
plant transport systems
3. Ring structures exhibit α- and β- isomerism (α – if OH grp at C1is below ring and on opposite
β glucose side of C6 and β – if OH grp at C1 is above ring and on same side as C6)
4. e.g. glucose, galactose and fructose
Features

1. Are made up of 2 monosaccharides joined by a glycosidic bond formed between two

monosaccharides by a condensation reaction that involves the loss of a water molecule
2. Can be split into their component monosaccharides is via hydrolysis reaction where, with the
addition of one molecule of water, the glycosidic bond can be broken
3. Have many hydroxyl groups which extend out of the ring
α glucose α 1-4 α glucose  form hydrogen bonds with water and hence are readily soluble in water and easily
glycosidic
bond transported in animal and plant transport systems
4. All are reducing sugars except sucrose
5. e.g. Sucrose (glucose + fructose)
Maltose (glucose + glucose)
Lactose (glucose + galactose)

Maltose

Compare the structures and properties of starch (amylose and amylopectin), glycogen and cellulose and explain how these are related to
their roles in plants and animals.
Polysaccharides:(C6H10O5)n are made up of many monosaccharides joined by glycosidic bonds formed between them by condensation
reactions which involve the loss of water molecules.
Point of Comparison Starch Glycogen Cellulose
Function Plant storage polysaccharide Animal storage polysaccharide Plant structural polysaccharide
Location Stored as granules in chloroplasts Stored in liver and muscle Cell walls of plant cells
cells
Monomer Made up of -glucose monomers Made up of -glucose Made up of β-glucose monomers
monomers
Bond between monomers In amylose: (1-4) glycosidic bond (1-4) glycosidic bond links β (1-4) glycosidic bond links
links monomers; monomers within a branch and monomers in a molecule
In amylopectin: (1-4) glycosidic bond (1-6) glycosidic bonds links
links monomers within a branch and (1- monomers at branch points
6) glycosidic bonds links monomers at
branch points
Orientation of monomer All glucose monomers in the chain have All glucose monomers in the Alternate β glucose monomers
the same orientation chain have the same rotated 180° with respect to each
orientation other
Structure of each molecule Amylose is a helical molecule while Helical and branched Long, straight chain
amylopectin is a helical and branched molecule, like amylopectin, but
molecule more extensively branched
Bonds between molecules No interchain hydrogen bonding No interchain hydrogen bonding OH groups projecting outwards in
both directions allow interchain
hydrogen bonding between parallel
chains  forming microfibrils

How the structures of starch and glycogen make them
good STORAGE molecules
1. Made up of many -glucose monomers that coil to
form helices
a. Hence is a large yet compact energy store as many
-glucose monomers can be packed per unit volume
b. Hence most OH groups are involved in
intramolecular hydrogen bonding within the helix
and few OH groups available for hydrogen bonding
with water.
Hence starch/glycogen is insoluble in water and the
Ψw of cells are unaffected by its presence. (Property
of starch/glycogen)
2. Amylopectin and glycogen are branched
a. Thus they have multiple branch ends which hydrolytic
enzymes can work on. Hence more glucose molecules
can be released at the same time and more ATP can
be generated by respiration per unit time.
How the structure of cellulose makes it a good
STRUCTURAL molecule
1. Adjacent glucose units are inverted 180 with respect to each other
and hence form a long, linear, unbranched molecule with free OH groups
projecting out in both directions which can hydrogen bond with OH groups of
other cellulose molecules lying parallel to it and form microfibrils.
Hence microfibrils have high tensile strength. (Property of cellulose)
2. As a macromolecule, cellulose has few OH groups available to hydrogen bond
with water as most are involved in interchain hydrogen bonding. Thus only the
surface of the microfibril has OH groups that can hydrogen bond with water.
Hence cellulose is insoluble in water and the Ψw of cells are unaffected by its
presence. (Property of cellulose)
3. The meshwork of microfibrils that form the cell wall
a. have a porous structure and hence the cell wall is freely permeable to water
and solutes and allows movement of substances across the cell wall.
b. Are strong and rigid and distributes stress in all directions to prevent the
plant cells from bursting due to osmotic stress.
4. Cellulases that hydrolyse cellulose are found in very few organisms. Thus
cellulose cannot be hydrolysed and used as a respiratory substrate and is a good
structural molecule.

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The Cell & Molecules of Life (9744) Carbohydrates 2018
Test Procedure Observations and Deduction
Benedict’s Test 1. Place 2 cm3 of test solution in a test tube. Presence of reducing sugar is indicated by formation of brick-red
for Reducing 2. Add equal volume of Benedict's reagent. precipitate.
Sugars 3. Shake the mixture.  colour of final suspension depends on amount of reducing sugar
4. Heat it by immersing the tube in boiling water bath for present (test is semi-quantitative)
3-4 minutes.  varies from green to yellow to brown to brick red

Test for Non- If a negative result for Benedict’s test is obtained for the Presence of non-reducing sugar indicated by:
Reducing test solution, then  A blue solution remains when Benedict’s test is first carried out.
Sugars 1. boil equal volume of test solution with dilute  After acid hydrolysis, Benedict’s test is carried out again
hydrochloric acid for about 1 minute to hydrolyse  colour of final suspension depends on amount of sugar
disaccharide to monosaccharides present
2. cool contents of tube.
3. neutralise acidic content with sodium bicarbonate *Both observations must be made to conclude presence of non-
solution. reducing sugars.
4. carry out Benedict's test for reducing sugar.
Iodine Test for 1. Add a few drops of iodine solution to 1 cm 3 of test Presence of starch is indicated by blue-black coloration.
Starch solution (or a piece of test specimen).
2. Observe any colour change.

(unbranched)

α glucose
(branched) monomer

α 1-6 glycosidic
bond at branch α 1-4 glycosidic
point bond within branch

α 1-6 glycosidic bond at branch

points
(extensively branched)

α 1-4
α glucose
glycosidic monomer
bond within
branch

(unbranched)

β 1-4 glycosidic bond

β glucose
monomer

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The Cell & Molecules of Life (9744) Lipids 2018

Lipids
 Can be classified into: triglycerides, phospholipids, steroids (cholesterol to be covered under cell membranes) and waxes (not in syllabus).

Describe the formation and breakage of an ester bond.

 To form triglycerides  Three non-polar, hydrophobic, hydrocarbon chains are joined to a glycerol backbone via the formation of 3 ester
linkages. An ester linkage is formed between a hydroxyl group (-OH) and a carboxyl group (-COOH) via a condensation reaction. One water
molecule is removed for each fatty acid joined to the glycerol.

Glycerol 3 Fatty Acids Triglyceride Water

Describe the structures and properties of glycerol & fatty acids

Molecule Structure Properties

Glycerol has 3 polar, hydrophilic hydroxyl groups which  Soluble in water
are able to form hydrogen bonds with water

Fatty acids when ionized in water, has charged COO- group
which can interact with water
(Note: for charged groups, the term used is
‘interact with water’, not ‘form hydrogen bond with
water’)
 Soluble in water
(if the fatty acid chains are short.)
However, as the length of the hydrocarbon tail of a fatty acid increases,
solubility decreases due to non-polar hydrophobic nature of the hydrocarbon
chains

Describe the structures and properties of a triglyceride and a phospholipid & explain how these are related to their roles in living organisms

Triglycerides
 Each triglyceride consists of
1. 3 long non-polar, hydrophobic hydrocarbon chains
2. joined to a glycerol backbone via ester linkages

 The long non-polar, hydrophobic hydrocarbon chains of the triglyceride

a) cannot form hydrogen bonds with water and
 hence the triglyceride is insoluble in water does not affect the water potential of the cell it is in
b) have a high proportion of C-H bonds from which energy in the form of ATP and metabolic water can be released during oxidation

This makes triglycerides a suitable energy store

 Other roles lipids:

1. Lipids are found beneath the layer of skin. They are poor conductors of heat and are able to provide thermal insulation to mammals
especially those in cooler climates.
2. Lipids are less dense than water and hence improve buoyancy in mammals, especially marine mammals like the whale.
3. Lipids form a protective layer around delicate internal organs of mammals. Hence they act as shock absorbers and protect organs from
mechanical damage.
4. Lipids can function as a reservoir for storage of fat soluble vitamins e.g vitamins A, D and K.
5. Lipids are insoluble in water hence osmotically inactive or without affecting water potential of mammalian cells.

 Triglycerides release twice as much energy on oxidation compared with an equivalent mass of carbohydrates as triglycerides contain proportionally
more C-H bonds and less O atoms compared to an equivalent mass of carbohydrates from which energy in the form of ATP can be released
during oxidation.

 In fact, lipids also produce more metabolic water per unit mass when compared with an equivalent mass of carbohydrates. This is especially
useful for desert animals which store fat and have limited access to water.

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The Cell & Molecules of Life (9744) Lipids 2018

Phospholipids
 Each phospholipid consists of
1. 2 long non-polar, hydrophobic hydrocarbon tails
2. joined to a to a glycerol backbone via ester linkages
3. with the third hydroxyl group of the glycerol backbone joined to a negatively charged (not polar!) phosphate group

 Major component of membranes of cells and organelles as

1. the non-polar hydrophobic hydrocarbon tails face inwards, away from the water and
2. the hydrophilic negatively charged phosphate group face outwards and interact with the aqueous environment
 hence giving rise to a phospholipid bilayer with a hydrophobic core that forms a selectively permeable membrane which
(i) regulates movement of substances by acting as a barrier to and ions, polar and large molecules
(ii) acting as a boundary between the intracellular & extracellular aqueous environment and
(iii) allows compartmentalization within a cell

(Note:
1. Since the phospholipid has both hydrophilic and hydrophobic regions, it is
referred to as being amphipathic
2. Unsaturated hydrocarbon chains with kinks prevent close packing of
phospholipids resulting in greater fluidity of the membrane and fewer
hydrophobic interactions between the phospholipids hence and allowing larger
transient pores to form in the bilayer
3. Additional small molecules, usually charged or polar, can be linked to the
phosphate group to form a variety of phospholipids

Carry out the emulsion test for fats

Emulsion Test If the test sample is a solution: Presence of lipids indicated by:
for Lipids 1. Add 2 cm3 of ethanol to two drops of test sample in a test tube. Mix well and allow  A homogeneous (or clear) solution is
to stand for 2 minutes. formed with ethanol and an emulsion
2. Decant the ethanol into another test tube containing 2 cm 3 of water. was formed when water was added.
If the test sample is a solid: If lipids are absent, a clear solution
1. Add 2 cm3 of ethanol to ground test sample in a test tube. remains.
2. Mix well to dissolve any lipids if it is present and allow it to stand for 2 minutes.
3. Decant the ethanol into another test tube containing 2 cm 3 of water.

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The Cell & Molecules of Life (9744) Proteins 2018

Proteins

Describe the structure and properties of an amino acid and describe the formation and breakage of a peptide bond

 Amino Acids – Basic structural unit of proteins

 Structure
o consists of an α-carbon atom covalently bonded to 4 groups
1) hydrogen atom, 2) amino group (-NH2), 3) carboxyl group (-COOH), 4) variable R group
 Properties of amino acids
o Classified according to their R groups as uncharged (non-polar or polar) or charged
o Exist as zwitterions in solution – carry both positive and negative charges
o Act as buffers
 Can donate or accept H+, therefore able to act as an acid or as a base – amphoteric
 Essential in biological systems – sudden change in pH could adversely affect performance of
proteins like enzymes

 Polypeptides
 Amino acids are joined by a peptide bond via a condensation reaction with the removal of one water
molecule.
 Further addition of amino acids results in the formation of a linear polymer called a polypeptide
o Regularly repeating part, the main chain, is referred to as the backbone.
o Variable part comprises the distinctive variable R groups
 polypeptide folds into a specific three-dimensional shape / conformation

 The nucleotide sequence in DNA determines amino acid sequence in polypeptide which determines types and locations of R groups which
determines R group interactions which determines 3D structure and function of protein. (See picture with 4 R gp bonds / interactions below)

Explain primary structure, secondary structure, tertiary structure and quaternary structure of proteins, and describe the types of bonds
(hydrogen, ionic, disulfide and hydrophobic interactions) that hold the molecule in shape.

 4 levels of organization in the structure of proteins

(a) Primary structure

 Refers to the number and sequence of amino acids in a single polypeptide chain.
 Linear structure maintained by peptide bonds
 The sequence of amino acids (and their R groups) in a polypeptide chain determines the
type and location of chemical bonds/interactions, and hence the 3D conformation and
characteristics of a particular protein.
(b) Secondary structure
 Structure formed by regular coiling or pleating of a single polypeptide chain.
 Maintained by hydrogen bonds
o between C=O and N-H groups of the polypeptide backbone.
o R groups are not involved
 Examples of secondary structures:
o -helix
 Made up of a single polypeptide chain which is wound into a coiled/spiral structure.
 A hydrogen bond forms between the C=O group of one amino acid residue and the
N-H group of another amino acid residue four amino acids away along the
backbone of a single polypeptide
 There are 3.6 amino acid residues in every turn of the helix β-pleated
o β-pleated sheet sheet
 Two or more regions/segments of a single polypeptide chain lying side by side
are linked together by hydrogen bonds
-helix
 A hydrogen bond forms between the C=O group of an amino acid residue
and a N-H group of another amino acid residue on an adjacent
segments along the backbone of a single polypeptide
 Chains may run parallel (same direction) or anti-parallel (opposite directions)
 Forms flat sheet which becomes folded
(c) Tertiary structure
 Structure formed by further extensive folding and bending of a single polypeptide Disulfide
chain, giving rise to the specific 3D conformation of a protein bridge
 Maintained by all 4 types of interactions between
o hydrogen bonds, ionic bonds, hydrophobic interactions and disulfide bonds sulfhydryl
o formed between R groups of amino acid residues within a polypeptide groups of
(d) Quaternary structure cysteine
 Refers to the association of two or more polypeptide chains into one functional residues
Hydrogen
protein molecule Ionic
bonds
 Maintained by all 4 types of interactions between bonds
o hydrogen bonds, ionic bonds, hydrophobic interactions and disulfide bonds polar R between
o formed between R groups of amino acid residues of different polypeptides groups oppositely
 Constituent chains of a multimeric protein can be identical or different. charged
R groups

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The Cell & Molecules of Life (9744) Proteins 2018

Describe the molecular structure of the haemoglobin and collagen and explain how the structure of each protein relates to the function it
plays:
Example Structure Function
Haemoglobin 1. Haemoglobin has a quaternary structure made up of 4 Fe2+ of haem group binds temporarily to O2, so 1 Hb
(globular protein) polypeptide subunits: 2 -globin subunits and 2 -globin molecule can carry up to 4 O2, at a time forming
transports subunits. Each subunit is made of globin polypeptide and a oxyhaemoglobin
oxygen in the prosthetic (non-protein) component called haem group. Each
blood haem group consists of a porphyrin ring and an iron ion (Fe2+)
**(The binding of
successive O2 molecules
facilitates binding of the 2. Each subunit is arranged so that most of its hydrophilic amino Thus haemoglobin is soluble in an aqueous
next. Binding of the 1st acid side chains are on external surface while its environment and can be transported in the blood while
O2 molecule increases hydrophobic amino acid side chains are buried in interior carrying O2 from lungs to tissues vice versa
the affinity of
haemoglobin for oxygen
and hence facilitates the 3. The 4 subunits held together by intermolecular interactions As a result binding of one oxygen molecule to one
binding of the 2nd O2 formed between R groups (hydrogen bonds, ionic bonds and haemoglobin subunit induces a conformational change
molelcule. Binding of the
2nd O2 molecule hydrophobic interactions). No disulfide bridges. in remaining 3 subunits so that their affinity for oxygen
facilitates the binding of increases. This is known as the **cooperative binding
the 3rd O2 molecule and of oxygen.
so on.)
Collagen 1. A tropocollagen molecule consists of three helical covalent cross-links
staggered
(fibrous protein) polypeptide chains (loose helices, not α-helices) wound around arrangement
 an essential each other like a rope. (has quaternary but no tertiary structure) of
component of tropocollagen
connective tissue 2. Each chain contains about 1000 amino acids and contain a
in the human repeating sequence, usually a repeating tripeptide unit: glycine-
body. X-Y, where X is usually proline, Y is usually hydroxyproline.
The tropocollagen molecule can form a tight, compact coil as
almost every third amino acid in each polypeptide chain is a Tropocollagen Fibril Collagen fibre
glycine, the smallest amino acid. This allows it to fit into the Bulky and relatively inflexible proline and
restricted space in the center of the triple helix. hydroxyproline residues confer rigidity on the molecule.

3. Extensive hydrogen bonds form between amino acid Increases tensile strength (ability to resist snapping
residues of adjacent polypeptides, hence interaction with due to stretching) and makes the molecule insoluble in
water molecules are limited. water

4. Adjacent tropocollagen molecules are arranged in a staggered Staggered/overlapping arrangement minimizes

manner points of weaknesses along fibrils

5. Covalent cross-links between lysine residues at C and N Greatly increases tensile strength.
ends of adjacent tropocollagen molecules results in the
formation of fibrils.

6. Bundles of fibrils unite to form long collagen fibres. Large size of collagen makes it insoluble, an important
property for a structural molecule

Fibrous protein (e.g. collagen) Globular protein (e.g. haemoglobin, amylase)

Shape Made up of long polypeptide chains forming long, straight Made up of polypeptide chains folded into roughly spherical
fibres shape
Solubility in Insoluble in water Soluble in water
H2O  as it is a large molecule and extensive hydrogen bonds  as polar R groups can form hydrogen bonds with water
have already formed between residues in different polypeptides molecules in the aqueous environment
Amino acid Less variety of amino acids are used to construct the protein. More variety of amino acids are used to construct the protein
sequence i.e. consists of repetitive regular sequence of amino acids. (eg i.e. consists of non-repetitive amino acid sequence
tripeptide, gly-X-Y repeats in collagen)
Length of Length of polypeptide may vary slightly between two samples Length of polypeptide is always identical between two samples
polypeptide of the same protein, yet protein is still functional. of the same protein, or else protein may not be functional.
Function Structural role Protein with metabolic role e.g. enzyme

Carry out Biuret test for proteins

Test Procedure Observations and Deduction

Biuret Test 1. Place 2cm3 of test solution in a test-tube Presence of protein indicated by a
(A test for 2. Add equal volume of 5% KOH solution and shake the mixture well purple colour developing slowly.
peptide bonds) 3. Add two drops of 1% copper sulphate solution, shaking well after each drop.

Denaturation of proteins
3D shape of a protein may be changed by: (a) Heat (affect hydrogen bonds, hydrophobic interactions) (b)Acids/Alkalis (affect hydrogen and ionic bonds)

NB: Strength of bonds: hydrophobic interaction<H bonds<ionic bonds <disulfide bridges <peptide bonds

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The Cell & Molecules of Life (9744) Enzymes 2018

Enzymes

Explain the mode of action of enzymes in terms of active site, enzyme-substrate complex, enzyme specificity and lowering of activation energy
using lock and key and induced fit hypotheses.

 Enzymes are biological catalysts – they speed up the rate of reactions and are chemically unaltered at the end of the reaction & thus can be reused,
and are effective in small amounts
 Most enzymes are globular proteins, each with an active site with specific 3D conformation that is complementary in shape and charge to a
specific substrate.
 The globular structure of enzymes contribute towards their solubility.

 2 models explain why enzymes are highly specific:

1. Lock & key hypothesis
 the enzyme’s (the lock) active site has a conformation which is
complementary in shape and charge to a specific substrate (the key)
 allowing the substrate to bind, hence forming an enzyme-substrate
complex
 products formed no longer fit the active site and will hence leave the active
site, making it available for another substrate to bind

2. Induced fit hypothesis

 binding of the substrate to the enzyme active site induces a conformational
change in the active site of the enzyme such that it now provides a more
precise fit for the substrate
 thus enzyme can perform its catalytic function more effectively

 In an enzyme-catalysed reaction:
 Effective collisions take place between the enzyme and substrate
 Resulting in the formation of an enzyme-substrate complex

 Amino acids residues that make up the enzyme have different functions:
1. Contact residues – found in the active site – help to position the substrate in the correct orientation via weak interactions such as
hydrogen bonds, ionic bonds & hydrophobic interactions.
2. Catalytic residues – found in the active site – have specific R groups which act on bonds in the substrate and help to catalyse the conversion
of substrate to product
3. Structural residues – interact with each other to maintain the overall 3D conformation of the protein
4. Non-essential residues – found on surface of the protein – no specific function

 Enzymes speed up metabolic reactions/allows metabolic reactions to proceed at

moderate temperatures by lowering activation energy (EA) of the reaction,
through:
1. proximity effects – temporary binding of substrates in close proximity in the
enzyme active site increases chances of a reaction (vs
depending only on random collisions between reactants in
the absence of enzymes)
2. strain effects – slight distortion of substrates as they bind to enzyme
strains bonds in substrates that need to be broken for
products to form
3. orientation effects – substrates are held by enzymes in their active sites in an
the correct orientation such that the bonds in
substrates will be exposed to chemical attack
4. microenvironment effects – enzymes create the appropriate
microenvironment for a reaction to occur e.g.
hydrophobic amino acids in enzymes create a
water-free zone that allows non-polar reactants to
react more easily
5. acid-base catalysis – R groups of acidic and basic amino acids in enzyme
active site facilitate reaction between substrates
Hence there will be a greater proportion of substrate molecules with energy
greater than the activation energy, allowing the reaction to proceed faster than
a uncatalysed reaction.

 Enzyme cofactors

1. inorganic ions (e.g. magnesium ions in PCR)

 mould enzyme or substrate and allows enzyme-substrate complex to form more easily

2. prosthetic group (e.g. haem group of cytochrome oxidase in electron transport chain in inner mitochondrial membrane accepts electrons from
cytochrome C and transfers them to oxygen to form water)
 permanently bound to enzyme
 transfers atoms/chemical groups between active site of enzyme and another substance

3. coenzymes (e.g. NAD transfers electrons in certain redox reactions in respiration)

 are organic molecules that are required by certain enzymes to carry out catalysis
 they bind to the active site of the enzyme and participate in catalysis but are not considered substrates of the reaction
 function as intermediate carriers of electrons or specific atoms that are transferred in the overall reaction

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The Cell & Molecules of Life (9744) Enzymes 2018

Following the time course of an enzyme-catalysed reaction by measuring the rates of formation of products (eg. using catalase) or rate of
disappearance of substrate (eg. using amylase)

Method Measuring rate of product formation Measuring rate of disappearance of substrate

2H2O2 2H2O + O2 Starch Reducing sugars

catalase amylase iodine
Example
1) blue-black in presence of iodine  incomplete digestion of starch
2) brown in presence of iodine  complete digestion of starch

Dependent
Volume of O2 evolved per unit time / over a fixed period of time Change in intensity of blue-black colouration of starch-iodine complex
variable
per unit time / over a fixed period of time
Concentration of substrate and enzyme kept constant Concentration of substrate and enzyme kept constant
Fixed
All other conditions that affect an enzyme-catalysed reaction, All other conditions that affect an enzyme-catalysed reaction, eg. pH,
variable
eg. pH, temperature kept constant temperature kept constant
Experimental
set-up Dropper
Drops of
iodine

amylase
White tile and starch

Note: Delivery tube may be attached directly to a gas syringe.

1. Add enzyme (amylase) to starch and start the stopwatch

1. Add enzyme (catalase) to H2O2 , mix and start the
stopwatch.
2. O2 evolved can be measured by the downward
Procedure
displacement of water in a graduated cylinder (as shown
above) or using a frictionless gas syringe.
3. Record volume of O2 evolved at fixed time intervals
immediately
2. Add fixed time intervals, transferred a drop of the reaction mixture
to a white tile with a drop of iodine
3. In the presence of starch, iodine turns from yellowish brown to
blue-black
4. Intensity of the blue-black colouration can be measured using a
colorimeter
5. Use a standard curve to convert the colorimeter reading to starch
concentration

Starch
concentration/
mol dm-3

Graph

Time / s

Rate Initial rate of reaction = Gradient of curve at time 0 sec Initial rate of reaction = Gradient of curve at time 0 sec

Intensity of blue-black colouration / concentration of starch decreases

Volume of O2 evolved increases with time with time
Trend
Rate of O2 production decreases with time Rate of change in intensity of blue black colouration / Rate of
decrease of starch decreases with time

 To investigate how factors like pH, temperature, [enzyme] or [substrate] can affect the rate of reaction:
o Vary independent variables e.g. pH / temperature / [enzyme] / [substrate]
o Keep all other variables constant.
o For each condition, plot a graph of amount of product formed vs time and obtain the initial rate of reaction.
o Plot a graph of rate of reaction vs the factor investigated (pH / temperature / [enzyme] / [substrate])

Notes:

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The Cell & Molecules of Life (9744) Enzymes 2018

Investigate and explain the effects of temperature, pH, [enzyme] and [substrate] on the rate of enzyme-catalysed reactions

Effect of temperature

As temperature. increases,
 Kinetic energy of enzyme and substrate molecules increases
 Frequency of effective collisions between enzyme and substrate molecules increases
 Rate of enzyme-substrate complex formation increases
 Number of substrate molecules with sufficient energy to overcome the activation energy barrier and
form products increases and rate of reaction increases.

Temperature coefficient, Q10 = Rate of reaction at (x+10)oC

Rate of reaction at x oC
When Q10=2, for every 10oC rise in temperature, the rate of reaction/ product formation doubles
Beyond the optimum temperature,
 kinetic energy of enzyme and substrate molecules continue to increases
 intramolecular vibrations increases
 hydrogen bonds, ionic bonds and hydrophobic interactions that maintain the 3D conformation of
the enzyme are disrupted
(* Covalent disulfide bonds are harder to break and thus can withstand higher temperature.
Hence enzymes with higher optimum temperature tend to have a larger proportion of disulfide bridges
or more intramolecular interactions.)
 specific conformation of active site is lost
 substrate no longer complementary to the shape and charge of active site and cannot bind to it
 rate of enzyme-substrate complex formation decreases and rate of reaction decreases.

Effect of pH

At optimum pH,
 conformation of enzyme active site is most ideal for substrate binding and rate of reaction is highest

As pH deviates from the optimum,

 excess H+ or OH- ions affects the ionisation of the R-groups of the amino acids residues as
excess H+ results in –COO- groups becoming -COOH and excess -OH- results in -NH3+ becoming –NH2
 thus ionic bonds and hydrogen bonds that maintain the conformation of the enzyme active site is
disrupted
 thus the interaction between substrate and catalytic residues in the active site of enzyme is disrupted
 rate of enzyme-substrate complex formation decreases
 rate of reaction/product formation decreases
(Note: if the change in pH affects the charges of the R groups of the
(1) catalytic residues in the active site, the catalytic activity of enzyme may be lost
(2) contact residues in the active site, this may affect the temporary binding between the
enzyme and substrate and thus no enzyme-substrate complex forms.
(3) structural residues, the tertiary structure of the protein and its active site can be affected and
this would denature the enzyme.)

Effect of [enzyme]

Initially, when [enzyme] is low, as [enzyme] increases,

 frequency of effective collisions between enzyme and substrate molecules increases
 rate of enzyme-substrate complex formation increases and rate of reaction increases

At linear portion of graph, [enzyme] is limiting

iIncreasing [enzyme] will result in a proportional increase in the rate of reaction

At curved portion of graph, rate of reaction is increasing at a decreasing rate. [Enzyme] is not the only
limiting factor. Some other factor is also limiting

At the plateau, [enzyme] is no longer the limiting factor (other factors are limiting)
 increasing [enzyme] will not affect the rate of reaction

Effect of [substrate]

Initially, when [substrate] is low, as [substrate] increases,

 frequency of effective collisions between enzyme and substrate molecules increases
 rate of enzyme-substrate complex formation increases and rate of reaction increases as active sites
of enzymes are readily available and substrate concentration is limiting
Beyond a certain [substrate],
 all active sites of enzymes are saturated with substrate at any one point in time
 [substrate] is no longer limiting and enzyme concentration is limiting, the rate of reaction will
remain constant(graph plateaus) and reaches maximum velocity (Vmax).

Michaelis constant (Km): [substrate] at which reaction proceeds at half its max. rate
 low Km – high affinity between enzyme & substrate
 high Km – low affinity between enzyme & substrate

Prepared by: Mrs Selvamani Nair & Mrs Wong Seok Hui Raffles Institution (Yr 5-6) 3
The Cell & Molecules of Life (9744) Enzymes 2018

Describe the structure of competitive and non-competitive inhibitors with reference to the binding sites of the inhibitor and explain the effects
of competitive and non-competitive inhibitors (including allosteric inhibitors) on the rate of enzyme activity
Competitive Inhibition Non-competitive Inhibition Allosteric Inhibition / Activation
Inhibitor / Activator binds to Active site Site other than active site Allosteric site
Shape and charge of inhibitor / Similar in conformation and charge Not similar in conformation and Not similar in conformation and charge
activator to substrate charge to substrate to substrate
Structure of enzyme  Can consist of 1 subunit with 1  Usually a 1 subunit with 1  Multimeric enzyme with multiple
active site active site active sites
 Presence of cooperative binding of
OR substrate to active site
 Has multiple allosteric sites
 Can be a multimeric enzyme with o But binding of 1 inhibitor /activator
multiple subunits, each with an is sufficient to inhibit /activate the
active site activity of the enzyme
Effect of inhibitor / activator on  Inhibitor is structurally similar  Inhibitor is not structurally  Inhibitor/ Activator is not
enzyme & the rate of the enzyme- to substrate & hence binds similar to substrate & hence structurally similar to substrate &
catalysed reaction reversibly to active site of binds to a site other than hence binds to allosteric site of the
enzyme the active site enzyme. This results in
Hence inhibitor competes with This results in a conformational change in enzyme.
substrate for active site of conformational change in Binding of inhibitor stabilises enzyme
enzyme the enzyme active site in an inactive state
This reduces the availability of Thus substrate cannot bind  to  shifts curve to the right
active sites for substrate active site  Binding of activator stabilises
binding  rate of reaction decreases enzyme in an active state
 rate of reaction decreases  shifts curve to the left
Effect of  [substrate] on the  At high [substrate], substrate is  Inhibitor binds to site other  Substrate binding stabilizes the
inhibition more likely to bind active site than active site and changes enzyme in the active conformation
than the inhibitor to form the conformation of the active and opposes the effect of the
enzyme-substrate site inhibitor. This allows Vmax to be
Hence the same Vmax can Hence the inhibitor effectively reached at high substrate
be reached at high [substrate] decreases the available concentration.
Thus the effects of inhibition [enzyme] as it forms an
can be overcome at high enzyme-inhibitor complex
[substrate] Hence the effects of the
inhibition cannot be
overcome by increasing
[substrate]
Graph demonstrating effect of
inhibitor / activator

Vmax remains the same Vmax decreases The enzyme freely oscillates between
Km increases Km remains the same the active form and the inactive form.

End-product Inhibition e.g. Synthesis of isoleucine from threonine.

 Regulation of a metabolic
pathway by the end-product of
the pathway
Initial substrate End-product
 End-product can function as an Intermediate A Intermediate B
allosteric inhibitor to an enzyme (Threonine) (Isoleucine)
Enzyme 1 Enzyme 2 Enzyme 3
present early in the pathway by
(threonine deaminase,
binding to its allosteric site and
an allosteric enzyme)
prevent further synthesis of the
Feedback Inhibition
product.
Isoleucine binds to allosteric site of enzyme  active site of enzyme changed, no longer binds to threonine

Note: Although enzyme inhibition can both be reversible & irreversible, we are more concerned with reversible inhibitors in our syllabus.

Prepared by: Mrs Selvamani Nair & Mrs Wong Seok Hui Raffles Institution (Yr 5-6) 4
The Cell & Molecules of Life (9744) Cell Structure 2018
Cell Structure

Use a graticule and stage micrometer to measure cells and be familiar with units (millimetre, micrometre, nanometer) used in cell studies
* Interconversion of units (mm, μm, nm)
1 mm = 103 micrometers (m)
1 m = 103 nanometers (nm)
Interpret & recognise drawings, photomicrographs & electron micrographs of the following membrane systems and organelles: rough and
smooth endoplasmic reticulum, Golgi body, mitochondria, ribosomes, lysosomes, chloroplasts, cell surface membrane (this is covered under
the cell membrane summary), nuclear envelope, centrioles, nucleus & nucleolus. Outline the functions of the organelles listed.

Membrane-bound organelles
Organelle Description Function
Nucleus . Nucleus  To contain the hereditary material
 Prominent, spherical organelle in  To control cell activities by synthesising
nuclear envelope eukaryotic cell mRNA which will be translated into
 Surrounded by a nuclear envelope (a proteins which are needed in the cell
double membrane) which is perforated
with pores & continuous with RER
nuclear pore
 Contains the nucleolus & chromatin
Nucleolus
nucleolus  To synthesise rRNA, a component of
 Non-membranous, sphere/s within ribosomes
chromatin nucleus  Assembly of large & small ribosomal
 Contains large amounts of DNA, RNA & subunits using rRNA synthesised in
protein nucleolus & proteins exported from
cytoplasm
[NB: Substances pass between nucleus & Chromatin
cytoplasm via the nuclear pores. They are  Hereditary material of the cell
1) free nucleotides & enzymes (for DNA  Are thin, elongated threads of DNA
replication & transcription), proteins (to make coiled around histone proteins
up ribosomal subunits) which enter the
 2 types of chromatin are present
nucleus 1. Euchromatin (lightly stained,
2) mRNA, tRNA and large and small ribosomal
transcriptionally active, exists in a
subunits which leave the nucleus]
diffused, extended state)
2. Heterochromatin (darkly stained,
transcriptionally inactive, usually found
along the edge of nucleus).
Endoplasmic Reticulum (ER)  Consists of the RER & SER
Rough ER Smooth ER Rough ER (RER)

 A network of membranous flattened  To transport of proteins which are

sacs called cisternae synthesised by the ribosomes on its
surface to the Golgi apparatus via
 Has ribosomes bound to the outer
transport vesicles
surface
 Continuous with the outer membrane  To allow proteins to fold into their native
flattened cisternae tubular cisternae
conformation in the cisternal space &
of the nuclear envelope
glycosylate them

1. The RER and SER together act as the
membrane factory of the cell by adding
membrane proteins and phospholipids to its own
membrane.
2. Protein channels on the RER surface
 Hold the ribosome in position
 Allow the entry of polypeptides synthesised on
the ribosomes on the surface into the lumen
Golgi Apparatus
‘trans’ face
Golgi vesicles
cisternae
‘cis’ face
Smooth ER (SER)
 A network of membranous tubular sacs
called cisternae
 Lacks ribosomes on outer surface
(Singular: cisterna, Plural:cisternae)
 Membrane-bound flattened sacs called
cisternae & associated Golgi vesicles
 Consists of a ‘forming’ or ‘cis’ face
where new cisternae are being formed
by fusion of transport vesicles from ER
& a ‘maturing’ or ‘trans’ face from
which Golgi vesicles continuously bud
off.
 To synthesise lipids and carbohydrates
 To detoxifiy drugs & poisons (Thus SER
abundant in liver)
 To glycosylate to proteins and lipids to
form glycoproteins and glycolipids
respectively
 To modify existing glycoproteins and
glycolipids by modifying/cleaving the
existing sugar chains
 To sort and package proteins into different
vesicles and target the proteins to different
parts of the cell or for secretion
 To form lysosomes
 To synthesises polysaccharides such as
pectin which is transported in vesicles to
the cell membrane.

Lysosome  Membranous sac containing hydrolytic
enzymes
single membrane [NB: The hydrolytic enzymes work best in the
acidic environment of the lysosme. Thus if a
lysosome bursts, the enzymes are not very
active as the cytosol has a neutral pH.
However, if many lysosomes burst, then the
cell will be destroyed.]
 To digest material engulfed by the cell
(phagocytosis)
 To release enzymes from cells
 To digest unwanted or worn-out
organelles (autophagy)
 To self-destruct a cell after its death
(autolysis)

Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6) 1

The Cell & Molecules of Life (9744) Cell Structure 2018
Organelle Description Function
Mitochondrion  Spherical or rod shaped structures  Acts as the site for certain stages of
DNA ribosomes surrounded by a double membrane aerobic respiration to generate energy in
1. the outer membrane is smooth the form of ATP
2. the inner membrane is highly 1) Inner mitochondrial membrane is highly
convoluted with infoldings called folded & hence increases surface
cristae area for oxidative phosphorylation
 Between the membranes is the 2) Mitochondrial matrix is the site of the
intermembrane space link reaction & the Krebs cycle
matrix
inner membrane  Cristae project into semi-fluid matrix
intermembrane space containing circular DNA, 70S
ribosomes, phosphate granules &
outer membrane
enzymes for aerobic respiration
ATP synthase complex  ATP synthase complex on inner
(appears as a stalked particle) membrane projects into matrix

Chloroplast  Lens-shaped structure surrounded by a  Chloroplasts contain chlorophyll which

double membrane convert solar energy to chemical energy
 Within the chloroplast is an internal through photosynthesis via
outer membrane ribosomes
membrane system which consists of 1) Site of light-dependent reactions
inner membrane (i.e. cyclic & non-cyclic
stroma flattened sacs called thylakoids (a
stack of thylakoids = granum) & photphosphorylation) which occurs in
DNA intergranal lamella the thylakoid membrane
 Fluid within chloroplast surrounding the 2) Site of light-independent reactions (i.e.
grana is called stroma (contains Calvin cycle) which occurs in the stroma
circular DNA, 70S ribosomes,
enzymes & starch grains) [NB: Like bacteria, both chloroplasts &
thylakoids ATP  Chlorophyll molecules are located on mitochondria contain circular DNA, 70S
intergranal synthase the thylakoid membrane. ribosomes. This led to the endosymbiont
lamella granum lipid starch on  ATP synthase complex on thylakoid theory which proposed that the ancestors of the
droplet grain membrane mitochondria and choloplasts were oxygen using
membrane project into stroma
non-photosynthetic prokaryotes and
photosynthetic prokaryotes respectively, that
were engulfed by an ancestral eukaryotic cell.]
Non membrane-bound Organelles
Organelle Description Function
Ribosome  Consists of a small & a large subunit  Act as the site for protein synthesis
 A complex of protein & rRNA
 May be found intact freely floating in
small subunit (40S) the cytosol or bound to ER (or outer
membrane of nuclear envelope as the [NB: Free ribosomes produce proteins that
large subunit (60S) ER is continuous with the nuclear function within the cytosol while bound
envelope) during translation. The small ribosomes synthesise proteins meant for
& large subunit only come together insertion into membranes, packaging within
during translation. organelles or secretion out of cell.]
Centrioles .  A pair of hollow cylinders made up of 9  To act as microtubule organising centre
triplets of microtubules (hollow tubes (MTOC) during spindle formation in cell
triplet of
made of the protein tubulin) each division
microtubules
 The two rod-like cylinders are positioned
at right angles to each other [NB: 1) During cell cycle, the centrioles replicate
 Found in a region called the & move to the opposite poles of the cell.
centrosome which is the microtubule They play a role in nuclear division in
organising centre (MTOC) animal cells by helping to organise the
cross section centrioles at right spindle fibres (which are made up of
angles to each microtubules)
lateral view other 2) In higher plants, centrioles are absent.]
The endomembrane system consists of the nuclear envelope, rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), Golgi
apparatus (GA), lysosomes and various vesicles and the plasma membrane.

DNA is transcribed in the nucleus to mRNAmRNA leaves the nucleus via the nuclear poresmRNA is translated into polypeptides on the ribosomes
of the RERpolypeptides enter the lumen of the cisternae of the RER where it undergoes modificationtransport vesicle buds off from the RER and
carry the proteins to the GA vesicle fuses with the ‘cis’ face of the GA and the proteins undergoes further modification, sorting and packing a
secretory vesicle containing the protein will bud off from the ‘trans’ face of the GA and be transported to and fuse with the cell surface membrane,
releasing the protein content of the vesicle by exocytosis. Microtubules direct the movement of the transport vesicle to the GA and the secretory vesicle
to the cell surface membrane.

Cell Theory states that:

1. Cells are the smallest unit of life. 2. All cells come from pre-existing cells. 3. Living organisms are composed of cells.
Draw plan diagrams of tissues and calculate linear magnifications of drawings
* When making drawings remember, STAMP
S: Size of drawing must occupy 2/3 of given space
T: Title must include : plane of section, name of specimen, name of tissue / cell,magnification e.g. T.S. of epidermal cell of onion (LP)
A: Annotation and Labels e.g. cytoplasm starts to pull away from cell wall (not just cytoplasm)
M: Magnification
P: Proportion and Lines: e.g. Cell wall cannot be too thick when drawing a plant cell. It must be proportional to the rest of the cell.

Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6) 2

The Cell & Molecules of Life (9744)
Cell Membranes
Explain the fluid mosaic model & the roles of constituent biomolecules (including phospholipids, cholesterol, glycolipids, proteins &
glycoproteins) in cell membranes
Plasma membrane or Cell surface membrane
 about 7.5nm thick
 said to have a fluid mosaic model structure
- ‘Fluid’ because the membrane is a dynamic structure where the phospholipids & proteins are able to move
(lipids can move both laterally & transversely (flip flop) while proteins move laterally due to weak interactions between the biomolecules)
- ‘Mosaic’ because of the random arrangement of proteins which are embedded in the phospholipid bilayer
glycoprotein
unilateral
protein (intrinsic
/ integral protein
transmembrane channel protein
(intrinsic / integral protein) has a
hydrophilic pore for movement of peripheral protein
ions and polar molecules
(extrinsic protein)
Components Characteristics of components
Phospholipid - Each phospholipid molecule is amphipathic
bilayer i.e. has 1 hydrophilic, negatively charged
phosphate head & 2 non-polar,
hydrophobic hydrocarbon tails all attached
to a glycerol backbone
- In an aqueous environment, they arrange to
form a phospholipid bilayer where the
phosphate heads interact with the aqueous
medium and the hydrocarbon tails form a
hydrophobic core in the interior of the bilayer
Cholesterol - Found in between phospholipid molecules in
membranes of eukaryotes
- Has a characteristic 4 ring structure
- Slightly amphipathic as it has a
hydrophilic, polar, hydroxyl group & a
hydrophobic 4 ring structure
- The hydroxyl group of cholesterol aligns with
the charged phosphate heads of the
phospholipids while the rest of it is tucked into
the hydrophobic core of the membrane.
Proteins - 3 types : unilateral, transmembrane &
peripheral
- Have domains that are hydrophobic(aas with
non-polar R gps) & hydrophilic (aas with polar
or charged R gps)
Thus are said to be amphipathic.
[NB:
Transport across membranes is vital to a cell,
1) for the intake of nutrients such as glucose
which acts as a respiratory substrate to
provide energy in the form of ATP for the
cellular activities;
2) for the secretion of synthesised products
such as hormones like insulin to the
bloodstream to maintain blood glucose
levels.
3) To generate ionic gradients essential for
ATP synthesis in the mitochondrion during
oxidative phosphorylation]
Glycoproteins Carbohydrate chains associated with
membrane proteins
Glycolipids Carbohydrate chains associated directly with
hydrophobic tails of membrane (& not the
phosphate head)
Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6)
Cell Membranes 2018
extracellular matrix
carbohydrate
chain
glycolipid
(carbohydrate
chain directly
attached to
hydrophobic tails
& not phosphate
head)
cytoplasm
cholesterol
Functions
-As a major component of cell membranes it
1) regulates movement of substances moving in and out of the cell by
acting as a barrier to and ions, polar and large molecules.
(i.e. contributes towards the selective permeability of the membrane)
2) acts as a boundary between the intracellular & extracellular aqueous
environment and
3) allows compartmentalization within a cell
(Note: Phospholipids in a membrane are held together by weak hydrophobic
interactions and van der Waals forces which contribute to the fluidity of the
membrane. The presence of unsaturated hydrocarbon tails with kinks will
increase the fluidity of the membrane.The charge, polarity and size of a
molecule will influence the ability to pass through the cell membrane.)
- Cholesterol regulates membrane fluidity i.e. it stabilises the membrane.
 The membrane is prevented from being overly fluid at warmer
temperatures as cholesterol restricts phospholipid movement through
its interactions with the phospholpids.
 The membrane is prevented from being overly firm at lower temperatures
as cholesterol prevents the close packing of phospholipids and hence
prevents solidification/crystallisation.
- Cholesterol stabilizes the lipid bilayer due to van der Waals interactions
between the rigid fused ring structure and the lipid bilayer
- Function as channels/carriers for facilitated diffusion and active transport
1) Channel Proteins
 have a hydrophilic channel/pore for the direct diffusion of ions or
molecules across the membrane from a high to a low solute
concentration. e.g. aquaporins
2) Carrier Proteins (have 2 alternative conformations)
 bind the solute on one side of the membrane and as a result the protein
undergoes a conformational change that allows access of the solute to
the opposite side of the membrane. e.g. glucose transporter
 some are pumps that usually use ATP to move solutes against a
concentration gradient (from a low solute concentration to a high solute
concentration). e.g. Na+ - K+ pump
The above two types of proteins are necessary for the movement of charged
particles (e.g. H+) and polar molecules (e.g. glucose, water). In contrast, non-
polar molecules can penetrate the hydrophobic core of the bilayer.
- Function as enzymes (e.g. acetylcholinesterase which are found on post-
synaptic membrane to hydrolyse neurotransmitter acetylcholine)
- Function as receptor proteins (e.g. insulin receptor) to which a specific ligand
will bind to. The formation of the ligand-receptor complex will initiate an
intracellular signaling cascade for signal transduction.)
- Function to stabilise membrane structure as they are non-covalently bound
to cytoskeleton (on cytoplasmic side) & extracellular matrix (on extracellular
side)
As the sugar component can be very diverse the carbohydrate chains can
- Function as markers/recognition sites in cell-cell recognition and
adhesion
e.g. allows cells to be attached to one another to form tissues and organs;
- Function as receptors
e.g. for specific chemicals like hormones
1
The Cell & Molecules of Life (9744) Cell Membranes 2018

Outline the functions of membranes at the surface of cells & membranes within the cell

1) Membranes are a selectively permeable barrier which act as a boundary a) between inside and outside of cell, (b) between organelle and
cytoplasm (e.g. Golgi apparatus & cytoplasm) & (c) between compartments within an organelle (e.g. mitochondrial matrix & intermembrane space).

2) Membranes allows for compartmentalisation which allow

(i) unique environments to be formed for highly specialised activities
(e.g acidic environment in lysosomes for hydrolytic enzymes to work)
(ii) spatial separation of biochemical processes & thus their sequential operation within a cell
(e.g. protein modification in RER and further protein modification, sorting and packaging in the GA)
(iii) accumulation of ions to high concentrations
(e.g.accumulation of a high concentration of H+ in the intermembrane space of the mitochondria enable a proton gradient to be established for
chemiosmosis)

3) Membranes act as a surface for chemical reactions to occur in a sequential manner

 membranes may have functionally-related proteins grouped together so that sequential biochemical processes can occur
(e.g. the thylakoid membranes of the chloroplast have electron carriers & ATP synthetase for chemiosmosis to occur.)

4) Membranes increase surface area for chemical reactions

(e.g. inner mitochondrial membrane is highly folded to hold more electron transport chains and ATP synthetase)

5) Membranes surface topography enable communication of cell with surroundings

 the unique combination of proteins/glycoproteins/glycolipids on surface of different cells enable
(a) cell-cell recognition and adhesion so that tissue formation is possible,
(b) ligands to recognize specific receptors so that signal transduction can occur.

Explain how and why different substances move across membranes through simple diffusion, facilitated diffusion, osmosis, active
transport, endocytosis and exocytosis.

Movement
Trpt
Type of ATP across
protein Something to note
transport reqmt conc.
reqmt
gradient
Simple no no down Definition: Net movement of molecules/ions from a region of high concentration to a region of low
Diffusion concentration, down a concentration gradient.
e.g. O2 diffuses from the lungs to the blood

Facilitated no yes down Definition: Net movement of molecules/ions from a region of high concentration to a region of low
diffusion concentration, down a concentration gradient through a transport protein .
Transport proteins facilitate diffusion of substances that are insoluble in phospholipids bilayer
e.g. 1) transmembrane hydrophilic channel proteins (e.g. aquaporins)
2) carrier proteins (e.g. glucose transporters)

Active yes yes up Definition: Energy (ATP) consuming transport of molecules or ions across a membrane through
transport a transmembrane carrier proteins called pumps against a concentration gradient.
e.g. Na+-K+ pump (e.g. in maintenance of polarised state of nerve cells)
Movement of each molecule or ion is in one direction (unlike diffusion which is reversible)

Bulk yes no down/up Buld transport is an active process as it requires ATP. However it is not active transport as it
transport does not transport molecules across a membrane through a transmembrane carrier protein.
2 types:
1)Exocytosis: Secretion of macromolecules (e.g. waste materials) to the exterior of the cell by
fusion of vesicle membrane with the plasma membrane
2)Endocytosis: Infolding or extension of cell surface membrane to form a vesicle or vacuole,
thus allowing cell to aquire macromolecules and particulate matter respectively.
a) Phagocytosis: solids taken into cell via a vacuole (e.g. white blood cells engulf bacteria)
b) Pinocytosis: liquids taken into the cell via vesicles (e.g. human egg cell takes up nutrients
from surrounding follicles)
Note: Receptor mediated endocytosis is a special type of pinocytosis that enables a cell to
acquire large quantities of a specific substance/ligand even though the ligand may not be present
at a very high concentration in the extracellular fluid. The ligands will bind to specific cell surface
receptor on the membrane and the membrane will invaginate and form a vesicle.

Osmosis no no down Definition: Net movement of water from a region of high water potential to a region of low water
potential down a water potential gradient through a selectively permeable membrane.
Note: Water movement can be directly across the membrane via transient pores (simple
diffusion) or through aquaporin channels (facilitated diffusion)

Investigate the effects on plant cells of immersion in solutions of different water potentials. (FOR PRACTICALS)

- Water potential of a solution  tendency for water molecules to leave a solution.

Water potential of a plant cell = Solute potential of cell contents + pressure potential of cell wall
Ψw ψs ψp
The SI unit is Pascal (Pa).
- Water potential of pure water is zero. This is the maximum for water potential.
When we add solute, (1) Ψw becomes –ve (2) concentration of solute increases
When we add more solute, (1) Ψw becomes more –ve (2) concentration of solute increases more

- At incipient plasmolysis, ψ p = 0.  the plasma membrane is about to pull away from the cell wall. Thus, ψ w = ψ s
- Net water movement occurs from a region of high water potential to a region of low water potential down a water potential gradient.

Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6) 2

Genetics & Inheritance (9744)
Describe the events that occur during the mitotic & meiotic cell cycle and the main stages of mitosis and meiosis (including the behaviour
of chromosomes, nuclear envelope, cell membrane and centrioles).
Interphase (before meiosis / mitosis)
1. Organelle synthesis occurs in G1 and G2 phases.
2. Semi-conservative DNA replication occurs in S phase.
Meiosis
Prophase I
1. Chromatin condenses to form chromosomes. Each chromosome
comprises 2 sister chromatids joined at the centromere.
2. Synapsis occurshomologous chromosomes pair up to form
bivalents.
3. Crossing over occurs between non-sister chromatids of
homologous chromosomes, forming chiasmata. Exchange of
corresponding alleles on non-sister chromatids occurs.
4. Centrioles move to opposite poles and spindle fibres start to form.
5. Nucleolus disappears & nuclear envelope disintegrates into vesicles.
Metaphase I
1. Homologous chromosomes align in pairs at the metaphase plate.
i.e. independent assortment occurs (the orientation of 1 pair of
homologues is independent of other pairs)
2. Each chromosome is attached to the kinetochore microtubules
from the pole it faces.
Anaphase I
1. Homologous chromosomes / homologues separate to opposite
poles
2. Each homologue is pulled by a shortening kinetochore
microtubule.
(* No division of centromere here.)
3. Non-kinetochore microtubules elongate & slide in opposite
directions  elongate the cell.
Telophase I
1. Each pole now has a haploid set of chromosomes.
2. Chromosomes decondense to form chromatin.
3. Spindle fibres disintegrate.
4. Nuclear envelope reforms & nucleolus reappears.
Cytokinesis
1. Animal cells: Cell membrane invaginates towards the equator of
the cell, forming a cleavage furrow. The cleavage furrow deepens
and is pinched into 2  2 daughter cells produced
2. Plant cells: Fluid-filled vesicles appear in the middle of the cell and
coalese to form a cell plate, separating the 2 daughter cells
Prophase II
1. Chromatin condenses to form chromosomes. Each chromosome
comprises 2 sister chromatids joined at the centromere.
2. Centrioles duplicate and move to opposite poles
3. Spindle fibres start to form.
4. Nucleolus disappears, nuclear envelope disintegrates.
Metaphase II
1. Chromosomes align at the metaphase plate in 1 row
(Sister chromatids may not be identical due to crossing over in
Prophase I. Orientation of sister chromatids of each chromosome is
independent of other chromosomes.)
2. Each chromosome is attached to kinetochore microtubules from
both poles.
Anaphase II
1. Centromere of each chromosome divides  each (non-identical)
sister chromatid now known as daughter chromosome.
2. Kinetochore microtubules shorten  pulls daughter
chromosomes, centromere first, to opposite poles.
3. Non-kinetochore microtubules elongate & slide in opposite
directions  elongate the cell.
Telophase II
1. Chromosomes decondense to form chromatin.
2. Spindle fibres disintegrate.
3. Nuclear envelope reforms & nucleolus reappears.
Cytokinesis
1. Produces haploid cell with half the number of chromosomes and
half the amount of DNA as compared to the parent cell (before S
phase).
Prepared by: Mrs Selvamani Nair Raffles Institution (Y5-6)
Cell & Nuclear Division 2018
Mitosis
Prophase
1. Chromatin condenses to form chromosomes. Each
chromosome comprises 2 sister chromatids joined
at the centromere.
2. Centrioles move to opposite poles and spindle fibres
start to form.
3. Nucleolus disappears & nuclear envelope
disintegrates into vesicles.
Metaphase
1. Chromosomes align at the metaphase plate.
2. Each chromosome is attached to 2 kinetochore
microtubules (1 from each pole) at the centromere.
Anaphase
1. Centromere of each chromosome divides  each
sister chromatid now known as daughter
chromosome.
2. Kinetochore microtubules shorten  pulls
daughter chromosomes, centromere first, to
opposite poles.
3. Non-kinetochore microtubules elongate & slide in
opposite directions  elongate the cell.
Telophase
1. Chromosomes decondense to form chromatin.
2. Spindle fibres disintegrate.
3. Nuclear envelope reforms & nucleolus reappears.
Cytokinesis
(see cytokinesis after meiosis I)
 Haploid cell: contains one complete set of chromosomes
i.e. it contains half the number of chromosomes as a diploid
cell. It contains one member of each homologous pair of
chromosomes (i.e. one chromosome from either parent)
 Diploid cell: contains two complete sets of chromosomes
i.e. chromosomes exist as homologous pairs. Each set of
chromosomes is from one parent.
 Homologous chromosomes:
Carry the same genes (e.g. hair colour gene) controlling the
same inherited characteristics at the same loci but may not
have the same alleles (e.g. brown and blonde hair colour
alleles)
 one is of maternal origin and one is of paternal origin
similar in size, shape, centromere position and staining
pattern
 Sister chromatids are the result of DNA replication and are
thus, genetically identical (i.e. have the same alleles).
Sister
chromatids
Non-sister
chromatids
Non-sister
chromatids
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Genetics & Inheritance (9744) Cell & Nuclear Division 2018

Significance of the mitotic cell cycle and the need to regulate it tightly
Significance
Production of genetically identical daughter nuclei with same number and type of chromosomes and the same alleles so that genetically
identical daughter cells can be produced for:
1. Growth
 increase number of cells by producing more cells genetically identical to existing ones
2. Regeneration and cell replacement
 damaged cells replaced by cells genetically identical to original ones, retaining the same function.
3. Asexual reproduction
 produces genetically identical offspring

Need for regulation

1. The cell cycle is tightly regulated as it is important for normal growth & development. Regulation is at certain control points known as
checkpoints which are at G1, G2 and M phase. These checkpoints are stop and go-ahead signals which determine whether or not the cell
cycle can proceed.
2. Cancer occurs when the dysregulation of checkpoints of cell division occur or cells escape the cell cycle control mechanism that normally
regulates their growth. This leads to uncontrolled division of cells (i.e. tumour formation) and possibly cancer.

Significance of meiotic cell cycle

1. For maintenance of chromosome number in every generation
Reduction division: production of 4 haploid gametes from 1 diploid parent cell
Chromosome number halved so that the chromosome number can be restored upon fertilisation
 Chromosome number of species remains the same after many generations

2. For genetic variation in offspring in every generation

1. Crossing over between non-sister chromatids of homologous chromosomes results in new combinations of alleles on chromatids.
(& eventually a variety of offspring)
2. Independent assortment of homologous chromosomes at the metaphase plate & their subsequent separation during metaphase I
& anaphase I respectively &
Random orientation of non-identical sister chromatids of each chromosome at the metaphase plate & their subsequent
separation during metaphase II and anaphase II respectively
 results in gametes with different combinations of maternal & paternal chromosomes. (& eventually in a variety of offspring)
3. Random fusion of gamete
 during sexual reproduction/fertilisation results in offspring with a variety of genotypes & possibly phenotypes (& hence a variety of
offspring)

Comparison between mitosis and meiosis

Feature Mitosis Meiosis

Location & cell type Somatic cells in all parts of the body
No of nuclear divisions One (Note: DNA replication occurs only once)
Prophase No synapsis/ homologues do not pair up;
No chiasma formation;
No crossing over of corresponding segments
of non-sister chromatids;
Metaphase
Chromosomes, each consisting of a pair of sister
chromatids, align individually on equator (i.e
form a single row)
Each centromere attaches to spindle fibres
from both poles
Anaphase Separation of centromere;
Separation of identical sister chromatids to
(is the least frequently opposite poles;
observed phase as it is (Note:
the shortest phase.) 1. Once the centromere separates, each sister
chromatid is called a chromosome
2. During anaphase, kinetochore microtubules
shorten while non-kinetochore microtubules
lengthen as they slide pass each other,
causing the cell to elongate.)
Telophase 2 daughter nuclei which are genetically
identical & have the same chromosome
number as parental cells
Result of nuclear 2 genetically identical daughter cells form;
division No variation occurs(in the absence of mutation);
Daughter cells have the same number of
chromosomes as parental cells  hence called
replicative division
NOTE:1.Some cells skip telophase I and cytokinesis, entering prophase II directly after anaphase I. 2.There is no such thing as interphase II.
Prepared by: Mrs Selvamani Nair Raffles Institution (Y5-6)
Precursor sex cells in reproductive organs that give rise to
gametes
Two (Note: DNA replication occurs only once)
Prophase I
Synapsis occurs/homologues pair up to form bivalents(tetrads)
Chiasma formation &
Crossing over of corresponding segments of non-sister
chromatids of homologous chromosomes (results in non-
identical sister chromatids with new combinations of alleles)
Prophase II Similar to prophase of mitosis
Metaphase I
Homologous chromosomes align in pairs at the equator
(ie. form 2 rows)
Centromeres of each chromosome attaches to spindle fibers
from different poles
Metaphase II  similar to metaphase of mitosis except that
chromosomes consist of non-identical sister chromatids
Anaphase I
NO separation of centromere;
Separation of homologous chromosomes
(i.e. pair of sister chromatids move to same pole);
Anaphase II
 similar to anaphase except that non-identical sister
chromatids separate
Telophase I
 2 daughter nuclei which are genetically different & each
has half the chromosome number as parental cells)
Telophase II
 4 daughter nuclei which are genetically different & each has
half the chromosome number as parental cells)
4 genetically different daughter cells form;
Genetic variation has occurred (in the absence of mutation);
Daughter cells with half the chromosome number as parental
cells hence called reductive division
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Genetics & Inheritance (9744) Cell & Nuclear Division 2018

Prepared by: Mrs Selvamani Nair Raffles Institution (Y5-6) 3

Genetics and Inheritance (9744) DNA replication, transcription, translation & mutations
Nucleic acid RNA (mRNA, tRNA & rRNA)
Pentose (five carbon) sugar Ribose
Nitrogenous Purines (2 rings) Adenine & Guanine
bases Pyrimidines (1 ring) Cytosine & Uracil
Complementary base pairing occurs between Adenine & Uracil ( 2 H bonds)
Cytosine & Guanine ( 3 H bonds)
Structure Single Stranded
Found in (location) Cytoplasm, Nucleus
Structure of a nucleotide (a nucleoside + phosphate group = nuceloside monophosphate)
Nitrogenous base: attached to C1
Phosphate group: attached to C5
OH group attached to C3: involved in phosphodiester bond formation
If H is attached to C2  deoxyribose sugar
If OH is attached to C2  ribose sugar
Structure of RNA and DNA
Evidence for semi-conservative replication
(a) A stock of parental E. coli were grown for
many generations in 15N medium as the
only source of nitrogen until 15N was
incorporated into the nitrogenous bases of
all bacterial DNA.
(b) The E.coli containing 15N-15N were then
transferred into a medium containing
only 14N. The transferred E. coli were
allowed to divide once and were then
collected. The DNA extracted and
centrifuged in CsCl were all hybrid (14N-
15
N) DNA. This excluded conservative
replication in which no hybrids form.
(c) Some of these cells were then allowed to
divide once more. The DNA extracted
and centrifuged in CsCl were half hybrid
(14N-15N) DNA and half “light” (14N-14N)
DNA. This excluded dispersive replication
in which no pure 14N-14N can be obtained.
Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6)
2018
DNA
Deoxyribose
Adenine & Guanine
Cytosine & Thymine
Adenine & Thymine ( 2 H bonds)
Cytosine & Guanine ( 3 H bonds)
Double Stranded
Nucleus
In DNA
* A:T = 1:1 and C:G = 1:1
* (A+G) = (C+T)
i.e. no.of purines = no.of pyrimidines
* Purines: 2 rings (baby’s are pure & go googooGAGA)
* Pyrimidines: 1 ring (CUT food on dinner plate)
* Constant width between sugar phosphate
backbone = 2nm
* 2 strands are anti-parallel: one strand runs
in the 5’ to 3’ direction, while the other
strand runs in the 3’ to 5’ direction DNA
is said to have directionality
* 1complete turn of the double helix has 10
base pairs and spans a distance of 3.4nm
* 1 DNA molecule is made up of 2 strands of DNA
3 hypotheses for DNA replication mechanism:
1) Semi-conservative replication
both strands separate by the breaking of
hydrogen bonds and each strand acts as a
template for the synthesis of a new strand
through complementary base pairing.
Thus each DNA molecule formed is a hybrid
consisting of 1 original strand and 1 newly
synthesized strand.
2) Conservative replication
2 parental strands re-associate after acting as
templates, thus restoring the original double helix.
The other DNA molecule consists of 2 newly
synthesized strands.
3) Dispersive replication
Parental DNA molecule is fragmented and
dispersed. Daughter molecules are madeup of a
mixture of old and newly synthesized parts.
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Genetics and Inheritance (9744) DNA replication, transcription, translation & mutations 2018

Gene Mutations
A gene mutation is an alteration in the sequence of nucleotides which may change the sequence of amino acids in a polypeptide chain. This may
change the 3D shape of the protein, affecting the protein function and subsequently affect the characteristics (phenotype) of the organism.
Type of mutation Substitution Inversion Insertion Deletion
Description Replacement of A segment of nucleotides One or several nucleotides One or several nucleotides are removed
one nucleotide separates from the allele are inserted into a from a sequence
by another and rejoins at the original sequence
position but is inverted
Result of mutation 1 codon 1 or more codons Shifts reading frame from Shifts reading frame from point of
changed changed point of mutation mutation
Effect on protein Minor/Major Minor / Major, depending Usually Major Usually Major
on whether a frameshift If the number of nucleotides inserted or deleted are a multiple of
occurs three, there will change the primary sequence but a frame shift will
not result.
1. Frame-shift mutation:
 due to insertion or deletion of a number of nucleotides that is not divisible by 3. Hence due to the triplet code, this would disrupt the
reading frame and produce a different and non-functional polypeptide
2. Silent mutation:
 is a point mutation that does not change the amino acid sequence in a polypeptide
 it can occur in the either coding or non-coding regions
 due to the degeneracy of the genetic code, more than one codon can code for the same amino acid, and hence even if the mutation
occurs in the coding sequence of a gene, the same polypeptide will be synthesized
 if the mutation occurs in the non-coding region, the same polypeptide will be synthesised.
3. Missense mutation
 is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid
 if the new amino acid has similar biochemical properties (e.g. charge, size) to the one that was replaced, the mutation is said to be
conservative
 if the new amino acid has different biochemical properties (e.g. charge, size) to the one that was replaced, the mutation is said to be
non-conservative
4. Nonsense mutation
 is a point mutation which results is a premature stop codon (UAG, UAA, UGA), causing the polypeptide to be truncated and
non-functional
Example of a disease due to a substitution mutation:
Name of disease Sickle-cell anaemia
Protein affected Beta-globin chain of haemoglobin (From HbA to HbS)
Description of change Change in DNA : CTC to CAC (substitution)
Change in mRNA : GAG to GUG
Change in amino acid : glutamate to valine
Effect of the change Charged and hydrophilic glutamate changed to non-polar and hydrophobic valine in HbS.
At low oxygen concentrations, HbS undergoes a conformation change which will cause the hydrophobic patches on
different HbS to stick together. This polymerization of HbS results in the formation of abnormal, rigid, rod-like fibres.
Shape of red blood cell distorted – sickle shaped.
Effects of disease Sickle red blood cells are more fragile and break easily.
This results in shortage of red blood cells and poor oxygen transport. This leads to anaemia, lack of energy and heart
failure.
Sickle red blood cells may also lodge in small blood vessels and interfere with blood circulation. This will lead to organ
damage.
Chromosomal aberrations
 Chromosomal aberrations can be due to variation in (A) chromosomal structure:
1. A deletion removes a chromosomal segment.
2. A duplication repeats a chromosomal segment.
3. An inversion reverses a segment within a chromosome.
4. A translocation moves a segment from one chromosome to another, non-homologous one.
Chromosomal deletions and duplications can result in phenotypic abnormalities due to the reduced or additional genes respectively.
Chromosomal inversions and reciprocal translocations can result in disease although the amount of genetic material remains the same as
the expression of a gene can be influenced by its new location.

 Chromosomal aberrations can also be due to variation in (B) chromosomal number:

1. Aneuploidy is a condition where the cell does not have a chromosome number that is a multiple of the haploid number. Chromosomes are present
in either extra or fewer copies than the wild type.
2. Aneuploidy is a result of a non-disjunction event where:
a) Homologous chromosomes do not move properly to opposite poles during meiosis I OR
b) When sister chromatids fail to separate properly to opposite poles during meiosis II.
So one gamete receives two of the same type of chromosome and another gamete receives no copy. If either of the aberrant gametes fuse with a normal
gamete at fertilisation, the zygote will have an abnormal number of chromosomes i.e. aneuploidy. Mitosis will subsequently transmit the anomaly to all
embryonic cells.

Non-disjunction can also occur during mitosis. If such an error occurs early in embryonic development, then the aneuploid condition is passed on to a
large number of cells where the severity of the effect is more pronounced.

 Down syndrome (Trisomy 21) is result of an extra chromosome 21 (a total of 3 copies), so each body cell has a total of 47 chromosomes.
Most cases result from non-disjunction during meiosis I. Individuals with Down syndrome have characteristic facial features, short stature, heart
defects, susceptibility to respiratory infection and mental retardation. Most individuals are sexually underdeveloped and sterile.
Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6) 2
 Genetics and Inheritance (9744) DNA replication, transcription, translation & mutations 2018

Process Replication Transcription Translation

Location Nucleus (also in mitochondria and Nucleus Cytoplasm
chloroplasts)
Begins at Origin of replication Promoter Start codon (AUG)
(AUG: Are U Good?)
Ends at Where 2 adjacent replication Termination sequence Stop codon (UAG, UAA, UGA)
(UAG: U Are Good
bubbles meet / Telomeres UAA: U Are Awful
UGA:U are Good & Awful)
Template DNA (both strands) DNA (template / non-coding strand) mRNA
Monomers Deoxyribonucleotides Ribonucleotides Amino acids
Complementary base-pairing Adenine & Thymine Adenine & Uracil Complementary pairing between codon
Cytosine & Guanine Thymine & Adenine and anti-codon
Cytosine & Guanine
Guanine & Cytosine
Enzymes Involved DNA polymerase, Helicase, RNA polymerase Aminoacyl – tRNA synthetase
Primase, DNA Ligase, (Poly A polymerase & endonuclease in Peptidyl transferase (a ribozyme)
eukaryotes)
Topoisomerase
Bonds within molecule Phosphodiester bonds, Hydrogen Phosphodiester bonds Peptide bonds
formed bonds
Ribosomes involvement No No Yes
Template strand is read in 3’ to 5’ direction 3’ to 5’ direction 5’ to 3’ direction
Molecule is synthesized in 5’ to 3’ direction 5’ to 3’ direction from the amino end to the carboxyl end
Proof reading Yes - -
Product (s) 2 DNA molecules mRNA,tRNA rRNA,snRNA etc. Polypeptide chain
Product destination Nucleus Cytoplasm Cytoplasm/ Cell membrane/Outside cell
Role of DNA
The main role of DNA is to store information and pass it on from one generation to the next.
It is a suitable store of information as:
a) It can be replicated accurately  daughter cells have identical copies of DNA as the parent cell
 Weak hydrogen bonding between the two strands allow them to separate and act as a template for new strand synthesis
(Adenine forms 2 hydrogen bonds with thymine and cytosine forms 3 hydrogen bonds with guanine through complementary base pairing)

b) It is a stable molecule  can be passed on to the next generation without loss of the coded information
Collectively, numerous hydrogen bonds hold the two strands of DNA together and adjacent nucleotides in each strand are joined by strong covalent
phosphodiester bonds

c)There is a backup of code

DNA is double stranded and one strand to serve as a template for the repair of the other if a mutation occurs on either one.

a) d) Coded information can be readily utilised/accessed

 Weak hydrogen bonding allows the template strand to separate from the non-template strand allowing transcription to take place
 Complementary base pairing allows the faithful transfer of info from DNA to RNA in transcription, which will be translated to protein subsequently

Role of mRNA:
1) Messenger RNA (mRNA) serves as a ‘messenger’ that, in eukaryotes, takes the information out of the nucleus via the nuclear pore to the
cytoplasm where translation takes place.
2) mRNA acts as a template for translation
3) As each codon within the coding region of the mRNA represents an amino acid in a polypeptide, the sequence of codons will determine the
polypeptide sequence.

Role of tRNA:
They bring in specific amino acids in a sequence corresponding to the sequence of codon in mRNA to the growing polypeptide.
It can facilitate translation due to:
1) its ability to bind to a specific single amino acid
2) the ability of the anticodon to base-pair with the mRNA codon

Role of rRNA:
1) rRNA associates with a set of proteins to form ribosomes.
2) rRNA is the main constituent of the interface between the large and small subunits of the ribosome
Thus the small ribosomal subunit can bind to the mRNA as complementary base pairing can occur between the rRNA in the mRNA binding site
of the small ribosomal subunit and the mRNA.
3) rRNA is the main constituent of the P site (peptidyl-tRNA binding site) and A site (amino-acyl tRNA binding site) on the large ribosomal subunit
Hence rRNA enables the binding of aminoacyl-tRNAs to the P site and A site
4) An rRNA molecule (peptidyl transferase) on the large ribosomal subunit also catalyses the formation of the peptide bond between the amino
group of the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site.

Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6) 3

Genetics and Inheritance (9744) DNA & Genomics (DNA replication) 2018
DNA REPLICATION
origin of replication (ori)
1. Before DNA replication, free deoxyribonucleoside
triphosphates are manufactured in the cytoplasm and 3
transported into the nucleoplasm via nuclear pores.
2. DNA replication occurs at S phase of interphase. 4&5
helicase single strand binding proteins
UNZIPPING OF PARENTAL STRAND
replication bubble
3. Replication begins at specific points of the DNA molecule 3’
each of which is known as an origin of replication (ori ). 5’ 5’
3’
4. Helicase binds to origin of replication. It disrupts 7&8 5’ RNA primer
hydrogen bonds between complementary base pairs, 3’ 3’
causing parental strands to unzip and separate. 3’ end
RNA primers are added by primase 5’ end 5’ of RNA
5. Single-strand binding proteins keep the strands apart so of RNA
that they can serve as templates for the synthesis of new primer
primer
strands.
6. Topoisomerase relieves “overwinding” strain ahead of DNA polymerase
replication forks by breaking, swiveling and rejoining DNA
strands.
9
ADDITION OF PRIMER

7. RNA primer is added to each template (parental) strand by

the enzyme primase. 10
23 - 13
8. RNA primer provides a free 3’ OH end for DNA
deoxyribonucleoside
polymerase to recognise and start DNA synthesis of the
triphosphate/ DNA
complementary daughter strand.
nucleotide
9. DNA polymerase can only add deoxyribonucleotides
(DNA nucleotides) to a pre-existing 3’OH end of a complementary base pairing
nucleotide.
parental
SYNTHESIS OF DAUGHTER STRANDS strand

10. DNA polymerase uses the parental strand as a template

and aligns the free activated deoxyribonucleoside DNA polymerase
pyrophosphate, PPi
triphosphates (dNTPs) in a sequence complementary to
that of the parental strand. phosphodiester bond forms
11. Adenine base pairs with Thymine and vice versa.
Guanine base pairs with Cytosine and vice versa.
12. DNA polymerase catalyses the formation of
phosphodiester bonds between adjacent daughter DNA
nucleotides of the newly synthesised strand.
13. Removal of the pyrophosphate (PPi) from the parental
deoxyribonucleoside triphosphate (dNTP) and the strand
subsequent hydrolysis of PPi provides the energy to drive
the polymerization reaction. 3’
14. As DNA polymerase moves along the template, it proof
reads the previous region for proper base pairing. Any 5’
leading strand
incorrect deoxyribonucleotide is removed and replaced
by the correct one.
15. The leading strand is synthesized continuously in the 5’ 5’
to 3’ direction. 16 & 17 5’ Okazaki fragments
16. The lagging strand is synthesized discontinuously. 3’
3’
Its synthesis is similar to leading strand, except that the Together the Okazaki 5’
lagging strand is synthesised in fragments known as fragments form the 3’
Okazaki fragments. Each fragment is initiated by an RNA lagging strand
primer before the addition of DNA nucleotides. 5’
17. A different DNA polymerase then removes the RNA
primer and replaces it with deoxyribonucleotides.
18. DNA ligase seals the nicks by forming phosphodiester
bonds between adjacent nucleotides of the each of the
DNA fragments on the new strand. a different DNA polymerase
replaces the RNA nucleotides with
END OF REPLICATION DNA nucleotides
17 & 18
19. Complementary parental and daughter strands rewind into
a double helix.
20. Each resultant helix consists of one parental strand and
DNA ligase seals the nicks by forming a
one daughter strand. Hence this is called semi-
phosphodiester bond between adjacent
conservative DNA replication. nick nucleotides of the 2 DNA fragments of
the new strand

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 Genetics and Inheritance (9744) DNA & Genomics (Transcription) 2018
TRANSCRIPTION. :the synthesis of RNA using a DNA template Promoter Transcription Unit Termination sequence
3 main components of a gene
(occurs in nucleus)
IN EUKARYOTES IN PROKARYOTES
INITIATION INITIATION
general transcription
1. RNA polymerase is a multimeric complex. factors
1. RNA polymerase is a multimeric (made up of many subunits) complex.
2. General transcription factors first assemble along 2. Sigma factor associates with core RNA polymerase forming the RNA
the promoter. polymerase holoenzyme
3. General transcription factors recruit RNA polymerase 3. As the holoenzyme scans along the DNA, its sigma factor recognizes
& position it correctly on the promoter. and binds to the promoter.
promoter
4. The complex of RNA polymerase and transcription 4. RNA polymerase
factors is called the transcription initiation complex. unzips the 2 strands of the
5. RNA polymerase unzips the 2 strands DNA double helix at
of the DNA double helix at promoter by breaking promoter by breaking
hydrogen bonds between base pairs. hydrogen bonds between
6. Only one strand is used as the template to synthesise the base pairs.
mRNA. 5. Only one strand is used as
(N.B. The template strand is also the non-coding strand) a template to synthesise mRNA.

IN EUKARYOTES AND PROKARYOTES

ELONGATION
1. Free ribonucleotides match up with the template DNA strand by complementary base pairing.
2. Cytosine base pairs with guanine, guanine base pairs with cytosine, thymine base pairs with adenine & adenine base pairs with uracil. mRNA
3. RNA polymerase catalyses the formation of covalent phosphodiester bonds between adjacent ribonucleotides. transcript
4. As each ribonucleoside triphosphate is brought in, 2 of its terminal phosphate groups are removed and the 5’ end of the resulting
ribonucleotide is added to the 3’ end of the growing RNA strand via the formation of a phosphodiester bond.
5. Thus the mRNA strand is synthesized & elongated in the 5’ to 3’ direction & the template DNA strand is read in the 3’ to 5’direction.
6. As the transcription complex continues to move down the DNA double helix, unzipping the 2 strands, and synthesizing mRNA, the
region of DNA that has just been transcribed, reanneals.
mRNA transcript

IN EUKARYOTES IN PROKARYOTES
TERMINATION TERMINATION
1. RNA polymerase transcribes a sequence on the DNA, which codes for a polyadenylation signal (AAUAAA) in hairpin loop
1. After transcribing through a
the pre-mRNA. termination sequence (in DNA), RNA
mRNA
2. Proteins (endonucleases) bind at a point (10 to 35 nucleotides) downstream of the polyadenylation signal to cut the transcribed terminator
and free the pre-mRNA from the polymerase. (an mRNA sequence) folds back
polyadenylation signal free pre-mRNA to form a hairpin loop. DNA
CUT 2. The loop acts as a termination signal
Pre-mRNA 5’________________AAUAAA_____________3’  5’________________AAUAAA__3’ that causes the mRNA and RNA
polymerase to be released.
POST-TRANSCRIPTIONAL MODIFICATION (Only in EUKARYOTES)
1. Addition of methylguanosine cap to 5’ end of pre-mRNA
 the 5’ cap protects mRNA from degradation by ribonucleases that degrade RNA form the 5’end, serves as
a recognition signal for the small ribosomal subunit to assemble & begin translation & facilitates the export of mature
mRNA form the nucleus) 5’ cap added, introns excised &
2. RNA splicing (which requires ATP) by spliceosomes which excise introns(non-coding seq.) & join exons(coding seq). exons joined, poly A tail added
3. Synthesis of poly A tail (polyadenylation) by the enzyme poly A polymerase which adds adenine nucleotides downstream
of polyadenylation sequence, AAUAAA.
protects mRNA from degradation by ribonucleases, making it a more stable template for translation and directs the export
of mRNA through nuclear pores into the cytoplasm.

Prepared by: Mrs Selvamani Nair Raffles Institution (Yr 5-6) 5

 Genetics & Inheritance (9744) DNA & Genomics (Translation) 2018
TRANSLATION.:the synthesis of a polpeptide using genetic information encoded in an mRNA molecule.
Amino acid activation: amino acid + tRNA aminoacyl-tRNA synthetase, aminoacyl tRNA Amino acid activation is NOT part of translation
ATP
but essential for translation to occur.
Each amino acid is covalently attached to the 3’ CCA stem of a specific tRNA by a specific aminoacyl-tRNA synthetase.
There are 20 different aminoacyl tRNA synthetases. Each enzyme has an active site which recognizes a specific amino acid & the unique identity sites at the 3’CCA stem & the anticodon loop of a
specific tRNA.
Thus, 20 different aminoacyl tRNAs can be formed.
INITIATION
(Pts 1-5 applicable to both EUKARYOTES & PROKARYOTES) ( is required)

1. Initiation factors facilitate the binding of the small ribosomal subunit to both mRNA and initiator tRNA.
IN EUKARYOTES IN PROKARYOTES

 Initiation factors and initiator tRNA (carrying methionine) bind to small ribosomal  Initiation factors bind to the small ribosomal
subunit. subunit and facilitate the binding of the small
 Small ribosomal subunit then recognizes & binds to the 5’ 7 methylguanosine cap ribosomal subunit to Shine-Dalgarno
of the mRNA & moves in the 5’ to 3’ direction along the mRNA in search of the sequence so that the start codon can be
start codon, AUG. correctly positioned before the initator tRNA
 The initiator tRNA associates with the start codon by complementary base pairing and large ribosomal subunit bind.
2. The large ribosomal subunit binds, completing the ribosome, resulting in the formation of the translation initiation complex.
3. The initiator tRNA will be positioned at the P site (peptidyl-tRNA binding site).
4. The A site (aminoacyl-tRNA binding site) will be vacant for the addition of the next aminoacyl tRNA molecule.
5. GTP is required for the initiation stage.
ELONGATION
(in both EUKARYOTES & PROKARYOTES) ( is required)
1. Codon recognition
3. Translocation  Anticodon of incoming aminoacyl tRNA
 Ribosome shifts one codon down mRNA in 5’ to 3’ direction. complementary base pairs with mRNA codon in
A site by forming hydrogen bonds.
 The tRNA from the P site is shifted to the E site (exit site)
and released into cytosol.

 The peptidyl-tRNA with growing polypeptide is translocated 2. Peptide bond formation

from A site to P site.  Peptidyl transferase in large ribosomal subunit
catalyses peptide bond formation between amino
 Empty A site is ready to receive the next incoming aminoacyl acid carried by tRNA in A site and methionine/ amino
tRNA, with anticodon complementary to mRNA codon acid at carboxyl end of growing polypeptide chain
exposed at A site. carried by tRNA in the P site.

 The process is repeated until a stop codon is reached.  The methionine/amino acid dissociates from the
(initiator) tRNA it was bound to.

TERMINATION
(in both EUKARYOTES & PROKARYOTES) ( is required)

1. When the stop codon (UAG, UAA, UGA) reaches the A site, release factors enter the A site.
2. Binding of the release factors causes the hydrolysis of the bond between the polypeptide chain & the tRNA at the P site.
3. The polypeptide is released from the ribosome as it completes its folding into it secondary & tertiary structure.
4. The ribosome disassembles into its subunits.
 In eukaryotes, transcription takes place in the nucleus and the pre-mRNA undergoes post-transcriptional modification within nucleus before being transported to the cytoplasm for translation.
In prokaryotes, mRNA can be translated while it is being transcribed.

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Genetics and Inheritance (9744) Viruses 2018
 Are viruses are considered living or non-living? Living as they contain genetic material. However, non-living because they have no cellular organization and only show characteristics of living
things when in host cell.
Characteristics of living things include1) metabolic activity 2) cellular organization 3) ability to reproduce and grow in numbers 4) ability to respond to stimuli and adapt to environment
 Why are viruses obligate parasites? This is because viruses, like obligate parasites, depend on host cells to complete their life cycle.

Structure of Viruses
Bacteriophages Animal Viruses
Size: 10-300nm Enveloped
T4 Lambda Influenza Human Immunodeficiency Virus
phage phage (HIV)
Genome  Double-  (-) strand RNA  (+) strand RNA
 Nucleic acid that codes for synthesis of viral components and stranded DNA viral genome is complementary to viral mRNA viral genome has the same
enzymes for viral replication & assembly  8 different segments of single stranded RNA associated with sequence as viral mRNA
 Can be either DNA/RNA, single/double-stranded nucleoproteins  2 identical copies of single
 Each RNA segment is packed with 3 polymerase proteins stranded RNA bound to
which come together to form an RNA-dependent RNA nucleocapsid proteins
polymerase enzyme complex which replicates and transcribes
the viral genome in the host cell
Capsid  Icosahedral  Present.  Present, conical shaped
 Protein coat that surrounds and protects viral genome capsid head Enzymes reverse transcriptase,
 Comprise subunits called capsomeres integrase and protease found in
capsid
Envelope  Absent  Glycoproteins embedded in envelope: haemagglutinin (80%) &  Glycoprotein embedded in
 Phospholipid bilayer surrounding the nucleocapsid neuraminidase (20%) envelope: gp41
 Derived from host cell membrane gp120 is attached to gp41
 Embedded with viral glycoproteins involved in host cell recognition
Icosahedral capsid head
containing double 2 copies of single
stranded DNA genome Reverse transcriptase stranded RNA genome,
Haemagglutinin
Envelope each associated with
Neuraminidase nucleocapsid proteins

Collar
Tail surrounded
by contractile Capsid
8 RNA segments,
sheath
each associated with
1) an RNA dependent Matrix proteins
Base plate RNA polymerase Integrase
2) nucleoproteins

Protease
Tail fibre Capsid Envelope
Tail pin
gp41
gp120
T4 phage Lambda phage Influenza Virus Human Immunodeficiency Virus
Antigenic Drift and Antigenic Shift (HIV)
Antigenic Drift : When the influenza virus replicates in its host cell, mutations frequently occur due to the poor proofreading mechanism of the viral RNA-dependent RNA polymerase and the fast
replication rate of the virus. Over time, there is an accumutation of mutations in the viral genome. Sometimes, these mutations produce viruses with modified** surface antigens (e.g.
glycoproteins such as haemagglutinin or neuraminidase) with different conformation. If these viruses infect a host that does not have the antibodies that recognise these modified surface antigens, the
host becomes susceptibleto the virus.

Antigenic Shift: When a bird strain of influenza A and human strain of influenza A infect a single cell of an intermediate host (e.g.a pig), genetic reassortment can occur. Thus when new viruses
are assembled in the host cell, a new combinations of RNA segments can come together. Sometimes, genetic reassortment produces viruses with new** surface antigens (e.g. glycoproteins such as
haemagglutinin or neuraminidase). If these viruses infect a human host the host becomes susceptibleto the virus, as the host will not have the antibodies that recognise these new** surface antigens,

Prepared by: Mrs Selvamani Nair, Mdm Sharon Cross and Mrs Wong Seok Hui Raffles Institution 1
Genetics and Inheritance (9744) Viruses 2018
Virus Life Cycle
Stages Bacteriophage Enveloped animal viruses
T4 phage Lambda phage Influenza HIV
(Lytic phage) (Temperate phage)
1. Attachment  Attachment sites on tail fibres adsorbs to complementary Enveloped viruses use viral glycoproteins to bind to specific receptor molecules on host cell.
Virus receptor sites on bacterial surface (e.g. E.coli)  Hemagglutinin binds to complementary  gp120 binds to complementary CD4 receptors on T
recognises and sialic acid receptor on host cell (e.g. helper cells or (macrophages) with the help of a co-
attaches to epithelial cells in respiratory tract) receptor.
host cell membrane
2. Penetration  Bacteriophage releases lysozyme which digests bacterial Release of capsid into host cell cytosol
Viral genome cell wall  Virus enters host cell by endocytosis (the  With the help of gp41, the viral envelope fuses with
introduced  This allows the release of molecules from the bacterium process involves invagination of membrane) host cell membrane  nucleocapsid is released into
into host cell which triggers a change in shape of the proteins in the base  Endocytic vesicle fuses with lysosome  cytosol
plate which causes the contraction of tail sheath which will which lowers the pH  causes viral envelope
drive the hollow core tube through cell wall to fuse with lipid bilayer of vesicle 
 When the tip of the hollow core tube reaches the plasma nucleocapsid is released into cytosol
membrane, phage DNA is injected into the bacterial cell (NB: HIV can also enter by endocytosis)
 The empty capsid remains outside Degradation of capsid to release viral genome (uncoating)
Capsid degraded by cellular enzymes and the Capsid degraded by cellular enzymes the 2 viral RNA
8 viral RNA segments that are released into strands and enzymes are released into the cytosol
cytosol enter the nucleus
3. Replication  Host cell  Linear phage DNA circularizes  Viral RNA-dependent RNA polymerase uses  Reverse transcriptase makes DNA strand using viral
Synthesis of macromolecular and inserted into host cell viral genome as a template to synthesise RNA as template to form a DNA-RNA hybrid. The RNA is
viral synthesizing genome by enzyme integrase mRNA then degraded and the 2nd DNA strand is made 
components & machinery is used  The integrated phage DNA is  mRNA double-stranded DNA molecule produced
viral genome to synthesise phage known as a prophage 1. enters cytosol translated into viral  Viral DNA enters nucleus  inserted into host cell
replication proteins  Expression of phage genes is structural components (Capsid proteins are genome by integrase  Viral DNA known as provirus
 Early phage repressed by phage repressor made in the cytosol. Envelope glycoproteins can remain latent for a long time
proteins: degrade proteins. Hence new phages are made in the RER & eventually are  Upon activation, viral DNA transcribed to viral RNA
host DNA are not synthesized embedded in host cell membrane) which enters cytosol
 Phage DNA  Prophage replicates along with 2. can also act as template for synthesis of new  Viral RNA can either act as mRNA and be translated into
synthesized using bacterial chromosome viral RNA genome in the nucleus. Viral RNA proteins or become part of the genome of the new virions
host cell nucleotides  During spontaneous induction, genome then exits nucleus.  mRNA
and early proteins cellular proteases are activated. 1. is translated to viral polyproteins
 Late phage They destroy the repressor 2. is translated into envelope glycoproteins gp120 and gp
proteins: are phage proteins 41 in the RER and eventually are embedded in the host
enzymes and  The prophage is then excised cell surface membrane.
structural from the bacterial genome
components  The replication phase of lytic
cycle then occurs.(see left)
4.Maturation  Phage DNA and capsid assemble into a DNA-filled head  Capsid proteins associate with host cell For HIV, maturation is completed only after release
Assembly of  Head, tail and tail fibers assembled independently & join in a membrane where viral glycoproteins are of virus.
complete specific sequence. inserted.  The viral RNA genome and polyprotein assembles at
viruses  Nucleoproteins associate with the RNA the cell surface membrane where viral glycoproteins
genome and then interact with capsid proteins have been inserted.
that have associated with the glycoproteins
embedded on the plasma membrane.
 This initiates the budding process.
5. Release  Phage lysozyme synthesised within the cell breaks down  Newly formed viruses bud off by evagination,  Newly formed viruses bud off by evagination, acquiring
the bacterial cell wall acquiring host cell membrane with embedded host cell membrane with embedded viral glycoproteins
 Bacterial cell membrane lyses and release the newly formed viral glycoproteins  Viral protease cleaves polyproteins, forming viral
virions  Neuraminidase facilitates the release of the enzymes and proteins.
new virions from the host cell membrane by  The viral RNA genome and enzymes are then
cleaving sialic acid from the host cell receptor. encapsulated by a protein coat to form a capsid
 The mature HIV virus (virion) is now able to infect
neighbouring cells.

Prepared by: Mrs Selvamani Nair, Mdm Sharon Cross and Mrs Wong Seok Hui Raffles Institution 2
Genetics and Inheritance (9744) Viruses 2018
Influenza Life Cycle HIV Life Cycle
(1) Hemagglutinin (5) Newly formed viruses bud off by evagination, (2a) With the help of
(2a) Virus enters host (1) gp120 binds to
recognizes & binds to acquiring host cell membrane with embedded gp41, the viral
cell by endocytosis CD4 receptors on T
sialic acid receptor on viral glycoproteins. Neuraminidase facilitates (1) envelope fuses
(which involves lymphocytes (or
host membrane the release of the new virions from the host cell with host cell
macrophages) with the
invagination of membrane. Host cell may or may not be lysed. membrane 
help of a co-receptor. (2a & b)
(1) membrane) nucleocapsid is
(5) (3a) Reverse transcriptase released into cytosol
makes DNA strand using viral
RNA as template  RNA (2b) Capsid
(3a) Viral RNA
degraded  2nd DNA strand degraded  viral
(4) Nucleoproteins Reverse RNA and
(2b) Endocytic associate with the RNA made  double-stranded RNA-DNA
transcriptase enzymes
vesicle fuses with (4) DNA molecule (ds DNA) hybrid
genome and then transcribes RNA released into
lysosome  which (2a) produced
into DNA
interact with capsid cytosol
lowers the pH  ds DNA
causes viral envelope proteins that have (3b) Viral DNA enters
to fuse with lipid associated with the nucleus  inserted into host (5) Newly formed
bilayer of vesicle  glycoproteins cell genome by integrase  viruses bud off
Translation
nucleocapsid is embedded on the Viral DNA known as provirus (3b) by evagination,
integrase integrates acquiring host
released into cytosol plasma membrane. This can remain latent for a long DNA into genome cell membrane
(2b) Lysosome initiates the budding time
with embedded
process. (3c) Upon activation, viral viral
DNA is transcribed to viral glycoproteins.
(3bii) RNA which enters cytosol Viral protease
(2c) (3bi) Viral
Viral RNA can either act as cleaves
mRNA RNA (3c) activation
mRNA and be translated into polyproteins,
(2c) Capsid degraded by proteins or become part of the Viral RNA forming viral
cellular enzymes and viral (3a) (3bi) mRNA can used genome of the new virions. enzymes and
RNA enters nucleus as a template for proteins.
synthesis of new viral (3d) mRNA is translated to (3d) The viral RNA
(3a) Viral genome used as a 1. viral polyproteins Viral RNA
Note: RNA genome in the translation genome and
template for mRNA synthesis by 2. envelope glycoproteins enzymes are
Only 2 out of the 8 RNA nucleus
RNA-dependent RNA gp120 and gp 41 in polyproteins Viral genome then
segments are shown
polymerase. the RER which protease encapsulated by
due to space
(3bii)mRNA then enters cytosol eventually are embedded proteins a protein coat to
constraints in the
 translated into viral structural in the host cell surface (4 & 5) form a capsid.
diagram)
components (Capsid proteins are membrane. The mature HIV
made in the cytosol. Envelope virus (virion) is
(4) Viral RNA genome and
glycoproteins are made in the now able to
polyprotein assemble at the cell
RER & are eventually embedded infect
surface membrane where viral
in host cell membrane) neighbouring
glycoproteins have been inserted.
cells.

Pathogenecity of Influenza Pathogenecity of HIV

When influenza will bind to sialic acid receptors on epithelial cells of respiratory tract When HIV binds to CD4 receptor on a T helper cell, a type of T lymphocyte
 
Influenza replicates within it and then buds off. Infected epithelial cells eventually lyse HIV replicates within it and then buds off. Infected T helper cells eventually lyse.
 
The build up of dead epithelial cells results in inflammation and symptoms of influenza appear With fewer T helper cells, the immune system is depressed & individuals are more susceptible to
runny nose & scratchy throat opportunistic infections. When infections become unmanageableAIDS  death

The epithelial layer weakens and the individual is more susceptible to bacterial infections like Virus able to avoid detection by immune system as it mutates at a high rate during replication
pneumonia surface proteins altered prevent recognition & elimination by immune system
 Treatment:1) antibiotics for bacterial infections Treatment: drug cocktail that targets (1) enzymes(RIP) i.e. enzyme inhibitors
2) antiviral drugs which target viral enzymes i.e enzyme inhibitors (2) glycoproteins (gp120) i.e. entry inhibitors
e.g:Tamiflu for some strains of influenza

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Genetics and Inheritance (9744) Viruses 2018
Life Cycle of Lytic Phage (e.g. T4)

Life Cycle of Temperate Phage (e.g. Lambda)

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The Cell & Biomolecules of Life | Genetics & Inheritance (9744) Bacteria 2018
Structure of Bacterial Cell

Chromosome: > circular DNA which contains essential genes for survival

Fimbria: > for attachment to

surfaces
70S ribosome:
> site of protein synthesis

Storage granule

Flagellum: > for motility

Capsule (distinct layer) / Slime layer (diffused):
> protects against phagocytosis

Peptidoglycan cell wall: > protects the cell from osmotic lysis
Cytoplasm
Plasmid:
Plasma Membrane:
> extrachromosomal circular DNA
> Phospholipid bilayer with the electron transport chains and ATP synthase are
> replicate autonomously
embedded to produce ATP
> genes may confer advantages
e.g. antibiotic resistance

Binary Fission – asexual reproduction produces genetically identical/clonal bacteria

1. DNA replication begins at the origin of replication (ori) where DNA is unzipped by breaking hydrogen bonds between bases of the 2 strands to form a
replication bubble
2. DNA replicates by semi-conservative replication where each original strand serves as template for synthesis of daughter strands by complementary
base pairing
3. 2 newly formed ori move to opposite poles of the cell and attach to the plasma membrane
4. Cell elongates to prepare for division.
5. DNA is circular with no free ends, and the 2 daughter DNA molecules will be interlocked with the completion of replication.
6. Enzyme topoisomerase cut, separate and reseal the two DNA molecules
7. Invagination of the plasma membrane and the deposition of new cell wall (division septum) eventually divide the parent cell into two daughter cells
 each inherits a complete genome (genetically identical)

Compare Binary Fission & Mitosis

 Produces genetically identical offspring: selective advantage in a stable, favourable environment as it allows successful genotypes to rapidly colonise a
habitat

Point of Binary Fission Mitosis

Comparison

End Product 2 genetically identical cells 2 genetically identical cells

Amount of DNA Same in each daughter cell as compared to parent cell Daughter cells have the same amount of DNA as parent cells
DNA Replication DNA replication occurs during binary fission DNA replication occurs during S phase of interphase before
mitosis
Behaviour of Attachment of chromosomes to plasma membrane No attachment of chromosomes to plasma membrane
Chromosomes Formation of entangled rings made up of 2 DNA DNA molecules are not entangled
molecules to be separated
No chromosome condensation into sister chromatids Chromosome condensation into sister chromatids
No specific positioning of chromosomes in the cell that Specific positioning of chromosomes in the cell characterize
characterizes the different stages the different stages
Spindle Fibres No spindle fibres involved Spindle fibres involved

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The Cell & Biomolecules of Life | Genetics & Inheritance (9744) Bacteria 2018
Genetic variation in bacteria arise from lateral gene transfer: Transformation, Conjugation & Transduction
 resulting in change of the bacterial cell’s genotype and phenotype

Transformation
Definition:
Transformation is the uptake of naked, foreign DNA from the surrounding environment, resulting in a change of the bacterial cell’s genotype and phenotype
Process:
1. Fragments of foreign naked DNA from dead lysed bacterial cells
2. Naturally competent bacteria with cell-surface proteins bind and transport DNA into the cell.
3. Artificially bacteria can be made competent through immersion in a medium with CaCl2 followed by a heat shock treatment
4. Foreign DNA incorporated into chromosome through crossing over at 2 homologous regions found on the bacterial chromosome
5. Result: recombinant cell
6. If different alleles for a gene were exchanged, the new allele will be expressed  permanent change in genotype & phenotype
7. Recombinant genome will be passed on to all subsequent offspring through binary fission
Conjugation
Definition:
Direct transfer of genetic material from one bacterial cell to another through a mating bridge between the two cells via the transfer of F plasmid from an F+
donor to F– recipient cell
Process:
1. Sex pilus (coded for by F factor) of F+ bacterial cell makes contact with a F- cell and retracts to bring the 2 cells closer
2. The hollow pilus then acts as a cytoplasmic mating bridge between the 2 cells
3. One of the 2 strands of the plasmid DNA is nicked and transferred from the F+ cell to the F- cell through the bridge
4. The single stranded F plasmid DNA circularizes in F - cell and is used as a template to synthesize a complementary strand for a double-stranded
plasmid DNA. The F- recipient cell is now a F+ cell
5. Replication of the plasmid occurs via rolling circle DNA replication occurs
a) One strand of ds F plasmid is nicked by a nuclease  free 3’OH end is then used as a primer for strand elongation by DNA polymerase using the
unnicked/intact strand as a template  elongation process is facilitated by the displacement of the 5’ end of the nicked strand and is transferred across
the mating bridge to the recipient bacterium  Upon completion of a unit length of the plasmid DNA (after 1 round), another nick occurs to release the
original strand
b) In the recipient cell, the single strand of F plasmid DNA re-circularises and serves as a template for the synthesis of a complementary daughter
stand to form a double stranded circular DNA.

Transduction
Definition:
Transduction is the process by which bacterial DNA from one host cell is introduced into another bacterial host cell by a bacteriophage due to aberrations in
the phage reproductive cycle
Generalised Transduction Specialised Transduction
1. A phage infects a bacterium, injecting its viral genome(DNA) 1. Temperate phage infects a bacterium, injecting its viral genome into the host
into the host cell cell
2. The bacterial DNA is degraded into small fragments, one of 2. The viral DNA is integrated into bacterial chromosome forming a prophage
which may be randomly packaged into a capsid head during 3. which may be improperly excised to include adjacent segment of bacterial
the spontaneous assembly of new viruses DNA and not the entire phage DNA during an induction event
3. Upon cell lysis, the defective phage will infect another 4. Hence phage-bacterium hybrid DNA may be packaged into a capsid head during
bacterium and inject bacterial DNA from the previous host cell the spontaneous assembly of new viruses
into the new bacterium 5. Upon cell lysis, the defective phage will infect another bacterium and inject
4. Foreign bacterial DNA can replace the homologous region in the bacterial DNA from the previous host cell into the new bacterium
recipient cell’s chromosome through homologous 6. New alleles from the previous bacterial cell can be incorporated into the genome
recombination, allowing the expression of a different allele of the new host by homologous recombination or integration of phage-
from the previous host bacterium hybrid DNA as defective phage enters the lysogenic cycle

Compare the similarities and differences between the mechanisms of transformation, generalized, specialised transduction and conjugation
Point of Comparison Transformation Generalised Transduction Specialised Transduction Conjugation
Type of donor cell / source Broken down DNA Bacteria cell infected by virulent Bacteria cell infected by F+ cell containing F plasmid
of DNA from lysed bacterial phage virulent template phage
cells

Agent mediating DNA Cell surface proteins Bacteriophage e.g. T4 phage Template bacteriophage e.g F factor on F plasmid, which
transfer or CaCl2 artificially, lambda phage codes for proteins involved in
which make cells formation of sex pili and
competent cytoplasmic mating bridge

Type of DNA transferred to
recipient cell
Homologous
recombination needed for
permanent expression of
foreign genes? (Yes/No)
Random fragments Random fragments of the DNA transferred is restricted F plasmid
of the bacterial bacterial genome small enough to bacterial genes adjacent to
genome; usually to fit into phage capsid; usually the integrated prophage and
from closely related from closely related species part of the viral genome
species (infected by same type of phage)
Yes Yes Yes No

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The Cell & Biomolecules of Life | Genetics & Inheritance (9744) Bacteria 2018
Regulation of gene expression in bacteria

Operon : is a cluster of genes with related functions, regulated in such a way that all the genes in the cluster are turned on and off together.
It includes a common promoter, an operator, and one or more structural genes that are controlled as a unit to produce a single polycistronic mRNA.
Promoter: RNA polymerase binding site, upstream of structural genes
Operator: repressor protein binding site to prevent RNA polymerase from binding to the promoter and intiating transcription
Polycistronic mRNA: A messenger RNA that contains the base sequence coding for the amino acids sequence of several proteins.
 A single mRNA contains multiple start codons (AUG) and stop codons (UAG, UAA, UGA) (one per polypeptide e.g. 3 sets for lac operon, 5 sets for trp
operon)
 Gives rise to a total of ___ different polypeptides which can be translated from a single mRNA, illustrating the polycistronic nature of the mRNA
Structural gene : Any gene that codes for a protein product that forms part of a structure or has an enzymatic function
Regulator gene: Any of several kinds of nucleotide sequences involved in the control of the expression of structural genes
 Codes for a protein involved in regulating the expression of other genes e.g. repressor, CAP
 Has its own promoter and terminator sequences
 Not within operon, usually far away, but gene products that control the expression are diffusible
Effector: a small molecule that binds to a specific protein, causing a conformational change and hence regulating its biological activity.
In this context, includes inducer (allolactose in lac operon) and corepressor (trptophan in trp operon)

Purpose/Advantages of Regulation

 Allows the bacteria to make economical use of energy and resources – prevents wastage/conserve resources i.e. relevant genes are expressed
only when necessary
o Especially since bacteria are able to use a variety of metabolites e.g. glucose is metabolized preferentially over lactose, thus it is not economical to
produce lac genes in the presence of glucose
o No need to synthesize a metabolite when it can be taken in from the surroundings
 Operons
o Can be turned ‘on’ or ‘off’ according to changes/ conditions of the environment
o Allows for functionally related proteins to be synthesized as a unit
 Enable bacteria to respond rapidly and appropriately to changes in environment
 All this provides a selective advantage to such bacteria, who can respond to and survive when there are changes in the environment

Lac operon Trp operon

Structure A cluster of 3 structural genes A cluster of 5 structural genes: trpE, trpD, trpC, trpB and trp A
 lacZ codes for beta-galactosidase: enzyme that  Code for enzymes in tryptophan synthesis
hydrolyses lactose into glucose and galactose  5 genes  5 polypeptides  3 enzymes (2 of the enzymes
 lacY codes for permease: facilitates movement of are dimers, trpA+trpB & trpD+trpE & trpC on its own)
lactose from outside of cell to inside of cell  Promoter  RNA polymerase binding site
 lacA codes for transacetylase: function remains  Operator  binding site for trp repressor complex with trp
unknown Operator within promoter
 Promoter  RNA polymerase binding site
 Operator  lac repressor binding site
Operator overlaps with promoter
Catabolite Activator Protein (CAP) binding site within
promoter
Regulatory gene Lac I gene that codes for lac repressor Trp R gene that codes for trp repressor
Purpose It regulates the production of inducible enzymes such It regulates the production of repressible enzymes for the
as beta-galactosidase and other proteins involved in synthesis of the amino acid tryptophan
the breakdown of lactose

Type of operon Inducible operon Repressible operon

Type of regulation
Effector
Type of metabolic pathway
Default state of repressor
Effect of effector on operon
Default state of operon
expression
Dual regulation - negative regulation by the lac
repressor and positive regulation by CAP
Inducer lactose, the substrate
Catabolic pathway: breakdown metabolites
Active
Turns on transcription of structural genes
Off
negative regulation by the trp repressor
Corepressor tryptophan, an end product
Anabolic pathway: synthesize metabolites
Inactive
Turns off transcription of structural genes
On

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The Cell & Biomolecules of Life | Genetics & Inheritance (9744) Bacteria 2018
Lac operon Trp operon

1. In the absence of lactose, a basal level of beta 1. When tryptophan is present in high concentrations in the
galactosidase and permease is present in the cell: cell
repression of lac operon by lac repressor is leaky 2. Tryptophan acts as a corepressor and binds to the
2. Lactose enters the cell by permease allosteric site of trp repressor
3. And is converted to allolactose by beta- 3. This causes a change in conformation of trp repressor
galactosidase and trp repressor become active
4. Allolactose acts as an inducer and binds to 4. The active repressor can bind to the operator
allosteric site of lac repressor. This causes a 5. This prevents binding of RNA polymerase to promoter
change in confirmation of lac repressor and lac 6. And prevents transcription of structural genes &
repressor becomes inactive expression of operon
5. The inactive repressor and can no longer bind 7. Synthesis of tryptophan is reduced/ stopped
to operator of lac operon (no longer
complementary in shape and charge)
6. Promoter site available for RNA polymerase to
bind
7. When glucose is absent  high levels of cAMP is
present  cAMP binds to CAP and activates CAP
 activated CAP binds to promoter of lac operon
which increases the affinity of RNA
polymerase to the promoter
8. Transcription frequency of structural genes lacZ,
lacY and lacA to produce beta-galactosidase,
permease and transacetylase respectively to
breakdown lactose thus increases.
Operon expression: 9. (Give one example of a product and what it does)
How it works Note: there will be a time lag for lac operon
genes to be expressed – time taken for
transcription of genes and subsequent
translation to form gene products

Lactose present, glucose present (cAMP level low):

little lac mRNA synthesized

Lactose present, glucose absent (cAMP level high):

abundant lac mRNA synthesized
Compare Viruses and Bacteria
Virus Bacteria
Cellular Acellular Is a cell
organisation Has no cell surface membrane, but may have a viral envelope Always has a cell surface membrane
Has no cell wall Has a (peptidoglycan) cell wall
Genetic material Genetic material is either DNA or RNA Has both DNA & RNA
Viral DNA or RNA may be single or double-stranded Bacterial DNA is double-stranded & circular
Macromolecular Has no ribosomes Has (70S) ribosomes
machinery
Does not Carries out metabolism such as
» respire » respiration
» feed » acquisition of food
» excrete/ have metabolic wastes while outside host cell » excretion of metabolic wastes
» grow Capable of cell growth on its own
Reproduction Can only replicate & assemble within host cell Reproduces either asexually or by binary fission
Size Much smaller: 10-300nm E. coli usually greater than 1µm
Pathogenicity Both are capable of causing disease in humans

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The Cell & Biomolecules of Life | Genetics & Inheritance (9744) Bacteria 2018
Effect of Mutations (using Lac operon as an example)

Category Observation Location of Mutation Details

Affects a single Truncated/ non- lacZ Permease and transacetylase coded by lacY and lacA will still be
structural gene functional/ absent beta- (or lacY or lacA) produced as the enzymes are translated independently because each
galactosidase has its own start & stop codon on the polycistronic mRNA
(or transacetylase or
permease)
Affects all 3 Enzymes not present, lacI  Change in conformation of DNA binding site of repressor  binds
structural genes even when lactose is irreversibly to / cannot dissociate from operator
equally present  indicates that  Change in conformation of allosteric site of repressor  cannot bind to
operon is switched off/ the inducer and become inactivated  remains bound to operator
not expressed Promoter RNA polymerase cannot bind to promoter  no transcription
Enzymes always present, lacI  Truncated/ non-functional/ absent repressor
even when lactose is  Conformational change in DNA binding site of repressor  cannot
absent  genes in bind to operator
operon are constitutively  (for trp operon) Conformational change in allosteric site of repressor 
transcribed cannot bind to corepressor to form repressor complex  cannot bind
to operator
Operator Repressor unable to bind to operator  RNA polymerase can continue
to bind to promoter & transcribe structural genes
Glucose present; lactose present Lactose used up
1. Glucose used in preference
over lactose
2. high glucose concn  ↓cAMP
3. cAMP is not bound to CAP,
(catabolite activator protein) Glucose used up; lactose present
4. CAP inactive and not bind to 1. Lac operon is expressed and β
CAP binding site at promoter galactosidase, permease and
5. RNA polymerase has low transacylase produced
affinity for promoter 2. Permease transports lactose into
6. Low frequency of transcription bacterial cell.
of lac operon 3. β galactosidase is produced that
hydrolyse lactose to glucose and
galactose
4. Glucose is used as a respiratory
substrate to allow for bacterial
growth.
Glucose used up; lactose present
1. No growth in bacteria
2. No glucose  cAMP
3. cAMP is bound to the allosteristic site of CAP, (catabolite
activator protein)
4. CAP is activated and bind to CAP binding site at promoter
At the same time
5. Allolactose binds to allosteric site lac repressor  change
conformation  inactive form  cannot bind to operator
Glucose used up 6. RNA polymerase binds to promoter
7. With CAP at promoter  increase frequency of transcription of
lac operon structural genes
8. Time taken for transcription and translation of lac operon

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 Respiration: C6H12O6 + 6O2  6CO2 + 6H2O
Aerobic respiration: (1) involves oxidation of glucose (2) produces 38 ATP mlcs per glucose mlc oxidised, CO 2 & water (3) requires oxygen

GLYCOLYSIS (in cytosol)

1 Glucose (6C)
* 1 glucose mlc  2 pyruvate mlcs
Phosphorylation Phosphofructokinase ATP * 2 NADH + net 2 ATP are produced
of sugars (PFK) ADP * O2 is not required for this step.
1 Phosphorylation of sugar
Fructose 1, 6-bisphosphate (6C)
2  addition of phosphate group from ATP activates the
Lysis sugars & commits it to the glycolytic pathway
2 Lysis
Glyceraldehyde-3-phosphate (3C) Dihydroxyacetone 1 mlc of fructose-1,6- bisphosphate (6C) lyses to form
3 phosphate 2 mlcs of glyceraldehyde-3-phosphate (G3P/TP)(3C)
(G3P) NAD
Oxidation by Pi  Hence the number of products formed (eg: ATP,
dehydrogenation NADH NADH, FADH2, CO2 etc.) in all subsequent rxns
1, 3-bisphophoglycerate (3C) (i.e. Link, Krebs, OP.) including glycolysis is doubled.
4
ADP Glycolysis 3 Oxidation by dehydrogenation
Substrate level  glyceraldehyde-3-phosphate (G3P/TP) undergoes
ATP
phosphorylation oxidation/dehydrogenation and phosphorylation
Glycerate-3-phosphate (3C) NAD is reduced to NADH
4 (GP) ADP 4 Substrate-level phosphorylation
Substrate level
ATP  enzyme-mediated ATP synthesis
phosphorylation
 involves transfer of a phosphate group (Pi) from a
Pyruvate (3C) substrate mlc in a metabolic pathway to ADP.
5 CoA  occurs during glycolysis and Krebs cycle
NAD
Oxidative Link Reaction
decarboxylation CO2 NADH LINK REACTION (in mitochondrial matrix)
Acetyl CoA (2C) * If O2 is present, pyruvate is actively transported
into the mitochondrial matrix via a transport protein
* 2 pyruvate mlcs (3C) undergo 5 oxidative
CoA
decarboxylation to form 2 acetyl CoA mlcs (2C)
* 2 NADH + 2 CO2 are produced
Oxaloacetate (4C) Citrate (6C) KREBS CYCLE (in mitochondrial matrix)
NADH * When 1 glucose molecule is oxidised, 2 Acetyl CoA
6 NAD mlcs form and enter the Krebs cycle. Thus 2 turns of
NAD the Krebs cycle is necessary to oxidise 1 mlc of glucose.
NADH
FADH2 Krebs Cycle * Acetyl CoA (2C) combines with oxaloacetate (4C) to
6 form citrate (6C)
FAD 5 * Citrate (6C) is converted to α-ketoglutarate (5C) by
ATP
5 oxidative decarboxylation
4 ADP CO2 * α-ketoglutarate (5C) then goes through a series of
enzymatic reactions (i.e 5 oxidative decarboxylation,
CO2
4 substrate level phosphorylation & 6 oxidation )
NADH
5 - ketoglutarate (5C) and is converted to oxaloacetate (4C).
NAD * When oxaloacetate (4C) is regenerated ATP, NADH &
FADH2 are also produced.
Oxidative Phosphorylation
* 6 NADH + 2 FADH2 + 2 ATP + 4 CO2 are produced

OXIDATIVE PHOSPHORYLATION
(on inner mitochondrial membrane)

* When molecular oxygen (O2) is available, NADH from

glycolysis, the link reaction & the Krebs cycle, donates
high energy e-(s) to the first electron carrier of the
electron transport chain (ETC) on the inner
mitochondrial membrane.

* The e- first carrier is thus reduced & the NAD which is

regenerated can pick up e-(s) and protons from
glycolysis, the link reaction or the Krebs cycle.
The first reduced e- carrier then transfers the e- to the
next e- carrier & reduces it while the first carrier itself
becomes reoxidised.

* The transfer of electrons continues in this manner until

they combine with H+ & O2 (the final e- acceptor), to
form metabolic H2O in the matrix. This reaction is
catalysed by cytochrome oxidase.
(2e- + 2H+ + ½O2  H20)

* As e- are transferred down the increasingly electronegative electron carriers in the ETC, energy is released. This energy is used to
actively pump H+ from the mitochondrial matrix to intermembrane space.

* This creates a proton gradient across the inner mitochondrial membrane. The energy stored in the form of a H+ gradient across a
membrane is known as a proton-motive force.

* As protons diffuse through ATP synthase (which projects into the matrix) down the H+ concentration gradient into the mitochondrial
matrix, ATP synthase is activated and it phosphorylates ADP to ATP in the matrix.

Prepared by: Mrs Selvamani Nair (2018) Raffles Institution (Yr 5-6) 1
 CO2 ATP NADH FADH2
Glycolysis - 2 (net) 2 -
Link reaction 2 - 1x2=2 -
Krebs cycle 4 2 3x2=6 2
Sub-total 6CO2 4ATP 10 NADH 2 FADH2

Oxidative phosphorylation
* From 1NADH 3 ATP (or 2.5 ATP) form
* From 1FADH2 2 ATP (or 1.5 ATP) form
Thus from 10 NADH and 2 FADH2  (10 X 3 ) + ( 2 X 2 ) = 34 ATP form

Total ATP from the oxidation of 1 glucose molecule: 34 + 4 = 38 ATP

* The e-(s)from FADH2 are also transferred down the ETC. However, FADH2 releases the e-(s) lower in the ETC compared to NADH. Hence,
Less energy is released from FADH2 during e- transfer. The regenerated FAD then can pick up e-(s) and protons from the Krebs cycle.
* NAD : nicotinamide adenine dinucleotide (a coenzyme)
* FAD : flavine adenine dinucleotide (a coenzyme)
* NAD
70S ribosomes  a coenzyme and a mobile electron carrier
 carries electrons & protons (in its reduced form, NADH) from
organic molecules to electron carriers in ETC & reduces them
while, NADH itself is reoxidised to NAD+

pumping of * The number of ATP molecules produced per glucose molecule

protons can vary between 36 to 38. This is because the mitochondrial
membrane is impermeable to the NADH generated by glycolysis.
The H+ and electrons of the NADH are passed to either NAD or
FAD inside theATP
mitochondrion via a shuttle system, and if
synthase
passed to FAD, only 2 ATP will be produced instead of 3.

circular DNA ATP synthase * Chemiosmosis: an energy coupling mechanism that uses
A mitochondrion energy stored in the form of hydrogen ion gradient across a
membrane to synthesise ATP.

Anaerobic respiration: (1) involves oxidation of glucose in absence of oxygen (2) produces 2 ATP mlcs per glucose molecule

* In the absence of oxygen (O2), there is no

final e- acceptor to accept electrons from the
electron transport chain (ETC).

* Electron carriers remain reduced and so

NADH and FADH2 can no longer donate
electrons to the ETC. Hence oxidative
phosphorylation (OP) cannot occur .

* In absence of oxidative phosphorylation, there

there is no regeneration of NAD & FAD and
thus the link reaction & Krebs cycle cannot
occur (because there are no e- acceptors i.e.
NAD & FAD).

* In the absence of oxygen, glycolysis can still

occur as the NAD needed for glycolysis is
regenerated from fermentation reactions.
NAD NADH NADH NAD
* Alcoholic fermentation occurs in yeasts while
Ethanol Ethanal Lactate lactate fermentation occurs in muscles of
+ animals.
CO2
* Both fermentation reactions regenerate
NAD from NADH in order to keep glycolysis
alcoholic fermentation lactic acid fermentation going. ATP is only produced from glycolysis.
(in yeast) (in humans)
* In animals  pyruvate is reduced by e- (s)
from NADH in the presence of lactate
Lactate is transported in the blood
dehydrogenase to lactate.
to the liver where it is converted
In yeasts  pyruvate converted to ethanal
back to pyruvate which can then
and CO2 . Ethanal is then reduced by e- (s)
enter the link reaction again during
from NADH in the presence of alcohol
aerobic respiration. dehydrogenase to ethanol.

* Thus pyruvate or ethanal are the final e-

acceptors during anaerobic respiration.

* Only 2 ATP mlcs. are produced per glucose

mlc. during anaerobic respiration. This is 19
times lower compared to aerobic respiration
which produces 38 ATP mlcs. per glucose mlc.

* Fermentation reactions occur in the cytosol.

Prepared by: Mrs Selvamani Nair (2018) Raffles Institution (Yr 5-6) 2
ATP : adenosine triphosphate

* ATP: universal energy currency

* Energy released from glucose oxidation during respiration is used to make ATP from ADP + Pi
* ATP made can then be hydrolysed to ADP + Pi, releasing energy in the process
* Removal of terminal phosphate group from ATP yields 30.6 kJ/mol of free energy
 This energy is useful for cellular work such as muscle contraction, maintenance of constant body temperature and active transport
* When ATP is hydrolysed, it is incorrect to say that a “high energy bond” is broken. Instead modification occurs to the molecule as a whole and
there is a net release of energy when the phosphate group is removed
* ATP is actually carrier of energy, does not store energy (compare: fats and glycogen which store energy)
* ATP is the same nucleoside triphosphate used to form RNA

Differences between Photosynthesis and Aerobic Respiration

Feature Photosynthesis Aerobic Respiration
Anabolic/ Catabolic Anabolic Catabolic
Synthesis of carbohydrate molecules from simple Breakdown of carbohydrate molecules into simple
inorganic molecules inorganic molecules
Energy storage Energy from light  stored in carbohydrates Energy from carbohydrates  incorporated into
ATP (for use in energy-requiring processes)
Oxygen Released Used
CO2 & H2O Used Released
Dry mass Increases Decreases
Conditions In cells possessing chlorophyll, and in the presence of light In all living cells, in the presence of oxygen
Organelle Chloroplast Mitochondrion (and cytosol)
Electron carrier NADP NAD, FAD
High [H+] Thylakoid space Intermembrane space
Major reactions Light-dependent reaction (cyclic & non-cyclic Glycolysis, link reaction, Krebs cycle, oxidative
photophosphorylation^) and Calvin cycle phosphorylation

Compare Photophosphorylation and Oxidative Phosphorylation

Feature Photophosphorylation Oxidative Phosphorylation
Location Both processes take place on membranes
Thylakoid membrane of chloroplast Inner mitochondrial membrane
Involvement of light Light energy needed for splitting of water Light energy not needed
Source of energy for Ultimately comes from light Comes from oxidation of glucose (chemical energy)
ATP synthesis
Energy conversion Light energy  Chemical energy Chemical energy (glucose)  Chemical energy (ATP)
Electron donors Non-cyclic: water. Cyclic: P700 in PSI. NADH and FADH2
Electron acceptors Non-cyclic: NADP. Cyclic: P700 in PSI. Oxygen (and it combines with H+ to produce metabolic water)
By-product Photolysis of water produces O2 as a by-product in H2O is produced when oxygen combines with electrons and
non-cyclic photophosphorylation H+ at the end of the ETC
Proton gradient Energy lost from the flow of electrons along ETC is used to actively pump protons across a membrane to generate a
proton gradient
ADP is phosphorylated to ATP via ATP synthase using energy directly from the flow of protons down their gradient in
chemiosmosis
Protons are pumped inwards from stroma across Protons are pumped outwards from matrix across inner
thylakoid membrane into thylakoid space mitochondrial membrane into intermembrane space

Distinguish between Calvin and Krebs cycle

Feature Calvin Cycle Krebs Cycle
Location Stroma of chloroplast Mitochondrial matrix
Redox reaction Reduction Oxidation (by dehydrogenation)
NADPH reduces GP NAD, FAD oxidize intermediates
Carbon dioxide Fixed by RuBisCO (1 molecule accepted by RuBP per Released by oxidative decarboxylation (2 molecules
cycle, catalyzed by RuBisCO) per cycle)
ATP Used in reduction of GP to G3P and regeneration of RuBP Synthesised by substrate-level phosphorylation
Substance regenerated Ribulose bisphosphate (5C) Oxaloacetate (4C)

Functions of NAD & FAD

1. The high-energy electrons yielded from the oxidation of organic food such as carbohydrates, fats and proteins in glycolysis, link reaction
and Krebs cycle are transferred to NAD and FAD, reducing them and forming NADH and FADH2
2. They are coenzymes which serve as mobile electron carriers which transport the high-energy electrons from organic molecules to the
electron transport chain in the inner mitochondrial membrane
3. By passing their electrons to the electron transport chain, NADH and FADH 2 are oxidized, regenerating NAD and FAD, allowing them to pick
up more electrons and protons from glycolysis, link reaction and Krebs cycle

Functions of ETC
1. Generating a proton motive force to produce ATP
2. Regeneration of coenzymes NAD and FAD so that they can pick up more electrons and protons from glycolysis, link reaction and Krebs cycle

Importance of Oxygen
1. By acting as the final electron acceptor at the end of the ETC where it combines with electrons and protons to form water, O 2 re-oxidises the
ETC so that the electron carriers NADH and FADH2 can continue donating their electrons to the chain, thereby allowing oxidative
phosphorylation to continue to generate ATP
2. NAD and FAD are regenerated when NADH and FADH2 donate electrons to the ETC, allowing NAD and FAD to pick up more electrons and
protons from glycolysis, link reaction and Krebs cycle
3. Reduction of oxygen to water removes H+ from the matrix, contributing to the generation of a proton gradient across the inner mitochondrial
membrane

Prepared by: Mrs Selvamani Nair (2018) Raffles Institution (Yr 5-6) 3
Photosynthesis ( 6CO2 + 6H2O  C6H12O6 + 6O2)
Light Dependent Reactions (on thylakoid membrane)
* Primary pigment: special chl a mlc, P680 & P700. P680 is found in the reaction centre (RC) of Photosystem II (PSII) & P700 in Photosystem I (PS I)
* Accessory pigments: other chl a, chl b mlcs & carotenoids (fd in the light harvesting complex (LHC)

Non-cyclic photophosphorylation

* When a photon of light is absorbed by an accessory pigment molecule in the light harvesting complex (LHC) of PS II, one of its electrons is
excited to a higher energy level. As the excited electron drops to its ground state, the energy released is passed on to the next pigment molecule. This
resonance transfer of energy continues until P680, the special chlorophyll a molecule in the reaction centre (RC) is reached.

* When P680 absorbs the energy from the accessory pigments of the light harvesting apparatus, it loses an electron, leaving an electron hole in PSII.
The displaced electron is accepted by a primary electron acceptor (X) in the reaction centre.

* The electron hole in PSII is filled by an electron released from the splitting of water in an enzyme-catalysed reaction in the thylakoid space. During the
splitting of water, the H+ released contributes to a high concentration of H+ in the thylakoid space while the O atom combines with another O atom,
forming molecular oxygen (O2) as a by-product

* The electron from the primary e- acceptor (X) is then passed down a series of increasingly electronegative electron carriers (of the 1st ETC)
losing energy during the transfer. The energy lost during this electron flow is used to actively pump H+ from the stroma to the thylakoid space,
generating a proton gradient across the membrane. Chemiosmosis occurs when H+ diffuse down the proton gradient back into the stroma via ATP
synthase, & ADP is phosphorylated to ATP.

* Meanwhile, PSI loses an electron in a manner similar to PSII. When P700 absorbs the energy from the accessory pigments in the light harvesting
apparatus, it loses an electron, leaving an electron hole in PSI. The displaced electron is accepted by a primary electron acceptor (Y) in the reaction
centre. The electron hole in PSI is filled by the displaced electron from PSII when it reaches the end of the first electron transport chain.

* The electron from the primary electron acceptor (Y) is then is passed down a series of electron carriers of a 2nd ETC. (Energy is not released during
electron transfer down this 2nd ETC.). The electron is finally accepted by NADP (the final electron acceptor) which is reduced to NADPH (NADP+e-
+H+NADPH) by NADP reductase which is found on the thylakoid membrane.

* The ATP & NADPH produced during non-cyclic photophosphorylation will be used in the Calvin cycle.

Cyclic photophosphorylation

* In cyclic photophosphorylation, electrons displaced from P700 of PSI & accepted by the primary electron acceptor Y are transferred to the middle
of the 1st ETC. The electron is transported down the ETC & is finally recycled back to PSI.

* Energy lost during electron transfer is coupled to the formation of ATP in a manner similar to non-cyclic photophosphorylation.

* Only PSI is involved & only ATP is produced during cyclic photophosphorylation. NADPH is not produced. The ATP produced is used in the Calvin
cycle.

Y
X

Non-cyclic photophosphorylation:

Cyclic photophosphorylation:

Prepared by: Mrs Selvamani Nair (2018) Raffles Institution (Yr 5-6) 1
Light Independent Reactions / Calvin Cycle (in stroma)
* Substances required from light reaction: NADPH & ATP

* Carbon fixation: CO2 combines with RuBP (5C) in the presence of the enzyme
ribulose bisphosphate carboxylase (Rubisco) to form an unstable 6C compound
which breaks down into 2 molecules of GP/PGA (3C)

* Reduction and sugar formation: GP is reduced to G3P/TP/PGAL(3C). ATP and

NADPH are needed for the reaction. NADPH provides the reducing power for the
reaction

* Regeneration of RUBP: G3P molecules can either be converted to sugars and

then polymerized to starch or enter a series of reactions driven by ATP to
regenerate RuBP to allow CO2 fixation to continue.

* C & O atoms of sugar (C6H12O6) come from CO2 & H atoms come from NADPH

* Products of light independent reaction : 1) G3P (a triose sugar)

2) NADP & ADP (which are recycled to the
light reactions)
(Note: GP: Glycerate-3-phosphate / Glycerate phosphate;
G3P: Glyceraldehyde-3-phosphate; TP: Triose phosphate)

Some terms:
Phosphorylation = addition of a phosphate group to a molecule [eg: ADP + Pi (inorganic phosphate) ATP]
Photophosphorylation = formation of ATP from ADP + Pi using light energy in photosynthesis
Non-cyclic photophosphorylation = Electrons obtained from PS II  Primary electron acceptor (X) electron transport chain  PSI 
Primary electron acceptor (Y)  electron transport chain NADP.
Electron from the photolysis of water replaces the electron lost form PSII.
Cyclic photophosphorylation = Electrons that are raised to a higher energy level are lost from PSI, but are recycled back to PSI
through the 1st electron transport chain.
Together cyclic & non-cyclic photophosphorylation produce sufficient ATP & NADPH to drive the Calvin cycle.
Chemiosmosis: an energy coupling mechanism that uses energy stored in a proton gradient across a membrane to synthesise ATP.
Photoactivation: When a chlorophyll molecule absorbs light, the energy from this light raises one of its electrons to a higher energy level. That
chlorophyll molecule is said to be photoactivated.
Resonance transfer: When a chlorophyll molecule absorbs light, the energy from light raises one of its electrons to a higher energy level.
When the excited electron returns to its ground state, the energy released is transferred to another pigment molecule.
This is called resonance transfer.
Limiting factor: Any environmental factor that - by its decrease or S: Light saturation point: Light intensity beyond which an
increase, absence or presence - alters the growth, increase in light intensity will not increase the rate of
metabolic processes or distribution of organisms and photosynthesis
populations most significantly.
If you increase a particular variable and there continues to be a proportional
relationship between the values on the x & y axes, it is referred to as the
only limiting factor.
At P: light intensity is a limiting factor (note linear relationship between x and
y values)
At Q: light intensity is not the only limiting factor. Some other factor is also
limiting. (eg: CO2 concentration)
At R: light intensity is no longer limiting. (How do you know this? Even when
light intensity is increased, there is no increase in the rate of reaction.)
Some other factor is limiting.

Compensation point: is the light intensity at which the rate of

photosynthesis is equal to the rate of respiration.

At compensation point, all the CO2 produced during respiration is used in

photosynthesis and all the oxygen produced in photosynthesis is used in
respiration. Hence there is no net gain / loss of CO2.

Absorption spectrum: a record of the amount of light absorbed by each Action spectrum: graph showing the effectiveness of
pigment at each wavelength of light. different wavelengths of light in stimulating photosynthesis
(effectiveness determined by rate of photosynthesis)

The action spectrum is similar to (wavelengths of peaks and troughs) but does not exactly match the absorption spectrum of
chlorophyll a, because chlorophyll b and carotenoids, being accessory pigments, broaden the spectrum of wavelength over which
photosynthesis can occur by channeling energy absorbed to chlorophyll a

Prepared by: Mrs Selvamani Nair (2018) Raffles Institution (Yr 5-6) 2
3 KEY Factors Affecting The Rate Of Photosynthesis

1) light intensity 2) CO2 concentration 3) temperature

(other factors which may limit rate of photosynthesis include chlorophyll or oxygen concentration, specific inhibitors like herbicides, water, pollution etc.)
Below is the setup to measure rate of oxygen evolution by a water plant during photosynthesis. Only 1 limiting factor should be tested at a time.

Rate of Photosynthesis is proportional to the volume of gas evolved. Since bubbles of evolved gas are collected over a fixed duration of time,
Rate of Photosynthesis = collected volume (mm 3) = mm3 of evolved O2/min (at a known temperature, toC)
time (minutes)

Prepared by: Mrs Selvamani Nair (2018) Raffles Institution (Yr 5-6) 3
Distinguish between Light-Dependent and Light-Independent Reactions

Feature Light-Dependent Reaction Light-Independent Reaction/ Calvin Cycle

Location Thylakoid (thylakoid space, thylakoid membrane) & stroma Stroma
Requires light for photoactivation/ photoexcitation of electrons (both) and Does not require light
photolysis of water (non-cyclic photophosphorylation only)
Conditions Less enzyme-dependent More enzyme-dependent
Requires enzymes for photolysis of water and NADP+ reductase for Requires RuBisCO for carbon fixation
reduction of NADP (non-cyclic photophosphorylation only)
Reactions Cyclic and non-cyclic photophosphorylation  Carbon fixation
involved  Photoactivation  Reduction
 Photophosphorylation (including chemiosmosis)  Regeneration of RuBP
 Photolysis (non-cyclic only)
 Reduction of NADP+ (non-cyclic only)
Reactants H2O, NADP+, ADP, Pi NADPH, ATP, CO2
Products (NADPH, ATP) G3P, (NADP+, ADP)
By-products O2 -

Compare Non-cyclic and Cyclic Photophosphorylation

Features Non-Cyclic Photophosphorylation Cyclic Photophosphorylation

Similarities
Conditions
Calvin cycle requires 9 ATP &
6 NADPH  NADPH may be
oxidized back but ATP
insufficient
Pathway of electrons
Photosystems involved
First electron donor
Last electron acceptor
Products
High [H+] in thylakoid space
 Energy lost from the flow of electrons along an ETC is used to actively pump H+ across membrane to
generate a proton gradient
 ADP is phosphorylated to ATP via ATP synthase using energy directly from the flow of protons down their
gradient via chemiosmosis
 Both processes take place on membranes
When both ATP and NADPH are required When only ATP is required
(too much NADP+) (i.e. when NADP+ concentration is limiting, e.g. when
there is a high NADPH to NADP+ ratio)
One direction from water through two ETC to Cyclical passing from PSI to ETC back to PSI
NADP+ via 2 photosystems
PSII and PSI PSI only
Water P700 in PSI (no photolysis of water)
NADP+ P700 in PSI
ATP, NADPH, O2 ATP only
Active transport of H+ ions by electron carriers of Active transport of H+ ions by electron carriers of ETC
ETC; Photolysis of water only
(H2O  2e– + 2H+ + ½ O2)

Describe the role of NADP in photosynthesis

1) NADP+ is a coenzyme which carries both protons and high energy electrons
2) NADP+ is the final electron acceptor in the non-cyclic light dependent reaction in the thylakoid membrane
3) Electrons carried in reduced NADP (NADPH) are used in the Calvin cycle in the stroma of the chloroplast to reduce glycerate phosphate (GP)
to glyceraldehyde-3-phosphate (G3P)
4) When GP is reduced to G3P, NADP is regenerated to carry out its role as an electron carrier from the light dependent reactions

Structure of chloroplast

Some Questions:

Q: What contributes to high H+ concentration in the thylakoid space?

1) proton pump (which actively pumps H+ into the thylakoid space)
2) photolysis of water (catalysed by enzymes on inner thylakoid membrane)
3) lack of permeability of thylakoid membrane to H+ (due to its hydrophobic core)
4) reduction of NADP to NADPH occurs in the stroma & hence reduces the H+ concentration in the stroma thereby ensuring the
steepness of the H+ gradient across the membrane

Q: Describe the function of the thylakoid membrane in photophosphorylation.

1) Provides a large surface area to embed many photosynthetic pigments for light absorption
2) Maintains the sequential arrangement of the electron carriers of electron transport chain for the flow of electrons
3) Maintains proton gradient for ATP synthesis since the hydrophobic core of the membrane is impermeable to protons and this is
essential for chemiosmosis
4) Allows of many ATP synthase to be embedded so ATP can be produced as protons flow down their gradient via chemiosmosis from
thylakoid space to stroma

Prepared by: Mrs Selvamani Nair (2018) Raffles Institution (Yr 5-6) 4
Genetics and Inheritance (9744) Organisation and Control of Prokaryotic and Eukaryotic Genomes 2018
General structure of a eukaryotic and prokaryotic cell:
Feature Eukaryotic cell Prokaryotic cell (bacteria)
Cell size Larger: 10-100µm in diameter Smaller: 0.5 - 5µm in diameter
Nucleus Nucleus with nuclear envelope present; No true nucleus / No nuclear envelope
Genetic material Linear DNA associated with many proteins; Circular DNA associated with few histone-like proteins;
Found in membrane bound nucleus; Found in a region of the cytoplasm known as the nucleoid region;
No plasmids Plasmids present
Ribosome for 80S; 70S;
protein synthesis Ribosomes may be attached to ER or free in cytoplasm No ER present. Ribosomes free in cytoplasm.
Organelles Many; Few;
Many membrane bound organelles present; No membrane bound organelles;
Cell walls Composed of cellulose in plants & chitin in fungi Composed of peptidoglycan or murein

Structure of the genome of a prokaryotic and eukaryotic cell:

Feature Structure of Eukaryotic Genome Structure of Prokaryotic Genome
Size 107-1011 base pairs (larger) 104-107 base pairs (smaller)
Appearance Multiple, linear molecules Generally a single, circular molecule
Molecule Double helix DNA Double helix DNA
Association with proteins Yes – large amounts of it e.g. histones, scaffold proteins Yes – relatively less histone-like proteins
Level of DNA High: DNA double helix  negatively charged DNA associated with Relatively low: DNA double helix 
packing/coiling positively charged histones via (electrostatic attraction): DNA is wound folded into looped domains by protein-
around 8 histone proteins twice to form nucleosomes with linker DNA DNA associations which undergoes
joining adjacent nucleosomes, forming a 10nm fibre/chromation  which supercoiling with the help of DNA gyrase
coils around itself to form 30nm fibre/solenoid (interphase chromatin) and topoisomerase(See * below)
which forms looped domains when associated with scaffold proteins,
forming 300nm fibre  which supercoils chromosome (at metaphase) (See
* below)
Location Nucleus Nucleoid region – not membrane-bound
Extrachromosomal DNA Yes – if you consider mitochondria and chloroplast circular DNA Yes – plasmids (much smaller rings of
DNA)

Organisation of the genome of a prokaryotic and eukaryotic cell:

Organisation of Eukaryotic Genome Organisation of Prokaryotic Genome
Number of genes 25,000 4,500
Non-coding regions (between and within Common – about 98% Not common – typically less than 15%
genes)
i. presence of introns Many Rare
ii. presence of promoters Yes Yes
iii. presence of repeated sequences Many (e.g. telomeres & centromeres) Few
iv. presence of enhancers/ silencers Common Rare
Presence of operons Very few known ones Many

*** How telomerase works

DNA double helix
1. A short 3-nucleotide segment of RNA within Histones
telomerase binds to part of a DNA repeat in Nucleosome
the 3’overhang by complementray base pairing. (“bead”) Circular
Linker DNA chromosomal
(“string”) DNA
2. The adjacent part of the RNA within telomerase Nucleosomes (10-nm fiber)
is used as a template to synthesise a short
complementary 6-nucleotide DNA repeat.

3. Telomerase catalyses the formation of the

Looped
phosphodiester bonds between the existing Nucleosome domains
3’OH group of existing DNA overhang and 5’ 30-nm fiber or solenoid
phosphate group of incoming Protein scaffold
deoxyribonucleotide
4. After the repeat is made, telomerase Looped domains (300-nm fiber)
translocates 6 nucleotides to the right in the 5’
to 3’ direction of the DNA overhang and begins to Supercoiling
make another repeat. The above process is
repeated such that a series of tandem repeats are
made, elongating the telomere.
5. Then primase makes an RNA primer near the
end of the telomere. DNA polymerase adds
Metaphase eukaryotic chromosome Prokaryotic
nucleotides to the 3’OH end of the primer and chromosome
hence synthesizes a complementary strand .
* Packing of DNA in eukaryotic and prokaryotic
The nick is then sealed by ligase. The RNA
chromosomes:
primer is eventually removed.
The packing of the very long eukaryotic DNA ensures that
it fits into the nucleus and that it does not get
Newly synthesized RNA primer entangled and break.
strand to be removed

Prepared by: Mrs Jeanne Wan, Mrs Selvamani Nair, Mrs Wong S H Raffles Institution (Yr 5-6) 1
Genetics and Inheritance (9744) Organisation and Control of Prokaryotic and Eukaryotic Genomes 2018

Non-coding DNA (does not code for proteins or RNA )

Non-coding Regulatory Sequences Non-coding Repetitive DNA
Introns Promoter Enhancer Silencer Telomere Centromere
Structure Structure Structure Structure Structure Structure
- non coding - located just - usually located far - usually located far 1- found at both ends/terminals of linear, eukaryotic chromosomes 1- Constricted region
DNA upstream of the away from the away from the on chromosome
sequences transcription start promoter (usually promoter (usually 2- non-coding DNA made up of a series of tandem repeat sequences where kinetochore
found within a site of a gene much further much further (a specific sequence of 5
microtubules
gene, upstream or upstream or nucleotides occurring End of parental Leading strand attach during
DNA strands Lagging strand
specifically - has critical downstream) downstream) many times in a row) nuclear division
3
between exons elements 2- non-coding DNA
in a specific e.g.1. TATA box 3- in humans, made up of a
Last fragment Previous fragment
segment of at -25 sequence each repeat has series of tandem
DNA (also (i.e. located 25bp the sequence RNA primer repeat sequences
present in upstream of 5’ TTAGGG 3’ Lagging strand 5

pre- mRNA) 3
transcription start
Primer removed but Removal of primers and
site) 4- have a single cannot be replaced replacement with DNA
- only in e.g. 2. CAAT stranded region with DNA because where a 3 end is
no 3 end available
eukaryotes and GC boxes at their 3’ ends for DNA polymerase
available
5
are not critical in known as the 3’
3
determining overhang Second round
transcription (due to a limitation of replication
frequency of DNA polymerase, 5
this region of DNA New leading strand 3
does not have a New lagging strand
complementary 5
3
strand) Further rounds
of replication
Shorter and shorter
daughter molecules

Function Function Function Function Function Function

- enables a - recognition & - recognition & - recognition & 1-Telomeres ensure that genes are not eroded (≠ DNA not eroded) and vital 1- allow sister
process binding site for binding site for binding site for genetic information is not lost with each round of DNA replication due to chromatids to
called general activators (which repressors (which the end replication problem. adhere to each
‘alternative transcription are specific are specific As DNA polymerase requires a free 3’OH of a pre-existing strand to add other
RNA splicing’ factors which transcription transcription nucleotides, the last RNA primer on the lagging strand with DNA cannot be
to occur where then recruits factors) factors) replaced with DNA. Hence, the DNA molecule shortens with each round of 2- allow kinetochore
different exons RNA replication. Telomeres, which are non-coding sequences at the ends of proteins to attach
of a single pre- Polymerase to - increase - decrease linear chromosomes will be lost before any vital genetic information is. Since and which in turn
mRNA can be form the frequency of the frequency of telomeres are non-coding, they can be lost without any deleterious effect. allow spindle
joined such transcription transcription by transcription by fibres to attach so
that different initiation promoting the preventing the 2-Telomeres protect and stabilize the terminal ends of chromosomes by that sister
mature mRNAs complex which assembly of the assembly of the forming a loop using the 3’overhang. chromatids
are produced initiates transcription transcription This prevents single-stranded terminal end of one chromosome from / homologous
eg. mature transcription initiation complex initiation complex annealing to a complementary single-stranded terminal end of another chromosomes can
mRNA 1  (with the help of chromosome, prevent fusing of 2 chromosomes. align along the
exons 1, 2, 3; DNA bending The formation of the loop also prevents the cell’s DNA repair machinery metaphase plate
mature mRNA 2 -TATA box proteins that bend from detecting the chromosome as damaged DNA (i.e. double stranded and subsequently
 exons 1, 3 determines spacer DNA) breaks) and trigger apoptosis. be separated to
etc. precise location Activators and repressors are not enzymes & do opposite poles.
- one gene can of transcription not have an active site. Each has a DNA binding 3-Telomeres allow their own extension, as they have a 3’ overhang which
now code for start site domain that is complementary in shape & charge provides an attachment point for the correct positioning of the enzyme Ultimately, allow s
more than to a specific sequence of DNA, allowing them to telomerase. Although telomeres shorten with every round of DNA replication, proper alignment and
one bind to a specific enhancer / silencer sequence. telomerase activity in germ cells, embryonic stem cells and cancer cells can segregation of
polypeptide maintain telomere length. (See *** on pg1 on how telomerase works) chromosomes.

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Genetics and Inheritance (9744) Organisation and Control of Prokaryotic and Eukaryotic Genomes 2018
Purpose of regulation of gene expression:

 Cellular differentiation: regulation of genes  production of different proteins  cells have different ultrastructures that their functions i.e. in a multicellular organism, all somatic cells carry identical
genes but cells show a wide variation in structure and function
 Adapt to changes: vary according to circumstances and demand
 Conserve resources (transcriptional level control predominates as it is the most efficient mechanism with minimal wastage, especially in prokaryotes)
 More varied proteome despite limited genome size

Regulation of gene expression at various stages in eukaryotes

Genomic/DNA Eukaryotes (only)

(+) Histone acetylation

 addition of acetyl group to certain amino acids (e.g. lysine) in the histone by histone acetyltransferase/acetylase
1) Histone  removes +ve charge on histones decreases electrostatic attraction between –vely charged DNA and histones
acetylation &  promoter region is more accessible to RNA polymerase and general transcription factors  promotes transcription as it promotes assembly of transcription initiation
deacetylation complex
(Note: acetylation works in concert with chromatin remodeling complex)

(-) Histone deacetylation

 removal of acetyl groups from histones by histone deacetylase
 restores +ve charge on histones  restores tighter interaction between DNA & histones
 promoter region is less accessible to RNA polymerase and general transcription factors inhibits transcription as it prevents assembly of transcription initiation complex

2) Chromatin Chromatin remodeling complex are protein complexes which

remodeling  alter structure of nucleosomes temporarily
complex 1) (-) can cause DNA to be more tightly coiled around histones inhibits transcription as it prevents assembly of transcription initiation complex
2) (+) can cause DNA to be less tightly coiled around histones promotes transcription as it promotes assembly of transcription initiation complex

(-)DNA methylation (Once DNA methylation occurs in certain genes in a cell, it is usually permanent for the entire lifetime of the cell.)
3) DNA  addition of a methyl group by DNA methylases to selected cytosine residues inhibits transcription by
methylation 1) blocking the binding of transcription factors at the promoter and hence preventing the formation of the transcription initiation complex
2) recruiting DNA-binding proteins (e.g. transcriptional repressors, histone deacetylases and repressive chromatin remodeling complexes)

Transcription Eukaryotes Prokaryotes

Specific transcription factors (activators and repressors) Activators and repressors

1) Activators 1) Activator (in lac operon)

 bind to enhancers  is the activated Catabolite Activator Protein (CAP) which binds to the CAP binding
 promote assembly of transcription initiation complex as bending of spacer DNA site at the promoter of the lac operon and increases the affinity of RNA
allows binding of activators with RNA polymerase and/or GTFs at the promoter polymerase to the promoter
 transcription frequency increases  transcription frequency increases  Positive gene regulation
Regulatory (Bound activator may recruit histone acetyltransferase and chromatin remodeling (CAP is activated by binding to cAMP)
proteins complex to increase accessibility of promoter to general transcription factors and RNA
polymerase)

2) Repressors 2) Repressor (in both lac and trp operon)

 bind to silencers
 prevent assembly of transcription initiation complex as bending of spacer DNA
allows repressor bind to general transcription factors hence preventing activators from
binding to general transcription factors
 transcription frequency decreases
(Bound repressor may recruit histone deacetylase and repressive chromatin
remodeling complex to decrease accessibility of promoter to general transcription
factors and RNA polymerase)
 binds to the operator, preventing RNA polymerase from binding to the promoter
 transcription frequency decreases  Negative gene regulation
(Genes with related functions are clustered in an operon
 are controlled by a single promoter and operator region and
 transcribed into a single polycistronic mRNA and are expressed together)

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Genetics and Inheritance (9744) Organisation and Control of Prokaryotic and Eukaryotic Genomes 2018
Post- Eukaryotes (only)
transcriptional

- Addition of a 7-methylguanosine nucleotide is added to the 5’ end of the pre-mRNA

1) Addition of - occurs shortly after transcription begins i.e. it occurs co-transcriptionally
5’cap - (role) - 1. helps the cell to recognize mRNA (amongst other RNAs) so that subsequent steps such as splicing and polyadenylation can occur
- 2. signal to export mRNA out of nucleus
- 3. protects the growing pre-mRNA chain from degradation by ribonucleases
- 4. promotes initiation of translation as it is recognized by translation initiation factors that help recruit mRNA to small ribosomal subunit

1. When the introns (noncoding regions within a gene) are excised and exons (coding regions within a gene) are joined together by spliceosomes (a complex of snRNA and
2) Intron proteins: snRNPs) which recognize the sequences at intron-exon boundaries (points of excision)  so that functional proteins can be produced
splicing 2. Alternative splicing  where different exons of a single pre- mRNA can be joined together such that different mature mRNAs and so  different proteins can be produced

- When 3’end of pre-mRNA is cleaved by endonucleases to make it shorter and a poly-A polymerase recognises the polyadenylation signal (AAUAAA) and adds a long
3) Addition of sequence of adenine nucleotides to 3’ end of the pre-mRNA, forming a poly(A) tail
poly A tail - occurs immediately after transcription
(polyadenylation) - (role) - 1. signal to export mature mRNA out of nucleus
- 2. protects mature mRNA from degradation by ribonucleases  more proteins can be made
- 3. required, together with 5’cap for initiation of translation

Translation Eukaryotes

1) mRNA half- mRNA half-life is determined by the length of its poly-A tail. The longer the poly-A tail, the longer the mRNA can be used as a template to make proteins.
life The poly-A tail is removed by ribonucleases in the 3’ to 5’ direction until a critical length is reached which will triggers removal of the 5’cap and degradation of the mRNA from the
5’end too.

1) During translation initiation, small ribosomal subunit binds to 5’cap of mRNA

2) Formation of This can be prevented by binding of translational repressor to (1) 5’cap, (2) 5’UTR (untranslated region), (3) 3’ UTR
translation which interferes with the interaction between the 3’ poly-A tail, the 5’cap, initiation factors, and the small ribosomal subunit which are needed for translation initiation
initiation
complex 2) During translation initiation, translation initiation factors facilitate the binding of the small ribosomal subunit to the 5’cap
The availability of translation initiation factors is determined by whether or not they are phosphorylated. Some initiation factors are activated by phosphorylation while others
are inactivated by phosphorylation. Without activated translations initiation factors, translation cannot begin.

Post-translational Eukaryotes
1) Formation
of functional Covalent modification/cleavage (eg. attachment of prosthetic groups, glycosylation, disulfide bond formation) of polypeptides make them functional proteins
proteins

2) Regulation of Phosphorylation / dephosphorylation of translation initiation factors can activate / deactivate the protein and hence up / down regulate its activity.
protein activity

3) Protein Protein degradation by proteasomes determines how long a protein remains in a cell.
degradation Proteins targetted for degradation are tagged with ubiqutin (by ubiquitin ligase) and then recognised and degraded by the proteasome.

Causative factors which increase chances of cancer the chances of cancerous growth

A. Environmental factors  exposure to carcinogens (eg. tar in cigarette smoke, asbestos etc.) and ionizing radiation (e.g. uv radiation, X-rays) can cause mutations that lead to cancer
B. Loss of immunity due to infection with certain viruses  HIV can weaken the immune system and reduces the body’s ability to fight infections by other viruses that can cause to cancer.
For example, Karposi’s sarcoma-associated Herpes virus genome can integrate into part of the human genome that controls DNA
synthesis, or that affects cell division or that affects tumour suppression pathways and may lead to cancer
C. Genetic predisposition  due to gene mutations (in the germ cells) which we inherit from our parents (e.g. BRCA1 gene, a tumour suppressor gene)
D. Age  chances of getting cancer increases with age due to accumulation of mutations in a cell over a lifetime

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Genetics and Inheritance (9744) Organisation and Control of Prokaryotic and Eukaryotic Genomes 2018
Proto-oncogenes
1. code for proteins (e.g. growth factors, activators, growth signal transduction factors) that stimulate normal cell division/proliferation
2. when mutated, they are known as oncogenes which
(a) increase the amount of proto-oncogene’s protein product
(i) by a point mutation in base sequences of regulatory elements (e.g. stronger promoter created)
This can lead to increased frequency of transcription, and excess production of the proto-oncogene protein product (e.g. growth factor) and can lead to uncontrolled cell division
(ii) gene amplification, where the number of copies of a proto-oncogene in a cell is increased due to a mistake made during DNA replication
This can lead to excessive production of proto-oncogene protein product (e.g. growth factor) and can lead to  uncontrolled cell division
(iii) chromosomal translocation such that the proto-oncogene ends up under the control of a enhancer
This can lead to excessive production of proto-oncogene protein product (e.g. growth factor) and can lead to  uncontrolled cell division
(iv) by retroviral integration
1) can inactivate a silencer of a proto-oncogene
2) can result in the insertion of a viral enhancer that upregulates expression of a proto-oncogene
3) can insert a viral homologue of proto-oncogene
All 3 events can lead to excessive production of proto-oncogene protein product (e.g. growth factor) and can lead to  uncontrolled cell division
(b) increase the intrinsic activity of the proto-oncogene protein product
(i) by a point mutation within the proto-oncogene
This changes the amino acid sequence of the proto-oncogene protein (e.g. growth factor) which can then become hyperactive or more resistant to degradation and can lead to
 uncontrolled cell divison
3. e.g. ras gene: mutation in the ras gene results in a constitutively active Ras protein that irreversibly binds to GTP and increases cell division even in the absence of growth factors

Tumour suppressor genes

1. codes for protein products that inhibit cell division and prevent uncontrolled cell division or by activating cell cycle arrest, DNA repair and/or apoptosis.
2. when mutated, they are inactivated. Mutations could be in promoter or coding sequence of gene. Note that both copies of the tumour suppressor allele needs to be mutated for the effect of the
mutations to be observed.
3. e.g. p53 gene: codes for a specific transcription factor (an activator) that can activate genes involved in
(a) cell cycle arrest  gives the cell enough time to repair damaged DNA and prevent formation of mutant daughter cells
(b) DNA repair  prevents mutations that may lead to the formation of oncogenes or inactivated tumour suppressor genes
(c) initiating apoptosis (programmed cell death) when DNA damage is beyond repair  which will thus remove cells with damaged DNA with the potential to cause cancer

Gain-in-function and loss-of-function mutations

Gain-in-function mutation  e.g. when a proto-oncogene (e.g.ras) is mutated to form an oncogene
(dominant mutation)  mutation in just one copy of the allele results can result in uncontrolled cell division due to the increased synthesis/activity of a functional product (which was not produced
previously) due to mutation.
Thus the mutation is said to be dominant.
Loss-of-function mutation  e.g. mutations in tumour suppressor genes (e.g.p53)
(recessive mutations)  mutations in both copies of the allele necessary for the loss-of-function phenotype to be observed.
 even when one copy is mutated, the non-mutant copy still produces a functional gene product.
Thus the non-mutant copy will mask the effect of the mutant copy and hence the mutation is said to be recessive.

Why is development of cancer a multi-step process?

 The development of cancer requires the accumulation of mutations in the genes which control regulatory checkpoints of the cell cycle in a single cell
 This will disrupt the normal cell cycle, thus causing the cell to undergo excessive cell proliferation
 A gain-in-function mutation is a dominant mutation where mutation in just one allele of a proto-oncogene will result in its overexpression which will result in the production of excessive amounts of
growth factors or hyperactive/degradation resistant growth factors leading to excessive cell proliferation
 Loss-of-function mutation is a recessive mutation where mutations in both alleles of a tumour suppressor gene will result in the non-functional/missing protein which will disrupt their ability to
inhibit cell cycle, enable DNA repair and promote apoptosis
 Activation of the genes coding for telomerase result in telomeres being lengthened allowing the cell to dividing indefinitely as the chromosomes are prevented from shortening with each DNA
replication cycle. (  A single cell that is immortal and continues to divide will accumulate more and more mutations)
 Loss of contact inhibition* will enable the cells to grow into a benign tumour (mass of cells).
 Angiogenesis occur within the tumour so that the blood vessels formed can transport oxygen and nutrients for its growth.
 The presence of blood vessels can result in the formation of a malignant tumour capable of metastasizing to other parts of the body via the bloodstream to form secondary tumours
 As it takes years to accumulate these mutations, the chances of developing cancer increases with age.

 Not all cells need to be replaced at the same rate or at the same time. There are built-in controls in the cell cycle, (i.e. checkpoints), that ensure normal cell division
 Dysregulation of the checkpoints in the cell cycle can result in uncontrolled cell division leading to tumour formation and eventually cancer.

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Genetics and Inheritance (9744) Organisation and Control of Prokaryotic and Eukaryotic Genomes 2018

1. Histone acetylation/deacetylation 4. Activators bind enhancers 6. RNA splicing

Note:
1-3: Regulation at chromatin level

4-5: Regulation at transcriptional level

6-7: Regulation at post-transcriptional

level

8-10: Regulation at translation level

2. Chromatin
remodeling 11: Regulation at post-translational
complex level

5. Repressors bind silencers

3. DNA methylation 7. Alternative
splicing

8. mRNA ½ life
9. Availability of activated translation
initiation factors
10. Binding of
translational repressor

11. Protein degradation

Feature Normal Cells Cancerous Cells

Nuclei Normal nuclei with low nucleo-cytoplasmic ratio Abnormal nuclei with high nucleo-cytoplasmic ratio
Genes Proto-oncogenes, tumour suppressor genes not mutated Oncogenes and mutated tumour suppressor genes present
Cell division Undergo controlled cell division & proliferation Undergo excessive uncontrolled cell division & proliferation
Apoptosis Undergo apoptosis: divide for a certain number of times and then stop dividing Do not undergo apoptosis: can divide indefinitely
Contact inhibition Show contact inhibition: do not divide further when in contact with other cells  monolayer of cells Do not show contact inhibition multiple layers of cells
Differentiation Can differentiate to become specialized cells Do not differentiate
Cell adhesion/ metastasis Cell adhesion  formation of tissues and organs Can detach from surrounding cells and metastasize
Angiogenesis Do not stimulate new blood vessels Stimulate growth of new blood vessels

Prepared by: Mrs Jeanne Wan, Mrs Selvamani Nair, Mrs Wong Seok Hui Raffles Institution (Yr 5-6) 6
 Genetics and Inheritance (9744) Molecular Techniques 2019

Describe the principles and procedures of the polymerase chain reaction (PCR) (including its advantages and limitations)

Polymerase Chain Reaction PCR Components

 Amplifies DNA from a limited source of DNA so that there is sufficient amount for analysis. * Template DNA
DNA containing the target sequence to
* Process: (3 steps) be amplified
1. 1. Denaturation: Double stranded DNA denatures into single stranded DNA by heating to 95C
2. as weak hydrogen bonds between complementary bases of each strand is * Primers
3. broken due to increased molecular vibrations synthetic single-stranded DNA
fragment (20-30 nucleotides long)
2. Primer annealing: Each primer anneals specifically to the 3’end of each single stranded needed to initiate DNA synthesis by
target DNA sequence via complementary base pairing when the providing a free 3’OH group for Taq
temperature is lowered to 64C polymerase to bind to and extend
 2 different primers are required.
3. Extension: Taq polymerase synthesizes the complementary DNA strand from the free 3OH’ Each is complementary to the
end of the DNA primer by catalyzing the formation of phosphodiester bonds sequence at 3’end of each
between dNTPS when the temperature is increased to 72C single stranded target DNA
sequence.
* Advantages:
1. Only a minute amount of DNA is required to carry out PCR as with each round of PCR, the
number of copies of target DNA is doubled. Thus the number of desired sequence increases
exponentially and there will be sufficient DNA for analysis.
2. Use of thermostable (i.e. resistant to denaturation at high temperatures) Taq polymerase
allows PCR to be automated so DNA can be amplified very quickly.
TAC ATT
* Limitations:
1. Taq polymerase lacks 3’ to 5’ proofreading ability. Hence errors occurring early in the PCR
reaction will get compounded with each subsequent replication cycle.  are required in large excess to
2. Knowledge of sequences flanking (i.e. at the 3’ ends of) the target sequence is required in increase the likelihood of them
order to design appropriate primers. binding to target DNA sequences
3. Taq polymerase tends to ‘fall off’ the DNA template before chain extension is complete if the (relative to template strands binding
strand is too long. Hence there is a limit to the size of DNA fragment (~3kb) to be amplified. each other)
4. Minute amounts of contaminant DNA can be exponentially amplified along with target DNA
and affect the reliability of the results. * Taq polymerase
* Number of copies of double stranded  thermostable DNA polymerase
DNA strand
DNA = 2n which is resistant to denaturation at
(where n is the number of cycles) high temperature
* Number of (single) strands of DNA = 2n+1
(where n is the number of cycles) * Deoxyribonucleotides (dNTPs)
 substrates for DNA replication made
End of n cycle
1 2 3 4
up of dATP, dTTP, dCTP and dGTP
where n is
Copies of ds
DNA
2 4 8 16 * Buffer
Strands of ss  contains cofactor, Mg2+, for proper
4 8 16 32
DNA polymerase function
End of: 1st cycle 2nd cycle 3rd cycle 4th cycle

Describe the principles and procedures of gel electrophoresis .

Agarose gel electrophoresis
 separates DNA based on fragment size

* Steps:
1. A slab of agarose gel is placed in a buffer solution contains ions which allows the
conduction of electricity when the current is turned on.
2. The DNA sample is mixed with a dense loading dye containing glycerol & 2
coloured dyes. Glycerol makes the DNA sample denser than the buffer solution so
that the DNA sample can sink to the bottom of the well.
3. Since DNA is invisible, the dyes colour the DNA sample and will indicate if the DNA
has been loaded correctly into the well. (NB: Loading dyes do not bind to the DNA.)
4. One dye (corresponds to a 100bp DNA fragment) and often runs ahead of the DNA
sample and gives an indication of when electrophoresis must be stopped so that
the samples do not run out of the gel. The other dye (corresponds to a 1100bp
DNA fragment) and gives an indication of the position of the larger fragments on the
gel.
5. The 2 coloured dyes thus act as visual markers which help to monitor the progress
of the migration of the invisible DNA fragments in the gel during electrophoresis.
6. DNA samples are pipetted into the wells in the gel near the negative electrode.
7. A DNA ladder (i.e. DNA molecular weight markers) which contains DNA fragments of
known sizes, is run in one of the lanes and acts as a standard for which to compare DNA
fragments of unknown size in the sample. molecular
weight marker
8. Negatively charged DNA is attracted towards the positive electrode (anode) when acts as
subjected to an electric current. standard to
9. The agarose gel matrix made of a meshwork of polymer fibres which impedes compare to
fragments of
movement of longer fragments more than shorter fragments. The longer fragments unknown
thus migrate more slowly compared to shorter fragments, leading to a banding sizes
pattern observed on the gel.
The top line Note: Bands are only
10. Before the loading dye reaches the end of the gel, the current is turned off. corresponds to visible after treating
11. To visualize the bands, the gel can be treated with a staining dye that binds DNA fragments that are with staining dye
(e.g. ethidium bromide, a carcinogen) and fluoresces under uv light. 1100bp and the such as ethidium
bottom line bromide (visible
12. Thus a) the fragment size can be estimated (based on position of the band relative to corresponds to 100bp under UV light).
bands in the molecular weight marker) and fragments.
b) the amount of DNA can possibly be estimated (based on intensity & NB: The higher the concentration of agarose, the finer the
thickness of the band). pores in the meshwork.
Thus smaller fragments can be effectively separated .

Prepared by: Mrs Selvamani Nair & Ms Eva Hor Raffles Institution (2019) 1
 Describe the principles and procedures of Southern blotting and nucleic acid hybridisation

Southern blotting: a nucleic acid hybridisation technique used in RFLP analysis

Tool to detect specific nucleotide sequences within a sample of DNA

Steps:
1. (Continued from Gel electrophoresis)
1. Gel slab is placed on top of the sponge and under a nitrocellulose membrane. A stack
of paper towels placed on top of nitrocellulose membrane. These are placed in a tray of
alkaline solution. A heavy weight is placed above the paper towels.
2. Absorbent paper towels draw the solution towards themselves and the alkaline solution
denatures double-stranded DNA into single-stranded DNA.
3. Single stranded DNA on the gel is then drawn upwards onto the nitrocellulose
membrane and binds to the membrane (in exactly the same position as they were in
the gel).
4. Nitrocellulose membrane is removed and incubated with single-stranded radioactive
DNA probe which hybridises via complementary base pairing to the target sequence.
5. The excess unhybridised probes are washed off.
6. Autoradiography is performed placing X-ray film over membrane. Radioactive regions
exposes the film forming an image that correspond to the bands that have base-paired
with probe.

[Note: RFLP analysis is a principle/ way of analyzing data. PCR, gel electrophoresis and nucleic acid
hybridization are tools used in RFLP analysis.]

What are RFLPs?

 RFLP = Restriction Fragment Length Polymorphisms
 RFLPs are restriction patterns that arise from restriction digestion using a specific
restriction enzyme.
 RFLPs arise due to DNA polymorphisms which are small nucleotide differences in
different individuals at specific locus.
 Thus due to the polymorphic nature of DNA in different individuals, there will be
variations in the
1. number/location of restriction sites or
2. number of tandemly repeated nucleotide sequences. This will result in a
unique banding pattern amongst individuals.

 The DNA polymorphism in sickle cell anaemia is a single nucleotide polymorphism (SNP). There is a difference in a single base pair due to a
point mutation.
 In sickle cell anaemia this SNP is within the coding region. However the majority of SNPs used for RFLP analysis are found in non-coding
regions.
i) Use of RFLP analysis in disease detection e.g. sickle cell anaemia
 A difference in single nucleotide can result in a gain OR loss of a restriction site of an enzyme. Thus when a particular section of DNA in
individuals with the mutation and without the mutation is digested with the same restriction enzyme, DNA fragments of different lengths will
result. Analysis of the banding pattern that arises, will allow determination of the presence of disease-causing allele or the normal allele.
 e.g. in sickle-cell anaemia, the disease-causing mutation occurs at restriction site for Mst II within the -globin gene.
1. In the disease causing allele, the Mst II restriction site is eliminated.
2. In the normal allele, Mst II restriction site is retained.
Sickle cell mutation
(S-type DNA)

 How to determine the genotype of a person for -globin gene?

1. Digest genomic DNA from both samples with MstII to obtain different restriction fragments will arise. (i.e. cut both DNA samples with the
SAME restriction enzyme, Mst II)

2. Perform gel electrophoresis following by southern blotting (use of nitrocellulose membrane, radioactive probes and autoradiography).
The same single-stranded radioactive probe (which is complementary to part of the target sequence) will be used to detect both the
presence of A-type and S-type DNA. Hence the site that the probe binds on the DNA fragments should give different banding patterns on
the autoradiogram.

fragment of short long fragment (3)

intermediate fragment (2)
length (1)

Prepared by: Mrs Selvamani Nair & Ms Eva Hor Raffles Institution (2019) 2
i) Use of RFLP analysis in disease detection e.g. sickle cell anaemia (continued)
AA SS AS
A type DNA will indicate presence of normal β globin.
S type DNA will indicate presence of abnormal β globin (sickle cell).
Note that normal β globin is dominant over abnormal β globin (sickle cell).

AA indicates that the individual is homozygous dominant and is phenotypically normal.

(The individual has 2 bands. One fragment of intermediate length and one short fragment.)
SS indicates that the individual is homozygous recessive and is suffering from sickle cell anaemia.
(The individual has 1 band corresponding to the long fragment.)
AS indicates that the individual is heterozygous and is phenotypically normal.
(The individual has 3 bands. One long fragment, one fragment of intermediate length and one short fragment.)

ii) Use of RFLP analysis in DNA fingerprinting

 As no individuals (exception of twins) have the same genome, therefore they will not have the same DNA profile.
 The DNA profile is the restriction banding pattern that identifies individuals.
 How to carry out DNA fingerprinting:
1. Restriction digestion of genomic DNA by restriction enzymes followed by gel electrophoresis to separate DNA fragments
2. Southern blotting using of nitrocellulose membrane.
3. Nucleic acid hybridisation using radioactive probes complementary to the STRs
4. Visualisation of bands via autoradiography using X-ray films
 At a particular RFLP locus, different individuals will have different number of STR repeats, hence different length of fragment.
 RFLP alleles of each locus is inherited from parents. Therefore individuals with similar banding patterns should be closely related.

 These RFLP alleles are all from the same RFLP locus.
DNA from the crime scene (C) has 2 RFLP alleles, one with 11 repeats and the other with 9 repeats. Individual (A) has two RFLP alleles, one with 3
repeats and the other with 4 repeats. Hence Individual (A)’s DNA fingerprint is different from the DNA at the crime scene.

(Note: Instead of using steps 1 and 2 (above) to carry out DNA fingerprinting, the different STR loci can be amplified using PCR and multiple PCR
primers that anneal to flanking sequences of different STR loci. Then gel electrophoresis can be carried out to separate the DNA fragments. The
DNA can then be stained by ethidium bromide and the fragments can be visualised under uv light. This alternative method will also give rise to the
different banding patterns seen above.)

* Restriction
The greater the number
enzymes of RFLP
(molecular loci used in the DNA fingerprint of an individual, the more unique the fingerprint. In the CODIS (combined DNA
scissors)
1) areindex system)
isolated from used by the
bacterial FBI, 13 different STR loci are used to distinguish between different individuals.
cells
2) recognize & bind to specific palindromic base sequences (restriction sites) on DNA
3) create either sticky ends/blunt ends by cleaving of phosphodiester bonds between nucleotides in both strands of DNA
e.g. Alu I e.g. Eco RI

NNAGCTNN G AATTC
NNTCGANN CTTAA G

NNAG CTNN G AATTC

NNTC GANN CTTAA G
blunt ends sticky ends

4) protect bacterial cells from invading viruses by degrading the foreign DNA that enter them
5) do not cleave the bacterial DNA in the bacteria they are found in as the restriction sites in the bacterial DNA are methylated. Methylation protects
bacterial DNA from degradation.

Prepared by: Mrs Selvamani Nair & Ms Eva Hor Raffles Institution (2019) 3
Energy and Equilibrium (9744) Homeostasis & Cell Signalling 2019
Homeostasis and Cell Signalling

 Homeostasis refers to the maintenance of a stable internal environment independent of fluctuations in the external environment by self-
regulating & negative feedback mechanisms so that the organism can function optimally.
Self-regulation: where a corrective mechanism is triggered by the very entity which is to be regulated
(e.g. control of blood glucose levels is triggered by changes in blood glucose levels)
Negative feedback: a mechanism which brings about increasing stability of a system i.e. it removes any deviations from the set point
i.e. a change in a variable triggers a response that counteracts the initial change.
(e.g. when blood glucose level goes higher than set point, insulin is secreted to return glucose levels to set point).
 Hormones:
 secreted by endocrine glands (ductless glands) directly into the bloodstream
 effective in small quantities (as signal amplification, that occurs during signal transduction, will lead to the production of a strong cellular response)
 act on specific target cells which have specific cell surface receptors
 each type elicits different cellular responses & after having served their function, are rapidly broken down
 e.g. insulin & glucagon are hydrophilic peptide hormones that bind to the specific receptors on the cell membrane (e.g. RTK & GPCR)

 Pancreas:
 is an organ that is both an endocrine (islets of Langerhans) gland & an exocrine (acinar cells) gland
 the islets of Langerhans contain alpha cells which secrete glucagon and beta cells which secrete insulin into the bloodstream
(insulin and glucagon (which are protein in nature) are secreted constantly and work in an antagonistic fashion; it is their relative concentrations
and not their actual levels that are critical to maintain normal blood glucose levels at the set point which is 90mg/dL)

 Glucose:  Glycogen:
 key respiratory substrate  stored in liver and muscles
 Insulin triggers the conversion of glucose to glycogen
 Glucagon triggers the conversion of glycogen to glucose
(It is incorrect to say that insulin converts glucose to glycogen as insulin binds to the insulin receptor which triggers a signal transduction pathway
that eventually leads to the conversion of glucose to glycogen in the cell. Likewise, it is incorrect to say that glucagon converts glycogen to glucose.)
 A deviation from the set point i.e. stimulus (e.g. high blood glucose levels)
 is detected by detectors (e.g. beta cells in islets of Langerhans) (N.B. Sometimes detectors are referred to as receptors)
 which secretes an appropriate signal (e.g. insulin)
 which binds to the cell surface receptors** of the cell (e.g. cell surface insulin receptor, RTK) of the effector (e.g. liver/muscle cells)
 which brings about an appropriate response that restores the condition to the set point (e.g. blood sugar levels return to set point)
 this serves as negative feedback to detectors (e.g. beta cells) to decrease secretion of signal (e.g. insulin)

 Cell signaling (3 stages) and role of kinases and phosphatases in signal amplification:

1) Ligand-receptor interaction:
 ligand/signal/first messenger binds to a specific, ligand-binding site (which is complementary in shape and charge to the ligand) on the
extracellular domain of the cell-surface receptor to form a ligand-receptor complex.
2) Signal transduction & amplification:
 where binding of the ligand/signal to the protein receptor causes a conformational change in the intracellular domain of the protein receptor
which initiates the signal transduction. i.e. the signal is converted to a form that can bring about a specific cellular response.
 signal transduction usually occurs in a series of multiple catalytic steps in a signal transduction pathway
 the multiple catalytic steps allow amplification of the signal, where the number of activated molecules increases with each subsequent
step.(Hence signal amplification occurs during signal transduction.)
 the signal transduction pathway is mediated by intracellular signaling proteins (e.g. kinases) or small molecules (e.g. cAMP) or ions.
 kinases phosphorylate and activate proteins and are involved in multiple catalytic steps in a signal transduction pathway. Hence kinases
allows amplification of the signal
 phosphatases dephosphorylate and inactivate proteins and are involved in multiple catalytic steps in a signal transduction pathway.
By dephosphorylating and inactivating proteins, they can inhibit signal transduction.
3) Cellular response:
 where the transduced signal triggers specific changes in cellular function, metabolism, or development by changing gene expression by
targeting proteins such as gene regulatory proteins, ion channels, components of a metabolic pathway etc.

* Advantages of a cell signaling pathway:

1) Facilitates amplification of signal

 small number of signal molecules binding to the receptors can produce a large cellular response as the number of activated molecules
increases with each catalytic step in the pathway
2) One signal molecule can trigger many signal transduction pathways in a cell and elicit many different cellular responses
 when blood glucose levels are high, insulin can bind to the RTK and increase rate of glycolysis, glycogenesis, protein and lipid synthesis etc.)
3) Provides many checkpoints for regulation as cellular responses can be terminated/regulated at
(i) At Reception:
 extracellular first messenger can be degraded by enzymes in the extracellular space
 endocytosis of cell surface receptors to prevent ligand-receptor interaction can prevent signal transduction
 endocytosis of the entire ligand-receptor complex can prevent signal transduction
(ii) During Signal Transduction Pathway
 production of phosphatases dephosphorylate & inactive the relay proteins  inhibit further signal transduction
 production of inhibitors that bind to the intracellular domain of the ligand-receptor complex and /or any of the intracellular signal proteins in the
signal transduction pathway to prevent transduction of the signal.
4) One type of signal can allow the coordinated activation of many different cells simultaneously (e.g. insulin can bind to receptors on liver and
muscle cells and trigger signal transduction pathways in the cell.)
5) Ensures specific reactions are triggered as a specific signal will bind to a specific receptor and will elicit specific reactions in specific cell types.
6) A signal molecule can activate genes in nucleus upon binding to cell surface receptor without the need to move into nucleus.

Prepared by Mrs.S.Nair, Mrs.Wong SH & Mdm.S.Cross Raffles Institution 1

Energy and Equilibrium (9744) Homeostasis & Cell Signalling 2019
Outline how insulin and glucagon regulate the concentration of blood glucose through the respective tyrosine kinase receptor and G-
protein linked receptor

INSULIN
A stimulus An increase in blood sugar level above 90
mg/dL
 
is detected by a detector/receptor (which is a cell is detected by the beta cells of islets of
that detects the change) Langerhans of pancreas
 
which releases a ligand/signal (e.g. insulin/glucagon) which secretes insulin (1st messenger)
 
which recognises and binds to cell surface which recognises and binds to cell surface
receptors (e.g. RTK / GPCR) receptors known as insulin receptors (a
receptor tyrosine kinase, RTK) which exist
as linked dimers
 
on the effector (which is a cell where the cellular response on the liver (or muscle) cell
occurs).
 
Ligand-binding causes a conformational Ligand-binding causes RTK to undergo
change in the intracellular,cytoplasmic changes in conformation in its intracellular,
domain of the receptor, activating it cytoplasmic domain
This initiates signal transduction.
 
The signal is further transduced and This conformational change activates
amplified. tyrosine kinases in each of the subunits of
the receptor and triggers
crossphosphorylation /
autophosphorylation of tyrosine residues
i.e. where kinases on 1 subunit cross-
phosphorylates tyrosine residues on the
other subunit
 
Phosphorylated tyrosine residues serve as
docking sites for other relay proteins
 
Relay proteins activated by binding or via
phosphorylation by RTK
 
Relay proteins may be kinases which can go
on to phosphorylate other proteins when
activated
GLUCAGON
A decrease in blood sugar level below 90
mg/dL

is detected by the alpha cells of islets of
Langerhans of pancreas

which secretes glucagon (1st messenger)

which recognises and binds to cell surface
receptors known as glucagon receptors (a G-
protein coupled receptor, GPCR)

on the liver cell

Ligand-binding causes GPCR to undergo
changes in conformation in its intracellular,
cytoplasmic domain

This conformational change causes an inactive
G protein to bind to the GPCR, and release
its bound GDP and allow GTP to bind in its
place.
GTP binding causes a conformational change
in the G protein, activating it.
Activated G protein dissociates from the
receptor and translocates along the
cytoplasmic side of the cell membrane to bind
to and activate an enzyme, adenylyl cyclase
Adenylyl cyclase converts ATP to cAMP (2nd
messenger) which activates Protein Kinase A
Activated Protein Kinase A can phosphorylate
other proteins

 a phosphorylation cascade 

which results in the following cellular
responses
which eliminates the stimulus,
 
resulting in a diminished response.
Phosphorylation activates protein kinases in a
cascade which eventually activates glycogen
synthase which catalyses glycogen
synthesis from glucose. (i.e. increase
glycogenesis in liver & muscle)
Other cellular responses:
e.g. 1) Translocation of glucose
transporters
from the membrane of cytoplasmic
vesicles to the cell membrane.
This increases glucose intake into
cells
e.g. 2) Increased rate of glycolysis
e.g. 3) Increased lipid & protein synthesis
 
Blood glucose levels decreases
This is detected by the receptor (detector, i.e.
beta cells) which then decreases insulin
production
Protein kinase A activates phosphorylase
kinase which activates glycogen
phosphorylase which catalyses the
breakdown of glycogen to glucose (i.e.
glycogenolysis in liver & muscle cells)
Other cellular responses:
e.g. 1) Increased gluconeogenesis (synthesis
of glucose from non-carbohydrate
sources)
Blood glucose level increases

This is detected by the receptor (detector, i.e.
alpha cells) which then decreases glucagon
production

(Note: G protein has intrinsic GTPase activity

which can hydrolyse GTP to GDP and
inactivate the G protein, terminating the cellular
response.)

 Explain the role and nature of second messengers (including cAMP):

 small, non-protein, water-soluble molecules or ions
 can readily spread throughout the cell by diffusion and activate cellular proteins

Prepared by Mrs.S.Nair, Mrs.Wong SH & Mdm.S.Cross Raffles Institution 2

Energy and Equilibrium (9744) Homeostasis & Cell Signalling 2019
 can participate in pathways initiated by both GPCR and RTK.
e.g. cAMP  synthesised from ATP by adenylyl cyclase
activates protein kinase A which phosphorylate other proteins hence in a signal transduction pathway
(Note: First messenger is the ligand/signal molecule that binds to membrane receptor e.g. insulin, glucagon etc.)

Describe the molecular structure of the G-protein linked receptor and explain how its structure is related to the function it plays.

1. G protein coupled receptor (GPCR) is cell surface receptor that will bind to specific signal molecule and initiates the process of signal
transduction which converts the information in the signal from the outside of the cell into a cellular response within the cell.
2. GPCRs consist of a single polypeptide with an extracellular N-terminus, and an intracellular C-terminus.
3. GPCR is a globular, seven pass transmembrane protein with a tertiary structure.
4. GPCR consists of 7 α-helices connected by three intracellular and three extracellular peptide loops.
5. As a transmembrane protein that is embedded in a cell's plasma membrane, it is folded such that its amino acid residues with hydrophobic R
groups are interacting with the hydrophobic core of the phospholipid bilayer of the plasma membrane and
6. its amino acids with hydrophilic R groups are arranged within the interior of the protein and also interact with the aqueous interior and
exterior of the cell as well as the hydrophilic phosphate heads of phospholipid bilayer.
7. The extracellular loops have a ligand binding site at which a specific signaling molecule (e.g. glucagon) can bind to the GPCR.
8. The intracellular domain of GPCR has a G protein binding site that allows binding of a heterotrimeric G protein complex.
9. When a ligand binds to the ligand binding site at the extracellular side of a GPCR it causes a conformational change of the intracellular domain
at the cytoplasmic side of the GPCR,
10. The activated GPCR can then activate an associated G protein by exchanging its bound GDP for a GTP.

Prepared by Mrs.S.Nair, Mrs.Wong SH & Mdm.S.Cross Raffles Institution 3

The Cell and Biomolecules of Life (9744) Stem Cells 2019

Stems Cells

a) are unspecialized/undifferentiated i.e. they do not have any tissue-specific structure for it to perform a particular function
b) are able to differentiate to produce specialised cells upon receiving appropriate molecular signals (e.g. hormones, growth factors)
c) undergo extensive proliferation and self-renewal i.e. they can divide many times by mitosis, with the daughter cells possessing the same
developmental and replicative potential as the parent cell
d) can undergo
1) symmetrical division  produces 2 identical daughter stem cells to ensure a constant pool of stem cells
2) asymmetrical divisionproduces a) 1 daughter stem cell  to ensure a constant pool of stem cells
& b) 1 progenitor cell to replace a population of specialised cells in a specific tissue that died
occurs in the presence of appropriate molecular signals
e) can be
1) totipotent  can differentiate into all of the cell types that make up an entire organism including the extraembryonic tissue e.g. the placenta
 e.g. fertilised egg to 8 cell stage (These cells are zygotic stem cells.)
2) pluripotent  can differentiate into all of the cell types that make up an organism except the extraembryonic tissue such as the placenta
 e.g. inner cell mass of blastocyst (The cells in the inner cell mass are embryonic stem cells.)
3) multipotent  can develop into only a limited and related range of cell types and tissues in an organism
 e.g. haematopoietic stem cells, which are found in the bone marrow & give rise to all of the cells found in the blood, including
red blood cells, white blood cells, and platelets. Haematopoietic stem cells are adult stem cells. They are also found in babies.)
Note: 1) Stem cells can differentiate into different cell types due to the presence of molecular signals that cause differential switching on of genes.
2) A progenitor cell is an early descendent of a stem cell that can only differentiate. It cannot renew itself. e.g. lymphoid progenitor cells gives
rise to B cells, T cells and natural killer cells while myeloid progenitor cells give rise to red blood cells, monocytes, platelet producing cells,
neutrophils, basophils and eosinophils
3) Stems cells can continue to divide mitotically as they can express their telomerase gene which lengthens the telomeres of their
chromosomes and hence prevents them from reaching critical length and hence prevents the cell from undergoing apoptosis.

fertilized egg  2 cell stage  4 cell stage  8 cell stage  blastocyst  baby
blastocyst
cavity haematopoietic
stem cells in
trophoblast bone marrow are
multipotent adult
inner cell mass (pluripotent stem cells
totipotent zygotic stem cells embryonic stem cells)

Discuss the ethical implications of the application of stem cells in research and medical applications and how induced pluripotent stem cells
(iPSCs) overcome some of these issues. (procedural details of how iPSCs are formed are not required)
Ethical implications of the use of stem cells in therapy

Argument against using embryonic stem cells Argument for using embryonic stem cells

Some believe that the embryo has the status of a human being as it has Embryos are not equivalent to human life:
the potential to become one. Embryonic stem cell research is tantamount Embryos are not conscious, cannot feel and cannot survive outside the
to murder. womb.

Some object to extracting stem cells from an embryo to make
replacement body cells is treating the embryo as just a source of spare
parts.
Blastocysts are a cluster of human cells that have not differentiated into
distinct organ tissue, making cells of the inner cell mass no more "human"
than a skin cell. Some believe life only begins when the heartbeat
develops (during the fifth week of pregnancy) or when the brain begins
developing (at 54 days after conception).

Claims of the benefits of embryonic stem cell research are over-rated as Embryonic stem cells can potentially treat a wide range of diseases as
there are few (if any) examples of success in medical applications they have the potential to grow indefinitely in a laboratory environment
and can differentiate into almost all types of bodily tissue.

Adult stem cell treatment is established, have produced some results It is unethical not to use established protocols on embryonic stem
and there are fewer ethical issues involved. Thus adult stem cell research cell research to further embryonic stem cell research to help relieve
may be able to make greater advances if more money and resources human suffering.
were channeled into it instead of embryonic stem cell research.

Current benign applications may lead to abuse in the future. Once There is legislation on the period when ES cells can be extracted.
human status is denied to embryos, this precedent may extend to other eg: Current UK legislation does not allow use of embryos that are more
categories of human beings such as the profoundly disabled or the than 14 days old. In fact, ES cells are obtained earlier from blastocyst
elderly infirm. (between 3-8 days after fertilization).

Possibility of unforeseen consequences in treated patients such as More than a third of zygotes do not normally implant in the uterus.
possible risks of tumor formation, immunological reactions, unexpected Thus, far more embryos are lost due to chance than are proposed to be
behavior of the cells, and unknown long-term health effects. used for embryonic stem cell research.

As embryonic stem cell research is expensive, funds can be channeled to Surplus embryos created via in vitro fertility treatments are destroyed,
treat other more treatable diseases. or stored long past their viable storage life. These can be used for creating
new stem cell lines for research which would otherwise be destroyed.
For donors of eggs, embryos or tissues, there are issues of informed
consent, understanding of research aims and privacy.
Prepared by: Mrs. Selvamani Nair and Mr Low Chor Meng Raffles Institution 1
 The Cell and Biomolecules of Life (9744) Stem Cells 2019

Potential solution to overcome some of these issues

Induced pluripotent stem cells (iPSCs)

Induced pluripotent stem cells are pluripotent stem cells that can be generated directly from adult somatic cells (e.g. skin cells) Adult somatic cells are
not totipotent, pluripotent or multipotent. The non-pluripotent cell is therefore induced to become pluripotent. The iPSC technology was pioneered by
Shinya Yamanaka’s lab in 2006 that the introduction of four specific genes encoding transcription factors could ‘reprogramme’ some specialised cells to
become pluripotent so that they lose their specialised functions and behave in virtually the same way as embryonic stem cell.

Advantages of iPSCs Possible problems of iPSCs

 Since iPSCs can be obtained directly from adult tissues, it does not generate or  Low efficiency: in general, the conversion to
destroy any human embryos. adult somatic cells to iPSCs has been incredibly
 Adult tissue (e.g. skin cells) required to make iPSCs can be easily obtained from low. For example, the rate at which somatic cells
donor without risk to the donor were reprogrammed into iPSCs in was 0.01–
 In contrast to ES cells extracted from human embryos, iPSCs derived from a patient’s 0.1%.
own cells would open the possibility of generating lots of patient-specific cells, which  Genetic modification of adult somatic cells to
will not be rejected by the immune system (as it is not a foreign cell) upon obtain iPSCs may cause cancer by
transplantation. overexpression of proto oncogenes or switching
 Hence, there will also be no need to use immunosuppressant drugs after off of tumour suppressor genes.
transplantation and the problem of looking for a suitable donor for transplantation will  There are ethical concerns (e.g. lack of consent,
also be overcome. long term unexpected consequences) related to
 Further, it also allows the generation of pluripotent stem cell lines from patients with the creation of embryos and children from IPSC-
inherited diseases, in order to better understand why the diseases develop and derived sex cells.
use in personalized drug discovery efforts.
 An additional reproductive technology that may be enabled by iPSCs is the
generation of sex cells (sperm and eggs) for treating infertility.

Ethical complications are related to the means of obtaining stem cells (e.g. techniques involving the destruction of human embryos), human cloning
and the exploitation of embryo and egg donors. The use of iPSCs still appears to overcome many ethical issues and provides viable solutions
related to stem cell research.

Comparison of human embryonic stem cells and human adult stem cells

Embryonic stem cells Adult stem cells

Differentiation ability
Role in body
Sources
Advantages in research and
therapy development
Pluripotent: capable of becoming any cell in the
human body except extraembryonic tissue e.g.
placenta.
To develop the embryo into an entire human.
Unused IVF embryos which have been
donated, or embryos created for the purpose
from donated eggs and sperm.
 Embryonic stem cells make up a significant
proportion of a developing embryo and are
easier to isolate and grow ex vivo than
adult stem cells
 Have a strong ability to self-renew in the
laboratory and divide more rapidly than
adult stem cells, resulting in a constant
supply of ES cells.
Multipotent: typically only give rise to the cells of the tissue
in which they are found.
To replace specific cells in the body which die throughout
life due to wear and tear or injury and disease.
Bone marrow, muscles and skin; and from the foetus,
umbilical cord, placenta etc.
 If taken from the patient’s own body for use in therapies,
cells would be genetically identical to that of the patient,
avoiding the problem of immune rejection.
 There are less ethical considerations compared with
using embryonic stem cells.

 Pluripotency means that ES cells have

Disadvantages in research
and therapy development
the potential to produce any cell type in
the body, potentially allowing them to treat
a wider range of diseases.
 Genetically different to cells of potential  They usually only produce a limited number of
patients, so immune rejection could different cell types.
occur.
 Conditions supporting self-renewal in the laboratory
 Ethical issues over embryo destruction have only been identified for a few tissue stem cell
types, for instance skin and cornea.

 Are found in small numbers and are difficult to

isolate.

 Adult stem cells with genetic mutations from the

patient's own body will not be effective in treatment of
genetic disorders.

Prepared by: Mrs. Selvamani Nair and Mr Low Chor Meng Raffles Institution 2
 Raffles Institution Infectious Diseases 2019
Infectious diseases

Immune System

2 4
Innate Immune System (1st line of defense) Adaptive Immune System (2nd line of defense)
1 3
pathogen Barriers breach Cellular Components antigen Cell-mediated Response Humoral Response
encounter inflammation presentation

Phagocytes Mediated by cytotoxic T Mediated by antibodies

1. Skin cells that kill cells (secreted by plasma
 Acidic pH and inhibits microbial infected with cells) which target
growth 1.Macrophages Antigen intracellular pathogens extracellular pathogens
presenting
2. Mucous membranes 2.Dendritic cells cells (APCs) 5 removal of pathogen
 contain antimicrobial proteins, and generation of
e.g. lysozyme, which break immunological memory
3.Neutrophils
down bacterial cell walls
 release fluids e.g. saliva, tears
to wash away pathogens and
prevent their attachment

Properties of the innate (non-specific) immune response: Properties of the adaptive (specific) immune response:

1. is non-specific  attacks anything that is foreign or 1. is specific  recognises only a specific antigen on a
non-self pathogen

2. is rapid  responds as soon as pathogen is
encountered
3. has no memory  responds the same way to repeat
encounters with the same pathogen
2. takes time to develop  this applies to first exposure
to pathogen (i.e. primary immune response)
3. shows memory  responds quickly to repeat
encounters to the same pathogen (i.e. secondary
immune response occurs rapidly)

* Infection: process where a pathogen invades and multiplies in a host and causes ill health in the host

* Pathogens:

1. microorganisms that cause disease (impair normal function) by invading and multiplying in the host
2. e.g. bacteria, viruses, fungi, worms and protozoa
3. can be intracellular (i.e. in cells) or extracellular (i.e. in blood, tissue fluid and lymph i.e. in the humour)
4. have antigens on their surface antibody A
epitopes

* Antigens: antigen-
binding sites antigen
1. many different types can be found on one pathogen
2. triggers an immune response when specific parts of the antigen called epitopes
are recognised by immune cells or antibodies

* Epitopes: antibody B

1. parts of a single antigen

2. each have a specific conformation complementary in shape and charge to a specific antigen-binding site of an antibody or
T cell receptor or B cell receptor

* Cells of the Immune System:

1. Phagocytes

a) macrophages
 an antigen presenting cell (APC) which resides in tissues
 are produced when monocytes in the blood enter tissues and differentiate
b) dendritic cells
 an antigen presenting cell (APC) which resides in tissues
c) neutrophils
 found in blood

Prepared by: Mrs Selvamani Nair, Ms Michelle Nah, Mr Ngan Wei Yeong 1
 Raffles Institution Infectious Diseases 2019
2. Lymphocytes

T lymphocytes/cells
Originate from haematopoietic stem cells in bone marrow but
differentiate in the thymus to form naïve T cells
Each T cell has a specific T cell receptor (TCR) on its surface
A TCR can only recognise and bind to a specific,
complementary processed peptide of a peptide-MHC
complex on an antigen-presenting cell (APC)
naïve T cell
when activated by APC
B lymphocytes/cells
Originate from haematopoietic stem cells in the bone marrow and
differentiate in the bone marrow to form naïve B cells
Each B cell has a specific B cell receptor (BCR) on its surface
A BCR can recognise and bind to a specific complementary
unprocessed antigen of a pathogen.
Antigen binding site of the BCR and the specific antibody
produced in response to the antigen are the same.
naïve B cell
when activated by helper T cell

effector T cells memory T cells plasma B cells memory B cells

helper T cells cytotoxic T cells
(CD4+ T cells) (CD8+ T cells)
When a specific naïve T cell is activated by a specific antigen
presenting cell, it undergoes clonal expansion and
differentiation to form effector T cells (i.e. helper T and
cytotoxic T cells) and memory T cells
produce
antibodies
When a specific naïve B cell is activated by a specific helper T
cell, it undergoes clonal expansion and differentiation to form
effector B cells (i.e. plasma B cells) and memory B cells

Helper T cell: activates naïve B cell so that it can undergo
clonal expansion and differentiation
Cytotoxic T cell: involved in cell-mediated response and
hence protects against intracellular pathogens by killing
cells that contain pathogens
Plasma B cell: produces antibodies which are involved in the
humoral response and protect against extracellular pathogens
and toxins secreted by pathogens. Plasma B cells have an
extensive network of rough endoplasmic reticulum needed for
synthesis of large amounts of antibodies (globular proteins) which
are secreted by exocytosis.

Memory T cell: When re-exposed to the same pathogen, Memory B cell: When re-exposed to the same pathogen,
memory T cells will recognise it and undergo faster clonal memory B cells will recognise it and undergo faster clonal
expansion & differentiation into effector T cells, mounting a expansion & differentiation into antibody-secreting plasma B
faster and stronger secondary immune response cells, mounting a faster and stronger secondary immune
(compared to the primary immune response). Memory cells response (compared to the primary immune response). Memory
also confers long term immunity to a specific pathogen. cells also confers long term immunity to a specific pathogen.
* 5 steps in immune response:
1 2 3 4 5
Pathogen encounterInnate immune response Antigen presentationAdaptive immune responseRemoval of pathogen
and immunological
memory
1 * Physical and chemical barriers of the innate immune system prevent entry of pathogens
When these barriers are breached  the pathogen enters the body tissues
2
This will cause phagocytes such as macrophages in the body tissues
(A) to engulf the pathogens by phagocytosis and
(B) to induce inflammation  aim: to recruit more phagocytes

* (A) Phagocytosis by macrophage:

1. Bacterium becomes attached to membrane evaginations called
pseudopodia
2. Bacterium is ingested, forming phagosome which then fuses with
lysosome forming phagolysosome
3. Lysosomal enzymes digest the captured material and digested
products are released from the cell by exocytosis

* (B) How are more phagocytes recruited during inflammation?

Pathogen triggers macrophages at site of infection (i.e. infected tissues)
to release signalling molecules, chemokines and cytokines which (see table)
Effect of chemokines and Effects
cytokines on blood vessels
Vasodilation: widening of blood More phagocytes can be carried
vessels  increases blood flow to site of infection from the
(causing redness, heat) bloodstream
Vasopermeability: increase More phagocytes can be recruited
permeability of blood capillaries to site of infection from
Fig: Phagocytosis
bloodstream
Fluid accumulation at site of
infection (causing swelling)
Release of inflammatory
mediators (causing pain)
Phagocytes that are recruited to site of infection during inflammation are
(1) macrophages  carry out phagocytosis and clear pathogen or function as an antigen presenting cell (APC)
(2) neutrophils found in bloodmigrate to site of infectioncarry out phagocytosis and then die and form pus

* If a pathogen evades the innate immune system (i.e. the barriers and cellular components of the innate immune system
are breached)
 the adaptive immune system is activated by antigen presentation.
Prepared by: Mrs Selvamani Nair, Ms Michelle Nah, Mr Ngan Wei Yeong 2
 Raffles Institution Infectious Diseases 2019
3 * Antigen Presentation:

1. Antigen uptake:Phagocytic APC engulfs pathogen

2. Processing of antigen:antigen is ‘cut up’ into short peptides inside phagolysosme
3. Peptides of antigen bind to MHC protein forming peptide-MHC complex
4. Peptide-MHC complex is transported via vesicles fuses with the cell surface membrane of the APC.
5. The peptide on the MHC complex is now ready for presentation to the complementary T cell receptor (TCR) on a specific
naïve T cell. (See (*****) next to pt. 2. below to see what occurs next)
4. Vesicle with peptide-MHC
1. Phagocytic APC complex fuses with the
engulfs pathogen membrane of APC.
with pseudopodia 5. Peptide on peptide-MHC
complex ready for
presentation to naïve T cells

3. Processed peptide of
antigen binds
to MHC protein forming
peptide-MHC complex
2. Processing of antigen:
- Fusion of phagosome and Rough ER produces
lysosome to form phagolysosome MHC protein
- Antigen of pathogen processed into
short peptides in phagolysosome

Fig: Antigen presentation in a phagocytic cell

3 * How naïve cells become activated and the adaptive immune response:
4
pathogen 1. Unique B cell receptor (BCR) on a specific 1. An antigen presenting cell (e.g. dendritic cell) forms
BCR
naïve B cell binds to complementary antigen pseudopodia around the pathogen which fuse and encloses
of the pathogen the pathogen in a phagosome by phagocytosis.
2. Receptor-mediated endocytosis of the antigen- 2. The pathogen is processed
pathogen occurs as the cell surface presenting cell and a peptide of the antigen
naïve
membrane invaginates and pinches off as a (APC)
B cell binds to a MHC protein to form a
endocytic vesicle peptide-MHC complex which is
3. A specifc then presented on the surface of
3. The pathogen is processed and
naiveT cell the APC (*****)
a peptide of the antigen binds to a 4. The APC
with a
MHC protein to form a peptide- secretes
MHC complex which is then specific T cell
receptor (TCR) TCR cytokines that
4. A specifc displayed on the surface of the activates the
helper T cell naïve B cell (an APC) binds to the
complementary naïve naive T cells.
with a specific 5. The helper T
peptide-MHC T cell
T cell receptor cell secretes peptide-MHC
complex
(TCR) binds to TCR cytokines that complex of the APC.
the activates the
complementary specific B cell
peptide on the
peptide-MHC
complex of the
cytokines
specific naïve B helper T cell cytotoxic T cell memory T cell
cell
5. The specific activated naive T cells undergo clonal
5 6. The specific B cell undergoes expansion to form many identical daughter cells and
clonal expansion to form many plasma
differentiation into effector T cells (cytotoxic T cells and T
identical daughter cells and B cells
helper cells) and memory T cells
differentiation to form
antibody-secreting plasma B 6. The helper T cells secrete cytokines which
cells (effector cells) and (a) activate naïve B cells which form plasma B cells and
memory B cells (not shown) antibodies (b) stimulate macrophages to attack infected cells.(not shown)
7. The antibodies then destroy and clear the extracellular
pathogen by neutralisation, opsonisation and agglutination 7. The cytotoxic T cells kill cells infected with intracellular
(pg.4) pathogens like viruses by producing perforins and
granzymes (see pg. 4)
8. If the body is re-exposed to the same antigen, the memory
B cells, rapidly undergo clonal expansion and differentiation 8. If the body is re-exposed to the same antigen, the memory T
into many plasma B cells that can rapidly manufacture large cells rapidly undergo clonal expansion and differentiation
quantities of antigen specific antibodies into many helper T cells and cytotoxic T cells.

* Clonal selection is a process proposed to explain how a single B or T cell that recognizes an antigen that enters the body is selected
from the pre-existing cell pool of differing antigen specificities and then reproduced to generate a clonal cell population that eliminates
the antigen.
* Clonal expansion and differentiation: refers to the repeated division of cells by mitosis and specialisation of cells due to differential
switching on of genes respectively.

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* Humoral response:

Protect against extracellular pathogens and toxins (i.e. those found in the blood)

1. Neutralisation of pathogen:

binding of antigen binding site of prevents binding of pathogen/toxin to host cell

antibody to pathogen/toxin receptor and hence prevent entry into host cell

2. Opsonisation to facilitate phagocytosis:

after binding of antigen binding of Fc portion of antibody signals from the Fc receptors on the phagocyte
binding site of antibody to Fc receptors on phagocyte promotes phagocytosis of the pathogen
to pathogen

3. Agglutination

binding of each antigen clumping or promotes phagocytosis of the pathogen

binding site of an antibody to aggregation of (Thus agglutination reduces the number of infectious units to
2 pathogens simultaneously pathogens be dealt with and prevents the pathogen from spreading)

bacterium binding of
pseudopodia
one
of phagocyte
antibody to
2 bacteria
toxin virus
Fc receptor

Neutralisation Opsonisation Agglutination

* Cell-mediated response:

Protect against intracellular pathogens by killing cells that contain pathogens (e.g. a virally infected cell)

TCR on cytotoxic T cells recognise infected Cytotoxic T cells release

target cells which display short peptides 1. perforins that make pores in the infected cell’s membrane and
from antigen of pathogen presented on a 2. granzymes that diffuse through the pores and activate enzymes in
MHC the cell that triggers apoptosis of the virus infected cell

Perforin creates pores in infected cells for

granzymes to enter and activate enzymes in
infected cell that triggers apoptosis

apoptosis of
granzyme infected cell

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* Antibodies:
Disulphide
1. aka immunoglobulins bonds Antigen-
2. are globular proteins secreted by plasma B VH VH binding site
cells VL
VL Fab
3. made up of 4 polypeptide chains
2 identical light chains and 2 identical CL
heavy chains CL Light chain
 hence has a quaternary structure which is Hinge region
held together by ionic and hydrogen bonds, Variable (V)
hydrophobic interactions (not shown in figure) regions
and disulfide bridges between the R groups of CH CH
the amino acids of the 2 heavy chains and the Constant (C) Fc
heavy chains and the light chains regions

Fab: Fragment antigen-binding Heavy chain

Fc: Fragment crystallisable
VH: variable region of heavy chain
VL: variable region of light chain
CH: constant region of heavy chain Structure of IgG:
CL: constant region of light chain

* Antibody Structure and Function:

Structure
Antigen binding site of a specific antibody
is complementary in shape to a specific epitope of
an antigen due to the precise folding of the variable
heavy and light chains that gives rise to its unique 3D
structure
Fc region of antibody/constant region of heavy
chain has a conformation that is complementary in
shape to Fc receptors on phagocytes
Disulfide bridges between heavy and light chains /
two heavy chains
Each antibody has a hinge region
Each has two antigen binding sites
Constant region of heavy chains
Function
Hence antibodies can carry out neutralisation by binding to
specific epitope of an antigen of pathogen thus preventing
pathogen from binding to host cell receptors and infecting the
host cells and at the same time facilitates agglutination
Hence opsonisation can occur as once antibodies bind to the
pathogen, Fc regions of antibodies bind to Fc receptors of
phagocytes and promote phagocytosis
This gives stability to the quaternary structure by holding the
heavy and light chains together / heavy chains together
This give antibody flexibility when binding to
epitopes/antigen/pathogen that are variable distances apart
Each antibody can bind to two epitopes/antigens at the same time
which will cause pathogens to aggregate/agglutinate/clump
together to facilitate clearance by macrophages;
determine the class of antibody thus their different functions

* Antibody diversity:

1. involves 3 genes: 2 genes coding for light chains (kappa and lambda) &1 gene coding for heavy chains
2. due to a. somatic recombination b. somatic hypermutation c. class switching
3. occurs during B cell development
immature B cell during B cell maturation naive B cell after activation of naïve B plasma B cell
cell
1. somatic recombination 2. somatic hypermutation
3. class switching
in bone marrow
in lymph nodes

Heavy chain gene

1. has many different variable (V), diversity (D), joining (J) and constant (C) V gene D gene J gene C gene
gene segments segments segments segments segments
2. the V, D and J gene segments contribute toward the variable region of
the heavy chains (VH)
3. The C segments contribute to the constant region of the heavy chain (CH)

Light chain gene

1. has many different variable (V), joining (J) and constant (C) gene
V gene J gene C gene
segments
segments segments segments
2. the V and J gene segments contribute toward the variable region of the
heavy chains (VL)
3. The C segments contribute to the constant region of the light chain (CL)

* Somatic recombination:

1. There are multiple gene segments at heavy and light chain genes
2. Somatic recombination is a form of DNA rearrangement where various gene segments are joined together randomly,
and some intervening segments are enzymatically removed followed by rejoining of remaining sequences.
3. At the Ig heavy chain gene locus, one V segment, one D segment and one J segment are randomly joined to form a
single VDJ exon
4. At the Ig light chain gene locus, one V segment and one J segment are randomly joined to form a single VJ exon

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1. Somatic recombination in heavy chain gene

VDJ recombination occurs

at the heavy chain gene
DNA during B cell maturation in the
bone marrow

1.During D and J
rearrangement, one D
segment and one J segment
are joined and the intervening
DNA sequences are enzymatically
removed

2.During V and DJ
rearrangement, one V
segment is joined to the DJ
DNA
segment and the intervening
sequences are enzymatically
removed

pre-mRNA 3.The DNA segment then

undergoes transcription

4.The resulting pre-mRNA

undergoes RNA splicing
during which the introns are
mRNA excised and the VDJ exon and
C exon are joined

5.The mature mRNA with the

VDJC exons joined enters the
cytoplasm where it undergoes
translation to form the
heavy chain protein specific heavy chain protein
with the specific V, D, J and C
domains

2. Somatic recombination in light chain gene

VJ recombination occurs
DNA at the light chain gene
during B cell maturation in the
bone marrow

1.During V and J
rearrangement, one V
DNA segment and one J segment
are joined and the
intervening sequences are
enzymatically removed

2.The segment then

pre-mRNA undergoes transcription

3.The resulting pre-mRNA

undergoes RNA splicing
during which the introns are
excised and the VJ exon and
C exon are joined
mature mRNA
4.The mature mRNA with the
VJC exons joined enters the
cytoplasm where it
undergoes translation to
form the specific light chain
protein with the specific V, J
light chain protein and C domains

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* Somatic hypermutation:

1. Somatic hyper-mutation is random point mutation in the rearranged VDJ / VJ regions in activated B cells
2. Further diversifies variable regions of antibody for antigen binding (due to slight amino acid differences which result due to
the mutation)
3. It occurs during clonal expansion of the activated B cells
3. Some point mutations result in the B cells expressing low affinity Ig chains on their cell surface membrane and some point
mutations result in the B cells expressing higher affinity Ig chains on their cell surface membrane
4. B cells that express higher affinity BCR on their cell surface membrane are selected for clonal expansion & differentiation.
This is called affinity mutation
5. The resulting plasma B cells and memory B cells will have BCRs with higher affinity antigen binding sites for a specific antigen.
The plasma B cells will also produce antibodies with higher affinity antigen binding sites for a specific antigen.

* Class Switching:

1. Class switching is DNA rearrangement at the constant gene segment of the heavy chain gene locus in activated B
cells
2. Allows for the production of antibodies with same antigen binding site, but different function

Heavy chain gene

 VDJ recombination complete
 occurs after B cell activation

1. Before class switching, VH DNA

linked to Cµ. Hence Ig M
antibodies are produced.

2. During class switching, DNA

rearrangement occurs and VH
DNA becomes linked to
another constant gene
segment of the heavy chain
gene locus, for example, Cγ
Hence Ig G antibodies are
produced

3. Only the constant region of the

heavy chain (CH) of the
antibody changes, due to the
change of C gene segment
from Cµ to Cγ. Hence Ig G is
produced instead of Ig M.

* Active and Passive Immunity:

Immunity

Innate immunity Adaptive immunity

Active Passive

Natural Artificial Natural Artificial

e.g. when e.g. memory e.g. anitbodies e.g. when
infected by a cell production passed frommum to antiserum with
pathogen due to child via antibodies
vaccination placenta/breastmilk received from
another host

Active immunity Passive immunity

Immune Yes No
response
Antibody Produced by individual’s own immune system in Transferred to recipient without the participation of the
production response to antigens recipient’s immune system
Duration Long term immunity conferred since memory cells Short-lived since there is no formation of memory
are formed after primary immune response cells

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* Immunological Memory:

 When naïve B cells are activated, clonal expansion and differentiation will produce plasma B cells and memory B cells. Memory
B cells, can remain in the body for years (or even a lifetime).
 Immunological memory is established to ensure rapid re-induction of antigen-specific antibodies on subsequent encounters
with the SAME pathogen, thus providing long-lasting protection against it.
 Since the individual does not presents exhibit any symptoms of infection upon exposure, the individual is said to be immune.

Primary immune response Secondary immune response

Presence of lag period: 3-6 days between encountering antigen &
production of antibody (time required for clonal expansion &
differentiation of T cells to effector T cells, then B cells to plasma B
cells)
Response is weaker: antibody concentration rises gradually, and
peaking at a lower level after a longer time
No Memory
Response is faster: within hours (memory B & T cells
can be reactivated easily, leading to a faster response
to re-exposure to the same pathogen)
Response is stronger: antibody concentration peaks
at much higher level and quicker
Has memory: Confers long term immunity to the SAME
pathogen if it is encountered again

* Vaccination:

 Vaccination: the intentional administration of an antigen, usually a harmless form of a pathogen in order to induce a
specific adaptive immune response that protects the individual against later exposure to the pathogen due to the production
of memory cells. The individual should not develop disease symptoms.

 A form of artificial active immunity: immune response is activated artificially by introducing antigens into the body to initiate
primary immune response

 Uses the property of immunological memory to provide long-lasting protection against infectious diseases so that when
exposed to the actual pathogen, the memory cells trigger a secondary immune response that is faster and stronger

 How vaccines cause primary immune response (general)

 The vaccine contains an attenuated/ weakened/ killed form of pathogen:
 The modified pathogen is no longer able to cause disease (removal of virulence gene which allows pathogen to successfully
replicate & cause disease in host) but it still retains immunogenic effect (ability to elicit an immune response) because
characteristic surface antigens of the pathogen are retained and can be recognised by APCs
 Adaptive immune response occurs: specific naïve B and T cells are activated to become effector and memory B and T cells
respectively (i.e. primary immune response is triggered)

 How vaccines protect from actual pathogen

 If the previously vaccinated individual is exposed to the virulent pathogen with the SAME antigen, memory B & T cells will
quickly recognize the surface antigen of the pathogen and mount a faster and stronger secondary immune response
 Memory B cells rapidly undergo clonal expansion and differentiation and develop into antibody-secreting plasma B cells
 The plasma B cells produce a large number of antibodies which are able to bind to & inactive the virulent pathogen to prevent
it from infecting healthy host cells in the individual
 Hence vaccines confer long term immunity. However because memory cells may not survive a life time, booster shots may
be required.

* Example of a type Vaccine:

Live, attenuated vaccine

Definition Attenuation: weakening of pathogenic bacteria/ virus by making it less virulent, done through the removal of
the virulence gene
Advantages Closest thing to a natural infection  will elicit a strong immune response with just a small dosage & confer
longer-term protection
Disadvantages  Possibility of reversion to virulent form by mutation  can then cause disease
 Needs to be refrigerated to stay potent

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* Benefits and Risks of Vaccinations:

Benefits Risks
 Vaccines protect individuals against disease  While live, attenuated vaccines are
 Deaths due to illness can be prevented more effective than inactivated
 Long-term disabilities (e.g. infertility, deafness, blindness) due to diseases can vaccines, they pose the risk of
be prevented reversion to virulence to cause
disease
 Herd immunity  Some people may be allergic to
- If there are sufficient individuals in a population (95%) who are components in vaccines
immunised due to vaccinations, transmission of the disease in a  Immunity developed after vaccination
community is less likely due to the low chance of a an individual who is may not be as effective as natural
not vaccinated (e.g babies, elderly) coming into contact with an immunity due to a real infection.
infected individual. Hence the unvaccinated are protected because (Hence booster shots may be
transmission is prevented. needed.)
 Some diseases may be completely eradicated by vaccination, reducing  Some pathogens mutate very
human suffering & future costs of treatment e.g. Smallpox (unique quickly and a new vaccine is need
circumstances) every year
- Human was the only host  Excessive vaccination may reduce
- Virus did not mutate effectiveness of immune system to
- Infected people easily identified & isolated, no symptomless carriers respond to new infections
- Compulsory live, attenuated vaccine that was also heat stable; no boosters
required (note: some people are unsuitable for vaccines)

* Treatment of Bacterial Infections with Antibiotics:

 Definition
- Natural substances obtained from microorganisms e.g. certain fungi & bacteria
- Inhibit growth of bacteria or kill bacteria by disrupting metabolism of prokaryotic cells (make use of differences between
prokaryotic & eukaryotic cells to target bacteria without harming human cells)

 Role
- Prevent spread of bacteria within the body and hence aid recovery
- Prevent death as consequences may be fatal without treatment
- Prevent transmission of disease from individual to individual in a population

 Types
- Bacteriostatic: inhibits cell division of bacteria
- Bactericidal: kills bacteria when bacteria are in the process of undergoing cell division by binary fission
- Can disrupt different metabolic pathways e.g. cell wall synthesis, protein synthesis (translation), nucleic acid synthesis

 Disrupting cell wall synthesis

- Targets bacterial cell wall (usually made of peptidoglycan) because humans do not have cell walls
- E.g. penicillin (a ß-lactam antibiotic: four-membered, nitrogen containing ß-lactam ring structure)
1. Bactericidal: only effective when bacteria is growing/ making new cell wall as it disrupts cell wall synthesis
2. Penicillin acts as a competitive inhibitor & binds to active site of transpeptidase
3. Inhibition to formation of cross-links between adjacent chains of peptidoglycan
4. Bacterial cell wall becomes weakened when bacterial cells carry out division
5. An increase in turgor pressure against the weakened cell wall causes bacteria to swell & lyse

 Disrupting protein synthesis

- Ribosomes of prokaryotes are 70S while ribosomes of eukaryotes are 80S
- E.g. streptomycin: binds to small subunit (30S) of bacterial ribosome such that initiator tRNA cannot bind to small
subunit
- E.g. tetracycline: blocks aminoacyl-tRNA from attaching to the A site of bacterial ribosome

 Disrupting nucleic acid synthesis

- Enzymes used are different in conformation e.g. bacterial RNA polymerase ≠ human RNA polymerase
- E.g. rifampin: inhibits RNA synthesis by binding to bacterial RNA polymerase  preventing transcription

 Administration of antibiotics
- Consumption at evenly spaced intervals: maintain a concentration of antibiotic in the body that is lethal to the bacteria
(note: concentration will not increase as it is being metabolised as well)
- Complete course of antibiotics: antibiotics will kill all susceptible strains, so that the immune system can focus on
tackling the resistant strains (note: development of antibiotic resistance cannot be prevented, happens via spontaneous
mutation)

 Development of antibiotic resistance

- Failure to complete course of antibiotics  some bacteria survive spontaneous mutation in bacterial population
produces antibiotic-resistant strains  transfer of antibiotic-resistance genes from bacterium to bacterium via
conjugation/ transduction/ transformation
- When antibiotic is given, it acts as a selection pressure  those with antibiotic-resistance gene survive, reproduce and
pass the allele to daughter cells whereas those that are susceptible die  over a few generations, microevolution
occurs  increased frequency of antibiotic-resistant allele in population
- Examples of spontaneous mutations that can lead to resistance: mutations which led to the production of enzymes to
break down antibiotics, or membrane proteins that inactivate/ pump out antibiotics

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* Treatment of Bacterial Infections- Use of Virus (Bacteriophage) vs. Antibiotics:

 Virus / phage is very specific and will only attack a particular bacterial strain (vs. antibiotics which kill different types of
bacteria, including useful ones)
 As bacteria evolve resistance, viruses can also evolve to overcome resistance (vs. bacteria evolve resistance to antibiotics
over time)
 Viruses can replicate once they infect the bacteria
- Only a small quantity of virus needed, as it can replicate once in the host to produce more viruses
(vs. antibiotics are metabolised and eliminated from the body)
 Viruses stop reproducing once specific target bacteria are destroyed
 Less likely to cause side effects/ allergies to patient

* Treatment of Viral Infections: Design of Drugs

Target Aspect Possible Drugs

Attachment Drug that binds to specific host cell plasma membrane receptor  prevents virus from binding and
entering via endocytosis
Replication Drug that carries antisense RNA that will bind to viral RNA to form dsRNA  ribosome cannot bind 
translation cannot occur to replicate viral RNA
Nucleoside analogs (resemble nucleosides): impair virus’s ability to extend its RNA during replication as
incorporating analog into RNA prevents addition of next nucleotide
Important virus- Drugs that inhibit RNA-dependent RNA polymerase (in influenza)  prevents viral replication
specific Drugs that inhibit viral reverse transcriptase/ integrase (in HIV)  prevents integration of viral genome
enzymes/ into host cell chromosomes  prevents viral replication
proteins Drugs that inhibit viral protease (in HIV)  cannot hydrolyse polyproteins into smaller functional protein
components
Drugs that bind to viral polyproteins (in HIV)  cannot be cleaved by viral protease

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* Pathogens & Diseases:
Disease Influenza
Pathogen Influenza virus
Target cells Epithelial cells of respiratory tract: haemagglutinin on virus
binds to sialic acid receptor found on epithelial cell membrane
Transmission Droplets of moisture from lungs of infected person/ infected
bird droppings
Pathogenicity  Virus settles on mucous membrane lining the nose,
pharynx, trachea, bronchi
 Neuraminidase enzyme on surface of virus helps virus
penetrate mucoproteins (glycoproteins) in mucus layer
 Haemagglutinin binds to sialic acid receptors on cell
membrane of epithelial cells lining respiratory tract 
virus penetrates into host cells & replicates within them
 Incubation period: 24-48h, after which infected epithelial
cells are destroyed  leads to inflammation & build-up
of dead epithelial cells in airways
 Symptoms: runny nose, scratchy throat
 Viral replication  weakening of epithelial layer 
respiratory passage more susceptible to secondary
bacterial infections, leading to fatal diseases like
pneumonia
How viral infections lead to cell death
 Immune system recognises infected cells  lysed
 Budding off causes progressive loss of host plasma
membrane  cells eventually die
 Host cell transcription and translation mechanism has
been taken over by virus producing viral proteins,
compromising vital functions of the cell  cell death
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2019
AIDS (acquired immunodeficiency syndrome)
Human Immunodeficiency Virus (HIV)
T helper cells or macrophages of immune
system: gp120 on virus binds to CD4 receptor
on host cell surface membrane with the help of
a co-receptor
Primarily through unprotected sexual contact/
exposure to infected blood & blood products
Can also be from mother to child via the
placenta, during childbirth, or during
breastfeeding
 Once virus enters bloodstream, it targets T
helper cells & macrophages: has a very
strong affinity for & binds to CD4 receptors
 Virus infects T helper cells: infected T
helper cells destroyed  T helper cell
levels fall as more & more are destroyed
 Virus infects macrophages: macrophages
may survive HIV infection because they are
not lysed by the virus  act as reservoirs
 HIV can pass from cell to cell in an
individual, or to other individuals while
remaining undetected
 High rate of mutation of virus during
replication  altered proteins on surface of
virus  escapes recognition & elimination
by immune system  evolves rapidly
within body
 Increasing loss of T helper cells
 impaired immune responses
 increasingly susceptible to opportunistic
diseases
 Secondary infections e.g. tuberculosis
 death
 Integration of viral dsDNA into host
genome is random  may result in the
activation of a proto-oncogene to become
an oncogene/ inactivation of a tumour
suppressor gene  development of cancer
e.g. Karposi sarcoma is a rare skin cancer
that is more prevalent in AIDS patients
11
Tuberculosis
Mycobacterium tuberculosis
 Obligate aerobe: requires oxygen for aerobic respiration, survival &
growth
 Peptidoglycan cell wall + mycotic acid layer (rich in lipids)
Primary infection (pulmonary TB): lungs
Airborne: transmitted in fine aerosol droplets (from ruptured tubercle) when
an infected person with active TB sneezes/ coughs & an uninfected person
inhales the droplets [Spreads rapidly in overcrowded conditions]
 Once bacteria is inside lungs, alveolar macrophages phagocytose the
bacteria
 In the phagosome, bacteria inhibits fusion of phagosome with
lysosome  no phagolysosome formed, no lysosomal enzymes kill
the bacteria
 Bacteria survives, continues to multiply inside macrophage that
phagocytosed them + more macrophages are brought to infected
alveolus
 A tubercle (ball-like clustering of cells like macrophages) is formed. At
the centre of the tubercle, cell death by necrosis occurs  involves
rupturing of cell membrane & releasing of cell content (i.e. bacteria)
(vs. apoptosis: shrinking)
 Disease may be arrested at this stage & remain latent for years:
persons who have TB infection, but not TB disease, do not spread
disease to others (only sign is a positive reaction to tuberculin skin
test/ TB blood test)
 TB disease: tubercle ruptures  bacteria spills into a bronchiole &
spreads throughout the lungs  productive cough that facilitates
aerosol spread of bacteria
 Rupturing of tubercles  formation of cavities  lungs progressively
destroyed
 TB often first opportunistic infection to strike HIV-positive people: if
person has dormant TB, HIV likely to reactivate TB disease OR if
person is previously uninfected, made more susceptible
 Raffles Institution Infectious Diseases
Treatment  No treatment for most people: bed rest & perhaps
aspirin/ paracetamol to alleviate headaches & fever
 Antiviral drugs: oseltamivir (Tamiflu) & zanamivir
(Relenza)  neuraminidase inhibitors: halt spread of
virus in the body (suited towards Influenza A & B strains
only) + amantadine & rimantiadine  block a viral ion
channel: prevents virus from infecting cells (Influenza A
only)
 Antibiotics: prevent secondary bacterial infection like
pneumonia
Prevention Vaccination: contains purified & inactivated material from 3
common influenza viral strains
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2019
 High rates of virus reproduction & mutation
of virus  generally 3 agents administered
in combination
 Types of retroviral drugs include reverse
transcriptase inhibitors, protease
inhibitors and integrase inhibitors
(inhibit respective enzymes) & entry
inhibitors (block interaction between HIV
envelope and CD4/ co-receptor, or prevent
fusion of viral & host cell membranes 
block entry of HIV into cells)
 Sustained treatment  suppression of
viral replication  dramatically enhanced
life-expectancy of infected individuals
12
 6 months of daily treatment with a combination of at least 2
antibiotics: combination used to minimise risk of developing
resistance & achieve an additive effect against bacteria; skipping of a
dose/ stopping before complete course increase chances of
development of resistance  potentially serious, difficult to treat,
requires longer course
 Antibiotic inhibits synthesis of mycobacterial cell wall e.g. antibiotic
isionizid, or inhibits RNA synthesis during transcription e.g. antibiotic
rifampin (interferes with prokaryotic RNA polymerase)
Vaccination: BCG vaccine prepared from live, attenuated Mycobacterium
bovis (bovine TB); often used to prevent spread of TB among children