Heinemann Biology 2

Introduction • Summaries give an idea of the main information being discussed and should assist students when summarising information or when locating particular points of discussion. • The glossary at the end of the book can be used to check the meaning of important words. • A comprehensive index is included and Heinemann Biology 1 has an appendix containing a classification of organisms.

Heinemann eBiology Student CD accompanies the text and includes: • complete copy of the textbook (EINEMANN

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in electronic format • interactive tutorials, which model and simulate key biology concepts • interactive glossary • exam and test self-timer.

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The fourth editions of Heinemann Biology 1 and Heinemann Biology 2 have been developed to support the 2005 Biology Study Design. The content provides an exact match to the study design, and the fourth edition boasts a totally new layout and design with many outstanding new features. The authors have incorporated the very latest developments and applications of biology, presented in an Australian context. The textbooks contain the most up-to-date information available including the fast-moving areas of genetics, immunology and classification. Each book is divided into four Areas of Study corresponding to the Study Design, and these are further divided into chapters. The following features will ensure an enjoyment of biology and assist students in grasping the key concepts: • Each chapter opening includes key knowledge statements and outcomes. These help students unpack the Study Design and expand on what they are expected to know and be able to do. • Each chapter is further divided into clear-cut sections that finish with a set of summary points and key questions to assist students to consolidate the key points and concepts of that section. • Chapter review questions are found at the end of each chapter, to test students’ ability to apply the knowledge gained from the chapter. • The Area of study review includes a large range of exam-style questions plus a practice assessment task. This task is expanded further in the corresponding Heinemann Biology Student Workbook. • Biology in action boxes contain biology in an applied situation or relevant context. These include the nature and practice of biology, applications of biology and associated issues, and the historical development of concepts and ideas. • Extension boxes contain material that goes beyond the core content of the syllabus. These are intended for students who wish to expand their depth of understanding in a particular area. The material may be conceptual or contextual. • Technologies and techniques spreads are written by practising Australian scientists. New and emerging technologies and techniques are explained and discussed, and help bring modern biology to life while addressing this vital area of the Study Design. • Biofiles are snippets of information that add interest and relevance to the text.

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Support material for Heinemann Biology 1 and 2 Student workbooks Heinemann Biology 1 and 2 Student Workbooks provide outstanding support and guidance for students studying VCE Biology. Each is designed to be used in conjunction with the textbook and assist students to grasp the key concepts. They provide practical activities and guidance, and assessment practice and opportunities. Key features: • highly illustrated study notes covering the main points of each Area of Study • Multiple Intelligence Worksheets that cater for a range of learning styles • Practice Assessment tasks • Practical Activities that are relevant and useful.

Website support www.hi.com.au/biol/ Heinemann Biology 1 and 2 have comprehensive website support. This includes course advice, practical notes, ICT support for activities, and detailed answers to all textbook questions.

iii The chemical nature of cells

Contents

unit

area of study 01

3

area of study 02

Detecting and responding

Molecules of life Chapter

01

Chapter

Life at the molecular level Biologically important inorganic molecules Organic molecules Biological membranes Chapter Review

Chapter

03 09 14 19 26

02

Enzymes and cellular processes Biomolecules—synthesis and transport Chapter Review

5.1 5.2 5.3 5.4 5.5 5.6 5.7

Enzymes and other biomolecules 2.1 2.2

05

Homeostasis and regulatory mechanisms

The chemical nature of cells 1.1 1.2 1.3 1.4

Signatures of life

28 39 45

It’s easier being big Homeostasis—stability in the face of change Homeostatic mechanisms Regulatory pathways—roles of nerves and hormones Homeostasis in mammals and birds Regulating in changing conditions Plant regulation Chapter Review

Chapter

95 97 101 105 107 113 120 124

06

Detecting and responding to signals

Chapter

03

Energy transformations 3.1 3.2 3.3 3.4

Life needs energy ATP—energy from glucose Getting glucose Storing energy Chapter Review

Chapter

48 51 56 63 68

04

Detecting and responding Receptors Signal transduction Coordinating responses Sensing and responding in plants Chapter Review

Chapter

127 131 135 140 146 154

07

Pathogens cause disease

DNA, proteins and proteomes Life has a common origin Synthesis of polypeptides Protein formation Chapter Review

70 73 80 87

Area of Study review: Molecules of life

88

4.2 4.2 4.3

6.1 6.2 6.3 6.4 6.5

7.1 7.2 7.3 7.4

Infection and disease Organisms that cause disease Non-cellular pathogenic agents Controlling pathogens Chapter Review

Chapter

156 158 169 175 178

08

Defending self 8.1 8.2 8.3 8.4 8.5

Levels of defences Mammalian immunity is innate and adaptive Non-specific defences Specific adaptive immunity Why is the immune system so complex? Chapter Review

Chapter

180 183 185 190 197 202

09

Applications of immunology 9.1 9.2 9.3

Acquiring immunity Disorders of the immune system Frontiers of medicine Chapter Review

Area of Study review: Detecting and responding

iv

204 207 215 222 223

Contents

unit

4

area of study 01

area of study 02

Change over time

Heredity Chapter

10

Chapter

Chapter

229 236 242 245

Chapter

261 267

12

The continuity of life Cell division for gametes: meiosis When meiosis goes wrong Genes and development Chapter Review

Chapter

269 273 277 282 286

13

Inheriting variation Mutation: the source of variation Germ line and somatic mutations Identifying mutatons and their causes Chapter Review

Chapter

Chapter

Studying inheritance Dominant and recessive phenotypes Environment affects some phenotypes Single genes—monohybrid crosses Two genes—dihybrid crosses Testcrosses and phenotypic ratios Chapter Review

Chapter

18.1 18.2 18.3 18.4

312 315 319 322 325 328 331

Chapter

15

15.1 15.2 15.3 15.4 15.5

Linked genes Sex-linked inheritance Pedigree analysis Many genes Genes in populations Chapter Review

Area of Study review: Continuity and change

334 339 343 347 350 353 355

18

Evolution—genetic change over time Selection Selection in action Gene flow and genetic drift Chapter Review

Chapter

Linked genes, sex linkage and pedigrees

383 388 389 393 398 403

Change in populations 406 411 416 421 425

19

Patterns of evolution 19.1 19.2 19.3 19.4

Genotype, phenotype and crosses 14.1 14.2 14.3 14.4 14.5 14.6

17

17.1 Evidence of evolution from comparative anatomy 17.2 Genetic comparisons 17.3 Molecular evidence for evolution 17.4 Sequencing DNA 17.5 Looking back in time Chapter Review

289 295 303 305 310

14

361 366 369 374 381

Evidence of evolution from anatomy and molecules

Variation: alleles and mutations 13.1 13.2 13.3 13.4

Discovering the past How old is that fossil? The geological time scale Biogeography Chapter Review

Chapter 248 257

Cell reproduction 12.1 12.2 12.3 12.4

16.1 16.2 16.3 16.4

11

Molecular tools and techniques 11.1 Working with DNA 11.2 Applications of DNA profiling 11.3 Gene cloning and recombinant DNA technology Chapter Review

16

Evidence of evolution from the past

Molecular genetics 10.1 Genes and DNA 10.2 Gene expression 10.3 Gene regulation Chapter Review

Continuity and change

Divergent and convergent patterns of evolution Races and geographic variation Forming new species Reproductive isolation Chapter Review

427 433 436 441 446

20

Human evolution and intervention Humans are primates Hominid evolution Origin of modern humans Early humans in Australia Human intervention in evolution Chapter Review

448 451 455 460 462 467

Area of Study review: Change over time

468

Glossary

472

Index

482

20.1 20.2 20.3 20.4 20.5

v

Acknowledgements The authors and publisher would like to thank the following for granting permission to reproduce copyright material in this book: Authors’ contributions: pp 2, 12 (top), 66 (bottom), 67, 116, 128, 144, 145, 158, 168 (top), 182, 212, 216, 232, 234, 302, 351, 361,373, 376, 400, 417, 427 (photo from Dr Jane Melville, Museum Victoria), 432 (photo from Dr Marianne Horak); AAP, pp. 95 (left), 186, 372, 454, 461 (right); ANT Photo Library, p. 461 (left); Ardea, pp. 16, 27, 105, 444; Art Archive, p. 455; Auscape International, p. 56; Australian Picture Library, pp. 3 (top), 9 (right),11, 12 (bottom),13, 15 (both), 28, 35 (bottom), 43 (bottom), 149, 158, 73, 173 (bottom), 201, 204 (left); Australian Picture Library/Corbis, pp. 31, 95 (right), 99, 120, 157, 259, 412 (left), 412 (right); Australian Synchrotron Project Department of Innovation, Industry and Regional Development, p. 79; C. Banks, p. 134; Bayer, p. 166 (both); Biology Department, University of Melbourne, pp. 127 (left), 210; Cancer Council of Victoria, SunSmart campaign, p. 309; Department of Primary Industries 2005, Oriental Fruit Moth © State of Victoria, Department of Primary Industries

vi Molecules of life

2005, created by Alex ll’lchev, p. 129; Digital Vision, p. 400; Fairfax Photos, p. 113; Mark Fergus, p. 17; Bruce Fuhrer, pp. 10 (bottom), 164 (bottom); Getty Images, pp. 55, 69, 126; Harcourt Index, pp. 34, 46, 48 (bottom), 84, 124 (top), 141, 456; Dennis Kunkel, p. 54 (left); Tim Low, p. 409 (left, both); C. Marcroft, p. 131; Nature Picture Library, p. 272; Northside Productions, p. 167; PhotoDisc, pp. 33, 37 (top), 48 (top), 58 (inset), 64, 124 (bottom), 230, 245, 276, 286; Photolibrary.com/Science Photo Library, pp. 3 (bottom), 5 (bottom), 60, 69, 119, 133, 160, 165, 168 (bottom), 180, 204 (right); Photolibrary.com, pp. 9 (left), 148, 151, 213; Professor Frances Separovic, p. 22; Professor Loane Skene, p. 35 (top); Sport the Library, p. 81 (bottom), 127; Visuals Unlimited, pp. 110, 139, 163, 164 (top), 170 (bottom), Every effort has been made to trace and acknowledge copyright material. The authors and publisher would welcome any information from people who believe they own copyright to material in this book.

unit

3

area of study 01

Molecules of life outcome On completion of this unit the student should have acquired key knowledge related to the molecular basis of living organisms and be able to analyse and evaluate evidence from practical investigations related to biochemical processes.

0

chapter 01

The chemical nature of cells

key knowledge • chemical nature of cells including the basic structure of the cell • composition of organisms • properties of biologically important inorganic and organic molecules • structure and properties of membranes

chapter outcomes After working through this chapter you should be able to: • describe the structure of eukaryote cells and the functions of organelles • distinguish between an atom, an element, a molecule, an ion and a compound • list the elements commonly found in living organisms • distinguish between organic and inorganic molecules • suggest why carbon is a key element in organic molecules • describe the roles of biologically important inorganic molecules • outline the properties of water that are important to life • describe the basic structures of carbohydrates, proteins, nucleic acids and lipids • describe the molecular structure of cell membranes • outline the particular role of phospholipids in membranes • describe the different ways that molecules cross membranes.

1.1

Life at the molecular level Living organisms are amazingly diverse in appearance—from tiny diatoms to huge trees, from worms to kangaroos, from bacteria to fungi (Figure 1.1). But the closer you look at all of them, the more similar they become. All of these organisms are composed of cells, which are the basic functional units of all organisms. This is one of the fundamental principles of biology known as the cell theory. While cells share many common features, there are also differences between cells that are related to their particular roles in organisms.

Cells are the functional units of life If we are to understand life we need to understand how cells work. Cells are the basic functional units of living organisms. The cell theory is based on detailed microscopic and biochemical observations of cells from all types of organisms. It states that: • all organisms are composed of cells (and the products of cells) • all cells come from pre-existing cells • the cell is the smallest living organisational unit. All types of cells perform similar basic processes and many also carry out highly specialised functions. The activities of cells require considerable energy, and require the production of a variety of biological molecules that are assembled into new organelles, used for repair or exported from the cell. All these processes are catalysed by enzymes and are precisely regulated. Some biochemical processes involve hundreds of enzymes operating sequentially along a complex integrated chemical pathway; each step is tightly controlled.

(a)

Cell structure There is really no such thing as a typical cell. Cells are specialised for many different purposes and their structures reflect those purposes. However, there are some features that are shared by all cells. In all cells, the cytoplasm of the cell is enclosed within an outer plasma membrane (also referred to as the cell membrane or plasmalemma), which separates it from its environment, and all cells contain genetic material in the form of DNA, which carries hereditary information, directs the cell’s activities, and is passed accurately from generation to generation. There are two fundamentally different types of cells—prokaryotes and eukaryotes. Prokaryotes are bacteria and cyanobacteria. Prokaryote cells are small and lack membrane-bound organelles. They contain a single circular DNA chromosome. The plasma membrane is surrounded by an outer cell wall of protein and complex carbohydrate (murein). The composition of this cell wall is different from the cell walls of plants, which are largely cellulose, and fungi, where the cell walls contain chitin (a polysaccharide). Eukaryote cells are characterised by having an internal membrane system forming the nuclear membrane and many other organelles.

(b)

Figure 1.1 (a) A tree fungus and (b) a tiny marine planktonic unicellular alga (diatom).

3 The chemical nature of cells

Organelles are subcellular structures involved in specific functions of the cell (Figure 1.2). Many organelles are found in most cells. A brief summary of the structure and functions of the different organelles follows.

Eukaryote organelles Centrioles:

a pair of small cylindrical structures composed of microtubules. They are involved in the separation of chromosomes during cell division in animal cells and protists. They are not found in plant cells.

Chloroplast:

found in some plant cells; a green organelle (due to the abundant presence of chlorophyll) in which photosynthesis takes place. It is composed of many folded layers of membrane.

Cytoplasm:

the contents of a cell, other than the nucleus. It is more than 90% water and contains ions, salts, enzymes, food molecules and organelles.

Cytosol:

the fluid component of cytoplasm in which organelles are located.

Endoplasmic reticulum:

a network of intracellular membranes, which links with the plasma membrane and other membranous organelles. It may be rough (associated with ribosomes) or smooth (lacking ribosomes). It is involved with the production, processing, transport and storage of materials within the cell.

Golgi apparatus:

a stack of flat membrane sacs where the final synthesis and packaging of proteins into membrane-bound vesicles occurs before they are secreted from the cell. It is linked to the endoplasmic reticulum.

Lysosomes:

membrane-bound vesicles containing powerful enzymes that break down debris and foreign material; found in most animal cells.

Mitochondria:

organelles composed of many folded layers of membrane. Mitochondria are involved in the energy transformations that release energy for use by the cell.

Nucleus:

a large organelle, surrounded by a double-layered nuclear membrane containing pores that allow movement between the nucleus and the cytoplasm. It stains differently from cytoplasm and so often looks darker in prepared slides. The nucleus contains genetic material and controls cellular activities.

Plasma membrane:

(also called the cell membrane, cytoplasmic membrane or plasmalemma) a delicate bilayer of phospholipid molecules with associated proteins, enclosing the cytoplasm in all cells. It controls the movement of substances into and out of the cell and is responsible for recognition, adhesion and chemical communication between cells.

Plastids:

a group of organelles found only in plant cells, all of which develop from simple organelles called proplasts. Chloroplasts and amyloplasts are plastids. Amyloplasts store starch in roots or storage tissue, such as in potato tubers, and may be involved in geotropism and chromoplasts (which contain colour pigments and are found in petals and fruit).

Ribosomes:

tiny organelles located in the cytosol, sometimes associated with endoplasmic reticulum. They are sites of production of proteins.

Tonoplast:

the vacuole membrane in plant cells; regulates the movement of substances into and out of the vacuole.

Vacuoles:

membrane-bound liquid-filled spaces found in most cells in variable numbers. Plant cells typically have large fluid-filled vacuoles, containing cell sap, that provide physical support (turgidity) and storage. In other cells, vacuoles may be involved in intracellular digestion (food vacuoles) or water balance (contractile vacuoles).

Vesicles:

membrane-bound organelles often associated with transport within the cell.

Cell wall:

The cell wall is not an organelle, but it is an important component of plant and bacterial cells. In plant cells it is a non-living, cellulose structure outside the plasma membrane. It provides support, prevents expansion of the cell, and allows water and dissolved substances to pass freely through it.

4 Molecules of life

Plant cell

Animal cell ribosomes nucleus cell membrane cytoplasm

Golgi apparatus

cell wall cytoplasm vacuole

chloroplast

mitochondria

nucleus

vesicles

Figure 1.2 Features of plant and animal cells as seen under the electron microscope.

biology in action Tissue culture for burns The Tissue Culture Laboratory at Monash University grows skin cells into epithelial grafts for burn patients in hospitals around Australia. From a small piece (2 × 2 cm) of the patient’s own skin, it is possible to grow enough epithelial grafts to cover a whole person in 3 weeks. Individual grafts are typically 10 × 7 cm in size and are multi-layered, very much like normal epidermis. By 2005, more than 200 patients with burns to up to 96% of their total body surface area have been grafted with a total of almost 10 000 grafts. This laboratory has also developed the method of culture of cartilage cells (chondrocytes) from a small piece of the patient’s own cartilage (30–300 mg). Since 1997, more than 190 patients have been treated successfully with cultured chondrocytes to repair the articular cartilage in damaged knees. Research is now focused on developing successful culture methods of other cells for application in future cell therapies. Figure 1.3 Cultured sheet of epithelial tissue ready for grafting onto burns patient.

5 The chemical nature of cells

The composition of living organisms The similarities between different organisms become even greater when you look more closely at cells and the atoms and molecules they are composed of. All life is composed of the same few elements. There are 92 naturally occurring elements. Only 11 of these are found in organisms in more than trace amounts, and four of these— carbon (C), hydrogen (H), oxygen (O) and nitrogen (N)—make up 99% of organisms by weight (Figure 1.4). The similarities of all organisms at the molecular level points to their common origin. Understanding the structure and properties of these molecules and the ways they interact is fundamental to developing an understanding of biological processes and the functions of organisms. )NORGANIC / /

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Figure 1.4 Periodic table highlighting elements commonly found in living organisms: major elements (pink), other elements (yellow) and trace elements (green).

Atoms are the basic unit of all matter. Substances consisting of only one kind of atom are called elements. Molecules are two or more atoms (of the same or different kinds) held together by chemical bonds (see page 7) and a compound is a molecule containing more than one type of atom. Both living and non-living things are made from the same chemical elements, but there is a difference in the way that these elements are organised into larger molecules (Figure 1.5). Organisms contain complex chemical compounds containing carbon and hydrogen (and sometimes other elements, such as oxygen and nitrogen). These are called organic compounds because the first ones discovered were produced by organisms or found in them. Most large organic molecules are composed of many smaller organic molecules linked together. All other compounds are called inorganic compounds. Inorganic molecules that are important for living organisms include water, oxygen, carbon dioxide, nitrogen and minerals. The chemical reactions in cells occur in a water environment. In most cells, oxygen is required for the release of usable energy from food molecules. Carbon dioxide is the main source of carbon for the production of organic molecules. Nitrogen is a part of all proteins and nucleic acids. Minerals are found in structural components and many enzymes.

extension A little chemistry Just a little chemistry is useful to understand the activities of cells at the molecular level. Elements are made of atoms An atom has a nucleus (which is composed of positively charged protons and uncharged neutrons) and one or more negatively charged electrons in orbit around the nucleus. Atoms that have the maximum number of electrons in their outer ‘orbital’ are most stable. The first orbital (see the top row of the Periodic Table) can contain two electrons—hydrogen (H) has one electron and helium (He) has two. Helium has a full orbital and is stable, hydrogen has not and this makes it likely to combine with other atoms (Figure 1.6a). The second

HYDROGEN

(a)

NUCLEUS

and third orbitals (represented by the second and third rows in the Table) have a maximum of eight electrons. Neon (Ne) and argon (Ar) are the only stable atoms in these rows. Chemical bonding of atoms makes molecules Because atoms are more stable when their outer orbitals are filled, they tend to combine with other atoms to achieve this state, forming molecules. Molecules are two or more atoms held together by chemical bonds. Covalent bonds are created by sharing electrons between two atoms to achieve stability (Figure 1.6b and c). Compound molecules, such as methane CH4, are those involving the combination

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Figure 1.6 (a) Hydrogen, carbon and oxygen showing electrons in orbitals. (b) Hydrogen and carbon combining to form methane (a non-polar compound molecule held together by covalent bonds). (c) Oxygen and hydrogen combine to form water (a polar molecule held together by polar covalent bonds). (d) Sodium and chlorine form Na+ and Cl– held together by an ionic bond.

7 The chemical nature of cells

extension A little chemistry (continued) of different types of atoms (Figure 1.6b). Covalent bonds between carbon and hydrogen are energy rich, which is why hydrocarbons (such as petrol and gas) make good fuels. Sometimes in covalent bonds one atom attracts the shared electron more strongly than the other, resulting in a polar covalent bond. This is the case within water molecules where oxygen has a stronger attraction for the electrons causing the molecules to be polar, meaning that they are slightly positive at the hydrogen atoms and slightly negative at the oxygen molecule (Figure 1.6c). Polar molecules have a positive region and a negative region (Figure 1.6c), whereas non-polar molecules have an even distribution of charge and are electrically neutral (Figure 1.6b). Polarity of molecules is an important property in biology; for example, it governs the way that many molecules cross cell membranes. Individual water molecules are held together by hydrogen bonds. Hydrogen bonds are weak bonds between the slightly positive hydrogen atom of one polar molecule and the slightly negative region (usually an oxygen or nitrogen atom) of a different polar molecule.

question

Sometimes the attraction for an electron is so strong that the electron actually leaves one atom to become part of another, resulting in the formation of ions (Figure 1.6d). Ions are electrically charged atoms or group of atoms. The atom that loses electrons will be a positive ion (a cation) and the atom that gains electrons will be a negatively charged ion (an anion). Positive and negative ions often come together and are held by weaker ionic bonds that can be easily broken in biological systems. The bonds formed in molecular recognition processes that are important to many biological functions, such as signalling and recognising self (see chapters 6 and 8), usually include ionic bonds. The special role of carbon The Periodic Table (Figure 1.4) shows that carbon has four electrons in its outer orbital. This allows each carbon atom to combine with up to four other atoms, as shown in Figure 1.6b. This gives carbon the ability to form many different kinds and sizes of molecules with other atoms. This is why carbon is the key atom in organic molecules (Figure 1.5).

?

Using the same style as Figure 1.6, draw diagrams of carbon dioxide (CO2), hydrogen gas (H2) and oxygen gas (O2).

summary

1.1

• All life is composed of the same few elements—carbon, oxygen, hydrogen and nitrogen make up 99% of organisms by weight. • Biologically important inorganic molecules include water, oxygen, carbon dioxide, nitrogen and minerals.

• Atoms in molecules are held together by different kinds of chemical bonds. • The properties of these bonds explain the interactions between molecules in cells.

key questions 1 What are the three parts of the cell theory? 2 Name three features that all cells share. 3 Describe the major differences between prokaryote and eukaryote cells. 4 List the four main elements that are found in living organisms.

8 Molecules of life

5 Distinguish between organic and inorganic molecules. 6 Water, oxygen, carbon dioxide, nitrogen and minerals are inorganic chemical substances that are important to living things. Explain how these chemical substances are different to organic compounds.

1.2

Biologically important inorganic molecules Water—the medium of life Life began in water. Living organisms are usually 70–90% water and the chemical reactions in living organisms take place in a watery medium. Therefore, it is not surprising that the properties of water are important in many biological processes. Water molecues are polar molecules (Figure 1.6c). Hydrogen bonding between water molecules (see page 7) is responsible for many of the biologically important properties of water, such as its solvent properties, high heat capacity, high heat of vaporisation, cohesion and surface tension. Hydrogen bonds between water molecules makes them very cohesive; that is, they have a strong tendency to stick together. The cohesion of water molecules allows thin columns of water to be pulled up tree trunks over 100 m tall without breaking (Figure 1.7a). Water molecules in an overfilled glass also stick together, so that the water can rise above the lip of the glass. The hydrogen bonds between the water molecules at the surface prevent water from spilling over the edge. Bonds between water molecules also cause surface tension, which allows small insects to walk across the surface of water without breaking through the bonds between the water molecules and sinking (Figure 1.7b). (a) (b)

Water has a high heat capacity: it can absorb a great deal of heat with very little increase in temperature. Therefore, heat produced by the activity of cells can usually be absorbed easily without the cells heating up significantly (which can affect chemical processes in cells—see Chapter 2). Water also has a high heat of vaporisation: it requires large amounts of heat to evaporate (change from liquid to gas). So when you sweat, the evaporation of even small amounts of water takes considerable heat from your body and cools you substantially.

Figure 1.7 (a) The strength of the bonds between water molecules produces surface tension, which is strong enough to prevent an insect’s legs sinking between the water molecules. (b) The strong attraction (cohesion) between water molecules holds the water together and allows very thin columns of water to be drawn up the trunks of tall trees.

9 The chemical nature of cells

Water as a solvent

biofile Imagine drying out from about 85% water to just 3% water. This tiny tartigrade (water bear) can do this and survive, often as long as 6–10 years. If the environment dries or freezes, the one millimetre long tartigrade gradually dries out and lives in a state of suspended animation until water becomes available again.

Water is able to dissolve a large number of compounds because water molecules are polar. The compounds that dissolve in water are ionic compounds, meaning that they can split (ionise) into two charged particles (ions). For example, sodium chloride (NaCl or salt) in water ionizes to form Na+ and Cl–. Polar water molecules form weak hydrogen bonds with the ions, which keeps the ions apart and the NaCl in solution (Figure 1.8). (a)

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! pH is the concentration of H+ ions per litre of solution.

10 Molecules of life



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Figure 1.8 (a) Sodium chloride crystal with regularly arranged Na+ and Cl– ions held together by ionic bonds. (b) When water is added, the polar water molecules surround the ions. The slightly negative oxygen ends of water attach to Na+ and the slightly positive hydrogen ends of water attach to Cl–.

Water has a strong tendency to form as many hydrogen bonds as possible. It therefore tends to exclude non-polar molecules (molecules without charge, such as fats and oils) with which it cannot form hydrogen bonds. Because non-polar molecules do not interact with water, they are hydrophobic (‘water-hating’). Polar molecules react readily with water and are hydrophilic (‘water-loving’). Polarity and non-polarity of molecules are fundamental to the structure and properties of biological membranes (see page 20).

pH Water has a tendency to ionise—to split into H+ and OH– ions. pH is the concentration of H+ ions per litre of solution and is a measure of the acidity or alkalinity of a solution. The chemical reactions of cells are very dependent on pH. This is because the structure of proteins, particularly enzymes, are affected by even slight changes in pH (see page 31). Living cells have different ways of maintaining a relatively constant pH. One way is through the use of buffers, which are substances that act as a reservoir for H+ molecules, adding and removing them from solution to maintain a stable pH. The three main buffers in the body are bicarbonate buffers (important in buffering the pH of blood), phospate buffers (important in intracellular buffering) and protein buffers (such as haemoglobin, which has an important role in buffering blood). pH is measured on a logarithmic scale of 0 to 14 (Figure 1.11) where the lower the number the more acidic the solution. Pure water has a pH of 7, which means that there are equal proportions of H+ and OH–. Acidic pH values are less than 7, and a solution with a pH of 6 has ten times the H+ concentration of a solution with a pH of 7. Alkaline solutions have a pH that is higher than 7. Most biological fluids have a pH of between 6 and 8, but there are extremes such as the gastric juices in the stomach, which have a pH between 1 and 2.

HYDROCHLORIC ACID

LEMON JUICE

GASTRIC ACID

P(





BEER COLA



  HYDROGENIONS ;(=MOL, 

TOMATO JUICE



URINE

RAINWATER



 

INCREASINGACIDITY



 

TEARS BLOOD

SALIVA



BAKING SODA

HOUSEHOLD BLEACH

EGGWHITE SEAWATER



 



HOUSEHOLD AMMONIA



 

NEUTRALITY

Oxygen and carbon dioxide In most living organisms, oxygen is needed to release energy from food molecules. A constant supply of oxygen is therefore necessary to maintain the activity of cells. It is usually easy for organisms that get their oxygen from air because the atmosphere is 21% oxygen. However, oxygen is not very soluble in water, so organisms that get their oxygen from water are often small, flat and relatively inactive (Figure 1.12). Larger aquatic animals are highly adapted to be able to extract sufficient oxygen from water; for example, the gills of fishes have very large surface areas and they are very efficient at extracting oxygen from water moving across them.







 

SODIUM HYDROXIDE





  INCREASINGALKALINITY

Figure 1.11 Acidity and alkalinity are measured on a pH scale from 0–14. One division of the scale means a ten-fold difference; that is, a pH of 5 is ten times more acidic than a pH of 6.

Figure 1.12 A black and white Pseudoceros flatworm swimming over white coral.

The carbon of organic compounds is cycled from the atmosphere (see Heinemann Biology 1). About 0.035% of the atmosphere by volume is carbon dioxide and this carbon dioxide is the main source of carbon for the production of the organic molecules from which living organisms are built. The recycling of carbon in ecosystems is therefore important to the survival of all organisms. Photosynthetic organisms trap light and convert carbon dioxide to sugars, some of which are eaten by animals. Carbon dioxide is released back into the atmosphere as an end-product of energy-releasing processes (cellular respiration) in most organisms, and as a result of the decay of organic material by microorganisms.

11 The chemical nature of cells

Nitrogen Nitrogen is a component of all proteins and is therefore required by organisms in large amounts. The atmosphere is largely (78%) nitrogen gas (N2); however, most organisms are unable to use nitrogen in this form. Certain bacteria and cyanobacteria are able to fix nitrogen; that is, they can convert atmospheric nitrogen into compounds, such as nitrates, that are usable by plants. The most important nitrogen fixing bacteria are the symbiotic bacteria found in the roots of plants, including legumes, casuarinas and acacias (Figure 1.13). Plants then absorb these nitrogenous compounds from the soil and use them to make amino acids. Heterotrophs obtain their amino acids by consuming plants and other organisms. They also produce nitrogen rich waste (manure), which has traditionally been used as a plant fertiliser.

Figure 1.13 Nodules containing nitrogen-fixing bacteria on acacia tree roots.

biology in action Industrial nitrogen fixation

Figure 1.14 Rice crop plants.

12 Molecules of life

Nitrogen-fixing organisms make life possible by trapping and ‘fixing’ nitrogen in a form that they, and other organisms, can use. Nitrogen fixation is a process where atmospheric nitrogen gas (N2) is ‘fixed’ by certain bacteria to form ammonium (NH4+) and eventually nitrate (NO3–), which are then absorbed by plant roots. These bacteria may live free in the soil or in association with plant roots. There are strong demands for agriculture to increase food production so we can feed the rapidly expanding human population. However, agricultural practices such as land-clearing have degraded soils. As a solution to both these problems, the prospect of ‘fixing’ nitrogen on a commercial scale is being vigorously pursued. The industrial production of nitrogencontaining fertiliser uses more energy than any other aspect of crop production. Producing 2.5 kg of ammonia fertiliser takes an amount of energy equivalent to burning 1000 kg of coal. Even for bacteria that do it naturally, nitrogen fixation requires a great deal of energy. A considerable effort is being made in the field of genetic engineering to produce new organisms that can fix nitrogen. For example, scientists are trying to introduce genes for nitrogen fixation into crop plants (Figure 1.14) that normally lack root nodules, such ‘BNF rice’ (biological nitrogen-fixing rice). There are many hurdles, such as the requirement that nitrogen fixation takes place under anaerobic conditions. However, the need is great, and as world rice production is second only to wheat production, the reward for success would also be great.

Minerals Mineral salts are naturally occurring inorganic compounds produced by the weathering of rocks. The water-soluble mineral salts produced are absorbed as ions into the roots of plants, making them available to be eaten by animals. Important minerals include phosphorus, potassium, calcium, magnesium, iron, sodium, iodine and sulfur. Minerals required by organisms in lesser quantities include boron, manganese, zinc, molybdenum, copper and chlorine. Humans require more than 20 minerals, some in only minute quantities. In organisms, mineral ions are found in the cytosol of cells, in structural components and in the molecules of many enzymes and vitamins. They may also be incorporated into other important organic compounds in cells. Phosphorus is present in the phospholipids of cell membranes and in ATP (adenosine triphosphate—an important energy carrier in cells, see page 51). Magnesium is an important constituent of chlorophyll, and iron is the central atom in every haemoglobin molecule in red blood cells. Calcium, potassium and sodium ions are important to the normal performance of cardiac muscle cells, and calcium and phosphorous are found in bones and teeth.

biology in action Too much or too little copper Copper is a cofactor in several enzymes, meaning that it is required for the enzyme to function efficiently. Because copper occurs widely in foods, a dietary deficiency of copper is rare in a balanced diet. However, too much or too little copper in the body can result from inherited copper management disorders, which cause serious problems. Wilson’s disease is a copper toxicosis disorder. Normally the liver functions as a copper storage organ and any excess copper is excreted in bile. People with Wilson’s disease have a defective protein needed to excrete copper. Copper therefore accumulates in the liver causing a very serious hepatitis-like disease that may be accompanied by neurological effects in some patients. The opposite problem occurs in Menkes disease—too little copper. Intestinal absorption of copper is defective, resulting in low copper

supply for the tissues and organs. Due to deficiencies in copperdependent enzymes, the major symptoms are found in connective tissues (arteries and bone) and in the brain. It usually leads to death due to the rupture of a weak major artery. Babies with Menkes disease have unusual ‘steely’ or ‘kinky’ hair. Interestingly, it was an observant Australian scientist who noted the similarity between the kinky hair of Menkes sufferers (Figure 1.15) and the wool of sheep grazing on copper-deficient soil. This astute observation led to the discovery that the cause of Menkes disease was too little copper. Both Wilson’s disease and Menkes disease are caused by defects in molecular copper pumps. The two proteins (the pumps) are very closely related, even though they are functionally opposite.

Figure 1.15 Babies with Menkes disease have unusual ‘steely’ or ‘kinky’ hair.

13 The chemical nature of cells

summary

1.2

• Living organisms are usually 70–90% water. The polar nature of water molecules explains many of its biologically important properties • Water is cohesive, has a high heat capacity and is a solvent for polar molecules. • Oxygen is needed in most organisms to release energy from food molecules.

• Atmospheric carbon dioxide is the main source of carbon, which is the key atom in organic molecules. • Nitrogen, which is a component of all proteins, is ‘fixed’ from the atmosphere by certain bacteria. • Minerals are required in lesser amounts and form important parts of organic molecules, such as enzymes and structural molecules.

key questions 7 Give two reasons why water is referred to as the medium of life. 8 Water molecules have a number of properties that make it important in biological processes. These include • cohesion • surface tension • high heat capacity. Explain what is meant by each term. Discuss a specific example to explain its importance. 9 a Use a single sentence to explain what is meant by pH. b What is the pH range for most biological fluids?

10 For living organisms, outline the role of a oxygen b carbon dioxide. 11 Explain the significance of carbon recycling in ecosystems. 12 a Outline the importance of nitrogen for living organisms. b What are nitrogen-fixing bacteria? Why are they important? 13 a Define ‘mineral salts’ b Include specific examples to explain the significance of minerals for living organisms.

1.3 ! Polymers are large organic molecules composed of many smaller molecules joined together.

Nucleic acids

Organic molecules The four main types of organic molecules are carbohydrates, lipids, proteins and nucleic acids (Figure 1.16). Large organic molecules, formed by joining together many smaller molecules, are known as macromolecules or polymers, (poly meaning ‘many’). Proteins

PHOSPHATE

PEPTIDEBONDS

Carbohydrates

NUCLEOTIDE SUGAR MONOSACCHARIDE

DISACCHARIDE

BASES FOURTYPES ! '

AMINOACID SUBUNITS

#

starch

4 Lipids

Figure 1.16 The structures of some organic molecules GLYCEROL

14 Molecules of life

POLYSACCHARIDES THREE MOLECULES OFFATTY ACIDS FATTYACID

cellulose

glycogen

Carbohydrates Carbohydrates are the most abundant organic compounds in nature. They • are an important source of chemical energy for living organisms • are used as energy reserves in plants and animals • form structural components such as cell walls • form part of both DNA and RNA • combine with proteins and lipids to form glycoproteins and glycolipids as in cell membranes. Carbohydrates are found on the surface of every cell in our bodies and are involved in a wide variety of interactions. Cell surface glycoproteins identify a cell as being of a particular type and are important in cell–cell communcation and adherance . Carbohydrates are compounds made of carbon, hydrogen and oxygen. In simple carbohydrates (such as glucose) the hydrogen and oxygen are present in the same proportions as in water: there are two hydrogens for each oxygen atom. The general formula is Cn(H2O)n (for example, glucose is C6H12O6). There are three main groups of carbohydrates—monosaccharides, disaccharides and polysaccharides (Figure 1.16). The basic subunits of carbohydrates are the simple sugars, called monosaccharides, meaning ‘single sugars’. For example, glucose is a simple sugar that is formed during photosynthesis. Common 6-carbon sugars include glucose, galactose and fructose. When two sugars are joined together they form a disaccharide (meaning ‘two sugars’) and a molecule of water is removed. Milk sugar (lactose) is made from glucose and galactose whereas cane sugar (sucrose) is made from glucose and fructose. When many sugars are joined together they form long chains or polymers called polysaccharides (‘many sugars’). Cellulose, the major component of plant cell walls, is the most abundant organic molecule on Earth (Figure 1.17). Starch is the polysaccharide used for energy storage in plants. In animals, the polysaccharide glycogen is used for energy storage. These three polysaccharides are each composed of glucose subunits, but they differ in a number of ways (Figure 1.16). Starch is a long chain molecule, glycogen has a branching structure and cellulose has additional bonds cross-linking between the subunits of the chain. Complex polysaccharides are those that consist of different monosaccharide subunits in the same molecule, such as murein found in the cell walls of bacteria. (a)

Figure 1.17 (a) Close up of cotton seed head. (b) Brightly coloured tunicate found in Sulawesi, Indonesia. Both cotton and the supporting skeleton of tunicates are largely made from cellulose.

(b)

15 The chemical nature of cells

Proteins

biofile Spider silks are proteins and are the strongest natural fibre known. The golden orb-weaver spider uses a silk ‘dragline’ to escape its web in case of danger. The dragline is a mixture of two types of proteins that are dry and essentially indestructible. They are also elastic and exceptionally strong, features that are directly attributable to the protein structure.

Proteins are more complex molecules than carbohydrates and make up over 50% of the dry weight of cells. All proteins contain carbon, hydrogen, oxygen and nitrogen. Many also contain sulfur, and often phosphorus and other elements. There are thousands of different kinds of proteins and their functions vary widely. Enzymes catalyse cellular reactions, hormones communicate information throughout the body of an organism, while other proteins form structural components of cells. Some proteins act as carrier molecules, such as haemoglobin which transports oxygen. Proteins may also form channels in membranes, which is important for the transport of certain molecules across membranes. Proteins are composed of chains of smaller subunits called amino acids. Because amino acids in proteins are linked by a certain kind of bond called a peptide bond, proteins are called polypeptides (polypeptide meaning ‘many peptide bonds’). There are 20 different amino acids commonly found in proteins. While carbohydrates and lipids are similar in most plants and animals, each kind of organism has its own unique proteins. The properties of many proteins are determined by their shape, which is determined by their amino acid sequence. There are four levels of protein structure—primary, secondary, tertiary and quaternary (see Figure 4.11, Chapter 4). Primary structure is the actual sequence of amino acids in a polypeptide. The particular sequence causes arrangements of the polypeptide into secondary stuctures, such as pleating or coiling, held by hydrogen bonds between the amino acids. The protein then folds into its distinctive threedimensional shape, which is its tertiary structure, usually fibrous or globular. Quaternary structure is when two or more polypeptide combine together, such as in the haemoglobin molecule.

Figure 1.18 Golden orb-weaver spider, UK’s heaviest spider, suspended from a ‘silk’ thread made from extremely strong protein.

Nucleic acids Nucleic acids are the genetic materials of all organisms and they determine the inherited features of an organism. There are two types of nucleic acid, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Both are made of long chains of subunits called nucleotides. DNA carries the ‘instructions’ required to assemble proteins from amino acid subunits. RNA molecules play major roles in the manufacture of proteins within cells. This process will be described in more detail in Chapter 2.4. The instructions are called genes and are found in chromosomes. DNA is accurately passed from cell to cell during cell division.

16 Molecules of life

biology in action Right- and left-handed molecules Before Louis Pasteur completed his famous work on microbes and the cause of disease (see page 157), he identified a fundamental principle of chemistry that has important implications in biology. He found that the same molecule can exist in two forms that are mirror images of each other (Figure 1.19): right-handed and left-handed molecules (like a pair of gloves). Labels of chemicals or drugs denote these forms as dextro- or d-molecule and laevo- or l-molecule (from the Latin, dextra meaning ‘turning to the right’ and laevus meaning ‘turning to the left’). These are referred to as optical or stereo-isomers and their study as stereochemistry. Why is stereochemistry important in biology? Molecules synthesised by organisms are usually of one form only and they can only utilise that form. Organisms usually make sugars in the d-configuration whereas proteins are made in the l-configuration. We can’t digest ‘wrong-handed’ sugars. One form of phenylalanine makes artificial sweeteners sweet and the other form is bitter. Thalidomide, the anti-morning sickness drug, caused its terrible effects on unborn babies in the early 1960s because, while one of its isomeric forms was effective against morning sickness, the other was teratogenic—causing serious damage in early embryonic growth. When chemicals are manufactured in a laboratory, both right- and left-handed forms are produced in equal amounts, and they are hard to separate on a commercial scale. Even if it were possible to administer only the correct form of thalidomide, it has been shown that the isomers are converted to each other in vivo (within the body), so both forms would be produced in the body and the teratogenic effect would still occur.

Figure 1.19 This left hand and its mirror image illustrate how the same parts of a structure, such as a hand or a molecule, can exist in two different shapes. In molecules these are known as stereoisomers.

Lipids Lipids are ‘fatty’ substances and are non-polar hydrophobic molecules. This gives rise to their critical role in living organisms—they can form an effective barrier between two watery environments. Lipids are the major component of cell membranes, whose role is to regulate movement into and out of the cell between the watery intracellular and extracellular environments. Lipids include fats and oils (important as energy-storing molecules), phospholipids (the important component of cell membranes) and steroids (hormones and vitamins). They are relatively small molecules and vary widely in structure. There are two general forms of lipids—simple and compound. Simple lipids are composed of carbon, hydrogen and oxygen, but in different proportions to carbohydrates (they have a much smaller proportion of oxygen). Simple lipids include fats, composed of fatty acids and glycerol (Figure 1.16), and steroids, such as cholesterol and the hormones cortisone and testosterone. Fatty acids may be saturated or unsaturated. A saturated fat has the maximum number of hydrogen atoms (it is ‘saturated’ with hydrogen atoms), with no double bonds ( –C=C– ) between carbon atoms in the chain (Figure 1.20). An unsaturated fat is one where there is at least one double bond between the carbon atoms in the chain. Steroids have quite a different structure to fats but they are also insoluble in water. Compound lipids contain fatty acids, glycerol, as well as other elements such as phosphorus and nitrogen. These include the phospholipids of biological membranes. Phospholipids have a hydrophilic end (the ‘phospho’ end) and a hydrophobic end (the lipid end). Their fundamental role in membrane function is described in the next section.

biofile Lorenzo’s oil, an unpalatable mixture of oils and the subject of a film by the same name, might just work after all. ALD (adrenoleukodystrophy) is a genetic defect, usually affecting males, which results in the progressive loss of ability to move, hear, speak and finally breathe. It involves the progressive loss of the myelin sheath that insulates nerve fibres (see Chapter 6) so they can no longer conduct action potentials properly. In a ten year scientific study, more than a hundred boys with the defect but not displaying any symptoms were given Lorenzo’s oil. Three quarters of the boys who took the oil regularly were still symptomless after ten years. However, of those who took the oil irregularly, only a third remained symptomless. ALD is associated with extremely high levels of long-chain fatty acids in the blood. The oil apparently blocks the enzymes that produce the long-chain fatty acids, bringing blood levels back to normal. How this prevents the development of symptoms is still unknown.

17 The chemical nature of cells

biology in action What’s so special about omega-3 fatty-acids? Omega-3 and omega-6 fatty acids are essential fatty acids, meaning that we need them but cannot synthesise them from other compounds, so we must obtain them from our diet. They are also both unsaturated fats, with at least one double bond between the carbon atoms in the chain. A double bond makes a ‘kink’ in the molecule shape, and the fatty acid does not align with adjacent fatty acids in membranes (Figure 1.20). A polyunsaturated fat has more than one double bond in the chain. The more kinks in the chains, the more flexible and permeable (leaky) the membrane. #//( #( #( #( #( #( #( #( #( #( #( #( #( #( #( #( #( #(

STEARICACID

A 3ATURATEDFATTYACID

Figure 1.20 Structures of saturated and unsaturated fatty acids. The –C=C– creates a rigid kink in the chain, whereas the other –C–C– bonds are free to rotate. (a) Stearic acid is saturated (no double bonds between carbon atoms). (b) Oleic acid is mono-unsaturated, meaning that it has one double bond. The omega carbon (ω) is at the opposite end to the acid group (COOH), so oleic acid is a omega-9 fatty acid. (c) Poly-unsaturated fatty acids: linolenic acid (an omega-6 fatty acid) and alpha-linoleic acid (an omega-3 fatty acid).

Figure 1.21 Fish contain high levels of omega-3 fatty acids. Research suggests that increasing our consumption of fish will lead to reduced mortality from several diseases.

18 Molecules of life

OLEICACIDOMEGA B -ONO UNSATURATEDFATTYACID

LINOLEICACIDOMEGA 

ALPALINOLEICACIDOMEGA 

C 0OLY UNSATURATEDFATTYACID

Omega is the name given to last carbon atom in a fatty acid chain (from the Greek alpabet, omega is the last letter) (Figure 1.20). An omega-3 fatty acid has a double bond (and kink) between the third and fourth carbon from the omega end, while omega-6 has a double bond between the sixth and seventh carbons. Research has shown that excessive amounts of omega-6 fatty acids (or a high omega-6 to omega-3 ratio in the diet) is linked to cardiovascular disease, cancer, inflammatory diseases and autoimmune diseases. Usual Western diets have an omega-6 :omega-3 ratio of about 10 :1, whereas diets of only 4 :1 or 2 :1 have been associated with reduced mortality from these diseases. Omega-3 fatty acids are found in fish, some seeds (such as flaxseed) and nuts. Omega-6 fatty acids are found in cereals, eggs, poultry and most vegetable oils. (See also Leaky membranes, lipids and metabolic rates, page 66).

summary

1.3

• Macromolecules (polymers) are large organic molecules formed by joining together many smaller molecules. • The four main types of organic molecules are carbohydrates, lipids, proteins and nucleic acids. • Carbohydrates are the most abundant organic compounds in nature. Their general formula is Cn(H2O)n. They are grouped into monosaccharides, disaccharides and polysaccharides and have many different properties. • Proteins are more complex molecules than carbohydrates or lipids, and make up over 50% of the dry weight of cells. All proteins contain carbon, hydrogen, oxygen and nitrogen; many also contain sulfur, phosphorus and other elements.

• Proteins are chains of amino acids known as polypeptides. The properties of proteins are determined by their shape, which is determined by their amino acid sequence. • The nucleic acids DNA and RNA are the genetic materials of organisms and they determine inherited features. • Lipids are non-polar hydrophobic molecules and can form an effective barrier between two watery environments. They have a much smaller proportion of oxygen than carbohydrates, and often contain other elements, such as phosphorus and nitrogen. • Lipids include fats and oils (important as energy-storing molecules), phospholipids (the important component of cell membranes) and steroids (hormones and vitamins).

key questions 14 a What is a polymer? b Distinguish between the terms monosaccharide, disaccharide and polysaccharide. 15 a In a discussion of proteins, what is meant by i amino acids? ii peptide bond? iii polypeptide? b Use a single sentence complemented by a simple diagram to explain each of the following terms: i primary structure ii secondary structure iii tertiary structure iv quaternary structure. Type of organic compound

Elements that make up compound

16 a What are the two different forms of nucleic acid? b Outline the role of each of these different kinds of nucleic acids. 17 a Define ‘lipid’. b Outline the differences between ‘simple’ and ‘compound’ lipids. 18 a Why are some fatty acids called ‘essential fatty acids’? b Outline the difference between omega-3 and omega-6 fatty acids. Why are they important. 19 Copy the following table and complete the summary of biologically important organic compounds.

Role of compound in living organisms

Carbohydrate

Examples of compound glucose

Protein DNA, RNA Lipid

1.4

Biological membranes Perhaps the most important part of a cell is the plasma membrane. It encloses the contents of cells and allows the cytosol (the liquid part of the cytoplasm) to have a different composition from the surrounding external environment by selectively regulating the movement of substances into and out of the cell. Most organelles of eukaryotes, including the nucleus, endoplasmic reticulum, mitochondria, chloroplasts, lysosomes and vacuoles, are also formed from

19 The chemical nature of cells

membranes. These membranes form discrete compartments within the cell and control the movement of substances between these compartments. As a result the chemical contents of various organelles are different. Membranes: • permit selective control of molecules entering and leaving cells • are active environments in which many essential chemical reactions of life occur • establish compartments within the cell, thereby separating hereditary material (DNA), cytosol, lysosomal enzymes, secretory products of cells, and energy-processing materials in mitochondria and chloroplasts • restrict movement of substances between one part of a cell and another, thereby permitting regulation of the many enzymatic processes that take place within the cell • have protein receptors involved in intercellular communication (directly between adjacent cells, and by hormones and nerves) • are involved in cell–cell recognition • produce electrical activity in excitable cells.

biofile Because of the fluidity of cell membranes, a lipid molecule may travel from one end of a bacterial cell membrane to the other in about one second.

Membrane composition The plasma membrane is 7–9 nm thick. (A nanometre, nm, is 10−9 of a metre.) It is somewhat thicker than the membranes of intracellular organelles; for example, nuclear and endoplasmic reticulum membranes are 5–7 nm thick. Otherwise, the basic structure of all biological membranes is the same. They are composed of two layers of phospholipid molecules, associated with other molecules including proteins, carbohydrates and cholesterol, as shown in the fluid-mosaic model (Figure 1.22). Phospholipid molecules have one end that

OUTSIDE THE CELL

HYDROPHYLICZONES OFPROTEINS HYDROPHOBICZONES

CARBOHYDRATE

Figure 1.22 Biological membranes are composed of a phospholipid bilayer with large protein molecules embedded in the bilayer. Some of these proteins provide channels for the passive and active movement of certain molecules across the cell membrane. Short carbohydrate molecules attached to the outside of the membrane are involved in cell recognition and cell adhesion.

PHOSPHOLIPID BILAYER

INSIDE THE CELL

PROTEINS

CHOLESTEROL

is hydrophobic (water-hating) and the other end hydrophilic (water-loving). This means that, when in contact with an aqueous solution, phospholipid molecules line up with their hydrophobic tails pointing away from the solution (Figure 1.23). The impermeability of membranes to water-soluble (polar) molecules is due to the phospholipid bilayer. Most other membrane functions are carried out by the proteins, which are located throughout the membrane; hence, the term ‘mosaic’.

20 Molecules of life

Figure 1.23 When in contact with an aqueous solution, phospholipid molecules line up with their hydrophobic ‘tails’ pointing away from the aqueous solution. (a) At an oil/water interface, this results in a monolayer. (b) In water, if the tails are short, the phospholipids spontaneously form a spherical ‘micelle’. (c) If the tails are longer, the phospholipids aggregate to form a bilayer membrane. Soaps and detergents cause fats to form micelles.

Membranes are fluid structures: individual lipid molecules (and some of the proteins) are free to move about within the layers. Membranes also contain large numbers of cholesterol molecules located between the phospholipid molecules, which makes the membrane less fluid and more stable. Without these cholesterol molecules, the membrane breaks down rapidly and releases its contents. Cholesterol also decreases the permeability of the membrane to small water-soluble molecules. Protein molecules in the membrane may cross both phospholipid layers, or be confined to only one layer (Figure 1.24a). Like phospholipid molecules, they are able to move about to some extent, but this movement may be limited to particular regions of the cell membrane. Proteins provide the channels through which water-soluble molecules and ions pass. Facilitated diffusion (passive movement) and active transport (requiring energy) occur through selective channels formed by membrane proteins. Membrane proteins may also be pumps that move ions across membranes, and enzymes that catalyse membrane-associated reactions. For example, the final digestion of some food molecules occurs as they pass through the membrane of cells lining the gut (gut epithelium). Carbohydrates associated with plasma membranes are usually found on the outer surface of the membrane, linked to protruding proteins. They play a role in recognition and adhesion between cells, and in the recognition processes that occur between cells and antibodies, hormones and viruses.

(a)

LIPID SOLUBLE MOLECULES ALCOHOL CHLOROFORM

SMALL UNCHARGED MOLECULES WATER UREA OXYGEN CARBON DIOXIDE

CERTAIN WATER SOLUBLE MOLECULES SOMEIONS ANIMOACIDS MONOSACCHARIDES

OUTSIDE CELL

OIL

WATER

(a)

MONOLAYER

(b) MICELLES

(c)

BILAYERMEMBRANE

MOST WATER SOLUBLE MOLECULES PROTEINS SUGARS IONS

PROTEIN CHANNELS

(b)

WATER OXYGEN

CARBONDIOXIDE

MONOSACCHARIDES NITROGENOUS WASTE EG AMMONIA UREA

AMINOACIDS

LIPIDS INSIDE CELL

PHOSPHOLIPID MOLECULE

VARIOUS IONS HYDROPHILIC gWATER LOVINGg END HYDROPHOBIC gWATER HATINGg END

PROTEIN MOLECULE

CARBOHYDRATE CHAINS

Figure 1.24 (a) Cells exchange many substances within their environment across the cell membrane. (b) Pathways for movement of substances across the cell membrane.

21 The chemical nature of cells

technologies and techniques Killer molecules —How toxins and antibiotics kill cells by Professor Frances Separovic

Professor Frances Separovic Professor Frances Separovic has a degree in mathematics, a PhD in physics and is now a biophysical chemist working on cell membranes at the University of Melbourne. This broad background undoubtedly helps her in the exciting multidisciplinary research in which she is engaged.

T

oxins, like melittin in the sting from the honeybee, and antibiotic peptides, like gramicidin A, can kill cells. How do they do this? To find out, I used nuclear magnetic resonance spectroscopy (NMR) and other biophysical techniques to study the structure and dynamics of membrane components in situ (as they naturally occur) and the effects of these molecules on cell membranes First, I determined the three-dimensional structures of melittin and the antibiotic gramicidin A in model cell membranes. Model membranes are made using phospholipids from cell membranes, which spontaneously form lipid bilayers in water, and incorporating the peptide under study into the lipid bilayers. Melittin is a 26 amino acid peptide that forms trans-membrane α−helical structures when in a lipid bilayer. These helices aggregate (come together) and form a pore in the membrane, which lyses the cell. Gramicidin A is made by the bacteria Bacillus brevis and is a 15 amino acid peptide of alternating l- and d-amino acids (see page 17). When incorporated in a lipid bilayer membrane, gramicidin A forms a β-helix.

Two gramicidin A molecules line up, span the membrane and together form an ion channel through which monovalent cations can pass. This upsets the ionic balance of a cell and can kill it. We are now using these techniques to study the three-dimensional structure of larger proteins, including membrane receptors, ion channels and other toxins. One biomolecular engineering application arising from this work has been the development of a tiny biosensor device for medical diagnostics. The gramicidin peptide, modified for use in a tiny device to identify the presence of particular molecules (Figure 1.25), is incorporated into a lipid bilayer membrane, which is supported on a gold electrode. A linker (shown in stick formation) is covalently attached to the gramicidin A and linked to a specific receptor. When a molecule of interest binds to the receptor, the ionic current across the membrane is disrupted and the device senses the presence of the molecule. The development of this device involved working with a team of synthetic chemists, biophysicists, biochemists, material scientists and electronic engineers.

Figure 1.25 A space-filling model of an ion channel formed by two molecules of gramicidin A as determined by NMR spectroscopy. The stick regions are added to link the channel to a receptor.

22 Molecules of life

Molecules crossing membranes The plasma membrane regulates the movement of molecules into and out of the cell (Figure 1.24b). This movement depends on the composition of the membrane and the surface area available for exchange (Figure 1.24a). One of the most important properties of membranes is their lipid nature, which makes them impermeable to most water-soluble molecules, ions (molecules with an overall positive or negative charge) and polar molecules (molecules with charged regions but no overall charge). These substances require specific channels (made from protein molecules) to pass through the plasma membrane. In summary: • Lipid-soluble substances of various sizes, such as chloroform and alcohol, are able to simply dissolve into the phospholipid bilayer and pass easily through membranes. • Tiny molecules, such as water and urea, can pass between the phospholipid molecules. • Small uncharged molecules, such as oxygen and carbon dioxide, can also pass through the phospholipid bilayer. • Larger water-soluble substances, including amino acids and simple sugars, pass through channels made from protein molecules. Protein channels may be selective for particular substances, and they may require the expenditure of energy for transport to occur.

biofile Alcohol enters your blood more quickly than most foods for two reasons. Alcohol does not need to be digested (broken down into a smaller molecules) and, because it passes through membranes easily, it is absorbed rapidly in the mouth and the stomach. Eating a meal before drinking alcohol reduces the efficiency and rate of alcohol absorption.

Diffusion All molecules in a solution move about at high speeds and in random directions. There are millions of collisions every second, which means the movement of individual molecules in any one direction is very slow. As a result of these movements, all molecules in the solution will become evenly dispersed throughout the space available. This random movement of molecules results in the net movement of molecules from a region of high concentration to a region of lower concentration—this is diffusion. Diffusion itself is a passive process; it is driven by the concentration difference and requires no further input of energy. The larger the difference in molecular concentrations—that is, the concentration gradient—the more rapid the rate of diffusion. Diffusion also occurs across membranes, provided the diffusing molecules can pass through the membrane. Lipid-soluble substances diffuse through the lipid bilayer. Tiny molecules and water molecules diffuse freely between the lipid molecules. Membrane proteins provide channels that allow all polar molecules and ions below a certain size to diffuse through. If the numbers of molecules (concentrations) are the same on both sides of a membrane, there will always be about the same number passing in either direction. That is, there will be no net movement from one side to the other. However, if the concentrations of a particular molecule are different on either side of the membrane, more molecules will move from the more concentrated region to the less concentrated region than in the opposite direction. There will be a net movement of molecules into the more dilute solution (until equilibrium is reached). For example, in active tissues, oxygen moves out of the blood into the surrounding fluid (interstitial fluid) and carbon dioxide moves into the blood by diffusion along concentration gradients. In the lungs, the reverse exchange takes place along the concentration gradients for oxygen and carbon dioxide between blood and air.

! Diffusion is the passive movement of molecules along a concentration gradient, from a region of high concentration to a region of low concentration

23 The chemical nature of cells

Osmosis ! Osmosis is the passive movement of water through a partially permeable membrane, from a region where there are more free water molecules to a region where there are fewer free water molecules.

Osmosis is a special case of diffusion that occurs across partially permeable membranes. Partially permeable (also sometimes called semipermeable or differentially permeable) membranes allow free passage of water molecules (and certain other molecules such as urea), but restrict the passage of most solutes. Because water molecules bind to solute molecules in solution, there are more free water molecules in a dilute solution than in a concentrated solution. When dilute and concentrated solutions are separated by a partially permeable membrane, free water molecules cross the membrane in both directions. Because there are more free water molecules in the less concentrated solution, there will be a net movement of water from the dilute to the concentrated solution (Figure 1.26). This is osmosis. It is the passive movement of water, through a partially permeable membrane, from an area where there are more free water molecules to an area where there are fewer free water molecules. In other words, it is water diffusing along its own concentration gradient.

FREEWATER MOLECULE

PARTIALLY PERMEABLEMEMBRANE Dilute sugar solution

Concentrated sugar solution

SUGAR MOLECULE HYDRATED SUGARMOLECULE

Figure 1.26 Osmosis is the net movement of free water molecules from a dilute solution through a partially permeable membrane to a concentrated solution.

HIGHCONCENTRATION OFFREEWATER MOLECULES

LOWCONCENTRATION OFFREEWATER MOLECULES

NETMOVEMENTOFFREEWATERMOLECULES

RATEOF TRANSFER THROUGH CHANNEL

#ALONE "ALONE

For example, absorption of water from food in the gut and reabsorption of water during urine formation in the kidneys both occur by osmosis. The opening and closing of stomata in leaves is the result of rapid osmotic movement of water into and out of guard cells (see Chapter 5, page 121).

Protein-mediated transport "INPRESENCEOF#

!ALONE CONCENTRATIONOFSUBSTANCE

Figure 1.27 Facilitated diffusion and active transport occur through protein channels. They both show: • selectivity—some substances (B and C) are transported, others (A) are not • saturation—no increase in rate of transfer after all the channels are occupied • competition—inhibition of transport by a related substance that can use the same channel (B in presence of C).

24 Molecules of life

Membrane proteins form selective channels or gates that permit or enhance the passage of specific ions and molecules. There are two means by which this transport can occur: facilitated diffusion and active transport (Figure 1.27). In each of these: • transport is more rapid than by simple diffusion • the channels are specific for particular molecules, so transport is selective— some substances are transported and others are not • the channels become saturated (fully occupied) as concentration of the transported substances increases • transport of one substance is inhibited by the presence of another substance able to use the channels as a result of competition for available channels. The principal difference between the two mechanisms is that active transport requires the expenditure of energy whereas facilitated diffusion does not. Consequently, active transport can move substances against a concentration gradient whereas facilitated diffusion cannot.

Diffusion is a slow process and, even if facilitated, it can only move substances down a concentration gradient. Many substances needed by organisms are required in much greater amounts than can be provided by diffusion alone, and these substances often need to be accumulated into cells against the prevailing concentration gradient. In this case, energy must be expended to actively move the required substances across cell membranes through protein channels. ‘Active’ means that energy is expended. Active transport mechanisms are important, for example, in the uptake of ions by the roots of plants and of digested food molecules from the gut of animals. It is worth remembering that there are no mechanisms for actively transporting water molecules across cell membranes. Net movement of water across membranes always occurs by osmosis.

summary

! Facilitated diffusion occurs through protein channels, is faster than diffusion and is passive.

! Active transport occurs through protein channels, is faster than diffusion, requires energy and can move molecules against a concentration gradient.

1.4

• Cells are composed of cytoplasm enclosed within an outer phospholipid plasma membrane. Organelles are subcellular structures involved in particular functions of the cell. • Most organelles of eukaryotes are formed from membranes, which form discrete sub-cellular compartments. • Phospholipid membranes are relatively fluid and selectively regulate the movement of substances into and out of the cell, and between the sub-cellular compartments. • Lipid-soluble substances are able to simply dissolve into and through membranes. Tiny molecules, including water, can pass between the phospholipid molecules. Small uncharged molecules can also pass through the phospholipid bilayer. Larger water-soluble substances may pass through selective protein channels.

• Diffusion is the passive movement of molecules along a concentration gradient, from a region of high concentration to a region of low concentration. Facilitated diffusion occurs through protein channels and is faster than simple diffusion. • Osmosis is the passive movement of water through a partially permeable membrane, from a region where there are more free water molecules (low solution concentration) to a region where there are fewer free water molecules (high solute concentration). • Active transport occurs through protein channels, is faster than diffusion, requires energy and can move molecules against a concentration gradient.

key questions 20 a What are the functions of cell membranes? b Explain why the structure of the membrane is described as a ‘fluid mosaic’? 21 You are asked to give a three-sentence summary to the class on ‘The structure of the plasma membrane’. What would you say? 22 Explain how the following affect the ability of a molecule to pass across a cell membrane: a size b charge c solubility (e.g., in lipids).

23 a Define diffusion. b ‘Diffusion is a passive process.’ Explain. 24 Explain the difference between: a diffusion and osmosis b diffusion and active transport c diffusion and facilitated diffusion d active transport and facilitated diffusion. 25 Why do cells such as those on the surface of a root expend energy to take up some substances?

25 The chemical nature of cells

01

key terms cell cell theory prokaryote eukaryote organelle centriole chloroplast cytoplasm cytosol endoplasmic reticulum Golgi apparatus lysosome

mitochondrion nucleus plasma membrane plastids ribosome tonoplast vacuole vesicle cell wall atom element molecule

compound organic compound inorganic compound pH hydrophobic hydrophillic polymer carbohydrate protein lipid nucleic acid omega-3 fatty acid

1 Classify the following terms as structures or processes: active transport, lysosome, diffusion, nucleus, mitochondrion, ribosome, osmosis, centriole. 2 You have been set the task of determining whether a sample contains plant or animal cells. What features of the sample would help you in this task? (Hint: You could look for structural or chemical features.) 3 a Find out why some elements important to the well-being of organisms are referred to as ‘trace elements’. b Which trace elements are important for normal functioning in i humans? ii plants? Include the role of the trace element in each case. 4 Prepare a simple diagram which illustrates the different forms of nitrogen in ecosystems and how it is made available to living organisms. 5 Carbohydrates are composed of carbon, hydrogen and oxygen, which are always in the same ratio in a given carbohydrate molecule. The general formula for carbohydrates is Cn(H2O)n. Use the information provided to write the correct formula for each compound: a glucose: C6H2nOn b maltose: CnH24On 6 a Make a list of the lipid-containing products in your pantry and refrigerator at home. For each item, state the percentage of i saturated fats ii polyunsaturated fats. b Find out which foods are high in omega-3 fatty acids and which are high in omega-6 fatty acids. 7 Prepare a ‘fact sheet’ that could be distributed by pharmacies informing the public about omega-3 and omega-6 fatty acids in the diet. Include definitions, clear explanations and diagrams. Discuss any benefits and disadvantages associated with the inclusion of these fatty acids in the diet.

omega-6 fatty acid phospholipid fluid-mosaic model diffusion concentration gradient osmosis partially permeable protein-mediated transport facilitated diffusion active transport

worksheet 01

8 Explain why tadpoles living in a puddle of water may die well before the water has completely dried up. 9 In the experiments shown below, what were the original concentrations of solutions A, B, C and D? Explain your reasoning. !

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SUGARSOLUTION

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10 Many biological functions depend on the properties of membranes. a Give two reasons why alcohol is absorbed more rapidly from the gut than most foods eaten at the same time. b Suggest why a breathalyser is able to give a relatively accurate indication of blood alcohol level. 11 Chloroform and ether quickly induce unconsciousness. What chemical property do they have which explains their rapid absorption and thus rapid effect? 12 If a drowning person inhales fresh water into their lungs, death occurs rapidly in about three minutes. If a drowning person inhales sea water instead of fresh water, death occurs more slowly taking about six to eight minutes. Use your understanding of osmosis to explain the difference between inhaling fresh water and sea water. You will need to consider the relative salt concentrations of sea water (1100 mOsm), blood (300 mOsm) and fresh water (0 mOsm). 13 Describe the biologically important properties of water.

26 Molecules of life

page 26.indd 26

20/3/06 9:31:16 AM

0

chapter 02

Enzymess and other biomolecules

key knowledge • enzymes—organic catalysts in biochemical processes, factors affecting enzyme action • biochemical processes—activation energy, anabolic and catabolic reactions • synthesis, packaging and transport of biomolecules • polymers including carbohydrates, polypeptides, nucleic acids and lipids • the roles of organelles and membranes

chapter outcomes After working through this chapter you should be able to: • define metabolism • state what an enzyme is and what it does • explain why enzymes are essential to the function of living organisms • explain the effect of enzymes on activation energy • describe five factors that affect the rate of an enzyme-catalysed reaction • list some uses of enzymes in industry • give examples of how lack of proper enzyme function can cause disease • describe, with examples, the roles of anabolic and catabolic reactions in cells • describe the roles of the nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus and lysosomes in protein production, handling and export • describe the roles of the endoplasmic reticulum and Golgi apparatus in the synthesis of other biomolecules.

2.1

Enzymes and cellular processes

! Enzymes are catalysts that speed up biochemical reactions.

Figure 2.1 The specialised cells in both these animals will be working in similar ways. Both will have high blood levels of adrenaline and high heart rates, but for very different purposes.

! Metabolism is the overall chemical activity of cells.

! A substrate is the molecule on which an enzyme acts.

28 Molecules of life

We know that cells are the basic functional units of living organisms, and that some are highly specialised. But what do cells do? Different types of cells carry out particular specialised functions, but certain basic processes must be performed by all cells. Cells must obtain nutrients, grow, maintain and repair themselves, provide energy for movement and metabolism, and eliminate wastes. These activities require the production of a variety of biological molecules (biomolecules), which are then assembled into new organelles or used for repair and maintenance of cells. Specialised cells include signalling cells and responsive cells. Signalling cells, such as adrenaline-releasing cells in the adrenal gland, produce and release signal molecules (in this case the hormone adrenaline, Figure 2.1). Responsive cells, such as heart muscle, produce specialised receptors to which the signal molecules (adrenaline) attach (causing increased heart rate). In this example, contractile proteins enable the heart muscle cells to shorten (contract). Particular proteins called enzymes control the synthesis of these various biomolecules and many other cellular processes, such as cellular respiration, which keep the cells alive. This chapter is about how enzymes are used in the metabolism of the cell, and about the production and handling of biomolecules.

Metabolism Metabolism is the overall chemical activity of cells. It includes the manufacture (synthesis) of organic molecules, various energy transforming and recycling processes, and the breakdown of unwanted substances. These chemical reactions involve hundreds of enzymes working in ‘chains’, where the product of one reaction is the substrate for the next enzyme. A substrate is the molecule on which an enzyme acts. These chains of reactions are known as metabolic pathways. There are more than 1000 enzymes inside each cell, and many different reactions may be taking place simultaneously. This is possible because cytoplasm is subdivided into organelles (page 39), and the enzymes are located at specific sites within cells. For example, the enzymes necessary for the breakdown of glucose to release energy in glycolysis are in the cytoplasm, but those required for aerobic respiration are in the mitochondria (see Chapter 3).

Enzymes are biological catalysts Enzymes act as catalysts for chemical reactions in the body. A catalyst speeds up (catalyses) chemical reactions that would otherwise take place, but much more slowly. They help reactions progress from the beginning to the end. If there is no enzyme present, a reaction would still run to completion but would take much longer. Enzymes: • are proteins • are substrate specific • can be reused over and over again • are needed in small amounts and are neither reactants nor products • make a reaction take place more easily (they reduce the activation energy, Figure 2.3) • can catalyse a reaction in either direction (most chemical reactions are reversible) • do not change the direction of a reaction • do not change the final amount of product. Enzymes increase the rate of chemical reactions by millions of times. For example, the combination of carbon dioxide with water is catalysed by the enzyme carbonic anhydrase. CO2 + H2O

H2CO3

Without the enzyme, this reaction might produce 200 molecules per hour. With the enzyme, 6 000 000 molecules are produced per second—the reaction is about 10 million times faster. Enzymes are not used up in the reaction and they remain the same at the end of the reaction, although they may have been temporarily altered during the reaction. Enzymes can therefore be used over and over again. Enzymes are proteins and their actions are generally specific, meaning that each enzyme usually catalyses only one type of reaction. This specificity is related to the three-dimensional structure of the molecules. The active site on an enzyme is the part of the molecule that interacts with the substrate. It has a shape that complements the shape of the binding site of the substrate; that is, they ‘fit together’ like pieces of a jigsaw puzzle. Starch-digesting enzymes cannot break cellulose apart because the glucose units in starch and cellulose are joined together differently, therefore they have differently shaped enzyme binding sites. The mechanism of an enzyme binding with a substrate has been referred to as a ‘lock-and-key’ interaction, though more recent evidence supports an ‘induced-fit’ mechanism (Figure 2.2). In the lock-and-key mechanism, substrate molecules have the right shape to fit an enzyme. In the induced-fit mechanism, the actual interaction between substrate and enzyme changes the shape of the enzyme to produce the right fit. (a)

3 3

3

3

biofile In one second, urease breaks down an amount of urea that would take 3 million years to break down spontaneously!

0

%

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Lock and key

(b) 3 3

%

3

3

0

% %

Figure 2.2 There are two major theories of enzyme action: (a) the lock-and-key and (b) the induced-fit mechanisms of enzyme–substrate interaction.

Induced fit

29 Enzymes and other biomolecules

Activation energy and enzymes Chemical reactions involve breaking and remaking chemical bonds. The molecules involved require a certain amount activation energy to get the reaction started, even if the reaction eventually releases energy (Figure 2.3). In any group of molecules, some will have more energy than others, and some of those will reach activation energy allowing the reaction to occur. Enzymes act by reducing the amount of activation energy required for a particular reaction to occur, allowing the reaction to take place more easily.

Figure 2.3 The addition of a catalyst (enzyme) reduces the amount of energy needed to initiate a reaction. Only the activation energy is changed, the change in free energy ( G) is not altered. 4OTALENERGY

ACTIVATIONENERGY WITHOUTENZYME WITHENZYME INITIALSTATE

'

FINALSTATE

0ROGRESSOFREACTION

Factors affecting enzyme activity %NZYME ACTIVITY 

CRITICALTEMPERATURE



Enzyme action involves specific binding between enzyme and substrate molecules. Because of this, any condition that changes the shape of an enzyme molecule will affect the activity of the enzyme and therefore the rate of the reaction. Chemical reactions are affected by the amount of substrate or enzyme present, or whether there is an accumulation of product. However, given unlimited time, the final amount of product is not affected by the amount of enzyme present.

Temperature 





 4EMPERATURE ª#

/PTIMAL TEMPERATURERANGE

Figure 2.4 The rate of enzyme activity increases with increasing temperature until the enzyme begins to break down (denature), in this case at approximately 45°C.

! Denaturation is an irreversible change in protein structure.

30 Molecules of life

Enzymatic reactions are affected by temperature (Figure 2.4). For a reaction to take place at all, the enzyme and substrate must first come into contact. Warming increases the rate of most chemical reactions, including enzymecatalysed reactions. This is because the extra heat energy is taken up by the molecules so they move faster, which increases the rate of interaction between substrate and enzyme. However, too much heat can damage the structure of an enzyme. Enzymes are proteins, and all proteins are denatured by heat when a certain critical temperature is reached. Denaturation is an irreversible change in protein structure (often a loss of the correct folding of the molecule). Boiling denatures most enzymes, which is why some vegetables are blanched in boiling water before being frozen. The boiling water deactivates enzymes that would otherwise cause the vegetable to deteriorate during storage. Most enzymes have an optimum temperature range, which is the temperature at which the enzyme’s catalytic activity is greatest. This is the

range immediately below the critical temperature at which denaturation of enzyme molecules begins to occur. It is a balance between the general increase in reaction rate with increasing temperature, and the increasing rate of denaturation of enzyme molecules that occurs around the critical temperature. Optimal temperatures for particular enzymes may vary in different species and are related to the normal body temperature of the organism (Figure 2.5). In the case of ectotherms, enzyme optimal temperatures are related to the environment in which the organism lives. %NZYMEACTIVITY TRYPSIN

Figure 2.5 Optimum temperatures for the enzyme trypsin isolated from different organisms are related to the optimum body temperature for each organism. Perch are warm-water fish. TROUT





PERCH







DOG







"ODYTEMPERATUREª#

Some bacteria, such as thermophilic (‘heat-loving’) bacteria living in hot springs (Figure 2.6), have enzymes that function at high temperatures. Some are active even at the temperature of boiling water. These bacteria are of particular interest to scientists because enzymes that operate in more ‘harsh’ environments could be useful for various biotechnological processes. The temperature range of the bacterium Thermus aquaticus is about 50–80°C. It is used to make Taq polymerase, a key component of the polymerase chain reaction (PCR). In order to copy DNA and amplify it using the PCR (page 252), an enzyme (DNA polymerase) that is active at high temperatures is needed. The DNA polymerase of Thermus aquaticus called Taq polymerase meets this criteria. This enzyme is widely used in medical diagnosis and forensics, and has become the basis of a US$300 000 000 industry.

Figure 2.6 The hot waters of Grand Prismatic Spring at Yellowstone National Park, USA.

pH The three-dimensional structure of proteins is affected by pH (page 10). In the case of enzymes, altering the pH may change the shape of the binding (active) site and so alter the ‘fit’ between enzyme and substrate. The optimal pH is that at which the fit is best and so the enzyme’s activity is greatest. For many enzymes this is a neutral or slightly acidic pH. In the case of digestive enzymes in humans, trypsin, which is released into the intestine, has a high optimal pH (alkaline pH), whereas pepsin, which is released into the acid environment of the stomach, has a very low optimal pH (Figure 2.7). %NZYME ACTIVITY

PEPSIN









UREASE





biofile In humans, the critical temperature at which enzymes are denatured is about 45°C. The inability of enzymes to function correctly is one of the reasons we feel sick and lethargic when running a high temperature. At very low temperatures enzymes may not work efficiently, which is why prolonged exposure to very low temperatures can be deadly. Cellular processes simply ‘grind to a halt’.

TRYPSIN











Figure 2.7 Enzyme activities in relation to pH values. The optimal pH for an enzyme is that at which the enzyme shows maximal activity.

P(

31 Enzymes and other biomolecules

Regulating enzyme affinity biofile If the lining of the stomach is damaged, the action of acid and the stomach enzyme pepsin can contribute to the formation of ulcers. Bacteria are also involved, particularly in the maintenance of chronic ulcers (page 160). Antacid medications raise the pH of the stomach contents to about 4, neutralising the acidity. This also reduces the activity of pepsin because its optimum pH is 1 (Figure 2.7).

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PARA AMINOBENZOICACID

SULFANILAMIDE

Figure 2.8 The molecular structures of para-aminobenzoic acid and sulfanilamide are very similar. These two compounds compete for the same site on a bacterial enzyme.

Cells can regulate the affinity of certain key enzymes for their substrate molecules. Affinity is the ease with which an enzymes binds with its substrate. Cells do this by attaching other molecules to the enzyme to alter the shape of its active site. This allows cells to increase or decrease the rate of a reaction in particular circumstances.

Chemical inhibition Other chemical substances can inhibit enzyme function by binding to the active site of the enzyme, or by combining with another part of the enzyme in such a way that the shape of the binding site is altered. The antibiotic sulfanilamide is effective against bacteria because it binds to the active site of the bacterial enzyme involved in the formation of folic acid (a Bcomplex vitamin). It is able to do this because the molecular structures of sulfanilamide and the normal substrate for the enzyme (para-aminobenzoic acid) are very similar (Figure 2.8). Sulfanilamides are useful in the treatment of a bacterial infections because they kill only bacterial cells. Mammalian cells are not affected by sulfanilamide because they do not use the enzyme to make folic acid; they obtain folic acid directly from food.

Amounts of reactants The rate of a reaction is also affected by the relative amounts of substrate or enzyme present and how much product has accumulated. In most chemical reactions, if there is more substrate present (Figure 2.9a), the net reaction will go to the right. However, if the product accumulates, the net reaction will slow and eventually begin to go to the left (Figure 2.9b). The direction of the reaction is not affected by the presence of enzyme.

Coenzymes and cofactors Coenzymes are very small organic molecules—less complex than proteins— that are associated with particular enzymes and are essential for their activity. They usually donate electrons (negative particles) or protons (positive particles). Many coenzymes cannot be synthesised by animals and must be obtained from plants or microorganisms. Most coenzymes are derived from vitamins, hence the vital requirement for small amounts of vitamins in the diet. Some enzymes require particular metallic cations known as cofactors, such as Cu2+ (page 13), in order to catalyse the reaction. Mg2+ helps to stabilise many enzymes.

Figure 2.9 (a) If there is excess substrate, the reaction tends to the right. (b) If there is a build-up of products, the reaction may go in the reverse direction.

32 Molecules of life

(a)

GLUCOSEGLUCOSEENZYME

ENZYMEANDSUBSTRATE

MALTOSEENZYME

(b)

GLUCOSEGLUCOSEENZYME

ENZYMEANDSUBSTRATE

MALTOSEENZYME

biology in action Using enzymes For food Humans have used enzymes to prepare food for hundreds of years. Rennin (extracted from the stomachs of certain mammals) is still used to clot milk in cheesemaking. Yeasts are used in baking, and also to ferment plant materials to make alcoholic beverages. Caribbean Islanders knew that pineapple would not only make meat more tender, but would also reduce indigestion after a large meal of meat. Today, the food industry can extract enzymes from organisms, purify them, and produce them in large quantities using genetic engineering techniques. Many meat tenderisers have extracts from pawpaw or banana juice as their active ingredient because these contain proteases, enzymes that act on proteins. Other proteases are used to clean contact lenses, to make infant formulas from cows’ milk and to manufacture meat extracts, such as those used in Bonox and Bovril. Lipases, enzymes that break down fats, are used in breadmaking to improve the handling of dough and to degrade the fats that have an effect on how the bread goes stale. Amylases (enzymes that degrade starch) have many uses, including converting starch to sweeteners. For cleaning Enzymes were introduced into biological laundry detergents in the mid 1960s to reduce the use of bleaching agents and phosphates. The main enzymes used are proteases, which act on organic stains such as human sweat, grass, blood and egg. In more recent years, lipases have been added. They remove stains caused by fatty substances such as oils, fats and lipstick. Amylases help remove starchy food deposits. For medical applications Pharmaceutical and scientific communities make use of enzymes to treat genetic disorders, extract medicinally important compounds such as heparin (a medication to prevent blood clotting), and in research and development. In the field of molecular biology, almost all processes in nucleic acid manipulation are performed with enzymes (e.g., restriction endonucleases and DNA polymerases).

biofile In Australia, washing powders containing protease enzymes were banned in the early 1970s because of concerns about the health of workers involved in their production. The enzymes (and the washing powders) were very fine powders and workers were exposed to airborne protein particles. Some of the workers developed respiratory allergies, and eye and skin irritations. Subsequently, codes of practice have been developed to reduce the amount of airborne enzymes in the workplace.

biofile Cellulases (enzymes that break down cellulose) are used to give the final appearance to denim. Denim was traditionally stonewashed with pumice stones to fade the surface of the garment. Now, a small amount of cellulase replaces much of this process and does less damage to both the garments and the machinery. This technique has become known as biostoning.

Figure 2.10 Enzymes are routinely used to produce both bread and wine. If the apples were to be commercially converted to juice, enzymes would be used to give a less cloudy product.

Enzymes and disease Several inherited diseases (often referred to as ‘inborn errors of metabolism’) involve an inability to manufacture a particular enzyme required to break down substances that are normally part of the diet. Three of these diseases are galactosaemia, lactose intolerance and phenylketonuria.

Galactosaemia The main sugar in milk is lactose, a disaccharide. During digestion, it is split into the monosaccharides glucose and galactose, which are then absorbed across the intestine wall. Glucose is directly available as a substrate for cellular respiration in cells. Galactose, however, must be converted by a series of enzymes into glucose-1-phosphate before it can be metabolised to release energy.

33 Enzymes and other biomolecules

Galactosaemia is an inherited condition and is due to an error (mutation) in the gene responsible for producing one of the enzymes needed to convert galactose to glucose-1-phosphate. Individuals have two copies of each gene; one received from each parent. If a child inherits two abnormal genes, they will be unable to make any of the enzyme and therefore will suffer from galactosaemia. If a child inherits one normal gene and one abnormal gene, then the normal gene generally seems to be able to produce sufficient enzyme to prevent a dangerous accumulation of galactose in blood and other tissues. For infants who suffer from galactosaemia, galactose accumulates in their blood and is present in their urine. Their livers become enlarged, their eyes develop cataracts, growth is slow and mental development is retarded. Quite often they do not live beyond infancy. The treatment for galactosaemia is simple and largely successful if it is commenced soon enough. All foods containing galactose, chiefly milk and milk products (Figure 2.11), must be excluded from the diet.

Lactose intolerance Most mammals produce the enzyme lactase only until they are weaned. Humans of Anglo-Saxon origin keep producing lactase throughout life. Indigenous Australians, and people from many parts of Asia, Africa, the Middle East and some Mediterranean countries, gradually produce less lactase as they get older and develop lactose intolerance. This means that lactose is not digested normally in the small intestine. When it passes to the large intestine, bacteria ferment it, causing excessive amounts of wind, bloating, pain and sometimes diarrhoea. Like galactosaemia, the treatment for lactose intolerance is the exclusion of all foods containing lactose from the diet.

Phenylketonuria

Figure 2.11 Foods containing lactose, such as milk, cheese and yoghurt, must be excluded from the diet of those suffering from galactosaemia and lactose intolerance.

Phenylketonuria (PKU) is a metabolic disorder in which the ‘missing’ enzyme is phenylalanine hydroxylase, which catalyses the conversion of phenylalanine (an amino acid) to tyrosine (another amino acid). When the enzyme is absent, very high levels of phenylalanine accumulate in blood and other tissues. Children born with phenylketonuria will suffer mental and physical retardation unless they are placed on a special diet soon after birth. They are kept on this tightly regulated diet until they are about 10 years of age, because the nervous system continues to develop during this period. After this, high blood levels of phenylalanine are less harmful and the diet can be relaxed to some extent. Since the late 1960s all newborn babies born in Victoria have been tested for this genetic condition by taking a blood sample from a heel prick, known as the Guthrie test.

Anabolic and catabolic reactions At a cellular level, metabolism involves both the ‘building up’ and ‘breaking down’ of molecules. Anabolism is the building of larger molecules from smaller molecules, which requires energy to construct the new bonds. Catabolism is the breakdown of macromolecules into smaller molecules, and the breaking of bonds releases energy. Both processes are central to the metabolism of cells.

34 Molecules of life

technologies and techniques Should scientists conduct research using your DNA? by Professor Loane Skene

I

f you were born in Victoria in the last 30 years, there will probably be a ‘Guthrie card’ with your blood on it at Genetic Health Services Victoria. The blood would have come from a heel prick soon after your birth to see if you have a genetic condition that might seriously harm your health, or even kill you, such as phenylketonuria (PKU), cystic fibrosis or hypothyroidism. Babies with these conditions can be treated and will develop normally if diagnosed early. Parental consent is required for testing, and nearly all parents consent because of the clear potential benefit to their child. Privacy issues arise because the cards are kept after the tests have been done. Retaining the cards is vital for quality assurance. Blood samples can be retested if new tests are developed later. A woman planning a pregnancy might want to know if an earlier deceased child had an undiagnosed genetic condition which might affect another child. Guthrie cards may be the only way to identify people killed in disasters like the Bali bombing or the tsunami in South East Asia. On the other hand, the cards contain sensitive personal information. With our current biochemical techniques, Guthrie card blood samples can be tested for about 30 genetic conditions. With the development and refinement of gene chip technology, there may be tests for hundreds of genetic conditions.

Many safeguards exist to protect privacy of card information. Only the mother’s name, hospital, baby’s sex and date of birth are on the cards—not the baby’s name, test results or medical information. Access is strictly controlled. More than two million cards are held in Victoria and fewer than a thousand have been accessed, for example, for use in research. This is subject to approval from a hospital ethics committee, and only an anonymous blood spot will usually be given unless specific consent is obtained from the parents to use the samples in research in an identified manner. Very occasionally, police have been given blood from the cards for testing, but this is conditional on obtaining a court order. How do you feel about your DNA being used by scientists in research if you have not been told about it? Are you happy to let this happen, knowing that it will benefit the community, provided your privacy is protected? Or are you so concerned about the risk of cards being misused that they should be destroyed after the first tests have been done, even if there are some benefits in retaining them? (In Western Australia, all cards were recently destroyed after fears that the police could use them and they are now kept for only two years.) How should the community balance individuals’ rights to privacy and potential community benefits?

Professor Loane Skene Professor Loane Skene is a lawyer specialising in Medical Law, particularly the legal regulation of genetic technology. She is also the Deputy Director of the Centre for Law and Genetics at the University of Tasmania and the University of Melbourne.

Figure 2.12 Guthrie card blood samples can be tested for approximately 30 genetic conditions.

35 Enzymes and other biomolecules

Trapping and releasing energy The most important anabolic reactions of living organisms are those of autotrophic organisms, which trap the radiant energy of the sun and use it to build up organic molecules in the process of photosynthesis. Heterotrophs cannot trap energy from the environment and they are entirely dependent on autotrophs for this. In contrast, both autotrophs and heterotrophs use similar catabolic reactions to release the chemical energy from organic molecules to fuel their cellular activities in the process of cellular respiration. These processes will be considered in more detail in Chapter 3.

Building up and breaking down polymers The synthesis and breakdown of polymers provides another example of anabolism and catabolism. Macromolecules (polymers) make up a large part of all organisms and are composed of many subunits joined together. The four major groups, polypeptides, lipids, polysaccharides and nucleic acids, are quite different types of molecules, but the general processes by which they are synthesised and broken down are similar. Building a macromolecule involves the synthesis of the individual subunits it is composed of, followed by their incorporation one at a time into the growing polymer. With many polymers, including proteins, polysaccharides and nucleic acids, the process of adding each subunit involves the removal of a molecule of water, a process known as dehydration (or condensation) (Figure 2.13). (a) 0OLYMERSUBUNITS

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(b) $EHYDRATIONSYNTHESIS (/

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Figure 2.13 (a) Macromolecules are made from many subunits joined together. (b) Subunits are joined together by the removal of a water molecule (dehydration). (c) Separation of subunits of polymers occurs by the addition of a water molecule (hydrolysis).

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(c) (YDROLYSISSPLITTING (/

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Organisms also need to be able to break down macromolecules. For example, during the digestion of food, macromolecules must be broken down into molecules small enough to cross membranes and enter the body. Also, when tissue is damaged or in the normal course of repair and maintenance, cells need to be able to remove unwanted or damaged macromolecules. The breakdown of polymers into subunits involves the reverse process—adding a molecule of water (Figure 2.13c), known as hydrolysis (‘hydro’ meaning water, ‘lysis’ meaning split).

36 Molecules of life

technologies and techniques Biopolymers— very versatile agents by Professor Tony Bacic

B

iopolymers are large molecules that have unique functional properties. They are either extracted from a wide range of renewable biological resources (such as seeds, fruits, crustacean shells, shark fins, bacterial fermentation) or are chemically synthesised from natural materials (such as sugars, starch, natural fats or oils). Biopolymers are used as stabilisers, thickeners, gelling agents, binders, dispersants, lubricants, adhesives and drug-delivery agents. They are used extensively in food products, medicines and other applications such as cosmetics, pharmaceuticals, printing inks, agricultural sprays and in drilling oil wells. The value of biopolymers lies in their texture modifying properties, which are due to the abiliy of the biopolymer chains to interact through hydrogen or ionic bonds (see page 8, Chapter 1) to form a 3-D network that prevents water from escaping. Depending on the extent and strength of these interactions, the solutions can range from viscous to gellike. You may have been given a Techni-ice polymer ‘ice block sheet’ from your doctor to use as a cold pack to reduce bruising. #//( /

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Professor Tony Bacic is a plant molecular biologist and biotechnologist at the University of Melbourne. He is an expert in the structure, function and chemistry of plant cell walls, and his research work has applications in the food and brewing industries.

Figure 2.14 Pectins are extracted from citrus fruit peels, crushed apples and many other fruits and vegetables. Sunflower heads (not seeds) are a good source of pectin.

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Major attractions for the industrial use of biopolymers • Biodegradable—provides solutions to ecological concerns resulting from the excessive use of non-biodegradable plastics. • Biocompatible—applications for medical uses • ‘Natural’ product—can be safely used with minimal regulatory hurdles in food applications.

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Figure 2.15 Pectins are highly complex polysaccharides found in cell walls.

37 Enzymes and other biomolecules

summary

2.1

• Metabolism is the overall chemical activity of cells, including synthesis of organic molecules, energy transforming and recycling processes, and breakdown of unwanted substances. • These reactions involve hundreds of enzymes working in ‘chains’. • Enzymes are proteins that act as biological catalysts to control the metabolism of the cell. • Enzymes make a reaction take place more easily. They do not change the direction of a reaction, are needed in small amounts and remain unchanged at the end of the reaction. • Each enzyme usually catalyses only one type of reaction. This specificity is related to the three-dimensional structure of the enzyme and substrate molecules. • Activation energy is the energy required to get a reaction started, even if the reaction eventually releases energy. • Enzymes act by reducing the amount of activation energy required for a reaction to occur.

• The affinity of an enzyme for a substrate can be regulated to alter the rate of a reaction. • Factors affecting enzyme action include temperature, pH, other chemicals, amounts of reactants and products, and the presence of coenzymes and cofactors. • Enzymes are important in both traditional and modern industries. • Enzymes play critical roles in metabolic pathways in living organisms. • Lack of a particular enzyme can result in disease. • Anabolism is the building of larger molecules from smaller molecules. It requires energy to construct the new bonds and often involves dehydration. • Catabolism is the breakdown of macromolecules into smaller molecules. The breaking of bonds releases energy and often involves hydrolysis.

key questions 1 What are the basic processes all cells must perform in order to live? 2 Explain why enzymes are referred to as biological catalysts. Give an example. 3 a What are enzymes? Provide examples of their functions. b To what major group of organic compounds do enzymes belong? c How do enzymes and their substrates relate to each other? d Describe how enzymes are involved in metabolic pathways. 4 What is meant by the ‘specificity’ of an enzyme? 5 Using Figure 2.2, describe the steps involved in an enzyme catalysing a reaction. 6 Why can enzymes be used over and over again? 7 a What is meant by the ‘activation energy’ of a reaction? b Explain what effect enzymes have on the activation energy of a reaction. 8 a Define ‘enzyme affinity’. b Describe how an enzyme’s affinity can be altered. c What effect does this have on an enzyme’s performance? 9 Explain, in terms of an enzyme’s active site, why pH and temperature can influence the rate of enzyme-controlled reactions. 10 List five factors that affect the rate of an enzyme-catalysed reaction. Describe the effect of each factor.

38 Molecules of life

11 Explain the difference between a ‘coenzyme’ and a ‘cofactor’. Outline their importance. 12 Describe two examples in which enzymes are used for commercial purposes. 13 Discuss a specific example of enzyme deficiency to show the importance of enzymes in metabolic pathways. 14 a Explain what is meant by an anabolic reaction. b Describe a specific example of an anabolic reaction, naming the group of organisms in which it occurs. 15 a Explain what is meant by a catabolic reaction. b Describe an example of a catabolic reaction, naming the group of organisms in which it occurs. 16 List the four major groups of polymers that make up living organisms. 17 a Outline the differences between ‘dehydration’ and ‘hydrolysis’. b Identify which of these processes represents anabolism and which represents catabolism. c Which of these processes is responsible for i constructing polymers? ii breaking down polymers?

2.2

Biomolecules—synthesis and transport The synthesis, packaging and transport of organic molecules in eukaryote cells involves the coordinated activity of a number of organelles, including the nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus and vesicles (Figure 2.16).

SECRETION

NUTRIENTS RIBOSOMES PROTEINS

AMINO ACIDS

NUCLEAR PORE

POLYSACCHARIDES GLYCOPROTEINS LIPOPROTEINS NUCLEUS

RIBOSOMES

'OLGI

PROTEINS

PLASMA MEMBRANE

SECRETORY VESICLES LYSOSOMES ROUGH ENDOPLASMIC RETICULUM

CARBOHYDRATE ANDLIPID BUILDINGBLOCKS

SMOOTH ENDOPLASMIC RETICULUM

Figure 2.16 Intracellular pathways and organelles involved in the synthesis of organic molecules.

Controlling cell functions—nucleus Except for the large vacuoles found in some plant cells, the largest organelle in a cell is usually the nucleus. Most plant and animal cells have one nucleus. Some cells have more than one nucleus; for example, rat liver cells and skeletal muscle cells. Others, such as human red blood cells and the sieve tube cells of flowering plants, lose their nucleus when they mature. Chromosomes, which are located within the nucleus, are usually not clearly visible except during cell division. The most obvious structure seen inside the nucleus of a non-dividing cell is the nucleolus (plural: nucleoli), a large, dark-staining body. There may be one or more nucleoli per nucleus, depending on the species. Nucleoli are rich in proteins and RNA, and are the site of synthesis of ribosomal RNA (rRNA). The nucleus is surrounded by the nuclear membrane (nuclear envelope), a double membrane containing many nuclear pores (Figure 2.17). Materials such as RNA pass between the nucleus and the cytoplasm through these pores. Cells use a wide array of very specific enzymes to manufacture the proteins, carbohydrates, lipids and nucleic acids they require. The instructions for building these enzymes, which are proteins, are contained in the DNA. The maintenance of cell structure and function is therefore under the control of DNA located in the nucleus, although the proteins are assembled in the cytoplasm. Messenger RNA (mRNA) is a copy of the DNA instructions for a particular protein. It is produced in the nucleus and then passes out into the cytoplasm.

N NE

Figure 2.17 An electron micrograph showing the nuclear envelope (NE; a double membrane) and nuclear pores (arrows) through which substances pass between the cytoplasm and the nucleus (N).

39 Enzymes and other biomolecules

biology in action Nucleus rules—OK? The role of the nucleus was clearly demonstrated in a set of experiments carried out in the 1930s using Acetabularia, a large single-celled marine alga consisting of a branching foot, stalk and cap. The nucleus is located in the foot. Two closely related species with different shaped caps are A. mediterranea and A. crenulata. When a cap is removed from either species, a cap of the same shape grows back (Figure 2.18b). In a reciprocal experiment (Figure 2.18c), caps were removed from individuals of each species, then the stems were cut and transplanted onto roots of the other species. The first caps that regrew were intermediate in form between the two species. When these were removed, all following regenerated caps had the form of the foot—the part of the cell that contained the nucleus. The nucleus was obviously controlling the functions of the cell.

(a)

(b)

Amediterranea

Acrenulata CAP

STALK NUCLEUS

Figure 2.18 (a) A single-celled Acetabularia about 1–2 mm high. (b) If the caps are removed, new caps grow similar to the original form. (c) If a foot of one species is grafted to the stalk of the other, the first cap that grows is intermediate in form. If this cap is removed, the cap that grows thereafter has the form of the species of the foot.

BRANCHING FOOT

(c)

Assembling proteins—ribosomes

Figure 2.19 An electron micrograph of rough endoplasmic reticulum (r.e.r.) in a protein-secreting cell.

40 Molecules of life

Ribosomal RNA passes from the nucleus into the cytoplasm where it becomes part of the protein-synthesising organelles called ribosomes. Messenger RNA also passes into the cytoplasm where it binds to ribosomes and initiates protein synthesis (Figure 2.19). The process of protein synthesis is considered in more detail in Chapter 4. Cells contain many thousands of tiny ribosomes, which are approximately 30 nm in diameter and only visible using an electron microscope. Depending on the type of protein they are making, ribosomes are found either free in the cytoplasm or attached to membranes (endoplasmic reticulum). Proteins produced by free ribosomes generally function as enzymes within the cytoplasm. Ribosomes bind to membranes when producing proteins that will be incorporated into membranes or that are destined for export from the cell.

Transporting and processing— endoplasmic reticulum Endoplasmic reticulum (ER) is an extensive network of interconnected membranous sacs (cisternae; see Figure 2.20) and tubules branching throughout the cell. In plants, ER membranes enclose a cavity (lumen) that is continuous between cells via the plasmodesmata (intercellular connections between adjacent plant cells). ER is connected to the nuclear membrane and is believed to arise from it. When the surface of the ER is covered with ribosomes, it is called rough endoplasmic reticulum (RER). In the absence of ribosomes, it is referred to as smooth endoplasmic reticulum (SER).

Figure 2.20 A diagram and an electron micrograph of a Golgi apparatus (G) showing vesicles forming at the ends of some cisternae.

(b)

(a)

CISTERNAE

G

VESICLESFORMING

Endoplasmic reticulum contains enzymes and proteins that are important in many aspects of metabolism including processing and sorting of proteins, adding sugar chains to proteins, and ensuring that proteins are correctly folded. They also have a critical role in regulating the concentration of cytoplasmic calcium which, in turn, regulates many cellular functions. As proteins are synthesised by ribosomes on RER, they pass into the lumen of the ER (Figure 2.21) to the Golgi apparatus for export from the cell. RER is abundant in cells that actively synthesise and export proteins, such as those pancreatic cells that secrete digestive enzymes.

INTERIOROFROUGH ENDOPLASMICRETICULATION

SUGAR MOLECULE

Figure 2.1 Proteins assembled on ribosomes of rough endoplasmic reticulum pass immediately into the lumen of the endoplasmic reticulum. Sugar molecules may be added to the protein within the endoplasmic reticulum to make glycoproteins.

GLYCOPROTEIN

PROTEIN RIBOSOME RECEPTORPROTEIN

BILAYER MEMBRANE

M2.!

CYTOPLASM

RIBOSOME

41 Enzymes and other biomolecules

SER is involved in the synthesis of other molecules, such as fats, phospholipids and steroids. Most cells have only small amounts of smooth endoplasmic reticulum, but it is abundant in certain specialised cells such as the steroid-secreting cells in the testis and adrenal gland.

Packaging for export—Golgi apparatus The Golgi apparatus (or Golgi complex) is a stack of cisternae and the matrix that encompasses it (Figure 2.20). The Golgi apparatus is the major site for the sorting of proteins to different sub-cellular compartments. Proteins synthesised by the RER reach the Golgi apparatus where they are often further modified before being packaged in vesicles for export from the cell. Additional processing of proteins, such as the addition of sugar molecules to form complex sugar-protein molecules called glycoproteins, can occur as a result of the action of enzymes located within the cisternae (Figure 2.21). Some polysaccharides are assembled in the Golgi apparatus for export from the cell via vesicles. Vesicles are membrane-bound organelles often involved in transport. When they reach the cell membrane, they fuse with it, releasing their contents to the outside of the cell by exocytosis (Figure 2.22).

EXOCYTOSIS SECRETION

POLYSACCHARIDES GLYCOPROTEINS LIPOPROTEINS

SECRETORY VESICLES

Figure 2.22 Secretory vesicles bud off the Golgi and move to the cell membrane where they release their contents by exocytosis.

biofile There is much research being conducted to try and understand more about the sugars that are attached to proteins and lipids during their passage through the Golgi apparatus, a process known as glycosylation. Some research groups are studying the enzymes involved and how glycosylation could be inhibited. Why the interest? Because it is these sugar molecules on the surfaces of cells that have been shown to be the cause of the early rejection of transplants of pig tissues.

42 Molecules of life

'OLGI

Secretory cells have a well-developed Golgi apparatus, whereas nonsecretory cells have small Golgi. Some products packaged by the Golgi, such as the enzymes found in lysosomes, are not intended for release from the cell. Plant cells must build cell walls, which lie outside the cell membrane. The Golgi apparatus in plant cells are largely engaged in producing and secreting a large array of complex polysaccharides (but not cellulose) to the outside of the cell. Several hundred different enzymes located within the ER and Golgi are involved. Because the composition of the cell wall is not the same in all regions of the cell, particular vesicles must be directed through the cell to deposit their contents at specific regions of the cell membrane. Cellulose, a major component of cell walls, is synthesised by an enzyme complex that forms a pore in the membrane. Synthesis occurs on the cytoplasmic side of the enzyme complex, and as the cellulose chains are assembled, they pass through the pore to be deposited outside the cell membrane into the cell wall.

Storage of biomolecules Organisms maintain stores of chemical energy in the form of carbohydrates and lipids in organelles such as starch grains, lipid droplets and glycogen granules. Starch, a complex carbohydrate, is stored in large grains in plant cells; for example, in cereals. Animal cells can contain granules of the complex carbohydrate glycogen and droplets of lipids. Some cells are specialised for storage, such as potato tuber cells and fat cells. Protein storage granules are abundant in the seeds of legumes, such as beans and peas. Animals do not store proteins, which is why it is important to eat a balanced diet containing all the essential amino acids, as these are necessary for protein production.

Removing wastes and debris Lysosomes Lysosomes are membrane-bound vesicles found in almost all types of animal cells (Figure 2.23). They are the sites of breakdown of debris within the cell, such as ‘worn out’ or damaged organelles, and of external debris or foreign microorganisms that might be harmful. Lysosomes are also involved in the breakdown of cells during programmed cell death (apoptosis), which occurs during development (see page 285). Powerful ‘digestive’ enzymes produced by ribosomes are transported to the Golgi in the endoplasmic reticulum. These enzymes must be contained safely within a vesicle or they would attack the cell itself. In the Golgi, the enzymes are packaged into transport vesicles that fuse with vesicles containing unwanted material, forming a lysosome. Digestion takes place within the lysosome. Useful small molecules may diffuse back into the cytoplasm and the residue will be retained in the lysosome or released from the cell by exocytosis.

Figure 2.23 An electron micrograph showing a lysosome (arrow) in the process of breaking down unwanted material.

Vacuoles Vacuoles are membrane-bound liquid-filled spaces found in most cells in variable numbers and sizes. In animal cells, food vacuoles contain enzymes and are involved with intracellular digestion. Contractile vacuoles in protozoa are involved in water balance, expelling excess water that enters by osmosis. Plant cells typically have large fluid-filled vacuoles that provide physical support (turgidity) and storage. In young plant cells, vacuoles are small and scattered. As the cells mature, the vacuole expands to occupy often 90% of the cell volume, with the cytoplasm forming a thin layer inside the cell wall. In mature plant cells, vacuoles tend to be long-lived and relatively static. The contents of the vacuole are relatively inactive. Movement of substances into (and occasionally out of) the vacuole is controlled by the vacuole membrane (the tonoplast). Salts are actively accumulated in the vacuole. Vacuoles often have a storage function and are a ‘dumping site’ for toxic substances, such as oxalic acid found in leaves of rhubarb and Dieffenbachia (Figure 2.24). Water-soluble pigments that are responsible for flower colour, such as anthocyanins, are found in the vacuole. The large vacuoles in cells of nonwoody plant tissues contribute to the support of the plant by taking up salts and swelling against the cell wall to produce turgor.

Figure 2.24 Eating the leaves of this well-known house plant Dieffenbachia can be fatal. Vacuoles in the leaf cells contain needle-like crystals of calcium oxalate, and other irritants and toxins. When the cells are broken during chewing they forcibly eject the crystals, which penetrate the lining of the throat, allowing the toxins to enter. In the wild, such an adaptation would deter herbivores from eating the plant.

43 Enzymes and other biomolecules

summary

2.2

• The synthesis, packaging and transport of organic molecules involves the coordinated activity of a number of organelles including the nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus and vesicles. • The maintenance of cell structure and function is under the control of DNA, located in the nucleus. DNA codes for the production of proteins, which include enzymes. • Messenger RNA is produced in the nucleus and passed into the cytoplasm where it binds to a ribosome and initiates protein synthesis.

• Proteins are manufactured at ribosomes, and transported and modified in the endoplasmic reticulum and Golgi apparatus before being directed to their final location either inside or outside the cell. • The endoplasmic reticulum and Golgi apparatus are also involved in the synthesis of other macromolecules such as nucleic acids, carbohydrates, steroids and lipids.

key questions 18 Proteins are a very important group of organic molecules. a What role does the nucleus play in protein synthesis? b Where is the nucleolus found and what is its purpose? c Through what structure do materials enter and leave the nucleus? d Where does protein synthesis occur in cells? e Describe the various functions of endoplasmic reticulum. f Explain the structural and functional differences between rough and smooth endoplasmic reticulum.

44 Molecules of life

g What occurs in the Golgi apparatus? h How are new proteins moved to the surface of the cell and secreted? 19 Describe some ways in which substances are stored in cells. 20 List the range of functions carried out by vacuoles. 21 Describe the function of vacuoles and lysosomes in removing wastes from cells.

02

key terms biomolecule enzyme metabolism substrate catalyst active site activation energy denaturation optimum temperature

critical temperature pH affinity coenzyme cofactor protease lipase amylase galactosaemia

lactose intolerance phenylkeronuria (PKU) anabolism catabolism dehydration hydrolysis nucleus nucleolus DNA

RNA messenger RNA ribosome endoplasmic reticulum Golgi apparatus lysosome vacuole

worksheet 04 1 Suggest the names of enzymes with the following substrate a maltose b lactose c sucrose d cellulose e protein (general) f lipid (general) g pectin 2 Consider the following incomplete graph for an enzymecontrolled reaction, where the enzyme is present at concentration x. Assume there is a fixed amount of substrate present. Amount of product

c Describe and account for all of your observations. d Dicing or grinding the liver will increase the rate of reaction observed. Explain why this is so. e Suggest a method that will be helpful in determining that the gas produced is oxygen gas. f Write out a balanced chemical equation for the reaction you observe. 5 The following graph illustrates the relationship between the concentration of an enzyme substrate and enzyme activity. RATEOF REACTION

Time

a Eventually the shape of the graph will change. Continue the line graph according to your expectations and explain what happens. b Redraw the graph for an enzyme concentration of 2x. 3 Pepsin is an enzyme which is released into the stomach of humans (pH 1, 37°C), where it breaks down proteins into polypeptides. Explain how you would expect the activity of pepsin to change as: a the temperature is increased from 37°C to 45°C b the pH is increased from 1 to 5. 4 Mammalian body cells produce hydrogen peroxide as a byproduct of metabolism. Hydrogen peroxide (H2O2) is highly toxic to cells. Consider the hypothesis: that mammalian liver cells produce an enzyme that breaks down H2O2 to produce water and O2. a Design an experimental procedure to test the hypothesis. b Share your design with other members of the class. In discussion with your teacher, organise to undertake the activity in class.



0

CONCENTRATION OFSUBSTRATE

a Describe the relationship between the substrate concentration and the rate of reaction from 0 to P units of substrate concentration. b What happens at and after point P? Explain. c The concentration of the enzyme is described as a limiting factor. Explain what this means. 6 Many laundry detergents advertised today boast the inclusion of enzymes as an active ingredient. Enter key words in a search engine to find a what kinds of enzymes are commonly added to laundry detergents b what kinds of stains/substances they remove c the effectiveness of detergents with enzymes compared with those without enzymes.

45 Enzymes and other biomolecules

7 Enzymes are vital ingredients in many commercial applications of our everyday lives. Choose one application from the list below and investigate the process involved, which enzymes are important and the role of the particular enzyme(s): • cheese making • yoghurt culture • manufacturing of beers and wines • meat tenderizer preparation • cleaning agents (e.g., for contact lenses) • bread-making.

8 Design and produce a fact sheet about a selected disease or condition caused by a particular enzyme deficiency. Include information about the • missing enzyme • role of the enzyme in normal metabolism • effects of the enzyme deficiency • treatment/management of the disease/condition. Examples: lactose intolerance, muscular dystrophy, Tay-sachs disease, Krabbe disease, phenylketonuria, galactosaemia, albinism.

46 Molecules of life

9 Imagine you are an observer inside the factory floor of a salivary gland cell. Use a flow chart to describe the steps involved in the production of the enzyme amylase, which begins the digestion of starch to simple sugars in the mouth. Start with the nucleus and provide instructions about how to build the protein, noting each of the significant sites within the cell. End the flow chart with the enzyme being secreted from the cell into the salivary gland. 10 Freshwater amoeba regulate their water balance by means of a contractile vacuole. This structure collects water and regularly discharges its contents to the external environment by fusing with the cell membrane. Under an electron microscope it can be seen that there is a close association between the vacuole and numerous mitochondria. Explain: a the need for a contractile vacuole b the presence of numerous mitochondria. 11 Prepare a concept map that summarises the synthesis and transport of biomolecules at the cellular level. Be sure to include the following: nucleus, ribosomes, endoplasmic reticulum, Golgi apparatus, mRNA, protein, exocytosis, lysosomes, vacuoles. 12 Revisit the Biology in Action box ‘Nucleus rules-OK?’ on page 40. a State the hypothesis being tested in the experiment. b Explain whether the experimental results support or negate the hypothesis.

0

chapter 03

Energy transformations

key knowledge • cells use chemical energy • the ATP/ADP cycle • similarities and differences in meeting the energy requirements of cells • energy transformations • main stages and sites of cellular respiration and photosynthesis • factors affecting the rates of cellular respiration and photosynthesis

chapter outcomes After working through this chapter you should be able to: • • • • • • • •

• • •

define energy and explain why cells need energy describe the type of energy used by cells define exergonic and endergonic reactions describe the ATP/ADP cycle and the role of ATP give the general formula for the complete breakdown of glucose during cellular respiration describe glycolysis, aerobic respiration and fermentation contrast the extraction of usable energy from glucose in the presence and absence of oxygen outline the process of photosynthesis, including light-dependent and light-independent (dark) reactions explain the difference between photosynthesis and chemosynthesis compare the roles of chloroplasts and mitochondria in energy transformations explain why animals largely store fat but plants largely store carbohydrates.

3.1

Life needs energy

Figure 3.1 Windfarms utilise the kinetic energy of fastmoving air to generate electricity.

Energy exists in many forms, such as light, heat, sound, mechanical, electrical and chemical energy. It cannot be created or destroyed, but it can be changed from one form to another. When we want energy for our homes or industry, an available source of energy is trapped and then changed into a usable form. Hydroelectricity is an example of this—the kinetic energy of fast-flowing water is trapped and converted into electrical energy by machines (Figure 3.1). Windmills act in a similar way using the kinetic energy of fast-moving air. Solar panels trap the radiant energy of the sun and use it to heat water or create electricity. Living organisms grow, move, respond and reproduce. Carrying out these activities, or doing work of any kind, requires energy. Living organisms are also vulnerable to damage from the wear and tear of daily life and from interactions with their environment. Unlike machines, living organisms are able to produce materials needed to maintain and repair themselves, but they need more energy to do so. What sort of energy do organisms use? How do they extract it from their environment? How efficient are the processes involved? How do plants and animals differ in the ways they get the energy they need? This chapter describes the similarities and differences in the ways that organisms trap, use, store and release energy.

What is energy?

! Energy is the capacity to do work

Energy is the capacity to do work and it comes in different forms. Kinetic energy is the energy of movement, such as the energy of waves (Figure 3.2), as seen in the tsunami off Indonesia in 2004, or the energy of random movement of molecules, which is known as heat. Potential energy is stored energy, such as the energy of water at the top of a waterfall or in a stretched rubber band, or the chemical energy held in the bonds between atoms in molecules. The energy of life is largely chemical energy.

Figure 3.2 Powerful large waves have an enormous energy of movement, kinetic energy.

48 Molecules of life

The laws of thermodynamics, whether in biological or physical systems, govern the transformation of energy. The first law is that energy cannot be created or destroyed, but it can be changed from one form into another. The second law of thermodynamics is that the entropy (or randomness) of the universe is increasing. All energy eventually becomes heat energy, which lies in the random movement of molecules.

Efficiency of energy conversions Whenever energy is converted from one form to another, some of the energy is always ‘lost’ to the surroundings. In cells, this energy is usually ‘lost’ as heat energy. It is referred to as ‘lost’ because usually heat is energy that is not available for work. For example, when we exercise we use the chemical energy of ATP (adenosine triphosphate, see page 51) to make our muscles contract. The more work we do, the more energy we use and the more energy is lost as heat, which warms up our muscles (and through blood warms the rest of our body). The continual generation of heat energy as a byproduct of chemical reactions allows endotherms (mammals and birds) to maintain stable body temperatures that are usually higher than their surroundings. The generation of heat from the work of cells can have dire consequences. Heat stroke is a potential problem for marathon runners. The muscular activity of running produces so much heat that the athlete may be unable to lose it fast enough to the environment. They use large amounts of body water to produce sweat in a vain attempt to lose heat by evaporation and cool down, but they become increasingly hotter. Extreme overheating and dehydration can be followed rapidly by collapse and even death. To prevent heat stroke, marathon runners have frequent drinks during the race. Given that in any energy transformation some energy will be lost, energy transformations have differing efficiencies. How efficient is the extraction of chemical energy from glucose by cells? Approximately 40% of the chemical energy released during the aerobic breakdown of glucose to carbon dioxide and water is converted to energy stored in the bonds of ATP molecules (page 51). How does this compare to other processes of energy conversion? The generation of hydroelectricity is 85–95% efficient: only 5–15% of the energy of fast-flowing water is lost, the remainder being converted into electricity. This is a very efficient energy conversion. Car engines are much less efficient. Approximately 75% of the chemical energy of petrol is lost to the surroundings as useless heat and sound energy (cars are hot and noisy!), and only 25% is successfully converted into the energy of motion of the car.

Cells use chemical energy Cells need energy to maintain and repair themselves, for movement and for reproduction. They need energy to build new molecules and break up old ones, to make muscles contract, to send nerve messages and create new cells. Regardless of the type of energy-requiring activity being carried out, cells always use chemical energy. For cells to function, organisms must first obtain energy from an outside source and transform it into chemical energy. What is chemical energy? In many chemical reactions, energy is needed to join atoms and molecules together to make new molecules. This energy is stored in the new bonds or connections that join the atoms together (page 8). If these new molecules are later broken apart, the energy of the bonds is released. This energy, stored in the bonds of molecules, is called chemical energy. Enzymes control all of these chemical reactions in cells (section 2.2).

! Chemical energy is the energy stored in the bonds of molecules.

49 Energy transformations

Organic molecules, such as glucose and proteins, have many energycontaining bonds that can be broken apart in cells to release energy. Plants and animals have different ways of obtaining glucose. Green plants are autotrophs and make their own glucose during photosynthesis, whereas animals are heterotrophs and obtain all their organic compounds from food.

(a) 4OTAL ENERGY INITIAL STATE

EXERGONIC

FINALSTATE

0ROGRESSOFREACTION

(b) 4OTAL ENERGY

FINALSTATE

ENDERGONIC

The facts about energy • Energy cannot be created or destroyed. • Free energy is energy that is available to do work. • When energy is transformed from one form into another, some energy is lost to the surroundings, usually in the form of heat energy, which cannot be used to do work in cells • Exergonic reactions result in a net release of energy. • Endergonic rections require an input of energy in order to occur. • All energy transformations obey the laws of thermodynamics: – The total energy in the universe is constant. – In the universe as a whole, the amount of free energy is declining.

INITIAL STATE 0ROGRESSOFREACTION

Figure 3.3 Change in free energy between initial and final state of a reaction is G. (a) Exergonic reactions release energy. (b) Endergonic reactions require the input of energy.

biofile Chemists use different words for energy movement in chemical reactions. They refer to exothermic reactions as those releasing heat and endothermic reactions as those requiring an input of energy.

summary

Exergonic and endergonic reactions Some chemical reactions release energy while others require an input of energy to occur. Exergonic reactions result in a net release of energy. Cellular respiration is an exergonic reaction that releases energy so that cells in the body can carry out their normal functions. Endergonic reactions require an input of energy in order to occur. Photosynthesis and protein synthesis are examples of endergonic reactions. Energy for photosynthesis is obtained from light, and energy for protein synthesis is chemical energy released by cellular respiration. The change in energy from initial state to final state is referred to as G (delta G). Note that the action of an enzyme affects only the activation energy; the overall energy change of the reaction, G, is unchanged. Reactions releasing energy (Figure 3.3a) are referred to as downhill or exergonic reactions. Reactions requiring an input of overall energy (Figure 3.3b) are referred to as uphill or endergonic reactions.

3.1

• Energy is the capacity to do work. • Energy cannot be created or destroyed, but it can be changed from one form to another. • In any energy transformation, some energy is usually lost as heat. • Organisms use chemical energy to do cellular work. • Chemical energy is the energy stored in the bonds of molecules such as glucose.

50 Molecules of life

• Cells store and release chemical energy by using enzymes to control chemical reactions. • Exergonic reactions result in a net release of energy and endergonic reactions require an input of energy in order to occur.

key questions 1 Define the term energy. 2 All cells need energy to live. For what is the energy required? 3 Describe some energy conversions that occur in living organisms. 4 Use a specific example to explain what is meant by the energy efficiency of a particular reaction.

5 a Explain what is meant by chemical energy. b How can chemical energy be released or made available to cells? 6 Use selected examples to explain the difference between exergonic and endergonic reactions.

3.2

ATP—energy from glucose While plants and animals obtain glucose in different ways, organisms use energy in similar ways. For immediately usable energy, organisms use the chemical energy carried in the phosphate bonds of ATP (adenosine triphosphate). This is the major source of available energy for virtually all cellular functions. ATP is a molecule with a high energy terminal phosphate bond that is easily broken by hydrolysis (page 36) to release a small ‘packet’ of energy, which can be used to carry out cellular work (Figure 3.4).

glucose

Harvesting the energy from glucose Glucose is a 6-carbon (6-C) energy-rich molecule. If it were broken down in a single step, most of the energy would be lost as heat (and cells would ‘cook’). Instead, glucose is broken apart in cells in a series of chemical reactions that involve transferring energy in small ‘packets’, each of which is used to produce ATP from ADP. The types of chemical reactions that occur and the amount of energy that can be harvested from glucose depends on whether oxygen is present or not. In the absence of oxygen, glucose passes down the anaerobic pathway (‘an’ meaning without,‘aero’ meaning air), in which glycolysis is followed by fermentation, and only two molecules of ATP are produced. If oxygen is available, glucose passes down the aerobic pathway, in which glycolysis

energy for work

energy

carbon dioxide and water

ATP/ADP cycle Cells store energy that can be used immediately as ATP. The energy used to make ATP is obtained by releasing the chemical energy stored in the bonds of glucose molecules (and other molecules) in a series of chemical reactions controlled by enzymes. The cells then ‘repackage’ the chemical energy into ATP molecules. These are used, one or more at a time depending on how much energy is required, to carry out all the energy-requiring processes of cells. Once an ATP molecule has given up its energy, it becomes ADP (adenosine diphosphate) plus inorganic phosphate. ADP can be ‘recharged’ to form ATP and then used again (Figure 3.4). The recharging process, like recharging a battery, requires some energy, but much less than it would take to make an entirely new ATP molecule. In biology there are many examples of such recycling mechanisms; organisms are efficient in their use of the limited resources that are available to them.

ATP

ADP adenosine phosphate energy-containing bond

Figure 3.4 The usable energy of ATP is contained in its three phosphate bonds. Usually the energy from only one bond is released for biologically useful work. The resulting ADP (adenosine diphosphate) is recycled using energy derived from the breakdown of glucose.

biofile ATP is only one of several biologically important energy-carrying molecules. Phosphocreatine (CP), which is found in striated muscle of vertebrates, is another. Splitting off the phosphate group from phosphocreatine by hydrolysis actually releases almost 50% more energy than the hydrolysis of ATP. This means that phosphocreatine can help to replenish supplies of ATP to working muscles. CP ↔ C + P + energy energy + P + ADP ↔ ATP Creatine is an amino acid, often taken as a diet supplement by athletes. The body can also make creatine, but most of it comes from eating meat and fish.

51 Energy transformations

is followed by the Krebs cycle and electron transport, with the overall production of about 36–38 molecules of ATP. Glycolysis was one of the earliest biochemical pathways to evolve in the early oxygen-poor atmosphere of the earth. It only releases about 2% of the available energy from glucose. With the evolution of photosynthetic prokaryotes (about 2.5 billion years ago), which release oxygen as a byproduct of photosynthesis (see page 57), the Earth slowly developed an oxygen-rich atmosphere (Figure 3.5). This set the stage for the evolution of the aerobic pathways of oxidative respiration and the extraction of up to 34 more molecules of ATP from each molecule of glucose. /XYGEN LEVELSIN ATMOSPHERE  

CH2OH O

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H

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with oxygen (cellular respiration)



AEROBIC RESPIRATION

sFIRST INVERTEBRATES sFIRSTLAND PLANTS sFIRST MAMMALS sFIRST FLOWERING PLANTS

CO 2 + H2O carbon dioxide and water

Figure 3.5 Oxygen levels in the Earth’s atmosphere were not always as they are today. The evolution of photosynthesis led to a dramatic change in the Earth’s atmosphere and allowed the evolution of aerobic respiration and more diverse species.

+ 36 ATP

CH3 CH3 H

H

C OH + CO 2

C OH C=O

H

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ethanol and carbon dioxide (in most plants, yeasts, bacteria)

lactic acid (in most animals)

no ATP produced

Figure 3.6 During glycolysis, glucose is split into two pyruvate molecules. In the presence of oxygen, pyruvate is broken down by aerobic respiration into carbon dioxide and water. If no oxygen is present, fermentation into lactic acid or alcohol occurs.

52 Molecules of life

Some biology books refer generally to all the energy-releasing processes in cells, both anaerobic and aerobic, as cellular respiration. Strictly speaking, ‘cellular respiration’ refers to the aerobic breakdown of glucose to drive the production of ATP; that is, the pathways that evolved when oxygen became available and which occur in mitochondria in eukaryotic cells. The general simplified formula for the complete aerobic breakdown of glucose is: INPUTS OUTPUTS glucose + oxygen → carbon dioxide + water + energy 6CO2 + 6H2O + 36−38 ATP C6H12O6 + 6O2 → An overview of the various cellular pathways involved the breakdown of glucose (and lipids) to produce ATP is shown in Figure 3.7.

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Glycolysis: anaerobic, occurs in the cytosol The first stage in the breakdown of glucose is glycolysis, which means the ‘lysis’ or splitting of glucose. One glucose molecule is converted via a number of steps into two pyruvate (pyruvic acid) molecules with a net direct yield of two ATP molecules and two electron carriers (or hydrogen acceptors) ‘charged’ with electrons and hydrogen ions (NADH) (Figure 3.7). This stage occurs rapidly and does not require oxygen. It takes place in the cytosol and is common to both aerobic and anaerobic pathways. A simple formula for glycolysis is:

Figure 3.7 Overview of the anaerobic and aerobic pathways for extracting energy from fuel molecules.

INPUTS OUTPUTS + Glucose + 2ADP + 2P + 2NAD → 2 pyruvate + 2ATP + 2NADH What happens next depends on whether or not there is an adequate supply of oxygen.

Aerobic respiration: occurs in mitochondria If oxygen is available, the next stages in the complete aerobic breakdown of glucose in eukaryote cells occur in mitochondria. Transferring the products of glycolysis (two pyruvate molecules and two charged electron acceptors) into the mitochondria ‘costs’ two ATP per molecule of glucose. (In prokaryotes, which do not have mitochondria, these processes occur in the cytosol and this ‘cost’ is not required.) Mitochondria are composed of two membranes. The inner membrane forms many folds called cristae, which provide a large surface area where chemical reactions take place (Figure 3.8). The number of mitochondria

53 Energy transformations

present in different cells is related to the rate of energy usage by the cell (Figure 3.9). Active cells, such as heart muscle cells, have many thousands of mitochondria. Within a cell, mitochondria are found clustered in regions of high metabolic activity, such as near the flagellum (tail) of a sperm, or near a cell membrane where nutrients are being actively transported.

Figure 3.8 The internal membrane of a mitochondrion is highly folded to provide a greater surface area for the enzymes involved in cellular respiration.

Figure 3.9 Mitochondria (red) are clustered around the cell membrane and nuclear membrane of this liver cell.

Krebs (citric acid) cycle

biofile Lipids are also energy-rich molecules that can be broken down to release energy and form ATP. They are processed along a different pathway in the cytosol that does not generate any ATP (as does glycolysis). The process is called β-oxidation, and its product is acetyl CoA, which can enter the Krebs cycle.

After transfer into the mitochondria, each 3-C pyruvate (Figure 3.7) is oxidised to 2-C acetyl, an electron carrier (or hydrogen acceptor) is ‘charged’ with electrons and a hydrogen ion, and CO2 is produced. Acetyl combines with coenzyme A to yield acetyl CoA, which enters the Krebs cycle. In each ‘turn’ of the cycle, one acetyl CoA enters the cycle and one molecule of ATP is formed. In addition, four electron carriers are ‘charged’ with electrons and hydrogen ions, and a further two molecules of CO2 are produced. Thus, two more ATP molecules are produced as two pyruvate molecules are broken down to form carbon dioxide molecules and hydrogen ions. No oxygen has been used yet.

Electron transport

biofile Cyanide used in mining operations has sometimes polluted waterways and killed many fish. Hydrogen cyanide is a poison that inhibits metal-containing enzymes (cytochromes) involved with cellular respiration in mitochondria. It blocks the enzyme involved in the transferance of electrons to oxygen, the last stage in the electron transfer chain, and brings the process to a halt. Death occurs because there is a lack of available energy in cells.

54 Molecules of life

The electron transport chain is a system of electron carriers (cytochromes) embedded into the inner membrane of a mitochondrion (and on the inner surface of the cell membrane in prokaryote cells). Electrons donated by the charged electron carriers from glycolysis and the Krebs cycle are passed from one compound to the next in the chain. At each transfer, some of their energy is used to pump hydrogen ions (protons) out of the mitochondrial matrix, creating a proton gradient that has potential energy. As the protons pass back into the matrix, this energy is used by ATP synthase to make ATP from ADP. In the final step of this sequence, the electrons are transferred to oxygen and used, together with hydrogen, in the formation of water (Figure 3.7). This stage requires oxygen and produces most of the ATP ultimately produced from glucose.

Fermentation: anaerobic, occurs in the cytosol If oxygen is not available, glycolysis is followed by fermentation and no more energy in the glucose molecule will be harvested—no further ATP is produced (Figure 3.7). Pyruvate is converted via an anaerobic pathway

to either lactic acid (in most animals) or alcohol and carbon dioxide (in most plants, and in microorganisms such as yeast and bacteria), with no ATP produced. Fermentation is necessary as it prevents the accumulation of pyruvate and thus allows glycolysis to continue (see page 53). Simple formulae for fermentation are: INPUTS OUTPUTS pyruvate (3C) + NADH → lactic acid (3C) + NAD+ pyruvate (3C) + NADH → ethanol (2C) + carbon dioxide + NAD+ The products of fermentation still contain considerable amounts of chemical energy. Animals are able to retrieve some of this energy by later recycling the lactic acid back into glucose compounds that can enter the aerobic pathway. Plants and microooganisms are not able to recycle alcohol, which can become toxic if it accumulates. This property of yeast is exploited in the beer, wine, and bread making industries.

!40STORES

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Value of the anaerobic pathway For each molecule of glucose, aerobic respiration produces almost 20 times the number of ATP molecules than are produced by glycolysis. Therefore, it is not surprising to find that the cells of animals and plants normally carry out aerobic respiration. Cells usually rely on the anaerobic pathway only if there is not enough oxygen available to continue aerobic respiration (Figure 3.10). However, in emergencies, the rapid rate of release of energy in glycolysis can be vital. For a cheetah, energy from glycolysis when chasing down a gazelle could be the difference between feast or famine; and for the gazelle, the difference between life and death. In short sprints, it is energy derived from glycolysis that gets athletes across the finishing line (Figure 3.11).

Oxygen debt in animals

S

S MIN TIMESINCE EXCERCISEBEGAN

Figure 3.10 The energy used by active muscles in the first 10 seconds of continuous exercise comes from ATP already present in the muscle cells, plus a small amount of ATP produced by glycolysis. After 40 seconds, most of the ATP needed is produced by glycolysis, which produces ATP more rapidly than cellular respiration. As exercise continues, most of the ATP used by the muscles comes from cellular respiration.

Prolonged anaerobic respiration in animals can result in the production of considerable amounts of lactic acid, which accumulates during heavy exercise. Lactic acid molecules still contain a lot of chemical energy and it would be wasteful to discard them. For this reason, the lactic acid passes to the liver where it is stored until oxygen becomes plentiful again (during the heavy breathing that follows strenuous exercise). Most are then ‘recycled’ back into glucose again for use in cellular respiration. The amount of oxygen required to fuel the recycling of lactic acid is known as the ‘oxygen debt’. Levels of the enzymes required to carry out the recycling of lactic acid increase as a result of regular anaerobic exercise.

Other substrates for cellular respiration Although glucose is most commonly used as the source of energy for cellular respiration, the processes of cellular respiration are quite flexible. Cells can also process other organic molecules, such as lipids and proteins, to enter the cellular respiration pathway and be broken down to generate ATP. When people diet to reduce their weight, most of the available glucose stores are used up first, then fat stores are used to provide the ATP needed for cells to continue functioning. When starved of food for a long period, even the proteins in muscles and other body tissues will be broken down to provide the energy necessary to survive. Fats provide more energy per gram (39 kJ) than either carbohydrates or proteins (about 17 kJ each).

Figure 3.11 Mark Lewis-Francis of Great Britain competing in the men’s 100 metre sprint.

55 Energy transformations

summary

3.2

• Organisms use energy in similar ways. • For immediately usable energy, all cells use the chemical energy carried in the terminal phosphate bond of ATP. • ATP in cells is replenished through the ATP/ADP cycle. • The energy used to make ATP can be obtained by releasing the chemical energy stored in the bonds of glucose molecules in a series of enzyme-controlled chemical steps generally referred to as cellular respiration. • The complete release of the energy of glucose involves anaerobic glycolysis followed by aerobic respiration. • Glycolysis takes place in the cytosol and generates two ATP molecules and two molecules of pyruvate per molecule of glucose. It does not require oxygen.

• Aerobic respiration takes place in mitochondria and generates 34–36 ATP molecules (plus CO2 and H2O) for every 2 molecules of pyruvate. • If no oxygen is available, pyruvate is fermented anaerobically in the cytosol to lactic acid (in animals) or alcohol and carbon dioxide (in plants and microorgansims), with no ATP produced. • Lactic acid is recycled back into glucose in the liver when oxygen becomes available again. The amount of oxygen required for this is the oxygen debt. • Other substrates, such as lipids and proteins, can also enter aerobic respiration pathways at various places and be broken down to produce ATP.

key questions 7 a What is ATP, and what role does it play in supplying energy within cells? b Use Figure 3.7 to help you explain the relationship between ADP and ATP. 8 Define glycolysis. 9 a Define aerobic respiration. b Summarise the process of aerobic respiration using a balanced chemical equation. 10 Use diagrams to help explain what happens in a glycolysis b Krebs cycle

! Autotrophs are producers—they can produce organic molecules from inorganic material

c electron transport. In each case state where in the cell the reaction occurs, how much energy is released and whether oxygen is required. 11 What are the products of fermentation in plants and animals? Write the word equations for the reactions involved in each case. 12 Suggest the advantage to a cell of converting pyruvate to lactic acid or ethanol when this yields no extra ATP. 13 Name two compounds other than glucose that can be broken down to release energy for ATP production. Are they equally energy-rich? Explain.

3.3

Getting glucose

Figure 3.12 Kangaroo Island western grey kangaroo feeding on eucalypt leaves.

! Heterotrophs are consumers—they must consume their organic molecules because they cannot make them from inorganic materials.

56 Molecules of life

Different types of organisms use glucose and ATP in similar ways to power cellular activities. However, they have very different ways of getting the glucose they need (Figure 3.12). Green plants are autotrophs and they make glucose during photosynthesis by combining carbon dioxide from the air with water. This method of making glucose uses energy that comes from the physical environment. Most autotrophs, including plants and algae, are photosynthetic (photo meaning ‘light’, synthesis meaning ‘putting together’), which means they get this energy from sunlight. Some prokaryotes are chemoautotrophs, getting the energy they need by carrying out energyreleasing reactions between inorganic molecules. From a global perspective, autotrophs trap the energy that is ultimately used by all organisms. Unlike plants, animals are unable to use simple inorganic compounds to make glucose, or other organic molecules, from which they could release energy. Instead, they get the organic compounds they need by eating other organisms or their products. For this reason, animals are called heterotrophs (hetero meaning ‘other’). In ecology, autotrophs are called ‘producers’ and heterotrophs are called ‘consumers’ to indicate their feeding roles.

Photosynthesis—converting solar energy The ultimate source of energy for virtually all living organisms is the radiant energy of sunlight. Green plants trap light energy and transform it into chemical energy by the process of photosynthesis. Light energy is trapped by chlorophyll (chloro meaning ‘green’, phyll meaning ‘leaf’), a green pigment molecule, and is used to form ATP molecules. These ATP molecules are then used to drive carbon dioxide fixation, which is the combination of carbon dioxide and water to form glucose and oxygen gas (Figure 3.13). Chlorophyll is found in plants, algae, purple and green bacteria, and cyanobacteria. In algae and the green parts of plants, such as the upper surface of a leaf, chlorophyll is in prominent green organelles called chloroplasts (Figure 3.14a). There may be several hundred chloroplasts in each cell. Chloroplasts are membranous structures about 6 µm by 3 µm, which are easily visible using a light microscope. They consist of an outer membrane, many internal layers of membrane that form sacs or lamellae, and a fluid matrix (Figure 3.14b). Chlorophyll is located on the surface of these internal membranes. The enzymes needed to carry out photosynthesis are located in the matrix of the chloroplast.

LIGHTENERGY

CHLOROPHYLL

WATER

FROMATMOSPHERE

OXYGEN

HYDROGENANDCARBONDIOXIDE

TOATMOSPHERE

GLUCOSEANDWATER CHEMICALENERGY

Figure 3.13 In photosynthesis, light energy trapped by chlorophyll splits water, releasing the oxygen to the air. The hydrogen then combines with carbon dioxide to form glucose.

(a) S

(b) chloroplast containing chlorophyll

stroma grana

G

G

mesophyll cell

photosynthesis

Photosynthesis is summarised by the following equation: INPUTS OUTPUTS light energy glucose + water + oxygen carbon dioxide + water chlorophyll 6CO2 + 12H2O

Figure 3.14 (a) Mesophyll cells in leaves and some stems contain many chloroplasts in which photosynthesis takes place. (b) An electron micrograph of chloroplast membranes. The light-dependent reactions take place in the grana (G) where the chlorophyll is located. The light-independent reactions take place in the stroma (S).

C6H12O6 + 6H2O + 6O2

Note that water is shown on both sides of the equation. This is because the water used in the reaction (left side) is split, and new water is formed (right side). Although the overall equation of photosynthesis seems simple, it is a complex pathway with many steps. These steps form two stages: the first stage are the light-dependent reactions, which take place on the inner membranes of the chloroplast. As suggested by their name, these reactions require light. Chlorophyll traps light energy and uses it to produce ATP and to split water into hydrogen ions and oxygen gas.

biofile In one leaf about the size of your hand, there can be up to 3–5 billion chloroplasts. This is about the same number as the total human population on earth.

57 Energy transformations

First stage:

biofile Attempts by chemists to develop a synthetic pathway that can trap solar energy and convert it to chemical energy as efficiently as photosynthesis have so far proved fruitless. It is a very complicated process. If we were able to develop an artificial photosynthetic system it could have practical applications, such as providing a new clean energy source or counteracting the build-up of carbon dioxide that contributes to global warming.

water

light energy

hydrogen ions + oxygen gas + ATP

chlorophyll Even though the whole process of photosynthesis takes place in sunlight, the second stage of photosynthesis does not require light, so it is referred to the light-independent reactions (or sometimes as the dark reactions). These reactions take place in the fluid matrix of the chloroplast. ATP made during the light-dependent stage provides the energy needed to combine carbon dioxide with hydrogen ions (also from the light-dependent stage) to form the energy-rich molecule glucose, and water. Second stage: ATP + hydrogen ions + carbon dioxide

glucose + water + ADP

extension Different pathways of photosynthesis Light-dependent reactions These reactions take place on the internal membranes (grana) of the chloroplast and are similar in all plants. Chlorophyll has a molecular structure that, when excited by light energy, initiates a series of steps that results in the formation of ATP molecules, at the same time splitting water molecules into hydrogen and oxygen. Oxygen gas is released as a by-product. Light-independent reactions in C3 plants These reactions take place in the fluid matrix within the chloroplast (stroma). Carbon dioxide diffuses into the leaf, into leaf cells and into

the chloroplasts, where it is captured in a complex pathway. This pathway is called the Calvin cycle, or C3 photosynthesis, because the first stable compound produced has three carbon atoms. Eighteen ATP molecules, made during the light-dependent stage, are needed to provide the energy to combine six carbon dioxide molecules with twelve hydrogen molecules (also from the light-dependent stage) to form one molecule of glucose and re-form six molecules of water. In C3 plants, up to 50% of the carbon dioxide captured in photosynthesis is released again before it can be converted to sugar! This is because under warm conditions, the enzyme that normally captures carbon dioxide in the C3 pathway reacts with oxygen instead.

(a) (b)

Figure 3.15 (a) Sugarcane plants carry out C4 photosynthesis, which for plants growing in a hot, sunny climate, such as that in many parts of Queensland, is a more efficient pathway for fixing carbon than the C3 pathway. (b) Cacti are CAM plants, adapted to conditions of high daytime temperatures, intense sunlight and low soil moisture.

58 Molecules of life

extension Different pathways of photosynthesis (continued) The efficiency of carbon dioxide capture in hot weather is reduced, so stomata must be open longer to obtain sufficient carbon dioxide, which increases water loss. Light-independent reactions in C4 plants To overcome the above problems with the C3 pathway, many species of plants native to hot climates, including maize, sorghum and sugarcane (Figure 3.15), have an additional process to capture carbon dioxide. This process is known as C4 photosynthesis because the first stable product produced (oxaloacetic acid) is a compound with four carbon atoms. Eventually, this four-carbon compound is converted to a molecule of carbon dioxide and a three-carbon compound, which enters the Calvin cycle. The enzyme used to capture carbon dioxide in C4 plants does not react with oxygen. This means that C4 plants are more efficient in warm conditions because they can capture more carbon dioxide in less time, and the stomata do not need to stay open for as long. Their

carbon-fixing pathway works well in warm environments with very high light intensity. C4 plants can produce two to three times as much sugar as a C3 plant on a hot, sunny day. However, on a milder day, a C3 plant is more efficient because its pathway uses less energy to capture the carbon dioxide. Light-independent reactions in CAM plants Another adaptation of plants to hot, dry climates is seen in plants that undergo CAM (crassulacean acid metabolism) photosynthesis. Plants such as pineapples and cacti close their stomata during the day and open them at night, at which time they take up carbon dioxide and convert it to four-carbon organic acids, which accumulate in the central vacuole. During the day, while the stomata are closed, carbon dioxide is released from these organic acids and used immediately for C3 photosynthesis. CAM plants are adapted to conditions of high daytime temperatures, intense sunlight and low soil moisture.

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Figure 3.16 Photosynthesis occurs in two linked stages. In the light-dependent reactions, energy trapped by chlorophyll is used to produce ATP and to split water into oxygen gas and hydrogen ions. In the light-independent reactions, the ATP is used to combine the hydrogen ions with carbon dioxide to manufacture glucose. Some water is re-formed.

59 Energy transformations

Factors affecting the rate of photosynthesis

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In various circumstances, any of the factors that influence photosynthesis (such as light intensity, carbon dioxide level or temperature) may limit the rate of photosynthesis. Photosynthesis will be limited by only one factor at a time, but if conditions in an individual chloroplast change, the particular factor that is limiting may also change (Figure 3.17). For example, carbon dioxide levels that are adequate (not limiting) in conditions of low light may become limiting if light intensity increases. Figure 3.17 The rates of photosynthesis at different carbon dioxide concentrations and increasing light intensity. At 0.03% carbon dioxide, the rate of photosynthesis is limited by light up to 25% of full daylight, after which carbon dioxide becomes the limiting factor. At 0.09% carbon dioxide, light is the limiting factor up to almost full sunlight level.

biology in action Selective poisons Poisons are used in agriculture because weeds and other pests reduce crop yields. They are also used to control the spread of disease in animals (including humans) and in plants. Many lethal poisons are effective because they interfere with the energy transformations of organisms. Some interfere with photosynthesis and are therefore selective for plants. For example, herbicides such as amitrole inhibit the production of chlorophyll, and others such as paraquat and diquat block the light-dependent reactions in the first part of photosynthesis. Other herbicides such as 2,4-D and 2,4,5-T act by disrupting the energy balance of the plant. Glyphosate inhibits enzymes that are needed for the normal growth of plant cells.

Figure 3.18 This strip of dead wheat was sprayed with the highly poisonous herbicide paraquat (Gramoxone), which blocks the light-dependent reactions of photsynthesis, to create a public footpath across the farmland.

Carbon dioxide

Rate of photosynthesis (P) or respiration (R) high

compensation point (P = R)

photosynthesis (P) P>R cellular respiration (R)

P80%)

agglutination, complement activation

IgM

10 days

produced early in infection response

agglutination, complement activation

IgA

6 days

found in exteral secretions, tears, saliva and milk

mucosal immunity

IgD

3 days

located on the surfaces of antibody-producing cells (B-cells)

development of the antibody response

IgE

2 days

produced in allergic reactions

attaches to mast cells

biology in action Blood groups—‘natural’ antibodies When plasma and red blood cells from different individuals are mixed together they sometimes clump (agglutinate) (Figure 8.15). On the basis of this clumping, blood groups of people can be grouped into four main categories (A, B, AB and O) according to the presence of genetically determined antigens on the surfaces of their red blood cells. Type A blood has A antigens on the red blood cells, type B has B antigens, type AB has both A and B antigens, and type O has neither A nor B antigens on the red blood cells. Agglutination indicates that there are antibodies that react with these antigens in blood plasma. But where do these ‘natural’ antibodies come from? We now know that their presence is not inherited, but is due to exposure to A-like and B-like antigens on the surfaces of bacteria in the gut. Antibodies are formed in the normal way if the antigens are not recognised as self. Donor cells

Figure 8.15 When blood cells and plasma from people of different ABO blood groups are mixed together, some combinations cause severe clumping (agglutination) of the red blood cells. Clumping occurs when the antigens on red blood cells interact with antibodies in plasma.

192 Detecting and responding

$ONORCELLS

Recipient serum AB

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Recipient serum AB

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T cells and cell-mediated immune response T cells are produced in the thymus gland (hence the name ‘T cells’) from precursor cells formed in the bone marrow. The thymus is located in the chest in front of the trachea. T cells are responsible for cell-mediated immune responses. They have antibody-like molecules known as T cell receptors located on their surface. These T cell receptors react with foreign antigens on eukaryotic cells, such as virus-infected cells and cancerous cells (which have new and therefore foreign antigens on their surface), and on the foreign cells of a transplanted organ. There are two types of T lymphocytes, both of which can distinguish between normal self tissues and tissues that are infected, damaged or from another organism. Helper T cells (TH cells) are regulatory cells that, when stimulated by antigen, produce and release cytokine molecules that control the development and function of other T and B cells, as well as phagocytes. Cytotoxic T cells (TC cells) are effector cells. When stimulated (activated) by antigen and cytokines, TC cells directly lyse (split open) or kill target cells, such as foreign or infected cells, on the basis of their particular antigen.

B cells and humoral response B cells are formed in the bone marrow and spleen. They are called B cells because they were first identified in the Bursa of Fabricius, a lymphoid structure located in the hind gut of birds. At any time there are about 2 × 1012 B cells circulating in the blood. They are small, non-dividing, relatively inactive cells.

IMMATURE "CELL

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PLASMA CELL4(

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Figure 8.16 Some B cells become plasma cells, which provide protection by secreting antibodies. Others become memory cells and are retained in lymph nodes.

193 Defending self

biology in action Parasites defend themselves too If parasites totally eluded all the host’s defences and were sufficiently virulent (able to grow rapidly at the host’s expense), they could kill the host on whom their survival depends. Conversely, if a parasite were too susceptible to its host’s defences it would quickly be destroyed. In these circumstances, well-adapted parasites strike a balance in a variety of ways. The more we can understand about parasite’s defences, the easier it will become to develop new drugs to counteract them. Antigenic variation Some parasites can ‘change their spots’. Several protozoan parasites evade an immune response by shedding their antigens upon entering the host. Other species change their antigens. Trypanosomes are protozoan parasites that are transmitted by the tsetse fly (Figure 8.17a). Infection by trypanosomes causes recurring bouts of sleeping sickness in humans. As they multiply in the blood and spread into the nervous system, they cause sleepiness, coma and ultimately death. Sleeping sickness is extremely difficult to treat effectively. But how does the parasite evade the immune system in the weeks before death occurs? Each trypanosome has one kind of surface protein, but a population of trypanosomes will contain individuals with different surface antigens. Upon infection, trypanosomes multiply in the host. At the same time their surface antigens induce the production of specific antibodies by the host, which will eliminate all trypanosomes carrying these surface antigens. However it takes some time for the host to manufacture the antibodies. In the meantime the trypanosomes ‘put on’ new surface antigens (they have hundreds of potential surface protein genes). Individuals carrying the new surface antigens will survive the antibodies and the parasite population will increase again until antibodies against the new surface antigens are produced, at which time the population will crash again. But by this time, gene shuffling will have produced ‘new’ individuals. These ‘boom-and-bust’ population cycles will continue until either the host dies or the parasite infection is finally wiped out (Figure 8.17b). The ability of parasites to vary their surface antigens in this way is called antigenic variation. Malarial parasites are also capable of antigenic variation, which is making the production of an effective vaccine very difficult. (a)

MITOCHONDRION

UNDULATING MEMBRANE

(b) ANTIBODY!

NUCLEUS FLAGELLUM 4RYPANOSOME

Figure 8.17 (a) The structure of a trypanosome protozoan parasite. (b) Antigenic variation in trypanosomes results in repeated cycles of infection by populations of trypanosomes with different antigens on their surface.

194 Detecting and responding

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biology in action Parasites defend themselves too (continued) Antigenic mimicry Some parasites can take up host molecules and insert them into their own surface layers so the host’s immune system can no longer recognise them as ‘not self’. Blood flukes, for example, can take blood group antigens and the major antigens for recognising ‘self’ from host red blood cells and incorporate them onto their outer surface. Evading macrophages Macrophages are important phagocytic cells that recognise and destroy a wide variety of microbes. However some microbes, such as leishmanias (protozoans that cause a disease with symptoms like malaria, Figure 8.18), when devoured by a macrophage, somehow evade enzymatic breakdown by lysosomes within the macrophage. They remain and grow inside the macrophage, avoiding detection and removal by the immune system. Some bacteria can avoid phagocytes by releasing an enzyme that destroys the component of the host’s complement that attracts phagocytes. Others can kill phagocytes by releasing a membrane-damaging toxin.

Figure 8.18 Light micrograph showing a macrophage that has engulfed several Leishmania donovani parasites (the small purple bodies). Leishmanian protozoans can hide inside macrophages and other tissue of the lymph system to protect themselves from the body’s immune defences.

Hiding in cells Some bacteria can get into the epithelial lining of the intestine (Figure 8.19), multiply inside these cells and transfer into neighbouring epithelial cells without entering the extracellular space where they would be vulnerable to detection by the host’s defences. Immune suppression A non-specific way of interfering with the immune response is to interfere with the normal function of the immune system itself. Most parasites seem to be able to disrupt the immune system of their host to some extent. It is this property of human immunodeficiency virus (HIV) that makes AIDS extremely difficult to treat. Disarming antibodies Some bacteria, such as Staphylococcus aureus, have receptors on their surface that disrupt the normal function of the host’s antibodies. These receptors can bind with the ‘stem’ end of the antibody instead of the usual antigen binding sites (Figure 8.14). This prevents the stem of the antibody, which normally communicates with various parts of the immune system, from initiating an immune response such as engulfment by a phagocyte.

195 Defending self

When a B cell meets and interacts with a specific antigen, the B cell becomes metabolically active and begins to divide (proliferate). To respond to most antigens, B cells require cooperation from TH cells. Two types of daughter cells are formed: plasma cells and memory cells. Plasma cells are essentially ‘factories’ specialising in antibody production. After about five to eight days, a mature plasma cell can produce up to 30 000 antibody molecules per second! Memory cells remain in lymphoid tissues for long periods (often for the lifetime of the animal) and are responsible for the immunity that follows many infectious diseases or vaccination. B cells are responsible for producing large quantities of antibodies when stimulated by particular antigens. This is the humoral immune response. It is primarily directed against invading bacteria and viruses, and their toxins. Antibodies released by B cells coat foreign organisms (due to antigen– antibody interactions) so that: • foreign material is recognised and engulfed by phagocytic cells; • activities of the foreign organism are inhibited; • complement proteins (see page 186) become attached and proceed to actively destroy foreign cells. 3UMMARYOFCELLULARANDHUMORALRESPONSES #ELLULARIMMUNITY

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Figure 8.19 The responses of the immune system involve the coordinated actions of T cells and B cells. The response is often initiated by dendritic cells presenting antigen to T cells able to recognise the antigen, causing them to proliferate and develop into helper T cells (TH cells) and cytotoxic T cells (TC cells). TH cells become activated and release cytokines. Cytokines help both T and B lymphocytes to proliferate (1 and 2), and B lymphocytes to differentiate into plasma cells, which produce antibody (3). Cytokines also activate phagocytes (4) increasing their efficiency in removing foreign material. TC cells lyse virus infected cells. Antibody with complement (C) kills bacteria, and antibody also attaches bacteria to the phagocyte membrane increasing phagocytosis.

Detecting and responding

ANTIBODY SECRETING CELLPLASMACELL



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summary

8.4

• Specific defence is provided by an elaborate immune system. • Vertebrate immune responses are characterised by specificity and memory. • Immune responses involve lymphocytes (a particular group of white blood cells formed in the bone marrow and spleen). • Immune responses include both humoral (‘blood-borne’) and cell-mediated mechanisms. In humoral immunity antibodies are released by B cells; in cell-mediated immunity, active destruction is carried out by T cells. • Antigens are molecules able to bind to antibodies or T cell receptors. They stimulate lymphocytes to begin the immune response.

• Antibodies are specific proteins produced by lymphocytes that are able to react with particular antigen molecules. • Blood groups refer to the antigens found on the surfaces of red blood cells. They are important in blood transfusions and pregnancy. ABO antibodies occur ‘naturally’. • TC cells act against virus-infected cells, cancer cells and transplanted tissue. TH cells assist B cells and TC cells. • B cells are lymphocytes that produce large quantities of antibodies when stimulated by particular antigens. This is the humoral immune response.

key questions 16 In terms of specific immune responses in vertebrates, explain what is meant by a specificity b memory. 17 a Name the two main lines of defence in the mammalian immune system. b Describe their respective roles. 18 Use a fully-labelled diagram to explain the relationship between antibodies and antigens.

19 20 21 22

Where are T cells originally formed and where do they mature? What type of responses are produced by T cells? Explain the purpose of T cell receptors. There are two kinds of T cell. Name them and describe the function of each. 23 What type of response is produced by B cells? 24 Draw a flow diagram to illustrate the role of B cells in defending the body against disease.

8.5

Why is the immune system so complex? Your immune system cannot predict which antigens you might encounter during your lifetime, and there is virtually no limit to the number of antigens in the environment. New organisms are evolving all the time and new molecules are being manufactured by humans. Your immune system has the difficult task of protecting you against an almost infinite and certainly unpredictable variety of potentially harmful antigens. In addition, you are made up of a huge array of cells and molecules which must not be subject to attack by your immune system or very serious damage can result (as in autoimmune diseases, see page 211).

197 Defending self

Clonal selection theory What is needed is an immense number of very specific antibodies. How does the immune system cope with this problem? Evolution has resulted in a novel solution, which lies in the structure of antibodies themselves. Antibody molecules are made up of a part that is constant and a part that is highly variable (Figure 8.14). The variable portion has a relatively small number of genes that are cut and shuffled (rearranged) freely and randomly to produce millions of different combinations. This allows the random production of innumerable antibodies using instructions encoded in only a modest amount of DNA. Each antibody is produced by only a small clone of cells. This explanation was developed by one of Australia’s most celebrated scientists, Sir Frank Macfarlane Burnet, and is known as the clonal selection theory. LYMPHOIDSTEMCELL INBONEMARROW

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Figure 8.20 The clonal selection theory proposes that there are many different immature lymphocytes present in the blood. When one is triggered by an antigen that reacts with it specifically, it is activated to form clones of similar cells.

198 Detecting and responding

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The clonal selection theory explains how a virtually unlimited array of antibodies, which will not react to ‘self’ can be produced, using a modest amount of DNA. During embryonic development, millions of different, randomly generated, tiny clones of lymphocytes (B cells and T cells) form, each with an antibody that will react to only a single antigen. If a B cell is stimulated by contact with the specific antigen with which it can combine, it responds by dividing and differentiating into both antibody-secreting plasma cells and memory cells. These remain in the blood and provide future immunity (Figure 8.16). Likewise, a T cell stimulated by contact with the specific antigen with which it can combine will multiply and develop into helper T cells and killer T cells. One problem that remains is that some of the random antibodies generated will inevitably react with ‘self’ antigens. Although most of these are removed or ‘silenced’ during their early development, some still remain. This is known as the ‘generation of self-tolerance’. The ‘learning’ process takes place during embryonic development and cells that would react with ‘self’ antigens do not survive. If a sample of cells from a donor organism is

grafted onto a developing embryo before its immune system has finished learning, the immune system will also learn to recognise the graft as self. When this embryo has grown to be an adult, a graft from the same donor organism onto the recipient organism will not be rejected.

summary

8.5

• The clonal selection theory explains how, using only a moderate amount of DNA, the immune system is able to respond quickly to an almost unlimited array of foreign antigens.

• Parasites show adaptations to evade their host’s defences, including: • antigenic variation and mimicry to evade detection; • evasion of macrophages and hiding inside the host’s cells; • suppression of the immune system; • disarming the host’s antibodies.

key questions 25 Explain what is meant by the statement: ‘A good parasite does not kill its host’. 26 Briefly describe three methods that parasites use to evade attack by the immune system.

27 Explain how an almost infinite number of antibodies can be produced using only a small amount of DNA.

199 Defending self

technologies and techniques Gene shuffling and cancer by Professor Suzanne Cory

H

ow are our lymphocytes able to recognise billions of different foreign antigens? According to the clonal selection theory this is possible by randomly rearranging a relatively small number of genes to produce billions of different combinations, each able to recognise a different antigen (see page 198). Lymphocytes are the only cells in our body that have the enzymatic machinery to ‘cut, shuffle and paste’ genes in this way. But these ‘genetic gymnastics’ in lymphocytes also have a darker side. Every now and then the ‘cut and paste’ job goes awry and pastes an antigen receptor gene next to a gene with potential to promote

cancer. This was first discovered in 1982 by scientists at The Walter and Eliza Hall Institute in Melbourne and simultaneously in two USA laboratories, where both were studying Burkitt’s lymphoma, an aggressive tumour prevalent in equatorial Africa and Papua New Guinea. The molecular biologists found that a gene called myc had been joined (translocated) to an antibody gene (Figure 8.21) and guessed that this was the root cause of the disease. The Hall Institute team provided the definitive proof by creating mice carrying a gene mimicking the myc translocation. Every mouse carrying this gene developed Burkitt’s lymphoma before it was 12 months of age.

chromosome A

chromosome A

chromosome B

chromosome B

chromosome translocation

Figure 8.21 A chromosome translocation results from the ends of a broken chromosome being joined to the broken ends of another chromosome rather than being rejoined correctly. This diagram shows how the myc gene and the antibody gene are joined after chromosome translocation.

200 Detecting and responding

antibody gene

antibody gene myc gene

myc gene

bio The myc gene plays a critical role in cell division and its expression is normally very tightly regulated. After the translocation, the powerful ‘on’ signals associated with the antibody gene ‘spill over’ onto the myc gene. The myc gene expression is no longer carefully checked, resulting in repeated cell divisions. The B lymphocyte carrying the translocated myc gene therefore starts dividing more often than its neighbours and eventually becomes a Burkitt’s lymphoma. Many other chromosome abnormalities in cancers have since been shown to activate oncogenes (cancer-provoking genes), most of which promote cell proliferation. In follicular lymphoma, the most common lymphoid tumour in Western society, the cause was found to be quite different. The Hall Institute group showed that bcl-2, the oncogene involved, acts by inhibiting

the intrinsic cell suicide program known as apoptosis (see page 284, Chapter 12). Apoptosis is essential for removing infected, damaged or unwanted cells, including those which have suffered mutations. Rather than promoting cell division, the bcl-2 oncogene slows cell death. This has the same outcome—too many cells. Activation of bcl-2 is particularly dangerous when combined with activation of myc, because mice carrying both genetic abnormalities all developed tumours before seven weeks of age (Figure 8.22). Bcl-2 has proved to have many relatives, some of which promote cell death rather than inhibiting it. By understanding in intimate molecular detail how this complex family operates, the Hall Institute team is hoping to develop more effective treatments for cancer.

 BCL 

 MYC



Professor Suzanne Cory Professor Suzanne Cory is the Director of The Walter and Eliza Hall Institute of Medical Research. She is one of Australia’s most distinguished molecular biologists. Her research has had a major impact internationally on the understanding of immunology and of the development of cancer. She is a Fellow of the Australian Academy of Science and a Fellow of the Royal Society.

#UMULATIVEMORTALITY

MYC







BCL 











Figure 8.22 Mice with an activated bcl-2 oncogene as well as an activated myc oncogene develop lymphoma much more rapidly than those with just one of these genes.

!GEDAYS

201 Defending self

key terms non-specific defence specific immunity innate acquired memory specificity lymphocytes cytokines interferon

complement NK cells phagocyte inflammation histamines mast cells platelets resolvins fever

pyrogens B cell T cell antibody antigen T cell receptors immunoglobulin blood groups cell-mediated immunity

helper T cells cytotoxic T cells humoral immunity plasma cells memory cells macrophage leucocytes glycoprotein

08 worksheet 21

1 Interferons produced by virus-infected cells provide some defence against invading pathogens. How is the action of interferons different from an immune response? 2 a People often confuse the ‘flu’ with the common cold. Find out the difference between influenza and a cold. b Explain why usually healthy people can suffer from influenza and colds many times. 3 Explain how a fever can contribute to non-specific defences. 4 How would you expect B cells and T cells to be involved in response to: a an infected cut on a finger? b virus-infected cells? 5 a Explain why many different types of B cells are needed. b Why is it important that TH cells do not change once they have left the thymus? 6 Immunoglobulins or antibodies are generally grouped into five classes. Copy and complete the following table to summarise the role of each class. Antibody type

Role

IgG IgM IgA IgD IgE

202 Detecting and responding

7 Cancer is characterised by cells that undergo unchecked reproduction, competing with body tissues for space and nutrients. Unlike normal body cells, cancer cells have surface antigens that are not recognised by the body as ‘self’. Leukaemia is a cancer of the white blood cells. In this condition the white blood cells of the bone marrow reproduce prolifically, which impairs their function in the defence system of the individual. One treatment for this cancer is radiation therapy to destroy a large percentage of the cancerous cells, followed by a bone marrow transplant. The stem cells of the donor marrow are critical to the patient’s recovery potential. a Suggest why radiation therapy is an important part of the treatment regime for leukaemia. b What are stem cells? How are they important in such a transplant? c Explain why it is important that the closest possible tissue match is made between a donor and the recipient for a bone marrow transplant to be successful. 8 Draw a concept map that summarises the functions of the body’s defence system. 9 Enter some of the key words into a search engine to discover novel defence mechanisms used by plants to manage pathogens. Write a short report on each.

0

chapter 09

Applications of immunology

key knowledge • actively and passively acquired immunity— vaccines and antibody serums • disorders of the immune response— hypersensitivity (allergens and allergic responses), autoimmunity, immunodeficiency and persistent inflammation (resolvins) • frontiers of immunology

chapter outcomes After working through this chapter you should be able to: • use examples to distinguish between active and passive immunity, and between naturally-acquired and artificially-acquired immunity • explain what vaccines, antibody serum and antitoxins are and how they work • list the types of disorders of the immune system that result in disease • give examples illustrating how hyperactivity of the immune response causes damage and disease • describe the roles of MHC and dentritic cells in recognising ‘self’ • describe how immune responses are involved in allergies, asthma, transplant rejection and autoimmune diseases • describe how the inappropriate persistance of the inflammatory response contributes to disease and the role of resolvins • explain the principle behind immunotherapy • describe the problem of development of drug resistance and what people can do to minimise this.

9.1

Acquiring immunity The beginning of the end for smallpox

Figure 9.2 Several smallpox pustules.

For centuries, smallpox and other infectious diseases ravaged human populations. Waves of epidemics would sweep through townships, often after the arrival of a traveller from a distant place. Strangers were considered dangerous. On the other hand, survivors of an epidemic seemed to be entirely safe when the disease reappeared. Servants were much more employable if they had smallpox scars because experience had shown that they would not catch this devastating disease again. It was also noticed that milkmaids (young women who milked cows by hand) who had previously been infected with cowpox, a disease similar to smallpox but relatively milder, did not succumb during smallpox epidemics. The English country doctor Edward Jenner conducted a detailed and rigorous study of the incidence of smallpox and concluded that cowpox somehow gave protection from smallpox. In 1796 he deliberately infected a small boy with material from a cowpox pustule, then six weeks later infected the boy with material from a smallpox pustule. The boy survived. Despite the public’s scepticism and outright fear of the consquences, Dr Jenner proceeded to deliberately infect the arms of his patients by contact with the milkmaids’ sores, thereby transferring future immunity to them. This was decades before Pasteur and Koch discovered the microbiological nature of infectious disease, and almost 100 years before the microscopic observation of bacteria and viruses. Jenner had made an historic advance in the treatment of infectious disease: he had shown that protection (later known as immunity) could be acquired by artificial means.

Figure 9.1 This cartoon drawn by James Gillray and published in 1802, eight years after Jenner’s first vaccination, was titled ‘The Cow Pock, or the Wonderful Effects of the New Inoculation’. It illustrated the anxiety of many people about this new treatment.

204 Detecting and responding

Almost a century after Jenner discovered that cowpox gave some protection against smallpox, the work of Louis Pasteur and Robert Koch led to some understanding of the relationship between microorganisms and disease (see page 157). They showed that it was possible to produce immunity against particular microbes. At the time, immunity was understood to be gained as the result of a successful battle against an invading microorganism, leaving the individual well prepared for any future encounter with the same microbe. The actual mechanisms involved in the development of immunity were not uncovered until well into the twentieth century.

biofile Formalin (formaldehyde in water) was used as a powerful antiseptic and disinfectant. It is also used to preserve organs and animals, and to fix tissue for microscopy. It acts by savagely denaturing proteins and is now known to be carcinogenic (to cause cancer).

Actively-acquired immunity We now know that Jenner’s treatment worked because the outer surfaces of smallpox and cowpox viruses have similar molecules, and the boy he treated with the cowpox virus developed an active immunity against these molecules. This method of treatment became known as vaccination (after vacca, which is Latin for cow). However, Jenner was fortunate with small pox. There is not a ‘cowpox equivalent’ for other dangerous diseases. Louis Pasteur (1822–1895), and then later Almroth Wright (1861– 1947), experimented with treating disease-causing bacteria with formalin to weaken or kill them, resulting in bacteria that would no longer produce disease but still be capable of provoking an immune response. They were successful in preparing such vaccines against cholera, anthrax, rabies and typhoid fever. In some diseases, such as diphtheria and tetanus, symptoms are caused by a toxin released by the infecting bacteria. Inactivated toxins (toxin plus formalin) are called toxoids, and they produce an immune response without eliciting the symptoms of the disease. Triple antigen is administered to many infants and young children to develop an immunity against diptheria, tetanus and whooping cough. Surviving an infection by bacteria or viruses results in a naturally-acquired active immunity that protects against further infection by the same organism (Figure 9.3). If you have had chickenpox or measles you are unlikely to catch them again because the immune system has memory (see page 193).

NATURALLYACQUIRED IMMUNITY INFECTION

DISEASE USUALLY ARTIFICIALLYACQUIRED IMMUNITY ANTIGENS STIMULATE IMMUNE SYSTEM

PRODUCTION OF

Vaccination Vaccination refers to the technique of inducing artificially-acquired active immunity by injecting a specific vaccine usually made of altered, weakened or dead microorganisms, or inactivated forms of the toxin released by certain bacteria (or, in the unusual case of smallpox, of a similar organism). The introduction of vaccination has dramatically altered the incidence of infectious diseases (Figure 9.4). Vaccines should be specific, safe and effective in initiating an immune response to give lasting protection against a particular disease-causing agent. Attenuated vaccines are a weakened form of the disease causing agent that is still able to reproduce, but which does not cause disease symptoms. The advantages of using attenuated vaccines is that a single dose will usually give long-lasting immunity because the microbe multiplies within the body for a period. The disadvantage is that it may cause disease in those with weakened immune systems, or cross the placenta in a pregnant woman and cause damage to the developing fetus. Examples of attenuated vaccines include those against measles, mumps, rubella and the Sabin vaccine against polio. Inactivated vaccines have sufficient immunogenic capacity to provoke an immune response, but they are to unable to replicate. They may be killed microbes or parts of them (protein or polysaccharide components). For example, a vaccine has been genetically engineered to produce a purified

MEMORY CELLS

PLASMACELLS THATPRODUCE

PASSIVE IMMUNITY ABILITYTO PRODUCE ANTIBODIES PERSISTS

SPECIFIC ANTIBODIES

FIGHTTHE INFECTION

Figure 9.3 The acquisition of active immunity can be natural or artificial. Passive immunity can provide a temporary defence against an invading organism.

205 Applications of immunology

(a)

(b) 80

Cases per 100000 people vaccine

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70 Measles

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First Hib vaccines approved for use in Australia

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Figure 9.4 (a) The introduction of vaccines in the United States of America caused a dramatic reduction in the incidence of several common infectious diseases. (b) The Haemophilus influenza type b bacterium (Hib) causes serous diseases such as meningitis, pneumonia and serious throat infections. In Australia, the introduction of the Hib vaccine and the commencement of the national vaccination program has led to dramatic reductions in the short- and long-term incidence of these infectious diseases.

component of the protein coat of the virus that causes foot-and-mouth disease. This method produces a vaccine that cannot be contaminated with live or virulent pathogens.

Passively-acquired immunity biofile Scientists in Melbourne have devised a totally synthetic and versatile vaccine that should be effective against viruses, harmful bacteria and tumours. The vaccine is peptide-based and different forms have been designed to trigger specific types of immune responses, such as humoral or cell-mediated responses. Unlike traditional vaccines, the synthetic vaccine does not contain any actual components of a real pathogen. It can be produced economically on a large scale with no risk of contamination.

206 Detecting and responding

It is too late to use artificial immunisation if a person has already been infected by a disease-causing microorganism, or been bitten or stung by a venomous animal. In these cases, it is sometimes possible to administer serum-containing antibodies (or antitoxins) that have been produced against the disease in another organism. These antibodies rapidly destroy the invading microorganism, or its toxin, before the disease has time to take hold. The invading organisms are often destroyed before the body’s own immune system can be mobilised, so the protection provided may be only temporary and there is no memory (as in actively-aquired immunity). Because the immunity provided does not involve the individual’s own immune system, it is referred to as passive immunity. Artificial passive immunity is provided, for example, by administering antibody serum to Rh negative mothers to prevent the occurrence of haemolytic anaemia of the newborn (see page 209). Injections of tetanus antitoxins are routinely used by doctors to protect against tetanus in patients at risk because of a deep and dirty puncture wound. Natural passive immunity occurs during pregnancy as a result of the mother’s antibodies crossing the placenta and during breast feeding. These passively-acquired antibodies provide protection to the baby while its own immune system is developing over the first three to six months.

Table 9.1 Different types of immunity Natural

Artificial

Active (has memory)

natural exposure to an antigen causing an immune response

deliberate exposure to an antigen causing an immune response

Passive (no memory)

natural transfer of antibodies, such as by mother to baby across placenta or in breast milk

deliberate administration of antibody serum or antitoxins to provide temporary protection

summary

9.1

• Immunity is active or passive, natural or artificial. • Naturally acquired active immunity results from surviving an infection by bacteria or viruses. • Artificially acquired active immunity arises as a result of the injection of a specific vaccine. • A vaccine is a preparation, usually made from a disease-causing agent, used to induce active immunity.

• Artificial passive immunity involves the administration of a serum containing antibodies (antibody serum) made in another organism. • Natural passive immunity occurs during pregnancy and breast feeding.

key questions 1 Explain the observation that milkmaids in the 1700s were protected from infection by smallpox. 2 Use a flow chart to summarise the method used by Edward Jenner to confer immunity from smallpox in his patients.

3 What distinguishes artificially-acquired active immunity from naturally-acquired active immunity? 4 Why is the injection of ready-made antibodies called ‘passive’ immunity?

9.2

Disorders of the immune system There are several kinds of immunological disorders. • Hypersensitivity—caused by excessive immune activity • Autoimmunity—caused by a misdirected attack against ‘self’ • Immunodeficiency—caused by reduced responsiveness of the immune system • Persistent inflammation—caused by absence of the normal dampening of the inflammatory response.

Hypersensitivity Overall, the immune system does a great job in protecting the body from invasion by pathogens. However, sometimes the immune system reacts to antigens that pose no real threat to the body, and the subsequent immune response can cause moderate to serious tissue damage and even death.

207 Applications of immunology

Allergies and allergens

Figure 9.5 The steps involved in an allergic response to pollen particles causing hay fever or asthma.

POLLEN ANTIGEN

Allergic reactions are due to a rapid and vigorous overreaction of our immune system to a previously encountered antigen—which is called an allergen because it produces an allergic response. This reponse of the immune system causes tissue damage or disease symptoms in the host. Hay fever is an example of such an overreaction. It is often triggered by pollen particles, which carry allergenic molecules. An initial exposure to pollen causes specific antibodies known as immunoglobulin E (IgE) molecules to be made in lymphoid tissue. These antibodies have two sites that bind specifically to a pollen allergen and another that binds to the surface of mast cells (Figure 9.5). The IgE molecules travel in the blood stream and attach in their thousands to mast cells in epithelial and mucosal tissues for several weeks. If the same pollen is encountered on a subsequent occasion, the pollen attaches to pairs of adjacent IgE molecules on the mast cells. This triggers the mast cells to release histamine and enzymes, which produce the symptoms of allergic disease.

MAKESSPECIFIC ANTIBODY

"CELL LYMPHOCYTE

ANTIBODIESCOMBINE WITHMASTCELL

HISTAMINE MASTCELL

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ANTIGEN CROSS LINKS ANTIBODIES ANTIGEN

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Fur

dog

horse

cow cat House dust

Food

Pollen

Figure 9.6 After animal fur, pollen is the next most common environmental cause of allergic reactions in humans.

208 Detecting and responding

The protein molecules on the surface of some types of pollen grains, which are necessary for recognition by the female parts of the flower and subsequent fertilisation, are the molecules responsible for provoking the allergic response. Treatment to desensitise people with pollen allergies involves the repeated administration of tiny amounts of pollen extracts under the skin. Many substances in our environment can initiate an allergic response in susceptible humans. Fur, pollen, house dust and foods such as peanuts, lobsters and monosodium glutamate (MSG) are typical allergenic substances. Their relative importance in causing allergies in people is shown in Figure 9.6. The most common pollen allergen in Australia and New Zealand is fresh grass pollen (allergens rapidly break down as pollen ages). A characteristic feature of pollen sensitivity is its seasonal pattern of occurrence, as pollen is most abundant in the atmosphere during spring and early summer. Levels of IgE antibodies vary according to the presence of the allergen. Just before the pollen season, IgE levels in hay fever sufferers are at their lowest. They may then increase two- or three-fold as pollen levels increase.

Antihistamines counteract the effects of histamine and therefore suppress some allergic symptoms. Other substances, such as cortisone, suppress the immune system itself and reduce the immune response in general. Asthma is another type of respiratory allergy. The symptoms of increased mucus secretion and bronchospasm (constriction of the airways) are largely caused by mediators other than histamine. Thus, antihistamines are not effective in treating asthma. Bronchodilating drugs that relax the constricted muscles and open the airways can be delivered by an inhaler. They are usually effective in relieving the bronchospasm, but they have no effect on the mucus secretions. In rare cases allergic responses can become life-threatening. Circulating histamine and other released mediators cause the bronchioles to constrict, blood vessels to dilate and loss of fluid to the tissues so that blood pressure falls dramatically—a condition known as generalised anaphylactic shock. Common causes are bee stings, peanuts and penicillin injections. For people allergic to lobster, the mere presence of a tiny piece of lobster in food can result in an allergic response so massive that life may be endangered. An anaphylactic reaction may be fatal within minutes because of insufficient blood flow to vital organs, but it can usually be controlled by an immediate injection of adrenaline. People at risk may carry an EpiPen (named after epinephrine, the US word for adrenaline) for use in an acute allergenic emergency.

biofile Hay fever and asthma are allergic diseases suffered by up to 20% of Victorian school children. These allergic reactions are caused by tiny particles contained in pollen grains. Rainfall causes pollen to burst and release these particles into the air, which is why asthma rates increase after rain.

Figure 9.8 Grass pollen, such as from rye grass (Lolium perenne), can cause asthma and hay fever.

biology in action A childhood with asthma—Maree’s story Growing up fifty years or so ago in a very small country town in New South Wales posed some difficulties for me as a child. I was diagnosed with asthma at 18 months of age. Being so young, it was difficult at first for doctors to diagnose the problem. Then there was the question of how bad an episode would have to be before my parents would eventually decide to make the journey, once again, by rough dirt road to the nearest doctor or hospital. Sitting hunched over the kitchen table, concentrating on dragging a miserly amount of air into my lungs, I would get increasingly panic stricken. Finally, the trip would be made and I’d be given an injection of adrenalin or tablets that made my heart pound and increased the oxygen level in my bloodstream. Nights with asthma seemed never-ending. I was sent to boarding school at 7 years of age for the sea air. There, I’d be propped up with pillows in the sick room, night after night, fighting for the next noisy breath. The rattle and wheezing would fill the darkness. I used to do sums in my head all night to try and distract myself and pass the time. In my last year of high school I spent thirteen weeks in hospital. My teachers said I wouldn’t pass HCE but I did. Doing all those sums in my head must have paid off! I survived childhood asthma and stopped having bad attacks when I was in my 20s. It is very rare that I suffer from asthma now, only occasionally after a chest infection.

Haemolytic disease of the newborn Discovery of the Rh blood groups provided the explanation for a once common problem: erythroblastosis fetalis or haemolytic disease of the newborn. This is characterised by severe jaundice, which is a deficiency of red blood cells and yellowing of the skin. It is caused by a massive lysis of red blood cells and the breakdown of released haemoglobin into yellow pigments in the blood. The Rh factor is an antigen on the surface of red blood cells; if you have it, you are Rh positive, if not, you are Rh negative.

Figure 9.7 Eight-year-old Maree at primary school in Newcastle, New South Wales.

biofile Australia has one of the highest prevalence rates of asthma in the world, ranking in the top three with the United Kingdom and New Zealand. One in four primary school children and one in seven teenagers currently suffer from asthma. Asthma is the most common cause of hospital admissions in children between the ages of five and fourteen years.

209 Applications of immunology

biofile Cosmetics, deodorants, shampoos and conditioners contain complex chemical combinations. Some of the ingredients in these products can be allergenic to certain people and some have been linked to cancer. There is very little or no knowledge about some of the chemical combinations used. So it is probably wise to read ‘enclosed information leaflets’ before throwing them away.

Unlike ABO groups (see page 192) the antibodies against the Rh antigen are not normally present in blood plasma, but Rh negative individuals recognise Rh factor as ‘foreign’. Problems arise during some pregnancies (Figure 9.9). If an Rh negative mother is carrying an Rh positive child, some of the baby’s blood cells may cross into the mother’s bloodstream, particularly during childbirth. If this happens, the mother will begin to produce antibodies to the Rh factor. If this sensitised mother becomes pregnant again with an Rh positive child, the leakage of only a few red blood cells through the placenta will trigger a rapid production of antibodies. Rh antibodies that pass through the placenta into the baby agglutinate the baby’s blood, causing severe anaemia. This often requires transfusion of the baby’s blood after it is born, or sometimes transfusion while the baby is still in the uterus. The severity of the hypersensitisation of the mother increases with each successive problem pregnancy. -OTHER RHESUSNEGATIVE

PLACENTA

"ABY RHESUSPOSITIVE

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THEMOTHERPRODUCES ANTIBODIESAGAINSTTHE BABYSRHESUSANTIGENS

!LATERPREGNANCYWITH ARHESUSPOSITIVEBABY

Figure 9.9 When an Rh negative mother is pregnant with an Rh positive baby, the mother may become sensitised to the Rh antigen. This can cause problems in a later pregnancy with an Rh positive baby.

THEMOTHERALREADY HASANTIBODIES

ANTIBODIESCROSS THEPLACENTA DURINGPREGNANCY

THEBABYSREDBLOOD CELLSCLUMPTOGETHER

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These days the blood group of a mother is always determined. If she is Rh negative the blood group of the baby will also be tested. If the baby is Rh positive, the mother will be given an injection of Rh antibodies (i.e. passive immunity, see page 206) shortly after giving birth. These antibodies bring about the destruction of any fetal blood cells that may have passed into her blood before they have time to trigger her immune system into producing Rh antibodies. To prevent hypersensitisation of the mother, this procedure must be carried out each time an Rh negative mother bears an Rh positive child.

210 Detecting and responding

Autoimmune disease—misdirected attack As we saw in the previous chapter, during development our immune system learns how to distinguish ‘self’ from ‘non-self’: to detect anything that is foreign and therefore potentially dangerous to our wellbeing. Sometimes this ability to recognise ‘self’ breaks down and our immune system begins to destroy our own body tissues. This results in illnesses called autoimmune diseases, which are characterised by the persistent presence of immune cells (T lymphocytes, B lymphocytes) against self-components (autoantigens) in the cells of different tissues. The T lymphocytes attack the tissues directly whereas the B lymphocytes act indirectly by secreting antibodies. Autoimmune diseases may be organ-specific. For example, in rheumatoid arthritis, T lymphocytes and antibodies attack the connective tissue producing arthritis-inflammation and swelling of joints (Figure 9.10). Particular autoimmune diseases and combinations of autoimmune diseases tend to run in families. Most autoimmune diseases are more common in females. Most diseases of the thyroid gland are autoimmune. In Hashimoto’s thyroiditis, T lymphocytes attack the thyroid cells leading to a deficiency of thyroid hormone (hypothyroidism). In Graves’ disease or thyrotoxicosis, antibodies attach themselves to the receptor on thyroid cells that normally receives a stimulation signal from the pituitary gland in the brain. This leads to overstimulation of the thyroid gland and too much thyroid hormone (hyperthyroidism). In pernicious anaemia, antibodies develop against parietal cells in the stomach, causing them to degenerate. This results in a marked reduction in the secretion of acid and intrinsic factor, a substance that is needed for the absorption of vitamin B12. Antibodies are also produced against intrinsic factor itself, blocking the effect of what little intrinsic factor is released. The result is a great reduction in vitamin B12 binding and absorption. A deficiency of vitamin B12 causes abnormal red blood cell formation, resulting in anaemia (a deficiency of normal red blood cells). In multiple sclerosis, T lymphocytes and antibodies develop against the myelin sheath, which insulates nerve axons (see Chapter 6). The resulting inflammation prevents the normal rapid conduction of action potentials in these nerves (see page 138). In type 1 diabetes, previously called juvenileonset diabetes, T lymphocytes and antibodies develop against the beta cells in the pancreas that secrete the hormone insulin (the pancreatic hormone that promotes uptakes of glucose into the liver and muscles. When the beta cells are killed off by this inappropriate immune attack, the lack of insulin leads to an increase in the level of circulating glucose in the body, which causes the symptoms and signs of diabetes. Autoimmune diseases may also be more generalised with many different tissues being affected, as in systemic lupus erythematosus. Where autoimmune diseases are focused on a specific organ, treatment may be relatively simple. It can involve the replacement of a hormone in cases involving damage to an endocrine gland. For example, diabetes results from damage to the pancreas and treatment involves administeration of insulin. Pernicious anaemia is due to damage to cells in the stomach lining and treatment involves administration of vitamin B12. Generalised autoimmune diseases, meaning those that occur widely throughout the body, usually require treatment with immunosuppressive and anti-inflammatory drugs. Figure 9.10 Rheumatoid arthritis is an autoimmune disease that causes swelling and pain in joints.

211 Applications of immunology

extension Recent advances in managing autoimmune diseases

Figure 9.11 In type 1 diabetes, T lymphocytes attack and destroy the insulin-producing beta cells in the islets of Langerhans in the pancreas. Islet comprising mainly beta cells (left). Islet being invaded by T lymphocytes (right).

Professor Len Harrison is Head of the Autoimmunity and Transplantation Division at the Walter and Eliza Hall Institute of Medical Research, Melbourne. His major current interest is the early diagnosis, prevention and cure of type 1 diabetes, for which he has received a number of national and international awards.

By Professor Len Harrison Autoimmune diseases are common and cause a lot of chronic illness in industrialised societies. They occur when the immune system is triggered to react against the body’s own tissues, as if they were foreign. The tendency to misdirect immune responses against ‘self’ is strongly influenced by genetic makeup. For example, the likelihood of autoimmune disease appearing in identical twins (with the same genes) is much higher than in nonidentical twins or in siblings. But genes alone are not enough. Many different environmental factors, such as micro-organisms, chemicals, drugs, ultraviolet light exposure and dietary components, appear to act as triggering factors. On the other hand, there is also evidence that in modern, ‘clean’ environments, with reduced exposure to micro-organisms soon after birth, the immune system may not be properly educated to regulate against autoimmune responses (this is called the ‘hygiene hypothesis’). Most autoimmune diseases probably have a long stage before symptoms appear. This is illustrated by type 1 diabetes. Children at risk of this autoimmune disease can be identified months to years before symptoms resulting from a high level of glucose in the body appear. This is done through a blood test for antibodies against ‘self’ antigens (autoantigens) in the insulin-secreting beta cells of the pancreas. A major auto-antigen targeted by the inappropriate immune response in type I diabetes is actually insulin itself. Advances in protein chemistry and genetic engineering have led to the identification of the auto-antigens (like insulin) in a range of autoimmune diseases. The cloning and production of auto-antigens as recombinant proteins has important applications in the treatment, diagnosis and prevention of autoimmune disease. For example, these days, people with type 1 diabetes inject themselves with pure, recombinant human insulin instead of impure insulin extracted from animal pancreas as in the past. Auto-antigen proteins are also used in tests for antibodies and immune cells so that doctors can identify who is at risk (has ‘pre-clinical’ diabetes), as well as to monitor the effects of treatment. Although auto-antigens are the targets of the autoimmune responses, they can, paradoxically, be used as therapeutic ‘tools’ to re-educate the immune system into making protective, ‘regulatory’ immune responses, rather than destructive responses. This natural vaccination approach to prevent autoimmune diseases works in animal models and is now being tested in clinical trials in humans; for example, type 1 diabetes trials of an intranasal insulin vaccine are being conducted by researchers based at the Royal Melbourne Hospital and Walter and Eliza Hall Institute of Medical Research.

Immunodeficiency There are two main forms of immunodeficient disease. Primary immunodeficiency is congenital, meaning that a child is born with the deficiency either as a result of a genetic defect or developmental abnormality. Examples are DiGeorge syndrome, where the thymus fails to develop therefore there are no T cells, and ‘bubble babies’, where neither B nor T cells develop. Secondary immunodeficiency is acquired as a result of severe stress or another disease, as in AIDS.

Bubble babies Severe combined immunodeficiency (Scid) is an inherited X-linked genetic disease, where bone stem cells are defective and neither B cells nor T cells are produced. With a non-existent immune system, even trivial infections are fatally to Scid babies. Usually these children must live in ‘bubbles’— tightly controlled sterile environments—in order to survive (Figure 9.12).

212 Detecting and responding

Conventional treatment has been a bone marrow transplant to completely replace their defective stem cells. This requires a heavy and lifelong regime of immunosuppressive drugs to prevent rejection of the transplanted bone marrow cells.

Gene therapy (see page 301) for these children has had encouraging outcomes. This involves extracting some of the baby’s defective bone marrow cells and infecting them with a genetically engineered virus carrying the normal form of the defective gene. These cells are then transplanted back into the patient. As the cells are their own there is no problem with rejection. After a few weeks, new normal cells are found in the circulation. These children have been able to leave their ‘bubbles’ and their newly developed immune systems appear to be working normally.

Figure 9.12 David Vetter was born with severe combined immunodeficiency (Scid) and lived all of his 12 years of life inside a sealed plastic bubble that protected him from infections.

AIDS Acquired immunodeficiency syndrome (AIDS) is the disease resulting from infection with human immunodeficiency virus (HIV). AIDS has spread rapidly in Africa and other parts of the world. The virus is present in the body fluids of infected individuals, and transmission from one person to another is almost always from the body fluids of one person (usually blood or semen) into the bloodstream of another. In Western society, the two behaviours most closely linked to the transmission of AIDS are anal intercourse (and therefore homosexuals are at particular risk) and the sharing of hypodermic needles. HIV is a retrovirus: an RNA virus that makes a DNA copy of its genetic information by reverse transcription. The HIV DNA is then inserted into the host DNA where it remains until the virulent phase of the disease is triggered. T4 lymphocytes (one of the helper T cells) are preferentially infected. Once triggered, the viral DNA begins to produce new viruses, which pass out and infect new T4 lymphocytes. Anti-HIV drugs target the replication process of the virus. There are two main mechanisms of action of these drugs. One mechanism involves inhibition of the enzyme that converts the viral RNA into the same form as human DNA to allow reverse transcription to occur. The other blocks the enzyme that cuts the proteins produced by reverse transcription into smaller pieces to construct new virus particles. This helps

213 Applications of immunology

to prevent infected cells from producing more virus particles. Death from AIDS is usually due to infections, such as pneumonia, that rage unchecked because the helper T cells are unable to activate normal immune responses to eliminate the infection.

Persistent inflammation Inflammation is a powerful non-specific defence response, switched on by damaged tissue or invading pathogens, which leads to a rapid development of an early immune response (see page 188, Chapter 8). However, recent research suggests that the continued presence of the cells and molecules of the inflammatory response may contribute to a wide variety of unrelated diseases—not causing the diseases but causing a continuing problem. When inflammation persists, surrounding healthy tissues can also become damaged. Inflammation normally subsides under the active influence of resolvins (see page 188), which are naturally occurring substances derived from omega-3 fatty acids that ‘turn off’ the inflammatory response. Evidence is now indicating that the inappropriate persistence of the inflammatory response contributes to a number of diverse disease conditions, including heart disease, rheumatoid arthritis, severe asthma, Alzheimer’s disease and cancer, and that treatment with anti-inflammatory drugs can be beneficial. Current anti-inflammatory drugs act to inhibit the development of inflammation, which has the unwanted side-effect of reducing one of our defences. For this reason it may be better to develop drugs that enhance the active resolution phase of inflammation.

summary

9.2

• Allergic responses, such as asthma, are the result of a vigorous overreaction of the immune system to a previously encountered antigen (allergen). • In autoimmune diseases, the ability to recognise ‘self’ breaks down and the immune system turns on its own body tissues. T lymphocytes attack the tissues directly whereas the B lymphocytes act indirectly by secreting antibodies.

• Immunodeficient diseases can be congenital as a result of a genetic defect or developmental abnormality, or acquired as a result of severe stress or another disease. • The inappropriate persistence of the inflammatory response contributes to a number of diverse disease conditions.

key questions 5 What is an allergen? 6 a Explain what is meant by an allergic response. b List the sequence of events that occur in the body during an allergic reaction. 7 a Outline the differences in the way that histamines and antihistamines affect the body. b Why are antihistamines sometimes administered to patients suffering allergic reactions? 8 Immune responses are an important part of the body’s defence mechanism. Explain how an oversensitive immune system—in terms of an allergic response—can be a potential danger to an individual. 9 a Explain what is happening in the body of an infant if it is suffering from haemolytic anaemia of the newborn.

214 Detecting and responding

b Use a flow chart to outline the key steps in the development of this disease. c How are women at risk of giving birth to children with this condition treated to avert the problem in future pregnancies? d Why does haemolytic anaemia not occur in infants born to Rh positive mothers? 10 a What is happening in the body of a person suffering an autoimmune disease? b Name two autoimmune diseases and outline how each disease affects the body. 11 In general terms, what effect does an immunodeficient disease have on the body? 12 a Use an example to explain how persistent inflammation can lead to disease. b Outline the role of resolvins in anti-inflammatory responses.

9.3

Frontiers of medicine Immunotherapy Immunotherapy can be used to produce a desirable change in immune function. For example, seriously hypersensitive reactions to particular allergens such as bee sting toxin (see page 22, Chapter 1) can be reduced by desensitisation. Beginning with extremely tiny amounts (remembering that even a tiny amount can kill if it causes an anaphylactic reaction), the offending allergen is repeatedly injected over a period of months in increasing amounts. This causes the formation of specific IgG antibodies against the allergen. If these IgG antibodies react with allergen before it binds to the IgE to cause the allergic response, this prevents an allergic response (Figure 9.13). During the course of treatment, the individual slowly becomes less and less sensitive to that particular allergen—desensitised. 

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Figure 9.13 Repeated tiny injections of an allergen causes an increase in circulating levels of specific IgG. When later challenged by the allergen, these IgG molecules bind with it before it can reach the IgE on the mast cells, thus preventing the allergic response.

Vaccines Australia is a much healthier place to live in than it was 100 years ago. In the early 1900s Australia was still prey to epidemics of infectious diseases like smallpox, polio, measles and whooping cough. These diseases either no longer exist in this country (polio) or the world (smallpox), or are much better controlled (measles and whooping cough), largely because safe and effective vaccines were developed. Vaccines stimulate the immune system to make us better prepared for an infection. Early vaccines such as the small pox vaccine (see page 204) were crude preparations containing the whole microbe, virus or bacterium. Our increased understanding of microbiology and immunology has allowed us to develop much purer vaccines comprising only the parts of the microbe necessary to induce the correct immune response. This part of the pathogenic microbe is known as the vaccine subunit and it is typically generated using molecular biology. The gene encoding the relevant part of the microbe is identified and used to direct expression of the protein subunit in an engineered host, such as Escherichia coli or a yeast.

215 Applications of immunology

technologies and techniques Vaccine technology by Professor Richard Strugnell

T

Professor Richard Strugnell Richard Strugnell is a Professor in the Microbiology and Immunology Department at The University of Melbourne, and is Deputy Director of the CRC for Vaccine Technology

Figure 9.14 Two different strains of Klebsiella pneumoniae and white discs containing the antibiotic kanamycin. The strain on the lower part of the plate is sensitive to the antibiotic; the other strain is resistant. Photo courtesy of Dr. Adam Jenney, CRC for Vaccine Technology.

216 Detecting and responding

he young scientists I work with at The University of Melbourne are immunologists and microbiologists studying pathogenic bacteria. The microbiologists are investigating the differences between bacteria that cause disease and their much less virulent counterparts. Virulent (meaning highly pathogenic) bacteria have usually acquired genes from another, often unrelated bacterium. These ‘imported’ genes are grouped together on ‘islands’ of DNA known as pathogenicity islands. Microbiologists identify and then study the individual genes in the islands to determine why one bacterium is more virulent than another. For example, in our studies of Klebsiella pneumoniae we have found a strain that is more common in large hospitals and appears to carry genes that make it stick more to cells and plastic. Case study The bacteria growing on the plate in Figure 9.14 are K. pneumoniae, a bacterium that infects older Australians, and those in hospitals. Two different strains of the bacterium are shown—one strain glistens and does not grow immediately around the white disc containing the antibiotic kanamycin, showing it to be sensitive to the antibiotic. The other strain grows right up to the kanamycin disc—it is resistant. Tests like this are used to identify resistance of disease-causing bacteria to antibiotics. In this case, the bacterium was engineered in the laboratory to be resistant to kanamycin, which is not used to treat patients.

Colonies of the antibiotic sensitive bacterium glisten or are ‘mucoid’ because these bacteria have an outer capsule composed of complex sugar molecules. The capsule makes the bacterium resistant to killing by phagocytes. This form of the bacterium is therefore potentially very harmful to someone in hospital. The kanamycinresistant bacterium has been engineered to lose its capsule and is no longer mucoid. The two forms of the bacterium (with capsule and without) are then ‘fed’ to phagocytes to test whether they can resist being engulfed and digested. The bacteria with the capsule cannot be eaten. In order to ingest the bacteria the phagocyte must produce ‘finger’-like processes that reach across the surface of the bacterium and take ‘firm hold’ of the bacterium. If the bacterium has a capsule, the ‘fingers’ cannot grasp its surface—they simply slide off leaving the bacterium free to replicate. By understanding how the process works we can hypothesise that development of a vaccine which comprises the capsule of K. pneumoniae, would be able to protect older people against potentially fatal infection by this organism. Unfortunately this is not quite so simple. Our surveys show that in a large teaching hospital in Melbourne, the K. pneumoniae that caused disease carried 60 different types of capsule, far too many to place in a vaccine. We are now searching for other components of the bacterium that might make good vaccine subunits.

The bacterium Haemophilus influenzae used to be a major cause of illness in young children. In the 1980s it was recognised that antibodies raised by a vaccine containing just the sugar capsule surrounding the bacterium could provide complete protection against H. influenzae. The development of the vaccine containing the capsule (called the Hib vaccine), and its now widespread implementation, has virtually eliminated H. influenzae as a problem.

Transplant rejection Mammalian cells have many antigens on their surface. Some of these are characteristic of the species and others are unique to the individual organism. The ‘self’ antigens are the products of a group of linked genes (see Chapter 15) called the major histocompatibility complex (MHC). They are found particularly on professional antigen-presenting cells, such as dendritic cells, phagocytes and B cells. Dendritic cells are found in many tissues and spend their time taking in and breaking down macromolecules into fragments, then presenting the antigenic-fragments on MHC cells at their surface. MHC molecules are the only molecules that can ‘show’ foreign antigen to T cells. Foreign antigen is only recognised by a T cell when it is presented by a MHC molecule. Because T cells regulate most immune processes (see page 196), including antibody formation and recognition of self, MHC molecules also play a key role in every aspect of immunity. MHC molecules themselves are the antigens that play the major role in transplant rejection. When tissues or organs are transplanted from one person to another, their MHC molecules are recognised as being ‘non-self’, which triggers the immune system to reject the foreign tissue. In a few days, if not prevented, the area is overrun by sensitised cytotoxic T cells (TC cells), NK cells and phagocytes that kill the transplanted tissue. Identical twins have identical MHC antigens, and therefore transplantation from one to the other is highly successful. In all other combinations, the similarity (compatibility) of MHC antigens between recipient and potential donor tissues must be determined. Often members of the same family will have a sufficiently similar pattern for transplantation to have a reasonable chance of success. Even when an excellent match has been found, it is still important to use immunosuppressive drugs, such as cyclosporine (produced by a fungus, see page 165), to prevent graft rejection by the TC cells . Because infection following surgery is also a concern with transplant procedures, a suppressed immune system will be less able to defend against it. Defence against bacterial infection is primarily a B cell response, so drugs that are more selective against T cells than B cells would be less likely to lower defences against infection. An example of this type of drug is cyclosporine—it interferes with cellular signalling and specifically prevents the proliferation of activated T cells. Alternatively, some means of making a transplant less ‘foreign’ would make it more acceptable to its new host. Perhaps we can learn something from the ways that parasites avoid defences (see page 194). An additional problem with immunosuppression is that it also inhibits the body’s normal immune surveillance, which constantly detects and destroys abnormal cells such as cancer cells. The incidence of cancer is higher in immunosuppressed patients.

4ISSUEORORGANTRANSPLANTED INTORECIPIENT

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Figure 9.15 Following successful transplant of tissue or an organ, rejection is common. This is due to the transplant recipient’s immune system detecting the foreign MHC molecules as ‘non-self’. Drugs such as cyclosporine can suppress the immune response and prevent rejection.

217 Applications of immunology

biology in action How does a fetus escape rejection?

Figure 9.16 Ultrasound scan of a 12-week-old fetus.

Sexual reproduction produces offspring that are genetically different to either parent. Transplanted tissue between a mother and her child will result in rejection, usually within about 10 to 14 days. T cells simply get into the graft tissue and kill the graft cells. So why is a developing fetus not recognised as ‘non-self’ and rejected by the mother’s immune system? It has been shown that the mother’s immune system is ‘aware’ of the presence of foreign cells, and small numbers of fetal cells do get into the mothers circulation, even though the placenta does an excellent job in keeping the mother’s T cells out and the baby’s cells in. So how does the fetus survive in the uterus for 9 months? Apparently, within two days of fertilisation, the tiny embryo releases immunosuppressive factors that suppress maternal cellular immune responses, thereby preventing maternal rejection of the embryo. Later, the outer layer of cells of a developing mammalian embryo, called the trophoblast, comes into contact with the maternal tissues of the uterus as the embryo implants and the placenta begins to form. Studies of this layer of the placenta suggest that it has reduced antigenic properties, meaning that the babies ‘self’ MHC molecules aren’t exposed. This may be due to the absence or masking of fetal ‘self’ antigens on the trophoblast cells and it reduces the likelihood of stimulating an anti-fetal immune response by the mother. The placenta also acts as a filter, preventing maternal cells from crossing the trophoblast and allowing only certain antibodies to cross from mother to fetus. Generally, any maternal antibodies formed against fetal antigens appear to be trapped by trophoblast cells of the placenta.

Resistance to antimicrobial drugs

Figure 9.17 The fungus growing on this plate is Penicillium chrysogenum (also known as Penicillium notatum). It produces penicillin, an antibiotic that prevents cell wall formation in bacteria.

218 Detecting and responding

Antimicrobial drugs are any chemicals that can be used to effectively treat microbial infections, whereas antibiotics are antimicrobials naturally produced by microorganisms. Most modern antibiotics are derived from microorganisms, such as bacteria and fungi, that live in the soil (Figure 9.17). They are useful because they are selectively toxic towards pathogenic organisms without causing significant damage to hosts. They may kill the pathogen or inhibit its growth. Biotechnology is used to produce antibiotics in sufficient quantities for therapeutic use. As scientists have discovered more about the molecular structures of useful drugs and the mechanisms of their action against pathogens, they found they could specifically alter the structure of these molecules to improve their effectiveness or to reduce unwanted side-effects. For example, since the 1960s this has led to the development of a whole family of penicillinlike drugs, each with its own particular properties. Effective antimicrobial drugs act at many levels on processes that are characteristic of microorganisms rather than vertebrates. In bacteria, they may prevent cell wall formation (e.g., penicillins), protein synthesis (e.g., tetracycline), nucleic acid synthesis, metabolic pathways (e.g., sulpha drugs) or damage membrane integrity. Doctors now have a vast array of drugs at their disposal and scientists can design new drugs needed for particular purposes (see page 85, Chapter 4). The development of drug resistance has become a huge problem, limiting the usefuless of all these drugs. There was a time when penicillin killed more than 97% of all Staphylococcus aureus ‘golden staph’ bacteria. Now over 90% are resistant to this drug.

Bacteria can resist antibiotics in a variety of ways that are genetically based. For example, they may reduce intake of the drug, alter the target molecule the drug attaches to, increase elimination of the drug from the cell or enzymatically deactivate the drug. If these resistant properties are present in members of the bacterial population, the bacteria possessing them will survive the drug treatment and their progeny will possess the same genes and therefore drug resistance. There is another way that drug resistance can spread through a bacterial population. Gene transfer can occur between bacteria, for example on a plasmid (a small piece of DNA that replicates independently, see page 262) during bacterial conjugation, and these plasmid genes can sometimes jump into and out of the chromosome. Many of the resistant genes are found on R plasmids (resistance plasmids), which can be transferred even to unrelated bacteria (Figure 9.18). 3PONTANEOUS MUTATION

(a)

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Figure 9.18 (a) Antimicrobial resistance can arise as a result of mutation and will then be transferred to progeny during replication. (b) Resistant genes carried on plasmids can be transferred independently to other unrelated bacteria.

#

Taking antibiotics exactly as prescribed, not missing doses and finishing the whole course is particularly important to minimise the spread of drugresistance. It is important to kill all of the bacteria, not just enough of the more susceptible ones so that you feel better. Also, because antibiotics do not act against viruses, taking them for viral infections such as colds and influenza just increases the rate of development of drug resistance and has no effect on the cold.

219 Applications of immunology

technologies and techniques The world problem of malaria by Sir Gustav Nossal Sir Gustav Nossal Australian of the Year in 2000, Sir Gustav Nossal is a leading international figure in the field of immunology. He has been Director of The Walter and Eliza Hall Institute and the Professor of Medical Biology at Melbourne University. Sir Gustav plays a strategic role in the World Health Organisation’s Global Polio Eradication Initiative and the Gates Foundation Vaccination Initiative. Sir Gustav Nossal provides guidance in the fostering of leadership and mentoring between scientists. Apical organelles microneme (EBA-175, AMA-1) rhoptries (RHOP H3, RAP-1,2)

E

ven though the disease has long since been eliminated in most affluent countries, malaria remains a huge global health problem. Two billion people live in areas at risk, there are 300 million attacks per year, and about two million die from the disease each year. Most of the deaths are African children under the age of five. There are basically three approaches to the control of malaria. The disease is caused by a single-celled parasite carried by mosquitoes, so the first approach is to minimise the risk of mosquito bites. Draining swamps and spraying with insecticides works, but these methods risk damaging the environment. Getting children to sleep Apical organelle dense granule (RESA)

Merozoite surface (MSP-1-5, GPI toxin)

under insecticide-impregnated bed-nets is an effective approach. Second, anti-malarial prescription medicines are used both for prevention and for treatment. The problem with this approach is that the parasites soon become resistant to the drugs, particularly if their use is widespread. This means that scientists simply have to invent new and better antimalarial drugs. The third and potentially the best approach is a malaria vaccine. A successful vaccine delivered to infants would prevent infection. If the vaccine was less than 100% effective, at least this would make the attacks less frequent and less severe. Much research is being conducted world-wide to develop such a vaccine, but the parasite has a number of tricks up its sleeve to foil the body’s immune defence system. Figure 9.19 illustrates one potential method—to develop a vaccine that stops the parasite getting into a red blood cell. Another approach complementary to this would be to stop the parasites that the mosquito injects under the skin from getting to the liver where they nest before hitting the blood cells. The most recent research into these areas has been quite encouraging.

Figure 9.19 A malaria parasite (right) about to invade a human red blood cell (left). Merozoite surface proteins are involved in attachment to the red blood cell and the apical organelles release their contents as the parasite enters, suggesting that they play a role in the invasive process. The initials refer to the names of a variety of merozoite proteins that are being tested as possible components of a malaria vaccine. The aim would be for antibodies to block the entry of the parasite into the red blood cell.

220 Detecting and responding

Managing AIDS As discussed in Chapter 7, viral infections are difficult to treat. But the severity and impact of the AIDS epidemic, which was first recognised in 1981 in the USA, led to a great deal of research. Now, for those AIDS sufferers with plenty of money (usually living in Western societies), there are ‘cocktails’ of medicines that can prolong and improve their quality of life. While treatment can have serious side-effects, in many cases it can reduce levels of HIV in the blood, halt the progress of the disease and allow some recovery of immune function. Drug regimes may include: • Attachment blockers—block T cell receptors preventing the HIV from attaching • Entry inhibitors—prevent the virus from fusing to and entering the cell • Reverse transcriptase inhibitors—prevent the synthesis of DNA from the viral RNA • Integrase inhibitors—prevent HIV DNA from integrating into the cell’s DNA • Protease inhibitors—prevent newly formed viral RNA from assembling the proteins of the viral coat. Unfortunately in the ‘developing world’ there is little access to these medicines for AIDS and for many other diseases. There are many who believe that the ‘developed world’ has a global responsibility to make available cheaper treatments and vaccinations to improve the health of all people.

summary





2.!

2.!

 $.!  VIRAL PROTEINS 

Figure 9.20 ‘Cocktails of drugs’ are now available for treating AIDS patients living in Western societies. They act to block binding of the virus to T cells (1), entry of the virus (2), transcription of RNA to DNA (3), integration of the viral DNA into host DNA (4) and synthesis of viral proteins (5). These drugs have dramatically reduced death rates and improved the quality of life for AIDS sufferers.

9.3

• Immunotherapy, such as desensitisation to particular allergens, can be used to change immune function. • Vaccines stimulate the immune system to make it better prepared for a future infection. • Organs transplanted from one person to another are recognised as being ‘non-self’ and trigger the immune system to mount a cell-mediated defence response against them. • Rejection of a fetus is usually prevented because the tiny embryo releases immunosuppressive factors, and the placenta selectively prevents the passage of cells and antibodies from mother to fetus.

• The development of drug resistance has become a huge problem in medicine, limiting the usefulness of many drugs. • As a result of a great deal of research, AIDS sufferers who can afford it have access to a variety of medicines that can significantly prolong and improve their quality of life. • Many diseases have long since been eliminated in most affluent countries, but remain serious global health problems.

key questions 13 In only two sentences, use a specific example to explain what is meant by immunotherapy. 14 a Outline the role that the major histocompatibility complex (MHC) plays in tissue or organ rejection after a transplant. b Explain why the success rate of tissue transplantation between identical twins is so high. 15 Describe some strategies used to overcome transplant rejection. 16 Explain why the incidence of cancer is higher for transplant patients using immunosuppressant drugs compared with the rest of the general community.

17 a What is an antimicrobial drug? b Describe some different ways in which antimicrobial drugs work. c Outline the problem of development of drug resistance in bacteria. 18 Outline the advantages of a malaria vaccine over more conventional management strategies.

221 Applications of immunology

09

key terms immunity vaccination toxoid naturally-acquired immunity artificially-acquired immunity

active immunity passive immunity antibody antigen hypersensitivity allergic response allergen

immunoglobulin mast cells histamines antihistamines ‘self’ ‘non-self’ autoimmune disease

1 Draw up a table summarising the preventable childhood diseases for which children in Australia are routinely immunised. Use the following headings: disease, type of pathogen, symptoms, treatment, prevention. 2 Research your own childhood immunisation record. a For which diseases have you been immunised? b State whether each of these diseases is bacterial or viral. c Make a list of the preventable childhood diseases which you actually contracted. d Are you likely to become infected again if exposed to the same pathogen? Explain. 3 Breast-fed babies tend to be healthier than bottle-fed babies. Give a reason why. 4 The following graph shows the levels of measles antibodies in a child from birth to seven years of age. ,EVELOFANTIBODIES ARBITRARYUNITS

immunodeficiency persistent inflammation MHC (major histocompatibility complex) transplant rejection drug resistance

worksheet 23

5 According to the World Health Organisation, smallpox has been officially eradicated for the last 30 years. Since it is no longer a threat, vaccination programmes for this disease have long ceased. If such a disease is no longer a threat, find out why soldiers assigned to duty in Iraq during the 2003 Iraqi Freedom campaign were routinely vaccinated against smallpox. 6 The oversensitivity of some individuals to the antigens on the surface of pollen grains is responsible for pollen allergy known as hay fever. Find out how the repeated administration of tiny amounts of pollen extracts under the skin of a hay-fever sufferer can be used as a treatment to desensitise patients to pollen. 7 a Use the internet to find out the link between omega-3 fatty acids and resolvins. b Outline the current dietary advice that seeks to both treat and prevent chronic inflammatory diseases. 8 The following graph shows the incidence of a disease in the USA from 1940 to 1980. #ASESPERPOPULATION   



222





  !GEYEARS







a This child had antibodies against the measles virus at birth. Explain why. b Why did the antibody levels drop off to zero in the months following birth? c The child was immunised against measles at one year old and again at four years old. i Explain why antibody production occurs more rapidly and to a higher level after the second vaccination compared with the first vaccination. ii Why don’t the antibody levels drop off to zero after immunisation occurs? d At six years of age antibody production increases dramatically once again, but no vaccination has occurred. Explain. Detecting and responding

   

















a Give two possible reasons for the rapid fall in incidence after 1946. b Give two possible reasons for the significant rise in incidence from 1965. 9 Use your knowledge and understanding of the concepts of major histocompatibility complex and recognition of ‘self’ and ‘non-self’ to compare the likelihood of successful organ transplanting between identical twins and non-identical twins.

3

unit

area of study 02

review w

Detecting and responding in

multiple choice questions 1 Homeothermic organisms are characterised by an ability to A maintain a relatively stable internal environment. B maintain a relatively stable core body temperature. C regulate body temperature in line with environmental temperature. D regulate external temperature. 2 In general, negative feedback systems operate as proportional control systems. This means that A the response acts to increase the original stimulus. B the stimulus acts to reduce the original response. C the magnitude of the response is related to the magnitude of the stimulus. D misalignment detectors have been stimulated to elicit a response. 3 Acetylcholine is a signaling molecule that A is released across a presynaptic membrane of a nerve terminal. B binds specifically to the receptor molecules on postsynaptic membranes of nerves or muscles or glands. C is rapidly inactivated after release from the presynaptic membrane. D all of the above. 4 The bending response of growing shoots towards a light source is referred to as A negative phototropism. B positive phototropism. C negative geotropism. D positive geotropism. 5 Infectious diseases A are caused by agents that can be passed from one individual to another. B are caused by cellular agents such as bacteria. C require a vector in order to be transferred from one individual to another. D need primary and secondary hosts to survive. 6 Chemical forms of defence against pathogens in plants include A the presence of the cell wall as a barrier to prevent entry of pathogens. B encapsulation. C the production of enzymes such as cellulases that digest the cell walls of fungi. D all of the above.

7 Malaria is caused by the Plasmodium protozoan. It relies on two hosts for completion of its life cycle, the Anopheles mosquito and humans. During a bite in search of food (blood), an infected female mosquito transfers the protozoan larvae into the bloodstream of a person. The larvae migrate to the liver where they feed and reproduce asexually for about fourteen days before returning to the bloodstream where they infect red blood cells. Here the parasite continues to grow, eventually bursting the red blood cells and spilling its tiny larvae contents. It is at this stage that the symptoms of fever, sweating, shivering and delirium occur. In the life cycle of the malarial protozoan A humans are the primary host. B humans are the vector. C asexual reproduction occurs only in humans. D the adult form occurs in the secondary host. 8 Specific immune responses in mammals involve a variety of specialised cells, proteins and processes that include A antigens, antibodies, interferons and histamines. B NK cells, phagocytes, complement proteins and IgE antibodies. C cell-mediated responses, humoral immune responses and interferons. D T-helper cells, T-cytotoxic cells, plasma cells, memory cells, antibodies and recognition of self and non-self antigens. 9 Naturally acquired active immunity is achieved as a result of A exposure to live or attenuated vaccines. B infection by particular bacteria or virus. C the administration of antibodies or antitoxin specific to a particular microorganism. D adequate breast-feeding in newborn infants. 10 Autoimmune diseases are characterised by A failure of the body to produce sufficient specific antibodies in response to the presence of antigens. B chronic inflammation. C the body’s failure to recognise ‘self’, resulting in the persistent presence of B and T lymphocytes that target ‘self’ tissue D lack of signalling molecules called resolvins.

223 Detecting and responding

short answer questions 11 Blood glucose levels in normal and diabetic individuals, after eating similar meals, are shown in the following graph. "LOODGLUCOSE CONCENTRATION 

e Explain why nerve impulses can only be transmitted in one direction f Using a table, outline the three differences between the nervous and endocrine systems. 13 In the following experiment the tips of growing shoots were cut off and placed on agar blocks in the light for an hour. The tips were then removed and the agar blocks placed off-centre on the cut shoots, this time in the dark. light

dark



4IME MEAL

a Which graph represents the diabetic individual? Explain your reasoning. b i Name the hormone that is produced in insufficient amounts in a diabetic individual. ii In which organ is this hormone produced in a normal individual? c i Draw the negative feedback model for the control of blood glucose levels in the body. ii Use the example of blood glucose level to explain what is meant by a ‘feedback mechanism’. d Explain how the hormones glucagon and insulin work in the body to regulate blood glucose concentrations. 12 Examine the following diagram of a neuron. !

a In which direction will electrical impulses flow along the neuron? b i Name the structure labelled A. ii Describe two functions of this structure. c When an action potential passes into a nerve terminal, vesicles containing neurotransmitter molecules move to the nerve cell membrane and release their contents to the outside. Neurotransmitter molecules diffuse across the narrow gap and bind to specific receptors on the membrane of the subsequent neuron. Name the junction between a neuron and the cell it stimulates. d Explain the similarity between neurotransmitters and hormones.

224 Detecting and responding

a Name the hormone responsible for the bending response in the shoots. b Explain what has occurred to cause this bending response. c What is the name of the response in which growing tips bend towards a light source? d Outline a simple experiment using growing shoots and tin foil to illustrate that light stimulates the bending response in shoots. e Name one other plant hormone and outline its function. f Plant roots grow downwards in response to gravity. What name is given to this response? 14 The following graph shows the relationship between increasing length of the dark period and flowering. Two groups of plants were grown under different day/night cycles. In one set of experiments, the light period was always a minimum 4 hours with the dark period varying between 4 and 20 hours. In the other set of experiments, the light period was always at least 16 hours and the dark period varied between 4 and 8 hours. )NCREASINGNUMBEROFFLOWERS  HOURLIGHTPERIOD

 HOURLIGHTPERIOD





  ,ENGTHOFDARKPERIODHOURS



a What is the general name of the responses shown by plants in relation to day/night length? b The use of the terms ‘long-day’ and ‘short-day’ plants is a misnomer. What environmental factor triggers the flowering response in flowering plants? c During which season—summer or winter—would you expect long-day plants to flower? Explain. d Use the graph to explain the effects on flowering of the length of the i dark period. ii light period. 15 A medical student studying the impact of antibiotics on pathogenic bacteria set up the following experiment. Three sterilised nutrient agar plates were prepared. Plate A remained sealed with nothing added to it. Plate B was exposed to bacterial spores, then sealed. Plate C was treated with three drops of a common antibiotic and then exposed to the bacteria in the same way as plate B, then it too was sealed. All three plates were incubated at 37°C for 24 hours and then examined for the growth of bacterial colonies. The results of the experiment are set out in the following diagram. BACTERIAL COLONIES

!

#

"

a Suggest an hypothesis that the medical student was testing. b i Which agar plate represented the control? ii Explain the significance of the control in this experiment.

c Explain whether or not the experimental results support the hypothesis. d Describe one other piece of information that would be useful to have before accepting the results of the experiment as reliable. e Distinguish between an antiseptic and a disinfectant. 16 Some native Australian plant species contain toxins that are unpalatable and so deter would-be consumers from taking too much. Fluoroacetate is one such toxin found in species of the genus Gastrolobium (family Fabaceae or pea family) and in some species of Acacia. Grazing livestock exposed to these plants are susceptible to poisoning. They suffer from weak, rapid heartbeat, muscular spasm, labored breathing, convulsions and finally death. Fluoroacetate is a neurotoxin. a Define signal transduction. b In terms of fluoroacetate poisoning, describe where the neurotoxin has its effect. c Death from fluoroacetate poisoning finally occurs as a result of asphyxiation due to failure to breathe. Explain why this occurs. 17 Hayfever is an allergic response in which the immune system overreacts to the presence of a previously encountered foreign antigen. The diagram below illustrates the key steps that occur in an allergic reaction. a Define the term ‘antigen’. b Identify structure M. c Where in the body are mast cells located? d Describe the event that causes mast cells to release substance N. e Describe two effects resulting from the release of substance N. f Resolvins are specialised signaling molecules. Outline their role.

PRODUCES

FOREIGNANTIGEN

"LYMPHOCYTE RECOGNISESANTIGENAS PREVIOUSLYENCOUNTERED

RELEASE

STRUCTURE-

MASTCELL

SUBSTANCE.

225 Detecting and responding

sample assessment task 1 A summary report of a practical activity related to bacterial response to chemical and/or physical stimuli. Blitzing bacteria—antibiotics and bacterial growth

sample assessment task 2 A web page or presentation in multimedia format on one aspect of the immune system. Defence against pathogens (www.ickyinfections.com) Design and construct a web page that will be informative to someone seeking information about how the immune system functions to defend the body against invading pathogens. The web page should provide detailed information about specific immune responses (both humoral and cell-mediated), including specialised structures and how they function to provide immunity against invading pathogens. In particular, you should include:

Purpose: To investigate the effect of antibiotics on bacterial growth. This is a first-hand data activity in which you will prepare and incubate bacterial cultures together with Mastrings (discs containing antibiotics) to investigate the effect of antibiotics on bacterial growth. The activity provides clear instructions as well as directed questions relating to the activity (including experimental design) and a simple guide to writing your summary report. See Heinemann Biology 2 Student Workbook page 79 for complete practical activity and sample summary report.

• key molecules and cells involved in specific immune responses; • an explanation of processes involved in immune responses to specific foreign antigens. Your web page should be informative and appealing with clear diagrams. Include a menu that provides links to different aspects of the body’s defence mechanisms, especially the immune system. Prepare a bibliography of all of your resources. The following example may give you some ideas.

IMOGEN’S INVINCIBLE IMMUNE SYSTEM www.ickyinfections.com Pathogens

Immune system responses that target specific pathogens involve specialised white blood cells or lymphocytes called T cells and B cells

Self/non-self Antigens Antibodies

T cells

Non-specific defence mechanisms

See Heinemann Biology 2 Student Workbook page 82 for sample web page.

226 Detecting and responding

unit

4

area of study 01

Heredity outcome On completion of this unit the student should be able to analyse and evaluate evidence for the molecular basis of heredity, and patterns of inheritance.

1

chapter 10

Molecular genetics

key knowledge • molecular genetics and the genetic code • genomes of organisms • genes as the individual units of inheritance • genes provide instructions to make polypeptides

chapter outcomes After working through this chapter you should be able to demonstrate that: • a chromosome is made of DNA, and genes are located on chromosomes • a gene is a sequence of nucleotides (containing the bases A, G, C, T) found in DNA and a genome is the whole set of genes in an organism • DNA replicates in cells, providing continuity from generation to generation • gene expression consists of two stages: transcription (synthesis of an RNA molecule complementary to a piece of DNA) and translation (synthesis by ribosomes of a polypeptide gene-product based on the code in the RNA molecule; the code specifies the order of amino acids in the polypeptide, and the function of proteins in cells) • gene expression is regulated (gene regulation); each cell of an organism has a complete set of genes but only a only a subset is switched on and used.

10.1

Genes and DNA Inheriting traits Throughout time people have wondered why offspring largely resemble their parents but also show differences. People became aware that many characteristics (traits) are due to heredity, that is, passed on from one generation to the next. Today, when a family meets with their doctor they may learn that a particular family illness is inherited. An explanation for the inherited illness, such as Marfan syndrome, comes from our understanding of genes and genetics at the molecular level.

biology in action It’s better to know Marfan syndrome has received publicity in sport magazines because of the sudden and untimely deaths of young elite athletes. Flo Hyman, the towering slam-spiking USA Olympic volleyball star who had led the USA women’s team to a silver medal at the 1984 Olympics and at the time was considered the world’s best volleyball player, died suddenly in 1986 during a game in Japan. At 31 years old and thought to be in perfect health, she suffered a cardiac arrest. She was found to have had Marfan syndrome. Marfan syndrome was named after the French doctor Antoine Marfan, who first described the condition in 1896. It is due to an altered (mutated) gene for fibrillin (on chromosome 15). It is inherited in 75% of cases; 25% of cases occur as a result of a spontaneous genetic change (mutation). Fibrillin is an important component of connective tissue. Most affected people will show some signs and complications of Marfan syndrome, but the severity of the syndrome varies widely. Individuals with Marfan syndrome are usually tall, thin and loose-jointed—useful attributes for playing sports such as basketball and volleyball, hence, the high number of Marfan people successful in these sports (Figure 10.1). One very telling feature is a large ‘wingspan’. In the general population, the distance from fingertip to fingertip of a person with arms spread is equivalent to that person’s height; in people with Marfan syndrome, the wingspan is substantially longer. They are also likely to have other problems, including hypermobility of joints and eyesight problems. Most life threatening is a weakness in the connective tissues of the heart and blood vessels, particularly the aorta. Unfortunately, some individuals with Marfan syndrome have died of a burst aorta before a diagnosis of their condition has been made. The incidence of Marfan syndrome in the population is thought to be about 1 in 10 000, but could be higher. It is possible to manage and correct Marfan symptoms with early diagnosis. Depending on the severity of the symptoms, treatment involves regular monitoring of the heart and aorta, eyesight and the skeletal system, so that medical intervention can be timely.

biofile In the USA, some basketball and volleyball clubs routinely test recruits who are very tall for Marfan syndrome.

Figure 10.1 Marfan syndrome. Andy Campbell, a former team member of the Canberra Cannons, shows the height and agility typical of people with this syndrome.

229 Molecular genetics

! A gene is a unit of heredity made up of a unique sequence of DNA. Together with proteins, it is organised into chromosomes.

! The whole set of genes in an organism is a genome.

Genes and genomes What do we know about genes today? We know that genes control the way an organism develops, grows and functions. We also know that DNA is the molecule of life that encodes the information on which organisms are built (Chapter 4). We can thus define a gene as a unit of heredity made up of a unique sequence of DNA. The whole set of genes in an organism, whether a bacterium, mushroom, eucalypt tree or wallaby (see page 232), is a genome. Each unique sequence of DNA carries a particular instruction for a cell (Figure 10.2). Nearly all genes provide instructions for making a particular polypeptide chain—a chain of amino acids, which determines the primary structure of a protein (page 16). Before we explore the mechanism of how genes provide instructions for cells, we need to revise the structure of DNA.

SPIDERGENOME

GENE

POLYPEPTIDE GENEPRODUCT

SILKPROTEINFORSPIDER WEBCONSTRUCTION

Genes and DNA Figure 10.2 Genes carry instructions for a cell. A spider’s web is made of the structural protein silk (a fibroin protein), which is the product of a gene.

aEND

aEND

NITROGEN CONTAINING BASE

SUGAR PHOSPHATE

You will remember from Chapter 4 that a DNA molecule is a double helix, made up of a series of chemical building blocks, called nucleotides, that form a polynucleotide chain (Figure 10.3). • Each nucleotide consists of: a phosphate group, a five-carbon sugar (deoxyribose) and one of four nitrogen-containing bases: adenine (A), guanine (G), thymine (T) and cytosine (C). The bases A and G are purines, because they have a double ring structure; T and C are pyrimidines, with a single ring. • Nucleotides and their particular bases can occur in any order within a strand: if a particular base is A, the next base in the sequence could be A, G, T or C. • There is direct pairing between A and T, and between G and C in the DNA molecule (the base-pairing rule). This complementary base pairing results in two polynucleotide strands joining together to form the doublestranded DNA molecule. An A of one strand pairs with a T in the other, and a G of one strand pairs with a C in the other (Figure 10.3). Given the base sequence of one strand we can determine the sequence of the other. • The two ends of a polynucleotide strand are referred to as the 5ʹ (five prime) and 3ʹ (three prime) ends. The 5ʹ end has a free phosphate group and the 3ʹ end has a free hydroxyl group. The two strands of a DNA molecule are referred to as being antiparallel, meaning that one strand runs 5ʹ → 3ʹ and the other 3ʹ → 5ʹ (Figure 10.3). Figure 10.3 The structure of DNA (see also Chapter 4). Two complementary strands forma double helix. The bases of each strand form a pair: A pairs with G and T pairs with C.

aEND

aEND

230 Continuity and change

Genes vary in size from about 100 to 2.5 million base pairs. The length of the sequence of DNA and the precise order of the base pairs in a gene are the critical factors that determine what the gene-product (nearly always a polypeptide) will be like and what it will do in a cell.

technologies and techniques Unlocking the secrets of the wallaby genome by Doctor Sue Forrest

T

here are exciting biological findings that could yield valuable results once the Tammar wallaby (Macropus eugenii) genome sequence is available. Knowledge of the genome sequence will help to unravel the biology of some interesting traits such as the regulation of lactation and diapause. For example, researchers noted that the lactating female wallaby is capable of feeding newborn and older joeys at the same time. Remarkably, each joey is delivered milk that has the correct composition for its particular stage of development. Understanding the control mechanisms involved here is of major interest to the dairy industry. In diapause, wallaby embryos develop to a stage of about 100 cells and then go into a state of suspended animation for 9–10 months until an environmental change spurs the older joey to leave the pouch. Leaving the pouch reduces the sucking stimulus and initiates the development of the ‘suspended’ early-stage embryo (blastocyst) into a full embryo. Understanding the mechanisms that control this phenomenon could lead to new treatments for infertility or methods of contraception in humans.

Sequencing the wallaby genome The wallaby genome is estimated to be 3600 Mb in size (Mb, Mega bases = one million bases), about 10% larger than the human genome. Sequencing this amount of DNA will require about 12 million chemical reactions. The project started with isolation of genomic DNA from a female wallaby (nicknamed ‘Matilda’). The methodology involves breaking the entire genome into DNA fragments at random (a shotgun approach). The fragments are a size that can be cloned (many copies made) and sequenced (using molecular tools described in Chapter 11). A collection of genomic clones is called a genomic library. Shotgun genomic libraries with clones of varying size from 2–10 kb (kb, kilo bases = one thousand bases) have been generated for Matilda. Isolating and sequencing these small random clones is progressing and will be complemented with some sequencing of larger clones (100–200 kb). The challenge will be assembling all of these different DNA regions to piece together the whole genome and alignment of the sequences to wallaby chromosomes. Bioinformatics, the use of computers to store large amounts of genetic information that researchers can access, will be an essential tool for assembly of Matilda’s genome.

Figure 10.4 Dr Sue Forrest and a Tammar wallaby.

Doctor Sue Forrest Doctor Sue Forrest is the Director of the Australian Genome Research Facility (AGRF), a Major National Research Facility. She works in the field of genomics with a focus on deciphering the genetic basis of common human disorders. Most recently, the AGRF has taken up the challenge and is driving the first large genome sequencing project ever undertaken in Australia—cracking the code of the Tammar wallaby (Macropus eugenii ) genome.

231 Molecular genetics

DNA and genes are packaged into chromosomes In eukaryotic cells, dark-staining thread-like structures, chromosomes, are located in the nucleus (Figure 10.5). Chromosomes consist of DNA and therefore carry genes. They constantly change their appearance during the life of a cell and come in various shapes and sizes. ACHROMOSOMECONSISTSOFSUPERCOILSOF$.!

NUCLEOSOME HISTONES

DOUBLE STRANDED$.!

DOUBLE STRANDED$.!WOUNDAROUNDSTRUCTURALPROTEINS

Figure 10.5 DNA is located in chromosomes. The chromosome shown here is at a stage where it is highly condensed. The diagram shows a section of the chromosome unravelled to show how DNA is wrapped within it. The DNA is coiled and supercoiled around structural proteins.

DNA is coiled and supercoiled around proteins biofile The muscle protein titin is coded by one of the largest known genes. It is approximately 280 000 DNA bases long.

The DNA molecule is extremely long. In humans, for example, the average DNA molecule is about 6.5 × 107 base pairs in length. The nucleus of a human cell is just 6 µm (6 × 10–6 m) in diameter, yet it contains 1.8 m of DNA. This is because the DNA is tightly packaged into chromosomes. In eukaryotes, DNA is coiled around small proteins (histones). In fact, eukaryotic chromosomes consist of about twice as much protein as DNA. Where the DNA is wrapped around a core of histone proteins it forms a particle about 10 nm in diameter called a nucleosome (Figure 10.4). The nucleosomes give the DNA strand the appearance of a string of beads. This arrangement of DNA wrapped around histones serves to package the DNA efficiently and to protect it from enzymatic degradation. When a eukaryotic cell is preparing to divide, chromosomes become very condensed. The nucleosomes themselves fold in a regular manner, producing supercoils (Figure 10.4). When chromosomes are highly condensed they are visible under a light microscope.

Each gene has a position on a chromosome ! The position of a gene on a chromosome is a site called a locus.

232 Continuity and change

Each DNA molecule contains many genes; for example, there are 5 770 genes in the fungus yeast and about 25 000 in humans. Each gene has a particular position, called a locus, on a specific chromosome. The genes of each DNA molecule are separated by regions called spacer DNA (Figure 10.6). Spacer regions include DNA that does not encode a protein product.

Spacer regions (Figure 10.6) may function in spacing genes apart so that enzymes or other molecules can interact easily with them.

'ENE!

SPACER

'ENE"

SPACER

'ENE#

Figure 10.6 A short stretch of double-stranded DNA is shown. Genes are highlighted in red and the spacer regions of DNA separating the genes are shown in blue.

How many chromosomes? All nucleated cells of an organism contain a fixed number of chromosomes. The number of chromosomes in somatic cells (all cells in a body except gametes) is characteristic of members of the species. The ploidy level of a cell is the number of chromosome sets that it carries. Gametes have nuclei that contain only one set of chromosomes (see Chapter 12), and they are called haploid (the number donated as n). Somatic cells are diploid (2n) because they contain two sets of chromosomes: one from each parent. The diploid chromosome numbers of organisms varies widely (Table 10.1). In humans the diploid number is 46. In some species of Australian ant (Myrmecia species), the diploid number is 2; each ant species has only one pair of chromosomes (n = 1). Some ferns have more than a thousand chromosomes in each somatic cell!

! The number of sets of chromosomes in a cell is the ploidy level: haploid = one set, diploid = two sets.

Table 10.1 Diploid numbers of chromosomes in various species Organism Animals Horse nematode worm, Parascaris equorum Vinegar fly, Drosophila melanogaster Koala, Phascolarctos cinereus Cat, Felis catus Human, Homo sapiens Chimpanzee, Pan troglodytes Platypus, Ornithorhynchus anatinus Dingo, Canis lupus subsp. dingo Plants and algae Garden pea, Pisum sativum Cabbage, Brassica oleracea All eucalypts, Eucalyptus spp. Blackwood, Acacia melanoxylon Pink rock orchid, Dendrobium kingianum Single-celled alga, Euglena gracilis Coconut palm, Cocos nucifera Fern, Ophioglossum reticulatum Fungi Mould, Penicillium Rust fungus, Puccinia graminis Edible oyster mushroom, Pleurotus ostreatus Edible mushroom, Agaricus bisporus Brewer’s yeast, Saccharomyces cerevisiae

Diploid number (2n) 2 8 16 38 46 48 52 78 14 18 22 26 38 90 596 1260

biofile A bacterium (prokaryotic cell) has a single chromosome, which is a circular molecule of DNA attached at a specific point to the plasma membrane. The DNA does not coil around clusters of proteins (nucleosome core particles) and condense as in eukaryotic chromosomes. However, with the aid of compression molecules, it is folded into a structure called a nucleoid.

2 6 22 26 30

233 Molecular genetics

biology in action Chromosome numbers of bush plants Chromosomes were first observed by Walther Fleming in 1882. While he was examining cells of a salamander larva under a light microscope, he saw minute threads in the nucleus. Since Fleming’s time, the chromosome number of many plants and animals has been documented. This information is important for crossing species to breed new varieties of plants for horticulture and for understanding evolutionary patterns. The family Proteaceae is a conspicuous and important part of the Australian bush, from heathlands to rainforests. It includes banksias, grevilleas, waratahs and the macadamia nut tree. The group (tribe Embothrieae) that includes all the Australian waratahs (Figure 10.7) and their relatives in South America is characterised by a diploid number of 22. In contrast, all grevilleas (Figure 10.8) and their relatives such as hakeas (tribe Grevilleeae) have one pair of chromosomes less, with a diploid number of 20. Chromosome number provides important evidence of the evolution and relationships of these plants.

Figure 10.7 Members of the family Proteaceae include the waratah, Telopea (tribe Embothrieae), with a diploid chromosome number of 22.

Figure 10.8 Grevillea (tribe Grevilleeae) with a diploid number of 20.

Synthesising new DNA The DNA molecule is passed on from one cell to another when cells divide (see Chapter 12). In order to be able to transmit the DNA molecule without losing it, cells must have a mechanism for accurately copying (replicating) their DNA.

DNA replication ! Replication is the synthesis of DNA on a DNA template (parent DNA molecule) to produce two identical molecules.

234 Continuity and change

DNA replication involves synthesising new DNA. During DNA replication, each strand of a parental DNA molecule acts as a template strand on which a new strand is synthesised. Because each daughter DNA molecule consists of one old and one newly synthesised strand (Figure 10.9), DNA replication is described as semi-conservative replication.

Separation of the two strands of DNA is achieved by enzymes that require ATP as an energy source. These enzymes break the hydrogen bonds holding the strands together. Other proteins bind to the single strands, keeping them apart and preventing them from rewinding. This ensures that the single-stranded template is available for the synthesis of a new complementary strand. A polynucleotide chain is built up during replication by adding complementary nucleotide units according to the sequence of the template strand. As the old strands ‘unzip’ (separate) from each other, a new strand is synthesised against each old strand (Figure 10.10a). The enzyme DNA polymerase is responsible for DNA synthesis by adding the correct nucleotides. However, it can only start by attaching nucleotides to a free 3ʹ OH group. Thus, to start synthesis, a short strand of RNA with a 3ʹ OH end, called a primer, is added to the single-stranded DNA template (Figure 10.10b). The RNA primer allows DNA polymerase to start work and the new strand is synthesised in the direction 3ʹ to 5ʹ. When synthesis has finished, the primer is removed by an editing enzyme. DNA replication is an extremely accurate process. DNA polymerase rarely adds the wrong nucleotides. Where this does occur, proof-reading and repair enzymes correct the error. Changes in DNA (mutations) only arise when these backup systems fail (page 295). (a)

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biofile The DNA polymerase enzyme adds nucleotides to a growing strand of DNA at a rate of 65 000 nucleotides per minute. This seems very fast until you think about how long it would take one DNA polymerase enzyme molecule to copy a gene of 2 500 000 base pairs: just over 38 minutes to replicate one strand of the DNA; over an hour and sixteen minutes for both strands.

Figure 10.10 (a) The synthesis of the daughter DNA sequence during replication. The complementary base-pair relationship in each strand of parental and daughter DNA is shown. (b) Initiation of DNA synthesis depends on a primer molecule of RNA first attaching to the DNA template. The primer molecule has a 3ʹ OH end, which allows DNA polymerase to start work to synthesis the new DNA strand in the direction of the 5ʹ end.

235 Molecular genetics

summary

10.1

• Many of the similarities and differences between individuals are determined by genes and passed on from generation to generation (inherited). • Genes are the units of inheritance, located on chromosomes and are particular sequences of DNA (deoxyribonucleic acid). The whole set of genes (all of its DNA) is an organism’s genome. • DNA contains the genetic code in all cellular organisms. In the double-stranded DNA molecule there are strict base-pairing rules between the two strands, with the base adenine (A) pairing with thymine (T), and guanine (G) with cytosine (C). • DNA is coiled around proteins and packaged into chromosomes, found in the nucleus in eukaryotic cells.

• The number, size and shape of chromosomes is characteristic of species. Somatic cells are diploid (with two sets of chromosomes) and gametes are haploid (with one set of chromosomes). • DNA is replicated and passed on when cells divide. During DNA replication, the two strands of DNA separate and each parental strand acts as a template strand on which a new strand is synthesised. • The enzyme DNA polymerase is responsible for adding the correct nucleotides to the 3ʹ end of the growing strands of DNA during replication.

key questions 1 Make a list of the following words and write next to each a short definition or explanation: genome, gene, DNA sequence, nucleotide base. 2 a Name the four bases found in DNA. b Which are purines and which are pyrimidines? 3 a What is meant by 5ʹ and 3ʹ when referring to a molecule of DNA? b One strand of DNA has the sequence ATTCCGTA. Write this out and under it write the sequence of the complementary strand. 4 Describe how DNA is organised into a chromosome.

5 Using Table 10.1, state whether the following are haploid or diploid, and give the chromosome number in the cells in each case: a sperm cells of a koala b skin cells of a cat c egg cells of a coconut palm d leaf cells of a garden pea. 6 a Describe the sequence of events that occurs when a strand of DNA replicates. Use a diagram in your answer. b What determines the sequence of bases in a new (daughter) strand of DNA? 7 a What is DNA polymerase? b What is its role? c What is the role of a primer?

10.2

Gene expression We know that a gene is a stretch of DNA. But how does the sequence of bases As, Gs, Cs and Ts in the DNA code for and result in a gene product (gene expression)? How is the code read? How is its message transferred from the chromosomes in the nucleus to the cytoplasm where ribosomes involved in protein synthesis are? How is the polypeptide gene product assembled? The overall process involves two major stages: 4RANSCRIPTION INNUCLEUS

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236 Continuity and change

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Transcription—reading the genetic code The first stage of gene expression involves transcription, which is ‘rewriting’ or copying of information from DNA to ribonucleic acid (RNA). The synthesised RNA is complementary to the sequence of one strand of the DNA. The particular RNA molecule involved in transcription is called messenger RNA (mRNA) because the molecule functions as a messenger, carrying a copy of the code into the cytoplasm. RNA is similar in structure to DNA (page 72). It is composed of many nucleotide units, each consisting of a sugar (ribose instead of the deoxyribose sugar in DNA), a phosphate group and a nitrogen-containing base (Figure 10.11a). The bases adenine, guanine and cytosine are the same as in DNA but the fourth base in RNA is uracil (U) rather than thymine (Figure 10.11b). The base-pairing rules for RNA are thus A to U and C to G. RNA is a singlestranded molecule consisting of a single chain of linked nucleotides rather than a pair of chains like the DNA molecule (Figure 10.11c). /(

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Synthesising messenger RNA Messenger RNA synthesis is controlled by the enzyme RNA polymerase. How does the RNA polymerase ‘know’ where to bind to DNA to help transcribe a particular gene? We have so-far defined a gene as a particular stretch of DNA. A typical gene consists of an upstream region (promoter region), coding segments (exons) and non-coding segments (introns): 0ROMOTER

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Figure 10.11 DNA and RNA differ in the following ways: (a) DNA contains the sugar deoxyribose, whereas RNA contains the sugar ribose; (b) DNA uses the bases adenine, guanine, cytosine and thymine, whereas RNA uses the bases adenine, guanine, cytosine and uracil; (c) DNA is a double-stranded helix, whereas RNA is single-stranded and does not form a double helix.

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237 Molecular genetics

The promoter region of a gene has a specific sequence recognised by RNA polymerase that initiates transcription. As RNA polymerase moves along the template strand of DNA, nucleotides are added to the growing RNA molecule according to the DNA–RNA base-pairing rules. Therefore, the single-stranded RNA molecule that is transcribed has a complementary sequence to the DNA template (Figure 10.12). Transcription always occurs in the same direction with respect to the DNA template strand. This RNA molecule is called a primary transcript. It is modified by enzymes that cut out the regions that corresponded to the introns (non-coding DNA) of the gene and join the remaining pieces back together. This shortened RNA molecule corresponding to the exon (coding) regions of the gene is the messenger RNA (mRNA). The mRNA molecule is complete when its two ends are modified. It is chemically ‘capped’ at the 5ʹ end. A tail of As (a poly-A tail) is added at the 3ʹ end. Each mRNA molecule produced then moves out of the nucleus through one of the many pores through the nuclear membrane and into the cytoplasm (page 39). 5'

3' C C A T C G C T A A A G C G T G G A

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Figure 10.12 During transcription the base sequence of the DNA template strand of a single gene is copied into mRNA. The DNA–RNA base-pairing rules are obeyed.

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Translation—assembling the protein product ! Translation is the synthesis of a polypeptide chain (protein) from messenger RNA (mRNA)

The second stage of gene expression is translation. In this process, the ‘instructions’ in the mRNA are read and a polypeptide (protein) product is assembled. The mRNA is transported to the ribosomes that are either free in the cytoplasm or located on the endoplasmic reticulum. The ribosomes provide a scaffold for the mRNA to assemble. At the ribosomes, the sequence of bases in the mRNA is ‘decoded’ to give the sequence of amino acids of the polypeptide.

Codons: a three-base code biofile The antibiotic tetracycline inhibits protein synthesis and thus the reproduction of bacterial cells. It works by blocking the binding of tRNA molecules to the bacterial ribosome. Eukaryotic ribosomes are structurally different from those of bacteria (prokaryotes), so our cells are not affected by the antibiotic.

238 Continuity and change

How does the information in DNA (genes) direct the assembly of a particular polypeptide? The base sequence of DNA links directly to the base sequence in mRNA, which in turn links directly to the sequence of amino acids in a polypeptide chain (Figure 10.13). As you studied in Chapter 4, the ‘instructions’ for assembling a polypeptide are coded as three base codons in the mRNA. Remember that for a genetic code based on triplets of four bases, there are 64 possible codons. Remember also that the code is degenerate because some amino acids are coded by more than one codon; for example, serine is coded by the four codons UCU, UCC, UCA and UCG. Other special codons do not code amino acids but

base sequence in DNA template strand

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TRANSCRIPTION (copying)

base sequence in mRNA

G C U A A A U C C U A G

TRANSLATION (change language)

amino acid sequence in polypeptide

Ala

Lys

Ser

stop translation

Figure 10.13 The base sequence of DNA is transcribed to produce a base sequence of mRNA, and the translation of that sequence produces an amino acid sequence in a polypeptide. UAG is a ‘stop’ instruction (codon) and does not specify any amino acid.

provide specific instructions, such as ‘start’ and ‘stop’. The codon AUG is used to ‘start’ translation (AUG specifies the amino acid methionine, which is the first amino acid in proteins). UAA, UAG and UGA are three codons that signal ‘stop’: no more amino acids are added to the polypeptide chain. The complete genetic code for the 20 common amino acids and also stop codons is summarised in Table 10.2 (see also Chapter 4). In summary, given an mRNA sequence we can determine the order of amino acids incorporated into the corresponding polypeptide (Figure 10.13). The code is universal, the same code applying from bacteria to humans, with few exceptions.

Table 10.2 The genetic code for 20 amino acids and STOP codons. The codon AUG is the usual START codon First position (5ʹ end)

Second position

Third position (3ʹ end)

U Phenylaline Phe Leucine Leu

C Serine Ser Ser Ser

A Tyrosine Tyr STOP STOP

G Cysteine Cys STOP Tryptophan (Trp)

U C A G

C

Leu Leu Leu Leu

Proline Pro Pro Pro

Histidine His Glutamine Gln

Arginine Arg Arg Arg

U C A G

A

Isoleucine Ileu Ileu Methionine

Threonine Thr Thr Thr

Asparagine Asn Lysine Lys

Serine Ser Arg Arg

U C A G

G

Valine Val Val Val

Alanine Ala Ala Ala

Aspartic acid Asp Glutamic acid Glu

Glycine Gly Gly Gly

U C A G

U

239 Molecular genetics

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A A A UU U

anticodon site

Transfer RNA molecules: the workers The ‘workers’ that assemble the polypeptide are transfer RNA (tRNA) molecules. Transfer RNA molecules are produced from tRNA genes by transcription (Figure 10.14). They are short single-stranded molecules of approximately 80 nucleotides in length, which fold to form a cloverleafshaped structure. There are many different types of tRNA, each produced by transcription from the DNA template of a different tRNA gene. The tRNAs act as transport molecules, bringing the correct amino acid to the ribosome for assembly into the polypeptide. To do this the tRNA has to be able to bind to a specific amino acid and to the appropriate bases in the mRNA. There are two specific sites in a tRNA where binding occurs: the amino acid attachment site and the anticodon site (Figure 10.15).

Amino acid attachment site The amino acid attachment site is where an amino acid attaches to the tRNA. For each tRNA this binding site is specific for a particular amino acid; for example, a glycyl tRNA will only attach the amino acid glycine. When a tRNA has an amino acid attached, it is said to be ‘charged’. The enzyme aminoacyl tRNA synthase catalyses the linking of an amino acid to its particular tRNA carrier.

Anticodon site An anticodon site is necessary to ensure that the correct amino acid is added to a growing polypeptide. This site is composed of three bases that are complementary to a triplet codon on the mRNA. For each tRNA, the anticodon site matches the amino acid attachment site according to the genetic code (Table 10.2). For example, a codon for glycine is GGU and the matching anticodon of the glycyl tRNA is CCA. The association of codon and anticodon occurs in the ribosome during translation (Figure 10.16). The order in which amino acids are added to the polypeptide is determined by the codon sequence of the mRNA. The mRNA is read in a 5ʹ–3ʹ direction as the ribosome moves along it. Individual codons are read from 5ʹ (first position, first base in the codon) to 3ʹ (third position, third base in the codon); for example, GCU can specify alanine (Ala), and AAA can specify lysine (Lys).

Summary of translation

C C A lysine

amino acid attachment site

Figure 10.15 This tRNA is charged with the amino acid lysine at the amino acid attachment site. The anticodon UUU will bind to the complementary sequence AAA in the mRNA.

240 Continuity and change

We have now looked at all stages of the polypeptide assembly process called translation. In the nucleus, the template strand of the DNA of a gene is copied into mRNA (Figure 10.17). The mRNA is a true messenger. It carries the message, written in codons, out through pores in the nuclear membrane into the cytoplasm to the ribosome. Here, the message is translated from the ‘language’ of base triplets into the ‘language’ of amino acids. The translation is accomplished by a molecule (tRNA), which ‘speaks’ both languages. Any given tRNA is able to interact with a specific amino acid and the appropriate codon. Different tRNAs bring the amino acids in the correct order to be joined together to form a polypeptide. A polypeptide will fold to form a complex three-dimensional structure determined by its amino acid sequence. Once translation is completed the new polypeptide is released into the cytoplasm (Figure 10.17). Some proteins consist of more than one polypeptide. In these cases the polypeptides associate in the cytoplasm to form the fully functional protein.

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Figure 10.16 A time-lapse representation of the translation of the mRNA sequence of Figure 10.13. Transfer RNAs bring specific amino acids to the ribosome. The amino acids are added to the polypeptide chain. For example: (a) the codon AAA pairs with the anticodon UUU which occurs on the tRNA that carries lysine; (b) lysine is linked by a peptide bond to the end of a growing polypeptide; (c) the tRNA drops away from the mRNA. It will go back into the cytoplasm to be charged with another molecule of lysine. Meanwhile, the next amino acid, serine, is added. (d) Translation ends when the ribosome reaches UAG because there is no tRNA that carries the corresponding anticodon. The polypeptide is released into the cytoplasm. (e) Finally the mRNA is released from the ribosome.

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241 Molecular genetics

summary

10.2

• A gene codes for a polypeptide. Amino acids linked by peptide bonds make up a polypeptide. One or more polypeptides make a protein. • A single strand of DNA acts as a template from which singlestranded ribonucleic acid (RNA) is transcribed. In RNA the base thymine (T) is replaced by uracil (U) and the sugar of the nucleotide units is ribose.

• During translation, the base sequence in messenger RNA (mRNA) is translated at ribosomes into an amino acid sequence in a polypeptide. The amino acids are specified by codons of three bases in mRNA. • Transfer RNAs (tRNAs) act as transport molecules to deliver the correct amino acid to ribosomes for assembly into a polypeptide sequence. The tRNA molecules function as adaptors, ‘plugging into’ a specific amino acid at one end and into the corresponding codon in the mRNA at the other.

key questions 8 a List the similarities between DNA and RNA. b Construct a table illustrating the differences between molecules of DNA and RNA. 9 Use a flow chart to summarise the steps involved in transcription and translation during protein synthesis. 10 Distinguish between a ‘codon’ and an ‘anticodon.’ In your answer include a definition, name the molecule on which each is located and outline the role of each. 11 How are the codons UAA and AUG different from all others that appear in the genetic code?

12 Draw a labelled diagram of a tRNA sequence that codes for the amino acid asparagine. (Hint: There are two possibilities.) 13 The sequence of a section of mRNA is 5ʹ CCCUAUAAGUAG 3ʹ. The code is read in triplets from the 5ʹ end of the sequence. Considering this sequence, work out: a the base sequence in the strand of DNA from which the mRNA sequence was transcribed. b the amino acid sequence in the polypeptide when the sequence is translated. 14 A typical gene (DNA sequence) has regions called introns and exons. Distinguish between an intron and an exon.

10.3

Gene regulation Some genes are expressed continuously Genes that encode proteins that are required constantly in cells are often expressed continuously, therefore transcription and translation are occurring most of the time. These genes are the so-called ‘housekeeping’ genes. They produce the proteins required for the basic functions of cells such as provision of energy, passage of molecules across the cell membrane, repair and maintenance of cellular organelles and cell division.

biofile Trypanosomes are parasites that cause African sleeping sickness. They have up to 1000 genes that encode proteins on their cell surface. Trypanosomes can express one of these genes at a time. When this occurs, one gene is active and the rest remain silent. During infection, the human immune system will produce antibodies as a defence against a coat protein. However, by gene switching to express a second type of protein, the parasite can overcome the human defence reaction and evade detection.

242 Continuity and change

Switching genes on and off While the expression of some genes is continuous, the expression of others is regulated in cells. Although all somatic cells in an organism carry identical gene sets, only a proportion of those genes are switched on in a given cell type or stage of development. A mammal such as Dolly the sheep is composed of 200 different types of cells (Figure 10.18). Dolly’s different cell types are distinguished by the set of gene products they produce. For example, the protein fibrillin is produced in connective tissue and bones and tyrosinase is produced in the skin.

white blood cells

biofile neuron

Study of a nematode has revealed that not all genes result in the production of a polypeptide. The nematode has a gene that encodes a short piece of RNA. This short RNA hybridises to a target mRNA and prevents translation. This is a newly discovered method of regulating gene expression (switching a gene off by blocking translation).

rod cell

epithelial cells

osteoblasts

Gene regulation involves processes that control gene expression, turning a particular gene ‘on’ or ‘off’. Some genes are controlled by other genes. The environment of a cell can also affect gene expression. Protein gene-products or ions may bind to DNA, or physical factors such as temperature may influence gene expression. In plants, genes that encode for the photosynthetic system are only expressed when there is sufficient light for photosynthesis to occur. Gene regulation is not as well understood in multicellular organisms such as plants and mammals as it is in bacteria, but similar processes do apply. In a prokaryote gene, there is a control region at the 5ʹ end of the gene that is the site for switching the gene on or off (Figure 10.19). For example, the promoter of gene A may be repressed by a protein produced by another, regulatory gene B. The repressor protein-product of gene B turns off gene A. In animals, steroid hormones have been shown to regulate the transcription of particular genes. In chickens, mature oviduct tissue responds to the hormone oestrogen by producing the egg white protein ovalbumin. Oestrogen binds first to a protein in the cytoplasm. The hormone–protein complex then enters the nucleus and binds to a specific DNA sequence in the ovalbumin gene. This results in the transcription of mRNA specific ovalbumin.

Figure 10.18 All of the somatic cells of Dolly the sheep are genetically identical. However, she has about 200 different cell types, including: a rod cell from the eye; a neuron (nerve) cell from the spinal cord; a bone cell (osteoblast) in the leg; white blood cells from the arteries; epithelial cells from the airways of the lungs.

! Gene regulation involves processes that control gene expression, turning particular genes ‘on’ or ‘off’.

TRANSCRIPTION transcription start point

stop signal

DNA molecule control region —regulates when and where the gene is activated

proteinencoding region

Figure 10.19 The structure of a gene. Notice the control region where transcription of the gene can be switched on or off.

243 Molecular genetics

extension Switching the lactose gene on and off in E. coli When lactose is added to its growth medium, the bacterium Escherichia coli switches on a gene and makes the enzyme β-galactosidase. The β-galactosidase enzyme splits the sugar lactose to produce the sugars glucose and galactose. In producing the enzyme, the bacteria can make use of the lactose available. Lactose is a sugar rarely encountered by bacteria, so β-galactosidase is not usually produced. When lactose is absent, a protein attaches to DNA and blocks the synthesis of mRNA for β-galactosidase. This protein is called a repressor because it prevents transcription (Figure 10.20). When lactose is present it binds to the repressor. This releases the repressor from the DNA, so mRNA is transcribed and β-galactosidase is produced (Figure 10.20). The response is rapid and dramatic. Within 10 minutes of lactose being added to the growth medium, around 6% of all the protein being made by the bacterial cells is β-galactosidase. When the lactose in the medium is used up, the repressor protein reattaches to the DNA, mRNA synthesis for β-galactosidase ceases and the concentration of the enzyme falls to un-detectable levels. The gene is ‘on’ or ‘off’ depending on the nutrients available to the cell.

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Figure 10.20 Regulation of β-galactosidase production in the bacterium E. coli. When lactose is absent the repressor protein blocks mRNA transcription. Therefore the gene is switched off. The enzyme is not produced when there is no need for it (no lactose substrate).

summary

10.3

• The expression of genes is regulated. Not all genes are switched on in any one cell at any one stage of development. • Some genes control the expression of other genes.

• The environment of a cell (light, temperature, ions, hormones) also can influence the expression of genes.

key questions 15 What is meant by ‘gene expression’ and ‘gene regulation’? 16 Explain why some genes are continually ‘on’, while others are not. Give an example of a ‘house-keeping’ gene.

244 Continuity and change

17 a Give an example of a physical environmental factor that can regulate gene expression (turn the gene off or on). b Give an example of a hormone regulating the expression of a gene.

10

key terms trait heredity gene genome nucleotide base adenine thymine guanine cytosine purine pyrimidine

complementary base pairing 5ʹ and 3ʹ ends antiparallel chromosome histone nucleosome somatic cell ploidy haploid diploid DNA replication template strand

semi-conservative replication DNA polymerase primer transcription RNA (ribonucleic acid) messenger RNA (mRNA) uracil RNA polymerase exon intron translation ribosome

polypeptide amino acid codon start codon stop codon transfer RNA (tRNA) anticodon gene regulation gene expression

worksheet 25 1 Complete the following table of chromosome number for the species of multicellular organisms shown. Species Horse

Diploid number

Haploid number

64

Dog

39

Chimpanzee

24 46

Rabbit

You will need to refer to Table 10.2 (page 239) to answer some of the following questions.

44

Eucalypt

11

Garden pea

14

Corn

20

3 Explain what is meant by DNA being coiled and supercoiled within a chromosome. Include a diagram in your answer. What benefit is there in DNA being packaged this way in a cell? 4 The literal meaning of transcription is ‘to write across’, and the meaning of translation is ‘to put into a different form’. Explain why each of these terms, transcription and translation, are appropriate to the steps in protein synthesis.

2 Explain what is meant by the ‘kangaroo genome project’. What information does it provide? How might this information be useful to humans?

5 The genetic code is described as • non-overlapping • unambiguous • redundant • universal. Explain what each of these features mean. 6 a Can a codon specify more than one amino acid? Explain. b Can an amino acid be specified by more than one codon? Explain. 7 Copy and complete the following table. Strand I of the DNA is the strand from which the mRNA is transcribed in this case. DNA strand I

T C T C C G T

DNA strand II

T T G A

mRNA Amino acid

A Meth

Arg

245 Molecular genetics

8 Synthetic mRNA molecules were made from a solution of 11 All cells in the human body are descended from the first cell, 80% adenine and 20% uracil. Proteins produced from these the fertilised egg. Yet in the mature adult only some cells mRNA molecules were found to consist of amino acids in the produce hormones, some digestive enzymes, some mucus. In ratio shown. terms of gene function, explain why this occurs. 64 : 16 : 4 : 1 lysine isoleucine tyrosine phenylalanine a What DNA triplet codons were probably specifying each amino acid? b Is the ratio in accordance with that expected from the given mixture of the bases? 9 The diagram shows the transcription and translation of a base sequence of DNA. DNA sequence

AAA

ACA

transcription mRNA sequence

UUU

amino acid sequence ???

Cys

CAG

AUC

GUC

Leu

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a Fill in the missing letters for the DNA sequence, the corresponding mRNA and the amino acid product. (Consult Table 10.2 for the genetic code and amino acids) b What will be the amino acid sequence if the sixth base of the sequence of the DNA is: i changed from A to G ii changed from A to C iii deleted? 10 Derive your own DNA sequence with a start codon, a stop codon and three codons in between for three different amino acids. Then alter this sequence so that: a each of the codons is different, but the amino acid sequence remains the same b each codon is altered by one base only, but all of the resulting amino acids are different Make sure you write down the starting DNA sequence, the transcribed mRNA sequence and the corresponding amino acids.

246 Continuity and change

12 Undertake research on one of the following statements. Prepare a short presentation on your topic and deliver this to your class. a The human genome project will lead to cures for all diseases. b The human genome project will allow couples to prevent genetic disease that run in the family being passed on to their offspring. c The human genome project can be used for the good or detriment of the human race.

1

chapter 11

Molecular tools and techniques

key knowledge • • • •

molecular tools and techniques amplification of DNA genetic profiling manipulation and modification of genomes

chapter outcomes After working through this chapter you should be able to: • appreciate how the study of molecular genetics has expanded and led to new tools and technologies, including restriction enzymes, gel electrophoresis, DNA profiling and DNA sequencing • explain the principle of the polymerase chain reaction (PCR) technique used to amplify (make many copies of) DNA, which allows researchers to work with DNA • demonstrate an understanding of what is meant by recombinant DNA technology and genetic transformation • give examples of how molecular techniques are applied in forensic science and paternity testing (DNA profiling), conservation biology (measuring genetic diversity in wild populations), agriculture (GM crops) and medicine (genetic diagnosis, production of human proteins).

11.1

Working with DNA Each cell contains the entire complement of an organism’s DNA (its genome), which includes thousands of genes. Today, biologists are able to isolate fragments of DNA. A single gene can be studied or the base sequence of an entire genome can be determined. Biologists use molecular tools to: • understand the way in which plants and animals, including humans, develop, function and evolve • investigate the molecular basis of disease • develop products for medicine and crops for agriculture • solve crime and paternity disputes • investigate endangered species for conservation management.

DNA profiling A number of methods have been developed that can be used to identify the DNA (genetic) profile of an individual. These same methods can also be used to measure genetic differences between individuals in a natural population.

Restriction enzymes ! A restriction enzyme is a bacterial enzyme that recognises a short sequence of bases in a DNA molecule and cuts the DNA at this recognition site.

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Continuity and change

One technique uses a set of enzymes that recognise and cut DNA into fragments at particular base sequences. These enzymes are called restriction enzymes (also called restriction endonucleases), which act as molecular scissors (Figure 11.1a). They are found naturally in bacteria. Different restriction enzymes recognise different sequences of bases and therefore produce different DNA fragments. For example, compare two individuals, A and B, who are genetically different. A base change has occurred in the DNA of individual B. This has disrupted the sequence where a particular restriction enzyme would normally recognise and cut the DNA. Thus, the DNA of individual B will have one less cut site. Following the mixing (digestion) of DNA with a number of different restriction enzymes, the DNA fragment sizes for individuals A and B will be different. The fragments can be separated using the technique of gel electrophoresis (see Figure 11.2). In summary, the different sequences of bases in individuals result in restriction enzymes cutting the target DNA at different sites. Thus, a DNA sample from an individual will result in a unique banding pattern on a gel, giving a DNA profile (DNA fingerprint) of that individual. (a) restriction enzyme

DNA

Figure 11.1 (a) Restriction enzymes are molecular scissors that cut DNA into fragments at particular sites. (b) A restriction enzyme recognises a specific site in a DNA molecule by its base sequence. At this recognition site the enzyme cuts the molecule. Different restriction enzymes recognise different sites, and thus cut the DNA into different sized fragments.

Cutting and joining DNA molecules

!

Molecular scissors Restriction enzymes are a basic tool of genetic engineering. Isolated from bacteria, they normally form part of the defence system of the bacterial cell. If foreign DNA, such as the DNA from a virus, enters the bacterial cell, the restriction enzyme will cut it up into small pieces. The restriction enzyme does not cut the bacterial DNA in the cell because the bacterial DNA is chemically modified and not recognised by the enzyme. Different bacterial species have restriction enzymes that differ in their recognition sequence (Figure 11.1b). Some enzymes recognise and cut four base pair sequences; others recognise six base pair sequences. The bacterium Haemophilus influenzae produces a restriction enzyme called HindIII, which recognises a specific six base pair sequence and cuts the sequence shown at the points indicated by the arrows: DNA (double stranded) Cut by restriction enzymes ↓ 5′–AAGCTT–3′ 3′–TTCGAA–5′ ↑

Two fragments of DNA

⇒

A TTCGA

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Restriction enzymes have many uses in molecular biology, so they are purified and sold by commercial suppliers. Joining sticky ends Once separated, DNA fragments from different sources can be joined (ligated) together. Some restriction enzymes make a staggered cut in DNA, leaving short single-stranded ends. These protruding single-stranded ends are ‘sticky’. If a molecule with sticky ends meets another piece of DNA with complementary sticky ends, the two pieces of DNA may join up, reforming hydrogen bonds. The enzyme DNA ligase is required to stabilise the recombined molecule. DNA ligase can be thought of as a ‘pasting’ enzyme. This is an important tool used in genetic engineering, when new DNA is inserted into an organism to make a genetically modified (GM) organism (see page 254). (Note that if the cut is not staggered the ends of the fragments are called blunt-ends and do not ligate).

Gel Electrophoresis Gel electrophoresis is a technique for separating fragments of DNA. The technique is also used to separate proteins of different size, charge and shape. Different sized molecules will migrate through a gel at different rates when driven by a drop in electric voltage. An electrophoresis gel has a jelly-like texture, and is composed usually of agarose (a purified form of agar) and ethidium bromide—a chemical stain for detecting DNA (Figure 11.2a). A DNA test sample can be cut into fragments with restriction enzymes. The size of the fragments will depend on the base sequence of that particular source of DNA. During gel electrophoresis, the sample of DNA is placed in a well in a gel. Other test samples and molecular weight standards, which are DNA segments of known size (length), can be added to other wells for comparison with the first sample. The gel is placed in an electrophoresis bath where it is covered in a controlled pH solution (Figure 11.2b). A power source is attached to the electrophoresis bath and it is switched on. DNA is negatively charged,

DNA profiling is characterising the genetic makeup of an individual by cutting DNA into fragments using restriction enzymes and examining the banding pattern on an electrophoresis gel.

biofile Variation in the genomes of individuals often occurs at the site of clusters of short repeated sequences that are a few letters long. These are called short tandem repeats (STRs). For example, the sequence TAACG may be repeated many times in a row in a non-coding region of DNA between two genes (called a spacer region). The number of repeats at any one site identifies an individual. Thus, differences between individuals are evident by the variable number of tandem repeats at a site (VNTRs). These variable regions result in much of the differences in the banding patterns of people who are DNA fingerprinted.

biofile A field trip to Yellowstone National Park in the 1960s radically altered the course of molecular genetics research. Thomas Brock, a bacteriologist from the University of Wisconsin–Madison, found bacteria in water taken from a hot spring. He found and named the new species Thermus aquaticus (Latin for ‘hot water’). Enzymes are normally denatured if heated to temperatures of 95°C for more than a few seconds. For T. aquaticus to survive in the hot springs, its enzymes, including DNA polymerase, need to tolerate these high temperatures. Therefore, the DNA polymerase from T. aquaticus (‘Taq polymerase’) has proved to be an ideal enzyme for PCR.

! Gel electrophoresis is the migration and separation of DNA fragments (or proteins) along a gel, driven by a voltage drop.

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Molecular tools and techniques

249

therefore it moves through the gel with the negatively charged current towards the positive terminal. The gel is like a molecular sieve. The smaller DNA segments move faster through the gel than the larger segments. After the power source is switched off, the DNA in the gel can be observed on an ultraviolet (UV) light box (Figure 11.2c). This is because the ethidium bromide, which binds to the DNA in the gel, fluoresces a bright pink colour when exposed to UV light. Bright pink bands are observed wherever there is DNA present in the gel. The size of the bands (DNA profile) of various samples can be determined by comparing them against the molecular weight standards (Figure 11.2d) that are also run out on the gel.

(a)

(b)

(c)

(d) Figure 11.2 DNA gel electrophoresis: (a) The gel is made in a mould. The two combs shown leave two rows of wells for samples and standards. (b) The gel is placed in an electrophoresis bath where it is covered with buffer solution. A power source is attached to the electrophoresis bath and it is switched on. (c) The DNA in the gel can be observed when the gel is placed in an ultraviolet light box. (d) This diagram of a gel shows fragments of digested DNA from four different plant samples. The left lane compares them with standards of DNA of known size. Which two plant samples are genetically identical?

DNA fragments from test samples

Molecular weight standards Size (bp)

1

1500 1000 900 800 700

2

3

4 Wells in agarose gel

600 500 400 300 200

100

The Southern blot technique

250

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Continuity and change

When a large sample of DNA (such as the total genome of an individual) is cut by restriction enzymes, hundreds of thousands of fragments may result. When so many segments are run on a gel by electrophoresis and stained with ethidium bromide, the result will be a smear because it is impossible to distinguish so many fragments. One technique used for large samples is called the Southern blot (named after Edward Southern who developed it). Southern blotting can be used to look for particular bands, that is, identify individual DNA sequences (fragments). The technique can also be used to determine the location of a specific gene among a whole genome.

The four steps of the Southern blot technique (Figure 11.3) are: 1 Cut the total genomic DNA with restriction enzymes and run out all of the bands. 2 Blot (pick-up) the smear of fragments onto a nitrocellulose filter paper (the Southern blot). 3 Probe the blot for a particular DNA region of interest. A DNA probe is a purified fragment of single-stranded DNA (25–1000+ bases in length) labelled with a radioactive isotope (e.g., 32P) or a fluorescent dye. Probes can be made from purified fragments amplified by PCR or made synthetically in a laboratory (up to about 100 bases). The machine that synthesises a probe is instructed to make a single DNA strand with a particular order of As, Ts, Cs and Gs. The probe will hybridise to a complementary region among the fragments of DNA in the sample. To locate a particular gene or DNA sequence, the researcher needs to know at least part of its sequence to construct the probe that will find it. 4 After the probe has hybridised with the DNA on the Southern blot, the fragment or gene of interest can be detected by the radioactive or fluorescent dye labelling.

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Figure 11.3 The technique of Southern blotting.

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Molecular tools and techniques

251

DNA amplification using the polymerase chain reaction (PCR)

gene A 1 copy of gene A

2 copies

4 copies

etc…

8 copies…

Figure 11.4 The polymerase chain reaction (PCR) results in the successive doubling of the number of copies of a stretch of DNA, such as gene A.

biofile PCR is an extraordinarily sensitive technique. In theory, a sample of one or two cells provides enough DNA for amplification. Scientists at the Victoria Forensic Science Centre have shown that merely touching an object deposits sufficient material for successful DNA amplification. In handling keys, opening a door or driving a car, the cellular material deposited by a criminal provides ample DNA for analysis following PCR.

! A primer is a short piece of DNA synthesised to be complementary to a stretch of DNA. The primer hybridises to the larger stretch of DNA. Primers are used in DNA replication techniques that use PCR and in DNA sequencing.

252

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Continuity and change

To work with DNA it is necessary to have more than a few molecules. The polymerase chain reaction (PCR) is a technique used to amplify (make millions of pure copies of) a piece of DNA in a test tube (Figure 11.4). It allows, for example, forensic scientists to amplify the DNA in traces of blood left at the scene of a crime. It is also used to amplify a particular gene from a sample of DNA fragments. Biologists developed the technique of PCR by studying how DNA is synthesised naturally in cells. The technique of (PCR) PCR is a chain reaction of DNA replication events. The method uses a complex mixture of ingredients, which is heated and cooled in cycles. At each cycle of synthesis, the number of copies of the DNA fragment is doubled. A large amount of DNA can be produced in a test tube in a few hours. The polymerase chain reaction (PCR) mixture contains four ingredients: • a sample of DNA, which acts as a template to make millions of copies • a source of the four nucleotides: A, T, C and G, which are the building blocks for DNA replication • a DNA polymerase (Taq polymerase), which is a heat-resistant enzyme • single-stranded DNA primers, which are synthetic, short pieces of DNA that are complementary to sequences of bases that flank the DNA region to be amplified. Primers specify the starting and finishing points for DNA replication (see Chapter 10). These ingredients are placed together in a plastic tube in a DNA thermocycler, a heating block that is able to change temperature very rapidly. The thermocycler initially heats to a temperature of 95°C. This breaks the hydrogen bonds and separates the strands of the DNA sample to make two single-stranded templates (Figure 11.5). The thermocycler then cools to a temperature of 50–60°C to allow the primers to bind to their complementary DNA sequence. These primers are typically 18–30 nucleotides in length. The two primers bind at the ends of the DNA that is to be amplified; one primer binds to each template strand. The temperature is then increased to approximately 72°C, the optimal temperature for the DNA (Taq) polymerase enzyme. The enzyme DNA polymerase begins to move along the template DNA, starting from the primer and adding nucleotides. Nucleotides are added at the 3′ end of the new strand according to the complementary base-pairing rules. Once this first round of DNA replication is complete, two double-stranded DNA molecules have been produced from each double-stranded DNA molecule added to the reaction mixture. The thermocycler then heats to 95°C and the next cycle of strand separation, binding of primers and DNA replication begins. Thermocyclers can be programmed to go through many cycles. Typically, 30–40 cycles are used to amplify a DNA sample.

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Figure 11.5 Synthesis of DNA by PCR. (a) All of the components of the PCR reaction mixture are present in the tube. (The four nucleotides, containing bases A, T, C and G, are added to the reaction mix although they are not shown here.) (b) Heating to 95°C separates the two strands of the template DNA. (c) Cooling to between 50°C and 60°C allows the two single-stranded primers to bind to their complementary DNA sequences in the template DNA. (d-e) As the temperature increases to 72°C, DNA polymerase begins synthesising a new strand of DNA from each of the two template strands. (f) Synthesis is stopped by increasing the temperature to 95°C. Heating to 95°C also separates the strands of the two doublestranded DNA molecules, ready to begin the next cycle of DNA synthesis.

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Molecular tools and techniques

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Genetically modified plants

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by Professor Adrienne Clarke

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Million hectares

technologies and techniques

Years • 15% growth from 2002 • 18 countries; USA (63%) Argentina (21%) Canada (6%) Brazil (4%) China (4%) South Africa (1%) Australia (