The Chemistry of Life and Biological Macromolecules

Every living thing, from a soil bacterium to a redwood to you, is a chemical system that obeys the same rules as a beaker of salt water. The difference is organization: a small set of elements, held together by a handful of bond types and dissolved in water, assembles into the four great families of large molecules that store energy, build structures, speed reactions and carry hereditary information. Master this chemistry now and almost every later topic in biology, from enzymes to genetics to ecology, becomes a matter of applying ideas you already own.

Section 1Atoms, bonds and the properties of water

Matter, elements and the atom

Matter is anything that takes up space and has mass. All matter is made of elements, substances that cannot be broken down into other substances by ordinary chemical reactions. About 92 elements occur naturally, yet only four, oxygen, carbon, hydrogen and nitrogen, make up about 96 percent of the mass of the human body. Calcium, phosphorus, potassium, sulfur, sodium, chlorine and magnesium account for most of the remaining 4 percent. Trace elements such as iron, iodine, zinc and copper are needed only in tiny amounts, but they are essential: iodine is built into thyroid hormone, and iron sits at the center of the heme group that carries oxygen in hemoglobin. A compound is a substance made of two or more elements in a fixed ratio, and it usually has properties quite unlike its parts. Sodium is an explosive metal and chlorine a poisonous gas, but sodium chloride is table salt.

The smallest unit of an element that keeps its chemical properties is the atom. Atoms have a dense central nucleus holding positively charged protons and uncharged neutrons, surrounded by a cloud of negatively charged electrons. Protons and neutrons each have a mass of about 1 dalton (1 atomic mass unit); an electron has about 1/1,840 of that mass, so electrons contribute almost nothing to mass but nearly everything to chemistry.

• The atomic number is the number of protons. It defines the element: every carbon atom has 6 protons.
• The mass number is protons plus neutrons. Common carbon has 6 protons and 6 neutrons, mass number 12.
• In a neutral atom, the number of electrons equals the number of protons.

Isotopes and radioactivity

Atoms of one element can differ in their number of neutrons. These forms are isotopes. Carbon-12 is about 99 percent of natural carbon, carbon-13 about 1 percent, and carbon-14 is present in trace amounts. Isotopes of an element behave almost identically in chemical reactions because chemistry depends on electrons, not neutrons. Some isotopes have unstable nuclei that decay spontaneously, giving off particles and energy; these are radioactive isotopes. Each decays at a fixed rate measured as a half-life, the time for half of a sample to decay. Carbon-14 has a half-life of about 5,730 years, which makes it useful for dating organic remains up to roughly 50,000 years old. In research, radioactive tracers let biologists follow atoms through metabolic pathways, as in the classic experiments that traced carbon through photosynthesis and showed that DNA, not protein, enters bacteria during viral infection.

Electrons, shells and valence

Electrons occupy energy levels, often drawn as shells around the nucleus. The first shell holds up to 2 electrons; the second and third each hold up to 8 for the elements that matter most in biology. The electrons in the outermost occupied shell are valence electrons, and they determine how an atom reacts. Atoms are most stable when their valence shell is full. Helium, neon and argon already have full shells and are inert. Other atoms reach stability by sharing, gaining or losing electrons. The number of unpaired valence electrons an atom usually shares gives its valence, the number of covalent bonds it typically forms: hydrogen 1, oxygen 2, nitrogen 3 and carbon 4. Remembering "HONC 1234" lets you check almost any biological structure for a missing or extra bond.

Covalent bonds and electronegativity

A covalent bond is a shared pair of valence electrons. Two hydrogen atoms share one pair to form H₂ (a single bond); two oxygen atoms share two pairs to form O₂ (a double bond). A molecule is two or more atoms held together by covalent bonds. Covalent bonds are strong, and the bonds within biological molecules do not break at body temperature without the help of enzymes.

Atoms differ in how strongly they pull shared electrons toward themselves, a property called electronegativity. Oxygen and nitrogen are highly electronegative; carbon and hydrogen are similar to each other and much less so. When two atoms of similar electronegativity share electrons, the sharing is equal and the bond is nonpolar covalent (C to C, C to H). When one atom is much more electronegative, the electrons spend more time near it, giving it a partial negative charge (written δ−) and leaving the other atom with a partial positive charge (δ+). Such a bond is polar covalent (O to H, N to H). The polarity of the O to H bonds in water is the single fact that explains most of what makes water special.

Ionic bonds, hydrogen bonds and weak interactions

When the difference in electronegativity is very large, one atom strips an electron from the other. Sodium gives up its single valence electron to chlorine; sodium becomes a cation (Na⁺) and chlorine an anion (Cl⁻). The attraction between oppositely charged ions is an ionic bond. Ionic compounds such as NaCl form crystals when dry, but in water the ions separate and are surrounded by water molecules, so ionic bonds are weak in the watery interior of a cell.

A hydrogen bond forms when a hydrogen atom that is covalently bonded to an electronegative atom (O or N) is attracted to another electronegative atom nearby. A hydrogen bond is about 5 to 10 percent as strong as a covalent bond, but biology runs on huge numbers of them. They hold the two strands of DNA together, shape proteins, and link water molecules into a constantly shifting network. Their weakness is a feature: hydrogen bonds can form and break quickly, letting DNA unzip for copying and enzymes bind and release substrates.

Van der Waals interactions are even weaker attractions that arise because electrons are always moving, creating fleeting regions of slight charge on any molecule. They matter only when molecules fit very closely, but many together are significant. Geckos cling to glass largely through van der Waals forces between millions of tiny hairs on their toes and the surface. The hydrophobic effect, the tendency of nonpolar molecules to cluster together in water, is not really a bond at all; it results from water molecules hydrogen bonding with each other and excluding the nonpolar substances. It is one of the main forces that folds proteins and builds membranes.

Molecular shape follows from bonding. When an atom forms covalent bonds, its valence orbitals rearrange into a geometry: carbon with four single bonds is tetrahedral, with bond angles of about 109.5 degrees, and water is bent, with an angle of about 104.5 degrees. Shape matters because molecules recognize each other by fit. Morphine and the body's own endorphins have similar shapes, which is why both bind the same opioid receptors in the brain.

Chemical reactions

A chemical reaction makes and breaks bonds, converting reactants into products. Matter is conserved: the same atoms appear on both sides of a balanced equation. Photosynthesis, for example, can be summarized as 6 CO₂ + 6 H₂O → C₆H₁₂O₆ + 6 O₂. Many reactions are reversible, and they proceed until the forward and reverse rates are equal, a state called chemical equilibrium. At equilibrium the concentrations stop changing, but they are not necessarily equal. Adding reactant or removing product pushes the reaction forward, an idea you will use often when you study metabolism.

Water: a polar molecule

Water (H₂O) is bent, with oxygen at the vertex. Oxygen pulls the shared electrons toward itself, so the oxygen end carries a partial negative charge and each hydrogen a partial positive charge. Because of this polarity, each water molecule can hydrogen bond with up to four neighbors. In liquid water these hydrogen bonds last only trillionths of a second, constantly breaking and reforming, but at any moment most molecules are hydrogen bonded to several others. Four emergent properties follow, and each is essential for life.

Four emergent properties of water

1. Cohesion and adhesion. Hydrogen bonds hold water molecules to each other, a property called cohesion. Cohesion gives water a high surface tension, which lets a water strider walk across a pond. Adhesion is the attraction of water to other polar surfaces. In plants, cohesion holds the column of water together inside the narrow xylem tubes while adhesion to the cell walls resists gravity, so evaporation from leaves can pull water up from the roots of a tree many meters tall.

2. Moderation of temperature. Water has a high specific heat: it takes 1 calorie to raise 1 gram of water by 1 °C, far more than for most substances. The reason is that much of the heat added first goes into breaking hydrogen bonds rather than speeding molecules up. Oceans and lakes therefore warm and cool slowly, stabilizing coastal climates, and the water that makes up 60 percent or more of a body buffers it against rapid temperature swings. Water also has a high heat of vaporization: about 580 calories are needed to evaporate 1 gram at body temperature. Because the fastest, most energetic molecules leave first, the remaining liquid cools. This evaporative cooling is why sweating cools you and why transpiration keeps leaves from overheating in the sun.

3. Floating ice. Most substances are densest as solids, but water is densest at about 4 °C. As water freezes, each molecule locks into hydrogen bonds with four neighbors in an open crystal lattice, so ice is about 10 percent less dense than liquid water and floats. Floating ice insulates the water below. If ice sank, lakes and seas would freeze from the bottom up and much aquatic life could not survive winter.

4. A versatile solvent. A solution is a uniform mixture of a solute dissolved in a solvent; when water is the solvent it is an aqueous solution. Water dissolves ionic compounds by surrounding each ion with a hydration shell, and it dissolves polar molecules such as sugars by hydrogen bonding with their polar groups. Substances that interact readily with water are hydrophilic; nonpolar substances such as oils are hydrophobic. Some hydrophilic substances, such as large proteins, do not truly dissolve but stay suspended as a colloid.

Chemists count molecules by the mole, which is 6.02 × 10²³ particles (Avogadro's number). One mole of a substance has a mass in grams equal to its molecular mass in daltons. Glucose (C₆H₁₂O₆) has a molecular mass of 180 daltons, so 180 grams is 1 mole. A 1 molar (1 M) solution has 1 mole of solute per liter of solution.

Acids, bases and pH

A small fraction of water molecules dissociate: one hydrogen leaves its electron behind and transfers to a neighbor as a proton, producing a hydronium ion (H₃O⁺, usually written H⁺) and a hydroxide ion (OH⁻). In pure water at 25 °C, [H⁺] = [OH⁻] = 10⁻⁷ M, and their product is always 10⁻¹⁴. An acid increases [H⁺] (hydrochloric acid releases H⁺ completely); a base decreases it, either by accepting H⁺ (ammonia) or by releasing OH⁻ (sodium hydroxide).

The pH is the negative base-10 logarithm of the hydrogen ion concentration: pH = −log[H⁺]. Pure water is pH 7, neutral. Acidic solutions are below 7 and basic (alkaline) ones are above. Because the scale is logarithmic, each unit is a tenfold change: pH 4 has 10 times the H⁺ of pH 5 and 1,000 times that of pH 7. Most cells keep their interior near pH 7, and human blood is held between 7.35 and 7.45. Stomach fluid is about pH 2, while the small intestine is slightly basic.

Living fluids resist pH change because of buffers, substances that take up H⁺ when it is in excess and release it when it is scarce. The most important buffer in blood is the carbonic acid and bicarbonate system:

CO₂ + H₂O ⇌ H₂CO₃ ⇌ HCO₃⁻ + H⁺

If H⁺ rises, the reaction shifts left as bicarbonate absorbs it; if H⁺ falls, carbonic acid dissociates to replace it. The lungs adjust CO₂ and the kidneys adjust bicarbonate, giving two levers for long term control.

Section 2Carbon and functional groups

Why carbon?

Apart from water, nearly every molecule in a cell is built on a skeleton of carbon. Compounds containing carbon bonded to hydrogen are called organic compounds; the name dates from a time when chemists thought only living things could make them. In 1828 a chemist synthesized urea from inorganic starting materials, and in 1953 Stanley Miller showed that amino acids form when electric sparks pass through a mixture of simple gases thought to resemble the early atmosphere. These experiments helped replace the idea of a special "vital force" with mechanism: life is built from the same atoms and obeys the same laws as nonliving matter.

Carbon's power comes from its four valence electrons. It forms four covalent bonds, so it can link to four other atoms in different directions. Carbon atoms bond readily to each other, producing chains, branches and rings of almost any length, and they can share one, two or three electron pairs. Each carbon with four single bonds sits at the center of a tetrahedron, giving organic molecules three dimensional shape. When two carbons are joined by a double bond, the atoms attached to them lie in one flat plane and the bond cannot rotate, which locks part of the molecule in place.

Hydrocarbons and carbon skeletons

Hydrocarbons contain only carbon and hydrogen. Their C to H bonds are nonpolar, so hydrocarbons are hydrophobic, and those bonds store a great deal of energy that is released when they are burned or, in cells, oxidized. The fatty acid tails of fats are long hydrocarbon chains, which is why fats are such rich fuel. Carbon skeletons vary in four ways: length, branching, the position of double bonds and the presence of rings.

Isomers

Isomers are compounds with the same molecular formula but different structures, and therefore different properties. There are three kinds that biology students must know:

• Structural isomers differ in the covalent arrangement of their atoms. Glucose and fructose are both C₆H₁₂O₆, but glucose has an aldehyde group and fructose a ketone.
• Cis trans isomers (geometric isomers) have the same bonds but differ in arrangement around a double bond. In the cis form, the two groups of interest are on the same side; in the trans form, on opposite sides. Retinal, the light-sensing molecule of the eye, switches from cis to trans when it absorbs a photon, and that change in shape starts vision.
• Enantiomers are mirror images that cannot be superimposed, like left and right hands. They arise around an asymmetric carbon, a carbon bonded to four different groups. Cells usually make and use only one enantiomer. Proteins are built from L amino acids, and most natural sugars are D sugars.

The chemical groups most important to life

The carbon skeleton gives a molecule its general shape, but small clusters of atoms attached to it, called functional groups, usually determine its chemical behavior. A functional group behaves much the same way whatever molecule it is on, so learning seven groups lets you predict the properties of thousands of compounds. The male and female sex hormones testosterone and estradiol share the same four-ring steroid skeleton and differ only in a few functional groups, yet they drive very different development.

At the near-neutral pH of a cell, carboxyl groups usually exist as ionized −COO⁻ and amino groups as −NH₃⁺. A free amino acid in the cytoplasm therefore carries both a positive and a negative charge, which makes it very water soluble. Phosphate groups carry two negative charges when at the end of a molecule, and the repulsion between closely packed phosphates is part of why ATP stores useful energy.

ATP: an important source of energy for cellular processes

Adenosine triphosphate (ATP) consists of the nitrogenous base adenine, the five-carbon sugar ribose and a chain of three phosphate groups. When water breaks the bond to the terminal phosphate (hydrolysis), ATP becomes ADP (adenosine diphosphate) plus inorganic phosphate (P_i), and energy is released. Cells couple this release to work: transferring the phosphate to another molecule changes that molecule's shape or reactivity, driving muscle contraction, active transport and synthesis. Cells regenerate ATP from ADP and P_i using energy from food, a cycle you will follow in detail in the chapter on cellular respiration.

Section 3Carbohydrates and lipids

Macromolecules, polymers and monomers

Carbohydrates, proteins and nucleic acids are polymers, long molecules built by linking many similar or identical building blocks called monomers. Lipids are large molecules too, but they are not true polymers. Cells use the same two reactions to build and dismantle all polymers. In dehydration synthesis (a condensation reaction), two monomers are joined by a covalent bond and a molecule of water is released: one monomer gives up a hydroxyl and the other a hydrogen. Hydrolysis is the reverse: water is added across the bond, breaking it. Digestion is mostly hydrolysis, as enzymes in your gut break starch, proteins and fats into monomers small enough to absorb. Both reactions are catalyzed by specific enzymes.

Monosaccharides and disaccharides

Carbohydrates include sugars and their polymers, and many have the general formula (CH₂O)_n. The simplest are monosaccharides, which carry a carbonyl group and several hydroxyl groups. They are classified by the position of the carbonyl, as aldoses (aldehyde, at carbon 1) or ketoses (ketone, inside the chain), and by the number of carbons: trioses (3), pentoses (5) and hexoses (6). Glucose and galactose are aldohexoses that differ only in the arrangement around one asymmetric carbon; fructose is a ketohexose. Ribose and deoxyribose are pentoses found in RNA and DNA. In water, most five and six carbon sugars form rings, because a hydroxyl group reacts with the carbonyl of the same molecule. Ring closure creates a new asymmetric carbon (carbon 1 in glucose) whose hydroxyl can point below the ring (the alpha form) or above it (the beta form). This tiny difference has enormous consequences.

A disaccharide forms when a dehydration reaction joins two monosaccharides by a glycosidic linkage. Maltose is glucose plus glucose (malt sugar, from starch breakdown). Sucrose, table sugar, is glucose plus fructose and is the form in which plants move sugar through their phloem. Lactose, milk sugar, is glucose plus galactose.

Polysaccharides: storage and structure

Polysaccharides are polymers of hundreds to thousands of monosaccharides. Their function depends on which sugar they use and how the units are linked.

• Starch is the main storage polysaccharide of plants, kept in plastids. It is made of alpha glucose. Amylose is unbranched and coils into a helix; amylopectin has branches every 24 to 30 units. Animals, including humans, have enzymes that hydrolyze the alpha linkages of starch.
• Glycogen is the storage polysaccharide of animals, stored mainly in liver and skeletal muscle. It resembles amylopectin but is more highly branched (about every 8 to 12 units), so enzymes can release glucose quickly from many branch tips at once. Your glycogen stores last only about a day without eating.
• Cellulose, the main component of plant cell walls, is the most abundant organic compound on Earth. It is made of beta glucose. Because of the beta linkage, every other glucose is flipped, and the chains stay straight. Parallel cellulose chains hydrogen bond to each other, forming strong microfibrils.
• Chitin forms the exoskeleton of arthropods and the cell walls of fungi. It is like cellulose, but each glucose carries a nitrogen-containing group. Chitin is tough and is also used to make absorbable surgical thread.

The enzymes that digest starch cannot break the beta linkages of cellulose, so for humans cellulose passes through the gut as insoluble fiber, which helps move material through the intestine. Cows, termites and other herbivores digest cellulose only with help from microbes in their gut that make cellulase.

Lipids: a diverse hydrophobic group

Lipids are grouped together not by a common monomer but by a shared property: they mix poorly, if at all, with water, because they consist mostly of hydrocarbon regions. The three most important kinds are fats, phospholipids and steroids.

A fat, or triglyceride, is built from one glycerol (a three-carbon alcohol) and three fatty acids. Each fatty acid is a long hydrocarbon chain, usually 16 or 18 carbons, with a carboxyl group at one end. Dehydration reactions link each fatty acid to glycerol by an ester linkage. Fats are excellent energy stores: a gram of fat stores more than twice the energy of a gram of carbohydrate (about 9 versus 4 kilocalories), because fats are rich in energy-storing C to H bonds and are stored without water. Adipose tissue also cushions organs and insulates the body.

A saturated fatty acid has no carbon to carbon double bonds; it holds as many hydrogens as possible and its chain is straight. Straight chains pack tightly, so saturated fats such as butter and lard are solid at room temperature. An unsaturated fatty acid has one or more double bonds, usually in the cis form, which puts a kink in the chain. Kinked chains cannot pack closely, so plant and fish oils stay liquid. Hydrogenation adds hydrogen to unsaturated oils to make them solid, and partial hydrogenation also creates trans fats, which have trans double bonds and straight chains. Trans fats raise LDL cholesterol and the risk of heart disease even more than saturated fats, and many countries have banned or sharply limited them. Omega-3 fatty acids, which have a double bond three carbons from the tail end, are essential fatty acids that the body cannot make.

Phospholipids and steroids

A phospholipid has only two fatty acids attached to glycerol; the third carbon carries a negatively charged phosphate group, usually linked to another small charged or polar molecule such as choline. The result is amphipathic: a hydrophilic head and two hydrophobic tails. In water, phospholipids self-assemble into a bilayer, heads facing the water on both sides and tails tucked inside, away from water. That bilayer is the basic fabric of every cell membrane, as Chapter 2 explains.

Steroids are lipids whose carbon skeleton is four fused rings. Cholesterol is a component of animal cell membranes, where it modulates fluidity, and it is the precursor from which animals make other steroids, including the sex hormones (estrogens, testosterone), the adrenal hormones (cortisol, aldosterone) and vitamin D. High blood levels of cholesterol carried in LDL particles contribute to atherosclerosis. Waxes, made of a fatty acid joined to a long chain alcohol, waterproof leaves, feathers and insect cuticles.

Section 4Proteins: structure and folding

The many jobs of proteins

Proteins account for more than half the dry mass of most cells, and they do nearly everything. Enzymes are protein catalysts that speed reactions without being used up. Structural proteins (collagen, keratin, silk) give strength; transport proteins (hemoglobin, membrane channels) move substances; contractile proteins (actin, myosin) produce movement; storage proteins (ovalbumin in egg white, casein in milk) hold amino acids; hormones such as insulin carry signals; receptor proteins detect them; and defensive proteins such as antibodies recognize invaders. All of this variety comes from one kind of polymer: a chain of amino acids folded into a specific shape.

Amino acids

An amino acid has a central carbon (the alpha carbon) bonded to four partners: an amino group, a carboxyl group, a hydrogen atom and a variable side chain, or R group. Cells use 20 standard amino acids, and the R group is what makes each one different. Side chains fall into broad classes:

Two amino acids are special. Cysteine has a sulfhydryl group, and two cysteines can form a covalent disulfide bridge. Proline's side chain loops back to bond its own amino nitrogen, making a rigid kink that often breaks helices. Humans can make about 11 of the 20; the other 9 are essential amino acids that must come from the diet.

Peptide bonds and polypeptides

A dehydration reaction joins the carboxyl group of one amino acid to the amino group of the next, forming a peptide bond. A chain of amino acids is a polypeptide. The repeating N to C to C sequence is the backbone, with the R groups sticking out from it. A polypeptide has direction: one end has a free amino group (the N-terminus) and the other a free carboxyl group (the C-terminus), and ribosomes always build chains from N to C. A protein is one or more polypeptides folded into a functional shape. With 20 choices at every position, a chain of just 100 amino acids could have 20¹⁰⁰ possible sequences, more than the number of atoms in the observable universe.

Four levels of protein structure

Primary structure is the sequence of amino acids, written from N to C terminus. It is dictated by the gene. Everything else follows from it, because the sequence determines which interactions are possible.

Secondary structure is local coiling or folding stabilized by hydrogen bonds between backbone atoms, specifically between the carbonyl oxygen of one peptide bond and the N to H hydrogen of another. Side chains are not involved. The two main forms are the alpha helix, a coil with a hydrogen bond between every fourth amino acid, and the beta pleated sheet, in which two or more stretches of chain lie side by side, connected by hydrogen bonds. Keratin of hair is mostly alpha helix; spider silk owes its strength to many beta sheets.

Tertiary structure is the overall three dimensional shape of one polypeptide, produced by interactions between R groups. Hydrophobic side chains cluster in the center, away from water (the hydrophobic effect), where van der Waals interactions hold them together. Polar and charged side chains form hydrogen bonds and ionic bonds with each other and with water. Covalent disulfide bridges between cysteines act like rivets that lock the shape.

Quaternary structure exists only in proteins made of more than one polypeptide, and it describes how those subunits fit together. Hemoglobin has four subunits (two alpha and two beta chains), each carrying a heme group; collagen is three helical chains wound into a rope like triple helix.

Sequence, shape and disease

Because shape depends on sequence, even one amino acid change can matter. In sickle cell disease, the sixth amino acid of the hemoglobin beta chain is valine instead of glutamate. Swapping a charged side chain for a hydrophobic one creates a sticky patch on the surface. When oxygen is low, the altered hemoglobin molecules stick together into long fibers that deform red blood cells into a sickle shape. Sickled cells clog small vessels and break down early, causing pain crises and anemia. People who carry one copy of the allele are partly protected from malaria, which explains why the allele is common where malaria is common.

Folding, chaperones and denaturation

A protein's shape is the lowest energy arrangement its sequence allows under cellular conditions, but reaching it is not always easy. Many proteins fold with the help of chaperonins (molecular chaperones), barrel shaped protein complexes that shelter a new polypeptide from the crowded cytoplasm while it folds. Chaperones do not specify the final shape; they prevent wrong interactions along the way.

If the pH, salt concentration or temperature changes enough, the weak bonds holding secondary, tertiary and quaternary structure break, and the protein unravels. This loss of shape is denaturation, and a denatured protein loses its function. The primary structure, held by covalent peptide bonds, survives. Cooking an egg denatures albumin, turning it from clear to white and solid. A high fever is dangerous partly because some proteins begin to denature. In a classic experiment, the enzyme ribonuclease was denatured with chemicals and then, when the chemicals were removed, refolded on its own into its active shape. That result showed that the information for folding lies in the primary structure. Today, computer models trained on known structures can predict how a sequence will fold with remarkable accuracy, though experimental methods such as X-ray crystallography and cryo-electron microscopy remain the gold standard.

Section 5Nucleic acids and the flow of information

Genes and the central dogma

The amino acid sequence of every protein is encoded in a gene, a stretch of deoxyribonucleic acid (DNA). DNA does not build proteins directly. In eukaryotes, it stays in the nucleus, and its information is first copied into messenger RNA (mRNA), a ribonucleic acid, in a process called transcription. The mRNA moves to the cytoplasm, where ribosomes read it and assemble the polypeptide in translation. When a cell divides, its DNA is copied by replication so each daughter cell receives a full set. This flow, DNA to RNA to protein, is the central dogma of molecular biology. There are exceptions: retroviruses such as HIV use the enzyme reverse transcriptase to copy RNA into DNA, and many RNA molecules (ribosomal RNA, transfer RNA, microRNAs) do their jobs without ever being translated.

Nucleotides

Nucleic acids are polymers called polynucleotides, built from monomers called nucleotides. Each nucleotide has three parts: a five-carbon sugar (a pentose), a nitrogenous base and one to three phosphate groups. The sugar plus base, without phosphate, is a nucleoside. The carbons of the sugar are numbered with primes (1′ to 5′) to tell them apart from the atoms of the base. The base attaches to the 1′ carbon and the phosphate to the 5′ carbon.

• The sugar in DNA is deoxyribose, which lacks the oxygen on the 2′ carbon. The sugar in RNA is ribose, which has an OH there. That extra hydroxyl makes RNA more chemically reactive and less stable than DNA, one reason DNA is the long-term storage form.
• The bases come in two families. Pyrimidines have a single six-membered ring: cytosine (C), thymine (T), found only in DNA, and uracil (U), found only in RNA. Purines are larger, with a six-membered ring fused to a five-membered ring: adenine (A) and guanine (G).

The sugar phosphate backbone

Nucleotides are joined by dehydration reactions that link the phosphate on the 5′ carbon of one nucleotide to the hydroxyl on the 3′ carbon of the next. The resulting phosphodiester linkage creates a repeating sugar phosphate backbone with the bases projecting from it. Like a polypeptide, a polynucleotide has direction: one end has a free phosphate on a 5′ carbon (the 5′ end) and the other a free hydroxyl on a 3′ carbon (the 3′ end). Sequences are written 5′ to 3′, and new nucleotides are always added to the 3′ end. The sequence of bases along a gene is unique to that gene and carries its information.

The double helix

RNA usually exists as a single strand, though it can fold back on itself where complementary stretches pair, giving molecules such as transfer RNA a precise three dimensional shape. DNA is a double helix: two polynucleotide strands wound around an imaginary axis, with the backbones on the outside and the bases paired in the interior. Several features are essential:

• The strands are antiparallel: they run in opposite 5′ to 3′ directions, like the lanes of a two-way road.
• Bases pair by hydrogen bonding in a fixed way, called complementary base pairing: A with T (two hydrogen bonds) and G with C (three hydrogen bonds). In RNA, A pairs with U. Each pair joins a purine to a pyrimidine, so the helix has a uniform width of about 2 nanometers.
• Because of pairing, the two strands are complementary: knowing one sequence tells you the other. That is the basis for accurate copying during replication. It also explains Chargaff's rules: in any double-stranded DNA, the amount of A equals T and the amount of G equals C.
• The helix makes one full turn about every 10 base pairs (3.4 nm). Regions rich in G and C, with three hydrogen bonds per pair, are harder to separate than regions rich in A and T.

Other jobs for nucleotides

Nucleotides are not only building blocks. ATP is the cell's energy currency; GTP powers protein synthesis and signaling; cyclic AMP is a second messenger inside cells; and the electron carriers NAD⁺ and FAD, central to respiration, are nucleotide derivatives. Some RNA molecules, called ribozymes, act as catalysts. The ribosome's peptide-bond-forming center is RNA, a clue that early life may have used RNA for both information and catalysis.

DNA, proteins and evolution

Because genes are passed from parents to offspring, closely related species share more of their DNA and protein sequences than distantly related ones. Comparing sequences, the field of molecular systematics, lets biologists build family trees of life. Human and chimpanzee genomes differ by only about 1 to 2 percent in the stretches that can be aligned. Modern genomics sequences entire genomes quickly and cheaply, and bioinformatics uses computers to store and compare that flood of data, opening approaches to medicine and evolution that were unimaginable a generation ago.

Words worth knowing

Cell Structure, Membranes and Transport

The cell is the smallest unit that is alive. A single bacterium can sense its surroundings, take in food, release waste and copy itself, and every organ of your body is an organized community of trillions of cells doing the same jobs in specialized ways. This chapter tours the cell from the outside in: how we see cells at all, how simple and complex cells differ, what each internal compartment does, how the cell holds its shape and attaches to its neighbors, and how its membrane decides what comes in and what goes out.

Section 1Microscopy and the cell theory

Seeing the invisible

Most cells are between 1 and 100 micrometers (µm) across, far below the roughly 0.1 millimeter limit of the unaided human eye. Cell biology therefore began with lenses. In 1665 Robert Hooke looked at thin slices of cork and named the tiny boxes he saw "cells"; they were the empty walls of dead plant cells. A few years later Antoni van Leeuwenhoek, grinding his own small, powerful lenses, became the first to see living single-celled organisms, which he called "animalcules," in pond water, and bacteria scraped from his own teeth.

Three parameters describe any microscope:

• Magnification is the ratio of the image size to the real size of the object.
• Resolution is the minimum distance two points can be apart and still be seen as separate. It measures clarity. Magnifying beyond the resolution limit just enlarges a blur.
• Contrast is the difference in brightness between parts of the sample. Most cells are nearly transparent, so contrast is often enhanced with stains or optical tricks.

Resolution is limited by the wavelength of what is used to form the image. Visible light has wavelengths of about 400 to 700 nanometers (nm), so a light microscope (LM) cannot resolve details much finer than about 0.2 µm (200 nm), roughly the size of a small bacterium. Light microscopes magnify usefully up to about 1,000 times. Their great advantage is that they show living cells.

Light microscopy techniques

In brightfield microscopy light passes straight through the specimen; unstained cells show little contrast, but stained cells show clearly, though staining usually kills them. Phase contrast and differential interference contrast optics convert tiny differences in density into differences in brightness, revealing living, unstained cells. In fluorescence microscopy, specific molecules are tagged with fluorescent dyes or with fluorescent proteins such as green fluorescent protein (GFP), which glow when lit with the right wavelength. Confocal microscopes use a laser and a pinhole to reject out-of-focus light, building sharp optical slices that can be stacked into three dimensional images. Super-resolution methods, developed in the last few decades, use clever control of fluorescent molecules to break the traditional 200 nm limit and resolve structures as small as 10 to 20 nm.

Electron microscopy

An electron microscope (EM) focuses a beam of electrons with magnets. Electrons have a much shorter wavelength than light, so the practical resolution of modern EMs is about 2 nm or better, a hundred times finer than the LM. Specimens must be fixed, dried or frozen and placed in a vacuum, so living cells cannot be observed.

• The scanning electron microscope (SEM) scans a beam across a sample coated with a thin film of metal. Electrons knocked off the surface are detected, producing striking images of surface topography that look three dimensional.
• The transmission electron microscope (TEM) sends electrons through a very thin slice of tissue stained with heavy metals that bind certain structures. It reveals internal structure, which is how most organelles in this chapter were first described.
• Cryo-electron microscopy (cryo-EM) flash-freezes samples in a thin layer of ice without fixatives, preserving molecules in their natural shape. Combining thousands of images, it can determine the structure of individual proteins and complexes at near atomic resolution.

Cell fractionation

Microscopes show structure, but biologists also need to know what each structure does. Cell fractionation breaks cells open and separates their parts by spinning them in a centrifuge at increasing speeds. Large, dense components such as nuclei settle (form a pellet) at low speeds; mitochondria and chloroplasts at higher speeds; small membrane fragments and ribosomes only in an ultracentrifuge spinning at up to 100,000 revolutions per minute or more. Testing the purified fractions showed, for example, that the enzymes of cellular respiration reside in mitochondria.

The cell theory

By the mid-1800s, microscopists had seen enough cells in enough organisms to generalize. Matthias Schleiden concluded in 1838 that plants are made of cells, and Theodor Schwann extended the idea to animals in 1839. In 1855 Rudolf Virchow added the crucial third idea, summed up in the Latin phrase omnis cellula e cellula, "every cell from a cell." Together these form the cell theory:

1. All living organisms are composed of one or more cells.
2. The cell is the basic unit of structure and function in organisms.
3. All cells arise from preexisting cells by division.

Modern biology adds that cells contain hereditary information (DNA) that is passed from cell to cell during division, and that all cells share the same basic chemistry. The third tenet also rejected spontaneous generation, the idea that life routinely arises from nonliving matter.

Why cells are small

Every substance a cell uses enters through its surface, and every waste leaves the same way. As a cell grows, its volume increases faster than its surface. For a cube with side length s, surface area is 6s² and volume is s³, so the surface area to volume ratio is 6/s. A cube 1 µm on a side has a ratio of 6; a cube 10 µm on a side has a ratio of only 0.6. A large cell would have too little membrane to supply its interior. Cells and organisms solve the problem in several ways: they stay small, they become long and thin (nerve cells), they fold their surfaces (the microvilli of intestinal cells increase surface area some twentyfold), and large organisms are built from many small cells rather than a few huge ones.

Section 2Prokaryotic and eukaryotic cells

Features shared by all cells

Every cell, whatever its type, has four things in common. It is bounded by a plasma membrane, a selective barrier that lets oxygen, nutrients and wastes pass while keeping the cell's contents in. Inside is the cytosol, a semifluid, jellylike substance in which subcellular components are suspended. Every cell carries chromosomes made of DNA that hold its genes. And every cell has ribosomes, the small complexes of RNA and protein that make proteins according to instructions from the genes.

Beyond these essentials, cells come in two fundamentally different organizations. Organisms of the domains Bacteria and Archaea are made of prokaryotic cells. Protists, fungi, plants and animals, which make up the domain Eukarya, are made of eukaryotic cells. The names come from the Greek karyon, kernel, meaning the nucleus: pro means before and eu means true.

Prokaryotic cells

The defining feature of a prokaryotic cell is that its DNA is not enclosed in a membrane-bounded nucleus. Instead, the main chromosome, usually a single circular DNA molecule, is concentrated in a region called the nucleoid, which has no membrane around it. Many prokaryotes also carry plasmids, small circles of extra DNA that often carry genes for antibiotic resistance and can be passed from cell to cell. Prokaryotes lack membrane-enclosed organelles; the region between the nucleoid and the plasma membrane is the cytoplasm, and their ribosomes are smaller (70S) than those of eukaryotes (80S). Most prokaryotic cells are 1 to 5 µm long, about a tenth the diameter of a typical eukaryotic cell.

Outside the plasma membrane, nearly all prokaryotes have a rigid cell wall. In bacteria the wall is made of peptidoglycan, a network of sugar polymers cross-linked by short peptides; archaeal walls lack peptidoglycan. Many prokaryotes also secrete a sticky outer layer of polysaccharide or protein called a capsule, which helps them stick to surfaces and resist the host's immune defenses. Hairlike appendages called fimbriae attach cells to surfaces and to each other, and longer pili (sex pili) pull two cells together to transfer DNA. Many prokaryotes swim with flagella, whip-like structures that rotate like propellers. Bacterial flagella are built from different proteins and work by a different mechanism than eukaryotic flagella, an example of similar functions evolving independently.

Eukaryotic cells

A eukaryotic cell has its DNA inside a nucleus bounded by a double membrane, and it is divided internally by membranes into many organelles, each with a specialized job. Compartmentalization lets incompatible processes run side by side: the acidic, enzyme-filled interior of a lysosome can digest material without harming the cytosol, and fatty acids can be built in one compartment while they are broken down in another. Internal membranes also provide huge surface area for enzymes and transport proteins. Eukaryotic cells are typically 10 to 100 µm in diameter, and they have a cytoskeleton of protein fibers that shapes them, organizes their interior and moves them.

Animal cells and plant cells

Animal and plant cells share most organelles: a nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, a cytoskeleton and a plasma membrane. They differ in a few telling ways. Plant cells have a rigid cell wall of cellulose outside the plasma membrane, chloroplasts for photosynthesis, a large central vacuole that can fill most of the cell's volume, and plasmodesmata, channels through the walls that connect neighboring cells. Animal cells have lysosomes as prominent digestive organelles (plants use their vacuole for much of this job), centrosomes with a pair of centrioles (absent from most plant cells), and, in some cells, flagella or cilia. Without a cell wall, animal cells rely on an extracellular matrix and on junctions with their neighbors for support.

Section 3The endomembrane system, mitochondria and chloroplasts

The nucleus: the cell's information center

The nucleus holds most of the cell's genes and is usually the most conspicuous organelle, averaging about 5 µm across. It is enclosed by the nuclear envelope, a double membrane perforated by nuclear pores about 100 nm wide. Each pore is lined by a large pore complex of proteins that regulates traffic: mRNA and ribosomal subunits move out, while proteins needed in the nucleus, such as DNA polymerases and histones, move in. On the inner side of the envelope, a meshwork of intermediate filaments called the nuclear lamina supports the nucleus and helps organize the DNA.

Inside, DNA is organized into chromosomes. Each chromosome is a single long DNA molecule wrapped around proteins, chiefly histones; the DNA-protein complex is called chromatin. A human body cell has 46 chromosomes. In a cell that is not dividing, chromatin is diffuse; as a cell prepares to divide, it condenses into the thick, visible chromosomes seen in mitosis. A dense region called the nucleolus is where ribosomal RNA (rRNA) is made and joined with proteins imported from the cytoplasm to assemble ribosomal subunits, which then exit through the pores.

Ribosomes: protein factories

Ribosomes are not membrane-bounded; each is a complex of rRNA and protein with a large and a small subunit. Cells that make lots of protein, such as pancreas cells that secrete digestive enzymes, have millions of ribosomes and prominent nucleoli. Ribosomes work in two places. Free ribosomes, suspended in the cytosol, make proteins that function there, such as the enzymes of glycolysis. Bound ribosomes, attached to the outside of the endoplasmic reticulum or nuclear envelope, make proteins destined for membranes, for packaging into organelles such as lysosomes, or for secretion from the cell. Bound and free ribosomes are identical; a ribosome binds the ER only when the protein it is making begins with a signal peptide that directs it there.

The endomembrane system

The endomembrane system includes the nuclear envelope, the endoplasmic reticulum, the Golgi apparatus, lysosomes, vacuoles and the plasma membrane. These membranes are related either through direct physical continuity or by the transfer of membrane segments as small vesicles, sacs made of membrane that bud from one compartment and fuse with another. Together they synthesize proteins, modify and sort them, make lipids, metabolize carbohydrates and detoxify poisons.

The endoplasmic reticulum (ER) is an extensive network of membrane-enclosed tubules and sacs called cisternae, often accounting for more than half the total membrane in a eukaryotic cell. Its internal space, the ER lumen, is continuous with the space between the two membranes of the nuclear envelope. The ER has two regions:

• Smooth ER lacks ribosomes. It synthesizes lipids, including phospholipids and steroids; the cells of the testes and ovaries that make sex hormones are packed with it. In liver cells, smooth ER enzymes detoxify drugs and poisons, mostly by adding hydroxyl groups that make them more soluble and easier to excrete. Repeated exposure to drugs such as alcohol makes smooth ER proliferate, increasing tolerance. In muscle cells, a specialized smooth ER (the sarcoplasmic reticulum) stores calcium ions and releases them to trigger contraction.
• Rough ER is studded with bound ribosomes. As a polypeptide grows, it is threaded into the lumen, where it folds into its shape. Many become glycoproteins as ER enzymes attach short carbohydrate chains. Rough ER also makes new membrane, inserting proteins into its own membrane and adding phospholipids. Finished proteins leave in transport vesicles that bud from a specialized region.

The Golgi apparatus: shipping and receiving

Transport vesicles from the ER travel to the Golgi apparatus, a stack of flattened membranous sacs (cisternae). The Golgi has a distinct polarity. The cis face, usually near the ER, receives vesicles; the trans face ships them out. As products move from cis to trans, mainly because the cisternae themselves mature, Golgi enzymes modify them, trimming and changing the carbohydrate chains of glycoproteins and making some polysaccharides, such as the pectins of plant cell walls. The Golgi then sorts products and targets them by attaching molecular tags, such as phosphate groups, that act like postal zip codes. Vesicles from the trans face go to lysosomes, to the plasma membrane for insertion, or to the surface for secretion.

Lysosomes and vacuoles

A lysosome is a membranous sac of hydrolytic enzymes that digest proteins, fats, polysaccharides and nucleic acids. These enzymes work best at about pH 5, which the lysosome maintains by pumping hydrogen ions in from the cytosol. If a lysosome leaks, its enzymes are not very active at the neutral pH of the cytosol, a built-in safeguard. Lysosomes carry out intracellular digestion. In phagocytosis, a cell engulfs a food particle or bacterium in a vacuole that fuses with a lysosome; macrophages destroy bacteria this way. In autophagy, a damaged organelle is wrapped in a membrane and digested, and its building blocks are recycled.

Vacuoles are large vesicles derived from the ER and Golgi. Food vacuoles form by phagocytosis. Many freshwater protists have contractile vacuoles that pump excess water out of the cell. Mature plant cells have a central vacuole enclosed by a membrane called the tonoplast. It stores ions, holds pigments that color petals, accumulates poisons that deter herbivores, and, most importantly, takes up water so that the cell grows larger with little investment in new cytoplasm.

Mitochondria and chloroplasts: energy converters

Mitochondria are the sites of cellular respiration, the oxygen-using process that releases energy from sugars, fats and other fuels and stores it in ATP. Nearly all eukaryotic cells have them, from one large mitochondrion to thousands, roughly matching the cell's activity; heart muscle cells are especially rich in them. A mitochondrion is enclosed by two membranes. The outer membrane is smooth. The inner membrane is folded into cristae, which greatly increase its surface area for the enzymes of the electron transport chain and ATP synthase. The inner membrane divides the organelle into an intermembrane space and the mitochondrial matrix, which contains enzymes of the citric acid cycle, mitochondrial DNA and ribosomes.

Chloroplasts, found in plants and algae, are the sites of photosynthesis, which converts light energy to chemical energy stored in sugar. They are members of a family of plant organelles called plastids, which also includes amyloplasts that store starch. A chloroplast is enclosed by two membranes separated by a narrow space. Inside lies a third membrane system of flattened sacs called thylakoids, often stacked like coins into grana (singular granum). The thylakoid membranes hold the green pigment chlorophyll and capture light. The fluid outside the thylakoids, the stroma, contains the chloroplast's DNA, ribosomes and the enzymes that build sugar.

The endosymbiont theory

Mitochondria and chloroplasts are not part of the endomembrane system; their proteins are made mostly on free ribosomes in the cytosol and on their own ribosomes inside. Their peculiarities point to a remarkable origin. According to the endosymbiont theory, an early ancestor of eukaryotic cells engulfed an oxygen-using bacterium that was not digested but became an endosymbiont, a cell living inside another cell. Over time the two merged into a single organism, and the endosymbiont became the mitochondrion. Later, one of these cells engulfed a photosynthetic cyanobacterium, which became the chloroplast of the lineage that gave rise to algae and plants. The evidence is strong:

• Both organelles are enclosed by two membranes, consistent with an engulfed cell inside a vesicle.
• Both contain their own circular DNA without histones, like bacterial chromosomes.
• Both have 70S ribosomes resembling bacterial ribosomes, sensitive to some of the same antibiotics.
• Both reproduce by a splitting process similar to binary fission, and cells cannot make them from scratch.
• Their DNA sequences are most similar to those of living bacteria: mitochondria to alphaproteobacteria and chloroplasts to cyanobacteria.

Peroxisomes

A peroxisome is a single-membrane compartment with enzymes that remove hydrogen atoms from various substrates and transfer them to oxygen, producing hydrogen peroxide (H₂O₂). Another enzyme, catalase, immediately converts the toxic H₂O₂ to water and oxygen. Peroxisomes break down long fatty acids into pieces that mitochondria can burn, and in the liver they detoxify alcohol and other harmful compounds.

Section 4The cytoskeleton and cell junctions

A dynamic scaffold

Early electron micrographs made the cytosol look like an empty soup in which organelles floated. Better methods revealed instead a network of protein fibers extending throughout the cytoplasm: the cytoskeleton. It gives mechanical support and maintains shape, which matters most in animal cells that lack walls. It anchors organelles in place, and it provides tracks along which motor proteins carry vesicles and organelles. It is constantly disassembled and rebuilt, letting cells change shape, crawl and divide. It has three kinds of fibers.

Microtubules

Microtubules, the thickest fibers (about 25 nm in diameter), are hollow tubes whose walls are made of the globular protein tubulin. Each tubulin unit is a dimer of alpha tubulin and beta tubulin. Microtubules have polarity: dimers are added and removed much faster at the plus end. They shape cells, serve as tracks for vesicles moving from the Golgi to the plasma membrane, separate chromosomes during cell division (as the mitotic spindle) and form the core of cilia and flagella. The drug colchicine, used to treat gout, binds tubulin and blocks microtubule growth; taxol, a cancer drug, does the opposite, freezing microtubules so dividing cells cannot complete mitosis.

In animal cells, microtubules grow out from a centrosome near the nucleus, a microtubule-organizing center that contains a pair of centrioles. Each centriole is a ring of nine sets of triplet microtubules. Plant cells and many fungi organize microtubules without centrioles.

Cilia and flagella are microtubule-containing extensions of some eukaryotic cells. They share a common structure: a core of nine doublets of microtubules arranged in a ring around two single microtubules in the center, the 9 + 2 pattern, covered by the plasma membrane. The assembly is anchored by a basal body whose structure (nine triplets, no center) is identical to a centriole. Arms of the motor protein dynein on each doublet "walk" along the neighboring doublet, using ATP; because the doublets are held together by cross-linking proteins, this walking makes the whole structure bend rather than slide apart. Flagella are usually few and long and move with a snake-like undulation (sperm); cilia are numerous and short and beat in an oar-like power stroke (the lining of the airways, which sweeps mucus out of the lungs, and the oviduct, which moves an egg toward the uterus).

Microfilaments

Microfilaments are the thinnest fibers (about 7 nm), solid rods built from a twisted double chain of actin subunits. They bear tension (pulling forces). A three dimensional network of microfilaments just inside the plasma membrane, the cortex, gives the outer cytoplasm a gel-like consistency and supports the cell's shape. Bundles of microfilaments form the core of microvilli, which increase the surface of intestinal cells. Microfilaments are best known for their role in movement. In muscle cells, thousands of actin filaments interdigitate with thicker filaments of the motor protein myosin, and myosin heads walk along the actin to contract the cell. In dividing animal cells, a ring of actin and myosin pinches the cell in two (the cleavage furrow). Cells such as amoebas and white blood cells crawl by extending pseudopodia as actin assembles at the leading edge, and in plant cells actin and myosin drive cytoplasmic streaming, a circular flow that distributes materials in large cells.

Intermediate filaments

Intermediate filaments range from 8 to 12 nm in diameter, between the other two. They are the most permanent part of the cytoskeleton and are specialized for bearing tension, like rope. Unlike the other two, they are built from a diverse family of proteins, and different cell types use different ones: keratins in epithelial cells (and in hair and nails), neurofilaments in nerve axons, vimentin in fibroblasts and lamins forming the nuclear lamina. They reinforce cell shape, anchor the nucleus, and in skin they connect to junctions between cells to spread mechanical stress. They are found only in some animals, including vertebrates.

Outside the cell: walls and matrix

A plant cell wall protects the cell, maintains its shape and prevents excessive uptake of water. It is built from cellulose microfibrils embedded in a matrix of other polysaccharides and protein. A young cell secretes a thin, flexible primary wall; between neighboring cells lies the middle lamella, rich in sticky pectins that glue cells together. When growth stops, many cells add a strong, often woody secondary wall between the plasma membrane and the primary wall.

Animal cells lack walls but secrete an elaborate extracellular matrix (ECM). Its main ingredients are glycoproteins and other carbohydrate-containing molecules. The most abundant is collagen, about 40 percent of the total protein in the human body, which forms strong fibers embedded in a network of proteoglycans, molecules with a small protein core and many carbohydrate chains that trap water. Glycoproteins called fibronectin bind the ECM to integrins, receptor proteins that span the plasma membrane and connect on the inside to microfilaments. Integrins transmit signals in both directions, so changes in the matrix can alter gene expression inside the cell and coordinate the behavior of all cells in a tissue.

Cell junctions

Neighboring cells in tissues adhere, interact and communicate through specialized junctions.

• Plasmodesmata are channels that pass through plant cell walls. The plasma membranes of adjacent cells line each channel, so the cytosol of the cells is continuous. Water, small solutes and even some proteins and RNA move from cell to cell this way.
• Tight junctions press the plasma membranes of neighboring animal cells tightly together, bound by specific proteins, forming a continuous seal around the cells that prevents leakage of fluid between them. Tight junctions between skin cells make us watertight, and in the gut and kidney they force substances to pass through cells rather than around them.
• Desmosomes (anchoring junctions) work like rivets, fastening cells together into strong sheets. Intermediate filaments of keratin anchor them on the inside. They are abundant in skin and heart muscle, tissues that are stretched and pulled.
• Gap junctions (communicating junctions) are channels formed by membrane proteins that connect the cytoplasm of adjacent animal cells, letting ions, sugars and other small molecules pass. They are functionally similar to plasmodesmata and are essential in heart muscle, where they let electrical signals spread quickly so the cells contract in unison.

Section 5Membrane structure, passive and active transport, bulk transport

The fluid mosaic model

The plasma membrane is only about 8 nm thick, yet it controls traffic into and out of the cell. It shows selective permeability: some substances cross easily and others hardly at all. The accepted description of its structure is the fluid mosaic model: a fluid phospholipid bilayer in which a mosaic of diverse proteins is embedded or attached. The phospholipids are amphipathic (Chapter 1), with hydrophilic heads facing the watery cytosol and extracellular fluid and hydrophobic tails facing each other in the interior.

The membrane is held together mainly by hydrophobic interactions, which are much weaker than covalent bonds. Phospholipids can therefore drift sideways within their layer, swapping places with neighbors millions of times per second, though they rarely flip from one layer to the other. Many proteins also drift, more slowly, and some are held in place by attachments to the cytoskeleton or the ECM. Membrane fluidity is essential: a membrane that is too fluid cannot hold proteins in position, and one that solidifies cannot function. Fluidity depends on:

• Temperature: as temperature falls, the membrane eventually solidifies.
• Fatty acid tails: unsaturated tails, kinked by cis double bonds, cannot pack closely and keep the membrane fluid at lower temperatures. Organisms adapt: winter wheat and cold-water fish increase the proportion of unsaturated phospholipids in their membranes as temperatures drop.
• Cholesterol, wedged between phospholipids in animal membranes, acts as a fluidity buffer. At warm temperatures it restrains phospholipid movement, and at cool temperatures it keeps them from packing tightly.

Membrane proteins and carbohydrates

Proteins determine most of a membrane's functions. Integral proteins penetrate the hydrophobic interior; most are transmembrane proteins that span the whole bilayer, with hydrophobic stretches (usually alpha helices of nonpolar amino acids) inside the membrane and hydrophilic regions exposed to the water on each side. Peripheral proteins are not embedded at all; they are loosely bound to the surface, often to exposed parts of integral proteins. Membrane proteins carry out six broad functions: transport, enzymatic activity, signal transduction (receptors), cell to cell recognition, intercellular joining and attachment to the cytoskeleton and ECM.

Cells recognize one another by binding to molecules, often containing carbohydrates, on the outer surface of the plasma membrane. Short, branched carbohydrate chains are covalently bonded to lipids, forming glycolipids, or to proteins, forming glycoproteins. Together they make a sugar coat, the glycocalyx, that differs from species to species, from individual to individual and from one cell type to another. The A, B, AB and O human blood types reflect variation in the carbohydrate part of glycoproteins on red blood cells. Membranes are therefore asymmetric: the inner and outer layers differ in their lipids, and carbohydrates face only the outside.

Permeability of the lipid bilayer

The hydrophobic core of the bilayer controls what can cross on its own. Small nonpolar molecules, such as O₂, CO₂ and steroid hormones, dissolve in the lipid and cross easily. Very small polar molecules such as water cross slowly. Ions and larger polar molecules such as glucose barely cross at all, because they are surrounded by water and repelled by the hydrophobic core. These substances cross through transport proteins. Channel proteins provide hydrophilic tunnels; aquaporins, for example, let up to 3 billion water molecules per second pass through each channel, and many ion channels open and close in response to signals (gated channels). Carrier proteins bind a specific substance and change shape to shuttle it across.

Passive transport: diffusion and osmosis

Diffusion is the net movement of a substance from where it is more concentrated to where it is less concentrated, the result of random thermal motion. Any substance diffuses down its own concentration gradient, independent of other substances, until it reaches dynamic equilibrium, when as many molecules cross one way as the other. Because no cellular energy is spent, diffusion across a membrane is called passive transport. Oxygen diffuses into a cell that is using it, and CO₂ diffuses out.

Osmosis is the diffusion of free water across a selectively permeable membrane. Water moves from the side with lower solute concentration (more free water) to the side with higher solute concentration, because solutes bind water molecules and reduce the concentration of free water. To predict osmosis, compare the solution outside a cell to the cell's interior using tonicity, the ability of a surrounding solution to make a cell gain or lose water. Tonicity depends on the concentration of solutes that cannot cross the membrane.

• An isotonic solution has the same nonpenetrating solute concentration as the cell; there is no net water movement. For human cells, 0.9 percent NaCl (normal saline) is isotonic.
• A hypertonic solution has more nonpenetrating solute than the cell; the cell loses water. An animal cell shrivels (crenation); in a plant cell, the plasma membrane pulls away from the wall in plasmolysis, usually lethal.
• A hypotonic solution has less solute than the cell; water enters. An animal cell swells and may burst (lysis). A plant cell swells until its wall pushes back, becoming firm, or turgid, which is the healthy state for most plant cells. If water is lost, plants wilt as cells become flaccid.

Organisms without walls that live in hypotonic or hypertonic environments must practice osmoregulation, the control of solute concentrations and water balance. The freshwater protist Paramecium lives in water strongly hypotonic to its cytoplasm and uses a contractile vacuole to pump the incoming water back out.

Facilitated diffusion

In facilitated diffusion, polar molecules and ions diffuse passively down their gradients with the help of transport proteins, either channels or carriers. It is still passive: no energy is spent, and the direction is always down the gradient. Because carrier proteins have a limited number of binding sites, facilitated diffusion saturates; once every carrier is busy, adding more solute outside does not speed transport. The GLUT family of glucose carriers moves glucose into most of your cells by facilitated diffusion.

Active transport

Some transport proteins move solutes against their concentration gradients, from low to high. This active transport requires energy, usually from ATP, and lets a cell keep internal concentrations very different from its surroundings. All active transport uses carrier proteins, not channels.

The best-known example is the sodium-potassium pump (Na⁺/K⁺ ATPase) of animal cells. In each cycle it binds 3 Na⁺ on the cytoplasmic side, is phosphorylated by ATP, changes shape and releases the sodium outside; it then binds 2 K⁺ outside, loses the phosphate, returns to its original shape and releases the potassium inside. The result is high K⁺ and low Na⁺ inside the cell. Because it moves 3 positive charges out for every 2 in, the pump also makes the inside of the cell negative relative to the outside.

That voltage across the membrane is the membrane potential, typically about −50 to −70 millivolts. It favors the movement of cations into the cell and anions out. Ions therefore respond to a combined electrochemical gradient: the chemical force of their concentration gradient plus the electrical force of the membrane potential. A transport protein that generates voltage is an electrogenic pump; in animals the main one is the sodium-potassium pump, and in plants, fungi and bacteria it is a proton pump that moves H⁺ out of the cell.

In cotransport (secondary active transport), a gradient created by a pump drives the uphill transport of another solute. A cotransporter lets an ion flow down its gradient and harnesses that energy to move a second solute against its gradient. In your intestine, a sodium-glucose cotransporter uses the inward Na⁺ gradient to pull glucose into cells even when glucose is already more concentrated inside. Plant cells use the H⁺ gradient from their proton pumps to load sucrose into the phloem the same way.

Bulk transport: exocytosis and endocytosis

Large molecules such as proteins and polysaccharides, and particles such as bacteria, cross the plasma membrane in bulk, packaged in vesicles. This also costs energy. In exocytosis, a transport vesicle from the Golgi moves along microtubules to the plasma membrane, the two membranes fuse, and the contents spill outside while the vesicle membrane becomes part of the plasma membrane. Pancreatic cells secrete insulin this way, and nerve cells release neurotransmitters.

In endocytosis, the cell takes in molecules and particles by forming new vesicles from the plasma membrane. There are three types:

• Phagocytosis ("cellular eating"): the cell wraps pseudopodia around a large particle, such as a bacterium, and packages it in a food vacuole, which fuses with a lysosome.
• Pinocytosis ("cellular drinking"): the membrane folds inward to take in droplets of extracellular fluid with whatever solutes they contain. It is nonspecific.
• Receptor-mediated endocytosis: specific receptors, clustered in coated pits lined with the protein clathrin, bind particular molecules (ligands), and the pit pinches off as a vesicle. This lets cells take up large amounts of specific substances even when they are scarce. Human cells take in cholesterol this way, as low-density lipoprotein (LDL) particles bound to LDL receptors.

Words worth knowing

Energy, Enzymes, Cellular Respiration and Photosynthesis

A living cell is a chemical factory that never shuts down: it pulls energy out of food or sunlight, stores it briefly in ATP, and spends it to build, move and copy itself. This chapter follows that energy from the laws of thermodynamics through the enzymes that control every reaction, the breakdown of glucose in cellular respiration, and the capture of light in photosynthesis. Once you see how these pathways connect, much of physiology, nutrition, agriculture and medicine becomes far easier to understand.

Section 1Energy, free energy and ATP

Metabolism and the forms of energy

Metabolism is the sum of all the chemical reactions in an organism, organized into metabolic pathways in which each step is catalyzed by a specific enzyme. Catabolic pathways break complex molecules down and release energy (cellular respiration is the classic example); anabolic pathways consume energy to build complex molecules, such as proteins from amino acids. The energy released by catabolism pays for anabolism.

Energy is the capacity to cause change, or to do work. Kinetic energy is the energy of motion, and thermal energy is the kinetic energy of randomly moving molecules; when it is transferred between objects we call it heat. Potential energy is energy stored by location or structure, such as water behind a dam or a proton gradient across a membrane. Chemical energy is the potential energy in the arrangement of atoms in molecules; glucose has much more of it than the carbon dioxide and water it can become.

The laws of thermodynamics

Thermodynamics is the study of energy transformations. An organism is an open system that exchanges energy and matter with its surroundings.

The first law of thermodynamics states that energy can be transferred and transformed but not created or destroyed. A plant converts light energy into chemical energy; an animal converts the chemical energy in food into movement, gradients, new bonds and heat.

The second law of thermodynamics states that every energy transfer or transformation increases the entropy (disorder, or the dispersal of energy) of the universe. No transformation is perfectly efficient; some energy is always lost as heat the cell cannot use. Living things build ordered structures only by taking in organized energy (food or light) and releasing heat and simple, low energy molecules to the surroundings, so the entropy of organism plus surroundings always rises.

Free energy and spontaneous change

To predict whether a reaction can occur without an outside input of energy, biologists use free energy (G), the portion of a system's energy that can do work when temperature and pressure are uniform, as they are in a cell. The change in free energy during a reaction is

ΔG = ΔH − TΔS

where ΔH is the change in enthalpy (total energy, essentially the energy in bonds), ΔS is the change in entropy, and T is the absolute temperature in kelvins. A reaction with a negative ΔG loses free energy and is spontaneous, meaning it is energetically favorable and can proceed without an energy input. In biology the word spontaneous says nothing about speed. The oxidation of a sugar cube in air has a large negative ΔG, yet the cube sits on the table for years, because a barrier called activation energy (Section 2) keeps the reaction from starting.

Reactions are classified by the sign of ΔG:

• An exergonic reaction releases free energy (ΔG is negative). The products have less free energy than the reactants. The complete oxidation of one mole of glucose, C6H12O6 + 6 O2 → 6 CO2 + 6 H2O, has a standard ΔG of about −686 kcal/mol (−2,870 kJ/mol).
• An endergonic reaction absorbs free energy (ΔG is positive). It is not spontaneous, and the energy must come from somewhere else. Photosynthesis, the reverse of glucose oxidation, is strongly endergonic; its energy comes from light.

At chemical equilibrium the forward and reverse reactions occur at the same rate, ΔG is zero, and the system can do no work. A cell at metabolic equilibrium would be dead, so living cells keep their pathways far from equilibrium: products are constantly pulled away by the next reaction and fresh substrates constantly arrive.

ATP: the cell's energy currency

Cells do three main kinds of work: chemical work (pushing endergonic reactions, such as building polymers), transport work (pumping substances across membranes against their gradients), and mechanical work (beating cilia, contracting muscle, moving chromosomes). Most of this work is powered, directly or indirectly, by ATP (adenosine triphosphate).

ATP is a nucleotide made of adenine, the sugar ribose, and a chain of three phosphate groups joined by phosphoanhydride bonds. These are often called "high energy bonds," but the bonds are not unusually strong. The three negatively charged phosphates repel one another, and the products of hydrolysis are far more stable. Removing the terminal phosphate yields ADP plus inorganic phosphate (P_i) and releases about 7.3 kcal/mol (30.5 kJ/mol) under standard conditions, and closer to 13 kcal/mol under cellular conditions.

Energy coupling and phosphorylation

Energy coupling is the use of an exergonic process to drive an endergonic one. The overall ΔG of two coupled reactions is the sum of their individual ΔG values, so as long as the sum is negative, the pair proceeds. The cell rarely just hydrolyzes ATP and lets the energy escape as heat. Instead, an enzyme transfers the terminal phosphate from ATP to another molecule, a step called phosphorylation. The phosphorylated intermediate is less stable (has more free energy) than the original molecule, so its next reaction becomes exergonic.

Consider the synthesis of glutamine from glutamate and ammonia (ΔG about +3.4 kcal/mol). The enzyme first phosphorylates glutamate using ATP, and the phosphorylated intermediate then reacts readily with ammonia. The coupled reaction has an overall ΔG of about −3.9 kcal/mol (+3.4 − 7.3). Transport and mechanical work follow the same logic: the sodium potassium pump changes shape when phosphorylated, and motor proteins such as myosin change shape as they bind and hydrolyze ATP.

ATP is a renewable resource. The ATP cycle regenerates ATP from ADP, an endergonic reaction paid for by catabolism. A working muscle cell recycles its whole ATP pool in about a minute, some 10 million molecules per second, and an adult turns over roughly his or her body weight in ATP each day. ATP is a currency spent as fast as it is earned; long-term storage is the job of glycogen, starch and fat.

Oxidation, reduction and electron carriers

Much of the energy flow in metabolism is the movement of electrons. A reaction that transfers electrons from one substance to another is an oxidation reduction reaction, or redox reaction. Losing electrons is oxidation; gaining electrons is reduction (the mnemonic is OIL RIG: oxidation is loss, reduction is gain). The electron donor is the reducing agent and the electron acceptor is the oxidizing agent. In biology, electrons often travel with protons, as hydrogen atoms, so a molecule that loses hydrogen atoms is usually being oxidized.

Electrons release energy when they move from a less electronegative atom to a more electronegative one. In glucose they are shared evenly between carbon and hydrogen; in CO2 and water they are pulled close to oxygen. The cell does not let them fall to oxygen all at once, as in a fire, but hands them in small steps to electron carriers.

The most important carrier is the coenzyme NAD⁺ (nicotinamide adenine dinucleotide, from the vitamin niacin). Dehydrogenase enzymes remove two hydrogen atoms from a fuel, give both electrons and one proton to NAD⁺, and release the other proton, forming NADH. A related carrier, FAD (from riboflavin), becomes FADH2. Photosynthesis uses NADP⁺, which becomes NADPH; NADH mainly feeds energy harvest, while NADPH supplies reducing power for synthesis.

Section 2Enzymes and their regulation

Activation energy and catalysis

Every reaction must pass through an unstable transition state in which old bonds are strained and new ones partly formed. The energy needed to reach it is the activation energy (E_A). At body temperature few molecules collide hard enough to get over this barrier, and heating the cell would denature proteins and speed every reaction indiscriminately.

A catalyst is a substance that speeds a reaction without being consumed. Biological catalysts are enzymes, nearly all of them proteins (a few are RNA molecules called ribozymes, such as the ribosomal RNA that forms peptide bonds). Enzymes speed reactions, often by factors of a million or more, by lowering the activation energy. They do not change ΔG, they do not change the position of equilibrium, and they cannot make an endergonic reaction exergonic. They simply let the reaction reach equilibrium faster, and because each enzyme catalyzes only one reaction or a small set of reactions, they let the cell decide which reactions happen.

The active site and induced fit

The reactant an enzyme acts on is its substrate. The substrate binds to a pocket or groove on the enzyme, the active site, usually formed by only a handful of amino acids brought together by the protein's folding. The shape and chemistry of the active site make enzymes highly specific: sucrase hydrolyzes sucrose but not maltose, even though both are disaccharides. When the substrate enters, the enzyme changes shape slightly so that the active site grips the substrate more snugly, an effect called induced fit. The older "lock and key" picture captures specificity but misses this flexibility.

Within the enzyme substrate complex, the active site lowers the activation energy in several ways:

• It acts as a template that holds two or more substrates in the correct orientation to react.
• It strains the substrate's bonds, bending it toward the transition state.
• It provides a favorable microenvironment, for example a pocket of acidic side chains that can donate protons.
• Amino acid side chains can participate directly, briefly forming covalent bonds with the substrate before the product is released.

The enzyme is unchanged by the reaction and may handle thousands of substrate molecules per second. The rate rises with substrate concentration until every enzyme molecule is busy; the enzyme is then saturated and the reaction runs at its maximum velocity, V_max. The substrate concentration giving half of V_max is the Michaelis constant, K_m; a low K_m means high affinity for the substrate.

Effects of the environment

Because an enzyme's activity depends on its shape, anything that changes shape changes activity. Each enzyme has an optimal temperature and optimal pH. Warming speeds collisions and the rate rises, until the weak bonds holding the protein's shape break and the enzyme denatures. Most human enzymes work best near 37°C, while enzymes of hot spring bacteria work best at 70°C or higher (one of them, Taq polymerase, makes the polymerase chain reaction possible). Pepsin in the stomach has a pH optimum near 2; trypsin in the small intestine works best near pH 8.

Many enzymes need nonprotein helpers called cofactors. Inorganic cofactors are metal ions such as zinc, iron, copper and magnesium. Organic cofactors are called coenzymes, and most vitamins are needed in the diet because they are coenzymes or their precursors: niacin for NAD⁺, riboflavin for FAD, pantothenic acid for coenzyme A, thiamine for the coenzyme of pyruvate dehydrogenase.

Enzyme inhibitors

Chemicals that selectively block enzymes are called inhibitors. If an inhibitor attaches by covalent bonds, the inhibition is usually irreversible; if it attaches by weak bonds, it is reversible.

• Competitive inhibitors resemble the normal substrate and compete for the active site. Because binding is reversible, adding more substrate can outcompete the inhibitor. A competitive inhibitor raises the apparent K_m (more substrate is needed to reach half speed) but leaves V_max unchanged. Statin drugs, which lower cholesterol, are competitive inhibitors of HMG-CoA reductase.
• Noncompetitive inhibitors bind elsewhere on the enzyme and change its shape so that the active site works less effectively. Adding substrate cannot overcome them. They lower V_max; in the classic pure form K_m is unchanged.
• Irreversible inhibitors bond covalently to the enzyme. Nerve gases such as sarin bind a serine in the active site of acetylcholinesterase; penicillin permanently disables the bacterial enzyme that cross links the cell wall; aspirin acetylates cyclooxygenase.

Allosteric regulation and feedback inhibition

Cells control which pathways run by switching enzymes on and off. In allosteric regulation, a regulatory molecule binds an allosteric site separate from the active site and stabilizes either the active or the inactive shape of the enzyme, which is usually built of several subunits. An activator stabilizes the active form and an inhibitor the inactive form; because binding is reversible, activity tracks the regulators' concentrations.

In cooperativity, substrate binding at one active site shifts the other subunits to their active shape, raising their affinity and giving an S shaped (sigmoidal) rate curve. Hemoglobin is not an enzyme, but its cooperative oxygen binding works the same way.

The most common control logic is feedback inhibition, in which a pathway's end product inhibits an early enzyme, often the first committed step. In some bacteria, isoleucine is made from threonine in five steps; accumulating isoleucine binds an allosteric site on threonine deaminase, the first enzyme, and shuts the pathway down until isoleucine is used up. Section 4 shows the same principle at phosphofructokinase.

Cells also regulate enzymes by adding or removing phosphate groups (Chapter 4), by controlling how much enzyme is made, and by localizing enzymes in particular compartments, such as the citric acid cycle enzymes in the mitochondrial matrix.

Section 3Glycolysis, the citric acid cycle and oxidative phosphorylation

An overview of cellular respiration

Cellular respiration is the catabolic process that extracts energy from organic fuel by oxidizing it, ideally all the way to carbon dioxide and water. In aerobic respiration the final electron acceptor is oxygen. Some prokaryotes carry out anaerobic respiration, using a different inorganic final acceptor such as sulfate or nitrate. Glucose is the fuel textbooks follow, but fats and proteins feed into the same pathways (Section 4). The overall equation for glucose is

C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + energy (ATP and heat)

In this reaction glucose is oxidized to carbon dioxide and oxygen is reduced to water. Respiration has three stages:

1. Glycolysis, in the cytosol, splits glucose into two molecules of pyruvate.
2. Pyruvate oxidation and the citric acid cycle, in the mitochondrial matrix (in the cytosol of prokaryotes), finish the oxidation of the fuel to carbon dioxide.
3. Oxidative phosphorylation, at the inner mitochondrial membrane, uses an electron transport chain and chemiosmosis to make most of the ATP.

ATP is made in two ways. In substrate level phosphorylation, an enzyme transfers a phosphate group directly from a phosphorylated substrate to ADP. This happens in glycolysis and in the citric acid cycle and accounts for only about 4 ATP per glucose. In oxidative phosphorylation, the energy of electrons passed down the transport chain builds a proton gradient that drives ATP synthase; this accounts for about 26 to 28 ATP per glucose, nearly 90 percent of the total.

Glycolysis

Glycolysis ("sugar splitting") is a sequence of ten enzyme catalyzed reactions in the cytosol that breaks one six carbon glucose into two three carbon molecules of pyruvate. It does not require oxygen and occurs in nearly every living organism, which suggests it evolved very early, before oxygen was abundant in the atmosphere and before eukaryotic organelles existed. Its steps fall into two phases.

In the energy investment phase (steps 1 to 5), the cell spends 2 ATP. Hexokinase phosphorylates glucose to glucose 6-phosphate, which traps it in the cell because the charged molecule cannot cross the membrane. After an isomerization to fructose 6-phosphate, the enzyme phosphofructokinase (PFK) adds a second phosphate, forming fructose 1,6-bisphosphate. This is the committed step of glycolysis and its main control point. Aldolase then splits the six carbon sugar into two three carbon sugars, glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate, which is converted to a second G3P.

In the energy payoff phase (steps 6 to 10), each G3P is oxidized by a dehydrogenase that transfers electrons to NAD⁺, producing NADH, while a phosphate from solution is attached. Two later steps transfer phosphates to ADP by substrate level phosphorylation; the last, catalyzed by pyruvate kinase, produces pyruvate. Because everything after the split happens twice per glucose, the payoff phase yields 4 ATP and 2 NADH.

The net result of glycolysis per glucose is 2 pyruvate, 2 ATP (4 made minus 2 invested) and 2 NADH. No carbon dioxide is released. Glycolysis captures only a small fraction of the energy in glucose; most of it remains in the two pyruvate molecules, waiting to be extracted in the mitochondrion.

Pyruvate oxidation

In eukaryotes, pyruvate enters the mitochondrion by active transport. There, a large multienzyme complex, pyruvate dehydrogenase, carries out three linked steps for each pyruvate: it removes the carboxyl group as CO2 (the first carbon dioxide released in respiration), oxidizes the remaining two carbon fragment while reducing NAD⁺ to NADH, and attaches the resulting acetyl group to coenzyme A, forming acetyl CoA. Per glucose, this link step produces 2 acetyl CoA, 2 CO2 and 2 NADH. Acetyl CoA is a crossroads molecule: it can enter the citric acid cycle for energy or, when energy is plentiful, be used to make fatty acids.

The citric acid cycle

The citric acid cycle (Krebs cycle) in the mitochondrial matrix completes the oxidation of the fuel in eight steps. First, the two carbon acetyl group joins the four carbon oxaloacetate to form the six carbon citrate. Two carbons are then removed as CO2, and the remaining four carbon molecules are oxidized and rearranged until oxaloacetate is regenerated. The succinate dehydrogenase step, the only one catalyzed by an enzyme in the inner membrane, transfers electrons to FAD.

Each turn of the cycle (one acetyl CoA) produces 2 CO2, 3 NADH, 1 FADH2 and 1 ATP (in animal cells, often made first as GTP and then transferred to ADP). Because one glucose yields two acetyl CoA, the cycle turns twice per glucose: 4 CO2, 6 NADH, 2 FADH2 and 2 ATP. Notice that the cycle itself does not use oxygen, but it stops quickly without oxygen because it depends on a supply of NAD⁺ and FAD that only the electron transport chain can regenerate.

The electron transport chain

By the end of the citric acid cycle, glucose has been completely oxidized to CO2, but only 4 ATP have been made. Most of the energy is held in the electrons carried by 10 NADH and 2 FADH2. The electron transport chain releases that energy in small, usable steps. In eukaryotes it is a series of protein complexes embedded in the folded inner mitochondrial membrane (the cristae, whose folding provides enormous surface area); in prokaryotes it lies in the plasma membrane. Most carriers are proteins with tightly bound prosthetic groups, such as iron sulfur clusters and the heme groups of proteins called cytochromes.

NADH donates its electrons to complex I (NADH dehydrogenase). FADH2, bound in complex II (succinate dehydrogenase), adds its electrons at a lower energy level. Both complexes pass electrons to ubiquinone (Q), a small, mobile, lipid soluble carrier that moves within the membrane. Q delivers electrons to complex III, which hands them to cytochrome c, a small mobile protein on the outer face of the inner membrane. Cytochrome c carries them to complex IV (cytochrome oxidase), which passes them to oxygen, the final electron acceptor. Oxygen picks up a pair of hydrogen ions and becomes water. Each carrier is more electronegative than the one before, so electrons fall step by step down an energy gradient of about 53 kcal/mol from NADH to oxygen.

The chain itself makes no ATP. Its job is to use the energy of falling electrons to pump hydrogen ions (protons) from the matrix across the inner membrane into the intermembrane space. Complexes I, III and IV act as proton pumps; complex II does not, which is one reason FADH2 yields less ATP than NADH.

Chemiosmosis and ATP synthase

Proton pumping creates a proton motive force: a higher H⁺ concentration and positive charge in the intermembrane space. Protons cannot cross the lipid bilayer, so their only route back is through ATP synthase. As protons flow through it, they turn a rotor and internal rod, and the rotation changes the shape of the catalytic knob in the matrix so that it joins ADP and P_i into ATP. Using a hydrogen ion gradient across a membrane to drive cellular work is called chemiosmosis; it also makes ATP in chloroplasts and powers bacterial flagella.

Each NADH supplies enough proton pumping to make about 2.5 ATP, and each FADH2 about 1.5 ATP. The two NADH from glycolysis are made in the cytosol and must pass their electrons into the mitochondrion through a shuttle; depending on the cell type the shuttle delivers them to NAD⁺ (full value) or to FAD (lower value). This is why the total is given as a range: about 30 to 32 ATP per glucose (older books quote 36 to 38, based on earlier estimates). Respiration captures roughly 34 percent of the energy in glucose as ATP; the rest is released as heat, which helps mammals and birds keep a constant body temperature.

Section 4Fermentation and the regulation of respiration

Why cells need to regenerate NAD⁺

Without oxygen, the electron transport chain stops and NADH accumulates. Glycolysis can still make 2 ATP per glucose without oxygen, but it needs a steady supply of NAD⁺, and the cell holds only a small pool. Cells solve this problem either by anaerobic respiration, in which a prokaryote uses a transport chain with a final acceptor other than oxygen, or by fermentation, in which NADH simply hands its electrons to pyruvate or a derivative of it.

Lactic acid and alcohol fermentation

Fermentation consists of glycolysis plus one or two reactions that regenerate NAD⁺ by transferring electrons from NADH to an organic molecule. It makes no additional ATP; its only purpose is to keep glycolysis running. The two most common types differ in what accepts the electrons.

In lactic acid fermentation, lactate dehydrogenase reduces pyruvate directly to lactate, oxidizing NADH to NAD⁺; no CO2 is released. Lactic acid bacteria do this when they turn milk into yogurt and cheese. Human skeletal muscle switches to lactic acid fermentation when it uses ATP faster than oxygen arrives, as in a sprint, and the liver later converts the lactate back to glucose. Lactate is not the cause of next day soreness, which reflects small injuries to muscle fibers.

In alcohol fermentation, pyruvate first loses CO2 to become acetaldehyde, which is then reduced by NADH to ethanol, regenerating NAD⁺. Yeasts carry out alcohol fermentation; the CO2 raises bread dough and the ethanol is the basis of brewing and winemaking.

Comparing fermentation, anaerobic and aerobic respiration

All three processes use glycolysis to make a net 2 ATP per glucose with NAD⁺ as the oxidizing agent. They differ in the final electron acceptor: an organic molecule in fermentation, oxygen or another inorganic molecule in respiration. Aerobic respiration yields about 16 times more ATP per glucose than fermentation, so fermenting cells must consume glucose much faster.

Organisms differ in their dependence on oxygen. Obligate anaerobes, such as the Clostridium species that cause tetanus and botulism, cannot use oxygen and are often poisoned by it. Facultative anaerobes, such as yeasts, E. coli and human muscle cells, switch between respiration and fermentation depending on oxygen supply. Obligate aerobes require oxygen.

Other fuels feed the same pathways

Glycolysis and the citric acid cycle are metabolic hubs. Free glucose is only one of many fuels. Starch and glycogen are hydrolyzed to glucose, and other sugars such as fructose and galactose are converted into glycolytic intermediates.

• Proteins are digested to amino acids, which are used mostly to build new proteins. Excess amino acids are deaminated: the amino group is removed and excreted (in mammals, as urea made in the liver), and the remaining carbon skeleton enters glycolysis or the citric acid cycle as pyruvate, acetyl CoA or a cycle intermediate.
• Fats are split into glycerol and fatty acids. Glycerol is converted to G3P, a glycolytic intermediate. Fatty acids are broken down by beta oxidation in the mitochondrial matrix into two carbon fragments that enter the cycle as acetyl CoA; each round of beta oxidation also yields one NADH and one FADH2. Because fatty acids are highly reduced (rich in C to H bonds), a gram of fat yields more than twice as much ATP as a gram of carbohydrate (about 9 versus 4 kcal per gram). The 16 carbon fatty acid palmitate yields about 106 ATP.

Regulation of cellular respiration

Cells are thrifty: they make ATP when it is needed and slow down when it is plentiful. The main control mechanism is feedback inhibition of key enzymes by allosteric regulators (Section 2). The most important control point is phosphofructokinase (PFK), which catalyzes the committed step of glycolysis.

• PFK is inhibited by ATP. When ATP is abundant, respiration slows.
• PFK is activated by AMP (and ADP). AMP accumulates when ATP is being used up quickly, so a high AMP level is the cell's signal to speed up.
• PFK is inhibited by citrate, the first product of the citric acid cycle. If citrate builds up in the cytosol, the cycle is running faster than needed, and glycolysis slows to match it. This synchronizes the two stages.

Other control points act later: pyruvate dehydrogenase is inhibited by acetyl CoA and NADH, and oxidative phosphorylation slows automatically when ADP is scarce. Hormones such as epinephrine (Chapter 4) add another layer by mobilizing glucose from glycogen during exercise or stress.

Section 5Photosynthesis: light reactions, the Calvin cycle, C4 and CAM plants

The process that feeds the biosphere

Photosynthesis converts light energy into the chemical energy of sugars. Plants, algae and cyanobacteria are photoautotrophs: they make organic molecules from CO2 using light, and they are the producers of almost every ecosystem. Heterotrophs, including animals and fungi, obtain organic molecules by eating other organisms, so they depend on photosynthesis too.

The overall equation is commonly written

6 CO2 + 12 H2O + light energy → C6H12O6 + 6 O2 + 6 H2O

or, simplified, 6 CO2 + 6 H2O → C6H12O6 + 6 O2. Experiments using water labeled with the heavy oxygen isotope ¹⁸O showed that the O2 released comes from water, not from carbon dioxide: water is oxidized, and its light-energized electrons reduce CO2 to sugar.

Chloroplasts and pigments

In plants, photosynthesis occurs mainly in the mesophyll cells of leaves. CO2 enters and O2 exits through pores called stomata. A chloroplast has two outer membranes around a fluid, the stroma, which holds the Calvin cycle enzymes. Inside are flattened membrane sacs, thylakoids, stacked into grana; their interior is the thylakoid space. The pigment chlorophyll sits in the thylakoid membranes.

Light travels in packets called photons; the shorter the wavelength, the more energy each photon carries. Visible light spans roughly 380 to 750 nanometers. Chlorophyll absorbs violet blue and red light most strongly and reflects green. An absorption spectrum plots how much light a pigment absorbs at each wavelength, while an action spectrum plots how effectively each wavelength drives photosynthesis; the two match closely in the blue and red.

The main pigment is chlorophyll a. Chlorophyll b and the yellow and orange carotenoids are accessory pigments that broaden the wavelengths captured; carotenoids also provide photoprotection by dissipating excess light energy. When a pigment absorbs a photon, an electron is boosted to an excited state. In isolated chlorophyll it falls back, releasing heat and red fluorescence; in the membrane, the energy is put to work.

Photosystems and linear electron flow

Chlorophyll is organized into photosystems: a reaction center complex surrounded by light harvesting complexes that funnel absorbed energy to a special pair of chlorophyll a molecules. There an excited electron is transferred to a primary electron acceptor, the step that converts light energy into chemical energy.

There are two photosystems, numbered in order of discovery rather than action. Photosystem II (PS II) has the reaction center chlorophyll P680 (absorbing best at 680 nm), and photosystem I (PS I) has P700. Their main path, linear electron flow, runs as follows:

1. A photon excites PS II, and an excited electron from P680 passes to its primary acceptor, leaving P680⁺, the strongest biological oxidizing agent known.
2. An enzyme complex on the lumen side splits water into 2 electrons, 2 H⁺ and an oxygen atom. The electrons refill P680⁺, the protons stay in the thylakoid space, and two oxygen atoms combine to form O2, released as a by-product.
3. The excited electrons travel down an electron transport chain: plastoquinone (Pq), the cytochrome complex, and plastocyanin (Pc). As they pass through the cytochrome complex, their energy is used to pump protons from the stroma into the thylakoid space.
4. Meanwhile, light energy excites PS I, whose P700 passes an electron to its primary acceptor. The resulting P700⁺ is refilled by the electron arriving from PS II through plastocyanin.
5. The re-energized electrons pass through a short chain including the iron protein ferredoxin (Fd) to the enzyme NADP⁺ reductase, which reduces NADP⁺ to NADPH on the stroma side.

The thylakoid space gains protons from water splitting and from pumping by the cytochrome complex. This gradient drives ATP synthase, whose catalytic knob faces the stroma, so ATP is made where the Calvin cycle needs it. Light-driven chemiosmotic ATP synthesis is called photophosphorylation.

Cyclic electron flow and the comparison with mitochondria

In cyclic electron flow, excited electrons from PS I return from ferredoxin to the cytochrome complex instead of going to NADP⁺. They pass back to P700, pumping protons on the way. Cyclic flow uses only PS I, produces ATP but no NADPH and no O2. It matters because the Calvin cycle consumes more ATP than NADPH (a ratio of 18 to 12 per glucose), and linear flow alone does not supply enough ATP.

Chloroplasts and mitochondria both make ATP by chemiosmosis with similar electron transport chains and ATP synthases. Mitochondria take high energy electrons from food and pump protons from the matrix into the intermembrane space; chloroplasts take electrons from water, energized by light, and pump protons from the stroma into the thylakoid space, where the pH falls to about 5 while the stroma rises to about 8.

The Calvin cycle

The Calvin cycle uses the ATP and NADPH from the light reactions to build sugar from CO2. It is anabolic, and like the citric acid cycle it regenerates its starting material with each turn. Its direct product is not glucose but a three carbon sugar, glyceraldehyde 3-phosphate (G3P). To make one net molecule of G3P, the cycle must fix three molecules of CO2 and turn three times. It has three phases.

1. Carbon fixation. Each CO2 is attached to a five carbon sugar, ribulose bisphosphate (RuBP). The enzyme is rubisco (RuBP carboxylase oxygenase), probably the most abundant protein on Earth. The six carbon product is so unstable that it immediately splits into two molecules of 3-phosphoglycerate (3-PGA). Three CO2 therefore yield six 3-PGA.
2. Reduction. Each 3-PGA is phosphorylated by ATP and then reduced by electrons from NADPH, forming G3P. For six 3-PGA, this uses 6 ATP and 6 NADPH and produces 6 G3P. One of the six is the net gain; it leaves the cycle to be used by the plant cell.
3. Regeneration of RuBP. The other five G3P (15 carbons) are rearranged in a complex series of reactions into three RuBP (15 carbons), at a cost of 3 more ATP. The cycle is ready to fix more CO2.

For each net G3P the cycle consumes 9 ATP and 6 NADPH. Making one glucose (two G3P) requires 6 CO2, 18 ATP and 12 NADPH. G3P is then used to make glucose, sucrose, starch and cellulose.

Photorespiration: rubisco's weakness

Rubisco evolved when the atmosphere held almost no oxygen, and it cannot fully tell O2 from CO2. When it adds O2 to RuBP, it forms a two carbon compound that the plant salvages at a cost of ATP, releasing CO2 without making sugar. This is photorespiration. It becomes severe on hot, dry days, when plants close their stomata to save water: CO2 in the leaf is used up, O2 accumulates, and heat further favors the reaction with oxygen. In some crops photorespiration drains off as much as half of the fixed carbon.

Most plants, including rice, wheat and soybeans, are C3 plants, so named because the first stable product of carbon fixation is the three carbon 3-PGA. Two groups of plants have evolved ways to keep rubisco supplied with CO2.

C4 plants: separating steps in space

C4 plants, such as corn, sugarcane and many grasses, have Kranz anatomy: bundle sheath cells packed around each vein, surrounded by mesophyll cells. In the mesophyll, PEP carboxylase adds CO2 to phosphoenolpyruvate (PEP), forming the four carbon oxaloacetate, usually converted to malate. PEP carboxylase has a high affinity for CO2 and none for O2, so it keeps fixing carbon even when stomata are nearly closed. The four carbon compound moves into the bundle sheath cells and releases CO2, keeping CO2 high around rubisco and minimizing photorespiration. Regenerating PEP costs extra ATP, a price worth paying in hot, sunny climates.

CAM plants: separating steps in time

CAM plants (crassulacean acid metabolism), such as pineapples, cacti and other succulents, open their stomata at night, when less water evaporates. They fix CO2 with PEP carboxylase into organic acids stored in vacuoles. During the day, with stomata closed, the acids release CO2 to the Calvin cycle in the same cells. CAM plants trade growth rate for water conservation.

Words worth knowing

Cell Communication and Cell Division

Your body contains tens of trillions of cells, and none of them acts alone: each one is constantly reading chemical messages and deciding whether to grow, divide, change its behavior or die. This chapter explains how cells receive and relay signals, how those signals control the cell cycle, what happens when that control fails in cancer, and how meiosis produces the genetic variety that sexual reproduction depends on.

Section 1Signals, receptors and signal transduction

Why and how cells talk

Cell signaling is ancient. Yeast cells of opposite mating types find each other by secreting chemical signals, and many bacteria use quorum sensing: they release small molecules whose concentration reports population density, and at high density the population switches on genes for biofilm formation, light production or toxin release. The signaling machinery of yeast and mammals is strikingly similar, suggesting it was present in their common ancestor.

In multicellular organisms, signals act over different distances:

• Direct contact. Cells connected by gap junctions (animals) or plasmodesmata (plants) pass small signaling molecules directly through the cytoplasm. In cell to cell recognition, membrane bound molecules on one cell bind receptors on an adjacent cell; immune cells and developing tissues communicate this way.
• Paracrine signaling. A cell secretes a local regulator, such as a growth factor, that acts on nearby target cells.
• Synaptic signaling. A nerve cell releases a neurotransmitter across a narrow synapse to a single target cell, a very fast, specific form of local signaling.
• Autocrine signaling. A cell responds to a signal it secreted itself, as some immune cells and many cancer cells do.
• Endocrine (hormonal) signaling. Specialized cells release hormones that travel through the blood to target cells anywhere in the body. Plants use hormones too, moving them through vascular tissue or diffusing them through the air (ethylene).

Whatever the distance, a cell responds only if it has the right receptor. Insulin floods through the entire bloodstream, but only cells bearing insulin receptors respond. This is the first source of specificity in signaling.

Three stages: reception, transduction and response

Much of what we know about signaling began with studies of how epinephrine (adrenaline) makes liver cells break down glycogen. Epinephrine activated the enzyme glycogen phosphorylase only in intact cells; the hormone never entered the cell, so something at the surface had to relay the message inside. From this work came a three stage model:

1. Reception. The signaling molecule, called a ligand, binds a receptor protein, changing the receptor's shape.
2. Transduction. The change in the receptor sets off a sequence of changes in a series of molecules, the signal transduction pathway, that converts the signal into a form that can bring about a response.
3. Response. The transduced signal triggers a specific cellular activity: activating an enzyme, rearranging the cytoskeleton, or turning on genes.

Receptors in the plasma membrane

Most signaling molecules are water soluble and too large or too polar to cross the membrane, so their receptors are transmembrane proteins. Ligand binding changes the receptor's shape or causes receptors to group together, and that change is passed inside. Three major classes are tested most often.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Each is a single polypeptide that crosses the membrane seven times. On the cytoplasmic side it works with a G protein, a protein that binds the energy-rich nucleotide GTP. When the G protein holds GDP it is inactive. When a ligand binds the receptor, the receptor changes shape and causes the G protein to swap GDP for GTP. The activated G protein (specifically its alpha subunit) moves along the membrane and activates an enzyme, such as adenylyl cyclase. The G protein is also a GTPase: it hydrolyzes its own GTP to GDP within seconds and so turns itself off, which keeps the response brief unless the signal continues. GPCRs detect hormones such as epinephrine and glucagon, neurotransmitters, odors and light (rhodopsin in the eye is a GPCR). About a third of all prescription drugs act on GPCRs.

Receptor tyrosine kinases (RTKs) are membrane receptors with enzymatic activity. A kinase is an enzyme that transfers phosphate groups from ATP to a protein; tyrosine kinases attach them to the amino acid tyrosine. An RTK has a ligand binding site outside the cell, one helix crossing the membrane and a tail inside with several tyrosines. When ligands bind two receptor monomers, the two associate into a dimer. Each tail then phosphorylates tyrosines on the other tail (autophosphorylation). The fully activated receptor is recognized by specific relay proteins, each of which binds a particular phosphorylated tyrosine and becomes activated in turn. Because one RTK can activate ten or more different pathways at once, these receptors suit processes that need several responses together, such as cell growth and division. Insulin and many growth factors act through RTKs, and abnormal RTKs that dimerize without a ligand contribute to some cancers.

Ligand-gated ion channels are receptors that act as gates. When the ligand binds, the channel opens or closes, letting specific ions such as Na⁺, K⁺, Ca²⁺ or Cl⁻ flow through. The resulting change in ion concentration or membrane voltage is itself the signal. These receptors are essential in the nervous system: acetylcholine released at a neuromuscular junction opens such channels in the muscle cell, and the influx of sodium triggers contraction.

Intracellular receptors

Some signals are small or hydrophobic enough to cross the membrane. Steroid hormones (such as testosterone, estrogen and cortisol), thyroid hormones and the gas nitric oxide (NO) bind receptors in the cytoplasm or nucleus. A hormone-receptor complex typically acts as a transcription factor, a protein that binds specific DNA sequences and turns particular genes on or off. Testosterone, for example, enters a cell, binds its receptor in the cytoplasm, and the complex moves into the nucleus to activate genes that produce male sex characteristics. Responses through intracellular receptors are usually slower (hours) but longer lasting than those through membrane receptors, because they require making new proteins.

Transduction: phosphorylation cascades

Transduction usually involves several steps, and at each step a protein changes shape, most often by phosphorylation. In a phosphorylation cascade, an activated protein kinase phosphorylates and activates a second kinase, which activates a third, and so on, until the last protein in the series (often an enzyme or transcription factor) carries out the response. Most cytoplasmic kinases phosphorylate serine or threonine rather than tyrosine. One of the best studied cascades is the MAP kinase pathway, which runs from growth factor RTKs through the small G protein Ras and a three kinase relay to transcription factors that promote cell division.

Signals must be switched off as well as on. Protein phosphatases remove phosphate groups and inactivate kinases, so the activity of each relay protein depends on the balance between its kinase and its phosphatase. When the signal molecule leaves the receptor, the phosphatases win, and the pathway returns to its resting state, ready for the next signal.

Second messengers

Many pathways also use small, nonprotein, water soluble molecules or ions called second messengers (the extracellular ligand being the "first messenger"). Because they are small, they spread quickly through the cytosol.

Cyclic AMP (cAMP) was the first second messenger discovered. Adenylyl cyclase, activated by a G protein, converts ATP to cAMP, and the cAMP concentration can rise twentyfold within seconds. cAMP's main target is protein kinase A (PKA), which phosphorylates other proteins. In the liver, PKA starts a cascade that activates glycogen phosphorylase. The enzyme phosphodiesterase quickly converts cAMP to AMP, ending the signal; caffeine inhibits phosphodiesterase, prolonging cAMP signals. Some G proteins (inhibitory G proteins) lower cAMP instead.

Calcium ions are an even more widely used second messenger. Cells keep cytosolic Ca²⁺ very low by pumping it out of the cell and into the endoplasmic reticulum. In one common pathway, a GPCR activates the enzyme phospholipase C, which cleaves the membrane phospholipid PIP2 into two second messengers: diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 diffuses to the ER and opens a ligand-gated calcium channel, releasing Ca²⁺ into the cytosol. Calcium, often bound to the small protein calmodulin, then activates many proteins, while DAG together with Ca²⁺ activates protein kinase C.

Section 2Cellular responses and apoptosis

Nuclear and cytoplasmic responses

Every signal transduction pathway ends by regulating some activity in the cell. Many pathways end in the nucleus: the final activated molecule is a transcription factor that switches genes on or off, so the cell begins or stops making particular proteins. Growth factors that stimulate cell division typically work this way, through the MAP kinase cascade. Other pathways act in the cytoplasm, changing the activity of proteins that already exist: opening an ion channel, activating a metabolic enzyme such as glycogen phosphorylase, or reorganizing the cytoskeleton. When yeast cells receive mating factor, for example, their cytoskeleton directs growth toward the source of the signal, producing projections called shmoos that grow toward a cell of the opposite mating type.

Amplification

Multistep pathways do something a single switch cannot: they amplify the signal. Each activated enzyme catalyzes many reactions before it is switched off, so the number of activated molecules grows at every step. In the epinephrine pathway, a few hormone molecules activate many G proteins, each adenylyl cyclase makes many cAMP molecules, each PKA phosphorylates many kinases, and so on. In the end, one epinephrine molecule can lead to the release of hundreds of millions of glucose 1-phosphate molecules from glycogen. This is why hormones are effective at extremely low concentrations, often less than a billionth of a mole per liter.

Specificity and coordination

The same signal can produce very different responses in different cells. Epinephrine stimulates glycogen breakdown in the liver, speeds and strengthens the heartbeat, and constricts some blood vessels while dilating others, because each cell type carries a different set of receptors, relay proteins and response proteins. Pathways can branch, converge, and interact with one another (cross talk), and scaffolding proteins hold several relay proteins together, increasing the speed and accuracy of signaling.

A response must also stop. Ligands leave their receptors, G proteins hydrolyze their GTP, phosphodiesterase destroys cAMP, phosphatases remove phosphates and calcium is pumped back into the ER, so the cell always responds to the current level of the signal.

Apoptosis: programmed cell death

One of the most dramatic responses to a signal is the decision to die. Apoptosis is a regulated, orderly self-destruction program. The cell shrinks, its chromatin condenses and its DNA is chopped into fragments, its membrane forms bulges called blebs, and the cell breaks into membrane-enclosed apoptotic bodies. The cell also flips the phospholipid phosphatidylserine to the outer face of its membrane, an "eat me" signal that tells phagocytes to engulf the remains. Because the contents never spill into the surrounding tissue, apoptosis does not cause inflammation.

Apoptosis is essential for normal life:

• Sculpting during development. Cells in the webbing between the developing fingers and toes die by apoptosis; when this fails, the digits remain joined. Many more neurons are produced in the developing brain than survive, and those that fail to make proper connections die.
• Removing dangerous cells. Cells with irreparable DNA damage, virus infected cells, and developing immune cells that would attack the body's own tissues are eliminated.
• Tissue turnover. Billions of cells die by apoptosis every day in an adult and are replaced by cell division.

The molecular basis of apoptosis was first worked out in the nematode Caenorhabditis elegans. Of the 1,090 somatic cells formed during the development of a hermaphrodite worm, exactly 131 die by apoptosis, always the same cells. Researchers identified genes required for this death: ced-3 and ced-4 (ced stands for "cell death") encode proteins that carry out apoptosis, while ced-9 encodes a protein on the outer mitochondrial membrane that acts as a brake. As long as Ced-9 is active, the cell lives; a death signal inactivates Ced-9 and releases the killing machinery. Ced-3 is a caspase, the main type of protease (protein cutting enzyme) that executes apoptosis.

The intrinsic and extrinsic pathways in mammals

Mammals use the same logic with many more players. Their apoptotic signals arrive by two main routes, both of which end by activating caspases.

The intrinsic (mitochondrial) pathway is triggered by signals from inside the cell, such as irreparable DNA damage (often detected through the protein p53), severe stress or the misfolding of proteins in the ER. Proteins of the Bcl-2 family control the outcome. Pro-apoptotic members such as Bax and Bak form pores in the outer mitochondrial membrane, while anti-apoptotic members such as Bcl-2 itself (the mammalian counterpart of Ced-9) block them. When pro-death signals win, cytochrome c leaks from the intermembrane space into the cytosol. There it combines with the protein Apaf-1 (the counterpart of Ced-4) to form a wheel-shaped complex called the apoptosome, which activates the initiator caspase-9. Notice that cytochrome c has two jobs: inside the mitochondrion it carries electrons in respiration (Chapter 3), and outside it triggers death.

The extrinsic (death receptor) pathway is triggered by signals from other cells. Immune cells such as cytotoxic T cells display a ligand (Fas ligand), and cells such as macrophages release tumor necrosis factor; these bind death receptors on the target cell, which recruit adapter proteins and activate the initiator caspase-8.

Both initiator caspases activate the executioner caspases (such as caspase-3), which cut hundreds of cellular proteins, activate nucleases that fragment DNA, and dismantle the cytoskeleton and nuclear lamina. Each caspase is made as an inactive precursor that must itself be cut to become active, and an active caspase can activate many more, so the process, once committed, proceeds rapidly and irreversibly.

Apoptosis compared with necrosis

Not all cell death is programmed. Necrosis is uncontrolled death from severe injury such as loss of blood supply, toxins or trauma. The cell swells, its organelles and membrane rupture, and the spilled contents trigger inflammation. A heart attack kills cardiac muscle mostly by necrosis.

Section 3The cell cycle and mitosis

Why cells divide, and what they must copy

"Every cell from a cell" is one of the founding ideas of biology. Cell division lets a single fertilized egg become a body of trillions of cells, replaces cells lost to wear and injury, and, in single-celled organisms, is reproduction itself. The life of a cell from its formation by division until its own division into two daughter cells is the cell cycle. To divide successfully, a cell must copy its DNA exactly and then separate the two copies so that each daughter cell receives one complete set.

A cell's total DNA is its genome. A human cell holds about 2 meters of DNA, packaged with proteins as chromatin and divided among chromosomes. Human somatic cells contain 46 chromosomes in 23 pairs; gametes (sperm and eggs) contain 23. Chromatin is spread out for most of the cycle and condenses into compact, visible chromosomes as the cell prepares to divide.

Before division, each chromosome is replicated. The duplicated chromosome consists of two identical sister chromatids, held together along their length by protein complexes called cohesins and most tightly at a constricted region called the centromere. On each side of the centromere sits a kinetochore, a protein structure where spindle microtubules attach. Counting is a frequent source of confusion: when sister chromatids are attached, they count as one chromosome. Only when they separate does each become a full chromosome.

Phases of the cell cycle

The cell cycle has two major parts. Interphase, usually about 90 percent of the cycle, is when the cell grows and copies its DNA. The mitotic (M) phase includes mitosis, the division of the nucleus, and cytokinesis, the division of the cytoplasm. Interphase has three subphases:

• G1 phase ("first gap"): the cell grows, makes proteins and organelles, and carries out its normal functions.
• S phase ("synthesis"): DNA is replicated, so each chromosome becomes two sister chromatids. The centrosome is also duplicated.
• G2 phase ("second gap"): the cell continues to grow and prepares for division.

The "gap" names are historical; the cell is busy throughout. For a typical human cell dividing in culture, the full cycle takes about 24 hours: G1 roughly 5 to 6 hours or more, S about 10 to 12, G2 about 4 to 6, and M less than 1 hour. Cells that are not dividing leave the cycle and enter a resting state called G0. Most adult nerve and muscle cells stay in G0 permanently, while liver cells can be called back into the cycle by growth factors after an injury.

The mitotic spindle

Many of the events of mitosis depend on the mitotic spindle, a structure of microtubules and associated proteins. In animal cells, spindle microtubules grow from the centrosome, a microtubule organizing region containing a pair of centrioles (centrioles are not essential; plant cells have centrosomes without them). The centrosome replicates during interphase, and as mitosis begins the two centrosomes move apart, each surrounded by a radial array of short microtubules called an aster. Three groups of microtubules form: kinetochore microtubules attach to chromosomes, nonkinetochore microtubules from opposite poles overlap in the middle of the cell, and astral microtubules anchor the poles to the cell cortex.

The stages of mitosis

Mitosis is a continuous process, but for study it is divided into five stages. The figure and table below summarize them; here is what happens and why.

Prophase. Chromatin fibers condense into discrete chromosomes, each consisting of two sister chromatids. The nucleoli disappear. The mitotic spindle begins to form as the centrosomes move apart.

Prometaphase. The nuclear envelope fragments, letting microtubules invade the nuclear area. Each chromatid now has a kinetochore, and some microtubules attach to them. Because the two sister kinetochores face opposite directions, each chromosome ends up attached to microtubules from both poles, and it is jerked back and forth in a tug of war.

Metaphase. The centrosomes are at opposite poles, and the chromosomes are lined up on the metaphase plate, an imaginary plane midway between the poles. This is usually the longest stage of mitosis, about 20 minutes, and it is the stage at which chromosomes are most condensed, which is why karyotypes are prepared from metaphase cells.

Anaphase. The shortest stage, often only a few minutes, begins when the enzyme separase cleaves the cohesins holding sister chromatids together. Each chromatid becomes a full-fledged chromosome. The kinetochore microtubules shorten by losing tubulin subunits at their kinetochore ends, and motor proteins at the kinetochore "walk" the chromosome toward the pole. At the same time, motor proteins push the overlapping nonkinetochore microtubules apart, elongating the cell. By the end of anaphase, each pole has an equivalent and complete collection of chromosomes.

Telophase. Two daughter nuclei form as nuclear envelopes reassemble from fragments of the parent's envelope and the endomembrane system. Nucleoli reappear, chromosomes decondense, and the spindle microtubules are depolymerized. Mitosis, the division of one nucleus into two genetically identical nuclei, is complete.

Cytokinesis

Cytokinesis usually begins during late telophase. In animal cells it occurs by cleavage. A shallow groove, the cleavage furrow, appears on the cell surface near the old metaphase plate. On the cytoplasmic side lies a contractile ring of actin microfilaments associated with myosin motor proteins. The ring contracts like a drawstring, deepening the furrow until the parent cell is pinched in two. Plant cells, which have rigid cell walls, cannot pinch. Instead, vesicles from the Golgi apparatus move along microtubules to the middle of the cell and fuse into a cell plate, which grows outward until it joins the parent's plasma membrane; a new cell wall forms between the daughter cells from the plate's contents.

Binary fission in prokaryotes

Bacteria and archaea reproduce by binary fission. Replication of the single circular chromosome begins at the origin of replication, and the two copies of the origin move toward opposite ends of the cell as the cell elongates. When replication is complete, the membrane pinches inward and a new wall forms. Prokaryotes have no spindle, but actin-like proteins help move the chromosomes and a tubulin-like protein (FtsZ) forms the dividing ring, hints of how the eukaryotic machinery evolved; some protists, such as dinoflagellates and diatoms, divide by intermediate mechanisms.

Section 4Regulation of the cell cycle and cancer

A control system with checkpoints

Skin and intestinal lining cells divide frequently, liver cells only when needed, and mature neurons not at all. The timing is set by a cell cycle control system of cytoplasmic molecules. Cell fusion experiments revealed it: fusing an S phase cell with a G1 cell made the G1 nucleus start replicating its DNA at once, and fusing an M phase cell with any other cell sent the second nucleus straight into mitosis.

The control system works like a timing device with built-in checkpoints, control points where stop and go signals regulate the cycle. Three are major:

• The G1 checkpoint (called the restriction point in mammals) seems to be the most important. If a cell receives a go-ahead signal here, it usually completes the whole cycle and divides. If not, it exits into G0. The checkpoint checks cell size, nutrients, growth factors and DNA damage.
• The G2 checkpoint checks that DNA replication is complete and that damage has been repaired before mitosis begins.
• The M checkpoint (spindle assembly checkpoint) delays anaphase until every chromosome is attached at its kinetochores to spindle microtubules from both poles. Unattached kinetochores send a "wait" signal that keeps an enzyme complex called the anaphase promoting complex inactive. Once all are attached, the complex triggers the destruction of the protein that has been inhibiting separase, separase cuts the cohesins, and anaphase begins. This checkpoint prevents daughter cells from receiving missing or extra chromosomes.

Cyclins and cyclin-dependent kinases

The cycle is driven by two kinds of regulatory proteins. Cyclin-dependent kinases (Cdks) are protein kinases that are present at fairly constant levels but are active only when attached to a cyclin, a protein named for its cyclically fluctuating concentration. Different cyclin-Cdk pairs act at different points in the cycle. The first to be discovered was MPF (maturation-promoting factor, also read as M-phase promoting factor), a complex of cyclin B and its Cdk. Cyclin is made steadily during interphase and accumulates; as it combines with Cdk, MPF activity rises and peaks at the start of mitosis. MPF phosphorylates many proteins, helping to fragment the nuclear envelope (by phosphorylating the lamins), condense chromosomes and assemble the spindle. During anaphase, MPF switches itself off by triggering the destruction of its own cyclin through the anaphase promoting complex. The Cdk persists, inactive, until new cyclin accumulates in the next cycle.

At the G1 checkpoint, cyclin D and its Cdks, made in response to growth factors, phosphorylate the retinoblastoma protein (Rb). Unphosphorylated Rb binds and holds a transcription factor called E2F; phosphorylated Rb releases it, and E2F switches on the genes needed for S phase. Rb is thus a brake on the cycle, and the cyclin-Cdk complexes release it.

Internal and external signals

The control system responds to signals from inside and outside the cell. A key internal signal is DNA damage. The protein p53 rises when DNA is damaged and acts as a transcription factor that switches on a Cdk inhibitor (p21), halting the cycle at G1; it also activates DNA repair genes and, if the damage is too great, triggers apoptosis. For this reason p53 is often called the "guardian of the genome."

External signals include growth factors, proteins released by certain cells that stimulate other cells to divide. More than 50 are known. Platelet-derived growth factor (PDGF), released by blood platelets at a wound, binds receptor tyrosine kinases on fibroblasts in connective tissue and stimulates them to divide and help heal the wound. Fibroblasts grown in a dish divide only if PDGF is added to the medium.

Two other external controls are visible in cultured cells. In density-dependent inhibition, crowded cells stop dividing: normal cells form a single layer and stop when they touch, and cells bordering a scraped gap divide until it is filled. In anchorage dependence, cells must be attached to a substrate, such as the extracellular matrix, to divide.

Cancer: cells that escape control

Cancer is a group of diseases in which cells escape the normal controls on division. Cancer cells do not show density-dependent inhibition or anchorage dependence; in culture they pile up in layers and keep dividing as long as nutrients are supplied. Many make their own growth factors, have abnormal signaling proteins that send "divide" signals without any factor, or have defective checkpoints. Cancer cells grown in culture can divide indefinitely, whereas normal mammalian cells stop after about 20 to 50 divisions.

The problem begins when a single cell undergoes transformation, the conversion to a cancerous cell. The immune system normally recognizes and destroys such cells, but if one evades destruction it may proliferate into a tumor, a mass of abnormal cells. If the abnormal cells remain at their original site, the lump is a benign tumor, which can usually be removed surgically. A malignant tumor invades surrounding tissues and can undergo metastasis, the spread of cancer cells through blood or lymph to form new tumors in distant parts of the body. Malignant cells often have abnormal chromosome numbers and recruit new blood vessels.

Genes that cause cancer: oncogenes and tumor suppressors

Cancer is fundamentally a disease of genes that control the cell cycle. Two broad classes are involved.

Proto-oncogenes are normal genes that code for proteins that stimulate cell division, such as growth factors, their receptors and relay proteins. A mutation that makes a proto-oncogene's product excessively active or abundant converts it into an oncogene (cancer gene). This can happen in three main ways: (1) a point mutation in the coding sequence produces a hyperactive or degradation-resistant protein; (2) gene amplification produces extra copies of the gene; or (3) a chromosomal translocation moves the gene next to an active promoter, or fuses it with another gene. Because the effect is a gain of function, a mutation in only one of the two copies is enough; oncogenes act like a car's accelerator stuck to the floor. The ras gene, which encodes the Ras G protein in the growth factor pathway, is mutated in about 30 percent of human cancers; the mutant Ras stays active and keeps signaling for division without any growth factor.

Tumor suppressor genes code for proteins that inhibit cell division, repair DNA or trigger apoptosis. Mutations that inactivate them contribute to cancer by a loss of function, so usually both copies must be inactivated before the cell loses control; they act like brakes that fail only when both are cut. The p53 gene (TP53) is mutated in roughly half of all human cancers, more than any other gene. Others include RB, whose loss in childhood causes the eye tumor retinoblastoma, and BRCA1 and BRCA2, which encode proteins needed to repair double-stranded DNA breaks.

Cancer develops in multiple steps

More than one mutation is needed to produce a cancer cell. In the best studied example, colorectal cancer, a normal lining cell first loses the tumor suppressor APC, producing a small benign growth (polyp). Over years, additional mutations, typically activation of the ras oncogene and loss of p53, convert it into a malignant tumor that can invade and metastasize. Cells along the way usually also activate the enzyme telomerase, which prevents the shortening of chromosome ends and lets them divide indefinitely. The requirement for several mutations, each occurring at a low rate, explains why the incidence of most cancers rises sharply with age.

About 5 to 10 percent of cancers involve an inherited predisposition. A person who inherits one mutant copy of a tumor suppressor gene starts with one "hit" already in every cell, so only one more mutation is needed in any cell. Women who inherit a mutant BRCA1 allele, for example, have a lifetime breast cancer risk several times that of the general population. Viruses can contribute too: human papillomavirus, which causes cervical cancer, makes proteins that inactivate p53 and Rb, which is why the HPV vaccine prevents most cervical cancers.

Section 5Meiosis and sexual life cycles

Heredity, genes and two kinds of reproduction

Offspring resemble their parents because they inherit genes, segments of DNA that are the units of heredity, each at a specific locus on a chromosome. In asexual reproduction, one parent passes copies of all its genes to its offspring by mitosis, producing a clone. In sexual reproduction, two parents give rise to offspring with unique combinations of genes, which requires a special division, meiosis, that halves the chromosome number.

Homologous chromosomes, ploidy and the karyotype

A karyotype is an ordered display of the chromosomes of a cell, usually prepared from cells arrested in metaphase and arranged in pairs by size, centromere position and banding pattern. In a human karyotype, the two chromosomes of each pair are homologous chromosomes (homologs): they have the same length, centromere position and staining pattern, and they carry genes for the same characters at the same loci. One homolog of each pair came from the mother and one from the father. The homologs may carry different versions (alleles) of a gene; for example, one may carry an allele for brown eyes and the other an allele for blue.

The two exceptions are the sex chromosomes, X and Y, which determine sex in humans. Females have a homologous pair of X chromosomes; males have one X and one Y, which share only a small region. The other 22 pairs are autosomes.

A cell with two sets of chromosomes is diploid (2n). For humans, n = 23 and 2n = 46. A cell with a single set is haploid (n); human gametes are haploid, containing 22 autosomes plus one sex chromosome. When a haploid sperm fuses with a haploid egg in fertilization, the resulting zygote is diploid. Meiosis is what keeps the chromosome number constant from generation to generation; without it, the number would double every time gametes fused.

Three types of sexual life cycle

All sexual life cycles alternate between meiosis and fertilization, but they differ in when these events occur and which stage is multicellular.

• Animals (gametic meiosis): meiosis in the gonads produces gametes directly, which undergo no further division before fertilization. The only haploid cells are gametes; the multicellular organism is diploid.
• Plants and some algae (alternation of generations): there are both diploid and haploid multicellular stages. The diploid sporophyte produces haploid spores by meiosis. A spore divides by mitosis into a multicellular haploid gametophyte, which makes gametes by mitosis. Fertilization yields a diploid zygote that grows into the next sporophyte.
• Most fungi and many protists (zygotic meiosis): the zygote is the only diploid stage. It undergoes meiosis immediately, producing haploid cells that grow by mitosis into a haploid multicellular (or unicellular) organism, which makes gametes by mitosis.

Note that either haploid or diploid cells can divide by mitosis, depending on the life cycle, but only diploid cells can undergo meiosis, because meiosis requires homologous pairs.

The stages of meiosis

Meiosis is preceded by interphase in which each chromosome replicates into two sister chromatids. It then consists of two consecutive divisions, meiosis I and meiosis II, producing four daughter cells, each with half as many chromosomes as the parent and each genetically unique.

Meiosis I separates homologous chromosomes.

• Prophase I is the longest and most complex phase, occupying 90 percent or more of the time of meiosis. Chromosomes condense, and each homologous pair comes together gene by gene in a process called synapsis, held by a zipper-like protein structure, the synaptonemal complex. The paired homologs (four chromatids in all) are called a bivalent or tetrad. Crossing over occurs: nonsister chromatids exchange corresponding segments of DNA, and the points where they cross remain visible as X-shaped regions called chiasmata after the complex disassembles. In humans, each pair typically has one to three crossovers.
• Metaphase I: the tetrads line up at the metaphase plate, with one homolog facing each pole. Both chromatids of one homolog attach to microtubules from one pole; those of the other homolog attach to the opposite pole.
• Anaphase I: the cohesins along the chromatid arms are cleaved, letting homologs separate and move toward opposite poles. Cohesins at the centromeres are protected, so sister chromatids stay together.
• Telophase I and cytokinesis: each half of the cell has a haploid set of chromosomes, but each chromosome still consists of two (no longer identical, because of crossing over) sister chromatids. In many species no DNA replication occurs before meiosis II.

Meiosis II separates sister chromatids. It closely resembles mitosis: in prophase II a spindle forms; in metaphase II chromosomes line up individually at the plate; in anaphase II the centromere cohesins are finally cleaved and sister chromatids move to opposite poles; in telophase II and cytokinesis, four haploid daughter cells form.

Comparing mitosis and meiosis

Three events are unique to meiosis, and all three occur during meiosis I: synapsis and crossing over in prophase I; the alignment of homologous pairs (rather than individual chromosomes) at the metaphase plate; and the separation of homologs rather than sister chromatids in anaphase I. Because meiosis I reduces the number of chromosome sets from two to one, it is called the reductional division; meiosis II, which separates sister chromatids as mitosis does, is the equational division.

Sources of genetic variation

Mutations are the original source of genetic diversity, creating different alleles. Sexual reproduction then shuffles those alleles into new combinations in three ways.

• Independent assortment of chromosomes. At metaphase I, each homologous pair orients randomly, with the maternal or paternal homolog equally likely to face either pole, independently of every other pair. With n pairs, the number of possible combinations in the gametes is 2ⁿ. For humans, 2²³ is about 8.4 million.
• Crossing over. By exchanging segments between maternal and paternal chromatids, crossing over produces recombinant chromosomes that carry combinations of alleles found in neither parent's original chromosomes. Because crossovers can occur at many points, this multiplies the possible gametes far beyond 8.4 million.
• Random fertilization. Any sperm can fuse with any egg. Even without crossing over, one human couple could produce a zygote with any of about 70 trillion (8.4 million × 8.4 million) chromosome combinations.

This variation is the raw material for natural selection (Chapter 8).

When meiosis goes wrong: nondisjunction

Occasionally the members of a homologous pair fail to separate in meiosis I, or sister chromatids fail to separate in meiosis II. This error is nondisjunction. One resulting gamete receives two copies of a chromosome and another receives none. If such a gamete joins a normal one, the zygote has an abnormal chromosome number, a condition called aneuploidy: a trisomy (2n + 1, three copies of one chromosome) or a monosomy (2n − 1). Most aneuploid embryos do not survive. The most common viable autosomal trisomy is trisomy 21, which causes Down syndrome; its frequency rises with maternal age, partly because human eggs remain arrested in prophase I from before the mother's birth until ovulation, and the cohesins holding the paired homologs together may deteriorate over decades. Aneuploidies of the sex chromosomes, such as XXY (Klinefelter syndrome) and a single X (Turner syndrome), are generally less severe. Chapter 5 returns to these disorders.

Words worth knowing

Mendelian Genetics and the Chromosomal Basis of Inheritance

Why do two brown eyed parents sometimes have a blue eyed child, why are color blind men so much more common than color blind women, and why does a single extra chromosome change the course of a whole life? The answers come from a set of rules first worked out in a monastery garden with pea plants and later traced to the behavior of chromosomes during meiosis. This chapter teaches you those rules, shows you how to calculate the odds of any genetic outcome, and then follows the many ways real inheritance bends and breaks the simple patterns.

Section 1Mendel's experiments and the laws of segregation and independent assortment

The problem Mendel solved

Before the 1860s, most naturalists explained heredity with the blending hypothesis: the traits of two parents mix in their offspring the way blue and yellow paint mix to make green. Blending has an obvious flaw. If it were true, variation would shrink every generation until a population became uniform, and a trait that disappeared in one generation could never come back. Farmers and breeders knew better. A white flowered plant could appear among the offspring of purple flowered parents, and a calf could resemble its grandmother more than either parent.

Gregor Mendel, a monk trained in physics and mathematics, replaced blending with a particulate hypothesis. He proposed that parents pass on discrete heritable factors, which we now call genes, and that these factors keep their identity from generation to generation even when they are hidden. He reached this conclusion not by clever argument but by counting thousands of offspring and treating the counts mathematically, an approach that was unusual for biology at the time.

Why the garden pea was a good choice

Mendel worked with the garden pea, Pisum sativum, and his choice of organism mattered as much as his method.

• Peas come in many varieties with clear either or characters: a character is a heritable feature such as flower color, and a trait is one variant of it, such as purple or white.
• Peas normally self fertilize, because the pollen producing stamens and egg producing carpel are enclosed together in the same flower. This let Mendel start with true breeding lines, plants that produce only offspring like themselves when they self.
• He could also force cross pollination by cutting away the immature stamens of one plant and dusting its carpel with pollen from another. That gave him full control over which plants mated.

Mendel also made two smart experimental decisions. He followed characters that came in only two clearly different forms, and he tracked them for at least three generations. The true breeding parents are the P generation (parental). Their hybrid offspring are the F₁ generation (first filial), and the offspring produced when F₁ plants self or cross with one another are the F₂ generation.

The monohybrid cross

In a typical experiment, Mendel crossed a true breeding purple flowered plant with a true breeding white flowered plant. Every F₁ plant had purple flowers. The white trait had not blended into a pale lavender; it had simply vanished. When he let the F₁ plants self pollinate, white flowers reappeared in the F₂ in roughly one quarter of the plants: 705 purple to 224 white, close to 3:1. A cross between parents that differ in one character, followed into the F₂, is a monohybrid cross, and the F₁ plants are monohybrids (heterozygous for one gene).

Mendel saw the same pattern for all seven characters in the table above. He called the trait that appeared in the F₁ dominant and the trait that was hidden in the F₁ and reappeared in the F₂ recessive. To explain the 3:1 ratio, he built a model with four parts. Translated into modern terms:

1. Alternative versions of a gene explain variation in inherited characters. These versions are alleles. The gene for flower color has a purple allele and a white allele. Each gene sits at a specific place, its locus (plural loci), on a particular chromosome.
2. An organism inherits two copies of each gene, one from each parent. The two copies sit at the same locus on a pair of homologous chromosomes. They may be identical, as in true breeding plants, or different, as in the F₁ hybrids.
3. If the two alleles differ, the dominant allele determines appearance and the recessive allele has no noticeable effect. By convention, a capital letter stands for the dominant allele (P for purple) and the lowercase letter for the recessive allele (p for white).
4. The two alleles for a character separate (segregate) during gamete formation and end up in different gametes. An egg or a sperm carries only one allele for each character. This is the law of segregation.

Vocabulary that makes problems easier

An organism with two identical alleles for a gene, such as PP or pp, is homozygous for that gene. One with two different alleles, Pp, is heterozygous. The genotype is the genetic makeup (PP, Pp or pp); the phenotype is the observable appearance (purple or white), which also includes physiological and biochemical traits. Two organisms can share a phenotype but differ in genotype: PP and Pp plants are both purple. That is why the F₂ phenotype ratio is 3:1 while the genotype ratio is 1 PP : 2 Pp : 1 pp.

A Punnett square is a grid that lists the possible gametes of one parent along the top and those of the other along the side. Each box is one possible fertilization, and because each gamete type is equally likely, each box is equally likely.

The testcross

A purple flowered plant could be PP or Pp. To find out which, breed it with a homozygous recessive plant (pp). This is a testcross. If all offspring are purple, the mystery parent was almost certainly PP, because a Pp parent would have given about half white offspring. If any white offspring appear, the parent must be Pp. The testcross works because the recessive parent contributes only recessive alleles, so the offspring phenotypes directly reveal the gametes of the unknown parent.

The dihybrid cross and independent assortment

Mendel next asked whether the alleles of two different genes travel together or separately. He crossed true breeding plants with round yellow seeds (RRYY) and true breeding plants with wrinkled green seeds (rryy). The F₁ were all round and yellow, and they were dihybrids, RrYy. Two hypotheses predicted different F₂ results.

• If the alleles stayed together as they had arrived from each parent (dependent assortment), an F₁ plant would make only two kinds of gametes, RY and ry, and the F₂ would show a 3:1 ratio of round yellow to wrinkled green, with no new combinations.
• If the alleles of each gene sorted independently, an F₁ plant would make four kinds of gametes in equal numbers, RY, Ry, rY and ry, and a 4 × 4 Punnett square would give 16 equally likely combinations.

Mendel's F₂ counts were 315 round yellow, 108 round green, 101 wrinkled yellow and 32 wrinkled green, very close to 9:3:3:1. The new combinations (round green and wrinkled yellow) proved that the two genes had sorted independently. Notice that each character on its own still gave 3:1: round to wrinkled was 423:133, and yellow to green was 416:140. The 9:3:3:1 ratio is simply (3:1) × (3:1). This led to the law of independent assortment: each pair of alleles segregates independently of each other pair during gamete formation.

Independent assortment has an important limit. It holds for genes on different chromosomes and for genes so far apart on the same chromosome that crossing over separates them about half the time. Genes close together on one chromosome are linked and tend to be inherited together (Section 4). Mendel was fortunate: the seven characters he reported are controlled by genes that either sit on different chromosomes or are far enough apart to behave as if they were.

The physical basis: meiosis

Mendel had no idea what his factors were made of. Decades later, cytologists watching cells divide noticed that chromosomes behave exactly the way Mendel's factors must. The match is the heart of the chromosome theory of inheritance.

• Segregation happens because homologous chromosomes, one from each parent, separate in anaphase I of meiosis. Each daughter cell receives one homolog, and therefore one allele, for every locus. (If crossing over has swapped the alleles between sister chromatids, the final separation of alleles occurs at anaphase II.)
• Independent assortment happens because each pair of homologs lines up at the metaphase plate in metaphase I independently of every other pair. Whether the maternal or paternal homolog of chromosome 1 faces a given pole has no effect on which homolog of chromosome 2 faces that pole.

Section 2Probability and genetic problems

Why genetics is a probability science

Segregation and fertilization are random events, much like coin tosses. A heterozygote makes P and p gametes in equal numbers, but which gamete reaches a particular egg is a matter of chance. That is why Mendel's counts were close to 3:1 but never exactly 3:1, and why a family with four children can easily have no affected children even when each child faces a one in four risk. A probability is a number from 0 (the event never happens) to 1 (it always happens), and the probabilities of all possible outcomes of an event add up to 1. Each new child, like each new coin toss, is an independent event: earlier outcomes do not change the odds for the next one.

The multiplication rule

The multiplication rule states that the probability that two or more independent events all occur together is the product of their separate probabilities. The key words are "and" or "both." For an Rr × Rr cross, the chance that the egg carries r is 1/2 and the chance that the sperm carries r is 1/2, so the chance of an rr offspring is 1/2 × 1/2 = 1/4. The same rule tells you that the chance that two children in a row will both be rr is 1/4 × 1/4 = 1/16.

The addition rule

The addition rule states that the probability that any one of two or more mutually exclusive events will occur is the sum of their individual probabilities. The key word is "or." Mutually exclusive means the events cannot happen together: a single offspring cannot be both Rr made by an R egg and r sperm and Rr made by an r egg and R sperm. Each of those ways has probability 1/4, so the probability of a heterozygous offspring is 1/4 + 1/4 = 1/2.

Solving multigene problems without giant Punnett squares

A trihybrid cross would need a 64 box Punnett square. Instead, because independently assorting genes behave like separate monohybrid crosses, break the problem into one gene at a time, find the probability for each, and multiply.

Worked example. In the cross AaBbCcDd × AaBbccDd, what fraction of offspring will be AaBbccdd?

• Aa × Aa gives Aa with probability 1/2.
• Bb × Bb gives Bb with probability 1/2.
• Cc × cc gives cc with probability 1/2.
• Dd × Dd gives dd with probability 1/4.
• Multiply: 1/2 × 1/2 × 1/2 × 1/4 = 1/32.

Worked example with "or." In the cross PpYyRr × Ppyyrr, what fraction of offspring will show at least two recessive traits? List the genotypes that qualify and calculate each one with the multiplication rule, then add. The qualifying genotypes are ppyyRr, ppYyrr, Ppyyrr, PPyyrr and ppyyrr, which give 1/16 + 1/16 + 2/16 + 1/16 + 1/16 = 6/16, or 3/8. The pattern is always the same: multiply within a combination, add across combinations.

The binomial expression: "exactly k of n"

Families raise a different kind of question: in a family of three children, what is the chance that exactly two are affected by a recessive condition when both parents are carriers? The two affected children could be the first and second, first and third, or second and third. Each specific birth order has probability (1/4)(1/4)(3/4) = 3/64, and there are three orders, so the answer is 3 × 3/64 = 9/64. The general form is the binomial expression:

P(exactly k of n) = [n! / (k!(n − k)!)] × p^k × q^n−k

Here p is the probability of the event in one trial and q = 1 − p. The fraction with factorials counts the number of orders. For "at least one" questions, the fastest route is often to subtract from 1: the probability that at least one of four children is affected is 1 − (3/4)⁴ = 1 − 81/256 = 175/256.

Conditional probability: the two-thirds rule

Sometimes information you already have changes the odds. Suppose two carrier parents (Aa × Aa) have a son who does not have the recessive disorder. What is the chance he is a carrier? Without any information, the probabilities are 1/4 AA, 1/2 Aa and 1/4 aa. But we know he is not aa, so only the AA and Aa outcomes remain, in a ratio of 1:2. The chance he is a carrier is therefore 2/3, not 1/2. This conditional probability appears constantly in genetic counseling problems involving the unaffected siblings of affected people.

Pedigree analysis

Geneticists cannot arrange human matings, so they study families that already exist. A pedigree is a family tree that shows how a trait passes through generations. Squares are males and circles are females; a filled symbol is an affected person; a horizontal line joins mates; a vertical line drops to their children, who are listed left to right in birth order. Generations are numbered with Roman numerals and individuals with Arabic numerals, so II-3 is the third person in the second generation.

To identify the mode of inheritance, look for a few decisive clues rather than trying every possibility at once.

Testing whether data fit a ratio: the chi-square test

Mendel's 315:108:101:32 is close to 9:3:3:1, but how close is close enough? The chi-square (χ²) goodness of fit test asks whether the difference between observed (O) and expected (E) counts is small enough to be explained by chance alone. The null hypothesis is that the data fit the predicted ratio and any deviation is random.

1. Calculate expected counts from the total and the predicted ratio. For 556 seeds and 9:3:3:1: 312.75, 104.25, 104.25 and 34.75.
2. For each class, compute (O − E)²/E and add them: 0.016 + 0.135 + 0.101 + 0.218 = χ² = 0.47.
3. Find the degrees of freedom: the number of classes minus 1, here 4 − 1 = 3.
4. Compare with the critical value at p = 0.05. If χ² is smaller than the critical value, you fail to reject the null hypothesis; the deviation is consistent with chance.

Mendel's value of 0.47 is far below 7.81, so the data fit independent assortment well. A large χ² does not prove the model wrong, but it signals that something other than chance, such as linkage or reduced survival of one genotype, is probably at work.

Section 3Extensions: dominance, multiple alleles, epistasis, polygenic traits

Mendel's rules still hold; the relationships get richer

Mendel chose characters in which one allele was completely dominant and each character was controlled by one gene with two alleles. Most traits are not that tidy. The patterns in this section can look as if they break Mendel's laws, but they do not. Alleles still segregate and genes still assort independently. What changes is the relationship between genotype and phenotype: how two alleles of the same gene interact, how many alleles exist in a population, how genes interact with one another and how the environment shapes the outcome.

Degrees of dominance

With complete dominance, the heterozygote and the dominant homozygote look the same. With incomplete dominance, the heterozygote has a phenotype intermediate between the two homozygotes. Crossing red snapdragons (C^RC^R) with white snapdragons (C^WC^W) gives all pink F₁ flowers. This might look like blending, but the F₂ settles the question: it contains red, pink and white in a 1:2:1 ratio, so the red and white alleles came through intact. In incomplete dominance the phenotype ratio equals the genotype ratio because every genotype has its own look. The molecular reason is usually dosage: one functional allele makes only enough red pigment to tint the petals pink.

With codominance, the heterozygote fully and separately expresses both alleles. The human MN blood group is the classic case. The M and N alleles specify slightly different forms of a glycoprotein on red blood cells. People who are MN have both forms on their cells, not an intermediate molecule. The AB blood type, discussed below, is another example.

Dominance can also depend on the level at which you look. In Tay-Sachs disease, children with two defective alleles cannot break down a membrane lipid (GM2 ganglioside) in brain cells and die in early childhood. At the level of the whole organism, the allele is recessive, because heterozygotes are healthy. At the biochemical level, heterozygotes have about half the normal enzyme activity, which looks like incomplete dominance. At the molecular level, both normal and defective enzyme molecules are made, which is codominance. Dominance describes phenotype, so it always depends on which phenotype you measure.

Multiple alleles

Any one diploid individual carries at most two alleles of a gene, but a population can carry many. The ABO blood group has three common alleles: I^A, I^B and i. I^A encodes an enzyme that adds the sugar N-acetylgalactosamine to a carbohydrate on the red cell surface, forming the A antigen; I^B encodes an enzyme that adds galactose, forming the B antigen; i encodes no functional enzyme. I^A and I^B are codominant with each other, and both are completely dominant over i. Six genotypes give four phenotypes.

Rabbit coat color shows a dominance series of four alleles: full color (C) > chinchilla (c^ch) > Himalayan (c^h) > albino (c). The Himalayan allele produces a temperature sensitive version of tyrosinase, the enzyme that starts melanin synthesis. The enzyme works only below body temperature, so pigment forms on the cool ears, nose, feet and tail. The same allele explains the color points of Siamese cats.

Pleiotropy and lethal alleles

Many genes affect more than one phenotypic character, a property called pleiotropy. The sickle cell allele changes one amino acid in the β chain of hemoglobin. In people homozygous for it, red cells sickle when oxygen is low, which leads to anemia, pain crises, organ damage, stroke risk and a damaged spleen. One base change, many effects. Marfan syndrome, a dominant disorder of the connective tissue protein fibrillin-1, affects the skeleton (long limbs), the eyes (dislocated lenses) and the aorta (dilation and dissection).

Some alleles are lethal when homozygous. In mice, the dominant yellow coat allele A^Y gives yellow fur in heterozygotes, but A^YA^Y embryos die. A cross of two yellow mice therefore gives yellow and agouti (wild type) offspring in a 2:1 ratio rather than 3:1, because the expected quarter of A^YA^Y offspring never appears. The A^Y allele is dominant for coat color but recessive for lethality, a reminder that dominance is defined per trait.

Epistasis: one gene masks another

In epistasis (Greek for "standing upon"), the phenotype produced by one gene depends on the genotype at a second gene. Labrador retrievers make a clear example. Gene B controls the type of pigment: B (dominant) makes black eumelanin and b makes brown. A second gene, E, controls whether pigment is deposited in the hair at all. Dogs with at least one E allele deposit pigment; ee dogs deposit almost none in the hair and are yellow whatever their B genotype. Gene E is said to be epistatic to gene B.

Cross two dogs that are both BbEe. The 16 combinations still form, in the familiar 9:3:3:1 genotype classes: 9 B_E_, 3 bbE_, 3 B_ee, 1 bbee. But the last two classes look the same, so the phenotype ratio becomes 9 black : 3 chocolate : 4 yellow. When a dihybrid cross gives a ratio whose numbers add to 16 but that is not 9:3:3:1, suspect two genes interacting.

The 9:7 ratio is worth understanding in terms of a pathway. Suppose a white precursor is converted to an intermediate by enzyme C and the intermediate to purple pigment by enzyme P. A plant needs at least one working allele of both genes (C_P_, 9/16) to make purple. Any plant that is cc, pp or both (3 + 3 + 1 = 7/16) is white. Crossing two different white strains (CCpp × ccPP) can therefore give all purple F₁ plants, a complementation result that tells geneticists the two white mutations are in different genes.

Polygenic inheritance and quantitative traits

Mendel's characters came in discrete either or forms. Height, skin color, blood pressure, birth weight and the number of grains on a wheat head vary continuously along a scale. These quantitative characters usually reflect polygenic inheritance, the additive effect of two or more genes on one phenotype.

A simplified model of skin pigmentation imagines three genes, each with a "dark" allele (A, B, C) that adds one unit of darkness and a "light" allele (a, b, c) that adds none. A person who is AaBbCc has three dark units. If two such people have children, offspring can carry anywhere from 0 to 6 dark alleles, giving seven phenotypic classes in the proportions 1:6:15:20:15:6:1 out of 64. Most children fall near the middle and few at the extremes. Add more genes and environmental effects such as sun exposure, and the steps blur into a smooth bell shaped curve.

Nature and nurture

Genotype rarely fixes a phenotype exactly. The range of phenotypes one genotype can produce across environments is its norm of reaction. Hydrangeas of the same genotype bloom blue in acidic soil and pink in neutral or basic soil, because soil pH controls how much aluminum the plant absorbs. Human height depends on nutrition as well as genes. Traits shaped by many genes plus the environment, such as heart disease, type 2 diabetes, many cancers and some mental illnesses, are called multifactorial.

Two more terms describe how consistently a genotype shows itself. Penetrance is the proportion of individuals with a genotype who show the expected phenotype at all; if only 80 percent of people with a dominant polydactyly allele have extra digits, penetrance is 80 percent (incomplete penetrance). Expressivity is the degree to which a phenotype is expressed in those who show it; in neurofibromatosis type 1, one family member may have a few pale brown skin spots while another has many tumors along nerves (variable expressivity).

Section 4Sex linkage, linked genes and recombination

The chromosome theory and Morgan's white eyed fly

Around 1902, Walter Sutton and Theodor Boveri independently noted the parallels between Mendel's factors and chromosomes: both come in pairs, both separate during gamete formation and both assort independently. The chromosome theory of inheritance proposed that genes occupy specific loci on chromosomes and that chromosome behavior in meiosis explains Mendel's laws. The first solid evidence tying a specific gene to a specific chromosome came from Thomas Hunt Morgan's work with the fruit fly Drosophila melanogaster, an ideal organism that breeds every two weeks, produces hundreds of offspring and has only four pairs of chromosomes.

Wild type flies have red eyes. (The wild type is the phenotype most common in natural populations; alleles for other phenotypes are mutant alleles.) Morgan found a male with white eyes and crossed it with a red eyed female. All F₁ flies had red eyes, so red was dominant. In the F₂, the familiar 3:1 ratio appeared, but with a twist: every white eyed fly was male. Half the F₂ males had white eyes and none of the females did. Morgan concluded that the eye color gene lies on the X chromosome and that the Y chromosome carries no allele for it. A male, with one X, shows whatever allele that X carries.

Sex determination

In humans and other mammals, females are XX and males are XY. The Y chromosome is small and carries relatively few genes, but one of them, SRY (sex determining region of Y), triggers development of testes in the embryo; without it, the gonads develop as ovaries. Other organisms use other systems.

Inheritance of X linked genes

Genes on a sex chromosome are sex linked; in humans, the term usually means X linked, because the X carries about 1,000 genes and most have nothing to do with sex. A male has only one copy of each X linked gene and is called hemizygous. That single fact explains the pattern of X linked recessive disorders.

• A male who inherits one recessive X linked allele shows the trait, because no second allele can mask it. A female must inherit two copies, which is much rarer. If an allele has frequency 1/10 in a population, about 1 in 10 males but only 1 in 100 females will be affected.
• A father passes his X to all his daughters and his Y to all his sons, so an affected father never passes an X linked trait to a son, but all his daughters become carriers.
• A carrier mother (X^AX^a) passes the allele to half her children: half of her sons will be affected and half of her daughters will be carriers.

Important X linked recessive disorders include red-green color blindness (about 8 percent of males of northern European ancestry), hemophilia A (lack of clotting factor VIII), Duchenne muscular dystrophy (absence of the muscle protein dystrophin, causing progressive weakness from early childhood) and G6PD deficiency (red cells that break down under oxidative stress).

X inactivation

Females have two X chromosomes, yet they do not make twice as much of each X linked protein as males. Early in embryonic development of a female mammal, one X in each cell condenses into a compact Barr body that lies along the inside of the nuclear envelope and is mostly silent. The choice of which X to inactivate is random and independent in each embryonic cell, but once made, all descendants of that cell keep the same inactive X. A female is therefore a mosaic of two cell populations. Inactivation depends on the XIST gene, which is expressed only from the chromosome that will become the Barr body; its RNA coats that X and recruits proteins that modify chromatin and shut it down.

The tortoiseshell or calico cat shows mosaicism on the outside. An X linked gene has an orange allele and a black allele. A heterozygous female has patches of orange fur where the black X is inactive and patches of black where the orange X is inactive (white patches come from a separate gene). Male cats with these colors are almost always XXY. In humans, a carrier female for an X linked condition can show mild symptoms if, by chance, most cells in a relevant tissue inactivated the normal X.

Linked genes

Each chromosome carries hundreds or thousands of genes. Genes on the same chromosome tend to be inherited together and are called linked genes. (Do not confuse linked genes, which share any chromosome, with sex linked genes, which are on a sex chromosome.) Morgan studied two Drosophila genes: body color (b⁺ gray, b black) and wing size (vg⁺ normal, vg vestigial). He crossed F₁ dihybrid females (b⁺b vg⁺vg) with tester males that were black and vestigial (bb vgvg). If the genes assorted independently, the four phenotype classes would appear 1:1:1:1. Instead he got:

• 965 gray, normal wings and 944 black, vestigial wings: the parental types, matching the combinations in the P generation.
• 206 gray, vestigial and 185 black, normal: the recombinant types, new combinations.

The excess of parental types meant the genes were on the same chromosome. But the recombinants meant the linkage was incomplete. Morgan proposed that crossing over between non-sister chromatids during prophase I of meiosis occasionally exchanges segments, creating chromosomes with new allele combinations. The recombination frequency is the percentage of offspring that are recombinant: (206 + 185) / 2,300 = 17 percent.

Genetic maps

Morgan's student Alfred Sturtevant reasoned that the farther apart two genes are on a chromosome, the more places a crossover can occur between them, and the higher their recombination frequency. He defined one map unit (now also called a centimorgan, cM) as a 1 percent recombination frequency and used the values to build a linkage map, a map of the relative order and spacing of genes. For example, the b to cn (cinnabar eye) distance is 9 map units, cn to vg is 9.5, and b to vg is 17. The order must be b, cn, vg, with cn in the middle. The b to vg value (17) is slightly less than the sum (18.5) because some chromatids have two crossovers between b and vg, which restore the parental combination and go uncounted.

Two rules keep map problems straight. First, recombination frequency can never exceed 50 percent. Genes far apart on the same chromosome cross over so often that they behave like unlinked genes and show a 1:1:1:1 testcross ratio; they are still physically linked but genetically unlinked. Second, in a three point cross, the rarest phenotype classes are the double crossovers; comparing them with the parental classes reveals which gene is in the middle. Map units measure recombination, not physical length, so they are only roughly proportional to base pairs, because crossing over is more frequent in some regions than others.

Section 5Chromosomal alterations and human disorders

Nondisjunction and abnormal chromosome number

Meiosis normally delivers exactly one copy of each chromosome to each gamete. Occasionally the members of a homologous pair fail to separate in meiosis I, or sister chromatids fail to separate in meiosis II. This is nondisjunction. One gamete gets an extra copy of a chromosome and another gets none. If an abnormal gamete is fertilized by a normal one, the zygote has an abnormal number of that chromosome, a condition called aneuploidy. A zygote with three copies of a chromosome (2n + 1) is trisomic; one with only one copy (2n − 1) is monosomic. Nondisjunction during mitosis early in development produces a mosaic, with a mix of normal and aneuploid cells.

Most aneuploid embryos do not survive; aneuploidy is a major cause of first trimester miscarriage. The few that survive to birth involve the smallest autosomes or the sex chromosomes. Sex chromosome aneuploidies are tolerated better than autosomal ones because X inactivation silences extra X chromosomes and the Y carries few genes.

About 95 percent of Down syndrome cases result from nondisjunction, most often in the mother's meiosis I. Human oocytes begin meiosis before birth and pause in prophase I for decades, which may let the proteins holding homologs together weaken; that may explain why risk rises from roughly 1 in 1,500 births at age 20 to about 1 in 100 at age 40. Roughly 4 percent of cases come from a translocation and about 1 percent are mosaics.

Polyploidy

Sometimes an entire set of chromosomes is added, producing polyploidy: triploid (3n), tetraploid (4n) and so on. Polyploidy is rare and lethal in animals with chromosomal sex determination but is common and important in plants. Bread wheat is hexaploid (6n), descended from three ancestral species. Triploid organisms are usually sterile because three sets cannot pair evenly in meiosis; growers exploit this to produce seedless bananas and seedless watermelons.

Changes in chromosome structure

Breakage of a chromosome can produce four kinds of structural change.

• A deletion removes a segment. Deletion of part of the short arm of chromosome 5 causes cri du chat syndrome ("cry of the cat"), with intellectual disability and a high pitched, cat like cry in infancy.
• A duplication repeats a segment, often after unequal crossing over. Duplications are an important raw material of evolution because the extra copy can acquire a new function.
• An inversion reverses a segment within a chromosome. A pericentric inversion includes the centromere; a paracentric inversion does not. Carriers may be healthy but can produce unbalanced gametes when crossing over occurs within the inverted loop.
• A translocation moves a segment to a nonhomologous chromosome. In a reciprocal translocation two nonhomologous chromosomes exchange pieces. In a Robertsonian translocation the long arms of two acrocentric chromosomes (such as 14 and 21) fuse. A carrier of a balanced 14/21 translocation is healthy but can pass on an extra chromosome 21 arm, causing familial Down syndrome.

Exceptions to Mendelian inheritance

In genomic imprinting, certain genes are expressed differently depending on whether they were inherited from the mother or the father. Imprints are set during gamete formation by methylation of DNA and are reset each generation. A deletion in region 15q11 to q13 shows the effect vividly: if the deletion is on the chromosome inherited from the father, the child has Prader-Willi syndrome (poor muscle tone in infancy, then excessive eating and obesity, intellectual disability); if it is on the chromosome from the mother, the child has Angelman syndrome (severe intellectual disability, seizures, jerky movements and frequent laughter). The same deletion, two diseases, depending only on which parent it came from.

Genes in mitochondria and plastids are inherited outside the nucleus. Because the egg supplies almost all the cytoplasm of the zygote, mitochondrial genes pass from a mother to all her children and are not transmitted by fathers. Mitochondrial DNA mutations often affect tissues with high energy needs, such as the eye, brain and muscle (for example, Leber hereditary optic neuropathy). A cell can contain a mixture of normal and mutant mitochondria (heteroplasmy), so severity varies among siblings.

Words worth knowing

DNA Structure, Replication and Gene Expression

Every cell in your body carries about two meters of DNA folded into a nucleus a few micrometers wide, and nearly every time a cell divides it copies all three billion base pairs with fewer than one mistake per billion. That same molecule is read thousands of times a day to make the proteins that build and run you. This chapter explains how scientists proved DNA is the genetic material, how its structure makes copying possible, how genes are transcribed and translated into proteins, and what happens when the sequence changes.

Section 1The search for the genetic material and DNA structure

Protein or DNA?

By the 1930s, geneticists knew that genes sit on chromosomes and that chromosomes are made of two kinds of molecule: protein and DNA. Most scientists bet on protein. Proteins are built from 20 different amino acids and come in endless shapes, so they seemed rich enough to carry complex information. DNA, with only four kinds of nucleotides, looked too simple, perhaps just a structural scaffold. A series of experiments, mostly with bacteria and viruses, overturned that view.

Griffith and transformation

In 1928, Frederick Griffith was studying two strains of the bacterium Streptococcus pneumoniae in mice. The S strain made a smooth polysaccharide capsule that protected it from the immune system and was lethal. The R strain lacked a capsule, formed rough colonies and was harmless. Heat killed S cells were also harmless. But when Griffith injected a mixture of heat killed S cells and living R cells, the mice died, and he recovered living S bacteria from their blood. Something from the dead S cells had permanently changed the R cells into S cells, and the change was inherited by their descendants. Griffith called this transformation: a change in genotype and phenotype caused by uptake of external DNA, although he did not yet know DNA was the agent.

Avery, MacLeod and McCarty

In 1944, Oswald Avery, Colin MacLeod and Maclyn McCarty purified the transforming principle from S cell extracts. They treated samples with enzymes that destroy specific molecules. Protein digesting enzymes and RNA digesting enzymes left transformation intact, but DNase, which destroys DNA, abolished it. Their conclusion that the transforming substance was DNA was strong, but many biologists remained skeptical, partly because they suspected tiny amounts of contaminating protein and partly because bacteria were not yet thought to have genes like those of complex organisms.

Hershey and Chase

The decisive experiment came in 1952 from Alfred Hershey and Martha Chase, using bacteriophage T2, a virus that infects Escherichia coli. A phage is little more than a protein coat around DNA, and when it infects a cell it reprograms the cell to make new phages. The question was which part enters the cell and carries the instructions. Hershey and Chase exploited a chemical difference: proteins contain sulfur (in methionine and cysteine) but almost no phosphorus, while DNA contains phosphorus (in its backbone) but no sulfur.

1. One batch of phages was grown with radioactive sulfur (³⁵S), labeling their protein. Another batch was grown with radioactive phosphorus (³²P), labeling their DNA.
2. Each batch infected separate cultures of E. coli. After a few minutes, the cultures were agitated in a blender to shake loose any phage parts outside the cells.
3. Centrifugation spun the heavier bacterial cells into a pellet and left the lighter phage coats in the liquid (supernatant).
4. In the ³⁵S batch, most radioactivity stayed in the liquid. In the ³²P batch, most radioactivity was in the pellet, inside the cells, and some appeared in the next generation of phages.

The phage DNA, not its protein, had entered the cells and directed the production of new viruses. DNA was the genetic material.

Chargaff's rules

Meanwhile, Erwin Chargaff analyzed the base composition of DNA from many species. He found two patterns, now called Chargaff's rules. First, base composition varies from one species to another, which is exactly the kind of variety a genetic material needs. Second, within any species the amount of adenine equals thymine and the amount of guanine equals cytosine. In human DNA, for example, A and T are each about 30 percent and G and C are each about 20 percent. Nobody could explain the second rule until the double helix was discovered.

Building the double helix

A DNA nucleotide has three parts: a five carbon sugar called deoxyribose, a phosphate group and a nitrogenous base. The bases come in two families. The purines, adenine (A) and guanine (G), have a double ring. The pyrimidines, cytosine (C) and thymine (T), have a single ring (in RNA, uracil replaces thymine). Carbons of the sugar are numbered with primes: the base attaches to the 1' carbon, and the phosphate attaches to the 5' carbon.

Nucleotides join through phosphodiester bonds: the phosphate on the 5' carbon of one nucleotide links to the hydroxyl on the 3' carbon of the next. The result is a sugar-phosphate backbone with bases sticking out to the side. A strand has direction. One end has a free phosphate on a 5' carbon (the 5' end) and the other a free hydroxyl on a 3' carbon (the 3' end). Sequences are written 5' to 3' by convention.

Rosalind Franklin, working with Maurice Wilkins, produced X-ray diffraction images of DNA fibers. Her best image showed an X pattern that indicated a helix, and her measurements showed that the helix had a width of about 2 nm and repeating features at 0.34 nm and 3.4 nm. She also concluded that the phosphates lay on the outside. In 1953, James Watson and Francis Crick used her data, Chargaff's rules and model building to propose the structure.

• DNA is a double helix: two strands wound around each other, usually right handed (the B form).
• The sugar-phosphate backbones are on the outside and the bases point inward, stacked like the rungs of a twisted ladder.
• The two strands are antiparallel: one runs 5' to 3' and its partner runs 3' to 5'.
• Bases pair by hydrogen bonds: A with T (two hydrogen bonds) and G with C (three hydrogen bonds). Each pair is one purine plus one pyrimidine, which keeps the width a constant 2 nm and explains Chargaff's equalities.
• Adjacent base pairs are 0.34 nm apart and one full turn contains about 10 base pairs (10.5 in solution) in 3.4 nm.
• The winding creates a major groove and a minor groove, where proteins can read the edges of the bases without unwinding the helix.

Watson and Crick noted that the pairing rules immediately suggest how DNA could be copied: each strand carries the information to rebuild its partner. Because the strands are complementary, knowing one sequence gives you the other: the partner of 5'-ATGCCA-3' is 3'-TACGGT-5', written by convention as 5'-TGGCAT-3'.

Packing DNA into chromosomes

A bacterium such as E. coli has one circular chromosome of about 4.6 million base pairs, which is supercoiled and packed with proteins into a region called the nucleoid. Many bacteria also carry small circular plasmids. A human diploid cell has 46 linear chromosomes totaling about 6 billion base pairs (3.2 billion per haploid set), which would stretch about 2 meters if laid end to end. Eukaryotic DNA is packed in several levels with proteins, together called chromatin.

During interphase, loosely packed euchromatin is accessible and can be transcribed, while densely packed heterochromatin (for example at centromeres, telomeres and the Barr body) is mostly silent. Cells change packing by chemically modifying histones, a topic in Chapter 7.

Section 2DNA replication and repair

Semiconservative replication

Watson and Crick predicted that each strand of the double helix serves as a template for a new partner strand. Under this semiconservative model, each daughter molecule would contain one old strand and one new strand. Two alternatives were possible: a conservative model, in which the parent helix stays intact and a completely new copy forms, and a dispersive model, in which every strand is a patchwork of old and new segments.

In 1958, Matthew Meselson and Franklin Stahl settled the question. They grew E. coli for many generations in a medium containing heavy nitrogen (¹⁵N), so all their DNA was dense. They then moved the cells to a medium with ordinary light nitrogen (¹⁴N) and extracted DNA after each round of replication. Density gradient centrifugation separated DNA by density. After one round, all the DNA formed a single band of intermediate (hybrid) density, which ruled out the conservative model. After two rounds, there were two bands, one hybrid and one light, which ruled out the dispersive model. Only the semiconservative model fit both results.

Getting started: origins and forks

Replication begins at specific sequences called origins of replication. The bacterial chromosome has a single origin, and replication proceeds in both directions until the two forks meet on the opposite side of the circle. Eukaryotic chromosomes are far longer and replication forks move more slowly (about 50 nucleotides per second in humans, compared with about 500 to 1,000 in bacteria), so each chromosome uses hundreds to thousands of origins. A human cell copies its 6 billion base pairs in a few hours this way. At each origin, proteins open a replication bubble, and at each end of the bubble is a Y shaped replication fork, where parental strands are unwound and new strands are made.

The proteins at the fork

Why replication is lopsided: leading and lagging strands

Every DNA polymerase has two firm limits. It cannot start a strand from scratch; it can only add nucleotides to an existing 3' hydroxyl, which is why a primer is needed. And it can add nucleotides only to the 3' end, so new DNA always grows in the 5' to 3' direction. The energy for each addition comes from the incoming nucleoside triphosphate (such as dATP), which loses two phosphates as pyrophosphate when it joins the chain; hydrolysis of the pyrophosphate makes the reaction strongly favorable.

Because the two parental strands are antiparallel, these rules force the two new strands to be made differently. On one template, polymerase can follow the fork as it opens, adding nucleotides continuously after a single primer. This new strand is the leading strand. On the other template, synthesis in the 5' to 3' direction means moving away from the fork. The polymerase must make the strand in a series of short pieces, each started from its own primer as the fork exposes more template. This is the lagging strand, and its pieces are Okazaki fragments, about 1,000 to 2,000 nucleotides long in bacteria and 100 to 200 in eukaryotes. DNA polymerase I then replaces each RNA primer with DNA, and DNA ligase joins the fragments into a continuous strand.

Eukaryotic differences

Eukaryotes use the same logic with different enzymes. A complex of DNA polymerase α and primase starts each strand or fragment; DNA polymerase ε extends the leading strand, and DNA polymerase δ extends the lagging strand. Primers are removed by an RNase and a flap endonuclease rather than by a polymerase I, and the gaps are filled and sealed in the same way. Behind the fork, histones are rapidly reloaded so the new DNA is packed into nucleosomes.

Proofreading: how replication stays accurate

Base pairing alone would allow about one error in every 10⁴ to 10⁵ nucleotides. Replicative DNA polymerases have a 3' to 5' exonuclease activity that lets them back up, remove a mismatched nucleotide they have just added and try again. Proofreading improves accuracy about a hundredfold. After replication, mismatch repair enzymes scan the new strand for remaining errors, recognize which strand is new (in E. coli because it is not yet methylated) and replace the wrong nucleotide. Together these steps bring the final error rate to about one in 10⁹ to 10¹⁰ nucleotides, roughly one mistake per human cell division.

The end replication problem and telomerase

Linear chromosomes have a problem at their ends. When the last RNA primer on a lagging strand is removed, there is no upstream 3' end from which polymerase can fill the gap. Each round of replication therefore leaves the daughter chromosome slightly shorter. Eukaryotic chromosome ends are capped by telomeres, repeated noncoding sequences (TTAGGG repeated hundreds to thousands of times in humans) bound by protective proteins. Telomeres act as buffers, so losing some sequence costs no genes, and they prevent cells from mistaking chromosome ends for broken DNA.

Telomeres shorten with each division in most somatic cells, which contributes to a limit of roughly 50 divisions for many human cells in culture. In germ cells and many stem cells, the enzyme telomerase restores telomere length. Telomerase is a reverse transcriptase that carries its own RNA template; it extends the 3' overhang of the chromosome with new repeats, after which primase and polymerase fill in the complementary strand.

DNA damage and repair

Beyond replication errors, DNA is constantly damaged. Each human cell suffers thousands of spontaneous lesions every day: bases lose amino groups (cytosine deaminates to uracil), purines fall off the backbone, reactive oxygen species oxidize bases, ultraviolet light fuses adjacent pyrimidines into thymine dimers, and ionizing radiation breaks both strands. Because the information is stored twice, once on each strand, most damage can be repaired by cutting out the damaged section and using the undamaged strand as a template. Cells have well over a hundred repair enzymes.

Section 3Transcription and RNA processing

From genes to proteins

How does a DNA sequence produce a trait? Early clues came from inherited metabolic diseases and from George Beadle and Edward Tatum, who studied mutant strains of the bread mold Neurospora that could not make particular nutrients. Each mutant lacked one enzyme in a biosynthetic pathway, which led to the one gene, one enzyme hypothesis. It was later refined to one gene, one polypeptide, since many proteins are not enzymes and many proteins have several polypeptide chains, and refined again once it became clear that some genes make functional RNAs that are never translated, and that one gene can give several polypeptides through alternative splicing.

The flow of genetic information is summarized by the central dogma: DNA is copied to DNA (replication), DNA is transcribed into RNA, and RNA is translated into protein. Retroviruses such as HIV add a reverse step, using reverse transcriptase to copy RNA into DNA. RNA is the intermediate because it lets a cell make many working copies of a gene without exposing the master copy, and because it allows each gene's output to be regulated separately.

Template strand and coding strand

For any given gene, only one strand of DNA is read. This is the template strand (also called the noncoding or antisense strand). The RNA is complementary to it and antiparallel to it, and is built 5' to 3' as the polymerase reads the template 3' to 5'. The other DNA strand, the coding strand (sense strand), therefore has the same sequence as the RNA, except with T in place of U. When geneticists publish a gene sequence, they write the coding strand, so you can read codons directly from it. Different genes on the same chromosome may use different strands as templates.

RNA polymerase and the stages of transcription

RNA polymerase pries the two DNA strands apart and links ribonucleotides in the 5' to 3' direction. Unlike DNA polymerase, it needs no primer, and it proofreads much less, so transcription makes more errors than replication; this is tolerable because each RNA is temporary and made in many copies. Transcription has three stages.

1. Initiation. RNA polymerase binds a promoter, a DNA sequence just upstream of the gene that marks where to start and which strand to read. The first nucleotide transcribed is called +1; positions before it are given negative numbers.
2. Elongation. The polymerase unwinds about 10 to 20 base pairs at a time, forming a transcription bubble, and adds about 40 nucleotides per second in eukaryotes. Behind it, the DNA rewinds and the new RNA peels away. Several polymerases can transcribe one gene at once, one behind another.
3. Termination. The polymerase stops and releases the RNA.

Bacteria compared with eukaryotes

Eukaryotic RNA processing

In eukaryotes, the initial transcript from a protein coding gene is a pre-mRNA (primary transcript). Before it leaves the nucleus, enzymes modify both ends and remove interior segments.

• 5' cap. Soon after transcription starts, a modified guanine nucleotide (7-methylguanosine) is attached to the 5' end through an unusual 5' to 5' triphosphate linkage.
• Poly-A tail. After the transcript is cleaved downstream of the polyadenylation signal, an enzyme adds 50 to 250 adenine nucleotides to the 3' end.

The cap and tail help export the mRNA from the nucleus, protect it from degradation by exonucleases, and help ribosomes attach to the 5' end in the cytoplasm. The mature mRNA also keeps untranslated regions (5' UTR and 3' UTR) at each end that are not translated but influence stability and translation.

Splicing

Most eukaryotic genes are interrupted. The segments that are expressed in the final mRNA are exons; the intervening sequences removed from the transcript are introns. A typical human gene has about eight introns, and introns make up a large share of the length of most genes. RNA splicing removes the introns and joins the exons. It is carried out by the spliceosome, a large complex of proteins and small nuclear ribonucleoproteins (snRNPs), each containing small nuclear RNA. The snRNAs base pair with conserved sequences at the intron boundaries, almost always GU at the 5' end of the intron and AG at the 3' end, and with a branch point adenine inside the intron. The intron is released as a looped structure called a lariat, and the exons are joined. The snRNAs themselves catalyze key steps, which makes the spliceosome a ribozyme, an RNA molecule that acts as an enzyme. Some introns even splice themselves out without any proteins.

Why genes are in pieces

Introns may seem wasteful, but split genes provide two major advantages. First, alternative RNA splicing lets one gene produce different mRNAs, and therefore different polypeptides, by including or skipping particular exons. More than 90 percent of human multi-exon genes are alternatively spliced, which helps explain how roughly 20,000 protein coding genes yield a much larger number of proteins. Second, exons often encode separate structural and functional regions of a protein called domains. Introns make it easier for crossing over to shuffle exons between genes (exon shuffling), which can create new proteins during evolution.

Section 4The genetic code and translation

Cracking the code

Four nucleotide bases must specify 20 amino acids. A code of one base per amino acid gives only 4 words, and two bases give only 16 (4²). Three bases give 4³ = 64 combinations, more than enough. Experiments confirmed that the code is read in nonoverlapping triplets called codons. In 1961, Marshall Nirenberg and Heinrich Matthaei added an artificial mRNA made only of uracil (poly-U) to a cell free protein synthesis system and obtained a polypeptide made only of phenylalanine, so UUU must code for phenylalanine. By the mid 1960s, all 64 codons had been deciphered.

The code has a handful of properties worth knowing precisely.

• Triplet and nonoverlapping: each codon is three bases, and successive codons do not share bases.
• Start and stop: AUG codes for methionine and also serves as the usual start codon. UAA, UAG and UGA are stop codons that code for no amino acid. That leaves 61 sense codons for 20 amino acids.
• Redundant (degenerate) but not ambiguous: most amino acids have more than one codon (leucine, serine and arginine have six each), but each codon specifies only one amino acid. Synonymous codons usually differ in the third base.
• Read in a fixed reading frame: the ribosome reads codons in groups of three from the start codon, so shifting the frame by one base changes every downstream codon.
• Nearly universal: the same code operates in bacteria, plants and animals, with minor variations in mitochondria and a few microorganisms. This is why a human gene inserted into bacteria can be expressed to make human insulin, and it is strong evidence that all life shares a common ancestor.

The players in translation

Transfer RNA (tRNA) molecules are the adapters that link codons to amino acids. Each tRNA is a single strand of about 80 nucleotides that folds, through internal base pairing, into a cloverleaf in two dimensions and an L shape in three. At one end of the L is the anticodon, a triplet that base pairs with a complementary codon in the mRNA; at the other end, the 3' end ending in CCA, is the attachment site for the amino acid. A tRNA with anticodon 3'-AAG-5' pairs with the mRNA codon 5'-UUC-3' and carries phenylalanine.

Each amino acid is joined to its correct tRNAs by a specific aminoacyl-tRNA synthetase. There are 20 of these enzymes, one per amino acid, and they spend ATP to form the bond; the resulting aminoacyl-tRNA (a "charged" tRNA) carries the energy that later forms a peptide bond. The synthetases are the real translators of the code, because they match the language of nucleic acids to the language of proteins, and they proofread their own work.

Cells have only about 45 kinds of tRNA, not 61, because of wobble: the pairing rules between the third base of a codon and the first base (5' end) of the anticodon are relaxed. For example, a tRNA whose anticodon has the modified base inosine (I) at the wobble position can pair with codons ending in U, C or A.

Ribosomes bring mRNA and charged tRNAs together and catalyze peptide bond formation. Each ribosome has a large and a small subunit, made of ribosomal RNA (rRNA) and proteins. Bacterial ribosomes are 70S (50S plus 30S subunits) and eukaryotic cytoplasmic ribosomes are 80S (60S plus 40S); S units measure sedimentation rate, so they do not add. A ribosome has an mRNA binding site and three tRNA binding sites.

• The A site (aminoacyl) holds the tRNA carrying the next amino acid.
• The P site (peptidyl) holds the tRNA carrying the growing polypeptide chain.
• The E site (exit) is where discharged tRNAs leave the ribosome.

Initiation

Initiation brings together the mRNA, the first tRNA and both ribosomal subunits. In bacteria, the small subunit binds the mRNA at a purine rich Shine-Dalgarno sequence just upstream of the start codon, which pairs with the 16S rRNA. In eukaryotes, the small subunit, already carrying the initiator tRNA, binds the 5' cap and scans down the mRNA until it reaches the first AUG in a favorable sequence context. The initiator tRNA carries methionine (a formylated methionine, fMet, in bacteria) and is the only tRNA that enters directly into the P site. Proteins called initiation factors help, using energy from GTP, and then the large subunit joins, completing the initiation complex with the A site empty and ready.

Elongation

Elongation adds amino acids one at a time in a three step cycle, each cycle moving the ribosome one codon toward the 3' end of the mRNA.

1. Codon recognition. An aminoacyl-tRNA, escorted by an elongation factor bound to GTP, pairs its anticodon with the codon in the A site. GTP hydrolysis increases accuracy by giving the ribosome a moment to reject a wrong pairing.
2. Peptide bond formation. The rRNA of the large subunit catalyzes a bond between the amino group of the new amino acid in the A site and the carboxyl end of the growing chain in the P site. The chain transfers to the tRNA in the A site. Because rRNA, not protein, does this chemistry, the ribosome itself is a ribozyme, and the catalytic activity is called peptidyl transferase.
3. Translocation. A second elongation factor, using GTP, moves the ribosome forward one codon. The tRNA carrying the chain shifts to the P site, the empty tRNA shifts to the E site and leaves, and the A site opens for the next codon.

The polypeptide grows from its amino (N) terminus to its carboxyl (C) terminus, and exits through a tunnel in the large subunit. Each amino acid added costs about four high energy phosphate bonds: two to charge the tRNA (ATP to AMP) and two GTPs during elongation. A bacterial ribosome adds about 15 to 20 amino acids per second; a eukaryotic ribosome adds a few per second.

Termination and what happens next

When a stop codon reaches the A site, no tRNA matches it. Instead a protein called a release factor binds the stop codon and causes a water molecule to be added to the chain instead of an amino acid. This hydrolyzes the bond between the polypeptide and the tRNA in the P site, freeing the polypeptide; the subunits and mRNA then separate, using more GTP. A single mRNA is typically translated by many ribosomes at once, spaced along it like beads, forming a polyribosome (polysome). In bacteria, ribosomes attach to an mRNA even while it is still being transcribed.

A new polypeptide begins folding as it emerges, often helped by chaperone proteins, and many undergo post-translational modifications: addition of sugars, lipids or phosphate groups, removal of the first methionine, or cutting into smaller pieces (insulin is made as a single chain, proinsulin, that is cut to give two linked chains). Proteins destined for secretion, for membranes or for lysosomes begin with a signal peptide of about 20 hydrophobic amino acids. As it emerges, a signal recognition particle binds it, pauses translation and docks the ribosome on the rough ER, where the growing chain is threaded into the ER lumen or membrane. Proteins made on free ribosomes stay in the cytosol or are imported into the nucleus, mitochondria or other organelles.

Section 5Mutations and their effects

What a mutation is

A mutation is a permanent change in the nucleotide sequence of DNA. Mutations range from a single base pair to large chromosomal rearrangements (Chapter 5). They are the ultimate source of all new alleles and therefore of the genetic variation on which evolution acts, yet they are also the cause of inherited diseases and of cancer. Where a mutation occurs matters. A germline mutation arises in cells that give rise to gametes and can be passed to offspring, so it will be present in every cell of a child who inherits it. A somatic mutation arises in a body cell; it affects only that cell's descendants and is not inherited, but it can contribute to cancer.

Point mutations: substitutions

A point mutation changes a single nucleotide pair. In a base pair substitution, one pair is replaced by another. Substitutions are classified chemically as transitions (a purine replaced by a purine or a pyrimidine by a pyrimidine, such as A to G) or transversions (a purine replaced by a pyrimidine or the reverse); transitions are more common. More useful for predicting consequences is the effect on the protein.

• A silent mutation changes a codon to another codon for the same amino acid, usually at the third position (AAG to AAA still codes for lysine). The protein is unchanged, although rarely the change affects splicing or translation speed.
• A missense mutation changes a codon so that it specifies a different amino acid. The effect depends on the substitution. A conservative change swaps an amino acid for a chemically similar one (leucine for isoleucine) and may be harmless. A nonconservative change at a critical position can destroy function.
• A nonsense mutation changes an amino acid codon into a stop codon, ending translation early and producing a truncated, usually nonfunctional protein. Cells often destroy such mRNAs through a surveillance process called nonsense mediated decay.

Insertions, deletions and frameshifts

Adding (insertion) or removing (deletion) nucleotide pairs is usually more damaging than a substitution. Because mRNA is read in triplets, inserting or deleting a number of nucleotides that is not a multiple of three shifts the reading frame, a frameshift mutation. Every codon downstream of the change is read in the wrong groups of three, so the amino acid sequence becomes garbled and a stop codon usually appears soon. Inserting or deleting exactly three nucleotides (or a multiple of three) keeps the frame and adds or removes amino acids. The most common cystic fibrosis allele, ΔF508, is such an in frame deletion: three base pairs are missing and the protein lacks a single phenylalanine at position 508, which causes it to misfold and be destroyed before reaching the cell surface.

Repeat expansions

Some genes contain short tandem repeats that can lengthen when DNA polymerase slips during replication. In Huntington disease, a CAG repeat inside the coding sequence of the huntingtin gene, normally fewer than about 27 copies, expands to 36 or more, adding a long stretch of glutamine that makes the protein toxic to neurons. In fragile X syndrome, the most common inherited cause of intellectual disability, a CGG repeat in the 5' UTR of the FMR1 gene expands to more than 200 copies, becomes heavily methylated and shuts the gene off. Repeats tend to grow from one generation to the next, so these disorders often begin earlier or become more severe in later generations, a pattern called anticipation.

Causes of mutations

Spontaneous mutations arise from normal processes: rare mispairing during replication that escapes proofreading, slippage at repeats, loss of purine bases and deamination of cytosine to uracil. Mutations caused by external agents, mutagens, are called induced mutations.

• Base analogs resemble normal bases but pair incorrectly; 5-bromouracil resembles thymine but sometimes pairs with guanine.
• Chemical modifiers such as alkylating agents add groups to bases and change their pairing.
• Intercalating agents such as ethidium bromide and acridine dyes slip between stacked base pairs, distort the helix and cause insertions or deletions (frameshifts).
• Ultraviolet light creates thymine (pyrimidine) dimers that block replication and lead to errors if unrepaired; this is the main cause of skin cancers.
• Ionizing radiation (X-rays, gamma rays) breaks DNA strands, including double strand breaks.
• Many chemicals become mutagenic only after liver enzymes modify them, as with benzo[a]pyrene in tobacco smoke, which binds guanine and causes characteristic mutations in the p53 gene of lung cancers.

Because most carcinogens are mutagens, chemicals are screened with the Ames test. It uses mutant Salmonella bacteria that cannot make the amino acid histidine. The bacteria are exposed to the test chemical, usually mixed with a liver extract to mimic metabolism, and plated on medium lacking histidine. Only bacteria in which a new mutation has restored the histidine gene can grow, so the number of colonies measures the chemical's mutagenic strength.

Mutations, function and dominance

Most mutations in coding regions that change a protein reduce or abolish its function. These loss of function mutations are usually recessive, since one working allele often makes enough product (Chapter 5). A gain of function mutation gives a protein a new or increased activity and is usually dominant; a single changed copy of the RAS gene that locks its protein in the "on" state can drive a cell toward cancer. A dominant negative mutation makes a faulty protein that interferes with the normal one, as when mutant collagen chains spoil the triple helices in some forms of osteogenesis imperfecta. In cancer biology, gain of function mutations turn growth promoting proto-oncogenes into oncogenes, while loss of function mutations disable tumor suppressor genes such as p53 and BRCA1.

Most new mutations, however, have little or no effect, because they fall outside genes, are silent or cause conservative changes. Each human is born with a few dozen new mutations not present in either parent. A small fraction of mutations are beneficial in a particular environment, and these are the raw material that natural selection can spread through a population.

Words worth knowing

Gene Regulation and Biotechnology

Every cell in your body carries the same genome, yet a neuron, a liver cell and a red blood cell precursor look and behave nothing alike. The difference is not which genes they have but which genes they use, when, and how much. This chapter explains how bacteria and eukaryotes switch genes on and off, how controlled gene expression builds an embryo, and how the tools that grew out of that knowledge (PCR, cloning, sequencing and CRISPR) now let us read and rewrite DNA in medicine, agriculture and forensics.

Section 1Gene regulation in bacteria: operons

Why regulate genes at all?

A bacterium such as Escherichia coli has roughly 4,300 protein coding genes, but it never needs all of their products at once. A cell that keeps making the enzymes to digest a sugar that is not present, or the enzymes to build an amino acid that is already plentiful, wastes resources and grows more slowly than a competitor that does not. Natural selection has therefore favored cells that match gene expression to their environment.

Bacteria can adjust metabolism in two broad ways. They can change the activity of enzymes that already exist, for example through feedback inhibition, in which the end product of a pathway binds an allosteric site on the first enzyme and slows it within seconds. Or they can change the amount of enzyme by controlling transcription of the genes that encode it. The second response is slower (minutes) but saves far more resources. In bacteria the main control point is the start of transcription, because transcription and translation are coupled in the cytoplasm and most bacterial mRNAs last only a few minutes.

The operon model

In 1961 François Jacob and Jacques Monod proposed that bacterial genes with related functions are often clustered and transcribed together under a single on and off switch. They called the unit an operon. An operon has three essential parts:

• A promoter, the DNA sequence where RNA polymerase (guided by its sigma factor) binds and begins transcription.
• An operator, a short DNA segment that usually overlaps or lies just downstream of the promoter. It is the switch: when a repressor protein binds the operator, it blocks RNA polymerase.
• The structural genes, the coding sequences for the enzymes or other proteins. They are transcribed as one long polycistronic mRNA that carries several coding regions, each with its own start and stop codon and ribosome binding site.

The repressor is encoded by a separate regulatory gene, which has its own promoter and is usually expressed continuously at a low level. Because the repressor is a diffusible protein, it can act on any copy of the operator in the cell. The operator, by contrast, is a DNA sequence that controls only the genes physically attached to it. This difference between a trans-acting factor (a diffusible product) and a cis-acting element (a DNA site) is one of the most useful ideas in all of gene regulation.

The lac operon: an inducible operon

E. coli prefers glucose, but it can live on lactose, the disaccharide of milk, by splitting it into glucose and galactose. The lac operon contains three structural genes. lacZ encodes β-galactosidase, which hydrolyzes lactose and also converts a little of it into the isomer allolactose. lacY encodes lactose permease, a membrane transporter that brings lactose into the cell. lacA encodes a transacetylase whose role in lactose use is minor. Upstream lies the regulatory gene lacI, which encodes the lac repressor.

When lactose is absent, the lac repressor is active and binds tightly to the operator. RNA polymerase can still sit at the promoter but cannot move into the genes, so almost no lac mRNA is made. When lactose enters the cell, the few molecules of β-galactosidase always present (a small leaky level of expression) convert some of it to allolactose. Allolactose is the inducer: it binds the repressor at an allosteric site, changes its shape and drops its affinity for the operator. The repressor falls off, and transcription begins. Because a small molecule switches the operon on, the lac operon is called an inducible operon. Inducible operons usually encode catabolic enzymes that break down a nutrient, and it makes sense to make them only when the nutrient is present.

Researchers often use IPTG, a synthetic look alike of allolactose, as an inducer. IPTG binds the repressor but is not broken down by β-galactosidase, so it keeps the operon switched on at a constant level.

Positive control: catabolite repression and CAP

Removing the repressor is necessary but not sufficient for strong expression. The lac promoter is weak; RNA polymerase binds it poorly on its own. Expression becomes high only when an activator helps polymerase bind. That activator is the catabolite activator protein (CAP, also called CRP). CAP can bind DNA only when it carries the small signaling molecule cyclic AMP (cAMP). When glucose is scarce, the enzyme adenylyl cyclase is active and cAMP levels rise; cAMP binds CAP, and the cAMP-CAP complex binds a site just upstream of the lac promoter, bends the DNA and contacts RNA polymerase, increasing transcription roughly fiftyfold. When glucose is plentiful, glucose transport keeps adenylyl cyclase inactive, cAMP falls, CAP leaves the DNA and lac expression stays low even if lactose is present. Glucose also reduces lactose uptake directly by inhibiting the permease, a process called inducer exclusion.

The result is a two input logic circuit. The repressor reports whether lactose is present; CAP reports whether glucose is absent. The cell commits to lactose only when it has lactose and lacks its preferred sugar. Because the lac operon and many other operons for alternative sugars all depend on cAMP-CAP, the cell can coordinate them as a group, an arrangement called catabolite repression. When E. coli is grown in a mixture of glucose and lactose, it uses up the glucose first, pauses briefly, then switches to lactose, giving the two phase growth curve (diauxie) that Monod first described.

Mutations that revealed the logic

Jacob and Monod worked out the system with mutants. A lacI^- mutant makes a nonfunctional repressor, so the operon is constitutive (always on). An operator constitutive mutant (lacO^c) has an altered operator that the repressor cannot bind, so it too is constitutive. A lacI^S (super repressor) mutant makes a repressor that cannot bind allolactose, so the operon can never be induced. To tell these apart they built partial diploid bacteria carrying a second copy of the lac region on an F plasmid. A normal lacI gene on the plasmid restored control to a lacI^- chromosome, because the repressor diffuses (trans acting, and the mutation is recessive). But a normal plasmid could not rescue a lacO^c chromosome, because an operator controls only the genes on its own DNA molecule (cis acting). The super repressor was dominant: its noninducible protein sat on both operators.

The trp operon: a repressible operon

The trp operon holds five genes (trpE, D, C, B and A) that encode the enzymes for making the amino acid tryptophan. Here the logic is reversed. The operon is normally on, because the cell usually needs tryptophan. The regulatory gene trpR makes a repressor in an inactive form (an aporepressor) that cannot bind the operator by itself. When tryptophan accumulates, tryptophan binds the repressor as a corepressor, activating it; the complex binds the operator and shuts transcription down. Because a small molecule turns the operon off, it is a repressible operon. Repressible operons usually encode anabolic enzymes that build a product, and it makes sense to stop building it when the product is abundant.

Attenuation: a second dial on the trp operon

The trp operon has a second control layer that depends on the coupling of transcription and translation. Before the first structural gene lies a leader sequence of about 160 nucleotides with four regions (1, 2, 3 and 4) that can pair in different ways. Region 1 encodes a short leader peptide with two tryptophan codons in a row. As soon as the 5' end of the transcript appears, a ribosome begins translating it.

• When tryptophan is plentiful, charged tryptophan tRNA is abundant, and the ribosome moves quickly through region 1 and covers region 2. Regions 3 and 4 then pair into a hairpin followed by a run of U's, which acts as a transcription terminator. RNA polymerase stops before reaching the structural genes.
• When tryptophan is scarce, the ribosome stalls at the two tryptophan codons in region 1. Region 2 is free to pair with region 3, forming an antiterminator that prevents the 3 to 4 terminator from forming, and RNA polymerase continues into the genes.

This attenuation fine tunes expression. Repression alone changes trp operon output about seventyfold; attenuation adds roughly another tenfold, so the combined range is several hundredfold.

Beyond operons

Operons are only part of bacterial control. Bacteria use alternative sigma factors to redirect RNA polymerase to whole sets of genes at once, for example the heat shock genes or the genes for spore formation in Bacillus. Some mRNAs contain riboswitches, folded regions that bind a metabolite directly and change their own shape to stop transcription or block the ribosome binding site, with no protein involved. Many bacteria also sense their own population density by releasing and detecting small signal molecules, a process called quorum sensing, and switch on genes for light production, biofilm formation or toxins only when enough cells are present.

Section 2Gene regulation in eukaryotes and epigenetics

Many control points, one goal

Eukaryotic cells face a harder problem than bacteria. A human has about 20,000 protein coding genes, and a multicellular body contains hundreds of cell types, each of which must express its own set of genes stably for years. A typical human cell expresses perhaps a third to a half of its genes, and the particular set defines what the cell is. Eukaryotic genes are also separated from ribosomes by the nuclear envelope, wrapped in chromatin, interrupted by introns, and transcribed one gene at a time rather than in operons. Each of these features creates an extra point where expression can be adjusted. Control is possible at every step from DNA to finished protein:

1. Chromatin: how tightly the DNA is packed and chemically marked.
2. Transcription: whether and how often RNA polymerase II starts at a promoter. This is still the most important step for most genes.
3. RNA processing: capping, polyadenylation and especially alternative splicing.
4. mRNA transport and stability: export from the nucleus and how long an mRNA survives in the cytoplasm.
5. Translation: whether ribosomes start on an mRNA.
6. Protein processing and degradation: cleavage, chemical modification, targeting and destruction.

Chromatin: packing as a control step

In the nucleus DNA wraps around octamers of histone proteins (two each of H2A, H2B, H3 and H4) to form nucleosomes, about 147 base pairs per core, like beads on a string. Nucleosomes fold further into fibers, loops and territories. Tightly packed heterochromatin is largely inaccessible to the transcription machinery and its genes are usually silent; looser euchromatin is where most active genes lie. Whether a gene sits in open or closed chromatin depends heavily on chemical marks on the histones and on the DNA itself.

Each histone has a flexible tail that sticks out of the nucleosome. Enzymes add and remove chemical groups on these tails:

• Histone acetylation: histone acetyltransferases (HATs) add acetyl groups to lysines. Acetylation neutralizes the positive charge of lysine, weakening the tail's grip on the negatively charged DNA and on neighboring nucleosomes. Chromatin opens, and acetylated lysines also serve as docking sites for proteins that promote transcription. Histone deacetylases (HDACs) remove the acetyl groups and help close chromatin.
• Histone methylation: methyl groups on lysines can either activate or repress, depending on which residue is marked. Trimethylation of lysine 4 on histone H3 (H3K4me3) marks active promoters; trimethylation of lysine 9 or lysine 27 (H3K9me3, H3K27me3) marks silenced regions.

DNA methylation works directly on the DNA. Enzymes called DNA methyltransferases add a methyl group to carbon 5 of cytosine, almost always where a C is followed by a G (a CpG site). Heavily methylated promoters are usually silent: methylated DNA attracts proteins that recruit histone deacetylases and compact the chromatin. Many promoters sit in CpG rich stretches called CpG islands that stay unmethylated in cells where the gene is active. Methylation patterns are copied when DNA replicates, because a maintenance methyltransferase recognizes a half methylated CpG on the new strand and methylates its partner. This copying lets a cell pass its pattern of silent genes to its daughters.

Transcription factors, promoters and enhancers

RNA polymerase II cannot recognize a promoter by itself. It needs general transcription factors (GTFs), a set of proteins that assemble at the core promoter. In many genes the core promoter contains a TATA box about 25 to 30 nucleotides upstream of the start site; the TATA binding protein, part of the factor TFIID, binds it and bends the DNA, and other factors and the polymerase join to form the transcription initiation complex. GTFs alone give only a low, basal rate of transcription. High rates, and the differences between cell types, come from specific transcription factors that bind control elements farther away.

Proximal control elements sit just upstream of the promoter. Distal control elements, grouped into enhancers, can lie thousands or even hundreds of thousands of base pairs upstream, downstream or inside an intron of the gene they control. Proteins that bind enhancers and stimulate transcription are activators; proteins that bind silencers and reduce it are repressors. A typical activator has a DNA binding domain that recognizes a specific sequence and an activation domain that contacts other proteins. DNA bending brings enhancer bound activators next to the promoter, where they contact the large mediator complex and the general factors, speeding assembly of the initiation complex. Activators also recruit histone acetyltransferases and chromatin remodeling complexes, opening the local chromatin. Insulators are boundary elements that keep an enhancer from acting on the wrong gene.

How can a few thousand transcription factors control 20,000 genes in hundreds of cell types? The answer is combinatorial control. Each enhancer contains binding sites for several different factors, and full activation requires a particular combination. A liver cell and a lens cell may both contain the same albumin and crystallin genes, but the liver cell has the right set of activators for the albumin enhancer and the lens cell has the set for the crystallin enhancer. A small number of factors, used in different combinations, can produce an enormous number of distinct patterns. Genes that must be expressed together, such as the genes a cell turns on in response to a steroid hormone, are coordinated not by being in an operon but by sharing the same control element in their enhancers, so that one activated factor binds all of them.

Control after transcription

Alternative splicing lets one gene make several proteins. By including or skipping particular exons, a cell can produce different isoforms from the same pre-mRNA. More than nine in ten human genes with multiple exons are alternatively spliced, which helps explain how a modest number of genes yields a far larger set of proteins.

Once in the cytoplasm, an mRNA's lifespan strongly affects how much protein it makes. Some mRNAs last minutes, others days.

Noncoding RNAs are a major regulatory force. MicroRNAs (miRNAs) are about 22 nucleotides long. A miRNA gene is transcribed into a precursor that folds into a hairpin; the enzyme Dicer trims the hairpin, and one strand is loaded into a protein complex (built around an Argonaute protein) called the RNA induced silencing complex. The miRNA then base pairs with matching sequences, usually in the 3' untranslated region of target mRNAs. Partial pairing blocks translation and speeds degradation; near perfect pairing triggers cleavage. One miRNA can regulate hundreds of mRNAs, and well over half of human protein coding genes are thought to be miRNA targets. Small interfering RNAs (siRNAs) are similar in size and use the same machinery but come from longer double stranded RNA, for example from viruses or experimentally introduced RNA. The silencing of genes by these small RNAs is called RNA interference (RNAi), and it is now used both as a research tool and in approved drugs. Longer long noncoding RNAs help organize chromatin, as XIST does in X inactivation.

Finally, cells control translation and protein lifetime. Initiation can be blocked globally, for instance by phosphorylating an initiation factor during stress, or for single mRNAs by proteins that bind regulatory sequences. Many proteins are inactive until cut or chemically modified. Proteins destined for destruction are tagged with chains of the small protein ubiquitin and fed into a proteasome, a barrel shaped protease complex. Timed destruction of regulatory proteins, such as cyclins, drives events like the cell cycle.

Epigenetics

Epigenetic inheritance refers to changes in gene expression that are passed through cell divisions (and sometimes across generations) without any change in the DNA sequence. DNA methylation and some histone marks are the best understood carriers of this cellular memory. When a skin stem cell divides, its daughters remain skin cells, partly because the pattern of silenced and open genes is copied along with the DNA.

Several classic phenomena are epigenetic:

• X chromosome inactivation. In female mammals one X chromosome in each cell of the early embryo is randomly chosen to be silenced. The chosen X expresses the long noncoding RNA XIST, which coats it and recruits repressive histone marks and DNA methylation. It condenses into a Barr body, and all descendants of that cell keep the same X silent. Females are therefore mosaics, as seen in the orange and black patches of calico cats.
• Genomic imprinting. For a small number of genes (on the order of 100 to 200 in humans), only the copy from one parent is expressed, because methylation marks laid down in egg or sperm silence the other. Loss of the same chromosome region on chromosome 15 causes Prader-Willi syndrome if the paternal copy is missing and Angelman syndrome if the maternal copy is missing.
• Environmental effects. Diet, stress, toxins and aging can shift methylation. In mice carrying a particular agouti allele, the mother's diet during pregnancy alters methylation of the gene and changes the coat color and obesity risk of her pups.

Section 3Development and differential gene expression

Three processes build a body

A human begins as a single fertilized egg, the zygote, and becomes a body of tens of trillions of cells. Three processes overlap to make this happen. Cell division increases the number of cells. Cell differentiation makes cells specialized in structure and function. Morphogenesis ("creation of form") shapes tissues and organs through cell movement, changes in cell shape, selective growth and programmed cell death. All three depend on cells expressing different genes from the same genome, a principle called differential gene expression.

The evidence that differentiated cells keep a complete genome came from nuclear transplantation. In frogs, nuclei taken from tadpole intestinal cells and placed into enucleated eggs sometimes produced swimming tadpoles. In 1996 the sheep Dolly was cloned from the nucleus of an adult mammary cell. These experiments showed that development does not usually remove genes; it changes which genes are used, and an egg's cytoplasm can, at least sometimes, reset a nucleus to an embryonic state.

Determination and differentiation

Before a cell looks specialized, its fate is often already fixed. Determination is the commitment of a cell to a particular fate; differentiation is the visible result, when the cell makes tissue specific proteins. Muscle offers the clearest example. Embryonic cells become myoblasts when they begin expressing MyoD, a master regulatory gene that encodes a transcription factor. MyoD activates more MyoD (a positive feedback loop that locks the decision in) and turns on genes for other muscle transcription factors, which in turn activate genes for myosin, actin and other muscle proteins. Myoblasts fuse into multinucleate muscle fibers.

How cells learn where they are

Cells acquire different fates through two kinds of information.

• Cytoplasmic determinants are maternal mRNAs and proteins placed unevenly in the egg. When the zygote divides, each daughter inherits a different share, so the nuclei in different cells are exposed to different regulators from the start.
• Induction is signaling between cells. Neighboring cells release or display signal molecules that change gene expression in target cells through signal transduction pathways. Induction is how the developing eye cup tells overlying ectoderm to form a lens, and how the notochord tells the ectoderm above it to form the neural plate.