BIO101 TOPIC WISE UPDATE UPDATED HANDOUTS highlights-2.pdf

BIO101 – Introduction to Molecular and Cellular Biology Topic Wise updated Handouts Prepared By: VU GLOBAL (Team) Psychology Department Contact us to join our esteemed network @ +96569002641 REMEMBER IN PRAYERS KEEP SMILING SAY NO TO CURRUPTION HELP TO OTHERS Regards, AARISH ALI & TEAM “VU GLOBAL” +96569002641 Topic No 01 What is Life? Anything is living if: ▪ It can acquire energy from the environment, e.g., plants acquire energy using sunlight and carbon dioxide and animals gain energy by eating plants like goats eat plants. ▪ It is capable of reproducing itself, e.g., all animals produce young ones like lions produce cubs. Plants also reproduce seeds to give rise to new plants. ▪ Mutating / changing itself: all organisms have a property of mutations, i.e., their heredity material – DNA changes itself during division or other times and the result is change in any characteristic of the organism. This characteristic may be beneficial or harmful; organisms survive better if mutation is beneficial and may die if it is harmful Biology – The Study of Life Biology (Bio – life; logos – study, reasoning); biology is hence the study of life or living organisms. Biology is about exploring the living part of the world, e.g., studying about animals, plants and even microorganisms is biology. Biology have many subdivisions; for example, anatomy – the study of structures, physiology – the study of functions, microbiology – the study of microorganisms and many more. The exploration of life helps in understanding the phenomena of nature and effective utilization and management of natural resources. We can find solutions to various problems for example treatment for various diseases could be discovered, methods for energy production from biological materials may be found, and e.g., few bacteria can produce fuel from grasses. Levels of Organization in Life ▪ Atoms ▪ Molecules o Micromolecules o Macromolecules ▪ Cells ▪ Tissues ▪ Organs ▪ Organ-systems ▪ Organisms ▪ Populations ▪ Communities ▪ Ecosystems ▪ Biosphere Atoms Greek: a, not; tom, to cut: The smallest component of an element that have all the properties of that element. In nature, 92 kinds of elements are present, out of which only 16 make the living organisms, called bioelements. Bioelements are Oxygen (O), Carbon (C), Hydrogen (H), Nitrogen (N), Calcium (Ca), and Phosphorus (P). These elements make 99% of living mass. Others ten elements make 1 % of total living mass named Potassium (K), Sulfur (S), Chloride (Cl), Sodium (Na), Magnesium (Mg), Iron (Fe), Copper (Cu), Manganese (Mn), Zinc (Nz), and Iodine (I). All living things consist of atoms, like all other forms of matter. Atoms consist of “subatomic particles”; charged or not charged. These include electrons which are negatively charged particles, protons which are positively charged and neutrons which have no charge. Protons and neutrons are present inside the center and the electrons revolve around these in orbits. Atoms do not live in isolation but join together to make molecules (compounds). Molecules Atoms join together by a process called “bonding” to each other to construct molecules. Bonding is of two types. o Ionic bonds o Covalent bonds In ionic bonding one atom gives one or more of its electron to the other atom which is called a donor and the other receive the electron called recipient. The donor atom then becomes positively charged and the recipient becomes negatively charged. In covalent bonding, however, the atoms share one or more of their electrons and these electrons revolve in the orbit of both atoms. Covalent bonding is more strong form of bonding. On the basis of their size molecules are categorized into micromolecules and macromolecules. Micromolecules are the molecules with low molecular weight, e.g., glucose, water. Macromolecules are the molecules with high molecular weight, e.g., proteins, carbohydrates and lipids. An organism consists of enormous number of biomolecules different types. Though some organisms are unicellular, i.e., consist of one cell only. Many other organisms are multicellular, i.e., these consist of many cells. Figure 1.1 Covalent Bond Vs. Ionic Bond Organelles Molecules make organelles. Organelles are sub-cellular structures, assemble together to make cells – the units of life, e.g., mitochondria, lysosomes, Golgi bodies, nucleus. For example, mitochondria of a cell (Singular: mitochondrion) is called “powerhouse” of the cell. This organelle is present in the cytoplasm of the cells and makes energy for the cells hence called “power house”. These are found in both plants and animals. Another example is nucleus present in almost all cells. Figure 1.2 Organelle: Nucleus Topic No 02 Cells – the basic unit of life All the living organisms consist of cells. Cells are called the basic units of life. Cells are specialized in their structure and functions. There are different types of cells present in the bodies of multicellular organisms. But some organisms like amoeba consist of only one cell. Cells are categorized based upon the placement of their nuclear material into prokaryotic and eukaryotic cells. Prokaryotic (Pro: old, Karyotic: related to nucleus) cells are those cells that do not have a true nucleus – it means that their nuclear material is not covered by a membrane. While eukaryotic cells have a true nucleus, i.e., their nuclear material is covered by a membrane called nuclear membrane. Sometimes a cell makes a whole unicellular organism, like Prokaryotes and Protists. A variety of cells makes a single, multi-cellular organism. Figure 2.1 Animal & Plant Cell Stem Cells These are the undifferentiated cells in which most of the genes are switched on and these have a potential to make almost all cells of the body. These cells are present in a few places in adult organism or present in the embryos. These are useful for human beings because these can be used in making organs of any type which may be damaged by for example a disease. Tissues These are the groups of similar kind of cells which perform a same function. The tissues perform a common function, specialized to the tissue. For example, epithelial tissue that makes the skin. Figure 2.2 Onion Epidermis (Tissue) Organs and organ systems Tissues group and work together to make a unit called organ. Different tissues in an organ work differently to perform collective function of the organ. For example in stomach, there are muscle tissues that contract and relax for grinding and there are secretory cells which secrete gastric juices to digest the food. Collective action is the secretion of gastric juices and its mixing. Figure 2.3 Human Digestive System In the simple to complex organisms, many organ-systems are present, for example, in humans digestive system, cardiovascular system, respiratory system and many more are present that work for a specific purpose. Digestive system consists of oral cavity, esophagus, stomach, intestine, pancreas, liver and rectum. Cardiovascular system consists of heart, vessels and blood. Organism Organ-systems join together to constructs organisms. In an organism, the organs and organ-systems coordinate to perform the activities of the whole organism. For example, in human’s brain control the activities of most of the organs and organ- systems. If a person is running; cardiovascular system provides it oxygen and nutrients, muscles contract and relax for movement and nervous system coordinate all of these functions. Topic No 03 Population: All organisms of a species living in an area at a particular time are called population, like all deer in a forest. Biologists study populations to explore the interactions between organisms. For example, interactions between male-male, female to female or else. Figure 3.1 Ducks in a local park Community & Ecosystem Different populations living in an area in a particular time, for example, in a forest plants, animals, algae, fungi live together are called a community. Populations interact with each other and also to the abiotic factors of the area to make Ecosystem, for example, a lake ecosystem. Figure 4 Plant community in a forest Biosphere The part of the world covered or inhabited by the living organisms is called biosphere. This is also called zone of life on Earth. Biosphere includes all ecosystems, like forests, lakes, oceans and valleys where biotic components exist. Topic No 04 What is Cell Theory? Cell theory in its modern form states: o All living organisms are composed of one or more cells. o Cells are the smallest living things, the basic unit of all living organisms. o Cells arise only by division in pre-existing cells. How Cell Theory Developed? o Cells were first described by Robert Hooke (Curator for instruments of Royal Society of London) in 1665. o Leeuwenhoek (Textile Merchant) observed tiny organisms in pond’s water, called them animalcules. o In 1809, de-Lamark proposed that nobody can have life if its parts are not cellular tissues. o Robert Brown discovered nucleus in the cell. o In 1838, Schleiden stated that all plants are aggregates of individual cells. o In 1839, Schwann stated that all animal tissues consist of cells. o In 1855, Virchow proposed that cells arise from pre-existing cells. o In 1862, Pasteur provided experimental proof for the above. Cellular Components Cells consist of several components. Some important and common ones are discussed below: o Cell membrane o Cell wall o Cytoplasm o Cytoskeleton o Organelles (e.g. nucleus, ribosomes) Figure 4.1 A Typical Animal Cell Cell Membrane o Cell membrane is the external most layer that covers the cell from outside. o Functions of the cell membrane are: It acts as a barrier, i.e., it separates the cell from environment. It provides protection to the inner parts of the cells including all the organelles. Another important function of cell membrane is transport of materials. Cell membrane manages the transport in and out of the cell. Figure 4.2 Structure of Plasma Membrane Topic No 05 Structure and Function of Cell Membrane Structure of cell membrane is described by Fluid Mosaic Modal. It consists of lipid bilayer, proteins and carbohydrates. Lipid bilayer provides it with fluidity, flexibility and transport of lipid like substances. Proteins are integrated inside the membrane or present on its peripheries called integrated proteins and peripheral proteins, respectively. Some proteins are trans-membrane, i.e., these are integrated and their ends (domains) are exposed from both intracellular and extracellular side of the membrane. These make channels for transport of materials, e.g., aquaporins are the protein channels for transport of water. Proteins and glycoproteins (carbohydrates attached to proteins) make receptors for message transmission. Cells carry out their message transmission with other cells or environment with the help of these glycoproteins mostly, we call these receptors. Movements across Cell Membrane ▪ Some molecules can pass directly e.g. few lipids. ▪ Other molecules need channels to pass through; channels are made up of proteins. There are different types of channels for different molecules like water channels and ion channels (Sodium channels, Calcium channels). Topic No 06 Cell Wall It is the outermost covering in many organisms surrounding the cell membrane. Prokaryotic cells, fungi and plant cells have a cell wall around their cell membrane. Cell wall makes the outermost covering in these organisms. Cell wall is tough in comparison to cell membrane; it is a rigid structure. Cell wall in plants consists of cellulose, hemicellulose, and pectin. Fungal cell walls consist of a long polymer called, chitin. Prokaryotic cell wall consists of a polymer, called peptidoglycan. Functions of the cell wall are protection, shape, strength and support. Plant cells have 2 types of cell wall, primary cell wall and secondary cell wall. Primary wall consists of mainly cellulose, hemicellulose and pectin. Secondary cell wall contains cellulose and some other molecules like lignin which make it stronger structure. Figure 6.1 Primary Cell Wall Figure 6.2 Secondary Cell Wall Cytoplasm Cytoplasm is a semitransparent substance present between plasma membrane and the nucleus. It contains water in which organic (e.g. proteins, carbohydrates) and inorganic materials (e.g. salts), which are partially or fully dissolved in this solution. Cytoplasm provides space for metabolic reactions (e.g. glycolysis). It also provides space for functioning of organelles and metabolic reactions. Topic No 07 Cytoskeleton Cytoskeleton is the skeletal framework of the cell – a network of filaments and tubules. There are three types of cytoskeletal elements called microfilaments, intermediate filaments and microtubules. Microfilaments are the smallest in their diameter. These help in movement of organelles and the cell. These consist of helical chains of a protein called actin e.g. in muscle cells these are highly modified. Intermediate filaments are intermediate in size. These consist of different proteins belong to a protein family called keratins. These filaments help in maintaining shape and placement of the cell and its various parts. These also provide protection to various parts of the cell particularly to the nucleus. Microtubules are largest in diameter, these filaments consist of a protein called tubulin which makes dimers and then long and large hollow tubes. These help in movement of the organelles inside the cells and also in movement of the cell itself. Cilia and flagella consist of microtubules. These filaments help in maintaining shapes of organelles and cell. For example these make the nuclear lamina, a layer that maintain the shape of the nucleus and give it support. Figure 7.1 Cytoskeletal fibers Topic No 08 Cell Organelles These are sub-cellular structures that perform a particular function. These include nucleus, mitochondria, endoplasmic reticulum and many more. Nucleus It is the organelle that contains genetic material. It is present in the center in animal cells usually. In plant cells, it is present on a side due to presence of a large vacuole. Nucleus is covered by nuclear membrane with nuclear pores. It is filled with a fluid called nucleoplasm. It also contains a denser body called nucleolus which is involved in ribosomal RNA production. Genetic material is present inside the nucleus in most of the eukaryotic cells, though, some cells have extra nuclear DNA. DNA is present in the form of chromosomes, which are visible during cell division. Ribosomes: Ribosomes are the protein making machinery of the cells. These are present free in cytoplasm or attached to endoplasmic reticulum. A large number of ribosomes are present in cells. Eukaryotic ribosomes are slightly different than prokaryotic ones in their size. Figure 8.1 Structure of Ribosome Mitochondria Mitochondria are called power house of the cell. These make energy for the cells in the form of ATP (Adenosine Tri Phosphate). ATP is the biological or chemical form of energy. Mitochondria have a double membrane, one is called outer and the other is inner membrane. Mitochondria are filled with matrix containing circular DNA molecule and other molecules including the enzymes. Mitochondria are self-replicating organelles. Figure 8.2 Structure of Mitochondria Plastids Plastids are the double membrane bound organelles, present in plants and in the other organisms which are producers such as algae. These are of three main types: ▪ Chloroplasts are present in the green parts of plants. These are green in color and their color is due to chlorophyll, the green pigment. These help in photosynthesis. ▪ Chromoplasts are the organelles present in the fruits and flowers of the plants. Beautiful colors of fruits and flowers are due to presence of Chromoplasts which contain red, yellow, orange and more colored pigments. ▪ Lecuoplasts are the plastids present in the roots and tubers. These are colorless pigments and their function is to store various materials in the roots and tubers, e.g., potatoes. Plastids have a double membrane system. Their membranes are called outer membrane and inner membrane. They have a membrane system called thylakoid. Stacks thylakoids are called grana. They have a matrix inside inner membrane which is called stroma. These organelles like mitochondria have their own circular DNA. These are self-replicating organelles. Figure 8.3 Structure of a Chloroplast (A kind of Plastid) Endoplasmic Reticulum Endoplasmic reticulum (ER) is a network of interconnected channels present inside the cells. This is of two types: Rough ER and Smooth ER. Rough endoplasmic reticulum is the rough due to the ribosomes present on its surface. This type is involved in protein modification. The other type is free of ribosomes so that shape of this one is smooth giving it its name. SER is involved in metabolism of lipids and carbohydrates. Figure 8.4 Endoplasmic reticulum Golgi Apparatus This is also called Golgi bodies or Golgi complex. This is also very important organelle of the cells. It was discovered by Camillo Golgi. Golgi apparatus consist of flattened disks called cisternae which are associated with endoplasmic reticulum. These are called the post office of the cell because these pack materials in the form of vesicles. For example the proteins formed by the ribosomes and modified by the endoplasmic reticulum enter in the cisternae and here these are packed in the vesicles and transferred to the part of the cell where these are required or secreted out of cells. Centrioles These are hollow and cylindrical bodies present near the nucleus of the animal cells. It is also present in some lower plants. A pair of Centrioles is collectively called centrosome. Their function is during cell division. These make the spindle fiber during cell division in animal cells. Vacuoles Vacuoles are membrane bound organelles present in most of the cells. Their major function is storage of various materials including food materials to waste materials. If these store food then these are called food vacuoles. Their size is from small to very large in different cells according to the requirements of the cells. In mature plant cells a single large vacuole is present. Contractile vacuole in unicellular fresh water organisms helps in removal of water from the body. Lysosomes Lysosomes are membranous sacs filled with enzymes. Lysosomes are spherical bag like structures that are bound by a single layer membrane. These are found in all eukaryotic cells and act as 'garbage disposal' or the 'digester' of the cell. These act as disposal system of the cell. They break down complex proteins, carbohydrates, lipids and other macromolecules into simpler compounds. These simple compounds are returned to the cytoplasm and are used as new cell building materials. They are used for digestion of cellular waste products, dead cells or extracellular material such as bacteria. Topic No 09 Genetics Genetics is a branch of biology in which we study the modes of inheritance that how genetics information is passed from one generation to next. Historic Perspective It has been long recognized that children inherit features from their parents. Humans were breeding 5000 years ago, these people recognized the genetic information is passed from one generation to next children offspring take features and characters from their parents. The prevailing view of hereditary was blending which basically implies that information once it is passed on to next generation it does not separate. For example we mix two dyes blue and red we will get purple dye. Once we formed the purple dye we cannot take out red or blue color separately. Mendel did a lot of experiments which proved that the principle of blending is not valid. Like If purple dye wants to reproduce purple dye can only contribute purple color to its offspring. Mendel proved that it was incorrect when the purple dye is going to reproduce it will give again the two characteristics the Red and Blue color will separate in the offspring. Important Definitions: A character is an observable feature such as flower color. A trait is a particular form of a character such as white flower. A heritable charter trait is one that is passed from parent to offspring. Topic No 10 What is Genetics? ▪ Genetics is the study of genes, heredity and variation. ▪ It is considered as a field of Biology. ▪ The principles of heredity were explained by Gregor Mendel in 1866. Figure 10.1 Figure 10.2 Gregor Mendel used garden pea as experimental plant for formulating the laws of heredity, following were the properties of garden pea. ▪ Seed in a variety of shapes and colors. ▪ Self, cross pollinate. ▪ Takes up little space. ▪ Short generation time ▪ Produces many offspring. Topic No 11 Common Genetics Terminologies What is Character: A heritable feature (skin color, height etc). What is Trait: variant for a character (i.e. brown, black, white etc). What is True-breed: all offspring of same variety. Different generations of a cross can be P generation (parents) F1 generation (1st filial generation) F2 generation (2nd filial generation) Pure Cross: A cross between a true breed plant/animal with another true breeds plant/animal is called pure cross Hybrid Cross: True breeding X True breeding WW X ww F1 generation X F1 generation Ww X Ww Genotype and Phenotype: Genetic make-up of an organism is called Genotype while physical appearance of an organism is called Phenotype. Figure 11.1 Genotype & Phenotype Dominant and Recessive: when one characteristic expresses itself over the other i.e. round over wrinkled was dominant in Gregor Mendel experiments while the trait that does not show through in the first generation is called as recessive trait i.e. wrinkled. Topic No 12 In this lesson we look at Mendel’s experiment and what was the Mendel’s question, how he designed the experiment and how he was able to conclude the experiment. Mendel’s Subject: Mendel was working on pea plants (Garden pea plants), easy to cultivate, short life-span and several other characteristic. Flowers are reproductive organ of these plants, as most flowers have both male and female reproductive organs. The ovary is the female sex organ as shown in Figure 12.1. The stigma is the part of the flower where pollen lands and extends a tube, pollen is sperm equivalent of the plants. Anthers are the male sex organ, these are attached to stamen which are filamentous structures and anthers produce pollen which can then land on stigma and fertilize the egg inside the ovary. So, Mendel use these plants it was easy to grow them as mentioned. Figure 12.1 Anatomy of a pea flower Mendel’s Technique: Mendel’s used a paint brush he rubbed it on the anthers and he collected pollen in the tip of the brush and later dusted on the stigma of the female part of the flowers, as a result the pollen fertilize the egg and resulted in seed which he could grow and look as the next generation of the cross of the breeding experiment between two types of flowers. Monohybrid Cross: “A monohybrid cross is the hybrid of two individuals with homozygous genotypes which result in the opposite phenotype for a certain genetic trait.” “The cross between two monohybrid traits (TT and tt) is called a Monohybrid Cross.” Monohybrid cross is responsible for the inheritance of one gene. It can be easily shown through a Punnett Square. Monohybrid cross is used by geneticists to observe how homozygous offspring express heterozygous genotypes inherited from their parents. Topic No 13 Mendel’s First Law of Segregation Mendel developed his genetic laws in 1866, using pea plants, but they were not rediscovered in the scientific literature until 1900. Mendel stated his laws in terms of "chance" or probability. In modern terminology, Mendel's First Law states that for the pair of alleles an individual has of some gene (or at some genetic locus), one is a copy of a randomly chosen one in the father of the individual, and the other if a copy of a randomly chosen one in the mother, and that a randomly chosen one will be copied to each child. He also said that each allele has an equal chance to be the one copied, and that the copying of alleles to different offspring or from different parents are independent. This very basic set-up underlies all of genetics. Since a parent has two alleles of each gene, the parent has 0.5 chance of passing one of the alleles to the offspring. For example, if a parent has a normal CF gene, and a mutant CF gene, he or she has a 0.5 chance of passing the mutant gene to the offspring. Likewise, he or she has a 0.5 chance of passing the normal gene to the offspring. Segregation of the sex chromosomes works the same way. In the case of X-linked genes, the mother has two alleles for each X-linked gene, therefore she has 0.5 chance of passing one of them to an offspring. The father, on the other hand, only has one X, and he only passes it to his daughters. Therefore, the chance that he will pass an allele of an X-linked gene on to a daughter is 1, and that he'll pass it to a son is 0. He passes his Y chromosome to each son. Experiment: Figure 13.1 Mendel’s Experiment (1st Law of Segregation) Topic No 14 Mendel’s Second Law OR Mendel’s Law of Independent Assortment Mendel's 2nd law states that during gamete formation the segregation of each gene pair is independent of other pairs. Mendel's 2nd law is often referred to as the principle of independent assortment. Both of Mendel's laws are about segregation, which is the separation of allele pairs. The law states that the separation of one pair of alleles isn't related to the separation of other pairs of alleles, and so is very important in Mendelian genetics. The only time there is an exception to this rule is when linkage is involved. Reference: Hartl D.L. and Ruvolo M. (2011) Genetics: Analysis of genes and genomes, page 91, 8th Edition, Published by Jones and Bartlett Learning Topic No 15 Diversity of Cells and Mitosis Animal Vs. Plant Cell Animal Cell o Cell membrane is outer most layer o Many small vacuoles o Nucleus in the center o Roughly round and/or have o variable shapes o Plastids are absent o Cilia present o Centrioles present Plant Cell o Cell wall is present outside the cell membrane o Have a large central vacuole o Nucleus on a side o Roughly rectangular shape due to presence of cell wall o Plastids are present o Cilia rarely present o Centrioles absent in most of the plants Figure 15.1 Animal and Plant Cell Prokaryotic Cell Vs. Eukaryotic Cell Prokaryotic Cell ▪ No defined nucleus but a nucleoid region ▪ No membrane bound organelles ▪ Small in size (e.g. 1-2 microns in bacteria) ▪ Cell wall consist of peptidoglycan (a polymer of amino acids and sugars) Eukaryotic Cell ▪ Defined nucleus with nuclear membrane ▪ Membrane bound organelles are present ▪ Larger than prokaryotic cells on average (20 microns of animal cells) Cell wall consist of cellulose (plants) and chitin (fungi) Topic No 16 Cell Division Division is the property of cells. Cells have to divide for various purposes, for reproduction or for growth and repair of the tissues. There are two types of the cells called somatic cells and germ line cells. Somatic cells are those which make all the tissues of the body except for some cells which are involved in reproduction. Few cells involved in reproduction and are meant for making gametes for reproduction. There are two types of the cell divisions called Mitosis and Meiosis. Mitosis is the division of the somatic cells and also serves as means of asexual reproduction. Meiosis is however involved in division of the germ line cells. Figure 15.2 Stages of Mitosis in Onion Root Tip Cells Cell Cycle Cells go through a cyclic process in which they pass by various phases over time. These phases include a phase of rest, high metabolic activity and division. It is also defined as the sequence of events or a cyclic process between divisions of the cell. The cell cycle consists of the following phases: ▪ Interphase (consist of Gap 1 phase, Synthesis Phase, Gap 2 phase) ▪ M Phase (Mitosis or Division Phase) ▪ Resting or G0 Phase (Gap 0, read as Gap not) Interphase Interphase consist of following phases. This is normally called as rest phase but actually it is a phase of high metabolic activity. Gap 1 Cells increase in size in Gap 1. They produce materials required for DNA synthesis. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis. Synthesis phase DNA replication occurs during this phase. An S phase check point checks that whether the synthesis of DNA is correctly done or not. If everything is correct cell continue to the next phase and if not then cell wither have to die or correct its errors. Gap 2 During the gap between DNA synthesis and mitosis, the cell will continue to grow. Cell prepares all the materials required for division, e.g. microtubule proteins. The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis) phase and divide. Cell Division or M- Mitosis Phase Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells. A checkpoint in the middle of mitosis (called Metaphase Checkpoint) ensures that the cell is ready to complete cell division. Resting or G0 (Gap 0) A resting phase in which the cell has leaves the cycle and has stopped dividing. G0 starts from G1 and cell may sustain in G0 may be for years. Topic No 17 Mitosis Mitosis is the cell division that results in two daughter cells which are like each other. Though, mitosis is a term that is used to describe the nuclear division. Cell division consists of following phases: Karyokineses – division of nucleus ▪ Divided into Prophase, Metaphase, Anaphase and Telophase Cytokinesis – division of cell ▪ Different in animal and plant cells Karyokinesis Prophase o Chromatin material condenses and chromosomes becomes visible o Each chromosome is replicated (duplicated) and consist of two sister chromatids o At the end of the stage nuclear membrane disappear o Centrioles move towards poles of the cell and microtubules starts forming Metaphase o Chromosomes arrange themselves at the equator o Kinetochores are attached to the microtubules Anaphase o Chromosomes starts moving towards the poles o The sister chromatids separate from each other, so each sister chromatid move towards a pole Telophase o Chromosomes (sister chromatids) reaches at the poles o Nuclear material starts de-condensing again o Nuclear membrane starts forming The nuclear division is complete, next is the Cytokinesis or division of the cell. Cytokinesis Cytokinesis in plant and animal cells is different from each other. In plant cells, a cell plate starts forming in the center of the cell and moves towards sides. The cell plate divides the cell into two cells. The cell plate consists of material produced by Golgi bodies in vesicles. This material contains the cell membrane and cell wall components. Cytokinesis in animal cells occurs in a different way. In animal cells a cleavage furrow is formed by invagination of cell membrane. This process occur with the help of cytoskeleton (microfilaments particularly). The furrows divide the cell into 2 daughter cells. Figure 17.1 Cleavage in Animal Cells Topic No 18 Importance of Mitosis Mitosis is the cell division which helps in development and growth processes of the cells and hence the tissues. Development of new cells is a requirement in many parts of the body like epithelia usually keep growing. Replacement of cells and wound healing is another requirement of the organisms which also need new cells. Regeneration is a capability of some organisms. They came make their lost parts. This process also needs mitosis. Mitosis also serves as a means of asexual reproduction in various organisms. Errors in Mitosis Cells divide correctly most of the times because of many check points at various phases but sometimes it may go wrong. If it happens due to any reason the result is usually serious problems. Uncontrolled division of the cells may result into abnormal tissue growth and cancer. Topic No 19 Cell Division – Meiosis (Reduction Division) MEIOSIS - CONSIST OF TWO DIVISIONS Meiosis occur in germ line cells to make gametes, gametes are formed for sexual reproduction. Meiosis is also called reduction division because it results in four daughter cells which are haploid. We know that chromosomes occur in pairs e.g. human have 46 chromosomes in 23 pairs which is called a diploid number. The chromosomes in each pair are called homologous because these are like each other and are complementary to each other. Meiosis makes cells that have a half number of the chromosomes in each daughter cell called a haploid number also. Diploidy and Polyploidy The condition of having two sets of chromosomes is called diploidy (2N number of chromosomes). The gametes formed by meiosis, hence, are called haploid (N number of chromosomes). For example, in humans 2N is 46 and N is 23 in each gamete, when gametes combine in fertilization the chromosome number is retained to 2N. Some plants have more than two sets of chromosomes and are called polyploids. This characteristic is called polyploidy. Figure 19.1 Homologous Chromosomes (A) Before Replication; (B) After Replication Phases of Meiosis ▪ Meiosis I o The reduction division o Chromosome number becomes half in each daughter cell (N) ▪ Cytokinesis I ▪ Meiosis II o Just like mitosis o Chromosome number remains same in daughter cells ▪ Cytokinesis II Topic No 20 Meiosis I ▪ Meiosis I consist of following phases: o Prophase I o Metaphase I o Anaphase I o Telophase I Stages of Meiosis I Prophase I is a long phase in meiosis. It consist of following stages: o It is marked by the pairing of homologous chromosomes (synapses) and recombination (exchange of the parts of chromosomes. o Paired chromosomes are called Bivalents or Tetrads. Figure 20.1 Chiasmata formation to Recombination Metaphase I o Chromosomes attach to the spindle fibers by kinetochore, one kinetochore per a chromosome and not per chromatid. o One homologue on each side so there is a 50-50 chance to get each parents chromosome. Anaphase I o The chromosomes move towards the poles. o A homologue, consist of two chromatids moves and sister chromatids do not separate. The result is half number of chromosomes towards each pole. Topic No 21 Telophase I and Cytokinesis o Chromosomes reach at poles, half on each side. o Cell divides and then starts meiosis II. Figure 21.1 Prophase I, Metaphase I & Anaphase I Events of Meiosis II ▪ Karyokinesis o Prophase II o Metaphase II o Anaphase II o Telophase II ▪ Cytokinesis o Each cell divides into two cells. At the end of meiosis each cell divides into 4 haploid cells. This division is different in males and females. In females, after first meiotic division (meiosis I), cytoplasm is unequally distributed and one large and other small cell are produced. The small cell is called a polar body. Then both of these cells carry out meiosis II. Polar body divides into 2 polar bodies. The large cell however is divided into one ovum (large) and another polar body. So that meiosis results into 1 ovum and 3 polar bodies. In males, both meiotic divisions’ results into equal sized cells called sperms. Comparison of Mitosis and Meiosis Mitosis o Cell divides into 2 daughter cells o Alike in males and females o Chromosome number remains equal (2N) in daughter cells Meiosis o One cell divides into 4 daughter cells o Different in males and females Chromosome number becomes half (N) in daughter cells Topic No 22 Importance of Meiosis Major advantage of meiosis is the genetic variations by recombination (crossing over). During prophase I of the meiosis crossing over takes place which result in genetic recombination. When gametes combine to make a zygote, more variations arise. This variation assures new combinations resulting in increase in adaptability of the organism. Errors in Meiosis Meiosis is a well regulated process but sometimes errors may arise which may lead to mostly serious disorders. The common cause of disorders in non-disjunction of the homologous pairs of chromosome abnormally. This may results into unequally distributed chromosomes in the gametes and when these fertilize, they give rise to individuals with disorders. For example, in Down’s syndrome the affected individual have 3 homologues in the 21st pair of chromosomes. Figure 22.1 Non-disjunction results into abnormal gametes Non-disjunction results into abnormal gametes, e.g., in above diagram N and N are normal and N +1 or N -1 are abnormal gametes Twins There are two types of twins. ▪ Fraternal twins o These are produced by separate eggs (also called dizygotic twins)/. These are produced if two eggs are released and fertilized. o These are genetically different from each other and may be both males, females and both male and female. ▪ Identical twins o These are produced by division in the same egg (also called monozygotic twins). These are produced by division in the zygote. o These are genetically same / identical and have same characteristics. Both of these are either males or females. Environment and twins Environment may affect even the identical twins. Identical twins may also have different characteristics if they are brought up in different environments. We know that the genes interact with environment to produce various characteristics. This characteristic also affects the twins. Topic No 23 Molecular Biology is the study of biological molecules related to genes, gene products and heredity. In the present age, world is in the midst of two scientific revolutions. One is information technology and the other is Molecular Biology. Both deal with the handling of large amounts of information. Molecular Biology has revolutionized the biological sciences as well especially in the fields of Health Sciences and Agricultural Sciences. Contribution of Molecular Biology ▪ The almost complete sequence of the DNA molecules comprising the human genome was revealed in the year 2003. So, in theory, science has made available all of the genetic information needed to make a human being. However, the function of most of a human’s approximately 35,000 genes remains a mystery. ▪ The other main arena where molecular biology has a massive impact is agriculture. New varieties of genetically engineered plants and animals have already been made and some are in agricultural use. ▪ So you can well imagine that how much important is this subject for you and for the economy of Pakistan. Topic No 24 Heredity Information Flow Reproducing itself is a property of life. Transferring characteristics to next generation is a property of living organisms. Heredity information flow in living organisms is carried out by the genes. The “Chromosome theory of heredity” states that the genes are present on chromosomes and are responsible for the transfer of characteristics from generation to generation. Genes are present in the form of DNA molecules, organized in a structure called chromosome (chromatin material). Chromatin - the Genetic Material Genetic material is present in the nucleus of the cell in eukaryotes and in the nucleoid region in prokaryotes. These are called chromatin material. Chromatin material is not visible during interphase (non-dividing state) of the cell. These become visible during cell division due to condensation of chromosomes. Functions of Genetic Material There are some important properties of genetic material, which are following: ▪ It replicates itself. ▪ It regulates the growth and development of the organism. ▪ It allows the organism to adapt to the environmental changes. Chromosomes - DNA – Genes Chromosomes consist of DNA molecule associated with proteins. In chromosomes, DNA is wrapped around proteins. Few of these proteins are called histones and few others. DNA is associated with histone and non-histone proteins in a chromosome. Introduction of DNA and Gene DNA is a macromolecule (large molecule) organized in structure chromosome. In prokaryotes DNA is a circular molecule. In eukaryotes it is a long linear molecule. Mitochondria and chloroplast also have their own circular DNA molecules. Gene is a length of DNA that codes for a peptide or protein. So that gene is a part or length of DNA. Condensation of Genetic Material Chromatin material condenses during prophase of mitosis in the form of chromosomes. Chemical analysis shows that chromosome consist of DNA and proteins. DNA is a long molecule about 2nm thick running continuously within each chromosome. Chemical analysis shows that DNA is acidic in nature. The Structure of Chromosomes Chromosome consists of a DNA molecule wound around proteins. DNA in human cell (All chromosomes) is about 6 feet long, packed in a microscopic nucleus of a cell. In one human chromosome, it is 1.7-8.5 cm long. How is this possible? The answer is “coiling” and “super-coiling”. The chromosome consists of highly condensed structure. If we can open this like a thread, then the long thread will appear like a flower like structure called solenoid which consists of many smaller units. These small units are called “nucleosomes”. A nucleosome is a length of DNA coiled around a set of proteins. The DNA coils around histones twice, which is up to 200 base pairs long. Two nucleosomes are connected to each other by a length of DNA, which is called “linker DNA” (up to 80 base pairs long). Figure 24.1 Chromosome coiling and nucleosome Figure 24.2 Two nucleosomes Topic No 25 Chemical Composition of DNA DNA is a complex macromolecule (large molecule). DNA stands for Deoxyribose Nucleic Acid. The smallest unit of DNA is called a “nucleotide”; nucleotides join to make polynucleotide. We can say that DNA consists of nucleotides joined together. Nucleotides Each nucleotide consists of: I. Deoxyribose sugar II. Phosphate group III. Nitrogenous base Figure 25.1 Structure of Nucleotide There are four nucleotides based upon four different nitrogenous bases attached to them. Nitrogenous bases are of two types: purines and pyrimidine. Purines include two bases Adenine and Guanine which have a double ringed structure. Pyrimidine bases include the other ones called Thymine and Cytosine that have single ringed structure. Topic No 26 Mechanism of Gene Action Genes express themselves by making proteins. Making the proteins by DNA occur by two processes called transcription and translation. Transcription is formation of a form of RNA from DNA called messenger RNA (mRNA). mRNA is formed inside the nucleus in eukaryotes and in nucleoid region in prokaryotes. The next process is translation, which is formation of a protein or peptide by mRNA with the help of another organelle called ribosome. Replication is another function of DNA. It is doubling of DNA molecule to make two copies of itself. Replication occurs before cell division to make copies of DNA for the daughter cells. Genetic code is a term used for the parts of DNA that code for proteins. A codon is a 3 nucleotides code for an amino acid, i.e., codon is a 3 nucleotide set of DNA molecule that codes for a protein. Transcription and Translation ▪ Transcription = DNA mRNA ▪ Translation = mRNA Protein The following scheme is called the central dogma of molecular biology / genetics DNA RNA Protein Transcription DNA mRNA The process of transcription involves an enzyme called RNA polymerase. One strand of DNA act as the template strand which is actually coded into the mRNA. Steps of transcription o RNA polymerase identifies and attaches to a region called promoter on the DNA upstream the gene. RNA polymerase open the double helix chain which results in the formation of transcription bubble. Topic No 27 Transcription Process ▪ RNA polymerase moves on the gene, the helix unwinds and make a complementary strand of RNA. This strand of mRNA protrudes out of transcription bubble. ▪ At end of the gene there is a stop sequence. Usually it is a series of GC base pairs followed by a series of AT base pairs. ▪ These sequences make a hair pin loop like structure which stops RNA polymerase from transcribing. ▪ Thymine is coded as uracil in mRNA. Transcription in Prokaryotes and Eukaryotes In prokaryotes, the mRNA directly moves into cytoplasm and its translation starts because there is no nuclear membrane, nucleoid region is continuous with cytoplasm. In eukaryotes, mRNA formed moves out of nucleus through nuclear pores and then it is translated in the cytoplasm with the help of ribosomes. Modification of mRNA in eukaryotes mRNA in eukaryotes has to travel from nucleus to cytoplasm, to protect it from the action of nucleases (the DNA cutting enzymes) and proteases (protein cutting enzymes), it is modified. On its 5’ end a cap of 7 methyl GTP is added; while on the 3’ end a poly A tail is added. Introns are also removed. Introns are DNA sequences in the eukaryotes which are non-coding and should be removed from the mRNA. The coding regions are called exons. Figure 27.1 mRNA in eukaryotes have regions – exons and introns Topic No 28 Translation mRNA Protein The process of translation consists of three major steps: initiation, elongation, and termination. Steps of Transcription ▪ In prokaryotes, translation starts while transcription is going on because there is no barrier between nucleoid and cytoplasm. ▪ In eukaryote, first introns are removed. Process of Translation Initiation: ▪ The mRNA binds to the small unit of ribosome. ▪ The large ribosomal subunit has 3 binding sites called E (Exit), P (Peptidyl), and A (Aminocyl). ▪ When the first codon (triplet code) is aligned at the P site then the large ribosomal subunit attaches to the small subunit. ▪ A tRNA carrying the amino acid methionine attaches to the start codon (AUG) on the messenger RNA. Elongation: ▪ A tRNA with its amino acid attaches to the A binding site. ▪ Peptide bond formation occurs between the methionine and the amino acid carried at the A binding site. ▪ Ribosome moves in the 3' direction down the messenger RNA by three bases, shifting the tRNA and polypeptide chain to the P Binding site. ▪ The A binding site is open and a vacant tRNA (without amino acid) is in the E binding site. ▪ Now, the next tRNA brings another amino acid and bind to A site. ▪ A peptide bond is formed between the second and this new (thirs) amino acid. ▪ Ribosome moves in 3’ direction and the vacant tRNA is released from the E site. ▪ This process continues until a stop codon arrives on mRNA. A Releasing factor comes and binds to the A site in place of stop codon. The polypeptide chain separates from tRNA and ribosome. Then ribosomal unit disassemble again. mRNA molecule also released which has been coded. Topic No 29 Nucleotide Nucleic acids are important group of biomolecules which are responsible for storage & transmission of hereditary information. Like proteins and polysaccharides, nucleic acids are also polymeric compounds. The repeating units in the nucleic acids are Nucleotides. There are two main types of nucleic acids, Deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) Figure 29.1 DNA Vs. RNA Topic No 30 The chemical structure of DNA DNA is a polymer of Deoxyribonucleotides. It is composed of three components: Deoxyribose, Nitrogenous Base, Phosphoric acid. Figure 4 Detailed about Chemical Structure of the DNA Topic No 31 Chemical Composition of RNA RNA (Ribonucleic acid) is a polymer of ribonucleotides. Each ribonucleotide is composed of three components: I. A ribose sugar II. A Nitrogenous Base III. A Phosphoric acid Figure 31.1 Chemical Composition of DNA and RNA Topic No 32 Chemical Composition of Protein (Summary) ▪ Proteins are polymers of amino acids ▪ They range in size from small to very large ▪ All the proteins are made up of twenty different types of amino acids. So these amino acids are called standard amino acids ▪ In a protein molecule, each amino acid residue is joined to its neighbor by a specific type of covalent bond which is called Peptide Bond ▪ Amino acids can successively join to form dipeptides, tripeptides, tetrapeptides, oligo peptides and polypeptides Figure 32.1 Amino Acid Structure Topic No 33 Carbohydrates: Sugar Polymers They act as source of energy that can be transported and have structural role as Monosaccharides, Disaccharides, Oligosaccharides and Polysaccharides. Monosaccharides Monosaccharides, also called simple sugars, are the simplest forms of sugar and the most basic units from which all carbohydrates are built. They are usually colorless, water-soluble and crystalline solids. Monosaccharides are produced by plants, all living cell have glucose. Figure 33.1 Structure of Glucose, Fructose and Galactose Topic No 34 Carbohydrates: Glycosidic Linkages Many of the carbon atoms to which hydroxyl groups are attached are chiral centers, which give rise to the many sugar stereoisomers found in nature. Stereoisomerism in sugars is biologically significant because the enzymes that act on sugars are strictly stereospecific. It is as difficult to fit the wrong sugar stereoisomer into an enzyme’s binding site as it is to put your left glove on your right hand. Monosaccharaides are colorless, crystalline solids that are freely soluble in water but insoluble in nonpolar solvents. Most have a sweet taste. In the open-chain form, one of the carbon atoms is double-bonded to an oxygen atom to form a carbonyl group; each of the other carbon atoms has a hydroxyl group. If the carbonyl group is at an end of the carbon chain (that is, in an aldehyde group) the monosaccharide is an aldose; if the carbonyl group is at any other position (in a ketone group) the monosaccharide is a ketose. The simplest monosaccharaides are the two three-carbon trioses: glyceraldehyde, an aldotriose, and dihydroxyacetone, a ketotriose. The aldopentoses D-ribose and 2-deoxy-D-ribose are components of nucleotides and nucleic acids. All the monosaccharides except dihydroxyacetone contain one or more asymmetric (chiral) carbon atoms and thus occur in optically active isomeric forms. The simplest aldose, glyceraldehyde, contains one chiral center (the middle carbon atom) and therefore has two different optical isomers, or enantiomers. One of the two enantiomers of glyceraldehyde is, by convention, one of these two forms is designated the D isomer, the other the L isomer. As for other biomolecules with chiral centers, the absolute configurations of sugars are known from x-ray crystallography. To represent three-dimensional sugar structures on paper, we often use Fischer projection formulas. In Fischer projection formulas, horizontal bonds project out of the plane of the paper, toward the reader; vertical bonds project behind the plane of the paper, away from the reader. The carbons of a sugar are numbered beginning at the end of the chain nearest the carbonyl group. Two sugars that differ only in the configuration around one carbon atom are called epimers; D-glucose and D-mannose, which differ only in the stereochemistry at C-2, are epimers, as are D-glucose and D-galactose (which differ at C-4). Some sugars occur naturally in their L form; examples are L-arabinose. In aqueous solution, aldotetroses and all monosaccharides with five or more carbon atoms in the backbone occur predominantly as cyclic (ring) structures in which the carbonyl group has formed a covalent bond with the oxygen of a hydroxyl group along the chain. The formation of these ring structures is the result of a general reaction between alcohols and aldehydes or ketones to form derivatives called hemiacetals or hemiketals. Two molecules of an alcohol can add to a carbonyl carbon; the product of the first addition is a hemiacetal (for addition to an aldose) or a hemiketal (for addition to a ketose). Addition of the second molecule of alcohol produces the full acetal or ketal, and the bond formed is a glycosidic linkage. The reaction can produce either of two stereoisomeric configurations, denoted α and β. Isomeric forms of monosaccharides that differ only in their configuration about the hemiacetal or hemiketal carbon atom are called anomers, and the carbonyl carbon atom is called the anomeric carbon. Topic No 35 Lipids Lipids are organic compounds that contain hydrogen, carbon, and oxygen atoms, which form the framework for the structure and function of living cells.” Properties of Lipids Lipids are oily or greasy nonpolar molecules, stored in the adipose tissue of the body. Lipids are a heterogeneous group of compounds, mainly composed of hydrocarbon chains. Lipids are energy-rich organic molecules, which provide energy for different life processes. Lipids are a class of compounds characterized by their solubility in nonpolar solvents and insolubility in water. Lipids are significant in biological systems as they form a mechanical barrier dividing a cell from the external environment known as the cell membrane. Lipids are also the building blocks of many hormones and are an important constituent of the plasma membrane. Lipids include fats, oils, waxes, phospholipids, and steroids. Examples of Lipids Waxes Waxes are another biologically important category of lipids. Wax covers the feathers of some aquatic birds and the leaf surfaces of some plants, where its hydrophobic (water-repelling) properties prevent water from sticking to, or soaking into, the surface. This is why water beads up on the leaves of many plants, and why birds don’t get soaked through when it rains. Steroids Steroids are another class of lipid molecules, identifiable by their structure of four fused rings. Although they do not resemble the other lipids structurally, steroids are included in lipid category because they are also hydrophobic and insoluble in water. All steroids have four linked carbon rings and several of them, like cholesterol, also have a short tail. Many steroids also have an –OH functional group attached at a particular site, as shown for cholesterol below; such steroids are also classified as alcohols, and are thus called sterols. Cholesterol The most common steroid, is mainly synthesized in the liver and is the precursor to many steroid hormones. Cholesterol also serves as the starting material for other important molecules in the body, including vitamin D and bile acids, which aid in the digestion and absorption of fats from dietary sources. It’s also a key component of cell membranes, altering their fluidity and dynamics. Of course, cholesterol is also found in the bloodstream, and blood levels of cholesterol are what we often hear about at the doctor’s office or in news reports. Cholesterol in the blood can have both protective effects and negative effects on cardiovascular health. Topic No 36 Fatty acids may be saturated or unsaturated. In a fatty acid chain, if there are only single bonds between neighboring carbons in the hydrocarbon chain, the fatty acid is saturated. Saturated fatty acids are saturated with hydrogen; in other words, the number of hydrogen atoms attached to the carbon skeleton is maximized. When the hydrocarbon chain contains a double bond, the fatty acid is an unsaturated fatty acid. Most unsaturated fats are liquid at room temperature and are called oils. If there is one double bond in the molecule, then it is known as a monounsaturated fat (e.g., olive oil), and if there is more than one double bond, then it is known as a polyunsaturated fat (e.g., canola oil). Saturated fats tend to get packed tightly and are solid at room temperature. Animal fats with stearic acid and palmitic acid contained in meat, and the fat with butyric acid contained in butter, are examples of saturated fats. Saturated Fats Unsaturated Fats Contains a single bond. Contains at least one double bond. Not to be consumed more than 10 percent of total calories Not to be consumed more than 30 percent of total per day. calories per day. Excessive consumption leads to heart diseases. Good for consumption, but excessive may increase cholesterol. Increases low- density lipoproteins (LDL), which is called Increases High-density lipoprotein (HDL), which is as bad cholesterol. commonly known as good cholesterol and also reduce low-density lipoproteins (LDL). Would not spoil quickly. Spoil quickly. Foods sources of saturated fats are whole milk, butter, Foods sources of unsaturated fats are walnuts, flax, cheese, margarine, coconut oil, vegetable oil, meat, peanut, avocado, sunflower oil, soybean oil, fish oil, canola fried foods, etc. oil, red meat, etc. High melting point. Low melting point. Solid state in room temperature. Liquid state in room temperature. Topic No 37 Study of Omics ▪ Genomics: all the genes ▪ Pharmacogenomics choice of personalized medicine ▪ Nutri-genomics choice of best diet ▪ Toxicogenomics prediction of toxicity ▪ Epigenomics: all epigenetic changes in genome ▪ Transcriptomics: all the mRNAs ▪ Proteomics : all the proteins ▪ Interactomics : all interactions between all proteins ▪ Metabolomics (or metabonomics): all metabolites Genome and Genomics The complete set of DNA found in each cell is known as the genome and study is called as genomics. Proteome and Proteomics The complete set of proteins found in each cell is known as the proteome. Proteins concentration (and activity) may be different than gene expression due to post-translational modification Metabolomics The complete set of metabolites found in each cell is known as the metabolome. Use of high-throughput mass spectrometry to analyze the metabolic components of cell. Metabolomics Useful for determining the effects of the environment or gene transformation on the metabolism of the plants/animals. Figure 37.1 Important branches of Omics Conclusion Genomics, proteomics and metabolomics will give an integrated, wholistic view of the cell. Topic No 38 Genomics, Proteomics and Metabolomics Genome and Genomics The complete set of DNA found in each cell is known as the genome and study is called as genomics. Proteome and Proteomics The complete set of proteins found in each cell is known as the proteome. Proteins concentration (and activity) may be different than gene expression due to post-translational modification Metabolomics The complete set of metabolites found in each cell is known as the metabolome. Use of high-throughput mass spectrometry to analyze the metabolic components of cell. Metabolomics Useful for determining the effects of the environment or gene transformation on the metabolism of the plants/animals. Conclusion Genomics, proteomics and metabolomics will give an integrated, wholistic view of the cell. Can be used to monitor or modify organisms in a comprehensive way. Bioinformatics - the key to understand the plethora of information and modeling the cell. Topic No 39 Genome Informatics Introduction Genome sequencing provides the sequences of all the genes of an organisms. A major application of bioinformatics is analysis of full genomes that have been sequenced. Genomics: It is the study of all of a person’s genes (the Genome), including interactions of those genes with each other and with the person’s environment. Genome Informatics: Genome Informatics is the field in which computational and statistical techniques are applied to derive biological information from genome sequences. Genome Informatics includes method to analyze DNA sequence information and to predict protein sequence and structure. Genome Analysis: ▪ Sequencing ▪ Assembly ▪ Repeat identification and masking out ▪ Gene prediction ▪ Looking for EST and cDNA sequences ▪ Genome annotation ▪ Expression analysis ▪ Metabolic pathways and regulation studies ▪ Functional genomics ▪ Gene location/gene map identification ▪ Comparative genomics ▪ Identify clusters of functionally related genes ▪ Evolutionary modeling ▪ Self-comparison of proteome Conclusion Sequencing and analysis of full genomes paves the way for future discoveries. Different model organisms can help explore our Genome and what matters most for us. Topic No 40 Prokaryotic Genome Prokaryotes are the organism whose genetic materials (DNA) is not enclosed in nuclear membrane (No membrane bound organelles). Figure 40.1 Prokaryotic Genome First prokaryotic genome sequence was that of Hemophilus influenzae, which paved the way for sequencing of many other organisms. Figure 40.2 Features of representative prokaryotic genomes Conclusion Prokaryotes are simple genomes, easy models to study biochemistry and molecular biology of life processes. Sequencing is done on economically important organisms. Topic No 41 Eukaryotic Genome Eukaryotes have larger genome as compared to prokaryotes. Eukaryotes have tandem repeats, introns in their protein-coding genes, heterochromatin and euchromatin region. Figure 41.1 Eukaryotic Genome (An animal cell) Conclusion Eukaryotes are distinguished by the presence of prominent nuclei, have larger genome, tandem repeats and introns in their protein-coding genes. Topic No 42 Viral Genomes Genomes of Viruses ▪ Viral genomes can be ▪ ssRNA ▪ dsRNA ▪ ssDNA ▪ dsDNA ▪ Linear ▪ Ciruclar Viruses Genomes ▪ A viral genome is the genetic material of the virus. ▪ Also termed the viral chromosome. ▪ Viral genomes vary in size -few thousand to more than a hundred thousand nucleotides. Viruses with RNA Genomes ▪ Almost all plants viruses and some bacterial and animal viruses ▪ Genomes are rather small (a few thousands nucleotides) Viruses with DNA Genomes ▪ Often a circular genome ▪ lambda = 48,502 bp Replicative form of Viral Genomes ▪ All ssRNA viruses produce dsRNA molecules ▪ Many linear DNA molecules become circular Viruses and Kingdoms ▪ Many plants viruses contain ssRNA genomes. ▪ Many fungal viruses contain dsRNA genomes. ▪ Many bacterial viruses contain dsDNA genomes. Figure 42.1 DNA, RNA & RNA-DNA Viruses Genomes in Virions: The genomes of viruses can be composed of either DNA or RNA, and some use both as their genomic material at different stages in their life cycle. However, only one type of nucleic acid is found in the virion of any particular type of virus. Figure 42.2 Viruses with number of genes Genome of Pox Virus ▪ Linear dsDNA 130-375 kbp; covalently closed termini. ▪ Large hairpin structure at eah terminus - up to 10 kb total at each end is repeat sequence. ▪ Encode 150-300 proteins. ▪ Coding regions are closely spaced, no introns. Coding regions are on both strands of genome, and are not tightly clustered with respect to time of expression or function. Topic No 43 Bacterial Genomes Genomes of Bacteria ▪ Small organisms carry high coding density (85-90%) ▪ 1 gene per 1000 bases in prokaryotes ▪ Large variation in genome size between bacteria Genomes of Bacteria – Large Variation ▪ Tremblaya princeps 140kb, 121 coding sequences ▪ Sorangium cellulosum ▪ 14000kb ▪ 11599 coding sequences Figure 43.1 Comparison of regulatory genes in bacterial genomes Figure 43.2 Distribution of genes among selected bacterial genomes and their sizes Conclusion ▪ Small organisms carry high coding density. ▪ Large variation in genome size between bacteria Topic No 44 Polymerase chain Reaction Molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production and health, environment and the industry related to these areas. Among the applications of molecular techniques is important to highlight the use of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral, bacterial, parasitic and fungal agents. This technique was developed by Kary Mullis in the mid 80's and since then it has been considered as an essential tool in molecular biology which allows amplification of nucleic acid sequences (DNA and RNA) through repetitive cycles in vitro. The mechanisms involved in this methodology are similar to those occurring in vivo during DNA replication. The amplification of specific nucleic acid sequences, even in the presence of millions of other DNA molecules, is achieved by thermo-stable DNA polymerase enzyme (as the name of this technique suggests: “polymerase chain reaction”) and specific primers. Primers are short sequences of DNA or RNA (oligonucleotides) that initiate DNA synthesis. Reference: Polymerase Chain Reaction Edited by Dr. Patricia Hernandez-Rodriguez, Chapter # 8: Polymerase Chain Reaction: Types, Utilities and Limitations, Page 157-158. Topic No 45 Steps of PCR PCR is a biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time. A typical PCR consists of: Initial Denaturation: The reaction temperature is increased to 95 °C and the reaction is incubated for 2–5 min (up to 10 min depending on enzyme characteristics and template complexity) to ensure that all complex, double-stranded DNA (dsDNA) molecules are separated into single strands for amplification. Cycling: 1. 1. Denaturation: The reaction temperature is increased to 95 °C, which melts (disrupts the hydrogen bonds between complementary bases) all dsDNA into single-stranded DNA (ssDNA). 2. Annealing: In annealing short DNA molecules called primers bind to flanking regions of the target DNA. The temperature is lowered to approximately 5 °C below the melting temperature (Tm) of the primers (often 45–60 °C) to promote primer binding to the template. 3. Extension: The temperature is increased to 72 °C, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended.DNA polymerase extends the 3′ end of each primer along the template strands. Repeat: Steps 1–3 repeated (“cycled”) 25–35 times to exponentially produce exact copies of the target DNA. Final elongation: Single step is performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Final hold temperature: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction. Figure 44.1 Three steps of PCR─denaturation, annealing, and extension─as shown in the first cycle, and the exponential amplification of target DNA with repeated cycling. Topic No 46 Types of PCR Multiplex PCR Multiplex PCR is an adaptation of PCR which allows simultaneous amplification of many sequences. This technique is used for diagnosis of different diseases in the same sample. Multiplex PCR can detect different pathogens in a single sample. Also it can be used to identify exonic and intronic sequences in specific genes and determination of gene dosage. Nested PCR This PCR increases the sensitivity due to small amounts of the target are detected by using two sets of primers, involving a double process of amplification. The first set of primers allows a first amplification. The product of this PCR is subjected to a second PCR using the second set of primers. These primers used in the second PCR are specific to an internal amplified sequence in the first PCR. Therefore, specificity of the first PCR product is verified with the second one. The disadvantage of this technique is the probability of contamination during transfer from the first amplified product into the tube in which the second amplification will be performed. Reverse Transcriptase PCR (RT-PCR) This PCR was designed to amplify RNA sequences (especially mRNA) through synthesis of cDNA by reverse transcriptase (RT). Subsequently, this cDNA is amplified using PCR. This type of PCR has been useful for diagnosis of RNA viruses, as well as for evaluation of antimicrobial therapy. It has also been used to study gene expression in vitro, due to the obtained cDNA retains the original RNA sequence. The main challenge of using this technique is the sample of mRNA, because this is considered difficult to handle by low level and concentration of mRNA of interest and low stability at room temperature together with sensitivity to action of ribonucleases and pH change. Semi-quantitative PCR This technique allows an approximation to the relative amount of nucleic acids present in a sample, as mentioned above. cDNA is obtained by RT-PCR when sample is RNA. Then, internal controls (that are used as markers) are amplified. The markers commonly used are Apo A1 and B actin. Amplification product is separated by electrophoresis. Agarose gel is photographed after ethidium bromide staining, and optical density is calculated by a densitometer. The disadvantage of the technique is possibility of nonspecific hybridizations, generating unsatisfactory results. Control of specificity is performed using highly specific probes for hybridization. Topic No 47 Applications of PCR During the past 30 years molecular techniques have been under development, however these have had a rapid and tremendous progress in recent year. Among molecular techniques, PCR and its different variations are highlighted as the most commonly used in laboratories and research institutes. Thus, these have contributed to identification and characterization of several organisms and understanding of physiopathology of diverse diseases in human, animal and plant. Also these have provided clues for future research directions in specific topics with impact in public health such as genetics and biochemistry of antimicrobial resistance. The following describes some applications of PCR and its variants in studies in human medicine, forensic sciences, and agricultural science and environment. Medicine Molecular biology techniques, particularly PCR, have had a major impact on medicine. The versatility of molecular techniques has allowed advances and changes in all fields of medicine. The following is an overview of the main impacts generated for molecular biology in medical sciences. Clinical microbiology has been transformed with the use of molecular technology because it has generated a benefit to the patient affected by infectious diseases. Molecular biology has allowed the development of clinical microbiology because it has been possible to identify microorganisms that are difficult to culture, that have many requirements of laboratory or dangerous for laboratory personnel. These problems have been reduced with the implementation of molecular diagnosis that provides high sensitivity, specificity, precision and speed with one small sample. These applications are transforming and complementing the work of biochemists, immunologists, microbiologists and other health professionals who see in the molecular tools new alternatives for a rapid diagnosis of microorganisms as well as for the determination of multiple factors associated with antibiotic resistance thus expanding the knowledge of microbial epidemiology and surveillance at the genetic level. The usefulness of PCR in identification of microorganisms has led to the selection and quality assurance of blood that blood banks are using for patients with different pathologies. The incorporation of molecular techniques has been of great importance in the identification and characterization of many viruses, including influenza, which through a rapid, sensitive, and effective molecular diagnosis has allowed inclusion of early treatment to benefit patients and control of a high impact infection. Forensic science In forensic pathology, classic morphology remains as a basic procedure to investigate deaths, but recent advances in molecular biology have provided a very useful tool to www.intechopen.com Polymerase Chain Reaction: Types, Utilities and Limitations 165 research systemic changes involved in the pathophysiological process of death that cannot be detected by morphology. In addition, genetic basis of diseases with sudden death can also be investigated with molecular methods. Practical application of RNA analysis has not been accepted for post-mortem research, due to rapid decomposition after death. However, recent studies using variants of conventional PCR (qPCR and RT-PCR) have suggested that relative quantification of RNA transcripts can be applied in molecular pathology to research deaths ("molecular autopsy"). In a broad sense, forensic molecular pathology involves application of molecular biology in medical science to investigate the genetic basis of pathophysiology of diseases that lead to death. Therefore, molecular tools support and reinforce the morphological and physiological evidence in research of unexplained death. Topic No 48 Blotting is used in molecular biology for the identification of proteins and nucleic acids and is widely used for diagnostic purposes. This technique immobilizes the molecule of interest on a support, which is a nitrocellulosic membrane or nylon. It uses hybridization techniques for the identification of the specific nucleic acids and genes. The blotting technique is a tool used in the identification of biomolecules such ad DNA, mRNA and protein during different stages of gene expression. Protein synthesis involves expression of a DNA segment which gets converted to mRNA to produce the respective protein. Molecules such as DNA, RNA and proteins are subjected to biochemistry analysis which are separated using blotting techniques. In the case of a cell, these molecules are present altogether and hence with the help of blotting scientists are able to recognize a specific molecule out of all others. Blotting is performed by allowing a mixture of molecules of interest pass through a block of gel which separates the molecules based on their molecular sizes. The hence processed molecules are required to be hard- pressed against a suitable membrane which will in turn transfer the molecules from the gel onto a suitable membrane (nylon, nitrocellulose or PVDF) via capillary action. After the molecules are transferred to the membrane their position does not change. Southern blotting was introduced by Edwin Southern in 1975 as a method to detect specific sequences of DNA in DNA samples. The other blotting techniques emerged from this method have been termed as Northern (for RNA), Western (for proteins), Eastern (for post-translational protein modifications) and South-western (for DNA-protein interactions) blotting. Subtypes of blotting such as northern, western & southern depend upon the target molecule that is being sought. When a DNA sequence is the foundation or code for a protein molecule, the particular DNA molecule of interest can be blotted using Southern Blotting technique. During gene expression, when the DNA is expressed as mRNA for a protein production, this process can be identified by Northern blotting. Finally, the coded mRNA produces the concerned protein, this protein identification can be done by Western Blotting. Blotting approaches are viewed as an aide to the gel electrophoresis which is generally applied for separation of DNA/RNA/protein and yields reproducible results attributed to their excellent resolving power. Thereby, specific molecules can be detected amid the combination of molecules that are subjected to the separation. Most of the methods have a general step wherein the molecules of interest are transferred once separated are transferred from the gel to a solid membrane phase which is accomplished by drenching in a solution across the gel and the membrane via penetrable paper. Many types of multifaceted apparatus are also provided from a lot of suppliers for electroblotting which is more specifically useful for transfer initiated from less porous polyacrylamide gels compared to commonly use porous agarose gels. In case of DNA and RNA the detection of specific sequences in the membrane are carried out via hybridization with nucleic acid labeled probes which in the case of proteins is replaced by the use of labeled antibody probes. The initially developed protocols applied radioactive probes labeled with, radioactive isotopes for detection purposes via implementation of autoradiography procedures. In this process using the pattern of decay emissions radiated from a radioactive material is applied to produce an image on an x-ray film which can also be made available as a digital image by application of scintillation based gas detectors or systems based on phosphorimaging. Keeping in mind the harmful effects of exposure to radioactivity other kinds of labeling systems have been developed which includes fluorescent and chemiluminescent reagents. Luminescence/fluorescence based methods are considered to be sensitive and background free thus generates more accurate results. Also, there are several other modifications that have been implemented in contrast to the original method like DNA probes are more commonly applied rather than RNA, the nylon membranes have replaced the traditional nitrocellulose, to avoid the renaturation of DNA sequences during transfer the transfer is carried out in alkaline which was supposed to be in neutral solution as per the original protocol. The DNA is subjected to acid treatment for reducing its size inorder to increase the transfer rate of larger fragments. The gel strips and tube gels applied in the original protocol are no longer used, instead gel formats are applied. Though there have been many changes in the original protocol still the modern day protocols retain most of the fundamental features of the original protocol. Southern blotting was applied in many important studies like the genetic mapping of the human genome which was based on blotting based detection of restriction fragment length polymorphisms. Also, the DNA fingerprinting was first developed via hybridization of the human DNA restriction digestion products with minisatellite probes. However, most of the primary applications of this method have been now substituted by DNA sequencing and polymerase chain reaction (PCR) as they provide more extensive facts or data and moreover are easy to implement. Nevertheless, blotting is still applied in a lot of arenas like in the measurement of copy number, analysis of long stretches of DNA that is difficult to be amplified or sequenced using PCR or DNA sequencing and in case of structural analysis of DNA wherein the physical forms of DNA are separated using two-dimensional gel electrophoresis and thereafter it is detected using blotting of specific components. Southern Blotting The first of these techniques developed was the Southern blot, named after Dr. Edwin Southern who developed it to identify specific DNA sequences. Southern blotting is a detection technique used to find the target DNA sequences in the DNA sample in the field of molecular biology. The process starts from electrophoresis of DNA molecules which are hybridized in a blotting membrane followed by a transfer step where DNA from gel is transferred onto the blotting membrane. Topic No 49 Principle Restriction endonucleases, which is an enzyme, is used to break the DNA into small fragments. These fragments are then separated using electrophoresis. The fragments achieved is then classified according to their size (kDa). Thus, DNA fragments are transferred to the blotting paper where it is incubated with probes. Probes used in the Southern blotting can be highly selective. They can selectively bind with a resolution of 1 in a million and the characteristics to bind to the intended target fragments. Materials Required Reagents ▪ The buffer used for electrophoresis is TAE or TBE. ▪ The agarose preferred should be of electrophoresis grade. ▪ For staining the DNA, ethidium bromide (0.5 µg ml–1, dissolved in H2O) is used. But there are other DNA staining dyes such as SYBR green which can replace ethidium bromide for safe handling. Note: Ethidium bromide being a mutagen requires careful handling. It is advised to wear gloves during its usage and follow the pertinent regulations for disposal of pipette tips. Avoid touching any objects with ethidium bromide contaminated gloves. ▪ 2Xand 20X SSC (the composition of 20X SSC includes 3.0 M NaCl and 0.3 M sodium citrate) ▪ 6X DNA loading buffer composed of 0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol in water. ▪ Suitable DNA markers of varying molecular weight also referred to as DNA ladders are used as standards for reference. ▪ The prehybridization and hybridization mixture consists of 0.5% SDS, 6X SSC, 5X Denhardt’s solution and 100 mg ml–1 sheared, denatured salmon sperm DNA or yeast tRNA. ▪ Denhardt’s solution widely used for hybridization is made up of 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin (BSA) ▪ Paraffin oil ▪ Cellulose nitrate or nitrocellulose membrane filter with uniform porosity. (e.g., Millipore 25 HAWP; nylon membranes used for blotting protocols are available under various trade names from commercial suppliers) ▪ RNase A (20 pg ml–1 in 2X SSC) for the specific cleavage is needed. ▪ Restriction enzyme and an appropriate buffer is used. ▪ Radioactively labelled RNA as a probe for specific detection where autoradiography is done. Note: Handling of radioactive probes must be careful which necessitates reliable safety measures and legal regulations. ▪ For detection of RNA labelled with tags such as 3H, 35S, 125I or 14C, there is a requirement of 2,5-Diphenyloxazole (PPO) in toluene at a concentration of 20 % wt/vol. Equipment ▪ The transfer from narrow strips of gel can be achieved by using three pieces of glass or Plexiglas having a size of 5 cm × 20 cm with the thickness similar to the thickness of the gel. ▪ There is a requirement of thick, dry filter paper (four to five in numbers) or paper towels of 10 cm × 18 cm in size. ▪ Hybridization vessel possess larger dimension (0.8 mm deep × 2 cm high × ~1 cm longer) than that of membrane used for hybridization and the material used for developing hybridization vessel is Perspex (note: Several alternative procedures are followed for hybridization) ▪ Four narrow pieces of Perspex possessing thickness similar to that of gel. The length of Perspex is sufficient to surround the gel at a spacing of ~3 mm ▪ A tray having 20–50 mm (approx.) depth and 20 mm (approx.) length and width larger than the gel ▪ A glass sheet with length sufficient to be placed on the tray and narrower to have a gap of 10 mm on each side ▪ Several thick pieces of filter paper having a large area as compared to gel. The length of filter paper is adequate to cover the glass sheet and can be dipped within the tray. ▪ A moistened piece of nitrocellulose membrane, having a wider area which can cover the entire gel. The nitrocellulose membrane is placed on the top of four strips made of Perspex. The moistening of nitrocellulose membrane is done using 2× SSC. ▪ Paper towels which are stacked one on top of other. ▪ Apparatus for casting gel. ▪ Gel tank for carrying out electrophoresis is necessary. ▪ Power supply for the entire device set up is required. Reagent Setup ▪ DNA: The entire procedure is initiated by employing enzyme digested DNA of varying concentrations which will quantify the optimum DNA concentration and specify restriction enzyme to be used. Generally, an amount of 1 µg of DNA derived from clones (e.g. from plasmid or bacteriophage clones) is adequate enough for plasmids having low- copy-number. We require larger amounts for carrying out the separation of complex DNA (e.g. genomic DNA). The advisable range to be considered would be 5–10 µg. ▪ The electrophoresis buffer used is TAE which has a composition of 40 mM Tris, 20 mM acetic acid, 1 mM EDTA with pH range of 7.4–8.2 which is normally made as stock concentration of 20X or TBE made up of 89 mM Tris, 2.5 mM EDTA, 89 mM borate, normally made as a 10 × stock). ▪ TAE is recommended to be best when we run gels for a shorter interval of time and when the recovery of DNA fragments from gel is to be carried out. ▪ TBE is considered to be a better buffer especially when we have to run the gels for a time period exceeding 2 h. Procedure Step 1: DNA purification To extract the DNA present inside the nucleus of a cell, we must first lyse the entire cell to enable the expulsion of the DNA. Incubating the cell culture with detergent lyses the entire cell. Now the lysed sample contains DNA, protein, and debris. Protein is lysed by adding the proteinase enzyme and incubated. DNA is purified and separated by alcohol precipitation and fibers are removed by using a buffer. Apart from standard manual isolation procedures, there are commercially available kits like GenElute™ for the isolation of DNA from a variety of sources such as mammalian cells, plants, bacteria, fungi. High purity of the isolated DNA is ensured in case of use of commercial kits. Step 2: Fragmentation The long nucleotide sequences should be broken into smaller fragments for the purification or identification process. This is done by the restriction endonuclease enzyme. All the reagent necessary for the digestion process should be kept on ice before setting up a restriction digestion reaction with suitable enzyme and appropriate DNA concentration. The components are added to the multiwall plates or microcentrifuge tubes (PCR tubes) and mixed by aspirating the contents with the help of a pipette slowly to avoid formation of any bubbles. It is to be taken care that the enzyme has to be added at the last step and until then it should be stored at −20 °C. To ensure complete digestion of the DNA a surplus amount of enzyme is supposed to be added as the fragments produced due to partial digestion can cause ambiguity in the results leading to inaccurate analysis of the subsequent blot to be analyzed. Nevertheless, the concentration of the enzyme added in total should not surpass one-tenth of the total volume of the digest, because the glycerol content generally found in stock of the enzymes has the capacity to impede digestion process when present at high concentration. Also, in order to minimize the probable errors due to pipetting master mixes should be prepared accordingly when there is a requirement of analysis of a large number of samples. The incubation of the digests is carried out at 37 °C either in an incubator or a water bath. A water bath is generally preferred. For DNA samples obtained from cloning 1–2 h deemed sufficient. In case of genomic DNA there is a requirement of overnight digestion wherein there is a chance that the enzyme is over in between. To ascertain that the overnight reaction is successful half of the enzyme is to be added at the beginning of the digestion reaction and the second half can be added in the morning and the incubation can be continued for an hour. Once the digestion is over it may be the case where a concentration step is required to ensure that suitable volume of DNA is present for loading into the gel which is generally fixed at 20 µl per well. This makes the total volume to be in 24 µl after the addition of 6X loading buffer. A standard method for concentrating the DNA samples involve precipitation in presence of ethanol after which the DNA sample is resuspended in ddH2O. The traces of ethanol should be removed completely which otherwise would lead to spilling of the samples out of wells once the samples are loaded. Step 3: Gel Electrophoresis Nucleic acids are negatively charged molecules. So they move towards the anode in an electrophoresis chamber. The movement of the DNA fragments differentiates the rate of the transport thus enabling the separation by size. The percentage of the gel that is to be used and size of the gel has to be determined. The percentage will decide on the size of the fragments that will be separated and the size of the gel on the other hand will give the range of fragments that are feasible to be resolved. Longer gels are generally needed in the case of separation of genomic DNA or multiple fragments that are having similar sizes in order guarantee appropriate separation. Generally, a 0.7–2% gel is considered to be adequate for most of the applications. However in case of some genomic DNA samples it may be required that a low percentage of gel is supposed to be run for the necessary separation of the fragments. When a less than 0.8% gel is required to be run a high base gel percentage is required (upto 2%) to provide support as low percentage gels are very fragile. It is necessary that the base gel is dispensed before the comb is positioned. As soon as the base gel is set the low percentage gel is poured on top and the comb is placed in a way that it does not touch the base gel at the bottom. ▪ A 1X electrophoresis buffer is made after dilution of the prepared stock solution in ddH2O and agarose is added to it in a conical flask which is kept under constant stirring to avoid formation of clumps. Thereafter, the ag

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