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Spontaneous generation, and how it was disproved - The old belief: life arises from non-living matter (maggots from meat, microbes from broth). - Francesco Redi (1668): meat in open jars grew maggots; meat in jars covered with gauze did not. Flies had to lay eggs, so maggots come from flies. - Lazzaro Spallanzani (1760s): broth boiled and sealed stayed sterile; broth left open became cloudy. Critics said sealing kept out a "vital force" in air. - Louis Pasteur (1859-1862): swan-neck flasks. Air could enter, but dust and microbes were trapped in the curved neck. The broth stayed sterile until the neck was broken. This answered the "vital force" objection. The three founders of cell theory - Matthias Schleiden (1838): all plants are made of cells. - Theodor Schwann (1839): all animals are made of cells too. - Rudolf Virchow (1855): "omnis cellula e cellula", every cell comes from a cell. Modern cell theory adds - Cells contain hereditary information (DNA) that is passed on at division. - All cells have essentially the same chemical composition and basic metabolism. - The activity of an organism depends on the activity of its cells. Earlier milestones - Robert Hooke (1665) saw the empty walls of cork and named them "cells". - Antonie van Leeuwenhoek (1670s) saw living bacteria and protists with single-lens microscopes. Clinical tip: Virchow also founded cellular pathology. Disease is a disturbance of cells, which is still the basis of histopathology. Exam focus: match each scientist to the experiment and to the conclusion. Remember that Pasteur's swan-neck flask is the decisive experiment against spontaneous generation.

Key terms - Magnification: how much bigger the image is than the object. - Resolution (resolving power): the smallest distance between two points that can still be told apart. Magnifying beyond the resolution limit gives "empty magnification", a bigger but blurrier image. - Contrast: the difference between an object and its background. Most cells are transparent, so they need stains or special optics. The resolution limit (Abbe) - d = 0.61 λ / NA, where λ is the wavelength and NA is the numerical aperture of the objective (n sin α). - Visible light (about 500 nm) and the best oil-immersion lens (NA about 1.4) give d ≈ 0.2 μm. - So a light microscope resolves cells, nuclei and mitochondria (just), but not ribosomes or membranes. - Electrons have a far shorter wavelength, so electron microscopes resolve about 1000 times finer detail. Light microscopy variants - Bright-field on stained sections (H&E: haematoxylin stains nuclei blue-purple, eosin stains cytoplasm pink). - Phase contrast and differential interference contrast: show living, unstained cells. - Fluorescence: labelled molecules (antibodies, GFP) emit light at a longer wavelength. Confocal microscopy removes out-of-focus light to give optical sections. Electron microscopy - Transmission EM (TEM): electrons pass through very thin sections stained with heavy metals. Shows internal ultrastructure (membranes, ribosomes, organelles). - Scanning EM (SEM): electrons scan the surface. Shows 3D surface topography. - Limits: the vacuum and fixation mean the sample must be dead. Preparation can create artefacts. Units to remember - Cells: 10-100 μm (eukaryotes), 1-2 μm (bacteria). - Viruses: 20-300 nm. - Membranes: about 7-8 nm thick. Exam trap: a bigger magnification does not show more detail once the resolution limit is reached.

The geometry - For a sphere of radius r: surface 4πr², volume (4/3)πr³. - Surface-to-volume ratio (SA:V) = 3/r. - Doubling the radius halves SA:V. The cell has twice as much volume to supply per unit of membrane. Consequences - Diffusion is fast over short distances but slow over long ones: the time scales with distance squared. Diffusion alone cannot supply the centre of a very large cell. - The nucleus controls a limited volume of cytoplasm. Very large cells are often multinucleate (skeletal muscle fibres). How cells and tissues get around the limit - Stay small and divide. - Flatten or elongate: squamous cells, neurons with long thin axons. - Fold the membrane: microvilli in the intestine and kidney tubule greatly increase the absorptive surface. - Compartmentalise: eukaryotes divide their volume into organelles, each with its own membrane surface for reactions. - Use transport systems: circulation, cytoplasmic streaming and motor proteins. Why prokaryotes are smaller - They have no internal membranes and rely on diffusion. Typical size is 1-2 μm, against 10-100 μm for eukaryotic cells. Exam focus: be ready to calculate SA:V for a cube (6/side) or sphere (3/r), and to explain why microvilli are an adaptation.

The early Earth - About 4.6 billion years old. The oldest evidence of life (microfossils, stromatolites) is about 3.5-3.8 billion years old. - The early atmosphere was reducing or neutral (little free oxygen), rich in water vapour, CO2, N2, and possibly CH4, NH3 and H2. Step 1: organic monomers from inorganic molecules - The Miller-Urey experiment (1953): water, methane, ammonia and hydrogen in a closed system, with electric sparks as "lightning". - After a week, amino acids and other organic molecules had formed. - Conclusion: the building blocks of life can form spontaneously under plausible early-Earth conditions. - Other possible sources: hydrothermal vents, meteorites (amino acids have been found in meteorites). Step 2: polymers - Monomers concentrate and polymerise on clay or mineral surfaces, or in evaporating pools. Step 3: self-replication, the RNA world - The problem: DNA needs proteins to be copied, and proteins need DNA to be made. Which came first? - RNA can both store information and catalyse reactions (ribozymes; the peptidyl transferase of the ribosome is RNA). - So RNA may have come first, later handing information storage to the more stable DNA and catalysis to the more versatile proteins. Step 4: compartments (protocells) - Lipids spontaneously form vesicles in water. A vesicle enclosing replicating RNA separates "self" from the environment, which allows selection. From prokaryotes to eukaryotes - The first cells were prokaryotic. Eukaryotes appeared about 1.8-2 billion years ago, probably through membrane invagination (nucleus, ER) and endosymbiosis (mitochondria and chloroplasts; see chapter 7). Exam focus: know what Miller-Urey showed and what it did not. It made monomers, not life. Know the evidence for the RNA world (ribozymes, the ribosome).

1. State the three principles of cell theory, and name who contributed each. 2. Describe Pasteur's swan-neck flask experiment. Which objection did it answer that Spallanzani's did not? 3. What is the difference between magnification and resolution? 4. Why can a light microscope not show ribosomes? 5. When would you use TEM, and when SEM? 6. Calculate SA:V for spheres of radius 1 μm and 10 μm. What does the result mean for the cell? 7. Give three ways cells or tissues overcome the surface-to-volume limit. 8. What did the Miller-Urey experiment show? What did it not show? 9. What observation supports the RNA world hypothesis? 10. Which property of lipids makes the first protocells possible?

1. All organisms are made of cells (Schleiden for plants, Schwann for animals); the cell is the basic unit of life; every cell comes from a pre-existing cell (Virchow). 2. Broth in a flask with a long curved neck stayed sterile although air could reach it, because microbes were trapped in the neck. It answered the claim that sealing kept out a vital force present in air. 3. Magnification is image size relative to object size. Resolution is the smallest separation at which two points are still distinguished. 4. Ribosomes (about 25 nm) are far below the light resolution limit of about 0.2 μm. 5. TEM for internal ultrastructure in thin sections; SEM for 3D surface topography. 6. 3 μm⁻¹ and 0.3 μm⁻¹. The larger cell has ten times less membrane per unit volume, so exchange limits its size. 7. Any three: staying small, flattening or elongating, microvilli, internal compartments, multiple nuclei, transport systems. 8. It showed that amino acids and other organic monomers can form spontaneously from simple gases and energy. It did not produce polymers, replication or cells. 9. RNA can catalyse reactions (ribozymes), and the ribosome's catalytic core is RNA. 10. Amphipathic lipids spontaneously form bilayer vesicles in water.

Basic structure, from inside out - Cytoplasm with the nucleoid (chromosome), 70S ribosomes, plasmids and storage granules. - Plasma membrane: phospholipid bilayer without sterols. It hosts respiration and ATP synthesis (bacteria have no mitochondria). - Cell wall of peptidoglycan: sugar chains of alternating NAG and NAM, cross-linked by short peptides. It gives shape and resists osmotic lysis. - Gram-negatives only: a periplasmic space, then an outer membrane. - Optional: capsule, flagella, pili (fimbriae), endospores. Shapes: cocci (spheres), bacilli (rods), spirilla and spirochetes (spirals); arrangements such as chains (streptococci) and clusters (staphylococci). Gram-positive envelope - Thick peptidoglycan (20-80 nm), with teichoic and lipoteichoic acids. - No outer membrane. Gram-negative envelope - Thin peptidoglycan in the periplasm. - An outer membrane whose outer leaflet is lipopolysaccharide (LPS). LPS has three parts: lipid A (endotoxin), the core, and the O antigen. - Porins let small molecules through; the outer membrane blocks many antibiotics and detergents. The Gram stain, step by step 1. Crystal violet stains all cells purple. 2. Iodine forms a complex with the dye. 3. Alcohol or acetone decolourises: it dehydrates and tightens the thick Gram-positive wall, trapping the complex. In Gram-negatives it dissolves the outer membrane and the thin wall lets the dye out. 4. Safranin counterstains the now colourless Gram-negatives pink. Result: Gram-positive purple, Gram-negative pink. Other structures - Capsule: polysaccharide layer; resists phagocytosis. - Flagella: rotary motor driven by the proton gradient. - Pili: adhesion. The sex pilus is used in conjugation. - Endospores (Bacillus, Clostridium): dormant, highly resistant to heat and disinfectants. - Some bacteria have no wall at all (Mycoplasma), so penicillins do not work on them. Clinical tip: lipid A of LPS triggers the massive cytokine release of Gram-negative septic shock. Penicillins and vancomycin target peptidoglycan, which human cells do not have. Exam trap: acid-fast bacteria (Mycobacterium) have a waxy mycolic-acid wall and stain poorly with Gram. They need the Ziehl-Neelsen stain.

The chromosome - Usually one circular, double-stranded DNA molecule (E. coli: about 4.6 million bp, about 4,300 genes). - No nuclear envelope. The DNA is compacted in the nucleoid by supercoiling (DNA gyrase, a topoisomerase) and nucleoid-associated proteins, not by histones. - Replication starts at a single origin and proceeds in both directions. - Genes are dense: about 88% of the genome codes for proteins, and there are almost no introns. - Operons: several genes transcribed together from one promoter as a single mRNA (the lac operon is the classic example). Transcription and translation are coupled because there is no nucleus. Plasmids - Small circular DNA molecules that replicate independently of the chromosome. - Not essential for normal growth, but they bring advantages in particular conditions. - Types by what they carry: - R (resistance) plasmids: antibiotic resistance genes, often several at once. - F (fertility) plasmid: genes for the sex pilus and conjugation. - Virulence plasmids: toxins, adhesins. - Metabolic plasmids: degrade unusual compounds. - Copy number can be low (1-2) or high (dozens). Mobile elements - Transposons ("jumping genes") move between the chromosome and plasmids and often carry resistance genes. - Integrons capture and express gene cassettes. They are one way multi-resistance builds up. Clinical tip: DNA gyrase is bacterial. Fluoroquinolones (ciprofloxacin) block it, stopping bacterial DNA replication. Exam focus: plasmids are the main vehicle of spreading antibiotic resistance. Be ready to explain why a plasmid with several resistance genes is so dangerous.

Vertical versus horizontal transfer - Vertical: from mother cell to daughter cells by binary fission. The daughters are clones. - Horizontal: between cells that are already living, possibly of different species. 1. Transformation - The recipient takes up naked DNA released by a dead bacterium. - Only "competent" cells can do it, naturally (Streptococcus pneumoniae, Haemophilus) or artificially in the lab (calcium chloride, electroporation). - The DNA recombines into the chromosome, or survives as a plasmid. - Griffith's experiment (1928): heat-killed virulent smooth (S) pneumococci turned live harmless rough (R) ones virulent. Avery, MacLeod and McCarty (1944) showed the transforming principle is DNA. 2. Transduction - A bacteriophage carries bacterial DNA from one host to the next. - Generalised transduction: during the lytic cycle a random piece of host DNA is packaged by mistake. - Specialised transduction: a temperate phage leaves the chromosome imprecisely and carries the adjacent genes (see the lysogenic cycle in chapter 3). - Clinical example: the genes for diphtheria and cholera toxin are carried by phages. 3. Conjugation - Direct contact through a sex pilus. The F⁺ donor passes a copy of the F plasmid to an F⁻ recipient, which becomes F⁺. - Transfer is one-directional and by rolling-circle replication, so the donor keeps its copy. - Hfr strains: the F plasmid is integrated into the chromosome, so chromosomal genes are transferred in order. This was used to map the E. coli chromosome by timing (interrupted mating). - Conjugative R plasmids spread resistance between species, for example among Enterobacteriaceae in hospitals. Exam focus: for each mechanism know what carries the DNA, whether contact is needed, and a medical example.

Virulence factors - Adhesion: pili and fimbriae, surface adhesins (for example uropathogenic E. coli binding the bladder epithelium). - Invasion: enzymes such as hyaluronidase, collagenase and coagulase. - Immune evasion: capsule (antiphagocytic), protein A (binds antibody Fc), antigenic variation, IgA protease. - Iron acquisition: siderophores. - Biofilms: communities embedded in matrix on catheters and prostheses. They are very tolerant to antibiotics. Toxins - Exotoxins: proteins secreted by Gram-positive and Gram-negative bacteria. - Highly potent and specific (tetanus, botulinum, diphtheria, cholera). - Many have an A-B structure: B binds the cell and A is the active enzyme. - They can be turned into toxoids for vaccines. - Endotoxin: the lipid A of Gram-negative LPS. - Released when the bacteria lyse. - Causes fever, and in large amounts septic shock. It cannot be made into a toxoid. Antibiotic targets (selective toxicity) - Cell wall synthesis: beta-lactams, vancomycin. - 70S ribosome: aminoglycosides, tetracyclines, macrolides. - DNA gyrase: fluoroquinolones. - Folate synthesis: sulfonamides, trimethoprim. The four mechanisms of resistance 1. Enzymatic inactivation of the drug: beta-lactamases (including extended-spectrum beta-lactamases and carbapenemases); aminoglycoside-modifying enzymes. 2. Altered target: a mutated penicillin-binding protein (PBP2a in MRSA); a methylated ribosome; mutated gyrase. 3. Reduced entry: loss of porins in Gram-negatives. 4. Active efflux: pumps that throw the drug out. How resistance arises and spreads - Spontaneous mutation, then selection by the antibiotic (vertical spread). - Acquisition of resistance genes on plasmids, transposons and integrons (horizontal spread; chapter 2 section on gene transfer). - Overuse and misuse of antibiotics speed up selection. Clinical tip: MRSA, VRE, and carbapenem-resistant Enterobacterales are the key hospital resistance threats. Prudent prescribing and hand hygiene are the main defences.

1. List five structural differences between prokaryotic and eukaryotic cells. 2. Describe the Gram-negative envelope from inside to outside. 3. Explain, step by step, why Gram-positive bacteria stay purple after the Gram stain. 4. Why is Mycoplasma resistant to penicillin? 5. What is a plasmid? Name three types by the genes they carry. 6. Compare transformation, transduction and conjugation: vehicle, contact needed, one example. 7. What did Griffith's experiment show, and what did Avery add? 8. What is an Hfr strain, and how was it used? 9. Compare exotoxins and endotoxin. 10. List the four mechanisms of antibiotic resistance, with one example each. 11. Why are biofilms on prosthetic devices hard to treat?

1. Any five: no nucleus; no membrane organelles; circular chromosome in a nucleoid; 70S ribosomes; peptidoglycan wall; smaller size; binary fission; no cytoskeleton of the eukaryotic type. 2. Plasma membrane, periplasm with thin peptidoglycan, outer membrane with porins and LPS (lipid A, core, O antigen). 3. Crystal violet and iodine form a complex. Alcohol dehydrates the thick peptidoglycan and traps it, so safranin makes no visible difference. 4. It has no cell wall, and penicillin targets cell wall synthesis. 5. A small circular DNA that replicates independently. R (resistance), F (fertility), virulence (and metabolic). 6. Transformation: free DNA, no contact, pneumococci. Transduction: a phage, no cell contact, toxin genes. Conjugation: a sex pilus, contact needed, R plasmid spread. 7. Griffith: heat-killed virulent bacteria transformed harmless ones. Avery: the transforming agent is DNA. 8. A strain with the F plasmid integrated into the chromosome. It transfers chromosomal genes in order, which allowed mapping by interrupted mating. 9. Exotoxins: secreted proteins, potent, specific, can become toxoids. Endotoxin: lipid A of Gram-negative LPS, released at lysis, causes fever and shock, no toxoid. 10. Inactivation (beta-lactamase); altered target (PBP2a in MRSA); reduced entry (porin loss); efflux pumps. 11. The matrix limits drug penetration and immune access, and the bacteria in it grow slowly, which makes them tolerant.

Components - Genome: DNA or RNA; single- or double-stranded; linear, circular or segmented. It is small, from a few genes to a few hundred. - Capsid: a shell of repeating protein subunits (capsomers). Genome plus capsid is the nucleocapsid. - Envelope (only some viruses): a lipid bilayer taken from the host's plasma membrane, nuclear membrane or ER while budding. It carries viral glycoproteins (spikes) that bind the receptor. - Some carry enzymes inside the virion, for example reverse transcriptase (HIV) or RNA polymerase (influenza). Capsid symmetry - Helical: subunits wind around the genome in a rod (tobacco mosaic virus; the nucleocapsid of influenza). - Icosahedral: 20 triangular faces. The most efficient closed shell from repeated subunits (adenovirus, herpesvirus capsid, poliovirus). - Complex: bacteriophage T4, with an icosahedral head and a helical tail with fibres. Enveloped versus naked viruses - Enveloped viruses: - fragile; destroyed by detergents, alcohol, drying and bile - usually spread by respiratory droplets, blood and sex - examples: HIV, influenza, coronavirus, herpes - Naked (non-enveloped) viruses: - resistant to drying, acid and detergents - often spread by the faecal-oral route - examples: norovirus, rotavirus, poliovirus, hepatitis A Size - 20-300 nm, below the light microscope's resolution. They are seen by electron microscopy. Clinical tip: alcohol hand rub inactivates enveloped viruses well but is weak against norovirus. Soap and water are needed in norovirus outbreaks.

The general replication cycle 1. Attachment: a viral protein binds a specific receptor. This decides tropism (which species and cells can be infected). Examples: HIV gp120 on CD4; SARS-CoV-2 spike on ACE2; influenza HA on sialic acid. 2. Entry: - Phages inject their genome and leave the capsid outside. - Enveloped viruses fuse with the plasma membrane, or are endocytosed and fuse from the endosome (often triggered by low pH). - Naked viruses are endocytosed and escape the endosome. 3. Uncoating: the genome is released from the capsid. 4. Replication of the genome and synthesis of viral proteins, using host ribosomes. 5. Assembly of new virions. 6. Release: - naked viruses by lysis of the cell - enveloped viruses by budding, which takes the envelope from a host membrane and does not always kill the cell at once The lytic cycle (virulent phages, for example T4) - The phage takes over the cell. Hundreds of new virions are made in about 20-30 minutes, and the cell bursts. The lysogenic cycle (temperate phages, for example lambda) - The phage genome integrates into the bacterial chromosome as a prophage. - It is copied with the chromosome at every division, without harming the cell. - Stress (for example UV damage) can induce excision and switch to the lytic cycle. - Lysogenic conversion: the prophage can give the host new traits. Diphtheria toxin, cholera toxin and botulinum toxin genes are all phage-carried. The animal-virus parallel - Latency: herpesviruses persist as episomes in neurons and reactivate later. - Integration: retroviruses (HIV) integrate their DNA copy into the host genome as a provirus. Exam focus: tropism is set by receptor binding. Know the receptor for HIV, influenza and SARS-CoV-2.

Double-stranded DNA viruses (herpes, adeno, papilloma) - Mostly replicate in the nucleus, using the host RNA polymerase II for transcription. - Large DNA viruses (herpes) encode their own DNA polymerase, which antivirals can target (acyclovir). - Exception: poxviruses replicate in the cytoplasm and carry all their own enzymes. Positive-sense single-stranded RNA viruses (polio, coronavirus, hepatitis C) - The genome is itself an mRNA and is translated at once. - It encodes an RNA-dependent RNA polymerase that makes a negative copy, which is then the template for new positive genomes. - The naked genome alone is infectious. Negative-sense single-stranded RNA viruses (influenza, measles, rabies) - The genome must first be copied into positive mRNA. Host cells have no enzyme for that, so the virion must carry its own RNA-dependent RNA polymerase. - The naked genome alone is not infectious. Double-stranded RNA viruses (rotavirus) - Carry their own polymerase and transcribe inside the capsid. Retroviruses (HIV) - Positive-sense RNA, but not used as mRNA directly. - Reverse transcriptase copies RNA into DNA. Integrase inserts the DNA into the host genome as a provirus. Host RNA polymerase II then transcribes it. The consequence for mutation rates - RNA polymerases and reverse transcriptase have no proofreading. RNA viruses and retroviruses therefore mutate very fast, which drives drug resistance, immune escape and the need for new flu vaccines every year. Exam focus: explain why a negative-sense RNA virus must carry its polymerase inside the virion, while a positive-sense one need not.

HIV (retrovirus) - Enveloped. Two copies of positive-sense RNA, with reverse transcriptase, integrase and protease in the virion. - gp120 binds CD4, then a co-receptor (CCR5 or CXCR4). gp41 drives membrane fusion. - Reverse transcription, then integration as a provirus, then transcription and assembly. The viral protease cleaves the precursor polyproteins during maturation. - Loss of CD4 T cells leads to AIDS. - Drug targets correspond to each step: reverse transcriptase, integrase and protease inhibitors, and entry blockers (CCR5 antagonist). Combination therapy prevents resistance. - People with a homozygous CCR5-Δ32 deletion are highly resistant to CCR5-tropic HIV. Influenza (orthomyxovirus) - Enveloped, negative-sense RNA in 8 segments. - Haemagglutinin (HA) binds sialic acid for entry. Neuraminidase (NA) cleaves sialic acid to release new virions; oseltamivir blocks it. - Replicates in the nucleus (unusual for an RNA virus). - Antigenic drift: point mutations in HA and NA cause seasonal epidemics and require yearly vaccines. - Antigenic shift: reassortment of segments between human and animal strains in the same cell creates a new subtype and can cause pandemics. Coronavirus (SARS-CoV-2) - Enveloped, large positive-sense RNA genome (about 30 kb). Unusually, it has a proofreading exonuclease. - The spike (S) protein binds ACE2. Host proteases (TMPRSS2) activate the spike for fusion. - The genome is translated directly into polyproteins, cut by viral proteases (a target of nirmatrelvir). - mRNA vaccines code for the spike. Herpesviruses (HSV-1/2, VZV, EBV, CMV) - Enveloped, large dsDNA, icosahedral capsid. They replicate in the nucleus. - Hallmark: lifelong latency with reactivation. HSV hides in sensory ganglia (cold sores, genital herpes); VZV causes chickenpox, then shingles; EBV hides in B cells. - Acyclovir is phosphorylated by the viral thymidine kinase, so it is activated only in infected cells, then blocks the viral DNA polymerase. Clinical tip: reactivation of latent herpesviruses (CMV, VZV) is a major problem in immunosuppressed and transplant patients.

1. Why are viruses considered to be at the boundary of life? 2. Name the two main capsid symmetries, with an example of each. 3. Why do enveloped viruses spread more easily by respiratory droplets than by the faecal-oral route? 4. List the six steps of the viral replication cycle. 5. Compare the lytic and lysogenic cycles. What is lysogenic conversion? 6. Why must a negative-sense RNA virus carry its own polymerase in the virion? 7. Why do RNA viruses mutate faster than DNA viruses? 8. Describe HIV entry, naming the proteins involved. 9. Distinguish antigenic drift from antigenic shift. 10. What is the receptor of SARS-CoV-2? 11. Why is acyclovir selective for infected cells?

1. They have genes and evolve, but have no cell, no metabolism and no ribosomes, and cannot replicate outside a host. 2. Helical (tobacco mosaic virus, influenza nucleocapsid) and icosahedral (adenovirus, poliovirus). 3. The lipid envelope is destroyed by bile, acid and drying, so enveloped viruses do not survive the gut or long exposure outside a host. 4. Attachment, entry, uncoating, replication and protein synthesis, assembly, release. 5. Lytic: the phage multiplies and lyses the cell. Lysogenic: the genome integrates as a prophage and is copied with the host. Lysogenic conversion: prophage genes give the host new traits (for example toxins). 6. The genome cannot be translated, and host cells have no RNA-dependent RNA polymerase to copy it into mRNA. 7. Their polymerases (RdRp, reverse transcriptase) lack proofreading. 8. gp120 binds CD4, then CCR5 or CXCR4. gp41 then drives membrane fusion. 9. Drift: small point mutations in HA and NA (seasonal epidemics). Shift: reassortment of whole segments between strains (new subtype, pandemics). 10. ACE2 (with spike activation by TMPRSS2). 11. It must be phosphorylated first by the viral thymidine kinase, which is present only in infected cells.

Building blocks - A nucleotide has three parts: - a sugar (2′-deoxyribose in DNA) - a phosphate - a nitrogenous base - Purines (two rings): adenine, guanine. Pyrimidines (one ring): cytosine, thymine (uracil replaces thymine in RNA). - Nucleotides are joined by phosphodiester bonds between the 3′-OH of one sugar and the 5′-phosphate of the next. Each strand therefore has a 5′ end and a 3′ end. The double helix (Watson and Crick, 1953, using Franklin's X-ray data) - Two strands, antiparallel (one runs 5′→3′, the other 3′→5′). - Complementary pairing: A–T (2 H-bonds), G–C (3 H-bonds). GC-rich DNA is more stable. - Chargaff's rules: A = T and G = C in double-stranded DNA. - B-form: a right-handed helix, about 10.5 bp per turn, 2 nm wide, with major and minor grooves. Proteins read the sequence mostly through the major groove. - Stability comes from base stacking as well as hydrogen bonds. Properties that follow from the structure - Each strand is a template for the other, so replication is semiconservative (Meselson-Stahl). - Heating separates the strands (denaturation, melting). Cooling lets them re-pair (renaturation, hybridisation), which is the basis of PCR, probes and FISH. DNA versus RNA - RNA has ribose (with a 2′-OH), uracil instead of thymine, and is usually single-stranded and folds on itself. - The 2′-OH makes RNA less stable and able to catalyse reactions. Exam focus: be able to write the complementary strand of a sequence with its polarity. The complement of 5′-ATGC-3′ is 3′-TACG-5′, written 5′-GCAT-3′.

The levels of packing 1. The double helix: 2 nm. 2. The nucleosome, "beads on a string", 11 nm fibre: - about 147 bp of DNA wrapped 1.7 times around a histone octamer (two each of H2A, H2B, H3, H4) - linker DNA joins the nucleosomes, and histone H1 binds the linker 3. The 30 nm fibre: nucleosomes stacked with the help of H1. Its exact structure in living cells is debated. 4. Loops anchored to a protein scaffold (cohesin and CTCF form loop domains). 5. The mitotic chromosome: maximum compaction, about 10,000-fold in total. Histones - Small, basic proteins rich in lysine and arginine. Their positive charge binds the negative DNA backbone. - Their N-terminal tails stick out of the nucleosome and carry chemical modifications. Euchromatin versus heterochromatin - Euchromatin: less condensed, stains lightly, gene-rich and transcriptionally active. - Heterochromatin: condensed, stains darkly, usually silent. - Constitutive heterochromatin: always condensed (centromeres, telomeres, repetitive DNA). - Facultative heterochromatin: condensed in some cells or stages only (the inactive X, the Barr body). The histone code - Acetylation of lysines (by HATs) neutralises the positive charge, loosens the DNA and favours transcription. Deacetylation (HDACs) represses it. - Methylation of lysines can activate or repress depending on the site. H3K4me3 marks active promoters; H3K9me3 and H3K27me3 mark silent chromatin. - Phosphorylation and ubiquitination add further signals. - The combination is read by proteins that remodel chromatin (for example HP1 binds H3K9me3). DNA methylation - A methyl group is added to cytosine in CpG dinucleotides by DNA methyltransferases (DNMT). - Methylated CpG islands in promoters silence genes. - The pattern is copied after replication (DNMT1), so it is inherited by daughter cells. This is the basis of epigenetic memory (see chapter 18). Clinical tip: HDAC inhibitors and hypomethylating agents (azacitidine) are used in some blood cancers to reactivate silenced tumour-suppressor genes.

Telomeres - Thousands of repeats of TTAGGG at each chromosome end, bound by a protective protein complex called shelterin. - The 3′ strand overhangs and loops back (the T-loop), hiding the end so it is not mistaken for a DNA break. The end-replication problem - DNA polymerase works only 5′→3′ and needs a primer. - On the lagging strand, the last RNA primer at the very end cannot be replaced. So each round of replication loses some telomeric DNA (about 50-200 bp). Telomerase - A reverse transcriptase that carries its own RNA template and adds TTAGGG repeats to the 3′ end. - It is active in germ cells, stem cells and most cancer cells, and is low or absent in most somatic cells. Consequences - In somatic cells, telomeres shorten with each division. When they are critically short, the cell stops dividing (replicative senescence, the Hayflick limit of about 50 divisions) or dies. - About 85-90% of cancers reactivate telomerase to become immortal. The rest use a recombination-based mechanism (ALT). - Short-telomere syndromes (dyskeratosis congenita) cause bone marrow failure and pulmonary fibrosis. Centromeres - A constricted region where sister chromatids are held together by cohesin until anaphase. - Made of repetitive alpha-satellite DNA and marked by a special histone H3 variant (CENP-A). - The kinetochore, a protein complex, assembles on the centromere and binds spindle microtubules. - Classification by centromere position: - metacentric (in the middle) - submetacentric - acrocentric (near one end: human chromosomes 13, 14, 15, 21, 22) - telocentric (at the end; not found in humans) Exam focus: explain the end-replication problem in two sentences, and why cancer cells need telomerase.

The nuclear envelope - An inner and an outer membrane separated by the perinuclear space, which is continuous with the ER lumen. - The outer membrane is continuous with the rough ER and carries ribosomes. - It separates transcription (nucleus) from translation (cytoplasm). This allows RNA processing (capping, splicing, polyadenylation) before export. The nuclear lamina - A meshwork of intermediate filaments (lamins A, B and C) lining the inner membrane. - It supports the shape of the nucleus, anchors chromatin (lamina-associated domains, mostly silent) and organises the pores. - Phosphorylation of lamins at mitosis breaks down the envelope; dephosphorylation rebuilds it. Laminopathies - Mutations in LMNA (lamin A/C). - Hutchinson-Gilford progeria: a point mutation creates an abnormal splice site. It produces "progerin", a truncated lamin A that stays farnesylated and distorts the nucleus. Children show premature ageing and die of cardiovascular disease in their teens. - Other laminopathies include forms of muscular dystrophy (Emery-Dreifuss) and cardiomyopathy. The nuclear pore complex (NPC) - A huge channel (about 120 MDa) of about 30 different nucleoporins in multiple copies, with eightfold symmetry. - Its central channel is filled with FG-repeat nucleoporins, which form a selective barrier. - Small molecules (under about 40 kDa) diffuse freely. Larger ones need transport receptors. Import and export - Import: proteins with an NLS (short stretches of basic residues, for example PKKKRKV in the SV40 T antigen) are bound by importins and carried through. - Export: proteins with an NES (leucine-rich) are carried out by exportins. mRNAs are exported as ribonucleoprotein complexes. The Ran cycle gives transport its direction - Ran-GTP is high in the nucleus, because RCC1, the exchange factor that loads GTP, is bound to chromatin. - Ran-GDP is high in the cytoplasm, because RanGAP, which triggers GTP hydrolysis, is cytoplasmic. - Import: importin plus cargo enters the nucleus; Ran-GTP binds importin and releases the cargo. - Export: exportin binds its cargo only together with Ran-GTP in the nucleus. In the cytoplasm GTP hydrolysis releases the cargo. - The energy comes from GTP hydrolysis, and the gradient keeps the process one-way. Exam focus: draw the Ran cycle, with where RCC1 and RanGAP sit and what happens to import and export cargo.

1. Write the complementary strand of 5′-AGGTC-3′ in the 5′→3′ direction. 2. Why is GC-rich DNA more stable than AT-rich DNA? 3. Describe the nucleosome: which histones, how much DNA, and the role of H1. 4. Compare euchromatin and heterochromatin. Give one example of facultative heterochromatin. 5. What is the effect of histone acetylation on transcription, and why? 6. Where does DNA methylation occur, and what does it usually do? 7. Explain the end-replication problem. 8. Why do most cancers reactivate telomerase? 9. What is a centromere made of, and what assembles on it? 10. What is the nuclear lamina, and what goes wrong in progeria? 11. How does the Ran cycle make nuclear import one-directional? 12. What type of signal targets a protein to the nucleus, and what binds it?

1. 5′-GACCT-3′. 2. G–C pairs form three hydrogen bonds (A–T only two) and stack more strongly. 3. An octamer of two each of H2A, H2B, H3 and H4, wrapped by about 147 bp of DNA. H1 binds the linker DNA and helps compaction. 4. Euchromatin: open, light-staining, active. Heterochromatin: condensed, dark-staining, silent. Facultative example: the inactive X (Barr body). 5. It favours transcription. Acetylation neutralises the positive lysines, loosening DNA binding and recruiting activators. 6. On cytosines in CpG dinucleotides, especially CpG islands in promoters. It usually silences genes. 7. DNA polymerase needs a primer and works 5′→3′, so the end of the lagging strand cannot be completed. Chromosome ends shorten with each replication. 8. To prevent telomere shortening, avoid senescence and become immortal. 9. Repetitive alpha-satellite DNA with the histone variant CENP-A. The kinetochore assembles on it and binds spindle microtubules. 10. A meshwork of lamins lining the inner nuclear membrane. In progeria an abnormal splice site produces progerin, a truncated, permanently farnesylated lamin A that deforms the nucleus. 11. RCC1 in the nucleus keeps Ran-GTP high there; RanGAP in the cytoplasm keeps Ran-GDP high there. Ran-GTP releases import cargo in the nucleus, and hydrolysis releases export cargo in the cytoplasm. 12. A nuclear localisation signal (a basic amino acid stretch), bound by importins.

Structure - Tubular, without ribosomes, continuous with the rough ER. - Some cells have very little; others are packed with it. Functions 1. Lipid synthesis - Phospholipids are made on the cytosolic leaflet of the ER, then flippases move some to the luminal leaflet. - Cholesterol synthesis (HMG-CoA reductase is an ER enzyme). - Ceramide, the precursor of sphingolipids (completed in the Golgi). - Lipids reach other organelles by vesicles or through membrane contact sites with lipid-transfer proteins (mitochondria, peroxisomes do not receive vesicles). 2. Steroid hormones: abundant in adrenal cortex, Leydig cells and ovarian cells, together with mitochondria. 3. Detoxification: cytochrome P450 enzymes hydroxylate drugs and toxins, making them more water-soluble for excretion. Conjugation (glucuronidation) follows. 4. Calcium storage: the ER pumps Ca²⁺ in (SERCA pump) and releases it through IP3 receptors or ryanodine receptors. In muscle this specialised smooth ER is the sarcoplasmic reticulum, which triggers contraction. 5. Glucose release in liver: glucose-6-phosphatase in the ER membrane removes the phosphate, so free glucose can leave the cell. Clinical tips - Barbiturates, alcohol and some anti-epileptics induce P450 enzymes and the smooth ER proliferates. This speeds up metabolism of other drugs, a classic source of drug interactions. - Glucose-6-phosphatase deficiency (von Gierke disease, glycogen storage disease type I) causes severe fasting hypoglycaemia. Exam focus: match each cell type to its SER role. Hepatocytes detoxify; adrenal and gonadal cells make steroids; muscle stores calcium.

Two destinations for newly made proteins - Free cytosolic ribosomes make proteins for the cytosol, nucleus, mitochondria and peroxisomes (these are imported after translation). - ER-bound ribosomes make proteins for the ER, Golgi, lysosomes, plasma membrane and secretion. - The ribosomes are identical. The protein being made decides where the ribosome goes. Co-translational import, step by step 1. Translation starts in the cytosol. The signal peptide (a hydrophobic stretch of about 15-30 amino acids at the N-terminus) comes out of the ribosome. 2. SRP binds the signal peptide and pauses translation. 3. SRP binds the SRP receptor on the ER membrane. Both are GTPases. 4. The ribosome is handed to the Sec61 translocon, and translation resumes. The chain is threaded into the lumen as it grows. GTP hydrolysis releases SRP. 5. Signal peptidase in the lumen cuts off the signal peptide. 6. The finished soluble protein is released into the lumen, where it folds. The signal hypothesis (Blobel, Nobel Prize 1999): the targeting information is written in the protein's own sequence. Membrane proteins and their topology - A hydrophobic stop-transfer sequence stops translocation. The translocon opens sideways and releases it into the bilayer as a transmembrane helix. - Single-pass protein: N-terminus in the lumen, C-terminus in the cytosol (type I). - An internal signal-anchor sequence can orient the protein either way. - Multipass proteins alternate start-transfer and stop-transfer sequences. - Topology is fixed at this moment and kept for the life of the protein. The ER lumen side becomes the outside of the cell after the vesicle fuses with the plasma membrane. Other ER-lumen events - Disulfide bond formation (protein disulfide isomerase), because the lumen is oxidising. - Folding helped by chaperones (BiP, calnexin, calreticulin). - Some proteins receive a GPI anchor. Exam focus: draw the SRP cycle with the order of events, and explain why the lumen side of the ER equals the outside of the cell.

N-linked glycosylation - A preformed oligosaccharide of 14 sugars (Glc3Man9GlcNAc2) is built on the lipid dolichol phosphate. - Oligosaccharyltransferase transfers it en bloc to asparagine in the sequence Asn-X-Ser/Thr, while the chain is entering the ER. - The sugars help folding, protect the protein, and act as labels for quality control and sorting. Quality control: the calnexin/calreticulin cycle 1. Glucosidases I and II remove two of the three glucoses. 2. The monoglucosylated protein binds calnexin (membrane) or calreticulin (lumen), lectin chaperones that help folding. 3. Glucosidase II removes the last glucose and the protein is released. 4. If still misfolded, the enzyme UGGT adds a glucose back, and the protein goes round again. 5. Folded proteins leave for the Golgi in COPII vesicles. ER-associated degradation (ERAD) - Proteins that never fold are trimmed of mannose, recognised, pulled back to the cytosol through a retrotranslocation channel, ubiquitinated, and destroyed by the proteasome. - Clinical example: in cystic fibrosis, the ΔF508 CFTR protein misfolds and is degraded by ERAD, so little reaches the membrane. Corrector drugs help it fold. The unfolded protein response (UPR) - Unfolded proteins pile up in the lumen and pull the chaperone BiP away from three sensors, which become active: 1. IRE1: splices the XBP1 mRNA, making a transcription factor that increases chaperones and ERAD components. 2. PERK: phosphorylates eIF2α, slowing down most protein synthesis to reduce the load. 3. ATF6: moves to the Golgi, where it is cleaved to release a transcription factor that increases chaperones. - Adaptive outcome: more folding capacity, less incoming protein. - If the stress persists: the UPR switches to apoptosis (through the CHOP factor). Clinical tip: chronic ER stress is involved in type 2 diabetes (beta cells making large amounts of insulin), neurodegenerative diseases and some liver diseases. Alpha-1 antitrypsin deficiency (Z mutant) aggregates in the hepatocyte ER, causing liver disease, while too little reaches the lung.

Structure and polarity - A stack of 4-8 flattened cisternae, usually near the nucleus and centrosome. - Cis face (entry): faces the ER and receives vesicles from the ERGIC. - Medial cisternae. - Trans face and trans-Golgi network (TGN): exit and sorting station. - Each cisterna has its own enzymes, so modifications happen in order. - Movement through the stack: the cisternal maturation model (the cisternae themselves move forward and mature, with resident enzymes recycled backward in COPI vesicles) is the most widely accepted. Modifications - N-glycan processing: mannose trimming, then addition of GlcNAc, galactose and sialic acid, which gives "complex" glycans. - O-glycosylation: sugars added to serine and threonine (mucins are heavily O-glycosylated). - Sulfation of proteoglycans and tyrosines. - Proteolytic cleavage of precursors (proinsulin to insulin mainly in secretory granules). - Sphingolipid and glycolipid synthesis. Sorting of lysosomal enzymes by mannose-6-phosphate (M6P) 1. In the cis-Golgi, a phosphotransferase recognises a surface patch on lysosomal hydrolases and adds GlcNAc-phosphate to mannose. 2. A second enzyme removes the GlcNAc, exposing M6P. 3. In the TGN, M6P receptors bind the enzymes, which are packaged into clathrin-coated vesicles. 4. The vesicles deliver them to late endosomes. The acidic pH releases the enzyme, and the receptor returns to the Golgi. Clinical example: I-cell disease (mucolipidosis II) - The phosphotransferase is missing, so no M6P is added. - The lysosomal enzymes are secreted outside the cell instead, and lysosomes fill with undigested material ("inclusion bodies"). - The enzymes are high in blood. Children have severe skeletal and developmental problems. Exit routes from the TGN - Constitutive secretion: continuous, default, not regulated (collagen, antibodies from plasma cells, new membrane). - Regulated secretion: stored in dense granules, released on a signal (insulin, neurotransmitters, digestive enzymes). - Lysosomal route via M6P. Exam focus: explain step by step how an enzyme gets into the lysosome, and what happens in I-cell disease.

1. List four functions of the smooth ER and the cell type where each is prominent. 2. Why do barbiturates change the metabolism of other drugs? 3. Describe the steps of co-translational import, naming SRP, SRP receptor, Sec61 and signal peptidase. 4. What decides whether a ribosome becomes bound to the ER? 5. How does a stop-transfer sequence produce a single-pass membrane protein? 6. Where is the N-linked oligosaccharide attached, and on which sequence? 7. Describe the calnexin cycle. What role does UGGT play? 8. What happens to CFTR ΔF508 in the ER? 9. Name the three UPR sensors and what each does. 10. What is the difference between the cis and trans faces of the Golgi? 11. Describe the mannose-6-phosphate pathway. What is missing in I-cell disease? 12. Compare constitutive and regulated secretion with examples.

1. Lipid and cholesterol synthesis (most cells); steroid hormones (adrenal, Leydig cells); detoxification by P450 (hepatocytes); calcium storage (muscle SR); glucose release via glucose-6-phosphatase (hepatocytes). 2. They induce P450 enzymes and SER proliferation, which speeds up the breakdown of other drugs. 3. The signal peptide comes out; SRP binds it and pauses translation; SRP docks on the SRP receptor; the ribosome passes to the Sec61 translocon and translation resumes into the lumen; signal peptidase removes the signal. 4. The presence of a signal peptide in the protein being made. 5. It halts translocation; the translocon opens sideways and the hydrophobic segment enters the bilayer as a transmembrane helix. 6. On asparagine in the sequence Asn-X-Ser/Thr. 7. After trimming to one glucose, calnexin/calreticulin bind and help folding. Removal of the last glucose releases the protein. UGGT re-glucosylates misfolded proteins so they re-enter the cycle. 8. It misfolds and is sent to ERAD, so it is degraded and little reaches the membrane. 9. IRE1 (splices XBP1 for more chaperones); PERK (phosphorylates eIF2α to slow translation); ATF6 (cleaved in the Golgi to release a transcription factor for chaperones). 10. Cis: entry face towards the ER. Trans: exit face with the TGN, where sorting happens. 11. A phosphotransferase adds GlcNAc-phosphate; its removal exposes M6P; M6P receptors in the TGN send the enzymes to endosomes. In I-cell disease the phosphotransferase is missing, so the enzymes are secreted. 12. Constitutive: continuous, not regulated (collagen, antibodies). Regulated: stored in granules and released on a signal (insulin, neurotransmitters).

COPII: ER to Golgi (anterograde) - Recruited by the GTPase Sar1. Sar1-GTP inserts into the ER membrane, then recruits Sec23/24 (the inner coat that selects cargo) and Sec13/31 (the outer cage). - Cargo receptors bind export signals on cargo proteins (for example a di-acidic signal). - Vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC). COPI: Golgi to ER (retrograde), and between cisternae - Recruited by the GTPase ARF1. - Retrieves ER-resident proteins that escaped. Soluble ER proteins carry the C-terminal KDEL signal and bind the KDEL receptor in the Golgi, which sends them back in COPI vesicles. ER membrane proteins carry a KKXX signal. - Carries resident Golgi enzymes backward, according to the cisternal maturation model. Clathrin: from the plasma membrane and the TGN - Clathrin triskelions (three legs) assemble into a lattice of pentagons and hexagons. - Adaptor proteins (AP) link clathrin to cargo receptors: - AP2 at the plasma membrane (endocytosis) - AP1 at the TGN (with M6P receptors going to endosomes) - The GTPase dynamin forms a ring around the neck of the bud and pinches it off. - The coat is removed quickly after budding (by Hsc70 and auxilin), so the vesicle can fuse. Memory aid: COPII goes forward ("two" goes out from the ER), COPI goes back, clathrin works at the two ends of the pathway (plasma membrane and TGN). Exam trap: the KDEL receptor retrieves escaped ER proteins from the Golgi. It is a retrieval system, not a retention one.

Rab proteins - A large family (about 60 in humans) of small GTPases. Each compartment has its own Rab set: Rab5 on early endosomes, Rab7 on late endosomes, Rab1 on ER-to-Golgi vesicles. - Active (GTP-bound) Rabs sit on the membrane and recruit effectors: tethering proteins (long coiled-coil proteins or multisubunit complexes) and motor proteins. - Rab identity changes as a compartment matures (Rab5 to Rab7 conversion as early endosomes become late endosomes). SNARE proteins - v-SNAREs on the vesicle (for example synaptobrevin, also called VAMP). - t-SNAREs on the target membrane (syntaxin and SNAP-25). - The helices of v- and t-SNAREs wind into a very stable four-helix bundle (trans-SNARE complex), pulling the membranes together until they fuse. - Pairing specificity adds a second level of address checking. - After fusion, the ATPase NSF with its adaptor SNAPs pulls the complex apart, so the SNAREs can be reused. Synaptic vesicle fusion - The SNARE complex is pre-assembled but held by complexin. Calcium entry binds synaptotagmin, which triggers fusion in less than a millisecond. Clinical tip: tetanus and botulinum toxins are proteases that cut SNAREs. - Botulinum toxin blocks acetylcholine release at the neuromuscular junction, causing flaccid paralysis. It is used therapeutically (Botox) for spasticity, dystonia and cosmetic purposes. - Tetanus toxin travels to spinal inhibitory neurons and blocks their release, causing spastic paralysis. Exam focus: explain the difference between the jobs of Rab (address and tethering) and SNARE (fusion) proteins.

Constitutive (default) exocytosis - Present in all cells, continuous and unregulated. - Delivers new lipids and membrane proteins to the plasma membrane. - Secretes extracellular matrix (collagen from fibroblasts), plasma proteins (albumin from hepatocytes) and antibodies (plasma cells). Regulated exocytosis - Only in specialised secretory cells. - Cargo is concentrated in secretory granules (dense-core vesicles) or synaptic vesicles, which wait near the membrane. - Release is triggered by a signal, usually a rise in cytosolic Ca²⁺. - Examples: - insulin from pancreatic beta cells (glucose raises ATP, K-ATP channels close, the cell depolarises, Ca²⁺ enters) - neurotransmitters at synapses - digestive enzymes from pancreatic acinar cells - histamine from mast cells - adrenaline from the adrenal medulla Polarised cells - Epithelial cells sort proteins separately to the apical and basolateral surfaces, keeping the two domains different. Tight junctions stop mixing. Membrane balance - Exocytosis adds membrane; endocytosis removes it. At a busy synapse, vesicle membrane is recycled by endocytosis within seconds. Clinical tip: in cystic fibrosis and in some diabetes treatments the defect or drug target lies in secretion. Sulfonylureas close the beta-cell K-ATP channel to trigger insulin exocytosis.

Phagocytosis ("cell eating") - Uptake of large particles (over 0.5 μm): bacteria, dead cells, debris. - Done mainly by professional phagocytes (macrophages, neutrophils, dendritic cells). - Triggered by receptors (for antibody Fc, complement, or "eat me" signals such as phosphatidylserine on apoptotic cells). - Actin-driven pseudopods surround the particle, forming a phagosome, which fuses with lysosomes (phagolysosome) for digestion. Pinocytosis ("cell drinking") - Non-specific uptake of extracellular fluid and dissolved molecules in small vesicles. - Macropinocytosis: large actin-driven ruffles take up big gulps of fluid (used by dendritic cells to sample antigens). Receptor-mediated endocytosis: the LDL example 1. LDL (carrying cholesterol esters) binds the LDL receptor on the cell surface. 2. The receptors gather in clathrin-coated pits (via AP2). Dynamin pinches off the vesicle. 3. The vesicle loses its coat and fuses with an early endosome. 4. The acidic pH (about 6) releases LDL from the receptor. 5. The receptor recycles back to the surface. LDL goes to the lysosome, where cholesterol esters are hydrolysed and free cholesterol is released. 6. Free cholesterol lowers the cell's own synthesis (HMG-CoA reductase) and LDL-receptor production. Other examples: transferrin (iron uptake, where the receptor and apo-transferrin both recycle), insulin receptor down-regulation, and viral entry. Caveolae - Small flask-shaped invaginations rich in cholesterol, sphingolipids and the protein caveolin. - Clathrin-independent. Abundant in endothelial cells, adipocytes and muscle. - Roles in transcytosis across endothelium, signalling and membrane tension buffering. Endosome maturation - Early endosome (Rab5, pH about 6.2) is the sorting station: recycle receptors or send cargo on. - Late endosome (Rab7, pH about 5.5). - Fusion with lysosomes (pH about 4.5-5). Clinical tip: in familial hypercholesterolaemia, LDL receptors are missing or defective. LDL stays in the blood, and patients (especially homozygotes) develop very high cholesterol, xanthomas and early coronary disease. Statins raise LDL-receptor numbers and lower LDL.

1. Which coat forms vesicles from the ER, and which small GTPase recruits it? 2. What does the KDEL receptor do, and in which vesicles? 3. What are the roles of adaptor proteins and dynamin in clathrin-coated vesicles? 4. Distinguish the jobs of Rab proteins and SNARE proteins. 5. Why does botulinum toxin cause flaccid paralysis, and tetanus toxin spastic paralysis? 6. Compare constitutive and regulated exocytosis, with an example of each. 7. Trace LDL from the blood to the release of free cholesterol in the cell. 8. What goes wrong in familial hypercholesterolaemia? 9. What are caveolae made of, and where are they abundant? 10. What triggers phagocytosis of an apoptotic cell?

1. COPII, recruited by Sar1. 2. It binds escaped ER proteins with a KDEL signal in the Golgi and returns them to the ER in COPI vesicles. 3. Adaptors (AP2, AP1) link clathrin to cargo receptors; dynamin pinches off the neck of the bud. 4. Rab: compartment identity, tethering and motor recruitment. SNARE: pairing between vesicle and target and driving membrane fusion. 5. Both cleave SNAREs. Botulinum acts at the neuromuscular junction (no acetylcholine release, flaccid paralysis). Tetanus acts on inhibitory spinal interneurons (no inhibition, spastic paralysis). 6. Constitutive: continuous (collagen, albumin). Regulated: stored and released on a Ca²⁺ signal (insulin, neurotransmitters). 7. LDL binds its receptor; clathrin-coated pit; vesicle; early endosome, where acid pH frees LDL and the receptor recycles; lysosome hydrolyses cholesterol esters to free cholesterol. 8. LDL receptors are missing or defective, so LDL is not cleared from blood; very high cholesterol and early atherosclerosis. 9. Caveolin, cholesterol and sphingolipids. Endothelium, adipocytes and muscle. 10. "Eat me" signals such as phosphatidylserine exposed on the outer leaflet.

Structure - Single membrane, 0.1-1 μm, very variable shape. - A V-type H⁺-ATPase pumps protons in, keeping the lumen at about pH 4.5-5. - Membrane proteins are heavily glycosylated (LAMP proteins) to protect them from their own enzymes. Enzymes - About 60 acid hydrolases: proteases (cathepsins), nucleases, lipases, glycosidases, phosphatases, sulfatases. - They reach the lysosome by the M6P pathway (chapter 5). - Safety feature: if the lysosome leaks, the neutral cytosol (pH 7.2) inactivates most enzymes. What lysosomes digest - External material from endocytosis and phagocytosis (heterophagy). - The cell's own components from autophagy. The storage disease principle - Usually autosomal recessive (loss of one enzyme; carriers are healthy because half the enzyme is enough). - The undigested substrate builds up. Which tissues are affected depends on where that substrate is normally turned over. - Main examples: - Tay-Sachs disease: hexosaminidase A is missing, and the ganglioside GM2 accumulates in neurons. Progressive neurodegeneration, cherry-red spot on the macula, death in early childhood. Frequent in Ashkenazi Jews. - Gaucher disease: glucocerebrosidase is missing, and glucocerebroside accumulates in macrophages ("Gaucher cells"). Hepatosplenomegaly, bone lesions, anaemia. Treated with enzyme replacement therapy. - Pompe disease (glycogen storage disease type II): acid maltase is missing, and glycogen accumulates in lysosomes of muscle and heart. Cardiomyopathy. - Mucopolysaccharidoses (Hurler, Hunter): GAGs accumulate. Coarse facies, skeletal deformities. - I-cell disease: all enzymes are missorted (chapter 5). Treatments: enzyme replacement therapy (the enzyme carries M6P so cells take it up), substrate reduction, stem cell transplantation, and gene therapy under development. Exam focus: explain why a lysosomal enzyme deficiency is usually recessive, and link each disease to the enzyme and substrate.

Types - Macroautophagy (usually just "autophagy"): a double membrane (phagophore) grows around cytoplasm and closes as an autophagosome, which fuses with a lysosome (autolysosome). - Microautophagy: the lysosome membrane itself invaginates and swallows small portions of cytoplasm. - Chaperone-mediated autophagy: proteins with a KFERQ motif are carried by Hsc70 across the lysosome membrane through LAMP-2A. Regulation - mTOR, the nutrient sensor, inhibits autophagy when food and growth factors are plentiful. - Starvation (low amino acids, low energy via AMPK) switches mTOR off, and autophagy starts. - ATG proteins (autophagy-related genes) build the autophagosome. LC3 is attached to the membrane and serves as a marker (LC3-II). Selective autophagy - Mitophagy: damaged mitochondria (which have lost membrane potential) accumulate PINK1 on their surface, which recruits Parkin, a ubiquitin ligase, and marks them for autophagy. Mutations in PINK1 and Parkin cause familial Parkinson's disease. - Aggrephagy (protein aggregates) and xenophagy (intracellular bacteria). Functions - Survival during starvation (recycling of amino acids and lipids). - Quality control: removal of damaged organelles and misfolded proteins. - Defence against intracellular pathogens. - Development and tissue remodelling. Autophagy and disease - Reduced autophagy contributes to neurodegeneration (Huntington's, Alzheimer's, Parkinson's), where aggregates build up. - In cancer it has two faces: it suppresses early tumours by removing damage, but helps established tumours survive stress. Exam focus: the Nobel Prize 2016 (Ohsumi) was for the genetic dissection of autophagy in yeast. Know the role of mTOR as the off switch.

Structure - Single membrane, 0.1-1 μm, with a granular matrix. In some species they contain a crystalline core of urate oxidase (humans have lost it, which is why we get gout). - No DNA and no ribosomes. All proteins are made on free cytosolic ribosomes and imported after translation, fully folded, using peroxisomal targeting signals: PTS1 is SKL at the C-terminus. - They divide by growth and fission, and can also form from the ER. Functions 1. Oxidation reactions: oxidases use O2 to oxidise substrates, making H2O2. 2. Catalase breaks H2O2 into water and oxygen, or uses it to oxidise other substrates (phenols, formaldehyde, ethanol). This is part of alcohol metabolism. 3. Beta-oxidation of very-long-chain fatty acids (over 22 carbons) and branched fatty acids. They are shortened here and finished in the mitochondria. 4. Plasmalogen synthesis: ether phospholipids abundant in myelin and heart. 5. Bile acid synthesis in the liver. Abundance: very abundant in liver and kidney. Peroxisomal disorders - Zellweger syndrome: mutations in PEX genes (peroxins), so peroxisomes cannot import enzymes and are "empty ghosts". Newborns show hypotonia, seizures, liver disease and craniofacial features; death in the first year. - X-linked adrenoleukodystrophy: ABCD1 transporter defect, so very-long-chain fatty acids cannot enter and accumulate. Demyelination of the brain and adrenal failure. The film "Lorenzo's Oil" is about this disease. Exam trap: peroxisomes and mitochondria both oxidise fatty acids, but only mitochondria couple it to ATP synthesis. Peroxisomes release the energy as heat and produce H2O2.

Structure - Outer membrane: smooth, permeable to small molecules through porins (VDAC). - Intermembrane space: where protons are pumped; contains cytochrome c. - Inner membrane: - folded into cristae, which increase its area - very impermeable - rich in the lipid cardiolipin - carries the electron transport chain and ATP synthase - Matrix: Krebs cycle enzymes, fatty acid beta-oxidation, the mtDNA and mitochondrial ribosomes. Number and shape - Hundreds to thousands per cell, most in cells with high energy demand (heart muscle, neurons, kidney tubule). None in mature red blood cells. Dynamics - Mitochondria are a dynamic network, not separate beans. - Fusion (mitofusins MFN1/2 for the outer membrane, OPA1 for the inner) mixes contents and dilutes damage. - Fission (DRP1, often at ER contact sites) allows distribution and isolates damaged parts for mitophagy. - ER-mitochondria contact sites exchange calcium and lipids. - Mutations in MFN2 (Charcot-Marie-Tooth 2A) and OPA1 (dominant optic atrophy) cause neurological disease. Functions beyond ATP - Apoptosis: the release of cytochrome c starts the intrinsic pathway. - Calcium buffering. - Heat production in brown fat (uncoupling protein 1). - Parts of haem and steroid synthesis. Endosymbiotic theory (Margulis): evidence - Double membrane (the inner one is the bacterium's, the outer one from the host). - Own circular DNA without histones, like bacteria. - 70S-type ribosomes, sensitive to some antibacterial antibiotics. - Division by fission, like bacteria. - The inner membrane contains cardiolipin, as in bacterial membranes. - DNA sequences closest to alpha-proteobacteria. - Most original genes moved to the nucleus over evolution; their products are imported back (through TOM and TIM complexes, with an N-terminal targeting sequence). Clinical tip: aminoglycosides and linezolid can affect mitochondrial ribosomes; some people with an mtDNA variant (m.1555A>G) become deaf after a single dose of aminoglycoside.

The mitochondrial genome - Circular, 16,569 bp, many copies per mitochondrion (and hundreds to thousands per cell). - 37 genes: 13 proteins (all subunits of the respiratory chain and ATP synthase), 22 tRNAs and 2 rRNAs. - Compact, no introns. It uses a slightly different genetic code. - Mutates about 10 times faster than nuclear DNA (reactive oxygen species nearby, less repair, no histones). - All other mitochondrial proteins (over 1,000) are encoded in the nucleus. So mitochondrial disease can also follow Mendelian inheritance. Maternal inheritance - The oocyte contains about 100,000 or more mtDNA copies. The few sperm mitochondria are marked and destroyed after fertilisation. - An affected mother passes the mutation to all her children. An affected father passes it to none. - The pedigree shows transmission through females only, affecting both sons and daughters. Homoplasmy and heteroplasmy - Homoplasmy: all copies are the same. - Heteroplasmy: a mixture of normal and mutant mtDNA. The genetic bottleneck - During oogenesis only a small number of mtDNA copies passes into each future oocyte. So the mutant proportion can differ widely between the children of a heteroplasmic mother. Replicative segregation - At each cell division mitochondria are distributed randomly, so the proportion of mutants drifts between cells and tissues. The threshold effect - Symptoms appear when the mutant proportion in a tissue exceeds a threshold (often 60-90%). - Tissues with high energy demand (brain, muscle, heart, retina, inner ear) are affected first. Examples - LHON (Leber hereditary optic neuropathy): usually homoplasmic point mutations in complex I genes. Acute painless loss of central vision in young men. - MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes): often m.3243A>G in a tRNA gene, heteroplasmic. - MERRF (myoclonic epilepsy with ragged-red fibres): tRNA-Lys mutation. Ragged-red fibres are muscle fibres packed with abnormal mitochondria. Clinical tip: mitochondrial replacement therapy ("three-parent IVF") transfers the nucleus of an affected woman's egg into a donor egg with healthy mitochondria. Exam trap: "maternal inheritance" does not mean all children are equally affected. Because of heteroplasmy and the bottleneck, severity varies widely.

1. How is the acidic pH of the lysosome maintained? Why is it a safety feature? 2. Why are most lysosomal storage diseases autosomal recessive? 3. Match the enzyme and substrate: Tay-Sachs, Gaucher, Pompe. 4. What controls the start of autophagy, and how does starvation switch it on? 5. How are damaged mitochondria recognised for mitophagy? Which disease is linked? 6. Give three functions of peroxisomes. 7. How do peroxisomal proteins reach the organelle? 8. What goes wrong in Zellweger syndrome and in X-linked adrenoleukodystrophy? 9. List four pieces of evidence for the endosymbiotic origin of mitochondria. 10. How many proteins does mtDNA encode, and what are they? 11. Explain heteroplasmy, the bottleneck and the threshold effect. 12. A man with LHON asks if his children will be affected. What do you say?

1. A V-type H⁺-ATPase pumps protons in. The enzymes are inactive at cytosolic pH, so a leak does little damage. 2. They are loss-of-function enzyme defects; one normal copy gives enough enzyme. 3. Tay-Sachs: hexosaminidase A, GM2 ganglioside. Gaucher: glucocerebrosidase, glucocerebroside. Pompe: acid maltase, glycogen. 4. mTOR inhibits autophagy when nutrients are plentiful. Starvation inactivates mTOR (and activates AMPK), starting autophagy. 5. PINK1 accumulates on depolarised mitochondria and recruits Parkin, which ubiquitinates them. Mutations cause familial Parkinson's disease. 6. Any three: oxidation producing H2O2; catalase breakdown of H2O2; very-long-chain fatty acid beta-oxidation; plasmalogen synthesis; bile acid synthesis. 7. Made on free ribosomes and imported after translation using a PTS (PTS1, SKL at the C-terminus). 8. Zellweger: PEX gene defects, no protein import. X-ALD: ABCD1 defect, very-long-chain fatty acids accumulate. 9. Any four: double membrane; own circular DNA; 70S-like ribosomes; division by fission; cardiolipin; genes closest to alpha-proteobacteria. 10. 13 proteins, all subunits of the respiratory chain and ATP synthase (plus 22 tRNAs and 2 rRNAs). 11. Heteroplasmy: mixed normal and mutant copies. Bottleneck: few copies pass into each oocyte, so the proportion varies between children. Threshold: disease appears only above a critical mutant fraction. 12. No. mtDNA is inherited only from the mother, so a father does not transmit it.

Assembly - G-actin (globular monomer) binds ATP and polymerises into F-actin (filament). - After incorporation, ATP is hydrolysed to ADP. ADP-actin binds less tightly, so older parts of the filament tend to depolymerise. - The plus end grows faster than the minus end. Treadmilling - At a certain monomer concentration, subunits add at the plus end and leave at the minus end at the same rate. - The filament keeps the same length but its subunits move through it, from plus to minus. Accessory proteins - Profilin: helps ATP loading and adds monomers to plus ends. - Thymosin β4: sequesters monomers. - Cofilin: severs ADP-actin filaments, speeding turnover. - Nucleators: - Arp2/3 makes branched networks (lamellipodia) - formins make long unbranched filaments (filopodia, stress fibres) - Capping proteins: stop growth. - Cross-linkers: fimbrin and villin (tight parallel bundles), α-actinin (looser contractile bundles), filamin (gel-like networks). - Spectrin: under the red cell membrane. Structures and functions - Cell cortex: a network under the plasma membrane that shapes the cell. - Microvilli: a core of 20-30 parallel filaments bundled by villin and fimbrin, anchored to the terminal web. They increase the absorptive surface. - Cell migration: lamellipodia and filopodia push forward, adhesions grip, and myosin II contracts the rear. - Contractile ring: actin and myosin II pinch the cell in two in cytokinesis. - Stress fibres, and the adherens junction belt. - Muscle contraction (sarcomeres; see Histology). Myosin motors - Move towards the plus end (except myosin VI). - Myosin II: contraction. Myosin V: transport of vesicles along actin. Drugs as tools: cytochalasin caps plus ends; phalloidin (death cap mushroom) stabilises filaments and, labelled, stains actin in the lab. Clinical tip: in hereditary spherocytosis, defects in spectrin or ankyrin weaken the red cell cortex; cells become spheres and are destroyed in the spleen.

Structure - α/β-tubulin dimers line up head-to-tail in protofilaments; 13 protofilaments form a hollow tube 25 nm wide. - Polar: the minus end (α-tubulin exposed) and the plus end (β-tubulin exposed, the growing end). Dynamic instability - Tubulin binds GTP. The GTP on β-tubulin is hydrolysed shortly after assembly. - A growing plus end has a "GTP cap". If the cap is lost, GDP-tubulin, which is curved, peels away and the microtubule shrinks fast (catastrophe). Regaining GTP-tubulin at the end stops shrinkage (rescue). - The result: individual microtubules switch between growing and shrinking, letting the cell explore space and reorganise quickly (for example to capture chromosomes in mitosis). The MTOC and the centrosome - Microtubules are nucleated by γ-tubulin ring complexes in the microtubule-organising centre. - In animal cells the main MTOC is the centrosome: a pair of centrioles (each made of 9 triplet microtubules) set at right angles, surrounded by pericentriolar material. - The centrosome duplicates once per cell cycle and forms the two spindle poles. Motor proteins - Kinesins: most move towards the plus end, which means outward, from the cell centre to the periphery (anterograde axonal transport of vesicles and organelles). - Cytoplasmic dynein: moves towards the minus end, inward (retrograde axonal transport, positioning the Golgi near the centrosome). - Both use ATP and "walk" with two heads. Cilia and flagella - Axoneme: nine outer doublets plus two central single microtubules, the "9+2" pattern. - Axonemal dyneins on the doublets slide them against each other. Nexin links convert sliding into bending. - The base is the basal body, a centriole-like structure (9 triplets, 9+0). - Motile cilia: airway epithelium (mucociliary clearance), fallopian tube, ependyma. Sperm flagellum. - Primary cilia: single non-motile (9+0) cilia on most cells, which act as sensory antennae. Drugs - Colchicine binds tubulin and prevents assembly (used in gout: it stops neutrophil migration). - Vinca alkaloids (vincristine) prevent assembly; taxanes (paclitaxel) stabilise microtubules. Both block the mitotic spindle and are used as anticancer drugs. Clinical tip: primary ciliary dyskinesia (Kartagener syndrome) results from defective dynein arms. It causes chronic sinusitis and bronchiectasis, male infertility, and in half of patients situs inversus, because cilia set left-right asymmetry in the embryo.

Structure - About 10 nm wide, between actin (7 nm) and microtubules (25 nm), hence the name. - Subunits are long fibrous proteins with a central α-helical rod. - Two monomers form a coiled-coil dimer; two dimers join antiparallel into a tetramer. Because of this antiparallel arrangement the filament has no polarity and no motor proteins. - Eight tetramers side by side form a unit-length filament; these join end to end, like a rope. - No nucleotide binding, so they are stable. They are regulated by phosphorylation (for example lamins at mitosis). The main classes and where they are found - Keratins (types I and II, acidic and basic): epithelia. Hair and nails are hard keratins. - Vimentin: cells of mesenchymal origin (fibroblasts, endothelium, leukocytes). - Desmin: muscle. - Glial fibrillary acidic protein (GFAP): astrocytes. - Neurofilaments: neurons. - Nuclear lamins (A, B, C): all nucleated cells, inside the nucleus. Junction anchoring - Keratins attach to desmosomes (cell-cell) and hemidesmosomes (cell-basement membrane), spreading mechanical stress across the whole epithelium. Diseases - Epidermolysis bullosa simplex: mutations in keratin 5 or 14. Basal keratinocytes break with minor friction, and the skin blisters. - Desmin myopathies: skeletal and cardiac muscle weakness. - Laminopathies, including progeria (chapter 4). - Alexander disease: GFAP mutations. Clinical tip: in pathology, intermediate-filament immunostaining helps classify tumours of uncertain origin. Keratin positive suggests carcinoma, vimentin sarcoma, desmin muscle tumour, GFAP glioma.

1. Compare the three cytoskeletal filaments by size, subunit, polarity and motors. 2. What is treadmilling? 3. What are the roles of Arp2/3, formins and cofilin? 4. Describe the core of a microvillus. 5. Explain dynamic instability and the GTP cap. 6. What is the structure of a centrosome? 7. In which direction do kinesin and cytoplasmic dynein move? Give a function of each in neurons. 8. Describe the axoneme of a motile cilium, and explain how it bends. 9. Why can primary ciliary dyskinesia cause situs inversus? 10. How do vincristine and paclitaxel act, and why do both block mitosis? 11. Why do intermediate filaments have no polarity? 12. Which intermediate filament protein would you stain for in a suspected carcinoma, sarcoma or glioma?

1. Actin: 7 nm, actin-ATP, polar, myosins. Microtubules: 25 nm, α/β-tubulin-GTP, polar, kinesins and dyneins. Intermediate filaments: 10 nm, various proteins, non-polar, no motors. 2. Monomers add at the plus end and leave at the minus end at the same rate; the length stays constant while subunits flow through. 3. Arp2/3 nucleates branched networks; formins nucleate unbranched filaments; cofilin severs ADP-actin filaments. 4. 20-30 parallel actin filaments bundled by villin and fimbrin, anchored in the terminal web. 5. GTP-tubulin at the plus end (the GTP cap) stabilises growth. If it is lost, GDP-tubulin peels off and the microtubule shrinks fast (catastrophe) until rescue. 6. Two centrioles (9 triplets each) at right angles, surrounded by pericentriolar material with γ-tubulin ring complexes. 7. Kinesin: towards the plus end, anterograde transport. Dynein: towards the minus end, retrograde transport. 8. Nine outer doublets and two central singlets (9+2). Axonemal dyneins slide doublets; nexin links convert sliding into bending. 9. Cilia at the embryonic node set left-right asymmetry; without motility, the side is random. 10. Vincristine prevents assembly; paclitaxel stabilises microtubules. Both destroy the dynamics that the spindle needs. 11. Dimers join antiparallel into tetramers, so both ends are identical. 12. Keratin (carcinoma), vimentin (sarcoma), GFAP (glioma).

Lipids - Phospholipids: amphipathic, with a hydrophilic head and two hydrophobic tails. In water they spontaneously form bilayers. - Glycerophospholipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol. - Sphingomyelin: a sphingolipid. - Cholesterol: inserted between phospholipids. - Glycolipids: sugars on the outer leaflet only (for example gangliosides in neurons). Membrane asymmetry - Outer leaflet: rich in phosphatidylcholine, sphingomyelin and glycolipids. - Inner leaflet: rich in phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol. - Maintained by flippases (ATP-driven, move lipids inward) and floppases. Scramblases mix the leaflets. - Loss of asymmetry has meaning: phosphatidylserine appearing outside is an "eat me" signal on apoptotic cells, and on activated platelets it supports clotting. Fluidity - Lipids move sideways fast (lateral diffusion) and rotate, but flip-flop between leaflets is very rare without enzymes. - Factors: - higher temperature increases fluidity - unsaturated fatty acids (with cis double bonds, kinked) increase fluidity - shorter chains increase fluidity - cholesterol buffers fluidity: it stiffens the membrane at high temperature and prevents freezing at low temperature - The transition temperature (Tm) is the temperature at which the bilayer changes from gel to fluid. - Lipid rafts: small, ordered domains rich in cholesterol and sphingolipids that gather certain proteins. Membrane proteins - Integral (transmembrane): span the bilayer, often with α-helices of about 20 hydrophobic amino acids, or β-barrels (porins). - Peripheral: attached to the surface or to other proteins, removable with salt. - Lipid-anchored: via a GPI anchor (outside) or fatty acids or prenyl groups (inside). - Mobility: many diffuse sideways (the Frye-Edidin cell fusion experiment proved it); others are held by the cytoskeleton or junctions. The glycocalyx - Carbohydrates on glycoproteins and glycolipids, all facing outward. - Roles: protection, cell recognition, adhesion, blood group antigens (ABO). Exam focus: explain the role of cholesterol as a fluidity buffer, and why asymmetry matters (phosphatidylserine exposure).

Simple diffusion - Direct movement through the lipid bilayer of small non-polar molecules (O2, CO2, steroid hormones, anaesthetic gases). - The rate is proportional to the concentration gradient, the area and the lipid solubility, and inversely to the membrane thickness (Fick's law). - Not saturable. Osmosis - Movement of water across a semipermeable membrane from low solute concentration to high. - Water crosses slowly through the bilayer, and quickly through aquaporin channels (kidney collecting duct, red cells). - Tonicity: - isotonic (0.9% NaCl): no net change - hypotonic: water enters and cells swell (red cells can lyse) - hypertonic: water leaves and cells shrink (crenation) Facilitated diffusion by carriers (transporters) - The solute binds, the carrier changes shape, and the solute is released on the other side. - Specific and saturable: the rate reaches a maximum (Vmax) when all carriers are busy, with Michaelis-Menten-like kinetics (Km). - Can be competitively inhibited. - Example: GLUT glucose transporters. - GLUT1 in red cells and the blood-brain barrier. - GLUT2 in liver and beta cells (high Km, works as a glucose sensor). - GLUT4 in muscle and fat, moved to the membrane by insulin. Facilitated diffusion by channels - Water-filled pores through which specific ions or water pass very quickly (up to 10⁸ ions per second, far faster than carriers). - Selectivity filter: size and charge decide which ion passes (the K⁺ channel strips water from K⁺ but not from the smaller Na⁺). - Gating (open and closed states): - voltage-gated (Na⁺ and K⁺ channels of the action potential) - ligand-gated (the acetylcholine receptor) - mechanically gated (hair cells in the ear, touch) - Leak channels are mostly open (K⁺ leak channels set the resting potential). Exam focus: draw the rate-versus-concentration curves for simple diffusion (a straight line) and carrier-mediated transport (saturating hyperbola).

Primary active transport: ATP-driven pumps - P-type pumps (phosphorylated during the cycle): - Na⁺/K⁺-ATPase: plasma membrane of all cells. - Ca²⁺-ATPases: PMCA in the plasma membrane and SERCA in the ER keep cytosolic Ca²⁺ very low (about 100 nM). - H⁺/K⁺-ATPase: gastric parietal cells, acid secretion; blocked by proton pump inhibitors (omeprazole). - V-type pumps: acidify lysosomes and endosomes. - F-type: ATP synthase in mitochondria, the same machine working in reverse to make ATP. - ABC transporters: a large family that uses ATP to move many substrates. - MDR1 (P-glycoprotein) pumps drugs out of cells; cancer cells that overexpress it become resistant to chemotherapy. - CFTR is an ABC protein that works as a Cl⁻ channel; its defect causes cystic fibrosis. Secondary active transport - A carrier couples the downhill movement of one ion (usually Na⁺) to the uphill movement of another solute. - Symport (co-transport, same direction): - SGLT1 in the intestine and SGLT2 in the kidney tubule carry glucose with Na⁺, absorbing glucose against its gradient. - The Na⁺-amino acid symporters. - The Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) in the loop of Henle, blocked by furosemide. - Antiport (exchange, opposite directions): - Na⁺/Ca²⁺ exchanger (NCX) in the heart. - Na⁺/H⁺ exchanger regulates intracellular pH. - Cl⁻/HCO3⁻ exchanger (band 3) in red cells. Transepithelial transport: glucose absorption in the gut 1. SGLT1 on the apical membrane brings in glucose with Na⁺ (secondary active). 2. GLUT2 on the basolateral side lets glucose out to the blood (facilitated diffusion). 3. The Na/K pump on the basolateral side removes Na⁺ and keeps the gradient going (primary active). Clinical tips - Oral rehydration solution contains glucose and salt together because SGLT1 absorbs Na⁺ with glucose, and water follows. This works even in cholera. - SGLT2 inhibitors (gliflozins) make the kidney excrete glucose. They lower blood glucose in diabetes and also protect heart and kidney.

The Na/K-ATPase - A P-type pump in all animal cells. It uses up to a third of a resting cell's ATP (more in neurons). - Each cycle: 3 Na⁺ out, 2 K⁺ in, 1 ATP hydrolysed. - The cycle: 1. Three Na⁺ bind on the inside. 2. ATP phosphorylates the pump. 3. The pump changes shape and releases Na⁺ outside. 4. Two K⁺ bind outside. 5. Dephosphorylation returns the pump to its first shape and K⁺ is released inside. - Electrogenic: it moves one net positive charge out per cycle, contributing a few millivolts directly. - Roles: - keeps intracellular Na⁺ low (about 12 mM against 145 mM outside) and K⁺ high (about 140 mM against 4 mM outside) - controls cell volume by preventing osmotic swelling - powers secondary active transport Ion concentrations (typical) - Na⁺: inside 12 mM, outside 145 mM. - K⁺: inside 140 mM, outside 4 mM. - Cl⁻: inside about 10 mM, outside about 110 mM. - Ca²⁺: inside 0.0001 mM, outside about 2.5 mM. How the resting potential arises 1. The pump builds the K⁺ gradient. 2. At rest the membrane is most permeable to K⁺ (K⁺ leak channels). 3. K⁺ diffuses out, leaving unbalanced negative charges (mostly proteins that cannot leave) inside. 4. The negative inside pulls K⁺ back. Equilibrium is reached when the electrical force balances the chemical gradient. 5. The equilibrium potential for K⁺ (Nernst equation): E_K = (RT/zF) ln([K⁺]out / [K⁺]in) ≈ 61.5 mV × log10(4/140) ≈ −95 mV. 6. The small Na⁺ permeability pulls the potential up, giving a real resting potential of about −70 mV (described by the Goldman-Hodgkin-Katz equation). Clinical tips - Digoxin inhibits the cardiac Na/K pump. Intracellular Na⁺ rises, the Na⁺/Ca²⁺ exchanger removes less Ca²⁺, and contraction strengthens. - Hyperkalaemia reduces the K⁺ gradient and depolarises cells, which can cause fatal arrhythmias. This is why plasma K⁺ is tightly monitored. Exam focus: explain why the resting potential is close to E_K, and why changing extracellular K⁺ changes it much more than changing Na⁺.

1. Which molecules cross a pure lipid bilayer easily, and which do not? 2. Which lipids are enriched in each leaflet of the plasma membrane? What does phosphatidylserine on the outer surface signal? 3. How does cholesterol affect membrane fluidity? 4. Distinguish simple diffusion from facilitated diffusion by carriers. Which one saturates, and why? 5. Why are channels faster than carriers? 6. What happens to a red blood cell in a hypotonic solution, and why? 7. Distinguish primary and secondary active transport, with an example of each. 8. Describe how glucose is absorbed across the intestinal epithelium. 9. Why does oral rehydration solution contain glucose? 10. What is the stoichiometry of the Na/K-ATPase, and why is it electrogenic? 11. Why is the resting potential close to the K⁺ equilibrium potential? 12. How does digoxin increase the strength of cardiac contraction?

1. Easily: small hydrophobic molecules (O2, CO2, steroids); slowly: small uncharged polar ones (water, urea). Not: ions and large polar molecules (glucose). 2. Outer: phosphatidylcholine, sphingomyelin, glycolipids. Inner: phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol. Outer phosphatidylserine is an "eat me" signal (apoptosis) and supports clotting on platelets. 3. It buffers fluidity: less fluid at high temperature, prevents freezing at low temperature. 4. Simple diffusion goes directly through the bilayer and does not saturate. Carrier transport is specific and saturates because there is a finite number of carriers (Vmax). 5. Channels are open pores; carriers must bind and change shape for every solute molecule. 6. Water enters by osmosis; the cell swells and may lyse (haemolysis). 7. Primary: uses ATP directly (Na/K-ATPase). Secondary: uses an ion gradient (SGLT1, the Na⁺/Ca²⁺ exchanger). 8. SGLT1 (apical, with Na⁺) brings it in; GLUT2 (basolateral) lets it out; the basolateral Na/K pump maintains the Na⁺ gradient. 9. SGLT1 absorbs Na⁺ together with glucose, and water follows the absorbed solutes. 10. 3 Na⁺ out, 2 K⁺ in per ATP; one net positive charge leaves per cycle. 11. At rest the membrane is mostly permeable to K⁺ via leak channels. 12. It inhibits the Na/K pump; intracellular Na⁺ rises, the Na⁺/Ca²⁺ exchanger removes less Ca²⁺, so more Ca²⁺ is available for contraction.

Milestones - 1866: Gregor Mendel, working on peas, shows that traits are inherited as discrete factors in predictable ratios. Ignored for 34 years. - 1900: Mendel's laws rediscovered (de Vries, Correns, Tschermak). - 1902: Sutton and Boveri: the chromosome theory of inheritance. Chromosomes behave in meiosis just as Mendel's factors do. - 1902: Archibald Garrod describes alkaptonuria as an "inborn error of metabolism", the first human recessive trait. - 1910: Thomas Hunt Morgan, Drosophila: the white-eye gene is on the X chromosome; genes lie on chromosomes. His student Sturtevant builds the first genetic map (1913). - 1928-1944: Griffith and Avery show that DNA is the transforming principle (chapter 2). Hershey and Chase confirm it in 1952. - 1941: Beadle and Tatum: one gene, one enzyme. - 1953: Watson and Crick, using Franklin's data: the double helix. - 1956: Tjio and Levan: humans have 46 chromosomes. - 1959: Lejeune: Down syndrome is trisomy 21. - 1977: Sanger sequencing. 1983: PCR (Mullis). - 2001-2003: the Human Genome Project completes the human sequence. 2022: the first fully complete, gap-free sequence (Telomere-to-Telomere consortium). The central dogma - DNA → RNA → protein (with reverse transcription as the exception from retroviruses). - The detailed mechanisms of replication, transcription and translation are not covered in these lectures. Genetics in medicine - Monogenic (Mendelian) diseases: one gene with a large effect (cystic fibrosis, Huntington disease). - Chromosomal disorders: numbers or structure (Down syndrome). - Multifactorial disorders: many genes plus environment (diabetes, hypertension). - Mitochondrial disorders (chapter 7). - Somatic genetic disease: cancer (chapter 19).

Size and content - About 3.2 billion bp per haploid genome, in 22 autosomes plus X or Y. - About 20,000 protein-coding genes, a number similar to much simpler animals. Complexity comes from regulation and alternative splicing, not gene number. - Protein-coding exons: about 1.5% of the genome. - Genes are split: exons are separated by introns, which are much longer. The average gene spans about 27 kb, but its mRNA is only a few kb. Categories of DNA - Unique-sequence DNA: genes and most regulatory elements. - Moderately repetitive DNA: - gene families (rRNA and histone genes) - transposable elements, about 45% of the genome: - LINEs (long interspersed elements, L1): about 17% - SINEs (short interspersed elements, Alu): about 10%, about a million copies - LTR retrotransposons and DNA transposons - Highly repetitive DNA (satellite DNA): - alpha satellite at centromeres - minisatellites (VNTRs, repeat units of 10-100 bp) - microsatellites (STRs, 1-6 bp repeats, very polymorphic, used in DNA fingerprinting and forensics) - Pseudogenes: gene copies that no longer work. Reassociation kinetics (the classical method) - Denatured DNA re-pairs faster when a sequence has many copies. This splits the genome into highly repetitive, moderately repetitive and unique fractions. Regulatory elements - Promoters, enhancers, silencers, insulators. Many lie far from their target genes. Other genomes in the cell - mtDNA (chapter 7). Exam focus: remember the orders of magnitude. About 3 × 10⁹ bp, about 20,000 genes, about 1.5% coding, about 45% transposon-derived.

The Human Genome Project - An international public project (1990-2003), with a parallel private effort (Celera, Venter). - Strategies: the public project used clone-by-clone mapping then sequencing; Celera used whole-genome shotgun sequencing. - Draft published in 2001, "finished" in 2003. Some gaps (centromeres, repeats) were closed only in 2022. - Findings: - about 20,000 protein-coding genes (initial guesses were 100,000) - the genome is mostly non-coding and repetitive - two unrelated people differ at about 1 in 1,000 bp (single nucleotide polymorphisms) plus structural variation - Ethical, legal and social issues (ELSI) were funded from the start: privacy, discrimination, consent. After the HGP - HapMap and 1000 Genomes: maps of common variation, which made GWAS possible (chapter 14). - ENCODE: catalogues functional elements. - Next-generation sequencing: a genome now costs a few hundred euros; exome and genome sequencing are in clinical use. Gene families - Groups of genes with similar sequences, produced by gene duplication followed by divergence. - Examples: - globin genes: the α-globin cluster (chromosome 16) and β-globin cluster (chromosome 11), expressed in sequence during development (embryonic, fetal, adult haemoglobin) - HOX genes: body plan - immunoglobulin and MHC genes - olfactory receptor genes (the largest family, about 400 functional genes) - Duplicated copies can: - gain new functions (neofunctionalisation) - share the original function - decay into pseudogenes - Unequal crossing over between similar family members causes deletions and duplications (for example α-thalassaemia and red-green colour blindness). Exam focus: explain how gene families arise, and one disease caused by recombination between family members.

1. Define gene, locus, allele, genotype and phenotype. 2. What did Morgan show with the white-eyed fly? 3. What did Garrod call alkaptonuria, and why was it important? 4. What percentage of the human genome codes for proteins? How many protein-coding genes are there? 5. Name the main classes of repetitive DNA, with an example of each. 6. Why are microsatellites (STRs) used in forensic DNA profiling? 7. What were the three main findings of the Human Genome Project? 8. How are gene families formed? Give two examples. 9. How can unequal crossing over cause disease?

1. Gene: a DNA sequence encoding a functional product. Locus: its position on a chromosome. Allele: an alternative form of a gene. Genotype: the alleles carried. Phenotype: the observable trait. 2. That the gene is on the X chromosome, so genes lie on chromosomes. 3. An inborn error of metabolism. It was the first human disease shown to follow recessive Mendelian inheritance. 4. About 1.5%. About 20,000. 5. Transposable elements (LINEs, SINEs such as Alu); satellite DNA (alpha satellite at centromeres); mini- and microsatellites (VNTRs, STRs). 6. They are highly polymorphic in repeat number, so a combination of several STRs is almost unique to each person. 7. About 20,000 genes, far fewer than expected; mostly non-coding and repetitive DNA; people are over 99.5% identical. 8. By gene duplication followed by divergence. Examples: globins, HOX, immunoglobulin, olfactory receptors. 9. Misaligned pairing between similar genes during meiosis produces one chromosome with a deletion and one with a duplication (for example α-thalassaemia, colour blindness).

The monohybrid experiment - P generation: true-breeding purple × true-breeding white. - F1: all purple, so purple is dominant. - F1 self-crossed gives F2: about 3 purple : 1 white. White reappears, so the factor for white was hidden, not lost. First law: segregation - Each individual has two alleles per gene, one from each parent. - The two alleles separate (segregate) during gamete formation, so each gamete carries only one. - Fertilisation restores the pair at random. - Chromosomal basis: homologous chromosomes separate in anaphase I of meiosis. The dihybrid experiment - Round yellow (RRYY) × wrinkled green (rryy) gives F1 RrYy, all round yellow. - F1 self-cross gives F2 in a 9 : 3 : 3 : 1 ratio: - 9 round yellow - 3 round green - 3 wrinkled yellow - 1 wrinkled green - The new combinations show that the two traits are inherited independently. Second law: independent assortment - Alleles of different genes assort independently into gametes. An RrYy plant makes four gamete types in equal proportions: RY, Ry, rY, ry. - Chromosomal basis: the random orientation of different homologous pairs at metaphase I. - The exception: genes close together on the same chromosome are linked and do not assort independently (chapter 14). Dominance in Mendel's sense - In his traits, the heterozygote looks like the dominant homozygote (complete dominance). Other patterns are discussed in chapter 12. Exam focus: link each law to its meiotic event. Segregation corresponds to separation of homologues in anaphase I; independent assortment to the random orientation of bivalents at metaphase I.

The Punnett square - Write the gametes of one parent on the top and of the other on the side. Each box is one equally likely offspring genotype. - Monohybrid Aa × Aa: - genotypes 1 AA : 2 Aa : 1 aa - phenotypes 3 dominant : 1 recessive The test cross - Cross the individual with a dominant phenotype (AA or Aa?) with a recessive homozygote (aa). - All offspring dominant: the parent is AA. A 1 : 1 ratio of dominant to recessive: the parent is Aa. - A dihybrid test cross (AaBb × aabb) gives 1 : 1 : 1 : 1 when the genes assort independently. The probability rules - Product rule (AND): the probability that two independent events both occur is the product. P(aa from Aa × Aa) = ½ × ½ = ¼. - Sum rule (OR): the probability that one of two mutually exclusive events occurs is the sum. P(heterozygote from Aa × Aa) = P(Aa) + P(aA) = ¼ + ¼ = ½. Using the rules on multi-gene crosses (the branch or forked-line method) - Example: from AaBbCc × AaBbCc, what is the probability of aabbcc? - ¼ × ¼ × ¼ = 1/64. - Probability of A_B_cc (dominant, dominant, recessive): ¾ × ¾ × ¼ = 9/64. - The number of gamete types from n heterozygous genes: 2ⁿ. The number of F2 genotypes: 3ⁿ. Binomial probability for families - The chance of exactly k affected children in n births, with p the chance per child: C(n,k) × pᵏ × (1−p)ⁿ⁻ᵏ. - Example: two carrier parents (p = ¼) with 4 children. Probability of exactly one affected: 4 × ¼ × (¾)³ = 0.42. - Each child is an independent event. "Chance has no memory": having one affected child does not change the risk for the next. Conditional probability (used in counselling) - The unaffected sibling of a child with a recessive disease has a 2/3 chance of being a carrier, not ½. We already know they are not aa, so only AA, Aa and aA remain, and two of those three are carriers. Exam trap: when the question is "an unaffected child", use 2/3, not ½, for carrier risk.

Sex chromosomes - Female 46,XX; male 46,XY. - The X is large (about 800 protein-coding genes); the Y is small (about 60), mostly for male functions. - Pseudoautosomal regions (PAR1, PAR2) at the tips of X and Y pair and recombine in male meiosis. SRY and sex determination - SRY (sex-determining region Y), on the short arm of the Y, encodes a transcription factor. - At about week 6-7, SRY switches on SOX9 in the bipotential gonad, which becomes a testis. - Sertoli cells make anti-Müllerian hormone, so the Müllerian ducts regress. - Leydig cells make testosterone, so the Wolffian ducts develop. DHT masculinises the external genitalia. - Without SRY, the gonad becomes an ovary and the female pathway follows. - Evidence: - 46,XX males carry an SRY translocated to an X (by abnormal crossing over outside PAR1) - 46,XY females have SRY deletions or mutations (Swyer syndrome) - Other routes of disordered sex development include androgen insensitivity (46,XY, female appearance, testes present). X inactivation (Lyon hypothesis, 1961) - In each female somatic cell, one X is inactivated early in embryonic life (around the blastocyst stage). - The choice is random in each cell, but then fixed and passed to all descendants of that cell. Females are therefore mosaics of two cell populations. - The inactive X condenses into the Barr body (sex chromatin) at the edge of the nucleus. - Rule: number of Barr bodies = number of X chromosomes − 1. So XY has 0, XX has 1, XXY has 1, XXX has 2, 45,X has 0. The mechanism - The X-inactivation centre (XIC) contains XIST, which is expressed only from the future inactive X. - XIST RNA coats that X in cis and recruits silencing complexes. Histone modifications (H3K27me3) and DNA methylation follow. - Some genes escape inactivation (about 15%), including those in the pseudoautosomal regions. This explains why 45,X (Turner) and 47,XXY (Klinefelter) individuals have a phenotype. Consequences - Tortoiseshell and calico cats: orange and black patches represent the two X-linked coat-colour alleles. - Female carriers of X-linked diseases show variable symptoms, depending on the proportion of cells expressing the mutant X. Skewed inactivation can make a carrier symptomatic (for example a female carrier of Duchenne or haemophilia). Exam focus: calculate the number of Barr bodies for any karyotype.

1. State Mendel's two laws and the meiotic event that explains each. 2. What is the F2 genotypic and phenotypic ratio of a monohybrid cross? 3. What is the F2 phenotypic ratio of a dihybrid cross, and why? 4. How would you tell whether a purple-flowered plant is PP or Pp? 5. From AaBbCcDd × AaBbCcDd, what is the probability of aabbccdd? 6. Two carriers of cystic fibrosis have three children. What is the probability that none is affected? 7. A healthy woman has a brother with cystic fibrosis. What is her chance of being a carrier? 8. What does SRY do? What happens if it is translocated to an X chromosome? 9. How many Barr bodies are in cells of a 47,XXY individual? And 45,X? 10. What is the role of XIST? 11. Why can a female carrier of an X-linked disease sometimes show symptoms?

1. Segregation: separation of homologues in anaphase I. Independent assortment: random orientation of bivalents at metaphase I. 2. 1 AA : 2 Aa : 1 aa; 3 dominant : 1 recessive. 3. 9 : 3 : 3 : 1, because the two genes assort independently (3:1 × 3:1). 4. A test cross with pp. All purple means PP; 1 : 1 means Pp. 5. (¼)⁴ = 1/256. 6. (¾)³ = 27/64 ≈ 0.42. 7. 2/3. Her parents are both carriers, and she is known not to be affected. 8. It switches on SOX9 and directs the gonad to become a testis. A 46,XX individual carrying SRY on an X develops as a male. 9. XXY: 1. 45,X: 0. 10. A non-coding RNA that coats the X it comes from in cis and recruits silencing machinery. 11. Skewed X inactivation: most cells may have inactivated the normal X.

Types of dominance - Complete dominance: Aa looks like AA (Mendel's peas). - Incomplete dominance: Aa is intermediate. - Snapdragons: red × white gives pink F1. The F2 is 1 red : 2 pink : 1 white; phenotype ratio equals genotype ratio. - Human example: familial hypercholesterolaemia. Heterozygotes have raised cholesterol; homozygotes much higher and earlier disease. - Codominance: both alleles are fully expressed in the heterozygote. - AB blood group: both A and B antigens on the red cells. - MN blood groups. - Sickle cell trait at the protein level: HbA and HbS are both present. - Overdominance (heterozygote advantage): Aa is fitter than either homozygote. Sickle cell carriers are protected against severe malaria. Dominance depends on the level of observation. For sickle cell: - Anaemia: recessive (only HbSS). - Sickling at low oxygen: dominant. - Haemoglobin molecules: codominant. Lethal alleles - Recessive lethal: aa dies. - Dominant with recessive lethal effect: yellow coat colour in mice. Yellow × yellow gives a 2 yellow : 1 non-yellow ratio instead of 3:1, because the homozygous yellow embryos die. - Human examples: achondroplasia (heterozygotes have short stature; homozygotes are usually lethal); Huntington disease is a late-acting dominant, not lethal before reproduction. Multiple alleles - A gene can have more than two alleles in a population, but any one person carries only two. - ABO blood groups: three alleles, Iᴬ, Iᴮ and i. - Iᴬ and Iᴮ are codominant with each other; both are dominant to i. - Genotypes and blood groups: IᴬIᴬ or Iᴬi is group A; IᴮIᴮ or Iᴮi is group B; IᴬIᴮ is group AB; ii is group O. - The alleles encode glycosyltransferases that add different sugars to the H antigen (N-acetylgalactosamine for A, galactose for B). The O allele encodes an inactive enzyme. - Other examples: HLA genes (hundreds of alleles), the CFTR gene (over 2,000 known variants), coat colour series in rabbits. Clinical tip: the Bombay phenotype (hh) makes no H antigen, so the person types as O whatever their ABO alleles, and can only receive Bombay blood. It is a classic case of epistasis. Exam focus: predict blood groups of children from parents' groups. For example, A × B parents can have children of all four groups if both are heterozygous (Iᴬi × Iᴮi).

Epistasis - One gene masks the phenotypic effect of another gene. The masking gene is epistatic; the masked one is hypostatic. - The dihybrid F2 ratio changes from 9:3:3:1 to a modified ratio, always summing to 16. Recessive epistasis (9 : 3 : 4) - Labrador coat colour: gene B (black dominant over brown) and gene E (pigment deposition). - ee dogs are yellow whatever their B genotype. - F2 of BbEe × BbEe: 9 black (B_E_), 3 brown (bbE_), 4 yellow (__ee). - Human example: the Bombay phenotype. hh masks ABO. Dominant epistasis (12 : 3 : 1) - Squash fruit colour: a dominant allele W gives white whatever the other gene. Duplicate recessive epistasis, or complementary genes (9 : 7) - Two genes in the same pathway: a recessive homozygote at either gene blocks the pathway. - Sweet pea flower colour; human congenital deafness. Two deaf parents with recessive deafness in different genes can have all children hearing (aaBB × AAbb gives AaBb). This is a complementation test. Other interactions - Pleiotropy: one gene affects many traits. Marfan syndrome (FBN1, fibrillin-1) affects the skeleton, eye lens and aorta. Phenylketonuria affects the brain and pigmentation. - Genetic heterogeneity: mutations in different genes give the same phenotype (locus heterogeneity; for example many genes cause retinitis pigmentosa), or different mutations in the same gene give different severity (allelic heterogeneity). - Polygenic traits: many genes of small effect plus environment (height, skin colour). They give continuous variation with a normal distribution. Penetrance - The percentage of individuals with the genotype who show the phenotype at all. - Complete penetrance (100%): everyone with the genotype is affected. - Incomplete (reduced) penetrance: some carriers are unaffected and the trait "skips" a generation. - BRCA1: lifetime breast cancer risk about 60-70%. - Polydactyly. - Age-dependent penetrance: Huntington disease (usually from about age 40). Expressivity - The degree to which the phenotype is expressed among those affected. - Variable expressivity: neurofibromatosis type 1. Some patients have only café-au-lait spots; others have many neurofibromas and complications. Marfan syndrome is similar. - Causes: modifier genes, environment, chance, and allelic differences. Exam trap: penetrance is all-or-none (affected or not); expressivity is how much. A person with mild NF1 shows penetrance but low expressivity.

Cytoplasmic (extranuclear) inheritance - Genes in mitochondria (and chloroplasts in plants) are inherited through the egg cytoplasm only. - Uniparental, maternal transmission; no segregation ratios; heteroplasmy and threshold effects (chapter 7). - Reciprocal crosses give different results, which is the classic test for it. Genomic imprinting - For a small number of genes (about 100-200 in humans), only one parental copy is expressed. The other is silenced by DNA methylation, depending on whether it came from the father or the mother. - The imprint is set in the germ line: erased in primordial germ cells, then re-established according to the sex of the individual. A man passes a paternal imprint on all his alleles, including those he inherited from his mother. - Imprinted genes cluster in regions controlled by imprinting control regions. Many are involved in growth. The parental conflict hypothesis says paternally expressed genes tend to promote fetal growth, and maternally expressed genes to limit it. - Example: IGF2 is expressed from the paternal allele only. Prader-Willi and Angelman syndromes: the classic pair (chromosome 15q11-q13) - The region contains paternally expressed genes (for example SNRPN and a small nucleolar RNA cluster) and a maternally expressed gene (UBE3A in neurons). - Prader-Willi syndrome: loss of the paternal contribution. - Causes: deletion on the paternal 15 (about 70%); maternal uniparental disomy, meaning both 15s from the mother (about 25%); imprinting defects. - Features: neonatal hypotonia and feeding problems, then hyperphagia and obesity from childhood, short stature, small hands and feet, hypogonadism, mild intellectual disability. - Angelman syndrome: loss of the maternal UBE3A. - Causes: deletion on the maternal 15 (about 70%); paternal uniparental disomy; UBE3A mutations; imprinting defects. - Features: severe intellectual disability, absent speech, ataxia, seizures, happy demeanour with frequent laughter, microcephaly. - Diagnosis: methylation testing of the region detects deletion, UPD and imprinting defects. Beckwith-Wiedemann and Silver-Russell (chromosome 11p15) are another imprinting pair, with opposite growth effects (overgrowth versus growth restriction). Exam focus: the same deletion gives Prader-Willi if paternal and Angelman if maternal. Uniparental disomy for the other parent gives the same disease as a deletion.

1. Distinguish incomplete dominance from codominance, with an example of each. 2. Why does a cross of two yellow mice give a 2:1 ratio? 3. A group A father and a group B mother have a group O child. What are the parents' genotypes? 4. Which blood groups are possible among children of an AB × O couple? 5. Explain the 9:3:4 ratio of Labrador coat colour. 6. Two deaf parents have only hearing children. How can this be explained? 7. Define pleiotropy and give an example. 8. Distinguish penetrance from expressivity, with an example of each. 9. How does reciprocal crossing distinguish cytoplasmic from nuclear inheritance? 10. What is genomic imprinting? At what stage is the imprint reset? 11. A child has maternal uniparental disomy of chromosome 15. Which syndrome is expected, and why? 12. What gene is responsible for Angelman syndrome?

1. Incomplete: the heterozygote is intermediate (pink snapdragons). Codominance: both alleles fully expressed (AB blood group). 2. The yellow allele is lethal when homozygous, so the 1/4 YY embryos die, leaving 2 Yy yellow : 1 yy non-yellow. 3. Iᴬi and Iᴮi. 4. A and B only (IᴬIᴮ × ii gives Iᴬi or Iᴮi). 5. ee masks the B gene (recessive epistasis): 9 B_E_ black, 3 bbE_ brown, 4 __ee yellow. 6. Complementation: their deafness is in different genes (aaBB × AAbb), so all children are AaBb and hear. 7. One gene affecting several traits. Marfan syndrome (skeleton, eye, aorta). 8. Penetrance: whether the phenotype appears (BRCA1 carriers not all developing cancer). Expressivity: its severity among the affected (NF1 variability). 9. In cytoplasmic inheritance the reciprocal crosses give different results, following the mother; in nuclear inheritance they give the same. 10. Parent-of-origin-specific silencing of some genes by methylation. It is erased and reset in the germ line. 11. Prader-Willi syndrome: there is no paternal copy of 15q11-q13, so the paternally expressed genes are missing. 12. UBE3A, expressed from the maternal allele in neurons.

Standard symbols - Square: male. Circle: female. Diamond: sex unknown. - Filled symbol: affected. Empty: unaffected. - Half-filled, or a dot in the centre: carrier (heterozygote, or X-linked carrier female). - A diagonal line through the symbol: deceased. - The proband (the index case who brought the family to attention): an arrow, often with a P. - Horizontal line between a male and female: a mating. A double line: consanguineous mating (related partners). - Vertical line down from the mating line to a sibship line; the children hang from it in birth order, left to right. - Twins: two lines from a single point. Monozygotic twins have an added horizontal bar between the lines. - A small triangle: miscarriage. Numbering - Generations: Roman numerals (I, II, III), the oldest at the top. - Individuals within each generation: Arabic numerals from left to right. So II-3 is the third person in generation II. Taking a family history - At least three generations. - Ask about first-degree relatives (parents, siblings, children) and second-degree relatives, miscarriages and stillbirths, consanguinity, ethnic origin, and age at onset and age at death. Pitfalls - Small families may not show the classical ratios. - Non-paternity, adoption, new mutations and reduced penetrance can make a pattern look different. - Variable expressivity may hide mildly affected relatives. Examine relatives when possible. Exam focus: be ready to draw a three-generation pedigree from a written family history, and to name each individual (for example III-2).

Autosomal dominant (AD) - Affected people usually have an affected parent; appears in every generation (vertical transmission). - Males and females equally affected. Male-to-male transmission occurs. - An affected heterozygote × unaffected partner: 50% risk for each child. - Complicating factors: new mutations (especially with advanced paternal age), reduced penetrance, variable expressivity, late onset. - Examples: Huntington disease, Marfan syndrome, neurofibromatosis type 1, achondroplasia, familial hypercholesterolaemia, adult polycystic kidney disease, BRCA1/2. Autosomal recessive (AR) - Affected people usually have unaffected carrier parents; appears in one sibship (horizontal pattern), often skipping generations. - Males and females equally affected. - Carrier × carrier: 25% affected, 50% carriers, 25% non-carriers for each child. - Consanguinity increases the risk, because relatives are more likely to carry the same rare allele. - Examples: cystic fibrosis, sickle cell disease, thalassaemias, phenylketonuria, Tay-Sachs, spinal muscular atrophy, most enzyme deficiencies. X-linked recessive (XLR) - Mostly males affected (hemizygous). Females are usually carriers. - No father-to-son transmission (a father gives his Y to sons). - Affected males are related through carrier females ("knight's move" pattern). - Carrier mother × normal father: 50% of sons affected, 50% of daughters carriers. - An affected father: all daughters are obligate carriers, no sons affected. - Examples: Duchenne and Becker muscular dystrophy, haemophilia A and B, red-green colour blindness, G6PD deficiency, fragile X (which has special features). X-linked dominant (XLD) - Affects both sexes, but females are about twice as often affected (and often more mildly). - An affected father passes it to all his daughters and none of his sons. - An affected mother passes it to 50% of her children of either sex. - Some are lethal in males (Rett syndrome, incontinentia pigmenti), so only females are seen, with an excess of miscarriages. - Example: X-linked hypophosphataemic rickets. Y-linked (holandric) - Only males, passed from father to all sons. Rare. Example: some forms of male infertility (AZF deletions). Mitochondrial - Both sexes affected, transmitted only through mothers to all their children, with variable severity (chapter 7). Example: LHON, MELAS. Exam focus: the single most useful discriminator is father-to-son transmission. If present, X-linked inheritance is excluded.

Types of twins - Monozygotic (identical): one fertilised egg splits. Genetically identical (apart from rare new mutations and epigenetic differences). Always the same sex. - The timing of the split decides the placenta and membranes: early split (days 1-3) gives two placentas and two amnions; days 4-8, one placenta and two amnions (most common); later, one amnion, and very late incomplete splitting gives conjoined twins. - Dizygotic (fraternal): two eggs fertilised by two sperm. Share on average 50% of their genes. Can be different sexes. Frequency rises with maternal age, family history and assisted reproduction. Concordance - Concordant: both twins have the trait. Discordant: only one does. - Comparing concordance between monozygotic and dizygotic twins: - MZ and DZ both high: shared environment dominates (for example measles in the past). - MZ much higher than DZ: genetic influence. - MZ below 100%: environment or chance also matters, even for strongly genetic traits. - Examples (approximate concordance, MZ vs DZ): - type 1 diabetes: about 40% vs about 5% - schizophrenia: about 45% vs about 15% - cleft lip: about 35% vs about 5% Heritability - The fraction of the variation of a trait in a population that is due to genetic differences. - Estimated from twin studies (for example Falconer's formula: H² ≈ 2 × (rMZ − rDZ), from correlations). - It refers to a population, not an individual, and depends on the environment of that population. Limits - Monozygotic twins may share more environment than dizygotic twins (treated more alike). - Twins raised apart (adoption studies) help separate genes from shared family environment. Exam trap: a heritability of 0.8 does not mean 80% of one person's trait is due to genes. It is a population measure of variance.

Genetic counselling: the process 1. Establish an accurate diagnosis (history, examination, tests). 2. Draw the pedigree and determine the mode of inheritance. 3. Calculate the risk. 4. Communicate the information, including the natural history of the condition, the risk, and the reproductive options. 5. Support the family's decision. Counselling is non-directive: the counsellor informs and supports but does not decide for the family. Recurrence risks, typical figures - AD, one parent affected: 50%. - AR, both parents carriers: 25% affected, and 2/3 carrier risk for a healthy sibling. - XLR, carrier mother: 50% of sons affected. - New dominant mutation (both parents unaffected and tested): low, but not zero because of germline mosaicism (a few percent). - Chromosomal: for trisomy 21 due to non-disjunction, the risk rises with maternal age; for a Robertsonian translocation carrier parent, it is much higher (chapter 15). - Multifactorial: empirical risks from population data (usually 2-5% for first-degree relatives). Carrier screening - Offered to populations with a high carrier frequency: thalassaemia in Mediterranean populations (common in Italy), Tay-Sachs in Ashkenazi Jews, cystic fibrosis. Prenatal screening (estimates risk, does not diagnose) - First-trimester combined test (weeks 11-13): nuchal translucency on ultrasound plus maternal serum free β-hCG and PAPP-A, combined with maternal age. - Non-invasive prenatal testing (NIPT), from week 10: sequencing of cell-free fetal DNA (from the placenta) in maternal blood. Very sensitive for trisomy 21 (about 99%), less for 18 and 13. A positive result must be confirmed by an invasive test, because the DNA comes from the placenta (confined placental mosaicism) and false positives occur. - Ultrasound for structural anomalies (weeks 19-21). Prenatal diagnosis (invasive, diagnostic) - Chorionic villus sampling (CVS): - weeks 10-13 - takes a biopsy of placental villi transabdominally or transcervically - earlier result - miscarriage risk about 0.5-1% - possible confined placental mosaicism - Amniocentesis: - from week 15-16 - samples amniotic fluid containing fetal cells (amniocytes) under ultrasound guidance - miscarriage risk about 0.1-0.5% - later result - Analyses: karyotype, rapid aneuploidy tests (QF-PCR, FISH), chromosomal microarray, or targeted testing for a known familial mutation. Pre-implantation genetic testing (PGT): embryos made by IVF are biopsied and tested before transfer. Ethical points: informed consent, the right not to know, confidentiality versus family members' interests, and testing of children for adult-onset diseases (generally deferred until adulthood). Exam focus: know the timing, the sample and the risk of CVS versus amniocentesis, and why NIPT is screening, not diagnosis.

1. What symbols indicate a carrier, a deceased person, a proband and a consanguineous mating? 2. List three features of an autosomal dominant pedigree. 3. Why does consanguinity increase the risk of autosomal recessive disease? 4. An affected man with an X-linked recessive disease has children with an unaffected non-carrier woman. What are the risks for his sons and daughters? 5. Which single pedigree feature excludes X-linked inheritance? 6. A trait passes from an affected father to all his daughters and none of his sons. What is the pattern? 7. What does it mean if monozygotic concordance is 40% and dizygotic 5%? 8. Why is heritability a population measure? 9. What is non-directive counselling? 10. Compare CVS and amniocentesis: timing, sample, miscarriage risk. 11. Why must a positive NIPT be confirmed by an invasive test? 12. Two unaffected parents have a child with a new dominant mutation. Is the recurrence risk zero? Why?

1. Half-filled symbol or central dot; a diagonal line through the symbol; an arrow; a double mating line. 2. Any three: affected in every generation; affected people have an affected parent; both sexes equally; male-to-male transmission; 50% risk to children. 3. Relatives are more likely to carry the same rare allele inherited from a common ancestor. 4. No sons affected (they get his Y); all daughters are obligate carriers. 5. Father-to-son transmission. 6. X-linked dominant. 7. A substantial genetic component, but the environment or chance also matters (MZ concordance is well below 100%). 8. It describes the proportion of variance due to genetic differences in a particular population and environment, not the causes of one person's trait. 9. The counsellor gives information and support but does not tell the family what to decide. 10. CVS: weeks 10-13, placental villi, about 0.5-1%. Amniocentesis: from week 15-16, amniotic fluid cells, about 0.1-0.5%. 11. The cell-free DNA comes from the placenta, which may differ from the fetus (confined placental mosaicism), and the test is a screening test with false positives. 12. No. There is a small risk (a few percent) from germline mosaicism in a parent.

Complete versus incomplete linkage - Complete linkage: genes so close that crossing over between them almost never happens. Only parental combinations appear. - Incomplete linkage: crossing over sometimes happens, producing some recombinant gametes. - Unlinked (independent assortment): 50% recombinants, the same as genes on different chromosomes. Crossing over - Happens in prophase I (pachytene) between non-sister chromatids of homologous chromosomes, visible as chiasmata. - Each crossover involves only two of the four chromatids, so even if a crossover always occurs between two genes, at most 50% of gametes are recombinant. The test cross that reveals linkage - Cross a double heterozygote (AaBb) with a double recessive (aabb). The offspring phenotypes show directly which gametes the heterozygote made. - Independent genes: four classes in 1 : 1 : 1 : 1. - Linked genes: two large parental classes and two small recombinant classes. Coupling and repulsion (phase) - Coupling (cis): AB / ab, meaning both dominant alleles on the same chromosome. - Repulsion (trans): Ab / aB. - The phase decides which classes are parental and which recombinant. Calculating the recombination frequency (RF) - RF = recombinant offspring / total offspring × 100. - Example: AB/ab × ab/ab gives 420 AB, 410 ab, 85 Ab and 85 aB. RF = (85 + 85) / 1000 = 17%. Morgan's discovery - In Drosophila, Morgan found that body colour and wing shape were inherited together more often than expected. He proposed that genes on the same chromosome are linked and are separated by crossing over. Exam trap: the maximum observable RF is 50%. Genes far apart on the same chromosome can look unlinked.

Map units - 1 map unit = 1 centimorgan (cM) = 1% recombination. - In humans, 1 cM corresponds on average to about 1 million bp, but this varies. There are recombination hot spots and cold spots, and recombination is higher in female meiosis. - The whole human genetic map is about 3,500 cM. Building a map (Sturtevant, 1913) - Measure the RF between each pair of genes. - Distances are approximately additive: if A–B is 5 cM and B–C is 3 cM, then A–C is 8 cM (or 2 cM if C lies between A and B). - Three-point test crosses order three genes at once. The rarest class is the double crossover; comparing it with the parental classes shows which gene is in the middle. Why long distances are underestimated - Double crossovers between two distant genes restore the parental combination, so they go uncounted. Mapping functions correct for this. - Interference: one crossover reduces the chance of another nearby. The LOD score (logarithm of the odds) - In human pedigrees, linkage between a disease locus and a marker is tested by comparing two hypotheses: - the probability of the observed family data if the loci are linked at recombination fraction θ - the probability if they are unlinked (θ = 0.5) - LOD = log10 of the ratio of these two probabilities. - LOD of 3 or more: odds 1000 : 1 in favour of linkage, accepted as significant. - LOD of −2 or less: linkage excluded at that θ. - LOD scores from different families add up, so small families can be combined. - The θ with the highest LOD is the best estimate of distance. Use: positional cloning - Linkage analysis in families located the genes for Huntington disease (chromosome 4, 1983), cystic fibrosis (chromosome 7, 1989) and Duchenne muscular dystrophy, before their products were known. Exam focus: know what a LOD of 3 means (1000:1 odds) and why scores can be added across families.

Why markers are needed - A useful marker must be polymorphic (many people heterozygous), easy to type, and located at a known place. RFLP (restriction fragment length polymorphism) - A variation that creates or destroys a restriction enzyme site, changing fragment lengths. - Detected by Southern blotting with a probe (now by PCR). - The first DNA markers for human linkage (Huntington disease, 1983). - Example: the sickle cell mutation destroys an MstII site in the β-globin gene. Microsatellites (STRs) - Short tandem repeats (for example CA repeats) with many alleles of different lengths. Highly informative; typed by PCR and fragment analysis. - Used for linkage, forensic profiling, paternity testing and tumour instability (chapter 19). SNPs (single nucleotide polymorphisms) - Single base variations, the most common type (millions per genome). Usually two alleles, but so numerous that dense maps are possible. - Typed in hundreds of thousands at once on SNP arrays. Genome-wide association studies (GWAS) - Compare the frequency of hundreds of thousands to millions of SNPs in cases versus controls from the population, rather than in families. - Based on linkage disequilibrium: SNPs close to a causal variant are inherited together with it in the population over many generations. - A strict significance threshold is needed because of multiple testing: p < 5 × 10⁻⁸. - Findings: thousands of loci with small effects for common diseases (type 2 diabetes, Crohn's disease, coronary disease). Individually weak, they can be combined into polygenic risk scores. - Limits: an associated SNP is often not the causal variant itself; much heritability is still unexplained; results depend on the ancestry of the population studied. FISH (fluorescence in situ hybridisation) - A fluorescently labelled DNA probe hybridises to its complementary sequence on chromosomes (metaphase spreads or interphase nuclei). The signal shows where the sequence is, a physical map. - Clinical uses: - microdeletions (22q11.2 DiGeorge syndrome) - rapid aneuploidy detection on interphase cells (13, 18, 21, X, Y) - translocations and gene fusions in cancer (BCR-ABL, HER2 amplification in breast cancer) Genetic map versus physical map - Genetic map: distances in cM from recombination. - Physical map: distances in bp, from FISH, clone maps, and finally the DNA sequence itself. Exam focus: distinguish linkage analysis (families, Mendelian disease, LOD score) from association studies (populations, common disease, linkage disequilibrium, p < 5 × 10⁻⁸).

1. Why can the recombination frequency not exceed 50%? 2. A test cross of AaBb (coupling) × aabb gives 45 AB, 45 ab, 5 Ab, 5 aB. What is the distance between the genes? 3. What do coupling and repulsion mean? Why does the phase matter? 4. A–B is 12 cM, B–C is 4 cM, A–C is 16 cM. What is the gene order? 5. Why are long map distances underestimated? 6. What is a LOD score, and what value is taken as proof of linkage? 7. Why can LOD scores from different families be added? 8. What is an RFLP, and how was it used in the sickle cell gene? 9. What makes microsatellites more informative than SNPs for family linkage? 10. What is the basis of a GWAS, and why is the threshold so strict? 11. Give two clinical uses of FISH.

1. Each crossover involves two of the four chromatids, so at most half the products are recombinant; and even multiple crossovers average 50%. 2. RF = 10/100 = 10%, so 10 cM. 3. Coupling: both dominant alleles on one chromosome (AB/ab); repulsion: on different chromosomes (Ab/aB). The phase decides which offspring classes are parental and which are recombinant. 4. A–B–C (A and C are furthest apart, 12 + 4 = 16). 5. Double crossovers restore the parental combination and are not counted. 6. The log10 of the odds of linkage versus no linkage. LOD of 3 or more (1000:1). 7. They are logarithms of independent probabilities, and logarithms of products add. 8. A variation that changes a restriction site and fragment length. The sickle mutation destroys an MstII site in β-globin, changing the fragment pattern. 9. They have many alleles, so most people are heterozygous and the parental origin of each allele can be traced. 10. Linkage disequilibrium between SNPs and causal variants in the population. The threshold (5 × 10⁻⁸) corrects for testing about a million SNPs. 11. Any two: microdeletions (22q11.2); rapid aneuploidy detection; cancer translocations and amplifications (BCR-ABL, HER2).

Making a karyotype 1. Take dividing cells: peripheral blood lymphocytes stimulated with phytohaemagglutinin; also bone marrow, amniocytes, chorionic villi, skin fibroblasts, tumour cells. 2. Culture for about 72 hours. 3. Add colchicine (or colcemid), which blocks spindle formation, so cells stop in metaphase when chromosomes are most condensed. 4. Hypotonic solution swells the cells and spreads the chromosomes; fix and drop onto slides. 5. Stain: G-banding (Giemsa after trypsin) is standard. Dark bands are AT-rich, gene-poor, late-replicating; light bands are GC-rich and gene-rich. 6. Photograph, arrange the chromosomes in pairs by size, centromere position and banding pattern. Chromosome shapes by centromere position - Metacentric: centromere in the middle (1, 3, 16, 19, 20). - Submetacentric: off-centre (most chromosomes). - Acrocentric: near one end, with tiny short arms carrying rRNA genes and satellites (13, 14, 15, 21, 22). Important for Robertsonian translocations. - Human chromosomes are never telocentric. Nomenclature (ISCN) - p = short arm, q = long arm. Bands are numbered from the centromere outwards: 15q11.2 means chromosome 15, long arm, region 1, band 1, sub-band 2. - Normal: 46,XX or 46,XY. - Trisomy 21 in a male: 47,XY,+21. - Turner: 45,X. - Deletion: 46,XX,del(5)(p15) (cri du chat). - Translocation: 46,XY,t(9;22)(q34;q11) (the Philadelphia chromosome). - Mosaic: 45,X/46,XX. Beyond the karyotype - FISH: specific sequences (chapter 14). - Chromosomal microarray (array CGH, SNP array): gains and losses down to tens of kb across the whole genome. Now the first-line test for intellectual disability and multiple anomalies. It cannot detect balanced rearrangements. Exam focus: know why colchicine is used, and what a microarray can and cannot detect compared with a karyotype.

Deletions - Loss of a segment: terminal or interstitial. - Effect: haploinsufficiency of the genes lost. - Examples: - cri du chat syndrome, 5p deletion: high-pitched cat-like cry, microcephaly, intellectual disability - Wolf-Hirschhorn syndrome, 4p deletion - microdeletion syndromes (below the resolution of a karyotype, detected by FISH or microarray): 22q11.2 deletion (DiGeorge/velocardiofacial: conotruncal heart defects, thymic hypoplasia, hypocalcaemia, cleft palate), Williams syndrome (7q11.23, elastin), Prader-Willi and Angelman (15q11-q13) - many microdeletions arise from non-allelic recombination between low-copy repeats that flank the region Duplications - Extra copy of a segment. Usually milder than deletions of the same region. - Example: Charcot-Marie-Tooth type 1A (PMP22 duplication on 17p12). Inversions: a segment turned 180° - Pericentric: includes the centromere. - Paracentric: does not include the centromere. - Usually balanced and harmless for the carrier, but crossing over within the inversion loop in meiosis produces unbalanced gametes, causing infertility, miscarriages or affected children. - Inversion of chromosome 9 around the centromere, inv(9), is a common harmless variant. Reciprocal translocations - Exchange of segments between two non-homologous chromosomes. About 1 in 500 people. - Carriers are usually healthy (balanced), but in meiosis the four chromosomes pair as a quadrivalent (cross shape). Depending on how they segregate, gametes can be balanced or unbalanced, causing recurrent miscarriage, infertility, or affected children. - In cancer: t(9;22) in chronic myeloid leukaemia creates the BCR-ABL fusion gene; t(8;14) in Burkitt lymphoma puts MYC next to the immunoglobulin heavy-chain enhancer. Robertsonian translocations - Fusion of two acrocentric chromosomes at or near the centromere, with loss of the short arms (which contain only redundant rRNA genes, so no harm). - Carriers have 45 chromosomes but are clinically normal. About 1 in 1,000 people. - Most common: rob(13;14) and rob(14;21). - Consequence: translocation Down syndrome. A carrier of rob(14;21) can produce a gamete with the fused chromosome plus a normal 21. The child has 46 chromosomes but three copies of 21q. - About 4% of Down syndrome is translocation Down syndrome. It is not related to maternal age, and the recurrence risk is high, so parents must be karyotyped. - Observed risk of a Down syndrome child: about 10-15% if the mother is the carrier, about 1-2% if the father. - A rob(21;21) carrier can only produce trisomy 21 or monosomy 21 (lethal) conceptions: 100% risk for live births. Other structures - Isochromosome: a chromosome with two identical arms from misdivision of the centromere, for example i(Xq) in some Turner syndrome. - Ring chromosome: both ends lost and joined. Exam focus: explain why the karyotype of the parents is essential after a diagnosis of translocation Down syndrome.

Terms - Euploid: exact multiple of the haploid number (23). Normal diploid = 46. - Polyploidy: - triploidy (69): usually from fertilisation of one egg by two sperm (dispermy). Seen in miscarriages and molar pregnancies; incompatible with long survival - tetraploidy (92) - normal in some tissues (hepatocytes, megakaryocytes) - Aneuploidy: not a multiple of 23. - Trisomy: 47 (one extra). - Monosomy: 45 (one missing). - Only monosomy X (Turner) survives among full monosomies. Autosomal monosomies are lethal. - Only trisomies 13, 18 and 21 (and sex chromosome aneuploidies) survive to birth. The chromosomes involved are the gene-poor ones. Nondisjunction - Meiosis I nondisjunction: homologues fail to separate. All four gametes are abnormal: two with both homologues (n+1), two with none (n−1). The n+1 gametes carry one of each parental homologue. - Meiosis II nondisjunction: sister chromatids fail to separate. Two gametes normal, one n+1 with two identical copies, one n−1. - Mitotic nondisjunction after fertilisation: produces mosaicism. Parental origin and maternal age - In trisomy 21, about 90% of the extra chromosomes come from the mother, mostly from errors in maternal meiosis I. - Why age matters: oocytes are arrested in prophase I from fetal life until ovulation, up to 40-50 years. The cohesin that holds chromosomes together degrades with time, and crossovers may be absent or badly placed. - Risk of Down syndrome at birth: about 1 in 1,500 at age 20, about 1 in 900 at 30, about 1 in 350 at 35, about 1 in 100 at 40, about 1 in 30 at 45. - The absolute number of Down syndrome births is higher among younger mothers, because they have most babies. This is why screening is offered to all ages. Exam focus: draw nondisjunction in meiosis I and meiosis II and give the resulting gametes.

Down syndrome (trisomy 21) - About 1 in 700 live births; the most common chromosomal cause of intellectual disability. - Karyotypes: about 95% free trisomy (47,+21, from nondisjunction); about 4% Robertsonian translocation; about 1-2% mosaic (milder). - Features: - hypotonia; flat face; upslanting palpebral fissures; epicanthic folds; small ears; protruding tongue; single palmar crease; sandal gap between toes; Brushfield spots on the iris - intellectual disability (variable, mild to moderate) - congenital heart disease in about 40-50% (atrioventricular septal defect most typical) - duodenal atresia; Hirschsprung disease - hypothyroidism, leukaemia (transient abnormal myelopoiesis and AML-M7 in infancy, ALL), early Alzheimer disease (the APP gene is on chromosome 21), atlanto-axial instability, hearing and vision problems - Life expectancy now about 60 years. Edwards syndrome (trisomy 18) - About 1 in 5,000 live births. - Growth restriction, clenched fists with overlapping fingers, rocker-bottom feet, prominent occiput, small jaw, heart defects, severe intellectual disability. Most die within the first year. Patau syndrome (trisomy 13) - About 1 in 10,000-15,000 live births. - Holoprosencephaly (failure of forebrain division), cleft lip and palate, microphthalmia, postaxial polydactyly, scalp defects (cutis aplasia), heart defects. Most die within the first year. Turner syndrome (45,X and variants) - About 1 in 2,500 female births; about 99% of 45,X conceptions miscarry. - Karyotypes: 45,X about 50%; mosaics (45,X/46,XX); isochromosome Xq; X deletions. Mosaicism with Y material raises the risk of gonadoblastoma, so the gonads are removed. - Features: - short stature (SHOX haploinsufficiency; treated with growth hormone) - gonadal dysgenesis (streak ovaries), primary amenorrhoea, infertility (oestrogen replacement for puberty) - neonatal lymphoedema of hands and feet; webbed neck (from fetal cystic hygroma); low hairline; broad chest with widely spaced nipples; cubitus valgus - coarctation of the aorta and bicuspid aortic valve; horseshoe kidney - intelligence usually normal (some visuospatial difficulties) - Why a phenotype at all? Genes that escape X inactivation (SHOX in the pseudoautosomal region) need two copies. Klinefelter syndrome (47,XXY) - About 1 in 600 male births; often undiagnosed. - Features: tall stature with long legs, small firm testes, hypergonadotropic hypogonadism (low testosterone, high FSH and LH), azoospermia and infertility (the most common chromosomal cause of male infertility), gynaecomastia, reduced body hair, learning and language difficulties, raised risk of breast cancer and autoimmune disease. - One Barr body. Treatment: testosterone; sperm retrieval (micro-TESE) with ICSI for fertility. Other sex chromosome aneuploidies - 47,XXX (triple X): usually tall, often undiagnosed, may have mild learning difficulties; fertile. - 47,XYY: tall, usually normal, may have learning and behavioural difficulties; fertile. Exam focus: for each syndrome, the karyotype, one major malformation, and one long-term complication.

Uniparental disomy (UPD) - Both homologues of a pair come from the same parent. The karyotype is normal (46 chromosomes). - Mechanisms: - trisomy rescue: a trisomic conception loses one copy; in one third of cases the copy lost is the one from the single parent, leaving UPD - monosomy rescue (duplication of the single copy) - gamete complementation (rare) - Types: heterodisomy (the parent's two different homologues, from meiosis I errors); isodisomy (two identical copies, from meiosis II errors or duplication). - Consequences: - imprinting disorders: maternal UPD 15 causes Prader-Willi; paternal UPD 15 causes Angelman (chapter 12) - recessive disease when only one parent is a carrier: isodisomy makes the child homozygous (the first case was cystic fibrosis with maternal UPD 7) Mosaicism - Two or more genetically different cell lines in one individual derived from one zygote. (Different from chimerism, which comes from two zygotes.) - Causes: mitotic nondisjunction after fertilisation; loss of a chromosome from a trisomic embryo; new mutation during development. - Somatic mosaicism: generally milder phenotype, depending on the proportion and distribution of abnormal cells (mosaic Down syndrome, mosaic Turner). - Germline (gonadal) mosaicism: a parent carries a mutation in part of their germ cells only. This explains a recurrence of an apparently new dominant or X-linked disease in siblings (for example Duchenne, osteogenesis imperfecta). This is why the recurrence risk is never zero. - Confined placental mosaicism: the abnormal cell line is only in the placenta. Relevant to CVS and NIPT (chapter 13). Fragile X syndrome - The most common inherited cause of intellectual disability (about 1 in 4,000 males, 1 in 8,000 females). - Gene: FMR1 at Xq27.3, with a CGG repeat in its 5' untranslated region. - normal: under about 45 repeats - intermediate (grey zone): about 45-54 - premutation: about 55-200. Unstable, can expand in the next generation. Carriers risk fragile X-associated tremor/ataxia syndrome (FXTAS, older males) and premature ovarian insufficiency (females). - full mutation: over 200. The promoter becomes methylated, FMR1 is silenced and its protein FMRP (an RNA-binding protein regulating synaptic translation) is absent. - Features in males: intellectual disability, autistic features, long face, large ears, prominent jaw, macro-orchidism after puberty, joint laxity. Females with the full mutation are affected in about half of cases, usually more mildly (X inactivation). - Inheritance quirks: - expansion from premutation to full mutation happens almost only through the mother; the risk rises with the size of the maternal premutation - anticipation: the disease becomes more frequent and severe over generations - the Sherman paradox: the risk depends on position in the pedigree, explained by progressive expansion - normal transmitting males carry a premutation, pass it to all daughters (who are not affected) and no sons - Name: under folate-deficient culture, the X shows a fragile site (a constriction) at Xq27.3. Diagnosis today is by PCR and Southern blot to size the repeat. - Other repeat expansion diseases with anticipation: Huntington disease (CAG), myotonic dystrophy (CTG), Friedreich ataxia (GAA). Exam focus: explain the premutation/full mutation distinction and why expansion occurs through the mother.

1. Why is colchicine added to cells for a karyotype? 2. Which chromosomes are acrocentric, and why do they matter? 3. Write the karyotype of a female with trisomy 18. 4. What is the difference between a balanced and an unbalanced rearrangement? 5. Why can an inversion carrier have recurrent miscarriages? 6. Why must the parents be karyotyped after a diagnosis of translocation Down syndrome? 7. What gametes result from nondisjunction in meiosis I versus meiosis II? 8. Why does the risk of trisomy increase with maternal age? 9. Give three features and two long-term complications of Down syndrome. 10. Which autosomal trisomy is associated with holoprosencephaly? 11. Why do 45,X women have a phenotype when normal women have one X inactivated? 12. What is the most common chromosomal cause of male infertility? Give the karyotype and hormone profile. 13. How can trisomy rescue cause uniparental disomy? 14. How can two unaffected parents have two children with the same new dominant mutation? 15. What is the difference between an FMR1 premutation and a full mutation?

1. It blocks the spindle, so cells accumulate in metaphase when chromosomes are most condensed. 2. 13, 14, 15, 21 and 22. They can form Robertsonian translocations. 3. 47,XX,+18. 4. Balanced: no material gained or lost (usually no phenotype). Unbalanced: gain or loss of material (usually clinical effects). 5. Crossing over within the inversion loop produces unbalanced gametes. 6. One parent may carry a balanced Robertsonian translocation, which gives a high recurrence risk (up to 100% for rob(21;21)). 7. Meiosis I: all four abnormal (two n+1, two n−1). Meiosis II: two normal, one n+1, one n−1. 8. Oocytes stay arrested in prophase I for decades; cohesion between chromosomes weakens. 9. Features: any three of hypotonia, flat face, upslanting eyes, single palmar crease, sandal gap, intellectual disability, heart defect. Complications: any two of leukaemia, hypothyroidism, early Alzheimer disease, atlanto-axial instability. 10. Trisomy 13 (Patau). 11. Some genes escape X inactivation (such as SHOX in the pseudoautosomal region) and need two active copies. 12. Klinefelter, 47,XXY: low testosterone, high FSH and LH. 13. A trisomic conception loses one of its three copies. If the lost one was the only copy from one parent, both remaining come from the other parent. 14. Germline mosaicism in one parent. 15. Premutation: about 55-200 CGG repeats, unstable, FMR1 still expressed (risk of FXTAS and ovarian insufficiency). Full mutation: over 200, methylated and silenced, causing fragile X syndrome.

The equations - For one gene with two alleles A (frequency p) and a (frequency q): p + q = 1. - Genotype frequencies: AA = p², Aa = 2pq, aa = q². Their sum: p² + 2pq + q² = 1. Assumptions (equilibrium holds only if) 1. Large population (no genetic drift). 2. Random mating (no assortative mating, no inbreeding). 3. No mutation. 4. No selection (all genotypes equally fit). 5. No migration (no gene flow). - One generation of random mating restores equilibrium after a disturbance. Calculating carrier frequency from disease incidence (the key exam skill) - Cystic fibrosis in a European population: about 1 in 2,500 births. - q² = 1/2,500, so q = 1/50. - p ≈ 1 (since q is small). - Carrier frequency 2pq ≈ 2q = 2/50 = 1/25. - So about 1 in 25 Europeans is a CF carrier. For rare recessive diseases, carrier frequency ≈ 2 × √(incidence). For X-linked recessive traits - The frequency of affected males equals q (they are hemizygous). - Affected females: q². Carrier females: 2pq. - Example: red-green colour blindness in about 8% of males, so q = 0.08 and about 0.64% of females are affected; about 15% of females are carriers. Using Hardy-Weinberg in counselling - A healthy person with no family history, whose partner is a known CF carrier: the risk of an affected child = 1 (partner carrier) × 1/25 (person is a carrier) × 1/4 = 1/100. Testing for equilibrium - Compare observed genotype counts with p², 2pq, q² (chi-square test). A deviation suggests selection, non-random mating, population mixture or genotyping error. Exam trap: for rare diseases, carriers vastly outnumber affected individuals (1/25 vs 1/2,500 for CF). Most disease alleles are hidden in heterozygotes, which is why selection against a recessive disease removes it only very slowly.

Mutation - The ultimate source of new alleles. The rate per gene per generation is low (about 10⁻⁵ to 10⁻⁶), so on its own it changes frequencies very slowly. - For severe dominant diseases, many cases are new mutations (for example about 80% of achondroplasia). Natural selection - Fitness: the relative ability to survive and reproduce. - Selection against a dominant allele: fast, because all carriers express it. - Selection against a recessive allele: slow when rare, because most copies hide in heterozygotes. - Mutation-selection balance: loss of alleles by selection is balanced by new mutation, giving a stable low frequency. - Heterozygote advantage (balancing selection): keeps a harmful allele common. - Sickle cell: HbAS carriers are protected from severe falciparum malaria, so HbS is common in malarial regions of Africa, the Mediterranean, the Middle East and India. - Thalassaemia and G6PD deficiency show the same pattern. - Proposed for cystic fibrosis (resistance to cholera or typhoid), less certain. Genetic drift - Random changes of allele frequency from generation to generation, because each generation samples only some of the parents' alleles. - Strong in small populations: alleles may be lost or fixed by chance. - Founder effect: a small group founds a new population, carrying by chance a high frequency of a rare allele. - Examples: Ellis-van Creveld syndrome in the Old Order Amish; Tay-Sachs and Gaucher in Ashkenazi Jews; many disorders in Finland (Finnish disease heritage); porphyria variegata in Afrikaners of South Africa. - Bottleneck: a large population shrinks drastically and loses variation, then regrows. Migration (gene flow) - Movement of individuals between populations brings new alleles and makes populations more similar. - Example: the gradient of blood group B frequency from Asia into Europe. Non-random mating - Assortative mating: partners choose similar phenotypes (height, deafness: many deaf people marry each other). Increases homozygosity for the trait. - Inbreeding (next note). Exam focus: explain why sickle cell disease is common in Africa, and why Tay-Sachs was common in Ashkenazi Jews. The first is heterozygote advantage; the second probably a founder effect with drift.

Coefficient of relationship and inbreeding - Proportion of genes shared by descent: - first-degree relatives (parent-child, siblings): 1/2 - second-degree (grandparent, uncle/aunt, half-siblings): 1/4 - third-degree (first cousins): 1/8 - Coefficient of inbreeding (F): the probability that a person is homozygous at a locus with both alleles identical by descent from a common ancestor. - Child of first cousins: F = 1/16. - Child of siblings or parent-child (incest): F = 1/4. - Child of second cousins: F = 1/64. Risks - The frequency of a recessive disease in the children of inbred unions is q² + Fpq (instead of q²). The rarer the allele, the larger the relative increase. - Rare recessive diseases have a high proportion of consanguineous parents. This is a clue to recessive inheritance in a pedigree. - The absolute excess risk of birth defects or serious recessive disease for first cousins is about 2-3% above the background of about 2-3%. So roughly double, but the majority of children are healthy. Clinical and social points - Consanguinity is common in some parts of the world (for example the Middle East, North Africa, South Asia), where it is culturally valued. - Counselling is non-directive: explain the risk, offer carrier testing for disorders common in the community, and extended family history. Exam focus: calculate F for a child of first cousins (1/16) and know that inbreeding increases homozygosity without changing allele frequency.

1. State the Hardy-Weinberg equations and five assumptions. 2. A recessive disease has an incidence of 1 in 10,000. What is the carrier frequency? 3. A healthy woman with no family history has a partner who is a CF carrier (population carrier frequency 1/25). What is the risk of an affected child? 4. An X-linked recessive trait affects 1 in 50 males. What proportion of females are affected, and what proportion are carriers? 5. Why does selection against a rare recessive allele change its frequency so slowly? 6. Explain why HbS is common in malarial regions. 7. What is the founder effect? Give an example. 8. What is the coefficient of inbreeding for a child of first cousins? 9. Does inbreeding change allele frequencies? What does it change?

1. p + q = 1; p² + 2pq + q² = 1. Large population, random mating, no mutation, no selection, no migration. 2. q = 1/100; carriers 2pq ≈ 2/100 = 1/50. 3. 1/25 × 1 × 1/4 = 1/100. 4. q = 1/50. Affected females q² = 1/2,500. Carrier females 2pq ≈ 2/50 = 1/25. 5. Most copies are in heterozygotes, who are not selected against. 6. Heterozygote advantage: HbAS carriers are protected against severe malaria, so they have higher fitness. 7. A small founding group carries by chance a high frequency of a rare allele. Examples: Ellis-van Creveld in the Amish; diseases of the Finnish heritage. 8. 1/16. 9. No. It increases homozygosity (and therefore recessive disease).

By cell type - Germline mutations: in gametes or their precursors. Passed to offspring and present in every cell of the child. - Somatic mutations: in body cells. Not inherited; affect only the descendants of the mutated cell. Cause of cancer and of mosaicism. By size - Genome mutations: changes in chromosome number (chapter 15). - Chromosome mutations: structural rearrangements (chapter 15). - Gene mutations: changes within a gene, from one base to many kb. Point mutations (base substitutions) - Transition: purine to purine (A↔G) or pyrimidine to pyrimidine (C↔T). The most common type. - Transversion: purine to pyrimidine or vice versa. Effect on the coding sequence - Silent (synonymous): the codon changes but the amino acid does not, because of the degenerate code (often at the third position). Usually harmless, but can affect splicing. - Missense: a different amino acid. - Conservative: similar chemical properties, often mild. - Non-conservative: different properties, often damaging. - Example: sickle cell disease, β-globin Glu6Val (GAG → GTG). A hydrophobic valine on the surface of haemoglobin causes polymerisation when deoxygenated. - Nonsense: a codon becomes a stop codon (UAA, UAG, UGA), giving a truncated protein. The mRNA is often destroyed by nonsense-mediated decay. Example: some β-thalassaemias (β⁰), many Duchenne mutations. - Stop-loss (readthrough): a stop codon becomes a sense codon, so the protein is extended. Insertions and deletions (indels) - Frameshift: insertion or deletion of a number of bases not divisible by 3, shifting the reading frame. All downstream codons change; a premature stop usually appears soon. Usually severe loss of function. - Example: Tay-Sachs (4-bp insertion in HEXA common in Ashkenazi Jews). - In-frame: a multiple of 3 bases, so one or more amino acids are added or removed. Example: the most common CF mutation, ΔF508 (now called F508del), a 3-bp deletion removing phenylalanine 508; the protein misfolds and is degraded (see also chapter 5). - Duchenne versus Becker muscular dystrophy: the reading-frame rule. Frameshift deletions in the dystrophin gene cause the severe Duchenne type (no protein); in-frame deletions cause the milder Becker type (shorter but partly functional protein). Other gene mutations - Splice-site mutations (chapter 18). - Promoter or regulatory mutations: change the amount of product. - Dynamic mutations: repeat expansions (fragile X, Huntington disease; chapter 15). By functional effect - Loss of function: less or no activity. Usually recessive (50% of normal enzyme is usually enough). Dominant when 50% is not enough (haploinsufficiency). - Dominant negative: the mutant protein interferes with the normal one, usually in multimeric proteins. Example: osteogenesis imperfecta (abnormal collagen chains disrupt the triple helix), some p53 mutations. - Gain of function: new or increased activity; usually dominant. Examples: achondroplasia (constitutively active FGFR3), Huntington disease (toxic protein), oncogene activation. Exam focus: classify a given DNA change (for example CAG → TAG) and predict its effect. That one is a nonsense mutation, giving a truncated protein.

Spontaneous mutations - Replication errors: - DNA polymerases insert a wrong base about once per 10⁵ bases; proofreading (3'→5' exonuclease) and mismatch repair lower the final error rate to about 1 per 10⁹-10¹⁰. - Tautomeric shifts: bases briefly take a rare form (enol or imino) that pairs wrongly, causing transitions. - Slippage at repeats (strand slippage): insertions or deletions in microsatellites. - Spontaneous chemical damage: - Depurination: loss of a purine base (thousands per cell per day), leaving an apurinic site. - Deamination: cytosine becomes uracil (recognised and removed). 5-methylcytosine becomes thymine, which is not recognised as foreign. This is why CpG sites are mutation hot spots (C→T transitions). - Oxidative damage from reactive oxygen species of metabolism: 8-oxoguanine pairs with A, causing G→T transversions. - Paternal age effect: sperm precursors keep dividing through life, so new point mutations increase with the father's age (about 2 new mutations per year of paternal age). Examples: achondroplasia, Apert syndrome. Induced mutations: chemical mutagens - Base analogues: 5-bromouracil (a thymine analogue that mispairs), causing transitions. - Base-modifying agents: - nitrous acid: deaminates bases - alkylating agents (ethyl methanesulfonate, nitrogen mustards, many chemotherapy drugs such as cyclophosphamide): add alkyl groups, causing mispairing and cross-links - hydroxylamine - Intercalating agents: flat molecules that slip between base pairs (ethidium bromide, acridine orange, some anticancer drugs such as doxorubicin), causing insertions and deletions, so frameshifts. - Bulky adducts: benzo[a]pyrene from tobacco smoke (becomes reactive after metabolic activation by cytochrome P450), aflatoxin B1 from mould (associated with a p53 codon 249 G→T mutation in liver cancer). Induced mutations: radiation - Ultraviolet light (UV-B mostly): forms pyrimidine dimers (thymine dimers, 6-4 photoproducts) between adjacent pyrimidines. Distort the helix and block replication. Cause of skin cancers; typical C→T mutations at dipyrimidine sites. - Ionising radiation (X-rays, gamma rays, particles): directly or by producing free radicals from water, causes single- and double-strand breaks and base damage. Double-strand breaks lead to deletions and chromosomal rearrangements. Effects are proportional to dose, with no safe threshold assumed for mutation (the linear no-threshold model used in radiation protection). Clinical tip: chemotherapy and radiotherapy work largely by damaging DNA, and for the same reason they can cause second cancers years later (for example therapy-related leukaemia after alkylating agents).

The Ames test (Bruce Ames, 1970s) - Uses Salmonella typhimurium strains with a mutation in a histidine synthesis gene (his⁻), so they cannot grow without histidine. - The strains are also made more sensitive: defective excision repair, and a more permeable cell wall. - Steps: 1. Mix bacteria with the test chemical and a rat liver extract (S9 fraction). The extract contains cytochrome P450 enzymes, which convert pro-mutagens into active mutagens as the human liver would (for example benzo[a]pyrene). 2. Plate on medium with only a trace of histidine. 3. Count colonies. Only revertants (his⁺ mutants) form colonies. 4. Compare with a control plate (spontaneous revertants). A dose-dependent increase means the substance is mutagenic. - Different strains detect different types of mutation (base substitution versus frameshift). - Limits: bacteria differ from human cells (no chromatin, different metabolism and repair). A positive result suggests carcinogenic risk; a negative one does not prove safety. Used as part of a battery of tests (mammalian cell tests, animal studies). DNA repair systems (outline) - Direct reversal: photolyase (not in humans) splits thymine dimers; O⁶-methylguanine methyltransferase (MGMT) removes methyl groups from guanine. - Base excision repair (BER): a glycosylase removes a single damaged base (uracil, 8-oxoguanine), then the backbone is cut, the gap filled and sealed. For small, non-distorting damage. - Nucleotide excision repair (NER): removes a stretch of about 24-32 nucleotides around bulky, helix-distorting damage (UV dimers, benzo[a]pyrene adducts). - Defect: xeroderma pigmentosum (autosomal recessive). Extreme sun sensitivity, freckling and skin cancers from childhood, often before age 10. - Mismatch repair (MMR): corrects replication mismatches and small loops after replication (MSH2, MLH1, MSH6, PMS2). - Defect: Lynch syndrome (hereditary non-polyposis colorectal cancer; chapter 19). - Double-strand break repair: - homologous recombination (HR): uses the sister chromatid as a template, accurate, in S and G2. Proteins include BRCA1, BRCA2. - non-homologous end joining (NHEJ): joins ends directly, fast but error-prone, throughout the cell cycle. - Other repair-defect syndromes: ataxia telangiectasia (ATM, a sensor of double-strand breaks), Fanconi anaemia (cross-link repair), Bloom syndrome (a helicase). The response to damage - Damage activates checkpoints (ATM and ATR kinases, p53). The cell cycle stops for repair; if damage is too severe, the cell dies by apoptosis. Note: the lectures cover repair only at this level; the molecular detail of each pathway is beyond the slides. Exam focus: describe the Ames test step by step and explain the role of the S9 liver fraction.

1. Distinguish germline from somatic mutations. 2. Distinguish a transition from a transversion. 3. What are silent, missense and nonsense mutations? Give an example of a missense mutation. 4. Why is a frameshift usually more severe than an in-frame deletion? Use dystrophin as the example. 5. What is a dominant negative mutation? 6. Why are CpG dinucleotides mutation hot spots? 7. How do intercalating agents cause mutations? 8. What damage does UV light cause, and which repair system removes it? 9. Why is a liver extract added in the Ames test? 10. Which disease results from defective nucleotide excision repair? 11. Name two repair pathways for double-strand breaks and state which is error-prone.

1. Germline: in gametes, inherited, in every cell of the offspring. Somatic: in body cells, not inherited, present only in descendants of the mutated cell. 2. Transition: purine to purine or pyrimidine to pyrimidine. Transversion: purine to pyrimidine or vice versa. 3. Silent: no amino acid change. Missense: a different amino acid. Nonsense: a stop codon. Missense example: Glu6Val in β-globin (sickle cell). 4. A frameshift changes every downstream codon and usually creates a premature stop, so no protein (Duchenne). An in-frame deletion removes only some amino acids, so a shorter partly functional protein (Becker). 5. The mutant protein interferes with the function of the normal protein, usually in multimers. 6. Methylated cytosine deaminates to thymine, which repair does not recognise as abnormal. 7. They slip between base pairs, causing insertions or deletions, so frameshifts. 8. Pyrimidine (thymine) dimers. Nucleotide excision repair. 9. To provide cytochrome P450 enzymes that convert pro-mutagens into active mutagens, as the human liver does. 10. Xeroderma pigmentosum. 11. Homologous recombination (accurate) and non-homologous end joining (error-prone).

The chemistry - DNA methyltransferases (DNMTs) add a methyl group to carbon 5 of cytosine, forming 5-methylcytosine, almost always at CpG sites. - De novo methylation: DNMT3A and DNMT3B set new patterns in development. - Maintenance methylation: DNMT1 copies the pattern onto the new strand after replication, because it recognises hemimethylated CpGs. This is how the mark is inherited through cell division. - Demethylation: passive (no maintenance during replication) or active (TET enzymes oxidise 5-methylcytosine). CpG islands - CpG dinucleotides are rare in the genome overall (methylated C deaminates to T over evolution), except in CpG islands: regions of about 1 kb rich in CpG, found in about 60% of promoters. - Usually unmethylated in active genes (including housekeeping genes). - Methylation of a promoter CpG island silences the gene: methyl-CpG-binding proteins (for example MeCP2) recruit histone deacetylases, which compact chromatin; methylation also blocks transcription factor binding. Biological roles - Cell differentiation: tissue-specific genes are methylated and silenced in other tissues. - Genomic imprinting (chapter 12). - X inactivation (chapter 11). - Silencing transposable elements to protect the genome. Reprogramming - Two waves of genome-wide demethylation: in the early embryo after fertilisation (except imprinted regions), and in primordial germ cells (including imprints, which are then reset by sex). Medical relevance - Cancer: global hypomethylation (genomic instability) together with hypermethylation and silencing of tumour suppressor promoters (for example MLH1 in sporadic colorectal cancer with microsatellite instability; BRCA1; p16). Drugs: DNMT inhibitors azacitidine and decitabine in myelodysplastic syndrome. - Rett syndrome: mutations in MECP2 (X-linked dominant, girls). - Fragile X: methylation silences FMR1 (chapter 15). - MGMT promoter methylation in glioblastoma predicts response to temozolomide. Environment and ageing - Methylation patterns change with age ("epigenetic clocks"), diet (folate supplies methyl groups), smoking and early-life stress. The Dutch Hunger Winter famine study found altered methylation (IGF2) decades later in people exposed before birth. Exam focus: explain how DNMT1 maintains methylation through cell division.

Chromatin states (see chapter 4) - Euchromatin: open, active. Heterochromatin: condensed, silent. Histone acetylation - Histone acetyltransferases (HATs) add acetyl groups to lysines in histone tails, which neutralises their positive charge, loosening binding to DNA. Chromatin opens, and transcription is activated. - Histone deacetylases (HDACs) remove them, closing chromatin and repressing transcription. - Bromodomain proteins read acetyl-lysine. - Drugs: HDAC inhibitors (vorinostat, romidepsin) in T-cell lymphoma. Histone methylation - Histone methyltransferases add one to three methyl groups to lysine or arginine. Does not change charge; the effect depends on which residue. - Activating: H3K4me3 (at active promoters), H3K36me3 (in transcribed gene bodies). - Repressive: H3K9me3 (constitutive heterochromatin, bound by HP1); H3K27me3 (facultative silencing, set by Polycomb complex PRC2, for example on the inactive X and HOX genes). - Demethylases (LSD1, JmjC family) remove them. - Nomenclature: H3K27me3 means histone H3, lysine 27, trimethylated. Other marks: phosphorylation (H3S10 in mitosis; γH2AX at DNA double-strand breaks), ubiquitination, sumoylation. The histone code hypothesis - Combinations of marks are read by proteins with specific domains (bromodomains for acetyl; chromodomains for methyl), which then recruit activating or repressing complexes. - Chromatin remodelling complexes (SWI/SNF) use ATP to slide or eject nucleosomes. SWI/SNF subunits are mutated in about 20% of human cancers. Non-coding RNAs - MicroRNAs (miRNAs): about 22 nucleotides. Processed by Drosha and Dicer, loaded into RISC (with Argonaute), bind the 3' untranslated region of target mRNAs by partial complementarity, causing repression of translation and mRNA decay. One miRNA regulates hundreds of mRNAs. - In cancer: some act as oncogenes (miR-21, miR-155), others as tumour suppressors (let-7; miR-15/16 deleted in chronic lymphocytic leukaemia). - Small interfering RNAs (siRNAs): perfect complementarity, cleave the target. Used as drugs (patisiran for transthyretin amyloidosis). - Long non-coding RNAs (lncRNAs, over 200 nucleotides): scaffolds and guides for chromatin complexes. Examples: XIST (X inactivation), HOTAIR (HOX silencing, raised in metastatic cancer). - piRNAs: silence transposons in the germ line. Exam focus: match each mark to its effect. Acetylation: active. H3K4me3: active. H3K9me3 and H3K27me3: repressed.

The splicing signals - 5' splice site (donor): the intron begins with GU (GT in DNA). - 3' splice site (acceptor): the intron ends with AG, preceded by a polypyrimidine tract. - Branch point: an adenine about 20-50 nucleotides upstream of the 3' site. - The "GU-AG rule". The spliceosome - Made of five small nuclear RNAs (U1, U2, U4, U5, U6) with proteins (snRNPs). - U1 binds the 5' site; U2 the branch point. Two transesterification reactions release the intron as a lariat and join the exons. - Autoantibodies against snRNPs (anti-Sm) are specific for systemic lupus erythematosus. Alternative splicing - Patterns: exon skipping (the most common), mutually exclusive exons, alternative 5' or 3' sites, intron retention. - Regulated by splicing enhancers and silencers in exons and introns, bound by SR proteins (enhance) and hnRNPs (repress). This varies by tissue and development stage. - Examples: - calcitonin gene: calcitonin in the thyroid, CGRP in neurons - membrane-bound versus secreted immunoglobulin - Drosophila sex determination - DSCAM in the fly: tens of thousands of isoforms from one gene - Explains how about 20,000 genes produce many more proteins. Splice mutations - Mutations of the invariant GU or AG destroy a splice site, causing exon skipping or intron retention, or the use of a nearby cryptic site. - Mutations creating a new splice site: activation of a cryptic site that is normally unused. - Exonic mutations, even silent ones, can disrupt splicing enhancers or silencers. - Examples: - β-thalassaemia: many mutations create cryptic splice sites in the β-globin gene (for example IVS1-110 G→A, common in the Mediterranean) - spinal muscular atrophy: SMN1 is deleted; the nearly identical SMN2 differs by a silent C→T change in exon 7 that weakens an enhancer, so most SMN2 transcripts skip exon 7 and produce an unstable protein - cystic fibrosis: some CFTR splice variants - Duchenne: mutations that disrupt the reading frame through splicing Therapy that targets splicing - Antisense oligonucleotides (ASOs): - nusinersen in spinal muscular atrophy blocks an intronic silencer in SMN2, forcing exon 7 inclusion, so more functional SMN protein - exon skipping in Duchenne (eteplirsen skips exon 51 to restore the reading frame, turning a Duchenne mutation into a Becker-like one) - Small molecules: risdiplam (oral) also modifies SMN2 splicing. Note: splicing is not a separate lecture but part of the epigenetics and gene expression slides; general transcription and translation are not covered (see the content flags). Exam focus: explain how nusinersen works on SMN2, and how exon skipping converts Duchenne into a milder Becker phenotype.

1. What is epigenetics? 2. Which enzyme maintains methylation through cell division, and how? 3. What are CpG islands, and what happens when their promoter CpGs are methylated? 4. How does DNA methylation change in cancer? 5. How does histone acetylation affect transcription? Why? 6. Which histone marks are associated with active genes, and which with silent genes? 7. How do microRNAs regulate gene expression? 8. Name a long non-coding RNA and its function. 9. What is the GU-AG rule? 10. How can a silent mutation cause disease? 11. What is the molecular difference between SMN1 and SMN2, and how does nusinersen work?

1. Heritable changes in gene expression without changes in DNA sequence. 2. DNMT1. It recognises hemimethylated CpGs after replication and methylates the new strand. 3. CpG-rich regions at about 60% of promoters. Methylation silences the gene. 4. Global hypomethylation with hypermethylation (silencing) of tumour suppressor promoters. 5. It activates. Acetylation neutralises the positive charge of lysines, loosening histone-DNA binding and opening chromatin. 6. Active: acetylation, H3K4me3, H3K36me3. Silent: H3K9me3, H3K27me3. 7. They bind the 3' UTR of target mRNAs (in RISC) and repress translation or cause mRNA decay. 8. XIST: coats the inactive X and silences it. 9. Introns begin with GU at the 5' splice site and end with AG at the 3' site. 10. By disrupting a splicing enhancer or silencer, or creating a cryptic splice site. 11. A silent C→T change in SMN2 exon 7 weakens an enhancer, so exon 7 is mostly skipped. Nusinersen, an antisense oligonucleotide, blocks an intronic silencer so exon 7 is included.

Proto-oncogenes and their normal roles - Growth factors: SIS (PDGF-B). - Growth factor receptors: EGFR, HER2 (ERBB2), RET, KIT. - Signal transducers: RAS (KRAS, NRAS, HRAS), BRAF, ABL. - Transcription factors: MYC. - Cell cycle regulators: cyclin D1, CDK4. - Apoptosis inhibitors: BCL2. How proto-oncogenes are activated (gain of function) - Point mutation: KRAS codon 12 mutations lock RAS in the active GTP-bound state (in pancreatic, colorectal, lung cancer). BRAF V600E in melanoma. - Gene amplification: many copies. HER2 in about 20% of breast cancers (treated with trastuzumab); N-MYC in neuroblastoma (poor prognosis). - Chromosomal translocation: - fusion gene: BCR-ABL in chronic myeloid leukaemia (the Philadelphia chromosome, t(9;22)), a constitutively active tyrosine kinase, treated with imatinib - promoter swap: MYC next to the immunoglobulin heavy-chain enhancer in Burkitt lymphoma, t(8;14); BCL2 in follicular lymphoma, t(14;18) - Viral insertion: retroviruses carrying oncogenes (v-src in Rous sarcoma virus was the first oncogene discovered), or inserting near a proto-oncogene. - One activated allele is enough, so oncogenes act dominantly in the cell. Inherited activating mutations are rare (RET in multiple endocrine neoplasia type 2 is the main example). Tumour suppressor genes (loss of function) - Gatekeepers: directly control cell proliferation or death. - RB1: controls the G1/S checkpoint by binding E2F (retinoblastoma, osteosarcoma). - TP53, "the guardian of the genome": responds to DNA damage by stopping the cell cycle (via p21) or causing apoptosis. Mutated in about 50% of all cancers. Germline mutation: Li-Fraumeni syndrome (sarcomas, breast cancer, brain tumours, adrenocortical carcinoma, leukaemia at young ages). - APC: degrades β-catenin (Wnt pathway). Germline: familial adenomatous polyposis (hundreds to thousands of polyps, colorectal cancer almost certain without colectomy). - NF1 (neurofibromin, a RAS inhibitor); VHL (von Hippel-Lindau: renal cell carcinoma, haemangioblastoma); PTEN; CDKN2A (p16). - Caretakers: maintain genome stability; their loss raises the mutation rate (next notes). - BRCA1, BRCA2 (homologous recombination), mismatch repair genes (MLH1, MSH2), ATM. Oncogenes versus tumour suppressors - Oncogene: gain of function, one allele (dominant at cell level), accelerator stuck on. - Tumour suppressor: loss of function, both alleles (recessive at cell level), brakes failing. - Yet inherited cancer syndromes caused by tumour suppressors show autosomal dominant inheritance in pedigrees. The predisposition is inherited dominantly, because the second hit is very likely (next note). Exam focus: for each gene, know its class, normal function, mechanism of activation or loss, and associated tumour.

Retinoblastoma: the model disease - A malignant eye tumour of young children (about 1 in 15,000-20,000). - Knudson compared ages at diagnosis: - hereditary cases (about 40%): bilateral or multifocal tumours, diagnosis earlier (usually before 1 year). The kinetics fit one additional event. - sporadic cases (about 60%): unilateral, single tumour, later diagnosis. The kinetics fit two events. - Interpretation: - hereditary: first hit (germline RB1 mutation) in every cell, second hit somatic in a retinal cell. With millions of retinal cells, a second hit is almost certain (penetrance about 90%), so inheritance looks autosomal dominant. - sporadic: both hits must occur somatically in the same retinal cell, which is rare. - Survivors of hereditary retinoblastoma have a high risk of second cancers, especially osteosarcoma (worse after radiotherapy), so radiotherapy is avoided where possible. - Every child with bilateral retinoblastoma, and about 15% with unilateral, has a germline mutation. Relatives are screened with regular eye examinations or genetic testing. Mechanisms of the second hit - A second point mutation or small deletion. - Loss of heterozygosity (LOH): loss of the remaining normal allele by: - deletion of a chromosome segment - loss of the whole chromosome (nondisjunction), with or without duplication of the mutant one - mitotic recombination - gene conversion - Promoter hypermethylation (epigenetic silencing; chapter 18). - LOH at a region in tumour DNA, compared with normal DNA of the same patient, is a signature that points to a tumour suppressor gene there. This is how many were found. Exceptions to two hits - Haploinsufficiency: one lost copy may be enough to give an advantage (some tumour suppressors, such as PTEN, show dosage effects). - Dominant negative mutant p53 inhibits the normal protein. The same model in other syndromes - Familial adenomatous polyposis (APC), Li-Fraumeni (TP53), hereditary breast and ovarian cancer (BRCA1/2), von Hippel-Lindau (VHL), neurofibromatosis type 1 (NF1). Exam trap: tumour suppressor mutations are recessive at the cellular level but inherited cancer predisposition is dominant in the family. Be able to explain why.

Microsatellite instability (MSI) - Cause: loss of mismatch repair (MLH1, MSH2, MSH6, PMS2). - Replication slippage errors at repeats are not corrected, so microsatellites change length (thousands of mutations). - Found in about 15% of colorectal cancers: - about 3% of all colorectal cancers: Lynch syndrome (hereditary non-polyposis colorectal cancer). Germline mutation in one MMR gene, autosomal dominant, with the second hit in the tumour. Colorectal cancer (often right-sided, younger age), endometrial cancer (the most common cancer in affected women), ovary, stomach, small bowel, urinary tract. Clinical criteria: Amsterdam criteria and revised Bethesda guidelines. - about 12%: sporadic, due to MLH1 promoter hypermethylation (often with BRAF V600E), usually in older patients. - Testing: immunohistochemistry of MMR proteins on the tumour, or MSI PCR. Universal testing of all colorectal cancers is now recommended. - Clinical relevance: MSI-high tumours carry many mutations, making many new antigens, so they respond well to immune checkpoint inhibitors (pembrolizumab). Lynch families need colonoscopy every 1-2 years from age 20-25. Chromosomal instability (CIN) - Frequent gains and losses of whole chromosomes or large segments, giving aneuploidy (typical of most solid tumours, about 85% of colorectal cancers). - Causes: defects in the spindle checkpoint, centrosome amplification, cohesion defects, telomere dysfunction, defective double-strand break repair. - Aneuploidy speeds up loss of heterozygosity for tumour suppressors. Homologous recombination deficiency - BRCA1 and BRCA2: hereditary breast and ovarian cancer syndrome (autosomal dominant). Lifetime breast cancer risk about 60-70% (BRCA1) and 45-70% (BRCA2); ovarian risk about 40% and 15%. BRCA2 also raises male breast, prostate and pancreatic cancer risk. - Without HR, double-strand breaks are repaired by error-prone pathways, causing rearrangements. - Synthetic lethality: PARP inhibitors (olaparib) block single-strand break repair. Normal cells survive by HR, but BRCA-deficient tumour cells cannot repair the resulting double-strand breaks and die. The first clinical application of synthetic lethality. Other caretaker defects - Xeroderma pigmentosum (NER), ataxia telangiectasia (ATM), Bloom syndrome, Fanconi anaemia (chapter 17). Telomere crisis - Critically short telomeres cause chromosome end-to-end fusions and breakage-fusion-bridge cycles, a burst of rearrangements before telomerase is reactivated (chapter 4). Exam focus: link each form of instability to its cause, a syndrome, and a therapeutic consequence (MSI to immunotherapy, BRCA to PARP inhibitors).

1. What is the difference between a proto-oncogene and an oncogene? 2. Give three mechanisms of oncogene activation, with an example of each. 3. Why is TP53 called the guardian of the genome? 4. Distinguish gatekeeper and caretaker tumour suppressor genes. 5. Why are tumour suppressor mutations recessive in the cell but inherited dominantly in families? 6. What evidence did Knudson use for his two-hit hypothesis? 7. What is loss of heterozygosity, and how can it occur? 8. Why do hereditary retinoblastoma survivors need long-term follow-up? 9. What causes microsatellite instability? Name the hereditary syndrome. 10. Why do MSI-high tumours respond to checkpoint inhibitors? 11. Explain synthetic lethality with PARP inhibitors in BRCA-mutated cancer.

1. A proto-oncogene is a normal growth-promoting gene. An oncogene is its overactive mutated form. 2. Point mutation (KRAS codon 12); amplification (HER2 in breast cancer); translocation (BCR-ABL in CML, MYC in Burkitt). 3. It detects DNA damage and stops the cell cycle or triggers apoptosis, preventing damaged cells from dividing. 4. Gatekeepers directly control growth or death (RB1, TP53, APC). Caretakers maintain genome stability (BRCA1/2, MMR genes). 5. Both alleles must be lost in a cell, but a person inheriting one mutant allele will almost certainly lose the second in at least one of many cells. 6. The age at diagnosis and number of tumours in hereditary (early, bilateral) versus sporadic (later, unilateral) retinoblastoma. 7. Loss of the remaining normal allele in a tumour: deletion, chromosome loss, mitotic recombination, gene conversion. 8. They have a high risk of second cancers, especially osteosarcoma. 9. Loss of mismatch repair. Lynch syndrome (HNPCC). 10. They carry many mutations, producing many neoantigens that T cells can recognise. 11. PARP inhibitors block single-strand break repair, creating double-strand breaks. Normal cells repair them by homologous recombination; BRCA-deficient cells cannot and die.