Cells

Cells are like tiny factories — each with specialized departments working together to keep the organism alive.

Info: Board Coverage AQA Paper 1 | Edexcel A Paper 1 | OCR (A) Paper 1 | CIE Paper 2

The cell theory, developed by Schleiden and Schwann (1838—1839) and later refined by Virchow, States:

1. All living organisms are composed of one or more cells.
2. The cell is the basic unit of structure and organisation in organisms.
3. All cells arise from pre-existing cells (biogenesis).

Light microscopy uses visible light ($\lambda \approx 400$—$700\ \mathrm{nm}$) focused through Glass lenses. The maximum resolving power of a light microscope is limited by diffraction:

$d = \frac{0.61\lambda}{n\sin\theta}$

Where $d$ is the minimum resolvable distance, $\lambda$ is the wavelength, $n$ is the refractive Index of the medium, and $\theta$ is the half-angle of the cone of light. For light microscopy, $d \approx 200\ \mathrm{nm}$Giving a maximum useful magnification of approximately $\times 1500$.

Electron microscopy uses a beam of electrons ($\lambda \approx 0.005\ \mathrm{nm}$) instead of Light, giving a resolving power of approximately $0.2\ \mathrm{nm}$ and effective magnifications up To $\times 2000000$.

Feature	Light Microscope	Electron Microscope
Resolution	$\approx 200\ \mathrm{nm}$	$\approx 0.2\ \mathrm{nm}$
Magnification	Up to $\times 1500$	Up to $\times 2000000$
Specimen	Living or dead	Dead only (vacuum required)
Contrast	Staining (dyes)	Heavy metal staining
Cost	Low	High

Transmission electron microscopy (TEM): electrons pass through thin sections; produces 2D images Of internal ultrastructure.

Scanning electron microscopy (SEM): electrons are scattered from the surface; produces 3D images Of surface features.

Magnification is defined as:

$\mathrm{Magnification} = \frac{\mathrm{Image\ size}}{\mathrm{Actual\ size}}$

Laser scanning confocal microscopy uses laser light and pinhole apertures to eliminate Out-of-focus light, producing sharp optical sections through thick specimens. This allows 3D Reconstruction without the need for physical sectioning.

### 1.3 Cell Fractionation

Cell fractionation is the process of breaking open cells and separating organelles by differential Centrifugation:

1. Homogenisation: cells are broken open in an isotonic, buffered, ice-cold solution. The isotonic condition prevents osmotic lysis or shrinkage; the cold temperature reduces enzyme activity that would degrade organelles; the buffer maintains pH.
2. Filtration: the homogenate is filtered through a gauze to remove debris.
3. Differential centrifugation: the filtrate is centrifuged at increasing speeds. At each step, the heaviest (densest) organelles form a pellet at the bottom of the tube:

Centrifugation Speed	Pellet	Supernatant Contains
Low ($1000\ \mathrm{g}$10 min)	Nuclei	All other organelles
Medium ($10000\ \mathrm{g}$20 min)	Mitochondria, lysosomes	Lighter organelles
High ($100000\ \mathrm{g}$60 min)	Ribosomes, microsomes (ER fragments)	Cytosol

All cells fall into one of two categories based on whether they possess a membrane-bound nucleus.

Definition. A prokaryotic cell lacks a membrane-bound nucleus and membrane-bound organelles. Its DNA is a single circular molecule located in the nucleoid region. Prokaryotes include bacteria And archaea.

Definition. A eukaryotic cell possesses a membrane-bound nucleus and membrane-bound Organelles. Its DNA is organised into linear chromosomes within the nucleus. Eukaryotes include Animals, plants, fungi, and protists.

Feature	Prokaryote	Eukaryote
Nucleus	Absent (nucleoid region)	Present, with nuclear envelope
DNA	Circular, naked; no histones	Linear, associated with histones
Ribosomes	$70\mathrm{S}$ ($50\mathrm{S} + 30\mathrm{S}$)	$80\mathrm{S}$ ($60\mathrm{S} + 40\mathrm{S}$)
Membrane-bound organelles	Absent	Present (mitochondria, ER, Golgi, etc.)
Cell wall	Peptidoglycan (bacteria)	Cellulose (plants) or chitin (fungi)
Size	$1$—$5\ \mu\mathrm{m}$	$10$—$100\ \mu\mathrm{m}$
Reproduction	Binary fission (asexual)	Mitosis, meiosis
Flagella	Simple, rotating	Complex, $9 + 2$ microtubule arrangement

### 2.2 Prokaryotic Cell Structure

Prokaryotic cells possess several structures not found in eukaryotes:

• Capsule (slime layer): a protective polysaccharide layer outside the cell wall; aids adhesion and protects against phagocytosis.
• Plasmid: small, circular, extra-chromosomal DNA that can carry genes for antibiotic resistance (R-plasmids). Plasmids replicate independently of the main chromosome and can be transferred between bacteria by conjugation.
• Pilus (plural: pili): protein filaments that attach bacteria to surfaces or to other cells. Sex pili are used in conjugation to transfer plasmids.
• Flagellum (plural: flagella): a rigid, rotating filament driven by a proton motor at the base, used for locomotion.

Prokaryotic cells also share some structures with eukaryotes:

• Cell membrane (phospholipid bilayer): controls exchange of substances.
• Cytoplasm: site of metabolic reactions; contains ribosomes and plasmids.
• Mesosomes: infoldings of the cell membrane — thought to be artefacts of fixation rather than genuine structures (not examined on most boards).

The nucleus is the largest organelle ($d \approx 5$—$10\ \mu\mathrm{m}$), enclosed by a nuclear Envelope consisting of two phospholipid bilayers. The envelope is perforated by nuclear pores (diameter $\approx 9\ \mathrm{nm}$) that allow selective transport of mRNA, tRNA, and proteins Between the nucleus and cytoplasm. Transport through nuclear pores is regulated and requires energy.

The nucleus contains:

• Chromatin: DNA loosely associated with histone proteins; condenses into visible chromosomes during cell division.
• Nucleolus: a dense region within the nucleus where rRNA is transcribed and ribosome subunits are assembled. The nucleolus is not membrane-bound.

Mitochondria ($d \approx 1$—$10\ \mu\mathrm{m}$Length up to $7\ \mu\mathrm{m}$) are the site of Aerobic respiration, specifically the Krebs cycle (matrix) and oxidative phosphorylation (inner Membrane/cristae). They have:

• Outer membrane: permeable to small molecules.
• Inner membrane: highly folded into cristae to increase surface area for the electron transport chain and ATP synthase. Impermeable to most ions.
• Matrix: contains the mitochondrial DNA (circular), $70\mathrm{S}$ ribosomes, enzymes for the Krebs cycle, and the link reaction.

The presence of circular DNA and $70\mathrm{S}$ ribosomes is strong evidence for the endosymbiotic Theory: mitochondria were once free-living prokaryotes that were engulfed by a larger cell.

The endoplasmic reticulum (ER) is a network of membrane-bound flattened sacs (cisternae) Continuous with the nuclear envelope.

• Rough ER (RER): studded with $80\mathrm{S}$ ribosomes on its outer surface. Functions in protein synthesis and folding (especially proteins destined for secretion or for the plasma membrane). Proteins enter the ER lumen where they fold and may be glycosylated.
• Smooth ER (SER): lacks ribosomes. Functions in lipid synthesis, steroid hormone synthesis, detoxification (liver cells have abundant SER), and calcium ion storage.

The Golgi apparatus consists of stacked, flattened cisternae with vesicles at the periphery. It Receives proteins from the RER via transport vesicles, then modifies, sorts, and packages them:

1. Cis face (receiving side): vesicles fuse with the Golgi, releasing their contents.
2. Modification: proteins may be glycosylated (sugar groups added), phosphorylated, or cleaved.
3. Trans face (shipping side): modified proteins are packaged into secretory vesicles for transport to the plasma membrane (exocytosis), lysosomes, or other destinations.

Lysosomes are membrane-bound vesicles ($d \approx 0.1$—$1.0\ \mu\mathrm{m}$) containing hydrolytic Enzymes (lipases, proteases, nucleases) optimised for pH $\approx 5$. They function in:

• Phagocytosis: engulfing and digesting pathogens (in phagocytes).
• Autophagy: breaking down worn-out organelles.
• Apoptosis: programmed cell death.

### 3.6 Other Organelles

• Ribosomes: $80\mathrm{S}$ in cytoplasm, $70\mathrm{S}$ in mitochondria and chloroplasts. Sites of translation.
• Centrioles: cylindrical structures of $9$ triplets of microtubules, found in animal cells (absent in most plant cells). Form the centrosome and organise the spindle during mitosis.
• Chloroplasts (plants only): double-membraned organelles containing thylakoids (site of the light-dependent reactions) and stroma (site of the light-independent reactions/Calvin cycle). Contain circular DNA and $70\mathrm{S}$ ribosomes.

Plant cells possess structures absent from animal cells:

• Cell wall: made of cellulose microfibrils embedded in a matrix of hemicellulose and pectin. Provides mechanical support, prevents osmotic lysis, and determines cell shape. Fully permeable.
• Middle lamella: pectin-rich layer between adjacent cell walls, cementing cells together.
• Permanent vacuole: a large, central, membrane-bound (tonoplast) compartment containing cell sap (a solution of sugars, amino acids, salts, pigments). Functions in turgor maintenance, storage, and waste isolation.
• Plasmodesmata: channels through the cell wall that allow cytoplasmic communication between adjacent plant cells.

Viruses are acellular — they are not cells and do not possess cytoplasm, ribosomes, or cell Membranes. A virion (virus particle) consists of:

1. Genetic material: either DNA or RNA (never both), single-stranded or double-stranded.
2. Protein coat (capsid): made of capsomere subunits; protects the genetic material and aids attachment to host cells.
3. Envelope (in some viruses): a phospholipid bilayer derived from the host cell membrane, with viral glycoproteins embedded.

Viruses are obligate intracellular parasites — they cannot replicate independently. They must Invade a host cell and hijack its metabolic machinery. The basic stages are:

1. Attachment: the virus binds to specific receptor proteins on the host cell surface.
2. Injection/entry: the viral genome enters the host cell (by endocytosis or fusion).
3. Replication: the viral genome is replicated using the host”s enzymes and nucleotides.
4. Synthesis: viral proteins are synthesised on the host’s ribosomes.
5. Assembly: new virions are assembled from the components.
6. Release: virions leave the host cell by lysis (killing the cell) or budding (enveloped viruses).

Viruses are not considered living organisms because they do not carry out respiration, nutrition, Excretion, growth, or response to stimuli independently.

The fluid mosaic model (Singer and Nicolson, 1972) describes the cell membrane as:

• Fluid: phospholipid molecules move laterally within the bilayer; their movement is constrained by hydrophobic interactions but is rapid and continuous. Cholesterol reduces fluidity at high temperatures and prevents crystallisation at low temperatures.
• Mosaic: proteins are embedded in or attached to the bilayer, creating a pattern of different molecules.

Component	Location	Function
Phospholipids	Bilayer	Form the basic structure; partially permeable barrier
Cholesterol	Interspersed between phospholipids	Regulates membrane fluidity and stability
Intrinsic (transmembrane) proteins	Span the bilayer	Transport proteins (channels, carriers); receptors
Extrinsic (peripheral) proteins	On one surface only	Enzymes; cell signalling; cytoskeleton attachment
Glycoproteins	Extrinsic, with carbohydrate chains	Cell recognition; antigenic markers
Glycolipids	Phospholipid with carbohydrate chain	Cell recognition; tissue compatibility

Simple diffusion: passive movement of small, non-polar molecules (O$_2$CO$_2$) down their Concentration gradient through the phospholipid bilayer. Rate depends on: surface area, Concentration gradient, temperature, distance.

Facilitated diffusion: passive movement of larger or polar molecules (glucose, amino acids, Ions) through transmembrane proteins. Two types:

• Channel proteins: water-filled pores selective for specific ions ($\mathrm{Na^+}$ $\mathrm{K^+}$, $\mathrm{Cl^-}$). May be gated (voltage-gated or ligand-gated).
• Carrier proteins: undergo conformational change to shuttle molecules across the membrane.

Osmosis: the net movement of water molecules across a partially permeable membrane from a region Of higher water potential to a region of lower water potential.

$\Psi = \Psi_s + \Psi_p$

Where $\Psi$ is water potential, $\Psi_s$ is solute potential (always negative), and $\Psi_p$ is Pressure potential.

Active transport: the movement of molecules against their concentration gradient, requiring Energy from ATP hydrolysis. Carried out by carrier proteins that act as pumps (e.g., the $\mathrm{Na^+/K^+}$ ATPase pump, which moves $3\ \mathrm{Na^+}$ out and $2\ \mathrm{K^+}$ in per ATP Hydrolysed).

Co-transport (secondary active transport): uses the concentration gradient of one molecule ( $\mathrm{Na^+}$) established by primary active transport to drive the transport of Another molecule against its gradient. This is the mechanism by which glucose is absorbed in the Ileum.

• Temperature: increasing temperature increases kinetic energy, making the membrane more fluid and more permeable. Above a critical temperature, proteins denature and the membrane may become fully permeable.
• Solvents: organic solvents (ethanol, acetone) dissolve the lipid bilayer, increasing permeability.
• pH: extreme pH denatures membrane proteins.

Mitosis produces two genetically identical daughter cells with the same diploid ($2n$) chromosome Number as the parent cell. It occurs in somatic (body) cells for growth, repair, and asexual Reproduction.

Phases:

1. Prophase: chromatin condenses into visible chromosomes (each consisting of two sister chromatids joined at the centromere). The nucleolus disappears. Centrioles move to opposite poles and the spindle forms.
2. Metaphase: chromosomes align at the metaphase plate (cell equator) attached to spindle fibres by their centromeres.
3. Anaphase: centromeres divide; sister chromatids are pulled to opposite poles by the spindle, now called chromosomes. The cell elongates.
4. Telophase: chromosomes decondense; the nuclear envelope reforms; the nucleolus reappears. Cytokinesis (division of the cytoplasm) follows.

### 6.2 The Cell Cycle

The cell cycle consists of:

• Interphase ($G_1$, $S$, $G_2$): cell growth and DNA replication. This accounts for approximately 90% of the cycle.
• Mitosis ($M$ phase): nuclear division.
• Cytokinesis: cytoplasmic division.

Regulation is by cyclins and cyclin-dependent kinases (CDKs). Cyclin concentration rises and Falls cyclically; when cyclin binds to CDK, the complex triggers the next stage of the cell cycle. Mutations in genes regulating the cell cycle (proto-oncogenes and tumour suppressor genes) can lead To uncontrolled division (cancer).

Microscopy calculations appear frequently in examinations. The fundamental relationship is:

$\mathrm{Magnification} = \frac{\mathrm{Image\ size}}{\mathrm{Actual\ size}}$

This can be rearranged to find any one variable when the other two are known. Units must be consistent — convert all measurements to the same unit ( $\mu\mathrm{m}$) before calculating.

Conversion factors:

$1\ \mathrm{mm} = 1000\ \mu\mathrm{m}, \quad 1\ \mu\mathrm{m} = 1000\ \mathrm{nm}, \quad 1\ \mathrm{nm} = 1000\ \mathrm{pm}$

Worked Example 1. A cell is observed under a microscope with a magnification of $\times 400$. The Image of the cell measures $4.8\ \mathrm{mm}$ across. Calculate the actual diameter of the cell.

$\mathrm{Actual\ size} = \frac{\mathrm{Image\ size}}{\mathrm{Magnification}} = \frac{4.8\ \mathrm{mm}}{400} = 0.012\ \mathrm{mm} = 12\ \mu\mathrm{m}$

Worked Example 2. A mitochondrion has an actual length of $5\ \mu\mathrm{m}$. If an electron Micrograph is taken at a magnification of $\times 50000$What will be the length of the mitochondrion In the image?

$\mathrm{Image\ size} = 5\ \mu\mathrm{m} \times 50000 = 250000\ \mu\mathrm{m} = 250\ \mathrm{mm} = 25\ \mathrm{cm}$

A scale bar on a micrograph provides a direct conversion between image distance and actual distance. If a scale bar of length $2\ \mathrm{cm}$ on the image represents $10\ \mu\mathrm{m}$ in reality, then Any measurement on the image is converted by multiplying by $\frac{10\ \mu\mathrm{m}}{2\ \mathrm{cm}} = 5\ \mu\mathrm{m\ cm^{-1}}$.

Worked Example 3. A micrograph shows a scale bar labelled $5\ \mu\mathrm{m}$ that measures $20\ \mathrm{mm}$ on the printed image. A cell on the same image measures $48\ \mathrm{mm}$ across. Calculate the actual cell diameter.

Scale factor $= \frac{5\ \mu\mathrm{m}}{20\ \mathrm{mm}} = \frac{5000\ \mathrm{nm}}{20\ \mathrm{mm}} = 250\ \mathrm{nm\ mm^{-1}}$.

Cell diameter $= 48\ \mathrm{mm} \times 250\ \mathrm{nm\ mm^{-1}} = 12000\ \mathrm{nm} = 12\ \mu\mathrm{m}$.

When working with TEM or SEM images, the magnification is often stated on the micrograph. To calculate Actual size from the image:

1. Measure the dimension of interest on the printed image (in mm).
2. Apply the magnification formula.

Worked Example 4. A TEM image is labelled $\times 200000$. A ribosome on the image measures $0.25\ \mathrm{mm}$ in diameter. Calculate the actual diameter.

$\mathrm{Actual\ diameter} = \frac{0.25 \times 10^{-3}\ \mathrm{m}}{200000} = 1.25 \times 10^{-9}\ \mathrm{m} = 1.25\ \mathrm{nm}$

This is consistent with the expected diameter of a ribosome ($\approx 20\ \mathrm{nm}$) only if the Measurement is of a sub-component. If the full ribosome is being measured, the student should recheck The image measurement. This highlights the importance of sanity-checking answers against known biological Dimensions.

## 8. Endocytosis and Exocytosis

Endocytosis is the process by which cells engulf substances by folding the membrane inward to form a Vesicle. It is an active process requiring ATP. Two main types are distinguished by the size of the Vesicle formed:

Phagocytosis (“cell eating”): the cell membrane extends pseudopodia around a solid particle (e.g., A bacterium or dead cell), enclosing it in a large phagocytic vacuole ($d \approx 1$—$5\ \mu\mathrm{m}$). This vacuole then fuses with a lysosome, whose hydrolytic enzymes digest the contents. Phagocytosis is Carried out by specialised cells: neutrophils and macrophages in mammals.

Pinocytosis (“cell drinking”): the cell membrane invaginates to form small vesicles ($d \approx 0.1\ \mu\mathrm{m}$) that take in droplets of extracellular fluid. This is a non-specific Process that all cells can perform.

Receptor-mediated endocytosis: a specific form of pinocytosis in which receptor proteins on the cell Surface bind specific ligand molecules (e.g., cholesterol-bound LDL, iron-bound transferrin). The Receptor-ligand complexes cluster in clathrin-coated pits, which invaginate to form clathrin-coated Vesicles. The clathrin coat is then removed and the vesicle fuses with an endosome, where the ligand Is released. This mechanism allows cells to concentrate specific molecules from the extracellular fluid With high selectivity.

Exocytosis is the reverse process: intracellular vesicles fuse with the plasma membrane, releasing Their contents to the extracellular space. This requires:

1. Vesicle transport: vesicles are moved along microtubules by motor proteins (kinesin and dynein) using ATP.
2. Docking: vesicles are brought to the plasma membrane and dock at specific sites.
3. Fusion: SNARE proteins on the vesicle (v-SNAREs) and on the plasma membrane (t-SNAREs) bind together, pulling the membranes into close apposition. Calcium ions ($\mathrm{Ca^{2+}}$) trigger the final fusion step.

Exocytosis is essential for secretion of hormones, neurotransmitters, digestive enzymes, and mucus, as Well as for insertion of new membrane proteins and lipids into the plasma membrane.

## 9. Osmosis and Water Potential Calculations

Water potential ($\Psi$) is measured in pressure units (kilopascals, $\mathrm{kPa}$). Pure water at Standard temperature and pressure has a water potential of $0\ \mathrm{kPa}$. The addition of solutes Lowers water potential (makes it more negative):

$\Psi = \Psi_s + \Psi_p$

Where $\Psi_s$ (solute potential, also called osmotic potential) is always zero or negative, and $\Psi_p$ (pressure potential) is positive in turgid plant cells and zero in animal cells and flaccid Plant cells.

Worked Example 1. A plant cell is placed in a solution with water potential $\Psi_{\mathrm{solution}} = -400\ \mathrm{kPa}$. The cell has a solute potential $\Psi_s = -700\ \mathrm{kPa}$ And a pressure potential $\Psi_p = +300\ \mathrm{kPa}$. Determine the direction of net water movement.

$\Psi_{\mathrm{cell}} = \Psi_s + \Psi_p = -700 + 300 = -400\ \mathrm{kPa}$

Since $\Psi_{\mathrm{cell}} = \Psi_{\mathrm{solution}} = -400\ \mathrm{kPa}$There is no net water Movement. The cell is in equilibrium with the external solution.

Worked Example 2. A plant cell with $\Psi_s = -900\ \mathrm{kPa}$ and $\Psi_p = +450\ \mathrm{kPa}$ is Placed in pure water ($\Psi = 0\ \mathrm{kPa}$). Describe what happens.

$\Psi_{\mathrm{cell}} = -900 + 450 = -450\ \mathrm{kPa}$

Water moves from pure water ($0\ \mathrm{kPa}$) into the cell ($-450\ \mathrm{kPa}$) down the water Potential gradient. As water enters, the pressure potential increases (the cell becomes more turgid). Equilibrium is reached when $\Psi_{\mathrm{cell}} = 0\ \mathrm{kPa}$I.e., when $\Psi_p = +900\ \mathrm{kPa}$.

Worked Example 3. A red blood cell (which has no cell wall and therefore $\Psi_p = 0$) is placed in A solution with $\Psi = -300\ \mathrm{kPa}$. The red blood cell has $\Psi_s = -300\ \mathrm{kPa}$. What Happens?

$\Psi_{\mathrm{cell}} = -300 + 0 = -300\ \mathrm{kPa}$

No net water movement — the solution is isotonic. If the solution had $\Psi = -100\ \mathrm{kPa}$ (hypotonic), water would enter the cell, causing it to swell and potentially burst (haemolysis). If the Solution had $\Psi = -500\ \mathrm{kPa}$ (hypertonic), water would leave, causing the cell to shrink (crenation).

When a plant cell is placed in a hypertonic solution (more negative water potential than the cell), water Leaves by osmosis. The protoplast (the living part of the cell inside the cell wall) shrinks and pulls Away from the cell wall. This is plasmolysis. The point at which the protoplast just begins to pull Away is the incipient plasmolysis point, at which $\Psi_p = 0$ and $\Psi_{\mathrm{cell}} = \Psi_s$.

Plasmolysis is reversible if the cell is returned to a hypotonic solution before permanent damage occurs.

## 10. The Cell Cycle in Detail

The cell cycle has three major checkpoints where the progression is assessed before the cycle is Allowed to proceed:

Checkpoint	Location	What is Assessed
$\mathrm{G_1/S}$	Late $\mathrm{G_1}$	Cell size, nutrient availability, DNA damage
$\mathrm{G_2/M}$	Late $\mathrm{G_2}$	DNA replication complete and accurate
M (spindle assembly)	Metaphase	All chromosomes attached to spindle fibres

At each checkpoint, cyclin-CDK complexes phosphorylate target proteins that either promote or inhibit Progression. If damage is detected, the checkpoint arrests the cycle to allow repair. If repair fails, Apoptosis (programmed cell death) may be triggered.

Cyclins are a family of regulatory proteins whose concentration fluctuates cyclically. Different Cyclins act at different stages:

• Cyclin D: rises in response to growth factor signalling; activates CDK4/6 to phosphorylate Rb protein, releasing E2F transcription factor and allowing transition past $\mathrm{G_1/S}$.
• Cyclin E: peaks at the $\mathrm{G_1/S}$ boundary; activates CDK2 for S phase entry.
• Cyclin A: rises during S phase and $\mathrm{G_2}$; activates CDK2 for DNA replication.
• Cyclin B: peaks during $\mathrm{G_2}$ and M phase; activates CDK1 (also called CDC2) for mitotic entry.

A cyclin-CDK complex is only active when both components are present. Cyclin concentration is regulated By synthesis (transcription and translation) and degradation (ubiquitin-proteasome pathway). This Ensures that CDK activity is strictly periodic.

Mutations in two classes of genes disrupt cell cycle regulation:

Proto-oncogenes code for proteins that promote cell division (e.g., growth factors, growth factor Receptors, signal transduction proteins, cyclins). A mutation that causes overexpression or Constitutive activation converts a proto-oncogene into an oncogene, driving uncontrolled Proliferation. For example, the ras proto-oncogene codes for a GTPase involved in signal transduction; A mutation that locks RAS in its active (GTP-bound) state causes continuous signalling to divide.

Tumour suppressor genes code for proteins that inhibit cell division or promote apoptosis. The Most well-known is TP53 (p53), which halts the cell cycle at $\mathrm{G_1}$ when DNA damage is Detected, allowing repair. If damage is irreparable, p53 triggers apoptosis. Mutations in TP53 are Found in approximately 50% of all human cancers.

Cancer requires mutations in multiple genes: activation of one or more oncogenes plus Inactivation of two or more tumour suppressor genes (Knudson’s two-hit hypothesis).

## 11. Meiosis: Overview and Comparison with Mitosis

Feature	Mitosis	Meiosis
Divisions	One	Two
Daughter cells	2, genetically identical ($2n$)	4, genetically distinct ($n$)
Synapsis/crossing over	Absent	Present (prophase I)
Homologous pairing	Absent	Present (bivalents at metaphase I)
Metaphase alignment	Individual chromosomes at equator	Bivalents at equator
Anaphase	Sister chromatids separate	Homologous chromosomes separate (Meiosis I)
Function	Growth, repair, asexual reproduction	Production of gametes for sexual reproduction

For a detailed treatment of meiosis with genetic cross analysis, see Genetics and DNA.

Meiosis is essential for sexual reproduction because it:

1. Halves the chromosome number ($2n \to n$) so that fertilisation ($n + n$) restores the diploid number.
2. Generates genetic variation through crossing over (prophase I) and independent assortment (metaphase I), which are the raw material for natural selection.
3. Prevents chromosome doubling with each generation, which would make cells non-viable.

Cell specialisation (differentiation) is the process by which cells become adapted to perform specific Functions. All specialised cells develop from unspecialised stem cells through differential gene Expression.

Specialised Cell	Adaptations	Function
Red blood cell	Biconcave disc shape; no nucleus; no mitochondria; contains haemoglobin	$\mathrm{O_2}$ transport
Sperm cell	Flagellum for swimming; many mitochondria; acrosome with digestive enzymes	Fertilisation
Root hair cell (plant)	Elongated projection; large surface area; thin wall	Mineral ion absorption
Palisade mesophyll cell	Elongated; many chloroplasts near upper surface; thin walls	Photosynthesis
Guard cell	Kidney-shaped; unevenly thickened walls; chloroplasts	Stomatal aperture control
Neurone	Long axon; dendrites; myelin sheath; synaptic terminals	Transmission of nerve impulses
Phagocyte (white blood)	Flexible membrane; lysosomes; lobed nucleus	Engulfing pathogens
Squamous epithelial cell	Very thin and flat; smooth surface	Gas exchange surface

Stem cells are undifferentiated cells with the capacity to divide and differentiate into Specialised cell types.

• Totipotent: can differentiate into any cell type, including extra-embryonic tissues. The zygote and early morula (up to the 8-cell stage in humans) are totipotent.
• Pluripotent: can differentiate into any cell type except extra-embryonic tissues. Embryonic stem cells (from the inner cell mass of the blastocyst) are pluripotent.
• Multipotent: can differentiate into a limited range of cell types within a given tissue. Adult stem cells (e.g., haematopoietic stem cells in bone marrow, which produce all blood cell types) are multipotent.
• Unipotent: can only produce one cell type, but retain the capacity for self-renewal.

Therapeutic uses: stem cells can be used to replace damaged tissues (e.g., spinal cord injury, Parkinson’s disease, type 1 diabetes, burns). Bone marrow transplants use haematopoietic stem cells to Repopulate the blood cell lineages after chemotherapy. Induced pluripotent stem cells (iPSCs) are adult Cells reprogrammed to a pluripotent state by introducing transcription factors (Oct4, Sox2, Klf4, c-Myc), Offering an ethically uncontroversial alternative to embryonic stem cells.

## Practice Problems

Problem 1

Describe the process of cell fractionation and explain why each step must be carried out under Specific conditions (cold, buffered, isotonic).

Answer. Cell fractionation involves three steps. (1) Homogenisation: cells are placed in a cold, Buffered, isotonic solution and broken open (e.g., with a blender or ultrasonication). The solution Must be cold to reduce the activity of enzymes that would degrade organelles; buffered to maintain a Constant pH, since enzyme activity is pH-dependent; and isotonic to prevent osmotic lysis (in a Hypotonic solution) or shrinkage (in a hypertonic solution) of organelles. (2) Filtration: the Homogenate is passed through a gauze to remove unbroken cells and large debris. (3) Differential Centrifugation: the filtrate is centrifuged at progressively higher speeds. At each speed, the Densest remaining organelles sediment into a pellet, which is removed. The supernatant is then Centrifuged at a higher speed to pellet the next densest organelles, and so on. This separates Organelles by size and density.

If you get this wrong, revise: Cell Fractionation

Problem 2

Explain three ways in which the structure of mitochondria is related to their function in aerobic Respiration.

Answer. (1) The inner membrane is folded into cristae, greatly increasing the surface area Available for the electron transport chain and ATP synthase enzymes, maximising ATP production. (2) The matrix contains the enzymes for the Krebs cycle and the link reaction, concentrating the Reactants and products of these pathways to increase reaction rates. (3) The outer membrane is Permeable to small molecules (pyruvate, $\mathrm{NADH}$), allowing substrates from the cytoplasm to Enter the mitochondrion. Additionally, the presence of circular DNA and $70\mathrm{S}$ ribosomes Supports the endosymbiotic theory, explaining how mitochondria retained their own genetic machinery.

If you get this wrong, revise: Mitochondria

Problem 3

Compare and contrast prokaryotic and eukaryotic cells. In your answer, refer to DNA structure, Ribosomes, and cell walls.

Answer. Prokaryotic cells are smaller ($1$—$5\ \mu\mathrm{m}$) than eukaryotic cells ($10$—$100\ \mu\mathrm{m}$). Prokaryotic DNA is a single circular molecule located free in the Cytoplasm (nucleoid), not associated with histone proteins. Eukaryotic DNA is linear, organised into Chromosomes within a membrane-bound nucleus, and associated with histone proteins. Prokaryotic Ribosomes are $70\mathrm{S}$ (composed of $50\mathrm{S}$ and $30\mathrm{S}$ subunits), whereas Eukaryotic ribosomes are $80\mathrm{S}$ (composed of $60\mathrm{S}$ and $40\mathrm{S}$ subunits). Prokaryotic cell walls contain peptidoglycan; eukaryotic cell walls (in plants) contain cellulose, And fungal cell walls contain chitin. Both types have a phospholipid cell membrane and cytoplasm, But only eukaryotes possess membrane-bound organelles (mitochondria, ER, Golgi).

If you get this wrong, revise: Prokaryotic and Eukaryotic Cells

Problem 4

Describe the roles of the Golgi apparatus in protein processing and transport. Why would a cell that Secretes large amounts of protein (e.g., a pancreatic acinar cell) have an exceptionally large Golgi apparatus?

Answer. The Golgi apparatus receives transport vesicles from the RER containing newly Synthesised proteins. It modifies these proteins by adding carbohydrate groups (glycosylation), Phosphate groups, or lipid groups, and may cleave the protein into its active form. It then sorts The modified proteins and packages them into secretory vesicles, which bud from the trans face and Move to the plasma membrane for exocytosis. A pancreatic acinar cell secretes large quantities of Digestive enzymes, so it requires an extensive RER for protein synthesis and a correspondingly large Golgi apparatus for the modification, sorting, and packaging of these proteins into secretory Vesicles. The size of an organelle generally reflects the cell’s functional demands.

If you get this wrong, revise: Golgi Apparatus

Problem 5

Explain the fluid mosaic model of cell membranes. How does cholesterol affect membrane fluidity at High and low temperatures?

Answer. The fluid mosaic model describes the cell membrane as a bilayer of phospholipid Molecules in which proteins are embedded (intrinsic/transmembrane proteins) or attached to the Surface (extrinsic/peripheral proteins). The bilayer is “fluid” because individual phospholipid Molecules can move laterally within their own monolayer by diffusion, and “mosaic” because the Various components create a heterogeneous pattern. Cholesterol is a small, hydrophobic molecule Interspersed between phospholipid tails. At high temperatures, it restricts the movement of Phospholipids, reducing fluidity and making the membrane less permeable. At low temperatures, it Prevents phospholipid tails from packing too closely and crystallising, maintaining some fluidity. Cholesterol therefore buffers membrane fluidity, keeping it within an optimal range.

If you get this wrong, revise: The Fluid Mosaic Model

Problem 6

Are viruses living organisms? Justify your answer with reference to the characteristics of life and The structure and behaviour of viruses.

Answer. Viruses are not considered living organisms. They possess genetic material (DNA or RNA) And can evolve through mutation and natural selection, but they lack the other characteristics of Life. They do not carry out respiration, nutrition, or excretion independently. They have no Metabolism of their own and cannot synthesise proteins or generate ATP without a host cell. They do Not grow or develop. They cannot reproduce independently — they require a host cell’s ribosomes, Enzymes, and nucleotides to replicate. Their acellular structure (no cytoplasm, no cell membrane, no Ribosomes) further distinguishes them from cells. While they are obligate intracellular parasites That can cause disease, they are more accurately described as infectious agents rather than living Organisms.

If you get this wrong, revise: Viruses

Problem 7

An electron micrograph shows a mitochondrion. The scale bar indicates that $1\ \mathrm{mm}$ on the Image represents $0.2\ \mu\mathrm{m}$ in reality. The mitochondrion measures $6.5\ \mathrm{mm}$ in Length on the image. (a) Calculate the actual length. (b) If the magnification of the micrograph was $\times 25000$What is the expected image length, and does it match?

Answer. (a) Actual length $= 6.5\ \mathrm{mm} \times 0.2\ \mu\mathrm{m\ mm^{-1}} = 1.3\ \mu\mathrm{m}$.

(b) Expected image length $= 1.3\ \mu\mathrm{m} \times 25000 = 32500\ \mu\mathrm{m} = 32.5\ \mathrm{mm}$.

The actual measurement on the image ($6.5\ \mathrm{mm}$) does not match the expected image length ($32.5\ \mathrm{mm}$) for the stated magnification. This indicates either the scale bar is not at the Same magnification as the image, or the stated magnification is incorrect. This discrepancy highlights The importance of using scale bars (which are always at the same magnification as the image) rather than Stated magnification values for calculations.

If you get this wrong, revise: Quantitative Microscopy and Magnification

Problem 8

Describe the role of ATP in the following cellular processes: (a) active transport, (b) exocytosis, (c) DNA replication. For each, explain what the energy from ATP hydrolysis is used for.

Answer. (a) Active transport: carrier proteins (pumps) use ATP to change conformation, moving Molecules against their concentration gradient. For example, the $\mathrm{Na^+/K^+}$ ATPase hydrolyses One ATP to move $3\ \mathrm{Na^+}$ out and $2\ \mathrm{K^+}$ in against their gradients. The energy From ATP is used to induce the conformational change in the pump protein.

(b) Exocytosis: vesicles are transported along microtubules by motor proteins (kinesin) that Hydrolyse ATP to “walk” along the microtubule. ATP is also required for SNARE-mediated fusion of the Vesicle with the plasma membrane. The energy drives the mechanical movement and the membrane Rearrangement.

(c) DNA replication: helicase unwinds the double helix by breaking hydrogen bonds (energy-consuming). DNA polymerase catalyses phosphodiester bond formation between nucleotides, using energy from the Nucleotide triphosphates (dNTPs have high-energy phosphate bonds). The energy released when two Phosphate groups are cleaved from each dNTP provides the energy for the phosphodiester bond.

If you get this wrong, revise: Cell Membranes and DNA Replication

Problem 9

A plant cell with $\Psi_s = -850\ \mathrm{kPa}$ is placed in a solution of sucrose with concentration $0.35\ \mathrm{mol\ dm^{-3}}$ at $20\ ^\circ\mathrm{C}$. (a) Calculate the water potential of the External solution. (b) Determine the direction of net water movement. (c) Calculate the pressure Potential at equilibrium.

Answer. (a) $\Psi_{\mathrm{solution}} = -iCRT = -1 \times 350 \times 8.314 \times 293 = -852605\ \mathrm{Pa} \approx -853\ \mathrm{kPa}$.

(b) Initially, $\Psi_p = 0$ (the cell is placed in the solution and has not yet adjusted), so $\Psi_{\mathrm{cell}} = \Psi_s + \Psi_p = -850\ \mathrm{kPa}$.

Since $\Psi_{\mathrm{solution}} = -853\ \mathrm{kPa} < \Psi_{\mathrm{cell}} = -850\ \mathrm{kPa}$ Water moves out of the cell (from higher to lower water potential).

(c) As water leaves, the cell loses turgor. At equilibrium, $\Psi_{\mathrm{cell}} = \Psi_{\mathrm{solution}} = -853\ \mathrm{kPa}$:

$-850 + \Psi_p = -853$So $\Psi_p = -3\ \mathrm{kPa}$.

A negative $\Psi_p$ means the protoplast is pulling away from the cell wall (incipient plasmolysis or Full plasmolysis, depending on the cell wall). The cell is plasmolysed.

If you get this wrong, revise: Osmosis and Water Potential Calculations

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The cytoskeleton is a network of protein fibres that provides structural support, enables movement, and organises intracellular transport.

Component	Diameter	Protein	Structure	Function
Microfilaments (actin filaments)	$\approx 7\ \mathrm{nm}$	Actin	Two intertwined helical chains	Cell shape changes, cell division (cleavage furrow), muscle contraction
Intermediate filaments	$\approx 10\ \mathrm{nm}$	Keratin, vimentin, lamin	Rope-like fibres of intertwined subunits	Mechanical strength (nuclear lamina, hair, nails)
Microtubules	$\approx 25\ \mathrm{nm}$	Tubulin ($\alpha$ and $\beta$)	Hollow cylinders of 13 protofilaments	Chromosome movement (spindle fibres), intracellular transport (tracks for motor proteins), cilia and flagella

Motor proteins move along cytoskeletal filaments, carrying cargo (vesicles, organelles, chromosomes):

Motor Protein	Track	Direction	Energy Source	Function
Kinesin	Microtubules	Towards (+) end (away from centrosome)	ATP	Transport of vesicles and organelles towards cell periphery
Dynein	Microtubules	Towards (-) end (towards centrosome)	ATP	Retrograde transport; movement of cilia and flagella
Myosin	Microfilaments (actin)	Towards (+) end	ATP	Muscle contraction; cytokinesis; cytoplasmic streaming

Cilia are short, hair-like projections ($\approx 10\ \mu\mathrm{m}$ long, $0.2\ \mu\mathrm{m}$ diameter) that beat in coordinated waves. They are found on epithelial cells lining the respiratory tract (moving mucus and trapped particles away from the lungs) and in the Fallopian tubes (moving the ovum towards the uterus).

Flagella are longer ($\approx 50$—$200\ \mu\mathrm{m}$) and occur singly (e.g., sperm tail).

Both have the same 9+2 arrangement of microtubules: 9 pairs of fused microtubules (doublets) form an outer ring, surrounding a central pair of single microtubules. Dynein arms between the outer doublets use ATP to slide the doublets past each other, causing the cilium or flagellum to bend.

Primary ciliary dyskinesia (Kartagener syndrome) is caused by defective dynein arms, resulting in immotile cilia. Symptoms include chronic respiratory infections (mucus not cleared), male infertility (immotile sperm), and situs inversus (organs on the wrong side of the body, because cilia are needed to establish left-right asymmetry during embryonic development).

The endomembrane system is a network of organelles that work together to synthesise, modify, package, and transport proteins and lipids:

$\text{Rough ER} \to \text{Vesicles} \to \text{Golgi apparatus} \to \text{Secretory vesicles} \to \text{Cell membrane}$

The Golgi apparatus consists of flattened membrane-bound sacs (cisternae) stacked on top of each other, with a cis face (receiving side, near the ER) and a trans face (shipping side, near the cell membrane).

Functions:

1. Protein modification: adding carbohydrate groups (glycosylation) to form glycoproteins. This occurs in a specific sequence: oligosaccharide chains are trimmed and extended as the protein moves through the Golgi cisternae.
2. Lipid modification: adding carbohydrates to lipids to form glycolipids.
3. Proteolytic processing: cleaving pro-proteins into their active forms (e.g., proinsulin $\to$ insulin + C-peptide).
4. Sorting and packaging: directing modified proteins to their correct destinations:

• Default pathway: proteins without a specific sorting signal are transported to the cell membrane in secretory vesicles and released by exocytosis.
• Lysosomal pathway: proteins tagged with mannose-6-phosphate are directed to lysosomes.
• Plasma membrane pathway: transmembrane proteins are incorporated into the membrane.

Lysosomes contain approximately 60 different hydrolytic enzymes (lipases, proteases, nucleases, glycosidases) that function optimally at pH $\approx 5.0$ (maintained by a $\mathrm{H^+}$-ATPase pump in the lysosomal membrane).

Lysosomal storage diseases result from inherited deficiencies in individual lysosomal enzymes, causing substrates to accumulate:

Disease	Deficient Enzyme	Accumulating Substance	Symptoms
Tay-Sachs disease	Hexosaminidase A	$\mathrm{GM_2}$ ganglioside	Progressive neurodegeneration, blindness, death by age 4
Gaucher disease	Glucocerebrosidase	Glucocerebroside	Enlarged liver and spleen, bone pain, anaemia
Niemann-Pick disease	Sphingomyelinase	Sphingomyelin	Hepatosplenomegaly, neurological deterioration

The cell cycle is regulated by cyclins and cyclin-dependent kinases (CDKs):

Cyclin	CDK Partner	Phase Controlled	Function
Cyclin D	CDK4/6	G1 $\to$ S	Phosphorylates Rb protein, releasing E2F transcription factor to initiate S phase
Cyclin E	CDK2	G1/S transition	Initiates DNA replication
Cyclin A	CDK2	S phase	Maintains S phase progression
Cyclin B	CDK1 (CDC2)	G2 $\to$ M	Triggers entry into mitosis (MPF = maturation-promoting factor)

Cyclin concentrations fluctuate throughout the cell cycle (synthesised and degraded at specific points), while CDKs are present at relatively constant levels. The cyclin-CDK complex is only active when both components are present.

Three major checkpoints ensure the cell cycle proceeds correctly:

1. G1 checkpoint (restriction point): checks cell size, nutrient availability, growth signals, and DNA damage. If conditions are not met, the cell enters $\mathrm{G_0}$ (quiescence). This is the most important checkpoint in mammalian cells.
2. G2 checkpoint: checks that DNA replication is complete and that there is no DNA damage. If DNA damage is detected, p53 (a tumour suppressor protein) halts the cycle and activates DNA repair enzymes.
3. M checkpoint (spindle assembly checkpoint): checks that all chromosomes are correctly attached to spindle fibres at the metaphase plate. If any chromosome is unattached, anaphase is delayed.

Apoptosis (programmed cell death) is an orderly, controlled process that eliminates damaged, infected, or unnecessary cells without causing inflammation (unlike necrosis, which is uncontrolled cell death that triggers inflammation).

Mechanism:

1. Internal signals: DNA damage activates p53, which upregulates pro-apoptotic proteins (Bax, Bak) that form pores in the mitochondrial outer membrane.
2. Cytochrome c release from mitochondria into the cytoplasm.
3. Cytochrome c binds to Apaf-1, forming the apoptosome, which activates caspase-9 (initiator caspase).
4. Caspase-9 activates effector caspases (caspase-3, -7), which cleave target proteins:

• Activate nucleases that fragment DNA into ladders of $\approx 200\ \mathrm{bp}$ multiples.
• Degrade cytoskeletal proteins.
• Cause membrane blebbing.

5. The cell breaks into apoptotic bodies, which are phagocytosed by macrophages.

External signals: death ligands (e.g., FasL on cytotoxic T cells) bind to death receptors (e.g., Fas) on the target cell, activating caspase-8 directly (extrinsic pathway).

Failure of apoptosis contributes to cancer (cells that should die continue to divide) and autoimmune diseases.

Bacterial cell walls are made of peptidoglycan (murein), a polymer consisting of:

• Glycan chains: alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues linked by $\beta$-1,4-glycosidic bonds.
• Peptide cross-links: short peptide chains (tetrapeptides) attached to NAM, cross-linked by a peptide bridge. This provides structural strength.

Gram-positive bacteria (e.g., Staphylococcus aureus) have a thick peptidoglycan layer ($20$—$80\ \mathrm{nm}$) containing teichoic acids. They retain the crystal violet stain in the Gram stain.

Gram-negative bacteria (e.g., Escherichia coli) have a thin peptidoglycan layer ($2$—$10\ \mathrm{nm}$) between the inner and outer membranes. The outer membrane contains lipopolysaccharide (LPS), which is an endotoxin. They do not retain crystal violet and stain pink/red with safranin.

Antibiotic	Target	Mechanism
Penicillin	Transpeptidase (cross-linking enzyme)	Binds to transpeptidase active site, preventing peptide cross-linking. Cell wall becomes weak; cell lyses due to osmotic pressure.
Vancomycin	D-alanyl-D-alanine terminus of peptide side chains	Binds to D-Ala-D-Ala, blocking transpeptidase access.
Bacitracin	Dephosphorylation of bactoprenol	Prevents recycling of the lipid carrier needed for peptidoglycan synthesis.

Penicillin resistance can arise through:

• $\beta$-lactamase production: an enzyme that hydrolyses the $\beta$-lactam ring of penicillin, inactivating it.
• Altered penicillin-binding proteins (PBPs) with reduced affinity for penicillin (e.g., MRSA — methicillin-resistant S. Aureus).
• Reduced permeability of the outer membrane (in Gram-negative bacteria).

## 18. Viruses: Structure, Replication, and Defence

Viruses are not cells. They are obligate intracellular parasites that consist of:

Component	Description	Function
Nucleic acid	Either DNA or RNA (never both); single-stranded or double-stranded; linear or circular	Carries genetic information
Capsid	Protein coat made of capsomeres (protein subunits) arranged with icosahedral, helical, or complex symmetry	Protects the nucleic acid; mediates attachment to host cell
Envelope (some viruses)	Phospholipid bilayer derived from the host cell membrane during budding	Helps the virus enter host cells; contains viral glycoproteins for receptor binding

1. Attachment: viral surface proteins bind to specific receptors on the host cell surface (this determines host specificity).
2. Entry: the virus (or its genome) enters the host cell by endocytosis or membrane fusion.
3. Uncoating: the viral capsid is removed, releasing the viral genome.
4. Replication: the viral genome is replicated using the host cell’s machinery (some viruses provide their own polymerases).
5. Synthesis: viral proteins are synthesised using the host cell’s ribosomes.
6. Assembly: new viral particles are assembled from the newly synthesised components.
7. Release: new virions leave the host cell by budding (enveloped viruses) or cell lysis (non-enveloped viruses).

Human Immunodeficiency Virus (HIV) is a retrovirus (RNA virus that reverse-transcribes its genome into DNA):

Step	Description
1. Attachment	gp120 glycoprotein on HIV binds to CD4 receptor and CCR5/CXCR4 co-receptor on T helper cells
2. Fusion and entry	Viral envelope fuses with the host cell membrane
3. Reverse transcription	Viral RNA is converted to DNA by reverse transcriptase (this enzyme has no proofreading activity, so mutations occur frequently — contributing to the virus’s ability to evade the immune system)
4. Integration	Viral DNA is integrated into the host cell’s genome by integrase, forming a provirus
5. Transcription	Host RNA polymerase II transcribes the proviral DNA into viral mRNA and genomic RNA
6. Translation	Viral proteins (gag, pol, env) are synthesised
7. Assembly	New viral particles are assembled at the host cell membrane
8. Budding	New virions bud from the host cell, acquiring an envelope

Why HIV is difficult to cure:

• The provirus is integrated into the host genome and can remain latent (inactive) for years, invisible to the immune system and to antiviral drugs.
• Reverse transcriptase has no proofreading activity, so the virus mutates rapidly, generating many variants that can escape immune detection and drug treatment.
• The virus targets T helper cells, which are essential for coordinating the immune response. Destroying T helper cells weakens the entire immune system.

Drug	Target	Mechanism
AZT (zidovudine)	Reverse transcriptase	Nucleoside analogue: incorporated into viral DNA by reverse transcriptase, causing chain termination (DNA synthesis stops)
Protease inhibitors (e.g., ritonavir)	HIV protease	Prevents cleavage of viral polyproteins into functional proteins
Reverse transcriptase inhibitors (non-nucleoside)	Reverse transcriptase	Bind to reverse transcriptase at a site other than the active site, causing conformational changes that inactivate the enzyme
Neuraminidase inhibitors (oseltamivir/Tamiflu)	Influenza neuraminidase	Prevents release of new influenza virions from infected cells
Aciclovir	Viral DNA polymerase (herpes viruses)	Nucleoside analogue; activated by viral thymidine kinase (selectively toxic to virus-infected cells); incorporated into viral DNA, causing chain termination

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## 24. Viruses: Structure and Replication

Viruses are not cells. They are obligate intracellular parasites consisting of:

Component	Description
Genetic material	Either DNA or RNA (single-stranded or double-stranded; linear or circular) — never both
Protein coat (capsid)	Made of capsomeres (protein subunits); protects the genome; determines host cell specificity
Envelope (some viruses)	Lipid bilayer derived from the host cell membrane during budding; contains viral glycoproteins (e.g., gp120/gp41 in HIV; haemagglutinin in influenza)

Viruses are not considered living because they:

• Do not have a cell membrane, cytoplasm, or ribosomes.
• Cannot carry out metabolic reactions independently.
• Cannot reproduce without a host cell.
• Do not respond to stimuli.
• Do not grow.

1. Attachment: the virus binds to specific receptors on the host cell surface (this determines host range and tissue tropism).
2. Entry: the virus enters the cell (by fusion with the membrane, endocytosis, or injection of nucleic acid).
3. Uncoating: the viral genome is released from the capsid inside the cell.
4. Replication: the viral genome is replicated using host or viral enzymes.
5. Synthesis: viral proteins are synthesised using host ribosomes.
6. Assembly: new viral particles are assembled from the synthesized components.
7. Release: new virions leave the cell (by lysis or budding). Budding (enveloped viruses) does not immediately kill the host cell; lysis (non-enveloped viruses) destroys the host cell.

Bacteriophages (phages) are viruses that infect bacteria:

Phage Type	Example	Replication Strategy
Lytic phage	T4 phage	Lytic cycle: phage attaches, injects DNA, hijacks host machinery to produce new phages, lyses the cell to release progeny (approx. 200 per infected cell)
Lysogenic phage	Lambda phage	Lysogenic cycle: phage DNA integrates into the bacterial chromosome as a prophage; the prophage is replicated along with the bacterial DNA; under stress (e.g., UV light), the prophage excises and enters the lytic cycle

Lysogenic conversion: when a prophage carries genes that change the phenotype of the host bacterium. For example, the cholera toxin gene in Vibrio cholerae is carried by a prophage (CTX$\phi$); Corynebacterium diphtheriae only produces diphtheria toxin when infected by the $\beta$-phage.

Feature	Prokaryotic Cell	Eukaryotic Cell
Size	1—5 $\mu\mathrm{m}$	10—100 $\mu\mathrm{m}$
Nucleus	No true nucleus; nucleoid region	True nucleus with nuclear envelope (double membrane with nuclear pores)
DNA	Circular; not associated with histones	Linear chromosomes; associated with histones (chromatin)
Membrane-bound organelles	None	Mitochondria, ER, Golgi, lysosomes, etc.
Ribosomes	70S (30S + 50S subunits)	80S (40S + 60S subunits)
Cell wall	Present in bacteria (peptidoglycan); absent in archaea	Present in plants (cellulose), fungi (chitin); absent in animals
Flagella	Simple, rotating (single protein, flagellin)	Complex, “9+2” microtubule arrangement
Reproduction	Binary fission (asexual)	Mitosis and meiosis (asexual and sexual)
Cell division	No spindle; no mitosis	Spindle fibres; mitosis
Metabolic diversity	Extremely diverse (anaerobic, aerobic, chemoautotrophic, photoautotrophic)	Mostly aerobic (obligate); some anaerobic (yeast, muscle)
Internal membranes	Mesosomes (infoldings of cell membrane)	Extensive (ER, Golgi, mitochondria, vesicles)
Cytoplasm	No cytoskeleton (in most bacteria)	Complex cytoskeleton (microfilaments, microtubules, intermediate filaments)
Gene organisation	Operons; no introns (in most)	Individual genes; introns and exons; split genes
Time to replicate	20—30 minutes (E. Coli)	8—24 hours (human cell in culture)

$\text{Magnification} = \frac{\text{size of image}}{\text{size of object}}$

Example: a cell is observed through a microscope at 400x magnification. The cell measures $4\ \mathrm{mm}$ on the micrograph. What is the actual size?

$\text{Actual size} = \frac{\text{image size}}{\text{magnification}} = \frac{4\ \mathrm{mm}}{400} = 0.01\ \mathrm{mm} = 10\ \mu\mathrm{m}$

1. Place a drop of water on a clean microscope slide.
2. Place the specimen on the drop (thinly section or single layer).
3. Add a coverslip at an angle to avoid trapping air bubbles.
4. Stain if necessary (e.g., iodine for starch; methylene blue for animal cells).

The mitotic index is the percentage of cells in a population that are undergoing mitosis at a given time:

$\text{Mitotic index} = \frac{\text{number of cells in mitosis}}{\text{total number of cells observed}} \times 100$

Example: In a sample of 200 cells, 12 are in prophase, 8 in metaphase, 4 in anaphase, and 6 in telophase.

Cells in mitosis $= 12 + 8 + 4 + 6 = 30$.

Mitotic index $= \frac{30}{200} \times 100 = 15\%$.

A high mitotic index indicates rapid cell division (e.g., in cancerous tissue or meristems). A low mitotic index indicates slow division or quiescence.

Component	Structure	Function
Phospholipids	Hydrophilic phosphate head, hydrophobic fatty acid tails; form bilayer	Form the basic structure; selectively permeable barrier
Cholesterol	Small, amphipathic molecule interspersed between phospholipids	Reduces membrane fluidity at high temperatures; prevents freezing at low temperatures; maintains membrane stability
Intrinsic (transmembrane) proteins	Span the entire bilayer; hydrophobic regions interact with lipid tails	Channel proteins (facilitated diffusion); carrier proteins (active transport and facilitated diffusion); receptors
Extrinsic (peripheral) proteins	On inner or outer surface; attached by ionic bonds or to intrinsic proteins	Enzymes; cell signalling; cytoskeleton attachment
Glycoproteins	Proteins with carbohydrate chains	Cell recognition (e.g., ABO blood group antigens); receptors; cell-cell adhesion
Glycolipids	Lipids with carbohydrate chains	Cell recognition; cell-cell signalling; tissue compatibility
Cholesterol	See above	See above

Factor	Effect	Explanation
Temperature (increase)	Increases fluidity	More kinetic energy; phospholipids move more; membrane becomes more permeable
Temperature (decrease)	Decreases fluidity	Less kinetic energy; phospholipids pack more tightly; membrane becomes less permeable
Saturated fatty acids	Decreases fluidity	Straight tails pack tightly together
Unsaturated fatty acids (cis double bonds)	Increases fluidity	Kinked tails prevent tight packing
Cholesterol	Buffers fluidity changes	Reduces fluidity at high temperature; increases fluidity at low temperature

Component	Description	Function
Cell wall	Made of peptidoglycan (murein); not cellulose or chitin	Provides shape; prevents osmotic lysis
Plasma membrane	Phospholipid bilayer (no cholesterol, unlike eukaryotes)	Selective permeability; site of respiration (no mitochondria); site of photosynthesis (in cyanobacteria, on thylakoids)
Cytoplasm	No membrane-bound organelles; no endoplasmic reticulum; no Golgi	Contains ribosomes (70S), plasmids, and metabolic enzymes
Ribosomes	70S (30S + 50S subunits); smaller than eukaryotic 80S	Protein synthesis
Nucleoid	Region of cytoplasm containing a single, circular DNA molecule (not enclosed in a nuclear envelope)	Contains the chromosome (genetic material)
Plasmids	Small, circular, extrachromosomal DNA molecules	Often carry antibiotic resistance genes; can be transferred between bacteria (conjugation)
Flagellum (plural: flagella)	Rotating filament (driven by proton motor at base)	Propulsion (swimming)
Pili (fimbriae)	Short, hair-like protein appendages	Attachment to surfaces; conjugation (sex pili transfer plasmids between bacteria)
Capsule (slime layer)	Polysaccharide or polypeptide layer outside the cell wall	Protection from phagocytosis; adhesion to surfaces; prevents desiccation
Mesosome	Infolding of the plasma membrane	May be involved in DNA replication and cell division (controversial; may be an artefact of electron microscopy preparation)

Step	What Happens	Result for Gram-Positive	Result for Gram-Negative
1. Crystal violet	Purple dye enters all cells	Purple	Purple
2. Iodine (mordant)	Forms crystal violet-iodine complex	Purple (trapped in thick peptidoglycan)	Purple
3. Alcohol/acetone (decolouriser)	Dissolves outer membrane (Gram-negative); dehydrates peptidoglycan (Gram-positive)	Remains purple (thick peptidoglycan retains dye)	Becomes colourless (dye washed out)
4. Safranin (counterstain)	Red dye stains decolourised cells	Purple (safranin has no visible effect)	Pink/red (safranin stains the cell)

Feature	Gram-Positive	Gram-Negative
Peptidoglycan layer	Thick (20—80 nm)	Thin (2—7 nm)
Outer membrane	Absent	Present (lipopolysaccharide layer)
Periplasmic space	Absent	Present
Teichoic acids	Present (in peptidoglycan)	Absent
Examples	Staphylococcus, Streptococcus, Bacillus	E. Coli, Salmonella, Pseudomonas

Component	Description
Genetic material	Either DNA or RNA (never both); single-stranded or double-stranded; linear or circular
Capsid	Protein coat that protects the genetic material; made of capsomeres (protein subunits)
Envelope (some viruses)	Lipid bilayer derived from the host cell membrane during budding; contains viral glycoproteins
Attachment proteins	Project from the capsid or envelope; bind to specific receptors on the host cell surface

Virus	Genetic Material	Envelope?	Host Cell	Disease
HIV	ssRNA (+ sense)	Yes	CD4+ T cells, macrophages	AIDS
Influenza virus	ssRNA (- sense, segmented)	Yes	Respiratory epithelial cells	Influenza (flu)
SARS-CoV-2	ssRNA (+ sense)	Yes	Respiratory epithelial cells (ACE2 receptor)	COVID-19
Tobacco mosaic virus (TMV)	ssRNA (+ sense)	No	Plant cells (leaf mesophyll)	Tobacco mosaic disease
Bacteriophage $\lambda$	dsDNA	No (tail)	E. Coli bacteria	Bacterial lysis
Adenovirus	dsDNA	No	Respiratory epithelial cells	Common cold, conjunctivitis
Herpes simplex virus (HSV)	dsDNA	Yes	Nerve cells (latent infection in ganglia)	Cold sores, genital herpes

Characteristic of Life	Do Viruses Show It?	Explanation
Cellular organisation	No	Acellular (no cytoplasm, no ribosomes, no cell membrane)
Metabolism	No	No independent metabolism; rely entirely on host cell machinery
Reproduction	No (not independently)	Can only replicate inside a living host cell
Response to stimuli	No	No homeostatic mechanisms
Growth	No	Assembled from pre-formed components; do not grow

Type	Source	Potency	Can Differentiate Into
Totipotent	Zygote; early embryo (up to 4-cell stage)	Can form ANY cell type AND extra-embryonic tissues (placenta)	All cell types + placental tissue
Pluripotent	Embryonic stem cells (ESCs) from the inner cell mass of the blastocyst (5—7 days)	Can form ANY cell type but NOT extra-embryonic tissues	All 220+ cell types in the body
Multipotent	Adult stem cells (bone marrow, umbilical cord blood, adipose tissue)	Can form a limited range of cell types within one tissue type	Bone marrow: RBCs, WBCs, platelets; Neural stem cells: neurons, astrocytes, oligodendrocytes
Unipotent	Some adult stem cells	Can form only ONE cell type	Satellite cells (muscle stem cells) $\to$ skeletal muscle cells only

Source	Advantages	Disadvantages
Embryonic stem cells (ESCs)	Pluripotent; unlimited self-renewal; can differentiate into any cell type	Ethical controversy (destruction of embryo); risk of teratoma formation; immune rejection
Adult stem cells	No ethical issues; patient’s own cells = no immune rejection; lower tumour risk	Limited potency; fewer in number; harder to isolate and culture
Induced pluripotent stem cells (iPSCs)	Pluripotent; patient’s own cells (no immune rejection); no embryo destruction	Risk of mutations from reprogramming; may not be fully equivalent to ESCs; expensive
Umbilical cord blood	Rich in haematopoietic stem cells; painless collection; lower immune rejection	Limited volume; only haematopoietic stem cells (not pluripotent)

Application	Stem Cell Type	Current Status
Bone marrow transplant (leukaemia)	Haematopoietic stem cells from bone marrow or cord blood	Well-established treatment
Skin grafts (burns)	Epidermal stem cells from patient’s own skin	Routine in specialist centres
Corneal repair (limbal stem cell deficiency)	Limbal stem cells from healthy eye or cultured	Available; good outcomes
Spinal cord injury	Neural stem cells or mesenchymal stem cells	Clinical trials ongoing
Parkinson’s disease	Dopaminergic neurons derived from ESCs or iPSCs	Clinical trials; promising early results
Type 1 diabetes	Pancreatic $\beta$ cells derived from stem cells	Research stage; clinical trials planned

Component	Protein	Diameter	Structure	Function
Microfilaments (actin filaments)	Actin (globular G-actin polymerises to filamentous F-actin)	~7 nm	Two helical chains of actin subunits twisted together	Cell movement (amoeboid movement, cytokinesis); cell shape; microvilli
Intermediate filaments	Various (keratin, vimentin, lamin, desmin, neurofilaments)	~10 nm	Rope-like fibres (more stable than microfilaments)	Mechanical strength; structural support; anchors organelles; nuclear lamina
Microtubules	Tubulin ($\alpha$-tubulin + $\beta$-tubulin dimers)	~25 nm	Hollow tubes of 13 protofilaments (tubulin dimers)	Cell shape; intracellular transport (motor proteins: kinesin and dynein walk along microtubules); spindle fibres in mitosis/meiosis; cilia and flagella

Motor Protein	Direction of Movement	Cargo	Role
Kinesin	Moves towards the (+) end of microtubules (away from the centrosome)	Vesicles, organelles, mRNA	Anterograde transport (e.g., moving neurotransmitter vesicles down the axon)
Dynein	Moves towards the (-) end of microtubules (towards the centrosome)	Vesicles, organelles	Retrograde transport; drives beating of cilia and flagella
Myosin	Moves along actin filaments	Vesicles, organelles	Muscle contraction; cytokinesis; cell crawling

Feature	Description
Appearance	Flattened sacs (cisternae) studded with ribosomes on the cytoplasmic surface
Function	Synthesis of proteins destined for secretion, the plasma membrane, or lysosomes; proteins enter the RER lumen during synthesis
Transport	Vesicles bud off from the RER and carry proteins to the Golgi apparatus
Location	Near the nucleus (continuous with the nuclear envelope)

Feature	Description
Appearance	Tubular network; no ribosomes
Functions	Lipid and steroid synthesis (e.g., cholesterol, steroid hormones); carbohydrate metabolism; detoxification (in liver cells, SER contains enzymes that break down drugs, alcohol, and other toxins); calcium storage (in muscle cells, SER is called sarcoplasmic reticulum)
Location	Throughout the cytoplasm

Feature	Description
Appearance	Stack of flattened membrane-bound sacs (cisternae) with vesicles budding off at the edges
Functions	Modifies proteins (glycosylation: adding carbohydrate groups); sorts proteins into different vesicles; packages proteins into secretory vesicles for exocytosis; forms lysosomes
Direction	Vesicles arrive from the RER at the cis face (receiving side); modified proteins leave from the trans face (shipping side)
Transport	Secretory vesicles carry proteins to the plasma membrane (exocytosis) or to lysosomes

Component	Description	Function
Collagen	Most abundant protein in the body; forms strong fibres	Provides tensile strength (resists stretching); major component of connective tissue (tendons, skin, bone)
Elastin	Elastic protein that can stretch and recoil	Provides elasticity (e.g., in arterial walls, lungs, skin)
Fibronectin	Glycoprotein that connects cells to the ECM	Links integrins (cell surface receptors) to collagen and other ECM components; involved in cell adhesion and migration
Proteoglycans	Protein core with long chains of GAGs (glycosaminoglycans, e.g., chondroitin sulfate, heparan sulfate)	Fill the spaces between cells; resist compression (especially in cartilage); bind growth factors and signalling molecules
Hyaluronic acid	Large GAG (not attached to a protein core)	Lubricates joints; provides hydration and turgor to tissues; component of the vitreous humour of the eye

Function	Description
Structural support	Provides a scaffold for tissues; determines tissue architecture
Cell signalling	ECM components bind to cell surface receptors (integrins); activate intracellular signalling pathways; influence gene expression, cell division, and differentiation
Cell adhesion	Cells attach to the ECM via integrins; provides anchorage
Filtration	Basement membrane (specialised ECM) filters blood in the glomerulus (kidney)
Wound healing	ECM provides a framework for tissue repair; fibroblasts deposit new collagen during scar formation

Apoptosis is an orderly, genetically programmed process of cell death that occurs during normal development and tissue homeostasis.

Step	Description
1. Signal	Cell receives a signal to undergo apoptosis (internal signal: DNA damage detected by p53 protein; external signal: death signals from immune cells)
2. Enzyme activation	Caspases (cysteine proteases) are activated (initiator caspases $\to$ executioner caspases)
3. Cell shrinkage	Cell loses water; becomes smaller; cytoskeleton breaks down
4. Chromatin condensation	DNA is cut into fragments by endonucleases (DNA laddering on gel electrophoresis)
5. Membrane blebbing	The plasma membrane forms irregular blebs (outward bulges)
6. Apoptotic body formation	The cell fragments into membrane-bound apoptotic bodies
7. Phagocytosis	Apoptotic bodies are engulfed and digested by neighbouring cells or macrophages (no inflammatory response because cell contents are not released)

Feature	Apoptosis	Necrosis
Cause	Genetically programmed; physiological or mild stress	Uncontrolled cell death; caused by severe injury, toxin, or hypoxia
Process	Orderly; energy-dependent (requires ATP)	Disorderly; energy-independent
Membrane integrity	Maintained until late; apoptotic bodies form	Lost early; cell contents leak out
Inflammation	No inflammation (cell contents are contained)	Inflammation occurs (cell contents released; attracts immune cells)
DNA fragmentation	Organised (laddering pattern)	Random (smear pattern)
Role	Essential for development; removes damaged or unwanted cells	Pathological; harmful to surrounding tissue

Example	Why Apoptosis Is Needed
Embryonic development	Tissue remodelling (e.g., separation of fingers and toes by apoptosis of webbing tissue)
Immune system	Removal of self-reactive T cells in the thymus (negative selection); removal of surplus B cells after infection
Skin	Shedding of dead skin cells from the epidermis
Menstrual cycle	Breakdown of the endometrium if implantation does not occur
DNA damage	If DNA damage is too severe to repair, p53 triggers apoptosis (prevents cancer)

Type	Resolution	Magnification	Specimen Preparation	What Can Be Seen
Light microscope	~200 nm	Up to ~1500x	Staining (e.g., methylene blue, iodine, eosin); sectioning	Cell structure (nucleus, chloroplasts, cell walls); living specimens (with special techniques)
Transmission electron microscope (TEM)	~0.1 nm	Up to ~1,000,000x	Specimen fixed, dehydrated, embedded in resin; ultra-thin sections (~100 nm); stained with heavy metals (uranium, lead)	Internal ultrastructure (organelles, membranes, ribosomes); very high resolution
Scanning electron microscope (SEM)	~10 nm	Up to ~100,000x	Specimen fixed, dehydrated; coated with gold/palladium	3D surface detail (external features of cells, pollen grains, insects); not internal structure
Laser scanning confocal microscopy	~200 nm	Up to ~1000x	Fluorescent staining; optical sectioning (no physical sectioning needed)	3D images of fluorescently labelled structures inside living cells

$\text{Magnification} = \frac{\text{Image size}}{\text{Actual size}}$

$\text{Actual size} = \frac{\text{Image size}}{\text{Magnification}}$

Unit	Symbol	Conversion
Metre	m	—
Millimetre	mm	$10^{-3}$ m
Micrometre	$\mu$M	$10^{-6}$ m
Nanometre	nm	$10^{-9}$ m

Stage	What Happens
Prophase	Chromosomes condense (become visible as two sister chromatids joined at the centromere); nuclear envelope begins to break down; nucleolus disappears; centrioles move to opposite poles; spindle fibres begin to form
Metaphase	Chromosomes line up at the metaphase plate (equator of the cell); spindle fibres attach to centromeres; chromosomes are at their most condensed (best stage to observe chromosomes under a microscope)
Anaphase	Centromeres divide; sister chromatids are pulled to opposite poles by the spindle fibres (shortening of microtubules); chromosomes are now individual chromatids (V-shaped)
Telophase	Chromatids arrive at the poles; they decondense (become chromatin again); nuclear envelope reforms around each set of chromosomes; nucleolus reappears; cytokinesis (cytoplasmic division) begins

Feature	Animal Cells	Plant Cells
Mechanism	Cleavage furrow: contractile ring of actin and myosin pinches the cell in two from the outside	Cell plate: vesicles from the Golgi apparatus fuse at the equator; form a new cell wall (middle lamella) between the two daughter cells
Result	Two identical daughter cells	Two identical daughter cells with new cell walls

Organelle	Function
Nucleus	Contains genetic material (DNA); controls cell activities via gene expression
Nucleolus	rRNA synthesis; ribosome assembly
Rough ER	Protein synthesis (for secretion, membrane, lysosomes)
Smooth ER	Lipid synthesis; detoxification; calcium storage
Golgi apparatus	Protein modification, sorting, packaging
Mitochondria	Aerobic respiration; ATP production
Chloroplasts (plants only)	Photosynthesis
Ribosomes	Protein synthesis (80S in cytoplasm; 70S in mitochondria/chloroplasts)
Lysosomes	Intracellular digestion; contain hydrolytic enzymes
Centrioles	Organise spindle fibres during cell division; form basal bodies of cilia
Cytoskeleton	Cell shape; movement; intracellular transport
Cell membrane	Selective barrier; cell signalling; cell recognition
Cell wall (plants, fungi)	Structural support; prevents osmotic lysis
Vacuole (plants)	Storage (water, ions, pigments); maintains turgor pressure
Peroxisomes	Breakdown of fatty acids ($\beta$-oxidation); detoxification of hydrogen peroxide

Feature	Prokaryotic Cell	Eukaryotic Cell
Size	1—10 $\mu$M	10—100 $\mu$M
Nucleus	No true nucleus (nucleoid region)	True nucleus with nuclear envelope and nucleolus
DNA	Single, circular chromosome; no histones	Multiple linear chromosomes; associated with histones
Ribosomes	70S (smaller)	80S (larger)
Membrane-bound organelles	None	Many (mitochondria, ER, Golgi, lysosomes, etc.)
Cell wall	Present (peptidoglycan in bacteria)	In plants (cellulose) and fungi (chitin); absent in animals
Plasma membrane	Present; no cholesterol	Present; contains cholesterol
Cytoskeleton	Simple (no microtubules, no intermediate filaments)	Complex (microtubules, microfilaments, intermediate filaments)
Reproduction	Binary fission (asexual)	Mitosis and meiosis; also meiosis (sexual reproduction)
Flagella	Simple, rotating (9+2 microtubule arrangement, but different protein composition)	Complex, 9+2 microtubule arrangement; beating motion
Plasmids	Present	Absent (in most eukaryotes)
Metabolic diversity	Very high (chemolithoautotrophs, photoautotrophs, heterotrophs)	Lower (mainly heterotrophic; some photoautotrophic)

Feature	Palisade Mesophyll	Spongy Mesophyll
Position	Upper layers of the mesophyll (near the upper epidermis)	Lower layers of the mesophyll (near the lower epidermis)
Cell shape	Columnar; elongated; tightly packed	Loosely packed; many air spaces (large intercellular air spaces)
Chloroplast density	Very high (many chloroplasts per cell)	Fewer chloroplasts per cell
Primary function	Photosynthesis (light harvesting)	Gas exchange (air spaces allow $\mathrm{CO_2}$ to reach palisade cells; $\mathrm{O_2}$ to diffuse out)

Cell Type	Location	Function
Root hair cell	Root epidermis (just behind the tip)	Increases surface area for mineral ion and water absorption from soil
Guard cell	Epidermis (flanking stomata)	Controls opening and closing of stomata (regulates gas exchange and transpiration)
Xylem vessel element	Xylem	Dead, hollow cells forming tubes for water transport
Companion cell	Phloem (adjacent to sieve tube elements)	Loads sucrose into sieve tubes via active transport; provides metabolic support
Sclerenchyma (fibres)	Scattered in vascular bundles; also in stems	Lignified, dead cells that provide mechanical strength and support
Collenchyma	Below the epidermis in young stems	Living cells with unevenly thickened cellulose cell walls; provides flexible support to young growing stems

Cell fractionation is the process of breaking open cells and separating the organelles by differential centrifugation.

Step	Description
1. Homogenisation	Cells are placed in a cold, isotonic buffer solution and homogenised (blended) to break the plasma membrane while leaving organelles intact
2. Cold temperature	Reduces enzyme activity; prevents organelle damage by proteases
3. Isotonic buffer	Prevents organelles from bursting (if too dilute) or shrinking (if too concentrated) by osmosis
4. Buffered pH	Maintains a constant pH; prevents enzyme denaturation and organelle damage
5. Filtration	Homogenate is filtered through gauze to remove debris (unbroken cells, connective tissue)
6. Differential centrifugation	Organelles are separated by spinning at increasing speeds; heavier/larger organelles sediment first

Centrifuge Speed	Time	Organelle Sedimented	Supernatant Contains
Low speed (~1000 g)	10 min	Nuclei	Mitochondria, lysosomes, microsomes, ribosomes (in solution)
Medium speed (~10,000 g)	20 min	Mitochondria, lysosomes	Microsomes (ER fragments), ribosomes (in solution)
High speed (~100,000 g)	60 min	Microsomes (rough ER, smooth ER), ribosomes	Soluble cytoplasm (enzymes, metabolites)

Phase	Key Events
G1 (Gap 1)	Cell grows; organelles duplicate; proteins are synthesised
S (Synthesis)	DNA replication occurs (each chromosome is copied to form two sister chromatids joined at the centromere)
G2 (Gap 2)	Cell continues to grow; synthesises proteins needed for mitosis (e.g., tubulin for spindle fibres)

Event	Description
Chromatin condensation	DNA coils and supercoils; chromosomes become visible as two sister chromatids joined at the centromere
Spindle formation	Centrioles (in animal cells) move to opposite poles; spindle fibres (microtubules) form from the centrioles and extend across the cell
Nuclear envelope	Breaks down and disappears
Nucleolus	Disappears

Event	Description
Alignment	Chromosomes line up at the metaphase plate (equator of the cell)
Attachment	Spindle fibres attach to the centromere of each chromosome via the kinetochore
Checkpoint	The spindle assembly checkpoint ensures all chromosomes are correctly attached before anaphase begins

Phase	Events
Anaphase	Sister chromatids are pulled apart to opposite poles by the spindle fibres (shortening of spindle fibres); the centromere divides first; each chromatid is now an independent chromosome
Telophase	Chromosomes arrive at the poles and begin to decondense (uncoil back into chromatin); the nuclear envelope reforms around each set of chromosomes; the nucleolus reappears
Cytokinesis	The cytoplasm divides: in animal cells, the cell membrane is pulled inwards by a contractile ring of actin filaments (cleavage furrow); in plant cells, a cell plate forms from vesicles containing cell wall material at the equator; the cell plate grows outwards to divide the cell into two daughter cells

Significance	Description
Growth	Produces new cells for tissue growth (e.g., bone elongation at the epiphyseal plate)
Repair	Replaces damaged or dead cells (e.g., skin cells, liver cells after injury)
Asexual reproduction	Produces genetically identical offspring in organisms that reproduce asexually (e.g., binary fission in bacteria, budding in yeast, runners in strawberry plants)
Genetic continuity	Produces daughter cells that are genetically identical to the parent cell (same chromosome number and same alleles)

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1. Confusing correlation with causation when evaluating experimental data and drawing conclusions.

2. Using anecdotal evidence or small sample sizes as though they constitute robust scientific data.

3. Writing vague answers without specific biological terminology. Use precise terms (e.g., ‘phospholipid bilayer’ not ‘membrane’).

4. Confusing DNA replication (S phase) with transcription or translation. Be clear about which process is being described.

Worked examples demonstrating the application of key concepts are covered in the detailed sub-pages linked above.

• Biology
• Cells
• Genetics and DNA