BFE Complete Notes - Updated.pdf

Lecture 1 Introduction to the course Purpose of Biology for Engineers “As engineers, we are always looking into how to modify or improve an existing system. We must hold the key to combining the fields of engineering and biology, and t...

Lecture 1 Introduction to the course Purpose of Biology for Engineers “As engineers, we are always looking into how to modify or improve an existing system. We must hold the key to combining the fields of engineering and biology, and this course is designed to provide a logical understanding about biology from an engineering perspective. So our aim is to understand the logical principles of biology, inspire the ideas from it to have a useful creativity in the engineering field” 2 2  Lecture 2 Bioinspiration: Examples of bioinspiration models used in engineering. In this lecture we are trying to discuss responses for few fundamental questions: 1. Why engineers must look at living things? Or What is the logic of asking engineers to look at biological systems 2. How biology can solve complex engineering problems? 3. Does biology helps engineers for better creativity and problem solving?? Livings things improve existing form and designs. It changes according to the environmental changes. This happens through evolution. Living things sense as well as produce and respond to signals. Living things can repair by itself. It stores information. They can also transfer information. At the molecular level, livings creatures on earth are similar, but a small change in their organization has resulted in its diversity. Scientists consider nature as the best engineer, because its designs are the perfect ones - survival of the fittest. (Discuss the examples shown in the video by Janine Benyus) 3 3  LECTURE 3 Building blocks of life: Elements of life and their bonding ability, importance of carbon, elemental replacement Chemistry of life: The atomic composition of the cell: H = 63%, O = 24% C = 10%, N = 1.4 %, P = 0.2 % & S = < 0.1% Trace amount: Ca, Cl, K, Na, Mg, Mn, Fe, Se, I etc Let us understand what an element is and where these elements related to life are present in the periodic table by simple pictures. 4 4 When we look at the composition of elements, C, N, O & H constitutes more than 95%. They are also lightest elements in the periodic table (As we go down in the periodic table, atomic number is going to increase so as atomic mass) Suppose we want to design a moving machine like car what are the critical things we look for before selecting material to construct a body of the car? One of the criteria should be Materials used should not be heavy (fuel efficiency is going to decrease). The backbone element of life is carbon and carbon is the appropriate element to become a backbone element of life, since no element is present above carbon in periodic table which can have similar properties and lighter than carbon. Important properties of these elements are their ability to form bond with other elements to form compounds/molecules. Let us see the molecular composition of the life. 80% is water & the dry weight of remaining 20% contains 50% protein, 15% carbohydrates, 10% lipids & fats & 15% nucleic acids. Formation of these molecules and the interaction between these molecules depends on the chemical properties of the important six elements we mentioned it before. In this course, we 5 5 just learn Chemistry to logically understand the structure and functions of Biomolecules and their interaction. Two important properties we are concentrating in this course to understand the elements used in life are valency and electronegativity. The Valency of an atom is unpaired electron in the outer orbital of the shell. This gives an opportunity for the element to combine with other element. Valency of H = 1, C=4, 0 = 2, N = 3 or 4, P = 3 or 5 and S = 2 (As shown in the figure), 4, 6 Covalent Bond: sharing of a pair of valence electrons by two similar or dissimilar elements. C−C single bond. Energy required to break them is equal to 80 Kcal/mol C=C double bond (more energy is required to break them compared to single bond between them) 6 6 C≡C triple bond (more energy is required to break them compared to double bond) Covalent bonds are very strong. Suppose if we compare covalent bond strength to say random energy fluctuation in daily life- random thermal fluctuations at room temperature are on the order of 0.6 kilocalories per mole. Covalent bonds are extremely stable, usually, unless something is attacking them and breaking them. 7 7  LECTURE 4 Different types of bonds and interactions in biological systems Atoms in a molecule attract shared electrons in varying degrees, depending on the element. The attraction of a particular element for the electrons of a covalent bond is called its electronegativity. The more electronegative this element is, the more strongly it pulls shared electrons towards itself. If an element bonded to more electronegative element, the electrons of the bond are not shared equally therefore, There exists a polarity between them. This type of bond is called polar covalent bond. Such bonds vary in their polarity depending on the relative electronegativity of the two elements. For example the bond between the hydrogen atom and oxygen atom in water molecule is quite polar. 8 8 How do we know that polarity exist between two atoms of elements? What is the measuring way? Please refer the figure above. Suppose if the difference in electronegativity between the two atoms is 0.5 and more, there exist polarity. Example: carbon and hydrogen C-H the difference in electronegativity is 0.4 so it is non- polar. Carbon and oxygen C-O The difference is 1.0 therefore polarity exist. Ionic Bond: transfer of electrons from one atom to another atom to form bond. The atom should form ions i.e. it should be in ionic state (positively charged or negatively charged) before it forms bond with another oppositely charged ion. The bonds are strong as long as it is not disturbed. If it is disturbed it becomes fragile. Ex: Once the water is added to NaCl, the ionic bond breaks. LECTURE 5 Water and phospholipids as well as their importance in the survival of life Water and Phospholipid 9 9  Life on earth began in water and evolved there for three billion years before spreading onto land. Although most of the water in liquid form, it is also in solid form and gaseous form. Water is the only solvent, it is present in all the three phases and interchange of the phases will enormously affect the life on the earth. We look into some of the important properties of water that make earth suitable for life. Polar covalent bonds in water molecules results in hydrogen bonding The hydrogen bonds form, break and re-form with great frequency. Each lasts only a few trillionths of a second, but the molecules are constantly forming new hydrogen bonds with a succession of partners. Therefore at any instant, all water molecules are hydrogen bonded to their neighbours. Cohesive and adhesive properties of water Water molecules stay close to each other as a result of hydrogen bonding. Although the arrangement of molecules in a sample of liquid water is constantly changing, at any given moment many of the molecules are linked by multiple hydrogen bonds. These linkages make water more structured than most other liquids. Collectively, the hydrogen bonds hold the substance together, a phenomenon called cohesion. 10 10 Adhesion, the clinging of one substance to another, also plays a role. Adhesion of water to cell walls by hydrogen bonds helps counter the downward pull of gravity Moderation of Temperature by Water Water moderates air temperature by absorbing heat from air that is warmer and releasing the stored heat to air that is cooler. Water is effective as a heat bank because it can absorb or release a relatively large amount of heat with only a slight change in its own temperature.How it can do that? The ability of water to stabilize temperature stems from its relatively high specific heat. The specific heat of water is 1 calorie per gram and per degree Celsius, abbreviated as 1 cal/g_°C. Compared with most other substances, water has an unusually high specific heat. Because of the high specific heat of water relative to other materials, water will change its 11 11 temperature less when it absorbs or loses a given amount of heat. We can trace water’s high specific heat, like many of its other properties, to hydrogenbonding. Heat must be absorbed in order to break hydrogen bonds; by the same token, heat is released when hydrogen bonds form. A calorie of heat causes a relatively small change in the temperature of water because much of the heat is used to disrupt hydrogen bonds before the water molecules can begin moving faster. And when the temperature of water drops slightly, many additional hydrogen bonds form, releasing a considerable amount of energy in the form of heat. What is the relevance of water’s high specific heat to life on Earth? A large body of water can absorb and store a huge amount of heat from the sun in the daytime and during summer while warming up only a few degrees. At night and during winter, the gradually cooling water can warm the air. This is the reason coastal areas generally have milder climates than inland regions. The high specific heat of water also tends to stabilize ocean temperatures, creating a favorable environment for marine life. Thus, because of its high specific heat, the water that covers most of Earth keeps temperature fluctuations on land and in water within limits that permit life. Floating of Ice on Liquid Water Water is one of the few substances that are less dense as a solid than as a liquid. In other words, ice floats on liquid water. While other materials contract and become denser when they solidify, water expands. How it helps for life? Water: The Solvent of Life 12 12  Water is a very good solvent. Many reactions take place in an organism. For almost all the reactions, water acts as a solvent. Possible Evolution of Life on Other Planets with Water Biologists who look for life elsewhere in the universe have concentrated their search on planets that might have water. To date, more than 200 planets have been found outside our solar system, and there is evidence for the presence of water vapor on one or two of them. In our own solar system, Mars has been most compelling to biologists as a focus of study. Like Earth, Mars has an ice cap at both poles. Lipids and phospholipids: Lipids are hydrophobic molecules. The hydrophobic behavior of lipids is based on their molecular structure. Although they may have some polar bonds associated with oxygen, lipids consist mostly of hydrocarbon regions. Lipids are varied in form and function. They include waxes and certain pigments, but we will focus on the most biologically important types of lipids: fats & phospholipids. 13 13  A fatty acid has a long carbon skeleton, usually 16 or 18 carbon atoms in length. The carbon at one end of the skeleton is part of a carboxyl group, the functional group that gives these molecules the name fatty acid. The rest of the skeleton consists of a hydrocarbon chain. The relatively nonpolar C¬H bonds in the hydrocarbon chains of fatty acids are the reason fats are hydrophobic. Fats separate from water because the water molecules hydrogen bond to one another and exclude the fats. In making a fat, three fatty acid molecules are each joined to glycerol by an ester linkage, a bond between a hydroxyl group and a carboxyl group. The resulting fat, also called a triacylglycerol, thus consists of three fatty acids linked to one glycerol molecule. The terms saturated fats and unsaturated fats are commonly used in the context of nutrition. These terms refer to the structure of the hydrocarbon chains of the fatty acids. If there are no double bonds between carbon atoms composing a chain, then as many hydrogen atoms as possible are bonded to the carbon skeleton. Such a structure is said to be saturated with hydrogen, and the resulting fatty acid therefore called a saturated fatty acid. An unsaturated fatty acid has one or more double bonds, with one fewer hydrogen atom on each double-bonded carbon. Nearly all double bonds in naturally occurring fatty acids are cis double bonds, which cause a kink in the hydrocarbon chain wherever they occur 14 14  Phospholipids are essential for cells because they make up cell membranes. Phospholipid is similar to a fat molecule but has only two fatty acids attached to glycerol rather than three. The third hydroxyl group of glycerol is joined to a phosphate group, which has a negative electrical charge in the cell. Additional small molecules, which are usually charged or polar, can be linked to the phosphate group to form a variety of phospholipids. The two ends of phospholipids show different behaviour toward water. The hydrocarbon tails are hydrophobic and are excluded from water. However, the phosphate group and its attachments form a hydrophilic head that has an affinity for water. These strange behaviour molecules are called amphipathic molecules. When phospholipids are added to water, they self-assemble into double- layered structures called “bilayers,” shielding their hydrophobic portions from water. At the surface of a cell, phospholipids are arranged in a similar bilayer. The hydrophilic heads of the 15 15 molecules are on the outside of the bilayer, in contact with the aqueous solutions inside and outside of the cell. The hydrophobic tails point toward the interior of the bilayer, away from the water. The phospholipid bilayer forms a boundary between the cell and its external environment; in fact, cells could not exist without phospholipids 16 16  Lecture 6 Carbohydrates Carbohydrates include both sugars and polymers of sugars. The simplest carbohydrates are the monosaccharides, or simple sugars; these are the monomers from which more complex carbohydrates are constructed. Disaccharides are double sugars, consisting of two monosaccharides joined by a covalent bond. Carbohydrates also include macromolecules called polysaccharides, polymers composed of many sugar building blocks. Monosaccharides (from the Greek monos, single, and sacchar, sugar) generally have molecular formulas that are some multiple of the unit CH 2O. Glucose (C6H12O6)(multiple of six), the most common monosaccharide, is of central importance in the chemistry of life. In the structure of glucose, we can see the trademarks of a sugar: The molecule has a carbonyl group (C_O) and multiple hydroxyl groups (— OH). Depending on the location of the carbonyl group, a sugar is either an aldose (aldehyde sugar) or a ketose (ketone sugar). Glucose, for example, is an aldose; fructose, an isomer of glucose, is a ketose. (Most names for sugars end in -ose.) Another criterion for classifying sugars is the size of the carbon skeleton, which ranges from three to seven carbons long. Glucose, fructose, and other sugars that have six carbons are called hexoses. Trioses (three-carbon sugars) and pentoses (five-carbon sugars) are also common. 17 17  A disaccharide consists of two monosaccharides joined by a glycosidic linkage, a covalent bond formed between two monosaccharides by a dehydration reaction. For example, maltose is a disaccharide formed by the linking of two molecules of glucose. Also known as malt sugar, maltose is an ingredient used in brewing beer. The most prevalent disaccharide is sucrose, which is table sugar. Its two monomers are glucose and fructose. Plants generally transport carbohydrates from leaves to roots and other nonphotosynthetic organs in the form of sucrose. Lactose, the sugar present in milk, is another disaccharide, in this case a glucose molecule joined to a galactose molecule. 18 18  Polysaccharides are macromolecules, polymers with a few hundred to a few thousand monosaccharides joined by glycosidic linkages. Some polysaccharides serve as storage material, hydrolyzed as needed to provide sugar for cells. Other polysaccharides serve as building material for structures that protect the cell or the whole organism. The architecture and function of a polysaccharide are determined by its sugar monomers and by the positions of its glycosidic linkages. Both plants and animals store sugars for later use in the form of storage polysaccharides. Plants store starch, a polymer of glucose monomers, as granules within cellular structures known as plastids, which include chloroplasts. Synthesizing starch enables the plant to stockpile surplus glucose. Because glucose is a major cellular fuel, starch represents stored energy. The sugar can later be withdrawn from this carbohydrate “bank” by hydrolysis, which breaks the bonds between the glucose monomers. Most animals, including humans, also have enzymes that can 19 19 hydrolyze plant starch, making glucose available as a nutrient for cells. Potato tubers and grains —the fruits of wheat, maize (corn), rice, and other grasses—are the major sources of starch in the human diet. Most of the glucose monomers in starch are joined by 1–4 linkages (number 1 carbon to number 4 carbon), like the glucose units in maltose. The simplest form of starch, amylose, is unbranched. Amylopectin, a more complex starch, is a branched polymer with 1–6 linkages at the branch points. Animals store a polysaccharide called glycogen, a polymer of glucose that is like amylopectin but more extensively branched. Humans and other vertebrates store glycogen mainly in liver and muscle cells. Hydrolysis of glycogen in these cells releases glucose when the demand for sugar increases. Structural Polysaccharides Organisms build strong materials from structural polysaccharides. For example, the polysaccharide called cellulose is a major component of the tough walls that enclose plant cells. On a global scale, plants produce almost 1014 kg (100 billion tons) of cellulose per year; it is the most abundant organic compound on Earth. Like starch, cellulose is a polymer of glucose, but the glycosidic linkages in these two polymers differ. The difference is based on the fact that there are actually two slightly different ring structures for glucose. When glucose forms a ring, the hydroxyl group attached to the number 1 carbon is positioned either below or above the plane of 20 20 the ring. These two ring forms for glucose are called alpha (α) and beta (β), respectively. In starch, all the glucose monomers are in the α configuration. The glucose monomers of cellulose are all in the β configuration, making every glucose monomer “upside down” with respect to its neighbors. The differing glycosidic linkages in starch and cellulose give the two molecules distinct three-dimensional shapes. Whereas certain starch molecules are largely helical, a cellulose molecule is straight. Cellulose is never branched, and some hydroxyl groups on its glucose monomers are free to hydrogen-bond with the hydroxyls of other cellulose molecules lying parallel to it. In plant cell walls, parallel cellulose molecules held together in this way are grouped into units called microfibrils. These cable-like microfibrils are a strong building material for plants and an important substance for humans because cellulose is the major constituent of paper and the only component of cotton. Enzymes that digest starch by hydrolyzing its α linkages are unable to hydrolyze the β linkages of cellulose because of the distinctly different shapes of these two molecules. In fact, few organisms possess enzymes that can digest cellulose. Animals, including humans, do not; the cellulose in our food passes through the digestive tract and is eliminated with the feces. Along the way, the cellulose abrades the wall of the digestive tract and stimulates the lining to secrete mucus, which aids in the smooth passage of food through the tract. Thus, although cellulose is not a nutrient for humans, it is an important part of a healthful diet. Most fresh fruits, vegetables, and whole grains are rich in cellulose. On food packages, “insoluble fiber” refers mainly to cellulose. 21 21  Some microorganisms can digest cellulose, breaking it down into glucose monomers. A cow harbors cellulose digesting prokaryotes and protists in its stomach. These microbes hydrolyze the cellulose of hay and grass and convert the glucose to other compounds that nourish the cow. Similarly, a termite, which is unable to digest cellulose by itself, has prokaryotes or protists living in its gut that can make a meal of wood. Lecture 7 Proteins and their structures Proteins are the most abundant biological macromolecules, occurring in all parts of the cell. Proteins also occur in great variety; thousands of different kinds, ranging in size from relatively small peptides to huge polymers with molecular weights in millions. Nearly every dynamic function of a living being depends on proteins. In fact, the importance of proteins is underscored by their name, which comes from the Greek word proteios, meaning “first,” or “primary.” Proteins account for more than 50% of the dry mass of most cells, and they are instrumental in almost everything the organism does. Some proteins speed up chemical reactions, while others play a role in defense, storage, transport, cellular communication, movement, or structural support. The basic building blocks of proteins are amino acids. Twenty different amino acids are commonly found in proteins. All 20 of the common amino acids are α-amino acids. They have a carboxyl group and an amino group 22 22 bonded to the same carbon atom (α carbon). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge, and which influence the solubility of the amino acids in water. The common amino acids of proteins have been assigned three-letter abbreviations and one-letter symbols, which are used as shorthand to indicate the composition and sequence of amino acids polymerized in proteins. The primary structure of a protein is simply the linear arrangement, or sequence, of the amino acid residues that compose it. Many terms are used to denote the chains formed by the polymerization of amino acids. A short chain of amino acids linked by peptide bonds and having a defined sequence is called a peptide; longer chains are referred to as polypeptides. Peptides generally contain less than 20–30 amino acid residues, whereas polypeptides contain as many as 4000 residues. We generally reserve the term protein for a polypeptide (or for a complex of polypeptides) that has a well-defined three-dimensional structure. 23 23 Secondary structure, are the result of hydrogen bonds between the repeating constituents of the polypeptide backbone (not the amino acid side chains). Within the backbone, the oxygen atoms have a partial negative charge, and the hydrogen atoms attached to the nitrogen have a partial positive charge; therefore, hydrogen bonds can form between these atoms. Individually, these hydrogen bonds are weak, but because they are repeated many times over a relatively long region of the polypeptide chain, they can support a particular shape for that part of the protein. One such secondary structure is the α- helix, a delicate coil held together by hydrogen bonding between every fourth amino acid. The other main type of secondary structure is the β- pleated sheet. In this structure two or more strands of the polypeptide chain lying side by side (called β strands) are connected by hydrogen bonds between parts of the two parallel polypeptide backbones. 24 24 Turns Composed of three or four residues, turns are located on the surface of a protein, forming sharp bends that redirect the polypeptide backbone back toward the interior. These short, U-shaped secondary structures are stabilized by a hydrogen bond between their end residues. Glycine and proline are commonly present in turns. The lack of a large side chain in glycine and the presence of a built-in bend in proline allow the polypeptide backbone to fold into a tight U shape. Turns allow large proteins to fold into highly compact structures. A polypeptide backbone also may contain longer bends, or loops. In contrast with turns, which exhibit just a few well-defined structures, loops can be formed in many different ways. Tertiary structure is the overall shape of a polypeptide resulting from interactions between the side chains (R groups) of various amino acids. One type of interaction that contributes to tertiary structure is—somewhat misleadingly—called a hydrophobic interaction. As a polypeptide folds into its functional shape, amino acids with hydrophobic (nonpolar) side chains usually end up in clusters at the core of the protein, out of contact with water. Thus, a “hydrophobic interaction” is actually caused by the exclusion of nonpolar substances by water molecules. Once nonpolar amino acid side chains are close together, van der Waals interactions help hold them together. Meanwhile, hydrogen bonds between polar side chains and ionic bonds between positively and negatively charged side chains also help stabilize tertiary structure. These are all weak interactions in the aqueous cellular environment, but their cumulative effect helps give the protein a unique shape. Covalent bonds called disulfide bridges may further reinforce the shape of a protein. Disulfide bridges form where two cysteine monomers, which have sulfhydryl groups (¬SH) on their side chains, are brought close together by the folding of the protein. The sulfur of one cysteine bonds to the sulfur of the second, and the disulfide bridge (¬S¬S¬) rivets parts of the protein together. All of these different kinds of interactions can contribute to the tertiary structure of a protein. 25 25  Tertiary Structure of a Protein. Quaternary structure is the overall protein structure that results from the aggregation of these polypeptide subunits. Example: collagen, which is a fibrous protein that has three identical helical polypeptides intertwined into a larger triple helix, giving the long fibers great strength. This suits collagen fibers to their function as the girders of connective tissue in skin, bone, tendons, ligaments, and other body parts. Collagen accounts for 40% of the protein in a human body. Hemoglobin, the oxygen-binding protein of red blood cells is another example of a globular protein with quaternary structure. It consists of four polypeptide subunits, two of one kind (α) and two of another kind (β). Both α and β subunits consist primarily of α-helical secondary structure. Each subunit has a nonpolypeptide component, called heme, with an iron atom that binds oxygen. 26 26 27 27 Lecture 8 Enzymes 28 28 29 29 30 30 CO2 Infer the flow of information in living systems through simple genetic experiments Flow of information in living systems: Mendelian model, Monohybrid 9 and Law of Segregation, Law of dominance, Test cross 10 Dihybrid cross, Dihybrid test cross, Law of independent assortment 11 Morgan concept on location of factors, recombination, crossing over 12 Recombination frequency, Map distance 13 Pedigree analysis 1 31 Class 9 Flow of information in living systems: Mendelian model, Monohybrid and Law of Segregation, Law of dominance, Test cross THE LOGIC OF MENDEL All of us have some characters similar to our parents or grand parents. Many of us likes to look like or behave like our parents. Our grand parentsgrandparents, most of them are farmers, selected the best seeds for their next season. All of them knew that the best seeds have the best characters. Characters are passed to next generation through seeds. Seeds comes from flowers. Bees visit flowers for honey. Flowers contain honey fine powders. Here we are trying to understand logically, how a normal man like Mendel who had lowest grades in Biology could do such a meticulously planned experiments? Above all he had failed for a teaching certificate in natural sciences! On the other hand Mendel studied physics, mathematics and chemistry along with important aspects of biology. This mathematical back ground enabled Mendel to plan his experiments, draw out a theory and experimentally evaluate it. We are logically going to understand what brought Mendel to understand the concept. Remember to consider the context that are looking at this from Mendel's perspective. He knew nothing about meiosis, never considered chromosomes or whether they had anything to do with what he was investigating. The word "gene" was invented after his death. All he did was breed peas, and do some high quality thinking. All this done between 1856 and 1863! It is also interesting to see what is Mendel’s previous knowledge? Sometimes early in his life he worked as a gardener and done the beekeeping. He also studied practical and theoretical philosophy and physics at the University of Olomouc, Czech republic. He happened to see the research of hereditary traits of plants and animals, in the department of natural history and agriculture. He also worked as a substitute high school teacher. He failed in getting a certificate for teacher and send to University of Vienna for further study on a sponsorship. He returned and again worked as physics teacher, but failed on the oral part of examination. Finally he was taken the superior priest of the monastery. MendelsMendel’s work can be divided into the following steps: (A) Preparation for experiments (B) Choice of experimental material (C) Planning and execution of experiment (D) Interpretation of experimental results and (E) Further testing of his observations. A. Preparation for Experiments 2 32 How do to an experiment? The laboratory should be accessible! That is Mendel approached the head of the department of Natural history and agriculture where he was working as a priest for permission to use the 2 hectar experimental garden intended to study differences in plants. His colleges conducted studies on the heredity of sheep. Why he wanted the entire full 2 hectershectares rather a few cents or pots ? pots? If we need to have a reliable results, the sample size should be very high ! high! Therefore how much land you need to cultivate around 20,000 pea plants? Ask a farmer !farmer! B. Choice of the experimental material First he decided to work on plants? Do you know why? Plants attain reproductive age very soon compared to many animals, the number of plantlets from a plant will be very high, Many plants 1 can be grown together and above all plants reproduce through seeds which we will get from flowers!. Now the question is which plant? Shall we take a mango tree? Here comes the advantage of critical observation. The feasibility. It is Pea plants, because very easy to cultivate, flowers are big (think why?), number of plantlets produced from one flower will be more and above all its generation time is less. But the most important thing? What is the aim of the experiment? To study variation in plants? So we need to study variation. Examples of variation? Flower color, seed color, seed size and shape, flower position etc. So the experimental plant should have easily recognizable variations. From the Mendel’s observations it is the Pea. C. Planning and execution of the experiment Selection of variations: He selected the following characters: 1. Seed shape [Spherical Vs Wrinkled]; 2. Seed color [Yellow Vs Green], 3. Flower color [Purple Vs white], 4. Pod shape [inflated vs constricted], 5. podPod color [green pod vs yellow], 6. Flower position [Axial vs Terminal] and 7. Plant size [Tall Vs Dwarf] (The first logic of Mendel: Easy recognizable separate characters for observation -that had well-defined, contrasting alternative traits) Why he selected seven characters? How did he do? He selected seven variations simultaneously in each experiment or separate experiments for separate variations? What you feel? He conducted different experiments for different characters first of all to see that whether he is getting the same kind of results in each case, secondly it is easy to follow the inheritance of one character at a time rather all together. That was the second logic of Mendel. This third logic of Mendel from the knowledge he may got from his practical knowledge on agriculture and theoretical knowledge he got from mathematics. Does Mendel randomly selected seeds? We should know which characters seeds are inherited? We are studying variation, should we select seeds which show consistent inheritance or variable inheritance? The choice should be of consistent inheritance. He knew that it is possible to raise plants with consistent inheritance by crossing sibling plants. That is Mendel used well defined seeds as the starting material in all his experiments involving thousands of plants. So if we want to test the fuel efficiency of 10 different bikes, we should 3 33 use the same experimental conditions in all the cases against one variability. This is the spectacular logic he applied and the basis of Mendel’s success. The fourth logic is experimental. How to do controlled breeding? We want to cross a tall plant with a dwarf plant. So we need to ensure that only the pollen grain of the tall plant is falling on the stigma of the dwarf plant and vice versa. How to do this? Simply bag the flower bud, cut the stamens of the experimental flower, take the pollen grain from the other flower using a brush, “paint” on the stigma of the initial flower, bag it again to avoid unwanted entry of other pollen grain. (See the animation). Get the seeds from the flower and observe for the variation intended to study! Here is the fifth logic. He just crossed only once. All other experiments involved self pollinationself-pollination. i.e., he allowed mixing of two different characters only once. Why not twice or thrice? As simple as this, first let us start from simple things! So do the cross only once, avoid complexity and evaluate the results. 2 4 34  Now comes the sixth and final logic. Looking behind a character how many generations we should follow after crossing? Again comes the simplicity and reproducibility. Just followed the first generation after crossing. So in short he started with the same experimental plan is as follows: Tall plants Dwarf plants True breeding Tall plant True breeding dwarf plant First filial generation (F1) Cross product (Self pollinate) Second filial generation (F2) Raise the plants look for variation how it was passed Do the counting for the variations, tabulate 3 5 35 Figure 1 The seven variations observed in Pea plant that Mendel followed: Are they easily recognizable? Figure 2 Flower structure of Pea: Observe the reproductive parts of the flower. 4 6 36 Photo of Mendel’s Experimental Model i.e. Green Peas: Mendel’s controlled experiments and elucidation of the results 7 37 MONOHYBRID CROSS AND SEGREGATION THE RESEATRCH METHOD OF MENDEL All of us now that pollen grains of one flower falls on the stigma of another flower or same flower. From the stigma the male nucleus of the pollen grain reaches the ovary and fertilizes an egg to produce a zygote. In plants fertilized zygote produces the seed and the ovary produces the fruit. The problem faced by Mendel is controlled pollination. Suppose he want to cross a tall plant with a dwarf plant. In this case the pollen grains of dwarf plant only should fall on the stigma of the dwarf plant or vice versa. How to achieve this? (Solve this problem with the help of the following figure and the animation provided) 5 8 38 EXPERIMENTS RESULTS OF MENDEL We have seen what Mendel’s logics were and how he executed the experiments. After his experiments the results were carefully tabulated by him. Analyze the following table showing the tabulation of Mendel 6 9 39 What you find here? (A) Only one character appeared in the F1 generation (B) Both characters were appeared in the F2 generation, but not in equal percentage (C) A character which disappeared in the F1, reappeared in F2 (D) The results are consistent in all the seven characters (E) There is no blending of characters The analysis of the results clearly indicate the following The character which appeared in the F1 is having the higher percentage in F2 i.e. almost three times to that of the disappeared character in F1. What you will conclude from these results? The character which is appeared in the F1 is also dominating in F2 also. This is the Dominant character. The other or alternative character which is disappearing in the F1 generation is also less in number compared to dominant character. This is Recessive character. (Law of Dominance). Many of us, including Mendel, expected a blended phenotype in F1. But it didn’t happen. Why? Can’t the characters blend? The experimental results in all the seven characters studied by Mendel didn’t showed any blending (i.e. when we cross a Tall and Dwarf plant we can expect a plant intermediate between tall and dwarf; but it didn’t happen). This further means that some “units” which functions as discrete particles are responsible for characters. Since the character which disappeared in the F1 reappeared in F2 it is logic to conclude that these units occur as pairs. It means that two discrete units are responsible for the “Tall” character. Otherwise any given individual can be homozygous (Dominant/Dominant or Recessive/Recessive) or heterozygous (Dominant/Recessive). We can express the alternate forms of characters (dominant or recessive) as alleles. Also we know at present that character means a gene. So alleles are alternate forms (in fact variables) of a gene. They can be 7 10 40  The factors can be represented as any letter forms (similar to polynomials). For example a tall plant can have TT or Tt. A dwarf plant can be represented as tt. This binomial expression is the genotype. In conclusion the “units” (or factors) are discrete, they never blend and are responsible for passing character from one generation to next ie inheritance. Why two units of inheritance? Not three or four? We have father and mother. i.e. we have characters both from our father and mother. A character is represented by two units. One unit inherited from father and one from mother. So the gamete contains one unit. This is the core idea of Mendel’s inheritance. At present the “units” proposed by Mendel is known as gene. The following figures illustrates a typical Mendelian cross. 8 11 41 Now study the following cross which can be represented in the form of a Punnett square (A simple grid representing all possible gametes and combinations. Given the credit to Reginald Crundall Punnett, a British geneticist) In conclusion the units of inheritance are never blended, but segregated independently during reproduction (Law of segregation). This is the second core idea of Mendel’s theory. Another illustration is here for you regarding the cross between a Tall plant and a Dwarf plant. This illustration represents Mendel’s core ideas. 9 12 42 13 43 Terminologies, Back cross and Test Cross, Dihybrid cross Verification of Mendel’s hypothesis: He did verification experiments for his hypothesis, as illustrated below. 10 14 44 Similarly the following figure illustrates a dihyrbid test cross 11 15 45 Mendel has modelled one experiment, he executed and finally he tested it. This type of cross is knows a test cross (Crossing the unknown genotype with the recessive parent). It can predict the genotype to be tested based on the phenotypic ratio of the cross output. Class 10 Dihybrid cross, Dihybrid test cross, Law of independent assortment ProbailityProbability laws govern Mendelian inheritance: The Study of dihybrid cross Initially Mendel did all his experiments by analyzing only one character at a time – monohybrid cross. Based on his results, he has tested his hypothesis of dominance and segregation. After this he wanted to study the inheritance of two characters at a time – the dihybrid cross The experiment is planned in such a way to analyze the following: Whether the alleles maintain the association they had in the parental generation: For this he crossed pure breeding spherical seed and yellow seed color pea plant with a wrinkled seed green seed color pea plant. If the alleles maintain the association, he is expecting only the parental types in the F2 generations (Why not F1 generation?) If the alleles maintain the association, the F1 gametes will be SY and sy. As a result the probability of Spherical and yellow seed peas: wrinkled and green seed peas will be 3:1 (Ie i.e.only two phenotypes). If they segregate independently he was expecting four different phenotypes. The experiment and the results are illustrated as below 12 46 He didn’t get a 3:1 ratio in F2 generation. New types were obtained in F2. It means that the alleles didn’t maintain the same association as seen in the parental types, rather they assorted independently (Law of Independent Assortment). Mendel and his Mathematics predictions. You have a 1 rupee coin and 5 rupee coin. You are going to toss it together. What is the probability of getting a tail in both cases? (Are the two events linked to each other or independent?) Probability of getting a 1 rupee tail = ½. Probability of getting a 5 rupee tail = ½ 13 47 Hence the probability of getting both tail = ½ X ½ = ¼ i.e. 25%. In a homozygote (SS), the probability of producing a S gamete is 1 In a heterozygoute (Ss), the probability of producing a S gamete is ½ and s gamete is also ½ Now consider the F2 generation. The probable gametes here are S and s. Hence the probability of getting SS is ½ X ½ = ¼ i.e. 25% are homozygous dominant The probability of getting ss is ½ X ½ = ¼ i.e. 15% are homozygous recessive Adding probabilities: What is the probability of getting Ss and sS? Probability of Ss (S from sperm and s from egg) = ½ X ½ = ¼ Probability of sS (s from sperm and S from egg) = ½ X ½ = ¼ Both Ss and sS are heterozygotes and will have the same phenotype. Hence added probability is ¼ + ¼ = ½ ie 50% will be heterozygotes. Now can we calculate the probabilities in dihybrid cross? In F2 generation, the probabilities are illustrated below 14 48 Now what is the probability of getting an SS homozygorehomozygote ? i.e. ¼ The probability of getting heterozygote (i.e. Ss or sS) is ¼ + ¼ = ½ The added probability ( i.e(i.e. spherical seed ) = ¾ Now calculate the probability of yellow seed using the above reasoning? It will be ¾ Hence what is the added probability of getting a spherical seed and yellow seed = ¾ X ¾ = 9/16 (Since both events are independent i.e. independent assortment) 15 49 What will be the probability of getting a yellow and wrinkled seed? Probability of yellow seed = ¾ Probability of wrinkled seed = ¼ Hence the added probability = ¾ X ¼ = 3/16 Using the same logic it is easy to calculate the probability of wrinkled yellow seed is 3/16 and wrinkled green seed is 1/16. Mendel did all these statistical problems. Because of his mathematical knowledge, he could easily predict, the ratio obtained in F2 generation of monohybrid and dihybrid crosses are simply a statistical event and the factors are independent of each other. You should understand both; i.e. doing a genetic problem by using probability and by using a Punnett square. It is also possible to test the dihybrid genotypes as illustrated below. Similar to monohybrid test cross, it is possible to predict the genotype of a phenotype by crossing with a true recessive parent. The prediction is based on the characteristic phenotypic ratio we will get in this cross. 16 50 51 Chromosomes and cell Division, Chromosomal Theory Mendel experiments: Did he predicted chromosomes? (Meiosis accounts for segregation) The segregation of Mendelian factors is because of meiosis Mendel proposed mechanisms of heredity. Mendel had no knowledge of chromosomes or meiosis. But he speculated that cells contained some type of factor that carried traits from one generation to the next. The scientific importance of Mendel’s work remained unrecognized for several years. Most probably Mendel believed that for each character there is a factor. Currently we know that this factor is a gene (or an allele) that is located on a chromosome. They show characteristic segregation and independent assortment are due to meiosis (Illustrated below). 17 52  So if we have a cell with a genotype Ss it should produce two types gametes, i.e. one type with S and the anotheranother type with s. Mendel said the two alleles will segregate. Our current knowledge is that meiosis accounts for segregation. This is exactly matches what Mendel speculated from his results. He also proved that each factor segregates when traits passed from one generation to another generation. What happens in mitosis? A cell with genotype Ss just produces two daughter cells with the same genotype as illustrated above and there is no segregation. Now you will see how alleles assort independently during meiosis when we consider a dihybrid cross 18 53 Mendelian principles doesn’t applies to all cases of inheritance. Whether all the inheritance follows Mendelian pattern? Human have several traits like hair pattern, skin color, tasting ability, shape of ear and so on. How many chromosomes we have? 23 pairs. So if Mendel’s rules we apply, we should have only 23 chromosomes. Hence it becomes clear that a chromosome can contain more than one factor. Now we have to think that who is the luckiest Man? It is Mendel. He selected seven characters. Each character was regulated by a gene and they were located in seven different chromosomes. Now we know that Pea plant has seven chromosomes. Suppose if the traits selected by Mendel resides on the same chromosome, he will not get a 9:3:3:1 ratio as expected. In short if we get a ratio of 9:3:3:1, we can assume that the genes we selected are located on different chromosome. If we are not getting this ratio, then genes may be on same chromosome. The genes on the same chromosome means that they are linked. The seven chromosomes of Pisum sativum. Luckily the seven genes for the selected traits by Mendel was located on seven different chromosomes leading to his success in modelling his hypothesis and successful testing. If it was not, Mendel might have failed in his efforts. Hence many people believes that Mendel was the luckiest person. 54 Class 11 Morgan concept on location of factors, recombination, crossing over 55 Two types of cell Division Morgan’s experiments 56 Morgan and his Drosophila: Mendel’s hypothesis is rejected in Morgan’s experiments [Chromosomal theory, Connecting Mendel to Morgan, Linkage and crossing over] Thomas Hunt Morgan and his students of Columbia University did pioneering works to explain heredity from the beginning of 1909. He explored the Mendel theories in Drosophila melanogaster, the fruit fly as the experimental organism. He selected fruit fly because of its small size, easy to grow and breed and its short generation time. Thomas H. Morgan correctly perceived that the success of genetic investigators depended critically upon the choice of the organism to be investigated. Much of the work in the early years had centred upon agricultural plants and animals: we knew how to grow successive generations of them, and the information had direct practical bearing. Morgan abandoned agricultural utility in favor of experimental utility-plants just took too long between generations, and they took up too much space. Morgan wanted an organism with which one could carry out many crosses, with many progeny, easily and quickly. With this in mind, he began to investigate the genetics of Drosophila. No genetic varieties were available in Drosophila, so Morgan set out to find them. He obtained his first mutant in 1910, from normal red eyes to white. At last he could set out to examine Mendelian segregation. MORGAN’S FRUIT FLY CROSSES First, Morgan crossed the white-eyed male he had found to a normal female, and he looked to see which trait was dominant in the F1 generation: all the progeny had red eyes. Now, would the white-eye trait reappear, segregating in the F2 progeny as Mendel had predicted? In the F2, there were 3470 red-eyed flies and 782 white-eyed flies, roughly a 3:1 ratio. Allowing for some deficiency in recessives, this was not unlike what Mendel’s theory predicted. But in this first experiment, there was a result that was not predicted by Mendel’s theory: all the white- eyed flies were male! At this point, Morgan had never seen a white-eyed fly that was female. Morgan preferred a straightforward test: if any of the F2 females carried the white-eye trait but did not show it, then it should be revealed by a test cross to the recessive parent. It was. Crossing red-eyed F2 57 females back to the original white-eyed male, he obtained 129 red-eyed females, 132 red-eyed males, and 88 white-eyed females, 86 white-eyed males. Again, this was a rather poor fit to the expected 1:1:1:1 ratio due to a deficiency in recessives. The important thing, however, was that there were fully 88 white-eyed female flies. Clearly, it was not impossible to be female and white-eyed. Why, then, were there no white-eyed females in the original cross? X AND Y CHROMOSOMES We know that in mammals and many other animals sex is determined by chromosomes i.e. XX will be female and XY will be male. Thus, sperm may contain either an X or a Y chromosome, while all the female gametes will contain a copy of the X chromosome. In forming a zygote, sperm that carry an X chromosome will produce an XX zygote (female), while sperm that carry a Y chromosome will produce an XY zygote (male). This simply model explained the 1:1 proportions of males to females usually observed, as well as the correspondence of sex with chromosome cytology. SEX LINKAGE This theory provided a really simple explanation of Morgan’s result, and he was quick to see it: what if the white-eye trait is resided on the X chromosome? Morgan had only to assume that the Y chromosome did not have this gene (it was later shown to carry almost no functional genes). Knowing from his previous crosses that white-eye is a recessive trait, the results he obtained could be seen to be a natural consequence of Mendelian segregation! Thus, a typically Mendelian trait, white-eye, is associated with an unambiguously chromosomal trait, “sex.” This result provided the first firm experimental confirmation of the chromosomal theory of inheritance. This association of a visible trait that exhibited Mendelian segregation with the sex chromosome (sex linkage) was the first case in which a specific Mendelian gene could be said to reside on a specific chromosome. It firmly established the fusion of the Mendelian and chromosomal theories, marking the beginning of modern genetics. 58  In the above cross, the normal allele is red, the recessive allele is white. Red is dominant over white. Whenever the white male is crossed with a true breeding red female the result is both male and female flies are red eyed. Whenever a red male is crossed with a true breeding white female all male offspring’s are whited eyed and female flies are red eyed. So the gene for the trait eye color in Drosophila resides on X linked chromosome. This inheritance is X linked recessive. We can show X linked inheritance in the pedigree chart illustrated below. Did you note three things (1) in females, both X chromosomes should carry the recessive allele for the expression of white eye color. Hence this is an X linked recessive trait. (2) Males have only one X chromosome. Hence the trait will express even if the X chromosome contains the recessive allele (3) X linked recessive traits are more frequently occurs in males compared to females. The reason is that males have only one X chromosome. Hence the recessive allele will express. Recombination of linked genes: crossing over From the independent assortment of chromosomes revealed that the traits that do not match those of either parent for e.g.: the cross between a pea plant with yellow-round seeds that is heterozygous for both seed color and seed shape (a dihybrid YyRr) and a plant with green- wrinkled seeds homozygous for both recessive alleles (test cross) 1:1:1:1 half of the offspring are called parental types and another with new combinations of seed shape and color are called recombinant type with 50% frequency of recombination. The proof that the genes were located on chromosomes was provided by single small fly. Thomas Hunt Morgan’s drosophila dihybrid experiments for the body color and wing size. Wild type flies have gray bodies and normal-sized wings. In addition to these flies, Morgan had managed to obtain, through breeding, doubly mutant flies black body and wings much smaller than normal, called vestigial wings. Mutant alleles are recessive to the wild – type alleles. Morgan wanted to know whether the genes for body color and wing size were genetically linked, and if so, how this affected their inheritance. The alleles for body color are b_ (gray) and b (black), and those for wing size are vg_ (normal) and vg (vestigial). Morgan mated true-breeding P (parental) generation flies—wild-type flies with black, vestigial-winged flies—to produce heterozygous F1 dihybrids (b_ b vg_ vg),all of which are wild-type in appearance. He then mated wild-type F1 dihybrid females with black, vestigial-winged males. This testcross will reveal the genotype of the eggs made by the dihybrid female. The resulting flies had a much higher proportion of the combinations of traits seen in the P generation flies (called parental phenotypes) than would be expected if the two genes assorted independently. Morgan thus concluded that body color and wing size are usually inherited together in specific (parental) combinations because the genes for these characters are near each other on the same chromosome. The Predicted ratios if genes are located on different chromosomes were 1:1:1:1. If the genes are located on the same chromosomes and parental alleles are always inherited together then the ratio is 1:1:0:0. However, both of the combinations of traits not seen in the P generation (nonparental phenotypes) were also produced in Morgan’s experiments, suggesting that the body-color and wing-size alleles are not always linked genetically. To understand this conclusion, we need to 59 further explore genetic recombination, the production of offspring with combinations of traits that differ from those found in either parent. Since most offspring had a parental (P generation) phenotype, Morgan concluded that the genes for body color and wing size are genetically linked on the same chromosome. However, the production of a relatively small number of offspring with non parental phenotypes indicated that some mechanism occasionally breaks the linkage between specific alleles of genes on the same chromosome. What Morgan expected is a 1:1:1:1 ratio (Recollect Mendel’s dihybrid test cross ratio) Total individuals = 965+944+206+185 = 2300 Parental types = 965 + 944 = 1909 Non parental types or recombinant types = 391 Recombination frequency =( Recombinant types/Total individuals) X 100 = (391/2300) X 100 = 17% (We can also write recombination frequency as 0.17 assuming that maximum recombination is 1) Now we have to see why the new phenotypes (non-parental phenotypes) occurs? The new phenotypes appear because of exchange of genes between homologous chromosomes that occurs during meiosis (swapping). This event is known as crossing over. Look at the following illustration to understand the process. 60 If we consider Morgan’s Drosophila testcross result offspring from the testcross for body color and wing size most of the offspring (>50%) had parental phenotypes and about 17% of offspring were recombinants. This suggested that the two genes were on the same chromosome. With these results, Morgan proposed that some process must occasionally break the physical connection between specific alleles of genes on the same chromosome. And this process is called crossing over which accounts for the recombination of linked genes. When replicating the homologous chromosomes are paired during prophase of meiosis I, an exchange of end portions of two non-sister chromatids takes places leading to crossover. Towards the genetic map The probability of recombination between two loci increases with distance. Morgan’s found recombination frequencies of many genes through experiments and used these frequencies to construct a genetic map or mapping the genes. A genetic map tells the distance between two genes. The following illustration helps us to find how to do a genetic map. It is measured in terms of centimorgan or cM. 61 The recombination frequencies can be used for making genetic maps. Morgan’s group conducted several crosses in Drosophila. After finding out the frequency he was able to apply for construction of genetic map because the more the distance between two loci, the more will be the recombination. It means the distance between genes can be calculated based on this. The unit is cM (Centimorgan) or map units (1cM = 1 map unit). The following illustration shows an illustration of genetic mapping by Morgan. 62 Flemming was a German military physician. He found cells contains the coloured genetic material, the chromosomes (Chrome = color; some = body). This is in fact the factor represented by Mendel. Even he discovered that chromosomes splits longitudinally during cell division (His illustration is given below). This is what happens during mitosis. We know, in meiosis the longitudinal splitting happens after crossing over. The chromosomal theory was not the work of a single scientist by Mendel or Morgan. Many people experimented over decades on it. Indeed, the first logic steps were initiated during 1860 by the mathematician Mendel and evolutionary biologist Charles Darwin. The probable mechanism of transmission from one generation to next was speculated by the discovery of chromosomes by Walther Flemming, a German biologist. Now to connect between chromosomes and heredity. This was done by Boveri, Sutton and Morgan during the dawn of 20th century. Thus the chromosomal theory came out which experimentally proved that chromosomes are responsible for transmission of trait from one generation to next. In fact Mendel was a Physicist (and philosopher), Darwin was a naturalist, Morgan was a zoologist. Above all Flemming was a military physician!! 63 Class 13 INHERITANCE AND PEDIGREE Life has evolved on earth gradually. Most of the life forms have two different sex, what we say is a male and a female or a + strain and a – strain. Why life preferred two genders? Male and Female? It would have been simple for the life if only one gender is existing and all of them will reproduce. The advantage lies on the recombination event. During sexual reproduction, the chromosome number of the gametes are reduced into half through meiosis. We have seen that meiosis accounts for segregation and assortment. We have also seen the non-parental genotypes appeared in the F2 generation of dihybrid cross. It means sexual reproduction gives an opportunity for variation through meiotic recombination. Hence life systems are not static, they are dynamic. They are evolving. The most perfect life machine will always be preferred by the nature. Others will disappear, the survival of the fittest. It means the best character is inherited over the generations. As we have seen with the Mendel and Morgan inheritance can be dominant or recessive Now let us go through the different types of inheritance. Before discussing that we should see how to represent the inheritance in the form of a diagram. This is known as pedigree. The basic rules of pedigree chart is give below. 64 Now calculate the probability of the children getting affected in a cross. The male can be homozygous dominant or heterozygous. If he is homozygous dominant, the probability of an unaffected child is zero. This probability will be 50% if he is heterozygous in each child birth. Now see the probability in the case of a recessive inheritance. The probability of getting the child having the recessive character is zero, because we know that the expression of a recessive character occurs only in case of homozygous condition of the alleles. But the unaffected child may be a carrier of the allele, even though he or she is not expressing the trait. The above types of inheritance illustrated here appears on both sexes. Hence the factor or the gene is resided on the autosomes. So an autosomal inheritance can be autosomal dominant or autosomal recessive. 65 Autosomal dominant trait 66 67 X Linked Recessive mode of inheritance Did you noted three things (1) In females, both X chromosomes should carry the recessive allele for the expression of white eye color. Hence this is an X linked recessive trait. (2) Males have only one X chromosome. Hence the trait will express even if the X chromosome contains the recessive allele (3) X linked recessive traits are more frequently occurs in males compared to females. The reason is that males have only one X chromosome. Hence the recessive allele will express. In some other cases, X linked characters may appear in dominant pattern also. Illustration is given below 68  Some of the traits only appear in males because the gene for this trait are located on Y chromosome. They are passed by father to all of his sons, but not daughter. The illustration is given below 30 69  The following table explains the features of different patterns of inheritance Diseases or traits can pass from one generation to another generation. Many of them follow typical Mendelian inheritance. The following table gives few examples 19 70 71 First observation for inheritance being related to a metabolic pathway Archibald Garrod was an English physician. He observed that many children exhibited a symptom - the urine turns dark brown immediately after urination, i.e. when exposed to air. Further analysis indicated that the frequency of the disease was more common in children of consanguineous (within the family marriage e.g. Cousin-Cousin, uncle-niece) marriages. Simultaneously, Garrod found the concepts involved in rediscovery of Mendelian inheritance inspiring. A pedigree chart allowed Garrod to determine that the couples had a recessive allele causing the child to be homozygous recessive. Biochemical composition of the urine was investigated by Garrod and observed the following pattern for normal individuals or heterozygous individuals. So in homozygous recessive cases the reaction is blocked. This was an indication that inheritance is responsible for turning the urine black. Garrod speculated this fact, but was unable to identify the enzyme or the gene. However an exact confirmation required several years of study. In 1958, the enzyme was identified –homogentisic acid oxidase and in 1996 the gene was identified. Chromosomes contain both proteins and DNA: What is the evidence that which chemical component carries the genetic information? Chromosome is a combination of two chemicals: DNA and Proteins: The chromosome is a dynamic structure in the sense that it condenses and expands during various stages of the cell cycle. Chromosome is a mixture of two different components (i) DNA and (ii) proteins in higher quantity compared to DNA. In fact, DNA is bound to proteins. This unique combination accounts for dynamicity of the structure. What we see or represent for a 1 72 chromosome is the most condensed state of chromatin fibers (DNA fiber). This can be visually seen during the metaphase of the cell division (Fig 1). Figure 1: Dynamicity of the chromatin fibers – they expand and contract according to the stage of the cell cycle: M=Mitotic phase (Division phase), G1 and G2 are gap phases and synthesis of the raw materials occurs during the Synthesis (S phase). [Figure adapted from Sadava et al, Life: The science of Biology, 9th edition, Page 211, Fig 11.8] The circumstantial evidences and logic to assume that DNA is the genetic material: Living creatures exhibit a great diversity. Similar traits are observed in living forms of the same kind (species), while differences are observed between different species. It means that the genetic composition of living forms of one kind differs from that of the other kind. This exactly means their amount will differ. So can we prove it? This theory was proven by Robert Feulgen, who developed a red colored dye which binds to DNA. It stains DNA material red inside the nucleus. So the intensity of the red color is an approximate estimate of the DNA it contains. This dye is known as Feulgen stain. The Feulgen staining techniques presented the following information: It was in the right position (inside the nucleus) and the color intensity varied between two species. The need of more cause and effect evidence! The Feulgen staining only provided a circumstantial evidence. We should prove with a cause and effect situation that DNA carries the genetic information and not the proteins. How to do it? 2 73 Frederick Griffith was a physician from England. He was working with bacteria, which are visible only under a microscope. Pneumonia was taking many lives during his time. So he wanted to develop a vaccine for pneumonia. He found that there exists two forms (strains) of the bacteria which causes pneumonia, Streptococcus pneumonia. These strains are: Smooth (S) and Rough (R) forms. Smooth forms care capable of causing the disease, while rough forms do not cause the disease. The reason is that the smooth forms are hidden inside a proteinaceous cover, so it can cheat the firewall (The immune system), while rough forms are not able to utilize that trick, as they lack the protein coat. Hence the firewall will definitely catch and eliminate them. Now he planned and executed the experiments as illustrated in figure 2. Figure 2: The experiments conducted by Griffith with the bacteria and mouse. [Figure adapted from Sadava et al, Life: The science of Biology, 9th edition, Page 268, Fig 13.2]. The above figure illustrates that in the experiment 4the material present in the S form transforms the material present in the R form. So we can say that some transforming principle is responsible for this change from an R form to S form of bacteria. How to identify this transforming principle? The scientific group led by Oswald Avery of Rockefeller University cracked this problem. Their experiment is illustrated in Figure 3. 3 74 Figure 3: Identifying the “transforming principle”. [Figure adapted from Sadava et al, Life: The science of Biology, 9th edition, Page 269, Fig 13.1] The above work was published without much impact in 1944 because, many were not aware of the fact that DNA is complex enough to give the diverse output. Moreover many were still wondering whether microscopic small creatures, like bacteria, has genes in it. 4 75 The impact of this work was intensified after another experimental work published in 1952 by Alfred Hershey and Martha Chase at Carnegie Laboratory of Genetics. They were trying to determine whether DNA or protein contains the genetic material by using a bacteriophage (a virus that attacks and kills the bacteria). Why they have selected a virus? Because virus is composed of just two components that we are trying to sort, the protein cover and the DNA inside it (Figure 4). Figure 4: Hershey and Chase experiment. [Figure adapted from Sadava et al, Life: The science of Biology, 9th edition, Page 271, Fig 13.4] 5 76  Transcription RNA (ribonucleic acid) is a key intermediary between a DNA sequence and a polypeptide. RNA is an informational polynucleotide similar to DNA, but it differs from DNA in three ways: RNA generally consists of only one polynucleotide strand. The sugar molecule found in RNA is ribose, rather than the deoxyribose found in DNA. Although three of the nitrogenous bases (adenine, guanine, and cytosine) in RNA are identical to those in DNA, the fourth base in RNA is uracil (U), which is similar to thymine but lacks the methyl (—CH3) group In the process of RNA synthesis, the information contained in DNA is transcribed into RNA. During transcription, the information in a DNA sequence (a gene) is copied into a complementary RNA sequence. The process occurs in the nucleus and the resulting RNA is carried to the cytoplasm, where the protein synthesis occurs. Figure 1- Sites of transcription and translation (protein synthesis) in a eukaryotic cell. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. Figure 2- The nucleotide Thymine (present only in DNA) is replaced by Uracil (present only in RNA). The pairing of the ribonucleotides obeys the same complementary base-pairing rules as in DNA, except that adenine pairs with uracil instead of thymine. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. 1 77 Single-stranded RNA can fold into complex shapes by internal base pairing. Three types of RNA participate in protein synthesis: Messenger RNA (mRNA) carries a copy of a gene sequence in DNA to the site of protein synthesis at the ribosome. Transfer RNA (tRNA) carries amino acids to the ribosome for assembly into polypeptides. Ribosomal RNA (rRNA) catalyzes peptide bond formation and provides a structural framework for the ribosome. Figure 3- Types of RNA. Transcription is responsible for the synthesis of mRNA, tRNA and ribosomal RNA (rRNA), who play important roles in protein synthesis. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. Transcription requires several components: A DNA template for complementary base pairing; one of the two strands of DNA The appropriate nucleoside triphosphates (ATP, GTP, CTP, and UTP) to act as substrates An RNA polymerase enzyme- Like DNA polymerases, RNA polymerases are processive; that is, a single enzyme–template binding event results in the polymerization of hundreds of RNA bases. But unlike DNA polymerases, RNA polymerases do not require a primer and do not have a proofreading function. Transcription occurs in three stages: a) initiation, b) elongation, and c) termination a) INITIATION- Transcription begins with initiation, which requires a promoter, a special sequence of DNA to which the RNA polymerase recognizes as a start site and binds very tightly. Eukaryotic genes generally have one promoter each, while in prokaryotes and viruses, several genes often share one promoter. Promoters are important control sequences that “tell” the RNA polymerase two things: 2 78  Where to start transcription Which strand of DNA to transcribe (which of the two strands of DNA will act as template for producing a single RNA strand) Part of each promoter is the initiation site, where transcription begins. Groups of nucleotides lying “upstream” from the initiation site (5′ on the non-template strand, and 3′ on the template strand) help the RNA polymerase bind. Figure 4- Initiation of RNA synthesis by RNA polymerase. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. b) ELONGATION- Once RNA polymerase has bound to the promoter, it begins the process of elongation. RNA polymerase unwinds the DNA about 10 base pairs at a time and reads the template strand in the 3′-to-5′direction. Like DNA polymerase, RNA polymerase adds new nucleotides to the 3′ end of the growing strand, but does not require a primer to get this process started. The RNA transcript produced is antiparallel to the DNA template strand. Because RNA polymerases do not proofread, transcription errors occur at a rate of one for every 104 to 105 bases. Because many copies of RNA are made, however, and because they often have only a relatively short life span, these errors are not as potentially harmful as mutations in DNA. Figure 5- Successive addition of ribonucleotides to the 3' growing end of the newly synthesized RNA transcript. 3 79  c) TERMINATION- Just as initiation sites in the DNA template strand specify the starting point for transcription, particular base sequences specify its termination. For some genes, the newly formed transcript falls away from the DNA template and the RNA polymerase. For others, a helper protein pulls the transcript away. Figure 6- Process of elongation and termination in transcription of RNA. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. Difference in transcription between Prokaryotes and Eukaryotes  Initiation: In bacterial cells, the holoenzyme (RNA polymerase plus sigma) recognizes and binds directly to sequences in the promoter. In eukaryotic cells, promoter recognition is carried out by accessory proteins (transcription factors) that bind to the promoter and then recruit a specific RNA polymerase (I, II or III) to the promoter. 4 80  RNA processing: In prokaryotes, several adjacent genes sometimes share one promoter; however, in eukaryotes, each gene has its own promoter, which usually precedes the coding region. Eukaryotic genes undergo a systematic process called RNA processing to produce a mature mRNA from pre mRNA. Eukaryotic genes may contain noncoding base sequences, called introns (intervening regions). One or more introns may be interspersed with the coding sequences, which are called exons (expressed regions). Both introns and exons appear in the primary mRNA transcript, called pre- mRNA, but the introns are removed by the time the mature mRNA—the mRNA that will be translated—leaves the nucleus (figure 1). Pre-mRNA processing involves cutting introns out of the pre-mRNA transcript and splicing together the remaining exon transcripts. Eukaryotic gene transcripts are processed before translation: The primary transcript of a eukaryotic gene is modified in several ways before it leaves the nucleus: both ends of the pre mRNA are modified, and the introns are removed. 5 81  MODIFICATION AT BOTH ENDS Two steps in the processing of pre mRNA take place in the nucleus, one at each end of the molecule. A G cap is added to the 5′ end of the pre-mRNA as it is transcribed. The G cap is chemically modified (methylated) guanosine triphosphate (GTP). It facilitates the binding of mRNA to the ribosome for translation, and it protects the mRNA from being digested by ribonucleases that break down RNAs. Figure 7- Addition of 5' cap. Image courtesy- Genetics- : A Conceptual Approach; by Benjamin A. Pierce 6 82 Figure 8- Addition of 50 to 250 adenine nucleotides at the 3 end is called Polyadenylation, which occurs after transcription is completed. Image courtesy- Genetics- : A Conceptual Approach; by Benjamin A. Pierce A poly A tail is added to the 3′ end of the pre-mRNA at the end of transcription. In both prokaryotic and eukaryotic genes, transcription begins at a DNA sequence that is upstream (to the “left” on the DNA) of the first codon (i.e., at the promoter), and ends downstream (to the “right” on the DNA) of the termination codon. In eukaryotes, there is usually a “polyadenylation” sequence (AAUAAA) near the 3′ end of the pre-mRNA, after the last codon. This sequence acts as a signal for an enzyme to cut the pre mRNA. Immediately after this cleavage, another enzyme adds 100 to 300 adenine nucleotides (a “poly A” sequence) to the 3′ end of the pre-mRNA. This “tail” may assist in the export of the mRNA from the nucleus and is important for mRNA stability. 7 83 Splicing- The next step in the processing of eukaryotic pre-mRNA within the nucleus is removal of the introns. If these RNA sequences were not removed, a very different amino acid sequence, and possibly a nonfunctional protein, would result. A process called RNA splicing removes the introns and splices the exons together. Figure 9- The process of splicing beta globin gene. (UTR- untranslated region). Alternative splicing Figure 10- Alternative Splicing Results in Different mRNAs and Proteins In mammals, the protein tropomyosin is encoded by a gene that has 11 exons. Tropomyosin pre-mRNA is spliced differently in different tissues, resulting in five different forms of the protein. Alternate splicing is a mechanism in which the differential splicing of the same pre- mRNA gives rise to different proteins, which may have different functions. It can be a deliberate mechanism for generating a family of different proteins from a single gene. For example, a single pre-mRNA for the structural protein tropomyosin is spliced differently in five different tissues to give five different mature mRNAs. These mRNAs are translated into the five different forms of tropomyosin found in these tissues: skeletal muscle, smooth muscle, fibroblast, liver, and brain. 8 84 DNA structure: Figure 1- Timeline of events in the discovery of DNA structure. Image courtesy www.genscript.com The race for DNA structure: The structure of DNA remained elusive until experimental evidence of many types were considered together in a theoretical framework. Figure 1 indicates the various experiments which acted as steps towards the discovery of the correct DNA structure. The most crucial evidence was obtained using X-ray crystallography. Some chemical substances, when they are isolated and purified, can be made to form crystals. The positions of atoms in a crystallized substance can be inferred from the diffraction pattern of X- rays passing through the substance. The events that provided information about this vital molecule are described in the following text. Chemical composition of DNA: Biochemists knew that DNA was a polymer of nucleotides. Each nucleotide consists of a molecule of the sugar deoxyribose, a phosphate group, and a nitrogen containing base. Figure 2- Chemical composition of DNA monomers. The only differences among the four nucleotides of DNA are their nitrogenous bases: the purines adenine (A) and guanine (G), and the pyrimidines cytosine (C) and thymine (T). Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. In 1950, biochemist Erwin Chargaff reported that DNA from many different species—and from different sources within a single organism— exhibits certain regularities. In almost all DNA, the following rule holds: The amount of adenine equals the amount of thymine (A= T), and the amount of guanine equals the amount of cytosine (G = C). 85 Figure 3- Chargaff's rule- In DNA, total abundance of purines is equal to total abundance of pyrimidines. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. In 1952, Rosalind Franklin was able to obtain an X-ray diffraction pattern for certain DNA fibers. This experiment provided the greatest help required by the scientists to deduce the DNA structure. Figure 4- The positions of atoms in a crystallized chemical substance can be inferred by the pattern of diffraction of X rays passed through it. The pattern of DNA is both highly regular and repetitive. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. Around the same time, Linus Pauling proposed a triple stranded helical structure for DNA. Linus Pauling had discovered the helical nature of protein folding and deduced that the same folding pattern may be followed by DNA. The structure proposed by Linus Pauling had the phosphate groups of the nucleotides facing inside the helical core. However, such a structure would result in the phosphate group repulsion (due to the negatively charged oxygen groups). Almost unbelievable that the man who had such a command over chemical bonds would get this wrong. Figure 5- DNA structure as proposed by Linus Pauling (Top view). The negatively charged oxygen atoms repel each other and would cause the strands to disassociate. Image courtesy- http://www.dnai.org/ 86 Double stranded structure discovery by Watson and Crick: The English physicist Francis Crick and the American geneticist James D. Watson, who were both then at the Cavendish Laboratory of Cambridge University, used model building to solve the structure of DNA. Watson and Crick attempted to combine all that had been learned so far about DNA structure into a single coherent model. Rosalind Franklin’s crystallography results (see Figure 4) convinced Watson and Crick that the DNA molecule must be helical (cylindrically spiral). Density measurements and previous model building results suggested that there are two polynucleotide chains in the molecule. Modeling studies also showed that the strands run in opposite directions, that is, they are antiparallel; that two strands would not fit together in the model if they were parallel. Figure 6- DNA model proposed by Watson and Crick made several assumptions. The nucleotide bases are on the interior of the two strands, with a sugar-phosphate backbone on the outside. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. Figure 7-To satisfy Chargaff’s rule (purines = pyrimidines), a purine on one strand is always paired with a pyrimidine on the opposite strand. These base pairs (A-T and G-C) have the same width down the double helix, a uniformity shown by x-ray diffraction. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. 87  Figure 8- In late February of 1953, Crick and Watson built a model out of tin that established the general structure of DNA. This structure explained all the known chemical properties of DNA, and it opened the door to understanding its biological functions. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. Important properties of DNA structure: 1. The double stranded helix has a uniform diameter 2. The two strands rum in opposite direction (antiparallel). 3. The backbone of each strand is made up of sugar phosphate groups linked by phosphodiester bonds 4. The two strands are held together by hydrogen bonding between the nitrogenous bases. Meselson and Stahl experiment: This experiment was instrumental in proving that DNA follows a semiconservative model of replication. A cell while undergoing division requires that old cell to produce copies of DNA that can be transferred to the new cells. The new cells receive a copy of the parent cell’s DNA. Figure 9- Watson and Crick suggested a semiconservative model of replication, wherein each parental strand acts as a template for synthesizing a new complementary strand. In this model, each daughter molecules consists of one old strand (from parent molecule) and one newly synthesized strand. Image courtesy- www.dnai.org Meselson and Stahl made clever use of radiolabelled (15N) heavy isotope of nucleotides and a density gradient of Cesium chloride (CsCl) to provide evidence for the semiconservative model of replication. They collected some of the bacteria after each division and extracted DNA from the samples. To separate the DNA from the cells at different generation on basis of density, they developed a density gradient solution in a test tube using CsCl. They found that the density gradient was different in each bacterial generation: 88  At the time of the transfer to the 14N medium, the DNA was uniformly labeled with 15N, and hence formed a single band corresponding with dense DNA. After one generation in the 14N medium, when the DNA had been duplicated once, all the DNA was of intermediate density. After two generations, there were two equally large DNA bands: one of low density and one of intermediate density. In samples from subsequent generations, the proportion of low-density DNA increased steadily. Figure 10- Meselson and Stahl experiment to prove that DNA replicates in a semiconservative manner. The researchers grew another E. coli culture on 15N medium, then transferred it to normal 14N medium and allowed the bacteria to continue growth. Image courtesy- Sadava et al, Life: The science of Biology, 9th edition. The results of this experiment can be explained only by the semiconservative model of DNA replication. In the first round of DNA replication in the 14N medium, the strands of the double helix—both heavy with 15 N—separated. Each strand then acted as the template for a second strand, which