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Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 28 Organelles of cells: Introduction : - The cell is the fundamental unit of life. - The modern ‘Cell theory’ states : i) All living organisms are com...

Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 28 Organelles of cells: Introduction : - The cell is the fundamental unit of life. - The modern ‘Cell theory’ states : i) All living organisms are composed of cells. ii) All new cells are derived from other cells. iii) Cells contain the hereditary material of an organism which is passed from parent to daughter cells. iv) All metabolic process take place within cells. Microscopy : 1. Light microscope : - It is the most common type of microscopes. - The degree of detail which can be seen with a microscope is called resolution or resolving power. This measures its ability to distinguish two objects which are close together. - The resolving power is inversely proportional to the wavelength of light being used. This means that the resolving power of any light microscope is limited because the wavelength of light has a fixed range. - At best it can distinguish two points which are 0.2µm apart and magnify around 1500 times. 2. Electron microscope : - It works on the same principal as the light microscope except that instead of light rays, a beam of electrons is used. - In practice it magnifies just over 500,000 times. - The image produced by the electron microscope cannot be detected directly by the naked eye. Instead, the electron beam is directed onto a screen from which black and white photographs, called photoelectromicrographs, can be taken. 3. A comparison of the light and electron microscope : Light microscope Electron microscope Advantage Disadvantage 0 Cheap to purchase Expensive to purchase Cheap to operate – uses a little electricity for a light bulb. Expensive to operate – requires up to 100,000 volts to produce the electron beam. Small and portable – can be used almost anywhere. Very large and must be operated in special rooms. Unaffected by magnetic fields. Affected by magnetic fields. Preparation of material is relatively quick and simple, Preparation of material is lengthy and requires requiring only a little expertise. considerable expertise and sometimes complex equipment. Material rarely distorted by preparation Preparation of material may distort it. Both living or dead material may be viewed. A high vacuum is required and living material cannot be observed. Natural colour of the material can be observed. All images are in black and white. Disadvantage Advantage Magnifies objects up to 1500x. Magnifies objecs over 5000,000x. Can resolve objects up to 200nm apart. Has a resolving power for biological specimens of around 1nm. The depth of field is restricted. It is possible to investigate a greater depth of field.  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 29 The Types of Cells : - Prokaryotic cells were probably the first forms of life on earth. There are no true nucleus and no **** membrane- bounded organelles Insert U.B. p39 fig 4.4 within a prokaryotic cell. This **** occurs only in bacteria and the blue- green algae Fig. 31 Structure of prokaryotic cell, e.g. a generalised bacterial cell. - The development of eukaryotic cells from prokaryotic ones involved considerable changes. The essential changes was the development of membrane- bounded organelles within the outer plasma membrane of the cell. - The presence of membrane- bound organelles confers four advantages : i) increase surface area for the metabolic process to take place; (enzymes are embedded in the membrane) ii) contain enzymes for a particular metabolic pathway; iii) control the rate of any metabolic reaction in an organelle as membrane of the organelle control the passage of reactants. iv) harmful substance can be isolated inside an organelle. - There are two main kinds of eukaryotic sells, they are plant and animal cells. Belows are the differences between them. Plant Cell Animal Cell Cellulose cell wall surrounds the cell membrane No cell wall (only a membrane surrounds the cell) Pits and plasmodesmata present in the cell wall Absent Middle lamella join cell walls of adjacent cells Cells are joined by intercellular cement Present of plastids e.g. chloroplast Absent Mature cells have a large single, central vacuole Vacuoles, e.g. contractile vacuoles, if present, are filled with cell sap small and scattered throughout the cell Tonoplast present around vacuole Absent Nucleus at edge of the cell Nucleus always in the central Lysosome not normally present Lysosomes always present Centrioles absent in higher plant Centrioles present Cilia and flagella absent in higher plant Present Starch grains used for storage Glycogen granules used for storage Only meristematic cells can divide Almost all cells are capable of division Few secretion are produced Wide variety of secretions are produced  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 30 Exercise : (95 I 1) Tabulate the major differences in cellular organization between prokaryotic and eukaryotic organisms. [4 marks] Ultra-structure of the cell : **** Insert BSI 3 rd Ed. P135 fig 5.10, 5.11 **** Fig. 32 Ultrastructure of a generalised animal cell as seen with the electron microscope. Fig. 33 Ultrastructure of a generalised plant cell as seen with the electron microscope. `  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 31 1. Cell membranes : - They are described as selectively permeable, since apart from small molecules, such as water, larger molecule e.g. glucose, amino acids, glycerol and ions can diffuse slowly through them. And they also exert a measure of active control over what substances they allow through. - As organic solvent (alcohol) penetrate membranes even more rapidly than water, this suggested that membranes have non- polar portions; in other words they contain lipids. - After careful chemical analysis, it is found that membranes are comprised almost entirely of proteins and lipids (phospholipids, glycolipids and sterols). - The fluid- mosaic model can be used to describe the detailed structure of plasma membrane. The fluid-mosaic model (Singer-Nicholson model ) : Ø It was put forward in the early 1970s by S.J. Singer and G.L. Nicholson. Ø The protein molecules vary in size and have a much less regular arrangement. Ø Some proteins occur on the surface of the phospholipid layer, while others extend into it, some even extend completely across it. Ø Viewed from the surface, the proteins are dotted throughout the phospholipid bilayer in a mosaic arrangement. Ø The hydrophilic phosphate heads of the phospholipids face outwards into the aqueous environment inside and outside the cell. Ø The hydrocarbon tails face inwards and create a hydrophobic interior. Ø Hydrophilic molecules will be repelled by the hydrophobic tails of the phospholipid, they can pass the membrane only through the pores formed by proteins that spans the membrane or by protein carriers. Ø The phospholipids are fluid and move about rapidly by diffusion in their own layers. Ø Membranes also contain cholesterol which disturbs the close packing of phospholipids and keeps them more fluid. This can be important for organisms living at low temperatures when membranes can solidify. Cholesterol also increase flexibility and stability of membranes. Without it membranes break up. Ø Glycoproteins and glycolipids are important recognition features of the cells. Fig. 34 The fluid-mosaic model of the plasma membrane. A Functional approach P27, fig2.22a - Function : separate contents of cells from their external environment; controlling exchange of substances between two cells; form separate compartments inside cells in which specialised metabolic pathways can take place, i.e. not interfering each other; as receptor sites for recognizing external stimuli; the glycoproteins on the surface act as cell identity markers, i.e. antigens; site for reaction to take place, e.g. protein on the membrane of chloroplast and mitochondria take part in the energy transfer system.  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 32 [Note] Cytoplasm = all living contents of the cell within the plasma membrane, exclude nucleus and large vacuole. Protoplasm = cytoplasm + nucleus. Protoplast = protoplasm + plasma membrane Exercise : (95 II 4) Using examples, describe the functions of cellular and subcellular membranes in living organisms. Relate these functions to the structure and composition of the membrane, whenever appropriate. [20 marks] 2. Endoplasmic reticulum (ER) : - It is a complex network of double membranes extending throughout the cytoplasm of all eukaryotic cells - It is an extension of the outer nuclear membrane - Types of ER : i) Rough ER : the ER lined with ribosomes; it is used for transporting the proteins synthesized from the ribosomes. ii) Smooth ER : they are lacking ribosomes and concerned with the synthesis and transport of lipids. - Functions of ER : Biosynthesis : the sER may assemble fats, steroids and carbohydrates and the rER produce proteins, especially enzymes Transportation : it is a complex network of passageways that extends throughout the cytoplasmic fluid, therefore, wastes and nutrients are transported intracellularly Support : it forms a sort of cytoskeleton that to maintain the shape of the cell Increase surface area of the cell : network- like ER provides a lot of surface area for the biochemical reactions to occur Storage : in striated muscle cells, there are a highly specialized sER called sarcoplasmic reticulum which stores calcium ions and is involved in the muscle contraction Detoxification : in the liver cells both rough and smooth ER are involved in detoxification of various drugs 3. Golgi apparatus : - It is a secretory organelle - It has a similar structure to sER but is more compact - It consists of flattened, membrane- bound sacs called cisternae, together with a system of associated vesicles called Golgi vesicles. - In plant cells a number of separate stacks called dictyosomes are found while in animal cells a single larger stack is thought to be more usual - At one end of the stack new cisternae are constantly being formed by fusion of vesicles which are probably derived from buds of smooth ER. This ‘outer’ or ‘forming’ face is convex, whilst the other end is the concave ‘inner’ or ‘maturing’ face where the cisternae break up into vesicles once more (forming lysosome or secretory vesicle)  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 33 - Function : Packaging : materials that are manufactured elsewhere in the cell move along the ER into Golgi apparatus where they are packaged and being pushed to the ends of the organelle and pinched off into small bubble- like secretion vesicles Glycosylation : many cell secretions are in the form of glycoproteins and, although considerable glycosylation (adding carbohydrates) takes place in ER, the finishing touches is a Golgi function Concentration : dilute secretion is firstly concentrated in the Golgi apparatus before discharged Transportation : when digested, lipid are absorbed as fatty acids and glycerol in the small intestine, they are resynthesised to lipids in the sER, coated in protein and then transported through the Golgi apparatus to plasma membrane where they leave the cell, mainly to enter the lymphatic system Lysosome formation Membrane differentiation : many membrane is synthesized at the ER, transferred to the Golgi apparatus where modification occur and fully modified is then added to the plasma membrane by fusion of Golgi vesicles during exocytosis Enzyme production e.g. the digestive enzymes of pancreas Making cell wall : secretes carbohydrates, those involved in cell wall formation. Fig. 35 Diagram of synthesis and secretion of a protein (enzyme) *** rd BSI 3 ed. P152 fig 5.29b *** 4. Lysosomes : - Size are similar to mitochondria, being 0.2- 0.5 μm in diameter - Functions : endocytosis : process whereby extracellular materials are brought into cells and then included inside the lysosome for digestion. exocytosis : the release of their enzymes outside the cell in order to break down other cells autolysis : the self- destruction of cell by release of the contents of lysosomes within the cell autophage : special type of controlled autolysis in which constituents are selectively degraded by inclusion within a specialised lysosome  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 34 *** rd BSI 3 Ed. P155 fig 5.32 *** Fig. 36 Three possible uses of a lysosome. ○ 1 endocytosis ; ○ 2 autophage; ○ 3 exocytosis. 5. Vacuoles : - It is a fluid- filled sac bounded by a single membrane (called tonoplast in plant cell) - It contains a solution of mineral salts, sugar, amino acids, wastes and sometimes also pigments, these substance are collectively called ‘cell sap’ - Animal cells contain small vacuoles but plant cells have large central vacuole - Functions: Support and cell growth : water enters the concentrated cell sap by osmosis, so turgor pressure builds up within the cell; osmotic uptake of water is also important in cell expansion during cell growth Store pigments : it sometimes contain pigments that responsible for the colours in flower, fruit, buds and leaves. This is important in attracting insects, birds and other animals for pollination and seed dispersal As lysosome : sometimes it may contain hydrolytic enzymes, after cell death the tonoplast losses its differential permeability and the enzymes escape causing autolysis Temporary stores for wastes and food 6. Mitochondria : - They are spherical or rod shaped scattering throughout the cytoplasm of all eukaryotic cells - Double membrane bounded, the outer of which controls the entry and exit of chemicals and the inner membrane is folded inwards, giving rise to cristae as to increase the surface area on which respiratory processes take place - Stalked elementary particles are spherical bodies located on the inner membrane. They contains enzymes for the phosphorylation of ADP (ATP formation process) - The remainder of the mitochondrion is the matrix, it is a semi- rigid material containing protein (include all the soluble enzymes of the Kreb’s cycle and those involved in the oxidation of fatty acids), lipids and traces of DNA. - Functions: Energy metabolism and respiration : the major function of mitochondrion is produce chemical energy (ATP) from food stuffs via Kreb’s cycle and respiratory chain. Mitochondria are the site of the terminal catabolism of foods ( the preliminary degradation of these compounds occurs in the cytoplasm) Heat production : energy of oxidation is dissipated as heat instead of being converted into ATP, so mitochondrion is the site to produce heat to maintain body temperature. Amine metabolism : some amines are metabolized in mitochondria  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 35 **** Fig. 37 Structure of mitochondria. (a) rd Insert BSI 3 Ed. p276 fig 9.12 Diagram ; (b) 3-D structure; ***** (c) Diagram of crista showing inner membrane particles; (d) Structure of inner membrane particle. Exercise : (99 II 2a) Illustrate the structure of a mitochondrion as seen under the electron microscope with a labelled diagram. [3 marks] 7. Ribosome : - It is a small (20nm in diameter) and non- membranous structure - It consists of two subunits, one large (called 70S) and one small ( called 80S) - It present in large numbers in both prokaryotic and eukaryotic cells - It may occur in groups called polysomes and may be associated with ER to form rER or occur freely within the cytoplasm. - It is made of roughly equal amounts of ribosomal ribonucleic acid (rRNA) and proteins - Functions : it acts as a binding site for protein synthesis 8. Nucleus : - Found in all eukaryotic cells except in mature phloem sieve tube elements and mature red blood cells of mammals. - The shape of the nucleus is sometimes related to that of the cell, but it may be completely irregular. - Almost all cells are mono- nucleate, but bi- nucleate cells (some liver and cartilage cells) and poly- nucleate cells (some white blood cells) also exits. - It is bounded by a double membrane (nuclear envelope). The envelope possesses many large pores which permit the passage of large molecules, such as RNA. - The cytoplasm- like material within the nucleus is called nucleoplasm. It contains chromatin which is made up of coils of DNA bound to proteins. The chromatin are the genetic materials of the cell. In a resting cell (interphase), it appears as a network of tiny granules. During cell division the chromatin granules are re- organized into filaments called chromosomes.  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 36 - Functions : contain genetic materials (chromatin) act as a control centre for the activities of a cell the nuclear DNA carries the instructions for the synthesis of proteins it is involved in the production of ribosomes and RNA it is essential for cell division 9. Nucleolus : - Appears as a rounded, darkly stained structure inside the nucleus. - One or more nucleoli may be present in a cell. - It stains intensely because of the large amounts of DNA and RNA it contains. - During nuclear division nucleoli seem to disappear, but this is because the DNA disperses. They reassemble after nuclear division. - Functions : making ribosomes Exercise : (90 I 2) Describe the relationships between the following pairs of cell organelles : (a) the nucleolus and the ribosomes [2 marks] (b) the endoplasmic reticulum and the Golgi apparatus [4 marks] 10. Cellulose Cell Wall : - It is a characteristic feature of plant cells - Consist of cellulose macrofibrils embedded in a matrix - The matrix is usually composed of polysaccharides, e.g. pectin or lignin. - Functions: provide support in herbaceous plants. provide mechanical strength, the strength may be increased by the presence of lignin in the matrix between the cellulose fibres permit movement of water through the plant, in particular in the cortex of root cell walls develop a coating of waxy cutin, the cuticle, to decrease water loss and risk of infection give shape of the cell sometimes cell walls are modified to act as food reserves, e.g. hemicellulose in some seeds cell walls possess minute pores through which plasmodesmata can pass, for living connections between cells, and allow all the protoplasts to be linked in a system called symplasm 11. Chloroplast : - It is the most common plastid (double membrane bounded organelle) in plant cells - It is bounded by double membrane, the chloroplast envelope. - The stroma is a homogenous matrix which contains enzymes for the carbon dioxide fixation processed (dark reaction) in photosynthesis.  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 37 - Grana are scattered within the stroma. Each granum consists of membrane- bounded disc- shaped vesicles (thylakoids) arranged like a pile of coins. The grana connected with each other by intergranal lamella. Both are the site for light reaction of photosynthesis. - The thylakoids are formed by double layers of thylakoid membrane. Such membranes contain all of the energy- generating system e.g. chlorophyll, electron transport chain and ATP synthetase. - A small amount of DNA is present within the stroma. This suggest that the chloroplasts are also a kind of semi- autonomous organelles. The chloroplasts might be the residue of primitive algae once lived symbiotically in the cells of non- green organisms. - Functions : Photosynthesis Synthesis of fatty acid : in the stroma, carbohydrates are converted to fatty acid in the aid of ATP and NADPH Reduction of nitrite to ammonia. *** rd BSI 3 Ed. P201 fig 7.7 *** Fig. 38 Chloroplast structure. The membrane system has been reduced in extent to make the diagram simpler. Exercise : (92 I 2) Match each of the functions from the following list with a correct letter from the Figure. (a) protein synthesis (b) Krebs cycle (c) electron transport (respiratory chain) (d) protein/ carbohydrate complex formation (e) generation of ATP (f) ribosome production Name each of the structures you have selected. [6 marks] (93 I 2) (a) Name all the cellular organelles which are surrounded by two layers of membrane. (b) One of these organelles is concerned with energy production. Draw a simple labelled diagram to show the structure of this organelle. (c) How is the structure of the organelle in (b) related to its function in cellular metabolism ? [7 marks]  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 38 (97 I 5) The electron micrograph below shows a portion of a cell. (a) Name organelle 1 and state the event occurring in it. [2 marks] (b) What is the functional relationship between organelle 1 and the Golgi apparatus ? [1 mark] (c) Structures 2 and 3 are parts of the Golgi apparatus. What is the relationship between structure 2 and 3 ? How does 3 perform its role in cellular activity ? [2 marks] (d) Calculate the diameter of organelle 4 at A- A. Show your working [2 marks]  Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 39 Summary :- Insert BSI 3rd Ed. p137 **** *** rd BSI 3 ed. P39 *** Buddhist Chi Hong Chi Lam Memorial College A.L. Bio. Notes (by Denise Wong) The Cell...... Page 40  162 BIOLOGY CHAPTER 10 CELL CYCLE AND CELL DIVISION 10.1 Cell Cycle Are you aware that all organisms, even the largest, start their life from a 10.2 M Phase single cell? You may wonder how a single cell then goes on to form such large organisms. Growth and reproduction are characteristics of cells, 10.3 Significance of indeed of all living organisms. All cells reproduce by dividing into two, Mitosis with each parental cell giving rise to two daughter cells each time they 10.4 Meiosis divide. These newly formed daughter cells can themselves grow and divide, giving rise to a new cell population that is formed by the growth and 10.5 Significance of division of a single parental cell and its progeny. In other words, such Meiosis cycles of growth and division allow a single cell to form a structure consisting of millions of cells. 10.1 CELL CYCLE Cell division is a very important process in all living organisms. During the division of a cell, DNA replication and cell growth also take place. All these processes, i.e., cell division, DNA replication, and cell growth, hence, have to take place in a coordinated way to ensure correct division and formation of progeny cells containing intact genomes. The sequence of events by which a cell duplicates its genome, synthesises the other constituents of the cell and eventually divides into two daughter cells is termed cell cycle. Although cell growth (in terms of cytoplasmic increase) is a continuous process, DNA synthesis occurs only during one specific stage in the cell cycle. The replicated chromosomes (DNA) are then distributed to daughter nuclei by a complex series of events during cell division. These events are themselves under genetic control.  CELL CYCLE AND CELL DIVISION 163 10.1.1 Phases of Cell Cycle A typical eukaryotic cell cycle is illustrated by human cells in culture. These cells divide once in approximately every 24 hours (Figure 10.1). G1 However, this duration of cell cycle can vary G 0 S from organism to organism and also from cell type to cell type. Yeast for example, can progress through the cell cycle in only about 90 minutes. sis The cell cycle is divided into two basic kine Cyto ase phases: oph ase e T el h s n ap pha G2 e z Interphase A a as et h M M op z M Phase (Mitosis phase) Pr Ph as The M Phase represents the phase when the e actual cell division or mitosis occurs and the interphase represents the phase between two successive M phases. It is significant to note Figure 10.1 A diagrammatic view of cell cycle indicating formation of two cells that in the 24 hour average duration of cell from one cell cycle of a human cell, cell division proper lasts for only about an hour. The interphase lasts more than 95% of the duration of cell cycle. The M Phase starts with the nuclear division, corresponding to the separation of daughter chromosomes (karyokinesis) and usually ends with division of cytoplasm (cytokinesis). The interphase, though called the resting phase, is the time during which the cell is preparing for division by undergoing both cell growth and DNA replication in an orderly manner. The interphase is divided into three further phases: z G1 phase (Gap 1) z S phase (Synthesis) z G2 phase (Gap 2) How do plants and animals continue to G1 phase corresponds to the interval between mitosis and initiation grow all their lives? of DNA replication. During G1 phase the cell is metabolically active and Do all cells in a plant divide all the time? continuously grows but does not replicate its DNA. S or synthesis phase Do you think all cells marks the period during which DNA synthesis or replication takes place. continue to divide in During this time the amount of DNA per cell doubles. If the initial amount all plants and of DNA is denoted as 2C then it increases to 4C. However, there is no animals? Can you increase in the chromosome number; if the cell had diploid or 2n number tell the name and the of chromosomes at G1, even after S phase the number of chromosomes location of tissues having cells that remains the same, i.e., 2n. divide all their life in In animal cells, during the S phase, DNA replication begins in the higher plants? Do nucleus, and the centriole duplicates in the cytoplasm. During the G2 animals have similar phase, proteins are synthesised in preparation for mitosis while cell growth meristematic continues. tissues? 164 BIOLOGY You have studied Some cells in the adult animals do not appear to exhibit division (e.g., mitosis in onion root heart cells) and many other cells divide only occasionally, as needed to tip cells. It has 14 replace cells that have been lost because of injury or cell death. These chromosomes in cells that do not divide further exit G1 phase to enter an inactive stage each cell. Can you tell how many called quiescent stage (G0) of the cell cycle. Cells in this stage remain chromosomes will metabolically active but no longer proliferate unless called on to do so the cell have at G 1 depending on the requirement of the organism. phase, after S phase, In animals, mitotic cell division is only seen in the diploid somatic and after M phase? cells. Against this, the plants can show mitotic divisions in both haploid Also, what will be the DNA content of the and diploid cells. From your recollection of examples of alternation of cells at G 1 , after S generations in plants (Chapter 3) identify plant species and stages at which and at G 2 , if the mitosis is seen in haploid cells. content after M phase is 2C? 10.2 M PHASE This is the most dramatic period of the cell cycle, involving a major reorganisation of virtually all components of the cell. Since the number of chromosomes in the parent and progeny cells is the same, it is also called as equational division. Though for convenience mitosis has been divided into four stages of nuclear division, it is very essential to understand that cell division is a progressive process and very clear-cut lines cannot be drawn between various stages. Mitosis is divided into the following four stages: z Prophase z Metaphase z Anaphase z Telophase 10.2.1 Prophase Prophase which is the first stage of mitosis follows the S and G2 phases of interphase. In the S and G2 phases the new DNA molecules formed are not distinct but interwined. Prophase is marked by the initiation of condensation of chromosomal material. The chromosomal material becomes untangled during the process of chromatin condensation (Figure 10.2 a). The centriole, which had undergone duplication during S phase of interphase, now begins to move towards opposite poles of the cell. The completion of prophase can thus be marked by the following characteristic events: z Chromosomal material condenses to form compact mitotic chromosomes. Chromosomes are seen to be composed of two chromatids attached together at the centromere. z Initiation of the assembly of mitotic spindle, the microtubules, the proteinaceous components of the cell cytoplasm help in the process.  CELL CYCLE AND CELL DIVISION 165 Cells at the end of prophase, when viewed under the microscope, do not show golgi complexes, endoplasmic reticulum, nucleolus and the nuclear envelope. 10.2.2 Metaphase The complete disintegration of the nuclear envelope marks the start of the second phase of mitosis, hence the chromosomes are spread through the cytoplasm of the cell. By this stage, condensation of chromosomes is completed and they can be observed clearly under the microscope. This then, is the stage at which morphology of chromosomes is most easily studied. At this stage, metaphase chromosome is made up of two sister chromatids, which are held together by the centromere (Figure 10.2 b). Small disc-shaped structures at the surface of the centromeres are called kinetochores. These structures serve as the sites of attachment of spindle fibres (formed by the spindle fibres) to the chromosomes that are moved into position at the centre of the cell. Hence, the metaphase is characterised by all the chromosomes coming to lie at the equator with one chromatid of each chromosome connected by its kinetochore to spindle fibres from one pole and its sister chromatid connected by its kinetochore to spindle fibres from the opposite pole (Figure 10.2 b). The plane of alignment of the chromosomes at metaphase is referred to as the metaphase plate. The key features of metaphase are: z Spindle fibres attach to kinetochores of chromosomes. z Chromosomes are moved to spindle equator and get aligned along metaphase plate through spindle fibres to both poles. 10.2.3 Anaphase At the onset of anaphase, each chromosome arranged at the metaphase plate is split simultaneously and the two daughter chromatids, now referred to as chromosomes of the future daughter nuclei, begin their migration towards the two opposite poles. As each chromosome moves away from the equatorial plate, the centromere of each chromosome is towards the pole and hence at the leading edge, with the arms of the chromosome trailing behind Figure 10.2 a and b : A diagrammatic (Figure 10.2 c). Thus, anaphase stage is characterised by view of stages in mitosis  166 BIOLOGY the following key events: z Centromeres split and chromatids separate. z Chromatids move to opposite poles. 10.2.4 Telophase At the beginning of the final stage of mitosis, i.e., telophase, the chromosomes that have reached their respective poles decondense and lose their individuality. The individual chromosomes can no longer be seen and chromatin material tends to collect in a mass in the two poles (Figure 10.2 d). This is the stage which shows the following key events: z Chromosomes cluster at opposite spindle poles and their identity is lost as discrete elements. z Nuclear envelope assembles around the chromosome clusters. z Nucleolus, golgi complex and ER reform. 10.2.5 Cytokinesis Mitosis accomplishes not only the segregation of duplicated chromosomes into daughter nuclei (karyokinesis), but the cell itself is divided into two daughter cells by a separate process called cytokinesis at the end of which cell division is complete (Figure 10.2 e). In an animal cell, this is achieved by the appearance of a furrow in the plasma membrane. The furrow gradually deepens and ultimately joins in the centre dividing the cell cytoplasm into two. Plant cells however, are enclosed by a relatively inextensible cell wall, thererfore they undergo cytokinesis by a different mechanism. In plant cells, wall formation starts in the centre of the cell and grows outward to meet the existing lateral walls. The formation of the new cell wall begins with the formation of a simple precursor, called the cell-plate that represents the middle lamella between the walls of two adjacent cells. At the time of cytoplasmic division, organelles like mitochondria and plastids get distributed between the two daughter cells. In some organisms karyokinesis is not followed by cytokinesis as a result of which multinucleate condition arises leading to the formation of syncytium (e.g., Figure 10.2 c to e : A diagrammatic liquid endosperm in coconut). view of stages in Mitosis  CELL CYCLE AND CELL DIVISION 167 10.3 Significance of Mitosis Mitosis or the equational division is usually restricted to the diploid cells only. However, in some lower plants and in some social insects haploid cells also divide by mitosis. It is very essential to understand the significance of this division in the life of an organism. Are you aware of some examples where you have studied about haploid and diploid insects? Mitosis results in the production of diploid daughter cells with identical genetic complement usually. The growth of multicellular organisms is due to mitosis. Cell growth results in disturbing the ratio between the nucleus and the cytoplasm. It therefore becomes essential for the cell to divide to restore the nucleo-cytoplasmic ratio. A very significant contribution of mitosis is cell repair. The cells of the upper layer of the epidermis, cells of the lining of the gut, and blood cells are being constantly replaced. Mitotic divisions in the meristematic tissues – the apical and the lateral cambium, result in a continuous growth of plants throughout their life. 10.4 MEIOSIS The production of offspring by sexual reproduction includes the fusion of two gametes, each with a complete haploid set of chromosomes. Gametes are formed from specialised diploid cells. This specialised kind of cell division that reduces the chromosome number by half results in the production of haploid daughter cells. This kind of division is called meiosis. Meiosis ensures the production of haploid phase in the life cycle of sexually reproducing organisms whereas fertilisation restores the diploid phase. We come across meiosis during gametogenesis in plants and animals. This leads to the formation of haploid gametes. The key features of meiosis are as follows: z Meiosis involves two sequential cycles of nuclear and cell division called meiosis I and meiosis II but only a single cycle of DNA replication. z Meiosis I is initiated after the parental chromosomes have replicated to produce identical sister chromatids at the S phase. z Meiosis involves pairing of homologous chromosomes and recombination between them. z Four haploid cells are formed at the end of meiosis II. Meiotic events can be grouped under the following phases: Meiosis I Meiosis II Prophase I Prophase II Metaphase I Metaphase II Anaphase I Anaphase II Telophase I Telophase II 168 BIOLOGY 10.4.1 Meiosis I Prophase I: Prophase of the first meiotic division is typically longer and more complex when compared to prophase of mitosis. It has been further subdivided into the following five phases based on chromosomal behaviour, i.e., Leptotene, Zygotene, Pachytene, Diplotene and Diakinesis. During leptotene stage the chromosomes become gradually visible under the light microscope. The compaction of chromosomes continues throughout leptotene. This is followed by the second stage of prophase I called zygotene. During this stage chromosomes start pairing together and this process of association is called synapsis. Such paired chromosomes are called homologous chromosomes. Electron micrographs of this stage indicate that chromosome synapsis is accompanied by the formation of complex structure called synaptonemal complex. The complex formed by a pair of synapsed homologous chromosomes is called a bivalent or a tetrad. However, these are more clearly visible at the next stage. The first two stages of prophase I are relatively short-lived compared to the next stage that is pachytene. During this stage bivalent chromosomes now clearly appears as tetrads. This stage is characterised by the appearance of recombination nodules, the sites at which crossing over occurs between non-sister chromatids of the homologous chromosomes. Crossing over is the exchange of genetic material between two homologous chromosomes. Crossing over is also an enzyme-mediated process and the enzyme involved is called recombinase. Crossing over leads to recombination of genetic material on the two chromosomes. Recombination between homologous chromosomes is completed by the end of pachytene, leaving the chromosomes linked at the sites of crossing over. The beginning of diplotene is recognised by the dissolution of the synaptonemal complex and the tendency of the recombined homologous chromosomes of the bivalents to separate from each other except at the sites of crossovers. These X-shaped structures, are called chiasmata. In oocytes of some vertebrates, diplotene can last for months or years. The final stage of meiotic prophase I is diakinesis. This is marked by terminalisation of chiasmata. During this phase the chromosomes are fully condensed and the meiotic spindle is assembled to prepare the homologous chromosomes for separation. By the end of diakinesis, the nucleolus disappears and the nuclear envelope also breaks down. Diakinesis represents transition to metaphase. Metaphase I: The bivalent chromosomes align on the equatorial plate (Figure 10.3). The microtubules from the opposite poles of the spindle attach to the pair of homologous chromosomes.  CELL CYCLE AND CELL DIVISION 169 Figure 10.3 Stages of Meiosis I Anaphase I: The homologous chromosomes separate, while sister chromatids remain associated at their centromeres (Figure 10.3). Telophase I: The nuclear membrane and nucleolus reappear, cytokinesis follows and this is called as diad of cells (Figure 10.3). Although in many cases the chromosomes do undergo some dispersion, they do not reach the extremely extended state of the interphase nucleus. The stage between the two meiotic divisions is called interkinesis and is generally short lived. Interkinesis is followed by prophase II, a much simpler prophase than prophase I. 10.4.2 Meiosis II Prophase II: Meiosis II is initiated immediately after cytokinesis, usually before the chromosomes have fully elongated. In contrast to meiosis I, meiosis II resembles a normal mitosis. The nuclear membrane disappears by the end of prophase II (Figure 10.4). The chromosomes again become compact. Metaphase II: At this stage the chromosomes align at the equator and the microtubules from opposite poles of the spindle get attached to the kinetochores (Figure 10.4) of sister chromatids. Anaphase II: It begins with the simultaneous splitting of the centromere of each chromosome (which was holding the sister chromatids together), allowing them to move toward opposite poles of the cell (Figure 10.4). 170 BIOLOGY Figure 10.4 Stages of Meiosis II Telophase II: Meiosis ends with telophase II, in which the two groups of chromosomes once again get enclosed by a nuclear envelope; cytokinesis follows resulting in the formation of tetrad of cells i.e., four haploid daughter cells (Figure 10.4). 10.5 SIGNIFICANCE OF MEIOSIS Meiosis is the mechanism by which conservation of specific chromosome number of each species is achieved across generations in sexually reproducing organisms, even though the process, per se, paradoxically, results in reduction of chromosome number by half. It also increases the genetic variability in the population of organisms from one generation to the next. Variations are very important for the process of evolution. SUMMARY According to the cell theory, cells arise from preexisting cells. The process by which this occurs is called cell division. Any sexually reproducing organism starts its life cycle from a single-celled zygote. Cell division does not stop with the formation of the mature organism but continues throughout its life cycle. CELL CYCLE AND CELL DIVISION 171 The stages through which a cell passes from one division to the next is called the cell cycle. Cell cycle is divided into two phases called (i) Interphase – a period of preparation for cell division, and (ii) Mitosis (M phase) – the actual period of cell division. Interphase is further subdivided into G 1, S and G2. G 1 phase is the period when the cell grows and carries out normal metabolism. Most of the organelle duplication also occurs during this phase. S phase marks the phase of DNA replication and chromosome duplication. G2 phase is the period of cytoplasmic growth. Mitosis is also divided into four stages namely prophase, metaphase, anaphase and telophase. Chromosome condensation occurs during prophase. Simultaneously, the centrioles move to the opposite poles. The nuclear envelope and the nucleolus disappear and the spindle fibres start appearing. Metaphase is marked by the alignment of chromosomes at the equatorial plate. During anaphase the centromeres divide and the chromatids start moving towards the two opposite poles. Once the chromatids reach the two poles, the chromosomal elongation starts, nucleolus and the nuclear membrane reappear. This stage is called the telophase. Nuclear division is then followed by the cytoplasmic division and is called cytokinesis. Mitosis thus, is the equational division in which the chromosome number of the parent is conserved in the daughter cell. In contrast to mitosis, meiosis occurs in the diploid cells, which are destined to form gametes. It is called the reduction division since it reduces the chromosome number by half while making the gametes. In sexual reproduction when the two gametes fuse the chromosome number is restored to the value in the parent. Meiosis is divided into two phases – meiosis I and meiosis II. In the first meiotic division the homologous chromosomes pair to form bivalents, and undergo crossing over. Meiosis I has a long prophase, which is divided further into five phases. These are leptotene, zygotene, pachytene, diplotene and diakinesis. During metaphase I the bivalents arrange on the equatorial plate. This is followed by anaphase I in which homologous chromosomes move to the opposite poles with both their chromatids. Each pole receives half the chromosome number of the parent cell. In telophase I, the nuclear membrane and nucleolus reappear. Meiosis II is similar to mitosis. During anaphase II the sister chromatids separate. Thus at the end of meiosis four haploid cells are formed. EXERCISES 1. What is the average cell cycle span for a mammalian cell? 2. Distinguish cytokinesis from karyokinesis. 3. Describe the events taking place during interphase. 4. What is Go (quiescent phase) of cell cycle? 172 BIOLOGY 5. Why is mitosis called equational division? 6. Name the stage of cell cycle at which one of the following events occur: (i) Chromosomes are moved to spindle equator. (ii) Centromere splits and chromatids separate. (iii) Pairing between homologous chromosomes takes place. (iv) Crossing over between homologous chromosomes takes place. 7. Describe the following: (a) synapsis (b) bivalent (c) chiasmata Draw a diagram to illustrate your answer. 8. How does cytokinesis in plant cells differ from that in animal cells? 9. Find examples where the four daughter cells from meiosis are equal in size and where they are found unequal in size. 10. Distinguish anaphase of mitosis from anaphase I of meiosis. 11. List the main differences between mitosis and meiosis. 12. What is the significance of meiosis? 13. Discuss with your teacher about (i) haploid insects and lower plants where cell-division occurs, and (ii) some haploid cells in higher plants where cell-division does not occur. 14. Can there be mitosis without DNA replication in ‘S’ phase? 15. Can there be DNA replication without cell division? 16. Analyse the events during every stage of cell cycle and notice how the following two parameters change (i) number of chromosomes (N) per cell (ii) amount of DNA content (C) per cell  Glossary Anaphase : The stage of mitosis or meiosis during which centromeres split and chromatids separate and chromatids move to opposite poles. (Back) Bivalent/ Tetrad : A homologous pair of chromosomes in the synapsed, or paired, state during prophase I of the meiotic division and it refer to the fact that the structure contains 4 chromatids. (Back) Cell Cycle : The cell cycle is the series of events that take place in a cell leading to its replication. These events have interphase—during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA—and the mitotic (M) phase, during which the cell splits itself into two distinct cells, often called "daughter cells". (Back) Centromere : It is the primary constriction in chromosome to which the spindle fibres attach during mitotic and meiotic division. It appears as a constriction when chromosomes contract during cell division. After chromosomal duplication, which occurs at the beginning of every mitotic and meiotic division, the two resultant chromatids are joined at the centromere. (Back) Chiasmata : X-shaped observable regions in diplotene in which nonsister chromatids of homologous chromosomes cross-over each other are called chiasmata. (Back) Chromatids : The copied arm of a chromosome, joined together at the centromere, that separate during cell division. (Back) Chromatin : Chromatin is the complex of DNA and protein that makes up chromosomes. It is found inside the nuclei of eukaryotic cells, and within the nucleoid in prokaryotes. The functions of chromatin are to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis, and to serve as a mechanism to control expression. (Back) Chromosomes : Thread like strands of DNA and associated proteins in the nucleus of cells that carry the genes and functions in the transmission of hereditary information. (Back) Crossing over : Crossing over is a process in which homologous chromosomes exchange genetic material through the breakage and reunion of two chromatids with the help of enzyme recombinase. This process can result in an exchange of alleles between chromosomes.(Back) Cytokinesis : The division of the cytoplasm of a cell following division of the nucleus that occurs in mitosis and meiosis, when a parent cell divides to produce two daughter cells. (Back) Diakinesis : This is the final stage of meiotic prophase I in which the chromatids break at the chiasmata and exchange their parts. During this phase the chromosomes are fully condensed and the meiotic spindle is assembled to prepare the homologous chromosomes for sepration. (Back) Diplotene : This is the stage of the first meiotic prophase, following the pachytene, in which the two chromosomes in each bivalent begin to repel each other and a split occurs between the chromosomes, which are then held together by regions where exchanges have taken place (chiasmata) during crossing over. (Back) G0Phase (Quiescent stage) : The G0 phase is a period in the cell cycle where cells do not divide further and exist in a quiescent state. This usually occurs in response to a lack of growth factors or nutrients. Cells in this stage remain metabiologically active but no longer proliferate. This is a very common phase for most mammalian cells. Cells that are permanently in the G0 phase are called postmitotic cells. (Back) G1 Phase : The G1 phase is a period in the cell cycle during interphase, after cytokinesis and before the S phase. During this phase the cell is metabiologically active, resulting in great amount of protein and enzymes synthesis, synthesize new organelles and continuously grows but does not replicate its DNA. (Back) G2 Phase : G2 phase is the final, and usually the shortest phase during interphase within the cell cycle in which the cell undergoes a period of rapid growth to prepare for M phase. During the G2 Phase the nucleus is well defined, bound by a nuclear envelope and contains at least one nucleolus. At the end of this phase is a control checkpoint (G2 checkpoint) to determine if the cell can proceed to enter M phase and divide. The G2 checkpoint prevents cells from entering mitosis with DNA damaged since the last division, providing an opportunity for DNA repair and stopping the proliferation of damaged cells so that the G2 checkpoint helps to maintain genomic stability. (Back) Homologous Chromosomes : Homologous chromosomes are chromosomes in a biological cell that pair (synapse) during meiosis and contain the same genes at the same loci but possibly different genetic information, called alleles, at those genes. (Back) Interphase : The interphase, though called the resting phase, is the time during which the cell is preparing for division by undergoing both cell growth and DNA replication in an orderly manner. The Interphase represents the phase between two successive M Phases. (Back) Karyokinesis : The indirect division of cells in which, prior to division of the cell protoplasm, complicated changes take place in the nucleus, attended with movement of the nuclear fibrils. The nucleus becomes enlarged and convoluted, and finally the threads are separated into two groups, which ultimately become disconnected and constitute the daughter nuclei. (Back) Kinetochore : These are disc shaped structures present on the sides of centromere. (Back) Leptotene : This is the stage of meiosis in which the chromosomes are slender, like threads. (Back) M Phase : The M Phase represents the phase when the actual cell division or mitosis occurs i.e., during which the chromosomes are condensed and the nucleus and cytoplasm divide. (Back) Meiosis : This is a special method of cell division, occurring in maturation of the sex cells, by means of which each daughter nucleus receives half the number of chromosomes characteristic of the somatic cells of the species. (Back) Metaphase : A stage in mitosis or meiosis during which the chromosomes are aligned along the equatorial plane of the cell. Metaphase chromosomes are highly condensed, scientists use these chromosomes for gene mapping and identifying chromosomal aberrations. (Back) Metaphase plate : The plane of the equator (a plane that is equally distant from the two spindle poles) of the spindle into which chromosomes are positioned during metaphase. (Back) Nonsister chromatids : Nonsister chromatids are not identical to each other as they represent different but homologous chromosomes and they will carry the same type of genetic information, but not exactly the same information. (Back) Pachytene : In meiosis, the stage following synapsis (zygotene) in which the homologous chromosome threads (synaptonemal complex) shorten, thicken, and continue to intertwine, and each of the conjoined (bivalent) chromosomes separate into two sister chromatids, which are held together by a centromere, to form a tetrad. During this phase the chromatids break up and corresponding regions of the nonsister chromatids of the paired chromosomes are exchanged in a process known as crossing over. (Back) Prophase : Prophase is the first stage of mitosis in which chromosomal material becomes untangled during the process of chromatin condensation and the centriole, begins to move towards opposite poles of cell. (Back) Sister chromatids : During S phase of the cell cycle the DNA is replicated and an identical copy of the chromatid is made. These identical copy of chromatids are called sister chromatids. (Back) S-Phase or Synthesis Phase : The S phase, short for synthesis phase, is a period in the cell cycle during interphase, between G1 phase and the G2 phase. In this phase DNA synthesis or replication occurs. (Back) Spindle fibres : It is a group of microtubules that extend from the centromere of chromosomes to the poles of the spindle or from pole to pole in a dividing cell. (Back) Synapsis : The pairing of homologous chromosomes along their length; synapsis usually occurs during prophase I of meiosis, but it can also occur in somatic cells of some organisms. (Back) Synaptonemal complex : A ribbon like protein structure formed between synapsed homologues at the end of the first meiotic prophase, binding the chromatids along their length and facilitating chromatid exchange. (Back) Telophase : The last stage in each mitotic or meiotic division, in which the chromosomes are assembled at the opposite spindle poles, nuclear envelope assembles around the chromosomes and nucleolus golgi complex and endoplasmic reticulum reform. (Back) Zygotene : This is the synaptic stage of the first meiotic prophase in which the two leptotene chromosomes undergo pairing by the formation of synaptonemal complexes to form a bivalent structure. (Back) 42636_06_p1-33 12/12/02 7:03 AM Page 1 C H A P T E R 6 The Structures of DNA and RNA T he discovery that DNA is the prime genetic molecule, carrying all O U T L I N E the hereditary information within chromosomes, immediately focused attention on its structure. It was hoped that knowledge DNA Structure (p. 2) of the structure would reveal how DNA carries the genetic messages that are replicated when chromosomes divide to produce two identical copies of themselves. During the late 1940s and early 1950s, several DNA Topology (p. 17) research groups in the United States and in Europe engaged in serious efforts — both cooperative and rival — to understand how the atoms RNA Structure (p. 25) of DNA are linked together by covalent bonds and how the resulting molecules are arranged in three-dimensional space. Not surprisingly, there initially were fears that DNA might have very complicated and perhaps bizarre structures that differed radically from one gene to another. Great relief, if not general elation, was thus expressed when the fundamental DNA structure was found to be the double helix. It told us that all genes have roughly the same three-dimensional form and that the differences between two genes reside in the order and number of their four nucleotide building blocks along the complementary strands. Now, some 50 years after the discovery of the double helix, this simple description of the genetic material remains true and has not had to be ap- preciably altered to accommodate new findings. Nevertheless, we have come to realize that the structure of DNA is not quite as uniform as was first thought. For example, the chromosome of some small viruses have single-stranded, not double-stranded, molecules. Moreover, the precise orientation of the base pairs varies slightly from base pair to base pair in a manner that is influenced by the local DNA sequence. Some DNA se- quences even permit the double helix to twist in the left-handed sense, as opposed to the right-handed sense originally formulated for DNA’s general structure. And while some DNA molecules are linear, others are circular. Still additional complexity comes from the supercoiling (further twisting) of the double helix, often around cores of DNA-binding proteins. Likewise, we now realize that RNA, which at first glance appears to be very similar to DNA, has its own distinctive structural features. It is principally found as a single-stranded molecule. Yet by means of intra-strand base pairing, RNA exhibits extensive double-helical character and is capable of folding into a wealth of diverse tertiary structures. These structures are full of surprises, such as non-classical base pairs, base-backbone interactions, and knot-like configurations. Most remarkable of all, and of profound evolutionary significance, some RNA molecules are enzymes that carry out reactions that are at the core of information transfer from nucleic acid to protein. Clearly, the structures of DNA and RNA are richer and more intricate than was at first appreciated. Indeed, there is no one generic structure for DNA and RNA. As we shall see in this chapter, there are in fact vari- ations on common themes of structure that arise from the unique physi- cal, chemical, and topological properties of the polynucleotide chain. 1 42636_06_p1-33 12/12/02 7:03 AM Page 2 2 The Structures of DNA and RNA DNA STRUCTURE DNA Is Composed of Polynucleotide Chains The most important feature of DNA is that it is usually composed of two polynucleotide chains twisted around each other in the form of a double helix (Figure 6-1). The upper part of the figure (a) presents the structure of the double helix shown in a schematic form. Note that if inverted 180° (for example, by turning this book upside-down), the double helix looks superficially the same, due to the complementary nature of the two DNA strands. The space-filling model of the double helix, in the lower part of the figure (b), shows the components of the DNA molecule and their relative positions in the helical structure. The backbone of each strand of the helix is composed of alternating sugar and phosphate residues; the bases project inward but are acces- sible through the major and minor grooves. a 3' 5' FIGURE 6-1 The Helical Structure of hydrogen bond DNA. (a) Schematic model of the double 1 helical turn = 34 Å = ~10.5 base pairs base helix. One turn of the helix (34 Å or 3.4 nm) spans approx. 10.5 base pairs. (b) Space-filling sugar-phosphate model of the double helix. The sugar and backbone phosphate residues in each strand form the backbone, which are traced by the yellow, gray, and red circles, show the helical twist of the overall molecule. The bases project inward but are accessible through major and minor grooves. A G C 3' 5' T 20 Å (2 nm) b 12 Å minor (1.2 nm) groove 22 Å major (2.2 nm) groove H O P C in phosphate ester chain C and N in bases 42636_06_p1-33 12/12/02 7:03 AM Page 3 DNA Structure 3 Let us begin by considering the nature of the nucleotide, the funda- mental building block of DNA. The nucleotide consists of a phosphate joined to a sugar, known as 2-deoxyribose, to which a base is attached. The phosphate and the sugar have the structures shown in Figure 6-2. The sugar is called 2-deoxyribose because there is no hydroxyl at position 2 (just two hydrogens). Note that the positions on the ribose are designated with primes to distinguish them from positions on the bases (see the discussion below). We can think of how the base is joined to 2-deoxyribose by imagin- ing the removal of a molecule of water between the hydroxyl on the 1 carbon of the sugar and the base to form a glycosidic bond (Figure 6-2). The sugar and base alone are called a nucleoside. Likewise, we can imagine linking the phosphate to 2-deoxyribose by removing a water molecule from between the phosphate and the hydroxyl on the 5 carbon to make a 5 phosphomonoester. Adding a phosphate (or more than one phosphate) to a nucleoside creates a nucleotide. Thus, by making a glycosidic bond between the base and the sugar, and by making a phosphoester bond between the sugar and the phosphoric acid, we have created a nucleotide (Table 6-1). Nucleotides are, in turn, joined to each other in polynucleotide chains through the 3 hydroxyl of 2-deoxyribose of one nucleotide and the phosphate attached to the 5 hydroxyl of another nucleotide (Figure 6-3). This is a phosphodiester linkage in which the phosphoryl group between the two nucleotides has one sugar esterified to it through a 3 hydroxyl and a second sugar esterified to it through a 5 hydroxyl. Phosphodiester linkages create the repeating, sugar-phosphate back- bone of the polynucleotide chain, which is a regular feature of DNA. In contrast, the order of the bases along the polynucleotide chain is irregu- lar. This irregularity as well as the long length is, as we shall see, the basis for the enormous information content of DNA. The phosphodiester linkages impart an inherent polarity to the DNA chain. This polarity is defined by the asymmetry of the nucleotides and the way they are joined. DNA chains have a free 5 phosphate or 5 hydroxyl at one end and a free 3 phosphate or 3 hydroxyl at the other end. The convention is to write DNA sequences from the 5 end (on the left) to the 3 end, generally with a 5 phosphate and a 3 hydroxyl. H H FIGURE 6-2 Formation of Nucleotide N by Removal of Water. The numbers of the N carbon atoms in 2’ deoxyribose are labeled in N O 2' deoxyribose A red. N N -O 5' O P OH HOCH2 4' 1' OH H base H H 3' HH 2' O- HO H phosphoric acid H2O H H N N N O A -O O N P OCH2 N H H HH O- HO H nucleotide (dAMP) 42636_06_p1-33 12/12/02 7:03 AM Page 4 4 The Structures of DNA and RNA TA B L E 6-1 Adenine and Related Compounds Nucleotide Deoxynucleotide Nucleoside Adenosine Deoxyadenosine Base Adenine Adenosine 5' -phosphate 5' phosphate Structurea NH2 NH2 O O N – – N N O P OCH2 O P OCH2 N O Adenine O Adenine OH OH N N H H H H N N H H H H HOCH2 O OH OH OH H H H H H OH OH M.W. 135.1 267.2 347.2 331.2 a At physiological pH, all of the hydroxyls bound to phosphate are ionized. Each Base Has Its Preferred Tautomeric Form The bases in DNA fall into two classes, purines and pyrimidines. The purines are adenine and guanine, and the pyrimidines are cytosine and thymine. The purines are derived from the double-ringed structure shown in Figure 6-4. Adenine and guanine share this essential structure but with different groups attached. Likewise, cytosine and thymine are 5' FIGURE 6-3 Detailed Structure of Polynucleotide Polymer. The structure O O- P shows base pairing between purines (in blue) 3' O O CH3 O N N and pyrimidines (in yellow), and the CH2 phosphodiester linkages of the backbone. T N N O HO N A N N O O O- O P CH2 O O O N O O P N CH2 -O C O O N G N N N N O O N O O- CH2 P CH 3 O O O O O N CH2 P T O -O N O A N N N O O O- O P CH2 N O O O O O CH2 P O -O C N N G O O N OH O 3' CH2 O O P -O O 5' 42636_06_p1-33 12/12/02 7:03 AM Page 5 DNA Structure 5 NH2 FIGURE 6-4 Purines and Pyrimidines. The dotted lines indicate the sites of attachment N N of the bases to the sugars. adenine N N N 7 6 N 5 1 purine 8 9 4 2 3 O N H N N NH guanine N NH2 N NH2 N cytosine N O 4 N 5 3 pyrimidine 6 2 1 O N H3C N thymine N O variations on the single-ringed structure shown in Figure 6-4. The figure also shows the numbering of the positions in the purine and pyrimi- dine rings. The bases are attached to the deoxyribose by glycosidic link- ages at N1 of the pyrimidines or at N9 of the purines. Each of the bases exists in two alternative tautomeric states, which are in equilibrium with each other. The equilibrium lies far to the side of the conventional structures shown in Figure 6-4, which are the pre- dominant states and the ones important for base pairing. The nitrogen atoms attached to the purine and pyrimidine rings are in the amino form in the predominant state and only rarely assume the imino configuration. Likewise, the oxygen atoms attached to the guanine and thymine normally have the keto form and only rarely take on the enol configuration. As examples, Figure 6-5 shows tautomerization of cytosine into the imino form (a) and guanine into the enol form (b). As we shall see, the capacity to form an alternative tautomer is a fre- quent source of errors during DNA synthesis. The Two Strands of the Double Helix Are Held Together by Base Pairing in an Anti-Parallel Orientation The double helix is composed of two polynucleotide chains that are held together by weak, non-covalent bonds between pairs of bases, as shown in Figure 6-3. Adenine on one chain is always paired with thymine on the other chain and, likewise, guanine is always paired with cytosine. The two strands have the same helical geometry but base pairing holds them together with the opposite polarity. That is, the base at the 5 end of one strand is paired with the base at the 3 end of the other strand. The strands are said to have an anti-parallel 42636_06_p1-33 12/12/02 7:03 AM Page 6 6 The Structures of DNA and RNA amino imino FIGURE 6-5 Base Tautomers. Amino K imino and keto K enol tautomerism. H H N N (a) Cytosine is usually in the amino form H but rarely forms the imino configuration. N N (b) Guanine is usually in the keto form but C C is rarely found in the enol configuration. O N O N keto enol H O O H N N N N G G H H N N N N N N H R H R H-bond donor H-bond acceptor orientation. This anti-parallel orientation is a stereochemical conse- quence of the way that adenine and thymine and guanine and cyto- sine pair with each together (see Figure 6-6). The Two Chains of the Double Helix Have Complementary Sequences The pairing between adenine and thymine and between guanine and cytosine results in a complementary relationship between the sequence of bases on the two intertwined chains and gives DNA its self-encoding