Cell & Molecular Biology


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Cell & Molecular Biology

  1. 1. Course Outline • This course involves a detailed study of 4 Units: • (1) Cell and Molecular Biology • (2) Environmental Biology • (3) Physiology, Health & Exercise • (4) Investigation • There is practical work within units (1) - (3), while unit (d) is based on research and experimentation entirely.
  2. 2. Overview • UNIT 1: Cell and Molecular Biology - The structure & function of prokaryotic and eukaryotic cells - The structure and function of cell components - Molecular interaction in cell events - Applications of DNA technology • UNIT 2: Environmental Biology - Circulation in ecosystems - Interaction in ecosystems - Human impact on the environment • HALF UNIT 3: Physiology, Health and Exercise - Exercise and the cardiovascular system - Exercise and metabolism • HALF UNIT 4: Investigation - A scientific investigation into a syllabus-related topic that is designed, carried out and written up as a 2000-2500 word report, worth 20% of the final marks.
  3. 3. Methods of Learning • Powerpoint presentations, directed reading and laboratory work • Practical work emphasises experimental skills used to investigate basic issues associated with each topic • You are also expected to devote considerable effort on the planning, evaluation and writing up of your investigations
  4. 4. Homework • Homework is set on a regular weekly basis. At least 3 hours per week in self study at home! - Short essay type answers like those in sections C and D of the external examination - Data Handling questions like those in sections B, C and D of the external examination and unit tests - Writing up learning outcomes to produce summaries - Revision for unit assessments, topic tests etc. - Planning, carrying out and writing up the investigation - Writing up class practical activities
  5. 5. Internal Assessment • End of unit tests Unit awards are obtained by taking a NAB at level C only. 65% is required to pass the test • Experimental reports You will carry out, record and write up, in the form of a report, one experiment. To achieve a course award in the final examination, all three unit tests and the experimental report must be passed • End of topic tests A/B tests are taken at the end of every unit and prelims are held during the year also
  6. 6. External Assessment 2 1/2 hour external examination with a total of 100 marks (worth 80%) SECTION A 25 Multiple choice Units 1 and 2 only 25 marks SECTION B 7 questions Short/Extended/data qu’s Units 1 and 2 only 55 marks SECTION C 4 questions Short answer Unit 3 only 20 marks
  7. 7. Career Opportunities • A good Advanced Higher pass will enhance an application for courses at College or University level, especially of Biological subjects … any course really (it shows that you have an excellent brain!) • In terms of University admission, Advanced Higher Biology is rated equivalent to A Level Biology, but the investigative research in Advanced Higher provides a very valuable opportunity at this level of study
  8. 8. Advanced Higher Biology UNIT 1 Cell & Molecular Biology
  10. 10. Prokaryotic and Eukaryotic Cells • All living creatures are made up of CELLS, small membrane bound units filled with aqueous solutions of chemicals, which have the ability to create copies of themselves by growing and dividing. [The sizes of cells and organelles]
  11. 11. • Living organisms can be classified into 3 major domains: Bacteria Archaea Plant cells Animal cells • Prokaryotes and Eukaryotes are 2 distinct cell types with STRUCTURAL differences PROKARYOTES EUKARYOTES
  12. 12. The Prokaryotic Cell • Simply stated, prokaryotes are molecules surrounded by a membrane and cell wall. 1 um
  13. 13. Prokaryotes • Lack a membrane bound nucleus enclosing the DNA • DNA is present as a single circular molecule called a BACTERIAL CHROMOSOME • DNA is naked having no associated histone proteins • No membrane bound organelles • Apart from the DNA nucleoid, there is little internal structure apart from dissolved substances and a large number of RIBOSOMES essential for PROTEIN SYNTHESIS • The cytosol is an effective site for bacterial cell metabolism. This allows bacteria to adapt quickly to changing nutritional conditions, but means the regulation of genetic and metabolic activity has to be tightly regulated. • Divide by BINARY FISSION • Some prokaryotic cells have external whip-like FLAGELLA for locomotion or hair like PILI for adhesion. • Prokaryotic cells come in multiple shapes: cocci (round), baccilli (rods), and spirilla or spirochetes (helical cells).
  14. 14. External Prokaryotic Structures Cell Wall • Contains PEPTIDOGLYCAN (only found in bacteria). Large complex molecule consisting of polysaccharide polymers cross- linked by short chains of amino acids Capsules • Sometimes the cell wall is further surrounded by a gelatinous polysaccharide sheath called an attach CAPSULE, GLYCOCALYX or SLIME LAYER Plasma Membrane • Basic structure of the phospholipid bilayer is the same for all bacteria Flagella Motile bacteria usually have long, thin appendages called FLAGELLA. These protein sub-units are used to propel bacteria through liquids
  15. 15. Pili or Fimbrae • A pilus (Latin; plural : pili) is a hairlike protein structure on the surface of a bacterial cell, required for bacterial conjugation (transfer of genetic material) • A fimbrium (Latin; plural: fimbria) is a short pilus that is used to attach the cell to a surface. Mutant bacteria that lack fimbria cannot adhere to their usual target surfaces and, thus, cannot cause diseases.
  16. 16. Spores & Cysts These are produced by some bacteria to survive unfavourable environmental conditions. Dormant forms are metabolically inactive and only germinate under suitable conditions ENDOSPORES: a dormant, tough, non-reproductive structure produced by a small number of bacteria. The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to ultraviolet and gamma radiation, desiccation, lysozyme, temperature, starvation, and chemical disinfectants. Endospores are commonly found in soil and water, where they may survive for long periods of time e.g. Clostridium (tetanus, gas gangrene), Bacillus (anthrax) CYSTS: also dormant, but unlike endospores are not resistant to heating at high temperatures
  17. 17. Classifying Prokarotes • Main method is using the GRAM’S STAIN • This separates bacteria into GRAM-POSITIVE (purple) and GRAM-NEGATIVE (red) depending on the percentage of PEPTIDOGLYCAN in the cell walls - GRAM-POSITIVE bacteria have a cell wall only 1 layer thick - GRAM-NEGATIVE bacteria have a cell wall several layers thick
  18. 18. Eukaryotes • More complex multicellular organisms e.g. plants, animals, fungi and also many single-celled organisms e.g. amoeba, yeast • Possess an NUCLEUS and other organelles all of which are surrounded by a MEMBRANE, which divided the cell up into compartments COMPARTMENTALISATION: very important ! ADVANTAGES: •Molecules are ‘concentrated’ together, increases rate of reactions •Keeps reactive molecules away from other parts of the cell that may be affected by them •Large work surface area … many enzymes are bound in membranes
  19. 19. Eukaryotes The basic eukaryotic cell contains the following: - membrane-bound nucleus - plasma membrane - glycocalyx (components external to the plasma membrane) - cytoplasm (semifluid) - cytoskeleton – microfilaments, intermediate filaments and microtubules that suspend organelles, give shape, and allow motion - presence of characteristic membrane enclosed subcellular organelles e.g. mitochondria, golgi, rER, sER etc
  20. 20. Plant & Animal Cells • For ANIMAL CELLS only: – Peroxisomes & Lysosomes often present – Some have microvilli on their surface – Centrioles organise spindle fibres during cell division • For PLANT CELLS only: – Cell walls made from cellulose – Communication with neighbouring cells occurs through plasmodesmata – Usually a large central vacuole – Photosynthesis occurs in cells containing chloroplasts [Stick in & label plant & animal cell diags]
  21. 21. Plasma Membrane Plasma Membrane A lipid/protein/carbohydrate complex, providing a barrier and containing transport and signalling systems.
  22. 22. Nucleus Nucleus Double membrane surrounding the chromosomes and the nucleolus. Pores allow specific communication with the cytoplasm. The nucleolus is a site for synthesis of RNA making up the ribosome
  23. 23. Mitochondria Mitochondria • Surrounded by a double membrane with a series of folds called cristae. • Functions in energy production through metabolism. • Contains its own DNA, and is believed to have originated as a captured bacterium.
  24. 24. Rough endoplasmic reticulum (RER) Rough endoplasmic reticulum (RER) • A network of interconnected membranes forming channels within the cell. • Covered with ribosomes (causing the "rough" appearance) which are in the process of synthesizing proteins for secretion or localization in membranes. Ribosomes • Protein and RNA complex responsible for protein synthesis
  25. 25. Golgi Apparatus Golgi apparatus • A series of stacked membranes. Vesicles (small membrane surrounded bags) carry materials from the RER to the Golgi apparatus. • Vesicles move between the stacks while the proteins are "processed" to a mature form. • Vesicles then carry newly formed membrane and secreted proteins to their final destinations including secretion or membrane localisation.
  26. 26. Centrioles Centrioles • Centrioles are found only in animal cells. They function in cell division.
  27. 27. Lysosymes Lysosymes • A membrane bound organelle that is responsible for degrading proteins and membranes in the cell, and also helps degrade materials ingested by the cell.
  28. 28. Peroxisomes Peroxisomes or Microbodies • Produce and degrade hydrogen peroxide, a toxic compound that can be produced during metabolism
  29. 29. Chloroplasts Chloroplasts • Surrounded by a double membrane, containing stacked thylakoid membranes. • Responsible for photosynthesis, the trapping of light energy for the synthesis of sugars. • Contains DNA, and like mitochondria is believed to have originated as a captured bacterium.
  30. 30. Vacuoles Vacuoles • Membrane surrounded "bags" that contain water and storage materials in plants.
  31. 31. Cell wall Cell wall • Plants have a rigid cell wall in addition to their cell membranes. They provide support for the plant.
  32. 32. Similarities between P & E cells • Prokaryotes & Eukaryotes are CHEMICALLY & METABOLICALLY similar: – Both have genetic material – Both have a cell membrane – Both have a cytosol – Both have ribosomes – Both contain nucleic acids, proteins, carbohydrates & lipids – Both use similar reactions for storing energy and metabolic activities e.g. building proteins
  33. 33. Differences between P & E cells • Main differences are STRUCTURAL: PROKARYOTES EUKARYOTES No membrane bound nucleus Membrane bound nucleus Cell walls made of peptidoglycan (Thickness of wall depends on whether the cell is Gram +ve or –ve) Cell walls, if present, made of cellulose (chitin in fungi) No membrane bound organelles Membrane bound organelles (compartmentalisation) Have pili & fimbriae (for adhesion) and flagella (for propulsion) Have cilia or flagella (for movement) Mucilaginous capsule No mucilaginous capsule present (numerous internal structures present including microtubules, ER, Golgi, secretory vesicles etc) Cell size ranges from 0.5um to 100um Cell size ranges from 10 – 150um
  34. 34. Comparison of Prokaryotic and Eukaryotic Cells PROKARYOTES EUKARYOTES Organisms Monera: Eubacteria and Archebacteria Protists, Fungi, Plants and Animals Level of organization single celled single celled (protists mostly) or multicellular usually with tissues and organs Typical cell size small (1 -10 microns) large (10 - 100 microns) Cell wall almost all have cell walls (murein) fungi and plants (cellulose and chitin); none in animals Organelles usually none many different ones with specialized functions Metabolism anaerobic and aerobic; diverse mostly aerobic Genetic material single circular double stranded DNA complex chromosomes usually in pairs; each with a single double stranded DNA molecule and associated proteins contained in a nucleus Mode of division binary fission mostly; budding mitosis and meiosis using a spindle; followed by cytokinesis
  35. 35. Cell Growth & The Cell Cycle • Living things can be distinguished from non-living things by their ability to REPRODUCE • This characteristic is based on cells being ability to DIVIDE
  36. 36. What is DNA and where is it stored? • The nucleus is a membrane bound organelle that contains the genetic information in the form of chromatin, highly folded ribbon-like complexes of deoxyribonucleic acid (DNA) and a class of proteins called histones.
  37. 37. Cell Cycle • Cell division allows organisms to grow, develop, to rweplace dead cells and to repair tissue • This is a CONTINUAL PROCESS • The length of the cell cycle depends on the type of cell and external factors e.g. temp, O2 supply etc • Bacterial cells – 20 mins • Liver cells divide only once a year or only if the need arises e.g. injury • Skin cells – all the time • Nerve & muscle cells don’t divide at all in a mature adult
  38. 38. The Cell Cycle • Stages in the Cell Cycle:
  39. 39. Cell Growth and The Cell Cycle • A eukaryotic cell cannot divide into two, the two into four, etc. unless two processes alternate: – doubling of its genome (DNA) in S phase (synthesis phase) of the cell cycle; – halving of that genome during mitosis (M phase) • The period between M and S is called G1; that between S and M is G2.
  40. 40. • For a new cell to be produced … – The quantity of DNA must double – DNA replication – Must be copied EXACTLY Due to the brief flurry of cytological activity during cell division, the cycle is divided up into 2 parts: INTERPHASE (G1, S, G2 phases) MITOTIC PHASE (M phase)
  41. 41. So, the cell cycle consists of: • G1 = growth and preparation of the chromosomes for replication • S = synthesis of DNA (and centrosomes) • G2 = preparation for • M = mitosis • When a cell is in any phase of the cell cycle other than mitosis, it is often said to be in Interphase.
  42. 42. What is (and is not) mitosis? • Mitosis is nuclear division plus cytokinesis, and produces two identical daughter cells during prophase, metaphase, anaphase, and telophase. • Interphase is often included in discussions of mitosis, but interphase is technically not part of mitosis, but rather encompasses stages G1, S, and G2 of the cell cycle.
  43. 43. Interphase • The cell is engaged in metabolic activity and performing its preparation for mitosis (the next four phases that lead up to and include nuclear division). • Chromosomes are not clearly discerned in the nucleus, although a dark spot called the nucleolus may be visible. • The cell may contain a pair of centrioles (or microtubule organising centres in plants) both of which are organisational sites for microtubules.
  44. 44. Prophase • Chromatin in the nucleus begins to condense and becomes visible in the light microscope as chromosomes. • The nucleolus disappears. Centrioles begin moving to opposite ends of the cell and fibres extend from the centromeres. • Some fibres cross the cell to form the mitotic spindle. • The nuclear membrane dissolves, marking the beginning of metaphase. • Proteins attach to the centromeres creating the kinetochores. Microtubules attach at the kinetochores and the chromosomes begin moving.
  45. 45. Metaphase • Spindle fibres align the chromosomes along the middle of the cell nucleus. This line is referred to as the metaphase plate. • This organisation helps to ensure that in the next phase, when the chromosomes are separated, each new nucleus will receive one copy of each chromosome.
  46. 46. Anaphase • The paired chromosomes separate at the kinetochores and move to opposite sides of the cell. • Motion results from a combination of kinetochore movement along the spindle microtubules and through the physical interaction of polar microtubules.
  47. 47. Telophase • Chromatids arrive at opposite poles of cell, and new membranes form around the daughter nuclei. • The chromosomes disperse and are no longer visible under the light microscope. • The spindle fibres disperse, and cytokinesis or the splitting of the cell may also begin during this stage.
  48. 48. Cytokinesis • In animal cells, cytokinesis results when a fibre ring composed of a protein called actin around the centre of the cell contracts pinching the cell into two daughter cells, each with one nucleus. • In plant cells, the rigid wall requires that a cell plate be synthesised between the two daughter cells.
  49. 49. Remember …! • Prophase • Metaphase • Anaphase • Telophase • Cytokinesis • Positive • Mental • Attitude • Towards • Calvin Klein
  50. 50. DNA Replication • Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle. • Recap the steps in DNA replication …. • A portion of the double helix is unwound by a helicase. • A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix. • Because DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase I then stitches these together into the lagging strand.
  51. 51. DNA Replication is Semiconservative • When the replication process is complete, two DNA molecules — identical to each other and identical to the original — have been produced. Each strand of the original molecule has remained intact as it served as the template for the synthesis of a complementary strand. • This mode of replication is described as semi-conservative: one-half of each new molecule of DNA is old; one-half new.
  52. 52. Interphase: G1, S and G2 phases • Lasts much longer than the M phase • Sometimes referred to as the ‘resting’ phase – this is UNTRUE as although it doesn’t look like much is happening, in biochemical terms, this is a very active period of CELL GROWTH & METABOLISM – Protein synthesis takes place – Cytoplasmic organelles are synthesised – The cell grows and replicates its chromosomes [only during S phase]
  53. 53. • Interphase is divided into 3 parts: (1) G1 – First ‘Gap’ phase (During this time the cell is very active, growing and carrying out metabolic processes) (2) S - DNA replication (The 'S' stands for synthesis as during this phase DNA is synthesized in the process of replication. Each chromosome becomes two sister chromatids) (3) G2 - Second ‘Gap’ phase (In this period mitochondria and other organelles are divided so that each daughter cell will have an equal number of organelles)
  54. 54. Mitosis: the M phase • Interphase is followed by M Phase which consists of mitosis and cytokinesis. • Mitosis is the division of the contents of the nucleus (PMAT), whilst cytokinesis (CK) refers to the division of the cytoplasm. • Cell division involves mitosis and cytokinesis. The growth of an organism and the replacement of its cells for tissue repair both depend on mitosis and cytokinesis.  
  55. 55. Control of the Cell Cycle • A central mechanism is used to assess the status of the cell as it progresses through the cycle. This system works through 3 main checkpoints: • G1 Checkpoint: towards the end of the S phase.Size of the cell is assessed - if sufficient growth has occurred i.e. cell large enough for division, then S phase can proceed • G2 Checkpoint: the success of DNA replication is monitored. If successful the cell cycle will continue to mitosis • M Checkpoint: during metaphase prior to anaphase and telophase triggers exit from from mitosis and cytokinesis and entry into next G1 phase for daughter cells
  56. 56. Abnormal Cell Division : Cancer cells • Normal cell development will break down if the control of cell division, cell growth or cell death fails • If cell division or cell growth fails, TUMOURS arise • These can either be benign - don’t cause serious problems and can be removed by surgery or malignant - enter the circulation, migrate and proliferate to form new tumours in new areas of the body. This is called METASTASIS
  57. 57. Causes of Cancer • Somatic Cell mutations • Proliferation genes (proto-oncogenes -> oncogenes) • Anti-proliferation genes (also known as Tumour- suppressor genes)
  58. 58. Mitotic Index • Fraction or percentage of cells in a given sample that contain condensed chromosomes i.e. the cells are undergoing mitosis and dividing http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/mitosis.htm
  59. 59. GLOSSARY • Checkpoints: Where stop and start signals regulate the cycle; register internal and external cell signals which report the state of crucial processes and if the cycle should proceed.
  60. 60. Chemotherapy • Chemotherapy is the use of anti-cancer (cytotoxic) drugs to destroy cancer cells (including leukaemias and lymphomas). There are over 50 different chemotherapy drugs and some are given on their own, but often several drugs may be combined (this is known as combination chemotherapy). • Chemotherapy may be used alone to treat some types of cancer. Sometimes it can be used together with other types of treatment such as surgery, radiotherapy, hormonal therapy, immunotherapy, or a combination of these.
  61. 61. How do chemotherapy drugs work? • Chemotherapy drugs interfere with the ability of a cancer cell to divide and reproduce itself. As the drugs are carried in the blood, they can reach cancer cells all over the body. The chemotherapy drugs are taken up by dividing cells, including some normal cells such as those in the lining of the mouth, the bone marrow (which makes blood cells), the hair follicles, and the digestive system. Healthy cells can repair the damage caused by chemotherapy but cancer cells cannot and so they eventually die. • Chemotherapy drugs damage cancer cells in different ways. If a combination of drugs is used, each drug is chosen because of its different effects. Unfortunately, as the chemotherapy drugs can also affect some of the normal cells in your body, they can cause unpleasant side effects. However, damage to the normal cells is usually temporary and most side effects will disappear once the treatment is over. • Chemotherapy is carefully planned so that it destroys more and more of the cancer cells during the course of treatment, but does not destroy the normal cells and tissues.
  62. 62. Multicellular Organisms • Multicellular organisms are created from a complex organization of cooperating cells. • Some cells provide protection; some give structural support or assist in locomotion; others offer a means of transporting nutrients. • All cells develop and function as part of the organized system -- the organism -- they make up. There must be new mechanisms for cell to cell communication and regulation. • In humans, there are 1014 cells comprising 200 kinds of tissues!
  63. 63. Cellular Differentiation • Each of us originated as a single, simple-looking cell -- a fertilized egg, or zygote -- so tiny that it can barely be seen without a microscope. (A human egg cell is about 1/100th of a centimetre in diameter, or a bit smaller than the width of a human hair.) • Shortly after fertilization, the zygote begins dividing, replicating itself again and again. Before long, a growing mass, or blastula, of dozens, then hundreds, then thousands of cells called stem cells forms; each stem cell is only one-fourth to one-tenth the diameter of the original zygote, but otherwise nearly identical to it
  64. 64. Cellular Differentiation • Every nucleus of every cell has the same set of genes. A heart cell nucleus contains skin cell genes, as well as the genes that instruct stomach cells how to absorb nutrients. • Therefore, for cells to differentiate, certain genes must somehow be activated, while others remain inactive. • Genes instruct each cell how and when to build the proteins that allow it to create the structures, and ultimately perform the functions, specific to its type of cell.
  65. 65. Gene Regulation in Bacteria • Bacteria adapt to changes in their surroundings by using regulatory proteins to turn groups of genes on and off in response to various environmental signals • The DNA of Escherichia coli is sufficient to encode about 4000 proteins, but only a fraction of these are made at any one time. E. coli regulates the expression of many of its genes according to the food sources that are available to it.
  66. 66. The lac operon • The best understood cell system for explaining control through genetic induction is the lac operon • Jacob & Monod (1961) - regulation of lactose metabolism in E.coli • Composed of 3 segments, or loci of DNA: 1. The REGULATOR - composed of gene that codes for a repressor protein which can repress the operon. 2. The CONTROL locus - consists of a promotor and the operator - can start transcription of the structural genes 3. The STRUCTURAL locus - contains structural genes encoding the enzyme β-galactosidase
  67. 67. The lac operon In an E. Coli cell growing in the absence of lactose, a repressor protein binds to the operator, preventing RNA polymerase from transcribing the lac operon's genes. The operon is OFF When the inducer, lactose, is added, it binds to the repressor and changes the repressor's shape so as to eliminate binding to the operator. As long as the operator remains free of the repressor, RNA polymerase that recognizes the promoter can transcribe the operon's structural genes into mRNA. The operon is ON
  68. 68. The lac operon Lactose absent :
  69. 69. The lac operon Lactose present :
  70. 70. Mammalian Cell Culture • The ability to grow cells in culture i.e. in the lab, is essential for biotechnology and research
  71. 71. Applications of cell culture 1 RESEARCH (small scale usage) • growing bacterial cells for basic gene manipulation • culturing mammalian cells to observe the effects of drugs and hormones on the functioning of cells e.g. cancer studies • producing new plants
  72. 72. Applications of cell culture 2 BIOTECHNOLOGY (large scale usage) • agriculture e.g. silage production • pharmaceuticals e.g. genetically engineered bacteria to produce insulin • food production e.g. brewing and baking • biodegradation e.g. sewage treatment
  73. 73. Conditions needed for cell culture In order for cells to grow, the conditions must be just right for each cell type. The cytologist must therefore consider the following: •Growth medium •Type of growth container or fermenter •Temperature •pH •Gas exchange •Aseptic conditions •Method for monitoring cell growth •Safety measures and implications
  74. 74. Aseptic conditions • To avoid contamination of growth media and cultures • All inanimate and living objects, including the atmosphere carry large numbers of microorganisms. • A variety of techniques can be used to provide these conditions: e.g. sterilisation of all utensils and media using heat. For example, using an autoclave (steam under pressure, necessary for bacterial spores) Growth of pure cultures
  75. 75. Microorganisms • They are everywhere! • They highly adaptable to their surrounding environment • They are relatively easy to culture • They incredibly diverse and are able to colonise very extreme conditions e.g. salt pans, hydrothermal vents in the ocean floor
  76. 76. Classes of Microorganisms • There are 2 recognised categories of micro-organism: 1. Unicellular Algae / PHOTOTROPHS: use sunlight to make their own food 1. Bacteria & Yeasts (Fungi) / HETEROTROPHS: need more complex media containing an organic carbon source and other compounds e.g. amino acids
  77. 77. Culture & Uses • Food industry - cheese production, baking, wine & beer • Chemical production e.g. acetone • Bases of food chains • Commensal bacteria in digestive tract • Production of therapeutic compounds e.g. insulin [See Scholar: Batch & Continuous culture]
  78. 78. Microbial Growth Culture Requirements • A few litres can be made in the lab • Thousands of litres can be made industrially • Micro-organisms are grown in a medium that supplies them with all nutrients necessary for growth. • This depends on … – the type of cell – the final purpose of the cell – the by-products
  79. 79. Microbial Growth Culture Requirements • Important factors that must always be considered are: – the nutrient media – temperature – pH – gaseous environment – light Unicellular algae, bacteria & yeast can be grown as batch cultures - no dilution is needed until max. density is reached. Growth can be limited by nutrient availability i.e. at the end of exponential growth
  80. 80. Nutrient Media • Chosen to imitate an organism’s natural environment • Generally supplies all the essential nutrients • A medium is classed as any solid or liquid preparation specifically for growth, storage or transport of micro- organisms • Must be at the correct pH and the correct gaseous concentration for the organisms to grow
  81. 81. Nutrient Media • There are 2 types of media commonly used: 1.1. Complex mediaComplex media - this has one or more crude sources of nutrients and their exact chemical composition and components are not known. Generally used for routine cultures 2.2. Defined mediaDefined media - otherwise known as synthetic media containing chemically known compounds and components which are in a relatively pure form REMEMBER: all media must be STERILESTERILE before use !!!
  82. 82. Mammalian Cell Culture • Many animal cells and tissues can be removed from an organism and cultured artifically. This allows the cell’s activities to be investigated e.g. control of the cell cycle
  83. 83. The process of culturing Mammalian Cells Once the cells are obtained from animal tissues or other cell lines they are placed in a flat culture vessel that lies on its side The cells stick or adhere to the inside of the vessel as they grow in the medium Most animal cells are ‘ANCHORAGE-DEPENDENT’ i.e. they need something to hold on to These cells usually form a monolayer that will eventually cover the entire surface of the medium
  84. 84. At this point, called confluence, it is necessary to subculture the cells into a fresh medium N.B.N.B. Cells that are associated with body fluids such as blood cells are NON-ANCHORAGE DEPENDENT and can be grown in suspension. Again, it is necessary to regularly subculture the cells into fresh medium N.B.N.B. All media and culture vessels are STERILISED to prevent the growth of micro-organisms
  85. 85. Mammalian Cell Growth Media • Contains … - mixture of glucose, amino acids, salts, water and antibiotics - sometimes BASIC GROWTH SERUM is added [This is animal serum prepared from blood and contains additional factors e.g. Platelet Derived Growth Factor,which enhances growth, 5-10% added or Fetal Bovine Serum (FBS) ] - pH indicator e.g. phenol red: this shows changes in pH due to waste production (pH decreases  red to yellow) * Finally, the media must be incubated at the appropriate temperature for the chosen cells e.g. human cells - 37o c *
  86. 86. Categories Of Mammalian Cell Cultures • There are 2 categories of animal cell cultures: (1)Primary cultures: • These cells are taken directly from fresh tissue. • The disadvantage is that the cells have a limited lifespan; the cells only divide so many times in culture, so therefore long term culturing is difficult
  87. 87. Process of Cell Collection • The cells are treated with a proteolytic enzyme e.g. trypsin, to separate out the fragments into single cells. • The advantage of this process is that cells can be collected and cloned. • This is useful to isolate a mutant cell line i.e. deriving secondary cell cultures otherwise known as ...
  88. 88. (2) Continuous Cell Lines • These cells have an acquired capacity for infinite growth and division [they are immortal] • They are derived from tumours or the cells have been transplanted [neo-plastic - produce cancer if transplanted into animals] so they have lost their sensitivity to factors associated with growth control. • Generally, these cells will lose their anchorage dependence facility and so are often easier to culture
  89. 89. Continuous Cell Lines • The advantage of using continuous cell lines is that they can be cloned. • This allows easy : – isolation of mutant cells – investigation of cell growth – production of hybrid cells in biotechnology This routine procedure is used to produce important pharmaceuticals e.g. vaccines and hormones
  90. 90. Bacterial & Fungal Cultures • Much easier to grow than mammalian cells ! • Bacteria and Fungi require much simpler growth media requirements and culture conditions compared to animal cells. [See previous notes]
  91. 91. Plant Tissue Culture • One major problem in plant breeding is that crosses can only be made between closely related parental types. This makes it very difficult to introduce new genes into a plant species. • The solution to this problem is PROTOPLAST FUSIONPROTOPLAST FUSION - protoplasts of different plants are mixed and fused together. These form a binucleate cell containing a nucleus from both parental types ProtoplastProtoplast = actively metabolising part of cell minus cell wall [cell wall digested by enzymes]
  92. 92. Process of Plant Cell Culture 1. Plant cells treated with cellulase & pectinase to remove the cell wall which is composed of cellulose, pectin and small amount of hemicellulose. These enzymes only break down the cell wall leaving the plasma membrane intact 2. The cells are then incubated with a mineral salt solution containing mannitol for several hours. This sugar exerts osmotic pressure causing PLASMOLYSIS leading to easier digestion 3. In order for the protoplasts to grow they must be put in a suitable medium to encourage cell wall growth
  93. 93. 4. EXPLANTSEXPLANTS (small pieces of young growing plant tissue e.g. root, shoot, bud or leaf) can be taken and grown in a suitable medium containing plant growth regulators (growth hormones e.g. auxins and cytokinins whuch cause tissue differentiation). Cell proliferation produces a CALLUSCALLUS (a mass of dividing, undifferentiated cells) 5. With continued sub-culturing and changing the balance of growth regulators, the new roots and shoots can be planted out to regenerate a complete plant !
  94. 94. Totipotency • All plant cells are totipotenttotipotent - they each have the ability to express the full genetic potential of that plant
  96. 96. Introduction • Living systems are composed of a limited number of elements namely… CARBON, HYDROGEN, OXYGEN, NITROGEN,CARBON, HYDROGEN, OXYGEN, NITROGEN, PHOSPHORUS & SULPHURPHOSPHORUS & SULPHUR • The carbon atom is of central biological importance as it can form 4 covalent bonds with other atoms • This allows a variety of complex molecules to be constructed • Many functional chemical groups are also associated with biological molecules as they are important in biological systems
  97. 97. Polymers • Many biologically important molecules are polymers composed of monomers linked together • Two monomers are joined together by removing water molecules. This is called a CONDENSATION reaction or DEHYDRATION synthesis • This can be reversed by adding (back) water -> HYDROLYSIS • This is an important feature of cell metabolism Dehydration Hydrolysis
  98. 98. • Making and breaking chemical bonds involves ENERGYENERGY • Synthesising more complex structures REQUIRES energy. These are called ANABOLIC or BIOSYNTHETIC reactions • If there is little overall change in energy, the reactions are reversible • Cell metabolism is tightly controlled to avoid energy chaos
  99. 99. Carbohydrates • Composed of CARBON, HYDROGEN & OXYGEN MONOSACCHARIDES ‘Single Sugars’ e.g. glucose, fructose - General formula (CH2O)n - classified by number of carbons they have n = 3 TRIOSE n = 5 PENTOSE n = 6 HEXOSE - structure can vary greatly depending on the number of C atoms and the arrangement of H and O atoms
  100. 100. Glucose (C6H12O6) • Hexose sugar • Can exist in different forms depending on the position of the carbonyl group (C=O) on the terminal carbon • Variations of C6H12O6 are called isomers • If OH group on C5 projects to the right = D Form (most common) on left = L Form D-GLUCOSE = straight chain form of glucose (C6H12O6)
  101. 101. • In solution, glucose adopts a cyclic form where C1 and C5 are linked by an oxygen atom giving a ring structure (see diagram) • Depending on the position of the -OH group on C1 whether: – (α) alpha - below C1 – (β) beta - above C1 • In solution the equilibrium proportions of the three forms are approximately 38% α to 62% β to 0.02% straight chain glucose at any given time
  102. 102. The Glycosidic Bond • 2 monomers (monosaccharides) can be linked by DEHYDRATION SYNTHESIS or the CONDENSATION REACTION, to give a disaccharide • The carbohydrate’s name is defined by the component monomers and the way the bond is arranged • Common disaccharides are : SUCROSE = Glucose + Fructose LACTOSE = Glucose + Galactose ANIMATIONANIMATION
  103. 103. Polysaccharides • Long chains of simple sugars e.g. starch, glycogen and cellulose • If the repeating monomers are the same, they form a homopolymer. If they are different they form a heteropolymer • Polysaccharides are insoluble in water and so make ideal storage compounds • The following three polysaccharides are all homopolymers of glucose but they have different functions and properties depending on their structure
  104. 104. 1. Starch • Found in plants • Helical arrangement of glucose • Storage polysaccharide of energy • Can be easily hydrolysed to release monomers of glucose for energy • Starch test: turns iodine from dark brown to blue/black
  105. 105. 2. Glycogen • Storage compound in animals, generally found in the liver • Polymer of glucose linked by α 1-6 bonds and α 1-4 bonds • Short term energy store • Plays a role in homeostatic control of blood sugar level • Remains dark brown with iodine
  106. 106. 3. Cellulose • Storage compound in plants • Parallel chain arrangement linked by β 1-4 glycosidic bonds and hydrogen bonding between parallel chains • Doesn’t stain with iodine • Very tough arrangement of fibres due to structural arrangement • most abundant organic material on Earth • Most animals lack cellulase, the enzyme needed to breakdown the component monomers
  107. 107. 4. Chitin – A homopolysaccharide similar to cellulose in structure. Component of many insect exoskeletons - very strong and rigid; also resistant to chemicals. 5. Glycosaminoglycans – A heteropolymer found in skin and connective tissue of vertebrates
  108. 108. Summary of Carbohydrate Functions • Immediate respiratory substrate e.g. glucose • Energy stores e.g. glycogen in mammals, starch in plants • Structural components e.g. cellulose in plant cell walls, chitin in insect exoskeleton, pentose sugars (ribose & deoxyribose in RNA & DNA) • Metabolites i.e. intermediates in biochemical pathways • Cell to cell attachment molecules e.g. glycoproteins or glycolipids on the plasma membrane • Transport e.g. sucrose in plant phloem tissue
  109. 109. Structure & Function of Lipids • Lipids are organic compounds found in every type of plant and animal cell. • They contain the elements CARBON, HYDROGENCARBON, HYDROGEN and OXYGENOXYGEN [but less O2 than in carbohydrates] • All lipids are INSOLUBLE in WATER • Lipids have many important functions: – In cell membrane structure - Mechanical Protection – Hormones - Electrical Insulation of Nerves – Energy storage molecules - Waterproofing & Buoyancy – Thermal Insulation
  110. 110. • FATS: Solid at room temperature – SATURATED FATSSATURATED FATS:: all available bonds are occupied by Hydrogen Most animal fats are saturated e.g. butter, lard • OILS: Liquid at room temperature – UNSATURATED FATS:UNSATURATED FATS: contain C-C double bonds in the molecule therefore kinks are introduced. Oils tend to be more available in plants e.g. sunflower oil, olive oil
  111. 111. Type of Lipids • 3 types of lipids which are important to cells: 1.Triglycerides • Most common type of lipid • 3 fatty acids and a glycerol molecule are linked by an ester bond formed during dehydration synthesis 2. Phospholipids • Same as triglycerides except one of the fatty acids molecules is replaced by a phosphate group (PO4 3 -) • The phosphate group is polar and so is attracted to water – therefore the phospholipid has two distinct ‘ends’ • A hydrophilic end (‘water loving’) that dissolves in water and a hydrophobic end (‘water hating’) that is repelled by water 3. Steroids • Very different structure – 4 carbon rings with variety of different side chains
  112. 112. Triglycerides cont. • The properties of triglycerides are determined by their constituent fatty acids • DEHYDRATION SYNTHESISDEHYDRATION SYNTHESIS occurs between the hydroxyl group of the glycerol molecule and the carboxyl groups of the fatty acid molecule producing an ester • Main function = ENERGY STORE e.g. camel hump • The form in which fatty acids are transported round the body and stored is adipose tissue ANIMATION
  113. 113. Phospholipids • Similar to triglycerides but one fatty acid is replaced by a phosphate group which often has other groups attached • Usually one fatty acid is saturated and one is unsaturated. Most common phospholipid in animal tissue is PHOSPHATIDYLCHOLINEPHOSPHATIDYLCHOLINE •The phospholipid has two distinctive ends: –HYDROPHILIC HEADHYDROPHILIC HEAD that dissolves in water –HYDROPHOBIC TAILHYDROPHOBIC TAIL that repels water This property causes phospholipids to spontaneously form bilayers
  114. 114. Functions of Phospholipids • Essential components of cells and organelle membranes • Components of lung surfactants
  115. 115. STRUCTURE & FUNCTIONS OF PROTEINS • Proteins are essential in biological systems as controlscontrols e.g enzymes and structural elementsstructural elements e.g. cytoskeleton • Proteins are heteropolymers as they are made up of different amino acids (20 different types) • The type and order of amino acids determines the structure and function of proteins allowing them to carry out many different roles
  116. 116. Amino Acids • Amino acids are characterised by the amino group (NH2) and the carboxylic acid (COOH) • These are attached to a central carbon atom which also carries a hydrogen • The side chains are variable, the ‘R’ group can be joined here
  117. 117. • At neutral pH, amino acids exist in ionised forms. Once joined, the charges on amino acids disappear. R GROUP • This gives the amino acid it’s unique chemical properties and specific shape. • The R group can be classified as acidic, basic, uncharged polar or non-polar
  118. 118. Types of Amino Acids Amino Acid Class Name Abbreviations R-Group Acidic Aspartic Acid Asp/D - CH2COOH Basic Lysine Lys/K - (CH2)4NH4 Uncharged Polar Serine Ser/S - CH2OH Non-polar Glycine Gly/G - H
  119. 119. The Peptide Bond • Proteins are made by joining amino acids together by an amide linkage / peptide bond • A chain of amino acids is called a polypeptide • The peptide bond is formed by DEHYDRATIONDEHYDRATION SYNTHESISSYNTHESIS or a condensation reaction between the carboxyl group of one amino acid and the amine group of the next amino acid • Amino acids joined in this way are called residues
  120. 120. The Peptide Bond • The Peptide bond is very strong • C-N bond is planar (flat) so peptide bond allows NO rotation • The single bonds either side DO allow rotation of the residues, so polypeptide chains are flexible ANIMATION
  121. 121. Protein Structure • Chemical bonding is critical in determining a protein’s shape and the different types of bonds are important for different levels of protein structure PEPTIDE BOND = COVALENT BOND = VERY STRONGPEPTIDE BOND = COVALENT BOND = VERY STRONG • In higher order protein structures, weaker interactions are important too.These include: – Non-covalent bonds – Hydrogen Bonds – Ionic bonds – Van der Waals interactions – Hydrophobic interactions between R groups
  122. 122. Primary Structure (1o ) • Primary structure refers to the "linear" sequence of amino acids. • The amino end or N terminus is positioned to the left. The carboxyl end or C terminus is positioned to the right N C Generally 3 or 1 letter abbreviations are used to denote amino acids when primary structures are drawn
  123. 123. Secondary Structure (2o ) • Secondary structure is "local" ordered structure brought about via hydrogen bonding mainly within the peptide backbone • A single polypeptide many contain several secondary structures • The most common secondary structure elements in proteins are the alpha (α) helix and the beta (β) sheet (sometime called b pleated sheet)
  124. 124. Tertiary Structure (3o ) • This describes the way in which the polypeptide folds to give the final structure of the protein. • The 3o structure is determined by hydrophobic interactions which place the amino acids non-polar R groups towards the centre of the molecule • In many proteins an additional important type of bond is the disulphide bond. This bond forms between sulphydryl (SH) groups on cysteine residues; so may be formed between 2 different polypeptides or within the polypeptide itself. • Within any tertiary structure, parts of the amino acid sequence may adopt an α-helix, β-sheet or more complex β sheet arrangements e.g. myoglobin
  125. 125. • The ion group is a prosthetic group – a non-protein group associated with a folded protein • If the attached group is : – CARBOHYDRATE = Glycoprotein – LIPID = Lipoprotein – NUCLEIC ACID = Nucleoprotein These are known as conjugated proteins
  126. 126. • As proteins have a relatively stable structure in a cellular environment, it is remarkable that the forces that hold them together can be easily disrupted if the chemical environment changes or the sequence of amino acids is changed
  127. 127. • Alpha Helix • The polypeptide chain is coiled into a right handed helix by Hydrogen bonding (stabilises the helix) between the NH group of the peptide and the C=O of the peptide bond, four residues away from it
  128. 128. Beta sheet • The polypeptide chains are linked together in a side by side configuration by hydrogen bonding. Beta sheets can be either parallel or anti-parallel depending on the orientation of the constituent parts
  129. 129. Quaternary Structure (4o ) • Proteins that are composed of 2 or more polypeptide sub-units
  130. 130. Nucleic Acids [revise Higher notes] • DNADNA and RNARNA are information carrying molecules – DNADNA: info storage & transmission – RNARNA: protein synthesis • Simple chemical structure based on a SUGARSUGAR PHOSPHATE BACKBONEPHOSPHATE BACKBONE • Coding part made of 4 nitrogenous bases which arrange themselves in pairs • This enables a massive variety and diversity of proteins to be built [Diagram of Nas/Nucleotides]
  131. 131. Nucleotides • Monomer of nucleic acid • Consists of 3 main parts : – a PENTOSEPENTOSE sugar (deoxyribose/ribose) – a PHOSPHATEPHOSPHATE group (PO4 2- ) – a nitrogenous base (PURINEPURINE or a PYRIMIDINEPYRIMIDINE) PURINE PYRIMIDINE double or fused ring structure single ring structure ADENINE, GUANINE CYTOSINE, THYMINE & URACIL (only found in RNA) N.B. Base Pairing: A always bonds with T (or U), G with C
  132. 132. Phosphodiester Bond • Chains of nucleotides (polynucleotides) formed by DEHYDRATION SYNTHESISDEHYDRATION SYNTHESIS reaction between the phosphate group of one nucleotide and the hydroxyl group on the sugar of another • This bonding gives polynucleotides a defined polarity reflecting the component nucleotides [Diagram of Phosphodiester bond]
  133. 133. Polynucleotides & Nucleic Acid Function • Polynucleotide chains provide the structural and functional basis for the encoding and decoding of genetic information. • The sugar phosphate backbone carries a sequence of bases that makes up the genetic code as a series of triplet codons • Complementary base pairing holds the key to copying genetic information in the processes of DNA replication and transcription • The bases fit together A-T(U) and G-C are joined together by HYDROGEN BONDINGHYDROGEN BONDING [Base pairing diagram][Base pairing diagram]
  134. 134. DNA • A double stranded helix composed of two polynucleotide chains that run in opposite directions (anti-parallel) • The bases fit across the right-handed helix; one purine pairing with its complementary pyrimidine • The helix is the only shape that accommodates the purine-pyrimidine base pair and maintains stable hygrogen bonds
  135. 135. RNA • 3 types of RNA which are SINGLE strandedSINGLE stranded but can fold to give 3D shapes or conformations: • mRNAmRNA - contains information transcribed from a DNA molecule and transports it to a ribosome • tRNAtRNA - collects amino acids and transports them to a ribosome to be fitted according to the messenger RNA code • rRNArRNA (ribosomal RNA) - provides a major structural support component of the ribosome
  136. 136. Polymerase enzymes • A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA • These are necessary for the following processes: 1) DNA REPLICATION:DNA REPLICATION: enables a complete copy of the genome to be passed on to each daughter cell during mitosis 2) TRANSCRIPTION OF DNA into RNA:TRANSCRIPTION OF DNA into RNA: : mechanism by which genes are expressed DNA polymerase
  137. 137. DNA Ligase • This enzyme forms phosphodiester bonds which are used to join DNA molecules or fragments together to produce recombinant DNA (recDNA) Both polymerases,ligases and restriction endonucleases (cut DNA) are important components of a genetic engineer’s ‘toolkit’. They are used to manipulate DNA
  138. 138. Cell Membranes • The cell membrane/plasma membrane represents the barrier that separates the cell’s contents from the surrounding environment and controls what moves in and out • In eukaryotic cells, membranes are also used to generate compartments within the cell, each with a specialised function e.g. golgi apparatus, endoplasmic reticulum, lysosomes etc
  139. 139. Membrane functions • Provides selectively permeable barriers • Compartmentalisation • Localises reactions in the cell • Transport of solutes often against the concentration gradient (active transport) • Signal transduction – receptor proteins on the membrane surface recognise and respond to different stimulating molecules, enabling specific responses to be generated within the cell • Cell to cell recognition – the external surface of the membrane is important as it represents the cell’s biochemical “personality”. In multicellular organisms this allows cells to recognise each other as similar or different, which is necessary for the correct association of cells during development.
  140. 140. Membrane Structure • The basic composition and structure of the plasma membrane is the same as that of the membranes that surround organelles and other subcellular compartments. • The foundation is a phospholipid bilayer – polar hydrophilic heads on the outer surface and hydrophobic non-polar fatty acid tails form the inner surface. The membrane as a whole is often described as a fluid mosaic – a two-dimensional fluid of freely diffusing lipids, dotted or embedded with proteins which may function as channels or transporters across the membrane, or as receptors.
  141. 141. The Plasma Membrane
  142. 142. • The idea that membranes were composed of phospholipids was first put forward in 1925. The currently accepted model for membrane structure was proposed by S.J. Singer (1971) as a lipid protein model and extended to include the fluid character in a publication with G.L. Nicolson in "Science" (1972) • The fluid mosaic model has 2 components, lipids and proteins. The lipids form the matrix bilayer of the membrane and the proteins carry out all of its functions • The membrane is not a static rigid structure, but a dynamic arrangement of lipids and proteins that drift laterally within it.
  143. 143. Types of Membrane Proteins • Proteins make up approximately 50% of the mass of the plasma membrane and can be classified into different groups depending on their arrangement in the membranearrangement in the membrane and/or their functionfunction • Proteins may be embedded in the lipid bilayer or attached to the surface • The embedded or INTRINSIC proteins may be transmembrane proteins (span the bilayer) or they may be linked to lipids on one side of the bilayer only • The peripheral or EXTRINSIC proteins are loosely attached to the membrane by ionic association with other proteins • Glycoproteins have carbohydrates attached to their extracellular domains.
  144. 144. Functions of Membrane Proteins • The main functions of these membrane proteins are as follows: –Transport –Cell recognition –Receptor sites –Enzymes –Intracellular Junctions
  145. 145. 1) TRANSPORT PROTEINS • Transport non-diffusable substances across the membrane. May be either: (a) Channel proteins – provide a ‘pore’ across the membrane through which molecules (usually small and charged) can diffuse (b) Carrier proteins – these are more specific with binding sites for only one solute • Both these proteins permit passive transport (with a concentration gradient this is called facilitated diffusion) • To transport molecules against the concentration gradient, special types of the carrier proteins are needed. These harness energy to drive the transport process during active transport e.g. sodium- potassium pump
  146. 146. 2) CELL RECOGNITION PROTEINS • Usually glycoproteins • The carbohydrate chain of the glycoprotein projects out of the cell enabling cell to cell recognition and serving as a cell “fingerprint” • Therefore, the immune system can recognise it’s own cells and organs e.g. ABO blood group antigens: – A = glycoprotein antigen A – O = no glycoprotein antigens
  147. 147. 3) RECEPTOR PROTEINS • These have a specific conformation (shape) that allows binding of a particular molecule (the ligand) • The binding of the ligand will then trigger a response in the cell
  148. 148. 4) ENZYMES • A protein that catalyses a specific reaction • Some receptor proteins have enzymatic activity; the cytoplasmic portion of the protein catalyses a reaction in response to binding a ligand
  149. 149. 5) INTRACELLULAR JUNCTIONS • Interactions between the plasma membranes of different cells is a frequent occurrence and takes place at cell junctions e.g. -> In PLANTSPLANTS • PLASMODESMATA – although each plant cell is encased in a boxlike cell wall, fine strands of cytoplasm, called plasmodesmata, extend through pores in the cell wall connecting the cytoplasm of each cell with that of its neighbors allowing direct exchange of materials
  150. 150. – In ANIMALSANIMALS, there are 3 types… • Spot desmosome – dense protein deposits that hold adjacent cells together by rivets. Mechanical strength is provided by the intracellular filaments passing from one desmosome to another • Tight junction – adjacent membrane proteins are bonded together preventing movement of materials in the space between the cells e.g. between epithelial cells lining the small intestine • Gap junction – doughnut shaped proteins from each cell joined together to form tiny channels allowing the passage of small molecules such as ions, amino acids and sugars
  151. 151. The Cytoskeleton • The eukaryotic cell is a 3D structure. It has a cytoskeleton anchored to proteins in the plasma membrane • These proteins both maintain shape and allow movement • The cytoskeleton is a dynamic structure, as the microfilaments and microtubules can depolymerise and repolymerise very easily MICROFILAMENTS MICROTUBULES INTERMEDIATE FILAMENTS
  152. 152. The Cytoskeleton • The cytoskeleton is made up of 3 components, in order of increasing diameter. They are … 1) Actin filaments/microfilaments 2) Intermediate filaments 3) Microtubules
  153. 153. 1) Microfilaments • These are composed of actin (protein) • They are arranged as 2 strands of protein molecules twisted together to give a rope-like structure about 7nm in diameter • These are present throughout the cell but are most highly concentrated just inside the plasma membrane • They are important in all cell movementcell movement and contractioncontraction Actin fibres in a cell stained with a fluorescent strain specific for actin
  154. 154. 2) Intermediate Filaments • These are about 10nm in diameter and are composed of tough fibrous protein strands twisted together • They are very stable structures in the cell and provide mechanicalmechanical strengthstrength to animal cells which lack the strong cell walls of plants • Intermediate filaments can be anchored between the membrane to provide extra support The nucleus in epithelial cells is held within the cell by a basketlike network of intermediate filaments made of keratins which have been stained here using a fluorescent stain
  155. 155. 3) Microtubules • These are hollow tubes (like straws) composed of tubulin protein (a globular protein) • The tubulin protein subunits of microtubules associate in a cylindrical arrangement to generate the final microtubule - a relatively rigid structure • Microtubules only form around a centrosome (organising centre) • The centrosome provides a “nucleus” from which the microtubules form. These are important in cell division as part of the spindle fibre network and can move components within the cell Microtubules growing in vitro from an isolated centrosome
  156. 156. Functions But the primary importance of the cytoskeleton is in cellcell motilitymotility. The cytoskeleton extends throughout the cytoplasm and determines the internal movement of cell organelles, as well as cell locomotion and muscle fibre contraction All of these components give mechanical supportmechanical support and shapeshape to the cell
  158. 158. Molecular Interactions In Cell Events • CATALYSISCATALYSIS: - The vast number of coordinated and complex biochemical reactions that occur in an organism is summarised as the cell METABOLISMMETABOLISM - The reactions are in ordered pathways, controlled at each stage by ENZYMESENZYMES - Through these metabolic pathways, the cells are able to transform energy, breakdown macromolecules and synthesise new organic molecules needed for life
  159. 159. Anabolic Reactions • Uses energy to SYNTHESISESYNTHESISE large molecules from smaller ones e.g. Amino Acids->Proteins • Also known as endothermicendothermic reactions ENDOTHERMIC REACTION
  160. 160. Catabolic Reactions • These release energy through the BREAKDOWNBREAKDOWN of large molecules into smaller units e.g. Cellular Respiration: ATP -> ADP + Pi • Also known as exothermicexothermic reactions EXOTHERMIC REACTION
  161. 161. Naming Enzymes • Enzymes are commonly named by adding a suffix "-ase" to the root name of the substrate molecule it is acting upon. For example, Lipase catalyzes the hydrolysis of a lipid triglyceride. Sucrase catalyzes the hydrolysis of sucrose into glucose and fructose • A few enzymes discovered before this naming system was devised are known by common names e.g. pepsin, trypsin, and chymotrypsin which catalyse the hydrolysis of proteins • Enzymes are also given a standard reference number (European Commission Number) to help characterise the 1500 or so enzymes
  162. 162. ENZYMES These catalyse a transfer of a phosphate group onto a molecule such as a carbohydrate or protein Kinases To hydrolyse ATP. Many proteins have an ATPase activity which is essential for their functionATPases To hydrolyse phosphodiester bondsNucleases To hydrolyse peptide bonds to breakdown proteins -> amino acids Proteases FUNCTIONNAME
  163. 163. Form & Function of Enzymes • Enzymes work by bringing about substrate(s) of a reaction close together in an active siteactive site so that bond breakage or formation occurs at atomic level • This is often facilitated (helped) by specific chemical effects such as the transfer of proteins or the alteration of charge distribution around the target atoms • The substrate and enzyme must fit together veryvery preciselyprecisely
  164. 164. The Catalytic Cycle • A cycle of events that describes an enzyme combining with a substrate, remaining unchanged by the reaction and being available at the end of the reaction to combine with another substrate molecule
  165. 165. The Catalytic Cycle of Sucrase Sucrase catalyses the hydrolysis of sucrose into it’s component monosaccharides, GLUCOSE &GLUCOSE & FRUCTOSEFRUCTOSE 1) At the start of the cycle, enzyme (E) and substrate (S) are available 2) The molecular interaction of enzyme and substrate at the active site forms the enzyme:substrate complex (ES) 3) Catalysis occurs, forming the enzyme:product complex (EP) 4) Products are released, leaving the enzyme free for the next substrate molecule E S ES EP
  166. 166. Model for Enzyme Action • A common model for enzyme action is the lock and keylock and key hypothesishypothesis • However, this model is a little misleading in that it tends to give the impression that enzymes are rigid structures, whereas in fact, they are quite flexible and can alter their conformation in response to the binding of other molecules • The currently accepted model for enzyme action is the INDUCED FIT MODELINDUCED FIT MODEL, in which conformational changes to the protein occur on binding of a substrate
  167. 167. The Induced Fit Model • The enzyme, HEXOKINASEHEXOKINASE, catalyses the transfer of a phosphate from ATP onto glucose • The active site and the two domains of the single polypeptide chain are clearly visible in the view of the backbone of the molecule • Think of the protein about to close around the substrate in the active site similar to the way your hand would close around a door handle • The effect of this is that glucose fits the active site more closely, and the binding of ATP is also enhanced [see diagram of ‘The catalytic cycle of hexokinase]
  168. 168. Control of Enzyme Activity • The activity of enzymes must be reguated in some way to avoid metabolic chaos • Regulation can be achieved through a number of different mechanisms • A major influence is the NUMBER OF ENZYMES MOLECULES in the cell, which is controlled at the level of gene expression • COMPARTMENTALISATION also enables the cell to keep sets of enzymes together and away from other enzymes • TEMPERATURE & pH also affect enzyme activity • Many enzymes also require CO-FACTORS to function
  169. 169. • However, the most effective way of enabling a fine control of enzyme activity is to alter the shape of the enzyme itself, and thus cause a change in its catalytic efficiency • Examples of this type of metabolic control include INHIBITORS,INHIBITORS, ALLOSTERIC EFFECTORSALLOSTERIC EFFECTORS,, COVALENTCOVALENT MODIFICATIONMODIFICATION and END-PRODUCT INHIBITION
  170. 170. Inhibitors • Enzymes reaction rates can be changed by competitive inhibition and non-competitive inhibition • Inhibitors can be either competitive or non- competitive
  171. 171. COMPETITIVECOMPETITIVE inhibitors compete for the active site of the enzyme, thus reducing its effectiveness – competitive inhibitors are usually similar in structure to the substrate and the enzyme is ‘fooled’ into accepting the inhibitor, which blocks the active site
  172. 172. E.G: An example for competitive inhibition is the enzyme succinate dehydrogenase by malonate. Succinate dehydrogenase catalyses the oxidation of succinate to fumarate.
  173. 173. NON-COMPETITIVENON-COMPETITIVE inhibitors bind at a different location and change the conformation of the enzyme, thus altering the shape of the active site and again reducing the catalytic efficiency
  174. 174. • Inhibition can either be reversible or non- reversible depending on how the inhibitor binds to the enzyme • Some inhibitors bind irreversibly with the enzyme molecules, inhibiting the catalytic activities permanently. The enzymatic reactions will stop sooner or later and are not affected by an increase in substrate concentration. These are irreversibleirreversible inhibitorsinhibitors. • Examples are heavy metal ions including silver, mercury and lead ions.
  175. 175. Allosteric Enzymes • These are enzymes that ‘change shape’ in response to the binding of a regulating molecule (often called a modulator or effector) • Allosteric modulators can be either positive or negative effectors of enzyme activity • They function by binding to allosteric sites that are distinct from the active site of the enzyme • Non-competitive inhibition is a form of allosteric regulation
  176. 176. Allosteric Enzymes
  177. 177. • In multi-subunit enzymes, the structure is more complex, and the enzyme often exists in 2 different conformational states: – ACTIVE and INACTIVE • These can be stabilised by binding the modulator – Positive modulators stabilise the active form of the enzyme – Negative modulators stabilise the inactive form • In addition to these modulators changing the activity of allosteric enzymes, sometimes the binding of the substrate itself to one active site enhances binding at the other active sites. This is known as COOPERATIVITY
  178. 178. Covalent Modification • Covalent modification of enzymes is another strategy used widely in metabolic regulation • One of the most common modifications is the addition of a PHOSPHATEPHOSPHATE group, which can alter the shape of a protein by attracting positively charged R-groups [phosphates carry 2 negative charges on the 2 single-bonded O atoms] • PROTEINPROTEIN KINASESKINASES add phosphate groups and PHOSPHATASESPHOSPHATASES remove them, thus the effect can be REVERSEDREVERSED • Some proteins are activated by phosphorylation, others are inactivated
  179. 179. • An example of phosphorylation activating an enzyme is the skeletal muscle enzyme GLYCOGEN PHOSPHORYLASEGLYCOGEN PHOSPHORYLASE • This enzyme releases glucose molecules from glycogen when heavy demands are placed on muscle tissue • This process is highly regulated. Traffic of sugar into and out of storage in glycogen is used to control the level of glucose in the blood, so glycogen phosphorylase must be activated when sugar is needed and quickly deactivated when glucose is plentiful
  180. 180. • Glycogen phosphorylase is present as an inactive non- phosphorylated form which is converted to the active phosphorylated form by the addition of a phosphate group to a serine residue in the protein by the enzyme PHOSPHORYLASE KINASEPHOSPHORYLASE KINASE • When the demand for glucose drops, PHOSPHORYLASEPHOSPHORYLASE PHOSPHATASEPHOSPHATASE removes the phosphate group and inactivates the enzyme However … glycogen phosphorylase is also regulated by an allosteric effect !
  181. 181. • Glucose and ATP act as negative modulators and AMP (adenine monophosphate) acts as a positive modulator – also causing the enzyme to shift to the active conformation • This is useful, because AMP is a product of ATP breakdown and will be more plentiful when energy levels are low and more glucose is needed • A further complication is that there is a hormonal control mechanism by adrenaline and glucagon
  182. 182. Proteolytic Cleavage • Another form of control by a covalent activating mechanism is proteolytic cleavage as found in the enzyme TRYPSINTRYPSIN • Trypsin is synthesised in the pancreas, but not in its active form as it would digest the pancreatic tissue • Therefore it is synthesised as a slightly longer protein called TRYPSINOGENTRYPSINOGEN, which is inactive • Activation occurs when trypsinogen is cleaved by a protease in the duodenum • Once active, trypsin can activate more trypsinogen molecules, resulting in an autocatalytic cascade that produces a large number of active trypsin molecules very rapidly
  183. 183. End-Product Inhibition • Metabolism is organised as a series of metabolic pathways, and control of these pathways is an important feature of cell biochemistry • One way in which control can be exercised is END-END- PRODUCT INHIBITIONPRODUCT INHIBITION • End-product inhibition is energetically efficient as it avoids the excessive (and wasteful) production of the intermediates of a pathway • This is a form of NEGATIVE FEEDBACKNEGATIVE FEEDBACK
  184. 184. For example, in the production of the amino acid isoleucine in bacteria, the initial substrate is threonine which is converted by five intermediate steps to isoleucine. As isoleucine begins to accumulate, it binds to an allosteric site of the first enzyme in the pathway thereby slowing down its own production. In this way, the cell does not produce any more isoleucine than is necessary.
  185. 185. The Sodium-Potassium Pump (Na+/K+ - ATPase) • A specific case of active transport • This is one of the best examples of active transport in animal cells • This pump transports Na+ ions out of the cell and K+ ions into the cell. Thus keeping the intracellular concentration of Na low compared to outside, and the intracellular concentration of K high • The pump is driven by hydrolysis of ATP • It uses about 30% of the energy available to any one animal cell! • The pump is a transmembrane carrier proteintransmembrane carrier protein made up of 4 subunits (2 large and 2 small)
  186. 186. • Structure: has 3 binding sites for sodium ions, 2 binding sites for potassium ions and a phosphorylation site to accept a phosphate from ATP • 2 different conformations of protein are possible. This is controlled by the phosphorylation state of the protein • Hydrolysis of one ATP molecule fuels the export of 3 Na+ ions and the import of 2 K+ ions • Can work as fast as 300 Na+ ions per second if required! [Sodium-Potassium Pump Diagram] Sodium Potassium Pump Animation
  187. 187. Cell Signaling Molecules • Cell-cell recognition • Although cells can act as self-contained units, they don’t exist in isolation • Even a unicellular organism must detect and respond to outside influences e.g. chemicals, light and other cells • In a multicellular organism, the organisation of tissues and systems brings more complexity • Therefore, it is essential that cells can COMMUNICATECOMMUNICATE to enable their activities to be fully coordinated
  188. 188. • Communication involves transmitting and receiving information • A SIGNALINGSIGNALING cell sends a signal and is received by a TARGETTARGET cell [Signal molecules can induce different responses in their target cells e.g. acetylcholine: causes cardiac muscle to relax, but skeletal muscle to contract ] • If a change in the form of a signal is required, it is called a SIGNAL TRANSDUCTIONSIGNAL TRANSDUCTION Analogy: Faxing a letter – conversion of a printed form of information into an electronic form – back into a printed form ANIMATION
  189. 189. Communication Systems ENDOCRINEENDOCRINE Secretion of a hormone into the bloodstream for dispersal. The signalling cell and the target cell can be far apart. Very slow method e.g. Insulin, Adrenaline PARACRINEPARACRINE Secretion of a local mediator. This affects cells in the immediate area of the signalling cell e.g. Histamine NEURONALNEURONAL Nerve cells or neurones elicit responses by the release of a neurotransmitter at synapses. Can signal over very long distances via a network of nerve cells. Very fast signalling e.g. GABA (Gamma-Amino-Butyric-Acid – an inhibitory neurotransmitter) CONTACTCONTACT DEPENDENTDEPENDENT Signal molecules in the plasma membrane of the signal cell interact with membrane bound receptors on the target cell. These signals are therefore restricted to cells which are in direct contact
  190. 190. Extracellular HYDROPHOBIC Signaling Molecules • Some small hydrophobic molecules can cross the plasma membrane and enter the cell by diffusion • Best known classes are the STEROIDSTEROID hormones e.g. cortisol & testosterone and the THYROIDTHYROID hormones e.g. thyroxine • The hormones can diffuse across the plasma membrane and bind to receptor proteins that are located either in the cytosol or in the nucleus itself • They work by activating GENE REGULATORY PROTEINSGENE REGULATORY PROTEINS in the cell, which stimulate transcription of particular sets of genes in the nucleus
  191. 191. The mode of action of cortisol: Cortisol is a steroid hormone that is released in the body in response to physical or psychological stress. The secretion of cortisol induces energy-directing processes for the purpose of providing the brain with sufficient energy sources that prepare an individual to deal with stressors. In addition to its role as a so-called "stress hormone", cortisol plays many key roles in almost every physiologic system. Regulation of blood pressure, cardiovascular function, carbohydrate metabolism, and immune function are among the best known functions of cortisol. Action of Cortisol animation [Diagram]
  192. 192. Extracellular HYDROPHILIC Signaling Molecules • In contrast to the hydrophobic signals, the majority of signaling molecules are either too LARGELARGE or too HYDROPHILICHYDROPHILIC to cross the plasma membrane • The receptor proteins for these signals must therefore present a binding site to the extracellular environment and elicit a response in the cytosol • There are 3 main classes of these cell surface transmembrane receptors all of which bind extracellular signal molecules, but generate intracellular responses in DIFFERENTDIFFERENT ways …
  193. 193. 1) ION-CHANNEL LINKED Receptor • These are also known as chemically-gated ion channels • They open pores through the protein in response to binding of a signal molecule • Ions flow through this ’gate’ generating an electrical effect • This type of receptor is found in excitable cells such as nerve and muscle cells
  194. 194. • A neurotransmitter (e.g. acetylcholine, noradrenaline) binds to this type of receptor, altering its conformation to open or close a channel (often through or near the receptor) to the flow of Na2+, K+, Ca2+, or Cl- ions across the membrane. • Driven by their electrochemical gradient (i.e. one side of the membrane has numerous ions, while the other side has few) the ions rush into or out of the cell, creating a change in the membrane potential due to the positive or negative nature of the ions. • This flow of ions through the channel can trigger a nerve impulse, or alternatively stop one from occurring.
  195. 195. 2) ENZYME LINKED Receptor • Found in all types of cells • Generate an enzyme activity (usually a KINASEKINASE activity) on the cytoplasmic end of the protein • This kinase activity causes the phosphorylation of other intracellular proteins, thereby activating them
  196. 196. 3) G-PROTEIN LINKED Receptor • Activate a GTP-binding protein (the G-protein) that sets off a chain of events in the cell • This group of receptors is the largest known, and many different signals and responses can be associated with G- protein activity • All have the same structural arrangement within the membrane – known as a seven-pass transmembrane protein • Several hundred types of receptor are known, which bind signals as diverse as peptide hormone, amino acids, fatty acids and neurotransmitters
  197. 197. • On binding the signal, the G-protein is activated by the binding of GTP • This activated protein diffuses away from the receptor protein site and activates its target protein • This may be an ion-channel protein or an enzyme such as adenylate cyclase or phospholipase C These enzymes catalyse the formation of small molecules known as secondary messengers which trigger the intracellular response to the original signal transduction event to the cell surface. G-Protein animation
  198. 198. The cyclic AMP (cAMP) signal transduction pathway • Adenylate cyclase activity generates cyclic AMP (cAMP), phospholipase C generates Inositol Triphosphate (IP3) • Second messengers are important parts of the signal transduction pathway, and can have many different effects • An outline of the cAMP pathway is shown below: [Insert cAMP pathway diagram]
  199. 199. Signal Transduction • Very complex area ! • Signals can be of many different types and can act either by diffusing across the plasma membrane (such as STEROID HORMONESSTEROID HORMONES e.g. testosterone and NITRICNITRIC OXIDEOXIDE) or by interacting with a receptor protein on the cell surface • The variety of signals, receptors and responses means that the system of signal reception and transduction can generate very specific effects in different types of cell
  200. 200. • The response of a cell to a signal can involve ion flow, activation of specific proteins, or changes in gene expression • These effects can be short-lived, as in the case of the generation of an action potential, or they may be permanent alterations that control the developmental fate of the cell • It is therefore clear that the idea of a cell as a self- contained unit is in fact very far from the reality of the situation - cells are constantly engaged in the exchange of information in the form of molecular signals and it is this that enables cells in multicellular systems to function in an integrated way.
  201. 201. Quiz 1 Quiz 2
  202. 202. APPLICATIONS OF DNA TECHNOLOGY One of the defining features of modern biology is the extensive use of the technology of gene manipulation. It is now possible to manipulate DNA directly to produce recombinant DNA. The manipulated genes can be replaced back into the original, or a different, organism to produce transgenic plants and animals.
  203. 203. Applications of DNA Technology 1) The Human Genome Project: genetic mapping, DNA sequencing, genome analysis/comparison 2) Human Therapeutics: detecting genetic disorders, gene therapy 3) Forensic Uses:DNA profiling 4) Agriculture: transgenic plants, production of BST
  204. 204. The Human Genome Project • The genome of an organism is it’s complete complement of genetic information • Completed in 2003, the international Human Genome Project was a 13-year project coordinated by the U.S. Department of Energy and the National Institutes of Health. • Project goals were to … - identify all the approximately 20,000-25,000 genes in human DNA, - determine the sequences of the 3 billion chemical base pairs that make up human DNA - store this information in databases - improve tools for data analysis - address the ethical, legal, and social issues that may arise from the project.
  205. 205. • The human genome project has been achieved using 3 approaches: a) GENETIC MAPPING b) PHYSICAL MAPPING c) DNA SEQUENCING • Firstly however, the desired DNA sequences must be amplified. The process used to do this is the POLYMERASE CHAIN REACTION (PCR)POLYMERASE CHAIN REACTION (PCR)
  206. 206. The Polymerase Chain Reaction (PCR) • Polymerase chain reaction (PCR) is a revolutionary molecular biology technique for enzymatically replicating DNA • The technique allows a small amount of the DNA molecule to be amplified many times in an exponential manner • PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, and paternity testing. [Insert diagram]
  207. 207. PCR product compared with DNA ladder in agarose gel DNA ladder (lane 1), the PCR product in low concentration (lane 2), and high concentration (lane 3).
  208. 208. Stages in PCR • PCR, as currently practiced, requires several basic components. These components are: • DNA template, which contains the region of the DNA fragment to be amplified • Two primers, which determine the beginning and end of the region to be amplified (primers = short lengths of a known DNA sequence) • DNA-Polymerase, which copies the region to be amplified • Nucleotides, from which the DNA-Polymerase builds the new DNA • Buffer, which provides a suitable chemical environment for the DNA- Polymerase • The PCR reaction is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction.
  209. 209. The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps: • (1) The double-stranded DNA has to be heated to 94-96°C in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. • (2) After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C) • (3) Finally, the DNA-Polymerase has to fill in the missing strands. It starts at the annealed primer and works its way along the DNA strand. This step is called extension. The extension temperature depends on the DNA-Polymerase Taq DNA Polymerase: This is a thermal stable enzyme isolated from thermophilic bacteria. This enzyme canonly synthesis DNA in one direction 3’ - 5’
  210. 210. Applications of PCR 1) MOLECULAR BIOLOGICAL RESEARCH - gene screening analysis (looking for a gene) and DNA cloning (copying particular DNA sequences) 2) GENETIC MAPPING STUDIES e.g. human genome project, sequence tagging on genome sites 3) CLINICAL & DIAGNOSTIC USES - screening and diagnosis of HIV; cancer (detects mutations of oncogenes); genetic disorders e.g. cystic fibrosis 4) GENETIC IDENTIFICATION and DNA TYPING - forensic and parentage testing; sex determination of pre-natal cells; classification of species 5) IDENTIFICATION of TRACE AMOUNTS of DNA - detection of contamination of foodstuff by: food-borne pathogens, genetically modified organisms in food products, presence of pork in beef etc
  211. 211. Nucleic Acid Hybridisation • Once a gene has been isolated from a complete genome as a piece of DNA; we may want to know from which chromosome gene it came from and where that chromosome is located; or from which cells of the organism the gene is transcribed; or to test a sample of human DNA for mutations in the gene suspected of causing an inherited disease • All these questions can be answered by taking advantage of the fundamental property of DNA : COMPLEMENTARY BASE PAIRINGCOMPLEMENTARY BASE PAIRING
  212. 212. • Remember, the 2 strands of DNA are held together by HYDROGEN BONDING. These bonds can be broken by heating to 90o c or altering the pH • These treatments release the single strands but DO NOT break the strong covalent bonds that link the nucleotides together • If the process is reversed i.e. slowly lowering the temperature and bringing the pH back to normal, the complementary strands will reform double helices - this is known as HYBRIDISATIONHYBRIDISATION • Using this technique, particular DNA sequences can be identified by hybridisation with the aid of a NUCLEIC ACIDNUCLEIC ACID PROBEPROBE
  213. 213. Nucleic acid hybridization (A) If the DNA helix is separated into two strands, the strands should reanneal, given the appropriate ionic conditions and time. (B) Similarly, if DNA is separated into its two strands, RNA should be able to bind to the genes that encode it. If present in sufficiently large amounts compared with the DNA, the RNA will replace one of the DNA strands in this region
  214. 214. A Nucleic Acid Probe - a short, single-stranded DNA or RNA molecule that has been radioactively labelled (e.g. 32-phosphate 32 P) and is used to identify a complimentary nucleic acid sequence
  215. 215. Genetic Linkage Mapping • Genetic maps are based on the recombination frequency between genetic markers during MEIOSIS [see Higher notes!] • These can be used to locate genes on particular chromosomes and establish the order of the genes and the approximate distance between them • This approach relies on having genetic markers that are detectable • Genetic markers are any gene that shows variation (different alleles). These include genes and other DNA sequences such as microsatellites, which are tandem repeats of units 2-4 bp in length. These units are also known as short tandem repeats and are distributed fairly evenly over the genome, and may even occur within genes. • Sometimes these are genes that cause disease, traced in a family by pedigree analysis
  216. 216. • The marker alleles must be HETEROZYGOUS so that meiotic recombination can be detected • NB: if 2 genes are on different chromosomes - they are unlinked and will sort independently during meiosis • If 2 genes are on the same chromosomes they are physically linked and a crossover between them during Prophase I of meiosis can generate non-parental genotypes • The chance of a crossover occuring increases as linked genes become further apart. In fact, they may behave as if they are essentially unlinked • Genetic mapping is used to produce a picture of the locations of the marker loci on the chromosome. However, it doesn’t provide the precise distances between the genes [Insert Genetic Linkage diagram]
  217. 217. Physical Mapping • A physical map is a more detailed map of a genetic map • As with genetic maps, construction of a physical map requires markers that can be mapped to an exact location on the DNA • Physical maps of the genome can be constructed in a number of ways, all of which aim to generate a map in which the distances between markers are known with reasonable accuracy
  218. 218. Restriction Mapping • Fragments of DNA are made by cutting with restriction enzymes or endonucleases • These are enzymes that cleave DNA at certain nucleotide sequences, thereby generating specific fragments • The recognition sequences where restriction enzymes are short (4,5 or 6 base pairs long) sequences that occur at defined positions in the DNA • Using a combination of these enzymes and measuring the size of fragments produced, the ‘puzzle’ can be pieced together to give the pattern of restriction enzyme recognition sites in the DNA • Defined fragments can then be identified either by their size or using a specific DNA probe to bind to its complementary map [electrophoresis or nucleic acid hybridisation] diagram
  219. 219. Restriction Mapping : An exampleAn example The most straightforward method for restriction mapping is to digest samples of the DNA with a set of individual enzymes, and with pairs of those enzymes The digests are then "run out" on an agarose gel to determine sizes of the fragments generated. If you know the fragment sizes, it is usually a fairly easy task to deduce where each enzyme cuts, which is what mapping is all about
  220. 220. Restriction Mapping : An exampleAn example To illustrate these ideas, consider a plasmid that contains a 3000 base pair (bp) fragment of unknown DNA. Within the vector, immediately flanking the unknown DNA are unique recognition sites for the enzymes Kpn I and BamH I. As illustrated in the diagram below, consider first separate digestions with Kpn I and BamH I :
  221. 221. – Digestion with Kpn I yields two fragments: 1000 bp and "big". Since there is a single Kpn I site in the vector, the presence of a 1000 bp fragment tells you that there is also a single Kpn I site in the unknown DNA and that it is 1000 bp from the Kpn I in the vector. The "big" fragment consist of the vector plus the remaining 2000 bp of the unknown – Digestion with BamH I yields 3 fragments: 600, 2200 and "big". The "big" fragment is again the vector plus a little bit (200 bp in this case) of unknown DNA. The presence of 600 and 2200 bp fragments indicate that there are two BamH I sites in the unknown. You can deduce immediately that one BamH I site is 2800 bp (600 + 2200) from the BamH I in the vector. The second BamH I site can be in one of two positions: 600 or 2200 bp from the BamH I site in the vector At this point, there is no way to know which of these alternativeAt this point, there is no way to know which of these alternative positions is correctpositions is correct
  222. 222. • The trick to determining where the second BamH I site is located is to digest the plasmid with Kpn I and BamH I together • This so-called double digest yields fragments of 600, 1000 and 1200 bp (plus the "big" fragment). The 600 bp fragment is the same as obtained by digestion with BamH I alone. The 1000 and 1200 bp fragments tell you that Kpn I cut within the 2200 bp BamH I fragment observed when the plasmid was cut with BamH I alone You already know where Kpn I cuts in the unknown DNA, and you therefore now know the location of the second BamH I site!
  223. 223. Chromosome Walking • Used to locate genes or other DNA sequences on a physical map or to locate genes associated with disorders – The marker DNA and target DNA must be linked – DNA probes used to locate and isolate multiple copies of DNA that have complementary sequences of DNA to the probe in libraries – 2 libraries are made, one from cloned fragments of the marker and one from cloned fragments of the target DNA – Different restriction enzymes are used so that the fragments in each library are different but overlap
  224. 224. Gel Electrophoresis • Since nucleic acids are negatively charged, they migrate toward the positive pole in an electric field • When the electric field is applied through the gel, molecular sieving takes place. Shorter chains move faster than longer ones. Thus, the chains are spread out in the gel according to their size. • Double-stranded DNA can be visualized by adding ethidium bromide, a flat aromatic chemical that fits between base pairs in the double helix. Only when bound to DNA does the ethidium bromide fluoresce orange when irradiated with UV
  225. 225. DNA Sequencing • The final stage of the genome project is to determine and assemble the actual DNA sequence itself. For this to happen: – DNA fragments must be generated – The sequencing technology must be accurate and fast – Computer hardware/software must be available to analyse the data
  226. 226. DNA Sequencing cont. • The technique used for sequencing is the Dideoxy Chain Termination method as developed by F. Sanger in the 1970’s • This method relies on making a copy of the chosen DNA template [See Student monograph for a more comprehensive explanation – pg.154 - 157]
  227. 227. Comparative Genome Analysis • In addition to mapping the human genome, the genomes of other species are also being mapped. These include species important to biological research and agriculture such as the mouse, chicken, pig, cow, rice, wheat, Caenorhabditis elegans (nematode), Drosophila melanogaster (fruit fly), Saccharomyces cerevisiae (yeast), Escherichia coli, and other prokaryotes. • The genomes of some of these organisms, such as E. coli, yeast, the nematode and the fruit fly have now been completely mapped and sequenced. These maps can be used to locate homologous genes in the human genome and to help in determining gene function.
  228. 228. • Comparative genome analysis is being used to find out more about evolution. The number of differences in an amino acid sequence can be used to calculate the time since two species diverged from a common ancestor. If there are lots of differences between the maps, it can be deduced that the species diverged longer ago than if there are only a few differences. This type of information is used alongside other methods of measuring the rate of evolution. • Gene maps can be used to predict gene order. If gene X is found next to gene Y and Z in one species, the likelihood is that it will be found next to the same two genes in another closely related species. Comparative maps will be used to find candidate genes for phenotypes mapped in species as diverse as chicken and human.
  229. 229. Human Therapeutics How is our knowledge of DNA technology being used today in human therapeutics? • Congenital abnormalities are genetically based diseases and there therefore inherited • 2 types: – MONOGENIC : – POLYGENIC : caused by a single gene defect (Cystic Fibrosis, Sickle Cell Anaemia, Haemophilia) caused by defects in several genes (Heart Disease, Diabetes, Obesity, some cancers)
  230. 230. Detecting genetic disorders Characteristics of a monogenic disease usually begins with the presentation of disease symptoms Step 1 : Trace disease through family using PEDIGREE ANALYSIS to determine if the faulty gene is dominant, recessive or X-linked (crucial for genetic counselling) Step 2 : Genetic maps are used to identify genetic markers, co- inherited along with the disease. Recombination frequencies give the distance of the marker to the diseased gene. The marker is then located on a more detailed physical map. The gene is then tracked down, characterised and sequenced, leading to accurate diagnostic procedures and potential new treatments
  231. 231. Cystic Fibrosis (CF) Affects 1 in 2000 Gene CFTR gene (Cystic fibrosis transmembrane conductance regulator) Protein coded Membrane protein (1480 a.a. long) 2 transmembrane domains Regulatory region 2 ATP binding sites Mutations Defective ion transport systems Result Epithelial surfaces are not fully hydrated causing sticky mucus accumulation in the lungs Symptoms Inflammation of lung tissue; bacterial infection; high salt content of sweat Gene found 1989 Chromosome 7
  232. 232. Duchenne Muscular Dystrophy (DMD) Affects 1 in 3500 boys Gene DMD gene Protein coded DYSTROPHIN (3685 a.a. in length) Mutations The cytoskeleton is not linked with the muscle cell membrane in muscle cells Result Muscle cells become permeable, extracellular fluid flows in & cells burst Symptoms Progressive failure of muscle growth & wastage leading to weakness, paralysis & respiratory difficulties Gene found 1987 Chromosome X
  233. 233. X linked inheritance of Duchenne Muscular Dystrophy Autosomal Recessive Inheritance of Cystic Fibrosis
  234. 234. Mini-project • Using either CF or DMD as a case study, write a report about the discovery and treatment of the disease making sure to : – explain the genetic mutation involved – describe the methods/tests used to detect genetic disorders such as Cystic Fibrosis and Duchenne Muscular Dystrophy; – explain the importance of genetic counselling; – explain how gene therapy could be used to treat genetic disorders; – include some analysis of results of gene therapy trials; – discuss the legal, moral and ethical issues for the future http://www.gig.org.uk & http://www.who.int/genomics/elsi/en/