Revision Notes - Cell and Molecular Biology (Trinity College Dublin)

lOMoARcPSD|49612230 Revision Notes 4bio1 Cell and Molecular Biology (Trinity College Dublin) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Caoimhe Rogers (rogerscaoimhe...

lOMoARcPSD|49612230 Revision Notes 4bio1 Cell and Molecular Biology (Trinity College Dublin) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 CELL THEORY:  All cells develop from pluripotent embryonic stem cells.  RNA polymerase transcribes DNA onto mRNA. Ribosomes translate mRNA into amino acids (Translation), amino acids come together to form growing polypeptide. This continues until a stop codon is reached.  Epigenetics: Alterations in gene expression that does not depend on changes in DNA sequences.  Polygamous DNA: DNA works with many partners, DNA methylation, for example, influences the way that genes are expressed without changing the underlying DNA sequence, and other epigenetic factors bind to histones to control when chromatin complexes open up and allow their DNA to be read.  23 pairs of chromosomes in the nucleus.  Methylation (Histones + Methyl Group + Genes) of DNA histones cause nucleosomes to pack tightly together, transcription factors cannot bind the DNA and genes are not expressed.  Histone acetylation (Histones + Acetyl Group +Genes) results in loose packing of nucleosomes (Histones + Genes), transcription factors can bind the DNA and genes are expressed.  Mitochondrion DNA comes from the mother, nucleus DNA comes from both parents.  GLYCOLYSIS: Phase 1 energy investment, Phase 2 Energy payoff. GLUCOSE -> 2PYRUVATE + 2H2O; 4ATP FORMED – 2 ATP USED -> 2 ATP; 2NAD+ + 4E- + 4H+ -> 2NADH + 2H+.  Glycolysis in cell plasma, citric acid cycle in mitochondrion, electron transport chain in mitochondrial membrane.  The Golgi apparatus process, sort and deliver proteins.  Exocytosis: materials transported out of the cell: signal picked up by ligand or receptor on cell membrane, signal transduced into cell. Proteins from Golgi apparatus stored in secretory (1) or transport (2) vesicles, (1) Secretory proteins, regulated membrane fusion (regulated secretion out). (2) Soluble proteins, unregulated membrane fusion, leave vesicle due to cell membrane proteins on vesicle. Constitutive secretion.  Biological membranes: - Segregation and protection of the cell from external environment. - Import and export; selective permeability to essential substance, transport via channels in membrane.  Lysosomes; Contain digestive enzymes for waste removal.  Cytoskeleton: Structural proteins give the cell characteristic shape and support. Important for motility and transport.  Cell Cycle and Cell division: M (mitosis) -> G1 (Cells increase in size in Gap 1, produce RNA and synthesize protein. An important cell cycle control mechanism activated during this period (G1 Checkpoint) ensures that everything is ready for DNA synthesis) or G0 (There are times when a cell will leave the cycle and quit dividing. This may be a temporary resting period or more permanent. An example of the latter is a cell that has reached an end stage of development and will no longer divide (e.g. neuron).) [ends here if G0] -> S (To produce two similar daughter cells, the complete DNA instructions in the cell must be duplicated. DNA replication occurs during this S (synthesis) phase) -> G2 (During the gap between DNA synthesis and mitosis, the cell will continue to grow and produce new proteins. At the end of this gap is another control checkpoint (G2 Checkpoint) to determine if the cell can now proceed to enter M (mitosis) and divide.) ->back to M.  Mitosis: Interphase (centrioles divide and proteins produced) , prophase (nucleus dissolves, chromatin condense to chromosomes, microtubule dissolve and spindles form), metaphase (spindles tense and chromosomes centre of cell), anaphase (spindles pull chromosomes apart), telophase (2 new nucleus form and membrane pinches apart). Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Apoptosis = programmed cell death, 2 pathways (intrinsic and extrinsic). Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 BIOLOGICAL MEMBRANES:  Cell = simplest collection of matter that can live (cells -> tissues -> organ).  Eukaryotic cell (standard mammalian cell).  Plasma membrane: - Keeps the cell together. - Separates living cell from non-living surrounds. - Is not solid, has tiny openings that let things in and out. - Exhibits selective permeability = allowing some substances to cross more readily than others. - Very important for life (nutrients in wastes out).  Biological membranes: - Barriers: Define the inside and outside of cell, prevent cellular molecules leaking out, prevent unwanted molecules getting in. Cellular membranes form closed compartments and have an internal and external face. o Plasma membranes: cytosolic face (into cytoplasm), exoplasmic face (outer environment facing). o Organelles: cytosolic face (into cytoplasm), exoplasmic face (into organelle). - Gatekeepers: Contain transport systems to import and export molecules (selective permeability). - Information and energy: signal transmission, energy transmission. o Proteins embedded in membrane, hold the membrane in a regular structure for easy bonding with other membranes. o Act as receptors for attachment to molecules that need to be taken up by the cell. o Act as carrier proteins to transport substances in or out of cell. o Passive transport (no energy, diffusion/facilitated diffusion) or active transport (requires energy). - Compartmentalization in cells: cell requires to compartmentalize intracellular activates, maintain a selective barrier to the exterior and transport substances in, out and around cell packets -> TRAFFICKING PROTEINS AROUND THE CELL IN MEMBRANE PACKAGES.  Membrane Structure: - Fundamental unit of biological membranes is an asymmetric phospholipid bilayer. - Other lipids are scattered between these phospholipid molecules. - Proteins are embedded and otherwise attached. - Carbohydrates (oligosaccharides, glycans) exist on the outer surface.  Plasma membrane: Separates cell from outside environment, maintain order and organization. Chemically achieve -> cytoplasm mainly water, protein and some nucleic acid, lipids won-t mix with these and provide separation.  Lipids: Diverse group of biochemical, contain carbon-hydrogen chains/rings, confers ridged structures, INSOLUBLE IN WATER, hydrophobic, non-polar. Common feature is solubility not structure. Lipids can have hydrophobic and hydrophilic portions (amphiphilic) -> ideal for membranes.  Phospholipids: - Hydrophilic choline and phosphate and glycerol head with hydrophobic fatty acid tail, polar head group consists of phosphoric acid conjugated to a base, linker molecule is glycerol a 3 carbon molecule. Phosphorylated alcohol ester provides the polar hydrophilic region - Two chains of fatty acids (C/H chain) in tail, on chain has a double bond which causes a kink -> important for structure of bi-layer. Fatty acid ester linkages provide for the hydrophobic core. o Fatty acids; even numbered hydrocarbon chains of variable length, terminate in a carboxyl group, can be esterified in a reaction with alcohol groups of glycerol. o Saturated fatty acids = no double bonds, anoic acids (straight and ridged). o Unsaturated = mono or polyunsaturated, enoic acids, one or more double carbon to carbon bond in a cis config with 120 degree kind in chain (flexible, membrane fluidity). - Amphiphilic lipid species- contains hydrophobic and hydrophilic portions. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 - When phospholipids get together: Micelle: circular (colloidal) arrangement of phospholipids, hydrophilic heads attract each other and mix with external soluble environment, hydrophobic tails attracted to each other. Alternative arrangement: - Phospholipid bilayer: Wants to form a circular/enclosed shape (energetically favourable), linear shape with exposed ends (energetically unfavourable) will not work. Phospholipids spontaneously form symmetric sheet-like bilayers, two molecules thick. Spontaneously seal to form closed structures that separate two aqueous compartments, very stable. Hydrophobic tails provide separation, hydrophilic heads allow mixing with the environment. - Movement: lateral diffusion, flexion, rotation, flip-flop (rare). Amphiphilic nature allows fluidity.  Trilaminate appearance (bilayer) on TEM: Railroad tracks, central electro-lucent zone bordered by defined areas of electron density.  Other lipids in membranes: Cholesterol (steroid molecule, up to 50% of animal plasma membrane), smaller and less amphipathic than phospholipid), sphingolipids (18 carbon back bones, important lipid in neural tissue), ceramides. Distributed asymmetrically in membrane.  Membrane Fluidity: lipids = rapid lateral motility, slow to flip flop. Distributed asymmetrically, lipid composition determines membrane fluidity. Saturated = less fluid (straight chains allow max interaction of fatty acid tails, solid at room temp), unsaturated = more fluid (bent chains keep tails apart, liquid at room temp, polyunsaturated have multiple double bonds.  Cholesterol aids membrane fluidity, bidirectional regulator, reduced membrane fluidity by reducing phospholipid movement at high temps, hinders solidification at low temps. Steroid, 3x6 carbon rings linked to cyclopentane ring.  Other hydrophobic lipids (cholesterol) become embedded in plasma membrane and provide extra stability.  Other components: Integral proteins (penetrate hydrophobic core, many transmembrane), peripheral proteins (not embedded in membrane, bound to exposed integral proteins or loosely to surface).  Two models of cell membrane: davson-daniellie model (protein layers on either surface of phospholipid bilayer) and fluid mosaic (current). Not all membranes have same % of proteins in them, membrane proteins not soluble in water.  Lipids are always moving sideways, proteins drift slowly, cholesterol acts as temp buffer (keep fluid), must be fluid to work.   Biological Membrane Composition: Ration of protein to lipid varies greatly between and within membranes (myelin sheath = low, mitochondrial inner membrane = high [protein: lipid]).  6 major membrane protein functions: 1. Transport (hydrophilic channel or carrier protein). 2. Enzymatic activity. 3. Signal transduction. 4. Intercellular joining (tight junctions formed between cells). 5. Cell-cell recognition. 6. Attachment to the cytoskeleton and ECM (stabilises location of certain proteins).  Diffusion- passive transport: tendency for molecules of any substance to spread evenly into available space. Any substance will diffuse down its concentration gradient, spontaneous, no energy needed small hydrophobic molecules will diffuse across the membrane.  Selective permeability: membrane proteins – key role in regulating transport. Uncharged polar molecules and ions need protein transporters. Transport proteins span the membranes, very specific, 2 types; channels and carrier. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Facilitated Diffusion – Passive Transport: diffusion of hydrophobic solutes across the membrane must be facilitated by transport proteins, channel proteins provide hydrophilic corridors (i.e. ion channels). Carrier proteins change shape to translocate across the membrane, triggered by the binding and release of transported molecule, passive because solute moves down concentration gradient.  Active transport: Uses energy: Movement of substances against concentration gradient across membrane. Low to high conc. requires work, cell uses energy, active. Active transporters are carrier proteins. Enables cell to maintain higher internal concentration of molecule compared to external environment.  Other components of membranes: - Membrane carbohydrates: face away from cytoplasm, attached to protein or lipid, blood antigens, glycoproteins (protein receptors), specificity for cell-cell or cell-protein interactions.  Transport across a membrane: regulation of transport across the cell membrane – essential to its existence (muscle cells), membrane is selectively permeable.  Active transport: ATP supplies energy, can transfer terminal phosphate group directly to transport protein -> transfer of energy -> phosphorylation. Eg. Sodium-potassium pump.  Sodium potassium pump -> electrogenic pump, generate voltage across membrane, more negative inside than out, electrochemical gradient.  Membrane potential; voltage across membrane, storage of electrical potential energy, can be tapped into by the cell to carry out work instead of using ATP. Diffusion of H+ ions down gradient can be coupled to active transport of sucrose against its gradient. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 IMAGING IN LIGHT MICROSCOPY USING FLURESCENCE:  Need thin layer of material to visualise for light transmission, get enough detail and also use cell culture.  Blood smear using Wrights staining.  Fixed samples: - Fixed samples as opposed to live imaging. - Main form is chemical fixative formaldehyde (cross link proteins). - Also use alcohol/acetone (dehydration/aggregation). - Stop breakdown of sample and preserve.  Fluorescence: - Fluorescent compound + absorbed light of right wavelength = light release at higher wavelength (less energy). - Photon release: electron excited from ground state by absorption of light, fluorescence observed as electron decays (photon release), and energy lost so light emitted at a longer wavelength.  Fluorescence imaging: - Allows for selective tagging of specific structures or proteins in cells in much greater detail. - Multiple probes can be used at the same time. - Can be done on fixed or live samples. - Can use antibodies, dyes and fluorescent proteins. - Linger wave = lower energy.  Fluorescein = a typical fluorescent probe.  Immunohistochemistry: can have dyes directly linked to primary antibody or to a secondary antibody which gives more flexibility and increased signal, to mark specific proteins like cell surface markers or a protein of interest, all on fixed cells.  Fluorescent stains: Can be used on both fixed and live cells; for organelles such as: 1. Dapi -> nucleus. 2. Mitotracker -> mitochondria. 3. Lysotracker -> lysosomes. 4. Phalloidin -> actin. 5. Cholera toxin -> cell surface.  Fluorescent proteins: such as in jellyfish. Green fluorescent proteins can be used as a marker for gene expression.  Mutagenesis followed by screening of expression libraries for the desirable qualities, mutants may be generated in vitro by random or site-directed mutagenesis. Key characteristics for practical use: 1. Brightness: QY and EQ. 2. Amyration rate. 3. Photostability. 4. Oligomeric nature and aggregation. 5. Spectra (colour). 6. Switchable.  GFP and modified GFP = popular fluorescent marker.  Co-localisation studies (ensure no spectral overlap): - Always do controls (each probed separately). - Ensure correct filter sets (spectral slit widths) used during acquisition. - Fluors should be carefully matched to the laser liners to get max excitation efficient high degree of separation between fluors. - Cannot be used to say two proteins are in contact, issues with resolution.  Using lasers on region of interest: looking for fluorescent tag to move back into area that is bleached. Used as a measure of mobility of tagged protein or organelle. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Phase contrast/DIC: Thin samples can lack contrast to pick out details, same properties that cause light to bend also delay the passage of light by a quester of a wavelength or so. Light from most objects passes through centre of the lens as well as to the periphery. If the light from an object to the edges of the objective lens is retarded by half wavelength and the light to the centre is not retarded at all the light rays are out of phase by half a wavelength. The cancel each other when the objective lens brings the image into focus. Reduction in the brightness of the objective is observed. Degree of reduction in brightness depends on the refractive index of the object. Polarized light can give similar effect. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Multiphoton microscopy is used for deeper imaging: - Uses 2 photons of higher wavelength to excite fluor, powerful pulsed lasers which emit in 800- 1300nm range. - Higher wavelength travels further through sample. - Light is focused to focal plane (reduced bleaching and removes need for confocal). - Brute force approach, imagining can be achieved to max depth of 600nm in rat or mouse brain. - 2 photon fluorescence excitation is proportional to the intensity squared, confining fluorescence generation to a small spot in the focal plane where the density of photons is high. This focal volume can be raster scanned to anywhere in the cuvette thereby creating a point-wise sequential 3D representation of fluorescence intensity.  Total internal reflection microscopy (TIRFM). - TIR of light has been used to study cell-substrate surfaces and to acquire detailed information about cell membranes. - Light beam propagated through medium of refractive index n1 (glass), meets and interface with a second medium of refractive index n2100), saucer shaped, forming face (cis), releasing face (trans).  Lysosomes: - Garbage disposal system of cell. - Degrade products of ingestion, such as bacterium that has been taken in by phagocytosis. - After bacterium is enclosed in vacuole, vesicles containing lysosomal enzymes (primary lysosomes) fuse with it. - pH becomes acidic, activates enzymes. - Vacuole becomes secondary lysosome and degrades bacterium. - Lysosomes degrade worn out organelles such as mitochondria.  Peroxisomes: - Contain at least 50 different enzymes, involved in a variety of biochemical pathways in different types of cells. - Organelles that carry out oxidation reactions leading to production of hydrogen peroxide, hydrogen peroxide harmful to cell therefor peroxisomes contain enzyme catalase, which decomposes hydrogen peroxide (converts it to water or uses it to oxidise another organic compound). - Variety of substrates broken down by oxidative reactions in peroxisomes including uric acid, amino acid and fatty acids. - Oxidation of fatty acid = important, provides major source of metabolic energy. - May resemble lysosomes but not formed in Golgi apparatus. - Distinguished by crystalline structure inside a sac which contains an amorphous grey material. - Self-replicating, like mitochondria. - Components accumulate at given sit and can be assembled into a peroxisome. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 - Look like storage granules but not formed in same way. - Enlarge and bud to produce new peroxisomes.  Mitochondria: - Structure: o Elongated cigar shaped membrane bound. o Number per cell varies widely depending on cell and tissue type (few to 2000 per cell). o Composed of compartments that carry out specialised functions: Outer membrane. Inner membrane folded into cristae. Intermembrane space. Cristae. Inner matrix. o Own genomes; circular DNA compliment (2-10 copies, 37 genes); replicates independently; homology to bacterial genomes. - Function: o Power plant of eukaryotic cells. o Harness energy obtained by combining oxygen with food molecules to produce ATP. o Aerobic respiration. o Calcium storage. o Regulation of programmed cell death (apoptosis)>  Ribosomes: - Site of protein synthesis. - Found attached to rough ER or floating free in cytosol. - Produced in a part of the nucleus call the nucleolus. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 THE CELLULAR CYTOSKELETON:  Function: - Tissue level: muscle movement: - Cellular level: Determines shape of cell; motility of the cells; cell adhesion; mitosis, meiosis. - Subcellular level: anchors organelles; organization of organelles; provides tensile strength; movement of chromosomes; organising cell polarity; intracellular movement of vesicles (endocytosis: clathrin mediated endocytosis and phagocytosis). - Dynamic, adaptable, stable strong. - The shape of the microvilli in the intestinal cell are supported by microfilaments, anchored to a network of intermediate filaments.  Main components: - Microfilaments: actin, 7nm. - Microtubules: tubulins (alpha and beta), 25nm. - Intermediate filaments: lamin, cell specific proteins (e.g. vimentin), 8-12nm.  Actin: - Molecular structure of actin: plus end and minus end, F and G actin. - Globular protein with an ATP binding site in the centre of the molecule. - G- Actin; the monomer will dimerize or form trimer -> serves as a site for nucleation and further growth of the actin protofilament. - G-actin forms F-actin (the filament) in the presence of ATP, Mg and K. - Concentration of G-actin is critical; above critical conc molecules will polymerize; below actin filaments will depolymerize. - ATP hydrolysis not requires for polymerization, required to promote depolymerisation (if it is converted to ADP). - Behaves like microtubules and their need for GTP hydrolysis to depolymerize. - Have polarity (like microtubules).  Motor Proteins: Myosin: - General structure: globular head and fibrillary tail; heavy chains and light chains; Head (motor domain with ATP-ase activity); ADP-straight.  Cell migration requires membrane protrusion at the cell front, force that projects the plasma membrane is largely provided by actin polymerization.  ARP2/3 complex needs to be activated by nucleation n-promoting factors (NPFs).  Leading edge: - WAVEcomplex (NPF) activated and recruited to lamellipodia by a combination of interactions with transmembrane receptors. - Not yet established whether these ligands forma an ordered pathway or whether they constitute parallel or alternative pathway. - ARP2/3 complex generates branched actin array; required for lamellipodium protrusion because it nucleates a new actin filament off the side of the existing filament. - Listeria monocytogenes ActA protein converts actin polymerization into a motile force; binds ARP2/3 complex and VASP.  Spectrin: theory: a mechanical scaffold which supports the membrane bilayer and controls the motility and perhaps the activity of membrane integral proteins, which are membrane channels, transporters and receptors.  Tubulin heterodimer = microtubule subunit (beta tubulin and alpha tubulin together). Then forms alternating chains, cylinder with lumen to form microtubule. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Microtubule geometry is not fixed, more flexible lateral contacts can accommodate between 11 and 16 protofilaments.  13 fold geometry is preferred because it the only geometry in which protofilaments run straight along the microtubule length as opposed to twisting around the microtubule -> processively tracing motor proteins remain on the same face of the structures.  MTOC microtubule organizing centre.  Gamma tubulin at the end of the tubule; role in nucleation microtubules; accessory proteins in gamma tubulin ring complex.  Polymerization of microtubules: breaking down; dynamic instability and tradmilling; GTP gap on top during growing, unstable bottom.  GTP cap has binding accessory proteins such as EB1; can influence binding of other proteins in a complex and the stability of the tubulin fibre; modulate interactions with organelles.  Cilia: - Dynein arms (ATPase activity, in the presence of ATP the can move from one tubulin to the other, enable the tubules to slide along one another so the cilium can bend) and nexin links. - Dynein bridges regulated so that sliding leads to synchronized bending. - Nexin and radial spoked; doublets are held in place so sliding in limited lengthwise, when subjected to enzyme digestion and exposed to ATP the doublets will continue to slide and telescope to x9 their length. - Primary purpose in mammalian cells is to move fluid, mucous or cells over their surface. - In lung trachea.  Centrioles: - Made of microtubules (like cilia and flagella). - Contain 9 sets of triplets and no doublet in the centre, triplets in the basal body turn into the cilium doublet. - Come in pairs, each organized at right angles to each other. - Organizes the spindle apparatus on which the chromosomes move during mitosis. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  5 different types of intermediate filaments: - 1 and 2: Acidic keratin and basic keratin, respectively. Produced by different types of epithelial cells. - 3: distributed in a number of cell types including: vimentin in fibroblasts, endothelial cells and leukocytes; desmin in muscle; glial fibrillary acidic factor in astrocytes and other types of glia and peripherin in peripheral nerve fibers. - 4: Neurofilaments H( heavy), M (medium) and L (Low), refers to molecular weight of the NF proteins. Another type: internexin and some nonstandard 4’s are found in lens fibers of the eye (filensin and phakinin). - 5: lamins which have a nuclear signal sequence so they can form a filamentous support inside the inner nuclear membrane, Vital to the re-formation of the nuclear envelope after cell division. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 THE INTRACELLULAR FLUID COMPOSITION:  Factors affecting simple diffusion: - Molecular radius. - Thermal energy. - Temperature. - Viscosity of diffusion medium. - Concentration gradient.  Diffusion in homogenous media: stokes-Einstein equation for diffusion coefficient D: D = -kT/6r𝜋ŋ, r = radius of molecule. Factors affecting: temp (increase, D increases), radius (increase, D decreases), viscosity (increase, D decrease).  Gradients of simple and complex molecules play a vital role in biology; gas exchange in lungs, salt-water balance in tissues, neurotransmitters, effects of hormones and growth factors.  Diffusion across barriers: Fick’s law describes the rate of diffusion across a membrane. First Law (J = flux/unit time [mol/s]) J = -DAΔC/ΔX (A = area across which diffusion is occurring, c = concentration gradient, x= distance). A increase J increase, C increase J increase, X increase J decrease.  Diffusion across Lipid Membrane barriers important considerations: - Chemical gradient. - Size of molecule. - Hydrophobic/ hydrophilic compatibility (partition coefficient). - Thickness of membrane (5nm). - Existence of pores/transporters for charged particles. - Channel open probability/ transporter saturation. - Electrochemical gradients. - Osmotic movement of water.  Net diffusion across plasma membrane (excluding electrical effect). - 5 nm lipid bilayer; lipids diffuse across, charged, hydrophilic cross via pores, channels and carriers. - Net diffusion: Q = ΔCPA/MWΔX. C increase Q increase, P (permeability) increase Q increase, A increase Q increase, MW (molecular weight) increased Q decrease, X increase Q decrease.  Osmosis is the diffusion of water down its concentration gradient.  Cell volume regulation: osmotic and hydrostatic pressure, ionic strength of a solution exerts an osmotic pressure, membrane is only permeable to water, at equilibrium hydrostatic pressure is opposite and equal to osmotic pressure. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Osmotic pressure: van’t Hoff’s Law, measured in atm (1atm = 760mmHg). Osmotic pressure (Π) = nCRT. n= number of dissociable particles per molecule, C is total solute conc, nxC converts molarity to osmolarity = number of osmotically active particles/litre.  Osmolartiy is a direct measurement of total solute concentration, body fluids typically in milliosmoles/l (mOsm), typically 300mOsm.  Osmotic pressure gradient of ECF and ICF: - ICF and ECF osmotic pressures need to be matched (iso-osmolar). - At sites of normal tomicity balance is achieved around 280 mOsm/L. Osmotic pressure of ECF and ICF 7 atm (5409mmHg).  Osmotic Activities of the ECF and ICF: - ICF: intracellular protein and K+ are responsible for the majority of the osmotic activity of the ICF. - ECF: Na+ and its anions are responsible for the majority of the osmotic activity of the ECF. - Absolute number of osmotically active particles in similar between the ECF and ICF, no net movement of water occurs in the resting state. - Changes in the ECF composition can however effect a net movement water in and out of cells. - Cells must adapt to maintain volume optimum as follow on the next slide.  Electrochemical gradient requires cell to actively maintain steady state volume across plasma membrane.  Intracellular pH Regulation: - Cid-base balance is critical for cell function (e.g. pH optima for enzymes). - Two main ICF buffers: protein buffer system and phosphate buffer system. - Excess H+ is also removed from cells via exchange transporters (e.g. Na+/H+#). - Such transporter are dependent on ion distribution and membrane potential.  Intracellular Protein as a buffer: - Amino-acid with acidic side chains (Glutamic acid, Aspartic acid) -> proton donors at pH 7.4. - Amino-acids with basic side chains (Arginine, Lysine, Histidine) -> proton acceptors at pH 7.4.  Phosphate buffer system: in acidic solution HPO4,2- accepts H+ (pH increases) in basic solution H2PO4 donates H+ (pH decreases).  ECF pH Regulation: - Bicarbonate is the major ECF buffer system. Haemoglobin is an important extracellular protein buffer in blood.  Why is maintaining pH important? - pH changes can cause conformational changes in proteins and can alter functions (enzyme activity is pH dependant). - Nervous system function is very sensitive to pH changes, can lead to seizures, come. - Can affect kidney function –K+ retention (hyperkalaemia) or K+ depletion (hypokalaemia). - Cardiac arrhythmia. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 PROTEINS (1+2):  4 main classes of biological molecules: - Carbohydrates – sugar polymer (polysaccharides), fuel for body. - Lipids- triglycerides, fatty acids, steroids and phospholipids, serve as energy stores and provide insulation, form cell membrane. - Nucleic acids – DNA and RNA, reproduction. - Proteins – Amino acid polymers, encoded by genes, functional complex molecules, molecular tools of the cell, 50% cell dry mass.  DNA (which replicates, genetic information genotype) -> RNA (during transcription, messenger RNA, mRNA) -> Inherited protein (physical manifestation of genetic information, physical trait = phenotype).  8 categories of protein: - Proteins are the key functional molecules of life. - Have many structures, resulting in a wide range of functions. 1. Enzymatic; selective acceleration of chemical reactions. 2. Structural; support. 3. Storage; storage of amino acids. 4. Transport; transport of other substances. 5. Hormonal; coordination of an organisms activities. 6. Receptor; response of cell to chemical stimuli. 7. Contractile and motor; movement. 8. Defensive; protection against disease.  Enzymatic proteins (lipase). - Function is to accelerate chemical reactions. - Act as catalysts- reagents that selectively speed up chemical reaction without being consumed by the reaction. - Example; digestive enzymes catalyse the hydrolysis of the o=polymers in food (lipase, lactose) [triglyceride to monoglyceride and fatty acids].  Structural: - Function: support. - Long chained molecules. - Example; silk fibroin silk fibres used by insects and spiders. - Are both strong and flexible. - Crosslinking and intertwining allows for many properties: o Flexible and elastic, wool. o Strong and ridged, collagen. - Collagen and elastin provide a fibrous framework in animal connective tissue, breaks down and disorganizes with age.  Storage: - Storage of biological reserves of amino acids and metal ions. - Metabolism of these proteins provides building blocks to make new proteins. - Example; ovalbumin in the developing embryo, casein in mike for baby mammals. - Ferritin the iron storage protein; iron = central atom of heme group and is an oxygen carrier, iron + water + oxygen = rust therefore ferritin acts as the iron buffer, ferritin protein consists of 24 subunits with iron stored in the centre of each. 2.5g iron in haemoglobin and 1g in ferritin.  Transport: - Transport of other substances. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 - Example; haemoglobin, protein of red blood cells, contains iron and transports oxygen from the lungs around to other parts of the body. 97% of RBC dry content is haemoglobin. - Example; transferrin, serum protein that transfers iron between cells, controlling the free iron in biological fluid.  Hormonal: - Coordination of an organisms activities. - Example; Insulin and glucagon – regulate the concentration of blood sugar levels, levels need to be kept at a constant concentration in the blood.  Receptor: - Allow cells to respond to chemical stimuli. - The stimuli that receptor proteins detect are called ligands (hormone proteins). - When a receptor bind to a ligand it usually changes the shape of the receptor (usually transmembrane). - Results of receptor binding its ligand is signal transduction. - Allows for a response to the external environment. - Example Insulin Receptor: Conformational change induced by insulin binding. - Example: Receptors at nerve endings, detect neurotransmitters e.g. Serotonin Rec. (Serotonin = mood, sleep, attention/learning).  Contractile and motor: - Movement. - Actin and Myosin; responsible for altering the shape of cells, the movement of muscle fibres.  Defensive: - Protect against disease. - Example; antibodies that help combat bacteria and virus. - Highly variable. - Disease XLA- no circulating Abs – susceptible to infection. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 - Protect against the effects of injury such as bleeding. - Example; Fibrinogen is converted to its insoluble form (fibrin) the enzyme thromboplastin. - Fibrin forms a fibrous meshwork which is the basis for a blood clot.  The genetic code. - Genes are made up of sets of 3 nucleotides (codons) which encode a sequence of amino acids that form a polypeptide. - DNA codes for RNA (base pair rule). - RNA codes for protein (3 nucleotides for 1 amino acid). - Protein (sequence of amino acids form a polypeptide). - 20 different amino acids with 64 different arrangements of nucleotides, 25000 different proteins.  What is the structure of a protein: - Each protein has a specific structure and function. - Protein shape is very important to its function, proteins interact with other proteins (e.g. antibodies). - Diverse functions = diverse structures. - Unique function = unique 3D shape. - Protein shape = conformation. - Function of a protein is inextricably linked to its shape.  Polypeptides: - Proteins; linear polymers of amino acids (aa), polymer of aa = polypeptide. - A protein consists of one or more polypeptides.  Amino Acid Monomers: - Amino acids: organic molecules possessing both carboxyl and amino groups, differ in their properties due to differing side chains (R groups), 20 different side chains = 20 different amino acids.  Subgroups of amino acids; - 20 naturally occurring amino acids. - Grouped into 4 subgroups, depending on side chains. - Polar and neutral; hydrophilic. - Polar and acidic; hydrophilic. - Polar and basic; hydrophilic. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 - Non polar hydrophobic. - Polar = spread of charge is not even, some atoms carry partial negative charge and others partial positive.  9 Non-polar amino acids: 1. Glycine 2. Alanine. 3. Valine. 4. Leucine. 5. Isoleucine. 6. Methionine. 7. Phenylalanine. 8. Proline. 9. Tryptophan.  6 Polar amino acids: 1. Serine. 2. Threonine. 3. Tyrosine. 4. Asparagine. 5. Cysteine. 6. Glutamine.  2 acidic: 1. Glutamic acid. 2. Aspartic acid.  3 basic: 1. Histidine. 2. Lysine. 3. Arginine.  Highlighted = essential amino acids, an organism cannot synthesize on its own.  Phenylketonuria (PKU). - Classical PKU caused by mutated gene for enzyme phenylalanine hydroxylase (PAH). - PAH converts essential amino acid phenylalanine to other essential amino acids, e.g. tyrosine. - Lack of PAH causes phenylalanine accumulation, lack of tyrosine (normal at birth with gradual loss of metal function, sever learning disabilities and seizures). - Treatment: intake of phenylalanine low, supplement diet with tyrosine. - Heel prick ‘Guthrie test’ detects at birth.  Amino acid polymers; - Amino acids are linked by peptide bonds. - Peptide bond is formed by a dehydration reaction – catalytic reaction. - Peptide bond = covalent bond.  Protein conformation and function: - Polypeptides: formed one at a time starting from N-terminus, range from a few monomers to 100 or more. - Unique sequences of aa’s = specific polypeptides, defined by genetic code. - Sequence of the aa polymer determine the 3D shape of the polypeptide. - Proteins are not just chains of aa’s, they are defined by their shape – interactions between backbone residues and R-groups. - A proteins specific conformation determines how it functions (Enzyme binds substrate).  2 models of protein conformation: ribbon and space-filling. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Four levels of protein structure: 1. Primary structure: is the unique sequence of amino acids in a polypeptide. 2. Secondary structure: o Results from interaction between atoms in the polypeptide backbone. (Beta sheets and alpha helixes). o Is the folding or coiling of the polypeptide into a repeating configuration. o Includes the alpha helix and beta pleated sheet. o Result of hydrogen bonding between the repeating backbones of the polypeptide. o Repeated coils or folds in patterns contribute to the overall conformation of a protein. o Hydrogen bonds:  Formed by the attraction between a partial positive charge on the H atom of the amino group and the partial negative charge on the O atom of the peptide bond.  Alpha helix: bonds are folded between repeating atoms on the same polypeptide chain (e.g. collagen).  Beta sheets: bonds are formed between polypeptide chains lysing side by side (e.g. silk fibroin). 3. Tertiary structure: o Overall 3D shape of a polypeptide, final shape of polypeptide. o Results from interactions between the side chains of the amino acids, hydrophobic and van der Waals interactions. o Hydrophobic interactions: Non-polar side chains groups are repelled by water, cluster together, force themselves into the core of a protein, and stabilize overall structure. o Van der Waals interactions: occur between hydrophobic non-polar side chains in close contact. o Ionic bonds/salt bridges:  Form between positively and negatively charged side groups.  Result for the neutralization of an acid and amine on side chinse.  Final interaction is ionic between positive ammonium group and negative acid group.  Any combination of the various acidic or amine amino acid side chains will have this effect.  One electron is completely transferred form one atom to the other.  The electrostatic attraction is what holds the charged ions together. o Disulphide bridges:  Form between 2 cysteine residues.  Disulphide bonds are formed by oxidation of the sulfhydryl groups (-SH) on cysteine.  Different protein chains or loops within a single chain are held together by the strong covalent disulphide bonds.  Example: insulin contains important disulphide bridges. 4. Quaternary structure: o Overall protein structure that results from the aggregation of two or more polypeptide subunits, two main categories; o Fibrous:  Trimer of alpha helical polypeptide chains.  I.e. Collagen; Rigid resistant to stretch; Functions = connective tissue in kin, bones, tendons, ligaments, 40% of all human proteins. o Globular:  4 polypeptide chains/subunits, primarily alpha helical.  i.e. Haemoglobin; 4 polypeptides bind together to form a round globular shape; functions carries oxygen. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Determining protein structure: X-ray crystallography: used to determine a protein’s three dimensional structure. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 METABOLISM (1+2):  Metabolism: sum of all chemical/molecular reaction in all cell types. Huge network of chemical reactions happening inside all cells, driven by enzymes.  Metabolic pathways: Begins with a specific molecule which is altered in a series of steps resulting in a certain product, each step is a chemical reaction.  Chemical reactions: - Involve the breaking and forming of bonds, requires energy. - Activation energy = initial investment of energy needed to start a reaction. - In most chemical reactions this is provided in the form of heat. - Humans need to maintain a temp of 37 degrees. - Enzymes provide the activation energy for reactions to proceed at normal temp in biological systems.  Enzymes: catalytic proteins: - A type of protein that acts as a catalyst speeding up chemical reactions without being consumed by the reaction. - Biological catalysts.  How do enzymes work; - 2nd law of thermodynamic; universe tends towards disorder; complex molecules have the potential to break down spontaneously. - They continue to exist because the initial activation energy required to breakdown can’t be reached to initiate a breakdown reaction. - An enzyme lowers the activation energy required for a reaction enabling the substrates to absorb enough energy to reach transition stage.  Entropy = measure of disorder.  Energy is the capacity to cause change, fundamental to all metabolic processes. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Energy cannot be created or destroy only transferred; kinetic energy (motion/heat), potential (position of an object), chemical (release from a chemical reaction), energy conversions increase entropy (a lot lost as heat).  Cells transform energy at they perform work.  Cells need continual free energy to function and survive.  Chemical reactions either store or release energy; - Endergonic reactions: absorb energy and yield product rich in potential energy (photosynthesis). - Exergonic reactions: release energy and yield products that contain less potential energy than their reactants (cellular respiration).  Biochemical energy transductions follow laws of thermodynamics; thermodynamics in biology considered in terms of change in free energy ΔG. Reactions can occur spontaneously is ΔG is negative (exergonic reactions) but most still require enzymes.  Enzymes as catalysts; - Bind substrates and lower the activation energy (ΔG) required for the acquisition of a transition state and subsequent generation of product. - Combination of enzyme and substrate (enzyme substrate complex) creates a reaction pathway where the transition state energy is lower than that of an uncatalysed reaction. - The active site environment stabilises/promotes formation of the transition state intermediate. - The transition state is very unstable and collapses to substrate or product. - The enzyme remains unchanged at the end of catalysis.  Enzymes active site; - The catalytic centre and is usually formed by only a few amino acids. - Not rigid. - Side chains of the aa’s in the enzyme interact with the chemical groups of the substrate – enzyme changes shape and fits even better around substrate, induced fit. - Induced fit brings the chemical groups of the active site into new positions enhancing the ability of the enzyme to catalyse the reaction.  Features of enzymes; - Many require non-protein helpers to aid catalysis; co-factors (usually metal ions) bind tightly to protein core and participate in redox reactions or alter substrate reactivity. - If co-factor is organics is a co-enzyme; most vitamins = co-enzymes (C and NAD+), frequently B vitamin derivatives operate in shutting of proton and functional groups. - Cellular enzymes can be activated by phosphorylation; the addiction of a phosphate group, enzyme that phosphorylate are called kinases.  Metabolic pathways divided into: 1. Catabolic pathways: transform fuels into cell energy, cells extract energy from energy containing nutrients (carbohydrates, fats, proteins). 2. Anabolic pathways: require energy, cells use energy to; synthesis biomolecules/macromolecules (proteins, lipids, carbohydrates); perform mechanical work (muscle contraction, cell division); move and actively transport ions and molecules inside and between cells (information transfer in nerve cells).  Cellular respiration: aerobic process by which the chemical energy stored in organic molecules (fuel) is converted to a form the cell can use to perform work). - Exergonic process – releases energy. - Released energy some stored as ATP, some released as heat. - ATP; shuttles chemical energy and drives cellular work, powers nearly all forms of cellular work and molecules are key to energy coupling (uses exergonic reactions to drive endergonic reactions). Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 - Most cell processes are not energetically favourable and thus require energy input to drive endergonic reactions.  ATP is the cellular energy source. - Adenosine triphosphate (ATP) + water = Adenosine diphosphate (ADP) and phosphate and energy (hydrolysis). - All three phosphate groups are negatively charged. - The like charges crowded together, mutual repression. Bonds are unstable and easily broken. - Broken by hydrolysis. - Once broken, energy released. - Energy coupling: catabolic pathways produce ATP; anabolic pathways use ATP. - Donates its free energy to endergonic work/synthesis processes in cell. - Usually involves transfer of a phosphate group from ATP -. Phosphoryl group transfer. - Heat energy released from ATP can also be harnessed to give the activation energy required to drive reactions.  3 main types of cellular work; - ATP dives endergonic reactions by phosphorylation, transferring a phosphate group to make molecules more active. - Chemical work. - Mechanical work. - Transport work.  The ATP cycle: - ATP is constantly recycled in cells, working muscle recycles all of its ATP once each minute 910 million ATP molecules spent and regenerated per second). - ATP -> energy from working cells + ADP +P -> ADP + P + energy from food -> ATP.  Cellular respirations: Aerobic harvesting of energy, make s ATP and consumes O2. - Glucose + 6O2 -> ATP + 6CO2 + 6H20 +heat. - Banks energy in ATP molecules - Breakdown of sugars and other food molecules in the presence of oxygen to carbon dioxides and water, generating ATP. - Cell taps energy from electrons falling from the organic fuels to oxygen (through the production of ATP). - Electrons lose potential energy during this transfer.  Redox reactions play a very important role in metabolism, occur by electron transfer, the loss of electrons by one chemical species and the gain of electrons by another.  Electron transfer is responsible for all the work done by cells, work accomplished by election motive force (emf), electrons from food.  Living cells have a biological circuit: - Oxidation of glucose and fats provide major source of electrons. - Electrons flow spontaneously through a series of intermediates to O2 (electron carriers). - O2 has a higher affinity for electrons than other electron carriers. - Resultant emf is harnessed and provided energy to a variety of proteins that do work.  Catabolic pathways are oxidative, anabolic are reductive.  Glucose oxidised to CO2 (glucose loses hydrogen atoms), hydrogen atoms added to O2, form water.  Dehydrogenase removes electrons (in hydrogen atoms) form fuel molecules (oxidation) and transfers them to NAD+ (reduction).  NAD is an important electron carrier in the process of oxidising glucose; key electron carriers in body are NAD, NADP, FAD, FMN, electron carriers are co-enzymes that undergo reversible redox reactions. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  NADH passes electron to an electron transport chains (ETC), electrons fall from carrier to carrier to O2, energy is realised in small quantities during this (controlled release of energy for synthesis of ATP).  3 main stages of respirations: Glycolysis, the citric acid cycle and electron transfer and oxidative phosphorylation.  Glycolysis: - Stage 1 of cellular respiration: metabolism of glucose to make simple 2 carbon Acetyl CoA. - Responsible for the largest flux of carbon in the cell. - In prokaryotic and eukaryotic cells. - Oxidises glucoses in a series of 10 steps to 2 molecules of pyruvate. - In cytoplasm of eukaryotic cells. - Does not require O2 (anaerobic). - Net production of 2 molecules of ATP. - Three stages (1 and 2 make the preparatory stage, 3 is the payoff phase): 1. Energy investment to be recouped later (1-3): Traps glucose in the cell and forms a compounds that can be readily cleaved into phosphorylated into 3 –carbon units. One molecule of glucose and 2 ATPs in, fructose 1,6 bisphosphate out. 2. Cleavage of six-carbon sugar to two three carbon sugars (4-5): Cleavage of fructose 1,6 bisphosphate. Fructose 1,6 bisphosphate in two molecules of glyceraldehyde 3 phosphate out. 3. Energy generated (6-10). ATP harvested. Two molecules of glyceraldehyde 3 phosphate in two molecules of pyruvate and 4 ATP molecules out. - Harvests chemical energy by oxidising glucose to pyruvate: ATP used to prime glucose molecules which is split into two molecules of pyruvate. 1. Hexokinase transfers a phosphoryl group to glucose, uses ATP, G-6-P cannot leak form the cell, the phosphoryl group de-stabilises the molecule, facilitating further metabolism. 2. Phosphogluco-isomerase isomerises G-6-P to fructose-6-P, the enzyme open the glucose ring (6C), catalyses the isomerisation, closes the fructose ring (5C). 3. Phosphofructo-kinase catalyses the phosphorylation of fructose to fructose 1,6 bisphosphate using ATP, important allosteric enzyme, central controlling role. 4. Aldolase splits fructose 1,6 bisphosphate into two 3C fragments glyceralderhyde-3-phospahte and dihydroxyacetonephosphate. 5. An isomerase reversibly converts DHAP to GAO (otherwise it would be lost)s Stages 4 and 5 reaction strongly favours DHAP production but proceeds because of product removal, net result is cleave of a 6C sugar into 2 molecule of GAP. 6. Glyceraldehydephosphatedehydrogenase (GAP dehydrogenase) catalyses the formation of an acyl phosphate. NAD oxidises the aldehyde to carboxylic acid and the acid is phosphorylated. GAP is first oxidised and second phosphorylated. An energetically favourable reaction is coupled to an unfavourable reaction. Oxidation gives 2NADH. GAP in 1,3 bisphosphoglycerate out. 7. 1,3 bisphosphoglycerateis energy rich, has a high phosphoryl transfer potential –can produce ATP. Phosphoglycerokinase causes 2 ATPs to be produced from the 2 three carbon molecules form stage 2. No net gain. 3-phosphoglycerate produced. 3-phosphoglycerate is converted to pyruvate in steps 8-10. 8. Phosphoglyceromutase relocate the remaining phosphate in preparation for the next reaction. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 9. Enolase causes a double bond to form by extracting a water molecule yielding Phosphoenolpyruvate (PEP), has high phosphoryl transfer energy (enol form is unstable). 10. Enzyme pyruvate kinase in last step. Produced more ATP by transferring a phosphate group from PEP to ADP, leaving stable ketone form. 2 ATPS produced per glucose molecule.  Net glycolytic reaction: Glucose + 2P + 2ADP +2NAD= -> 2 Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O.  The energy in A|TP can be used immediately but for the cell to use the energy in NADH, electrons form NADH much be passed down the ETC.  Glucose only releases 5% of its possible energy in glycolysis.  Substrate level phosphorylation: Glycolysis produces ATP by substrate level phosphorylation, a phosphate group is transferred from an organic molecule to ADP.  The Citric Acid Cycle: - Also called Kre’s cycle and tricarboxylic acid (TCA) cycle. - Complete the oxidation of organic fuel, generating many NADH and FADH2 molecules. - Stage 2 of the aerobic phase of cellular respiration. - Starts with entry of Acetyl CoA. - Pyruvate is end product of glucose catabolism (glycolysis), in present of O2 has to be converted to Acetyl CoA before citric acid cycle uses enzyme pyruvate dehydrogenase (PDH). - Pyruvate is chemically groomed for the citric acid cycle; enzymes process pyruvate, releasing CO2 and producing NADH and Acetyl COA. - Conversion of pyruvate to Acetyl CoA the junction between glycolysis and CAC, in Mitochondrion cytosol. - For each turn of the cycle:  2 CO2 molecules are released.  The energy yield is one ATP, three NADG and one FADH2. - Metabolic hub of the cell. - Entry point to aerobic metabolism for molecule that can be converted to Acetyl CoA or other components of the CAC. - Series of oxidation-reduction reaction. - Oxidises an Acetyl group to carbon dioxide and water. - Does not use O2. - Located within the mitochondria. - Harvest high energy electron and transfers them to electron carrier for use in oxidative phosphorylation. - CAC has 8 steps, each catalysed by a specific enzyme. - Acetyl CoA group (2C) joins the cycle by combining with oxaloacetate (4C), forming citrate (6C). - Sequential oxidation of 2 carbon units generates Co2, reduced electron intermediates, 1 molecule of guanosine triphosphate (GTP) and regenerates oxaloacetate, making the process a cycle. - High energy phosphate group transfer from GTP to ADP generates ATP (x2 per molecule of glucose). - NADH and FADH2 produced by the cycle relay electrons extracted from food to the electron transport chain. 8 steps: 1. Acetyl CoA (2C) condense with oxaloacetate to form 6C citrate; enzyme = citrate synthatse. (CoA-SH released). 2. Isocitrate (6C) formed from citrate; enzyme = isomerase (H20 released and used). 3. KEY EXERGONIC STEP Oxidation of Isocitrate (6C) to alpha-ketoglutarate (5C) – CO2 released; First oxidative decarboxylation; NADH formed; Enzyme = isocitrate dehydrogenase. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 4. 2nd KET EXERGONIC STEP addition of CoA and oxidation of alpha ketoglutarate (5C) to succinyl-CoA (4C), second CO2 released; 2nd oxidative decarboxylation; NADH formed; enzyme = alpha ketoglutarate dehydrogenase complex. 5. Conversion of succinyl-CoA (4C) to succinate (4C), removal of CoA -> energy from this forms 1 GTP with converts to 1 ATP (GTP returns to GDP); Enzyme = succinyl-CoA-synthetase. 6. Oxidation of succinate (4C) to fumarate (4C), removal of 2H and formation of 1 FADH2; Enzyme = succinate dehydrogenase (SDH) = flavoprotein. 7. Hydration = addition of H20 to fumarate (4C), forms malate (4C); Enzyme = fumarase. 8. Oxidation of malate (4C) to oxaloacetate (4C), removal of 2H, formation of NADH and H+; Enzyme = malate dehydrogenase. Cycle Starts Again.  Overview of energy conversions in CAC; - Acetyl group fed into cycle by combining with oxaloacetate. - 2 carbons emerge as CO2 waste. - Energy of oxidation is efficiently conserved so for 1 Acetyl CoA input  3NAD+ -> reduced to 3NADH  1FAD+ -> reduced to 1FADH.  1GTP -> 1ATP generated.  Net reaction of CAC (per Acetyl CoA) Acetyl CoA + 3NAD + FAD + GDP + P +2H20 -> 2CO2 + 3NADH + FADH2 + GTP +CoA.  Fate of NADH and FADH2: Move form CAC to Electron transport chain to produce ATP energy.  Each NADH generates 2.5ATP, each FADH2 generates 1.5ATP.  Electron transport chain (ETC) = staircase; in each of the drops energy is transferred to energy storing molecules ATP, NADH and FADH2.  Electron transfer and oxidative phosphorylation causing ATP formation.  ATP requirements in the body; - Daily requirement of 70kg. - Body reserve only 250g. - Solution: recycling of ATP from ADT (300 times per day per molecule). - Oxidative phosphorylation: the process of ATP formation due to electron transfer from NADH/FADH2 -> 02, couple the oxidation of carbon fuels to synthesis of ATP by generating a proton gradient, occurs in the inner mitochondrial membrane.  Electrochemical concepts: - Fundamental goal of CAC is to produce NADH+ H+ and |FADH2. - In the ETC electrons from NADH and FADH2 are transferred to produce 2H2O from O2. - ET potential of this reaction is generally described as the oxidation-reduction (or redox) potential. - Energy released by the redox reaction is used to pump H+ into the space between the mitochondrial membranes, this electron transfer potential is then converted to a phosphoryl transfer potential of ATP.  Overview of what happens in oxidative phosphorylation: - Electrons flow from NADH/FADH2 to O2 through series of complex electron carriers in 4 protein complexes on inner mitochondrial membrane. - Electron transfer accompanied by pumping of protons H+ from mitochondrial matrix to mitochondrial intermembrane space through 3 of the protein complexes. - Creates electrical potential – inside membrane negative, outside membrane positive. - ATP synthesis driven by proton flow back to matrix through the ATP-synthase complex –Complex V. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Electron transport chain; key components: - 3 electron driven protein pumps: 1. NADH-Q-oxidoreductase – Complex 1. 2. Q-cytochrome e oxidoreductase – Complex 3. 3. Cytochrome c oxidase – Complex 4. - 1 electron transfer only complex: Succinate Q reductase – Complex 2 (linked to SDH of CAC). - 2 electron carrier molecules: 1. Ubiquinone (Q) – 2 electron carrier. 2. Cytochrome c – Single electron carrier. - Final electron acceptor: Oxygen.  In addition of NAD and FAD other type of electron carrying molecule work in oxidative phosphorylation: 1. Ubiquinone – coenzyme Q or Q; lipid soluble, reduced form Q to QH to QH2. And 2 different types of iron containing proteins: 2. Cytochromes (cyt) = proteins with iron containing heme prosthetic groups, 3 types exist a, b, c, all integral membrane proteins except cyt C. 3. Iron sulfur (FeS) proteins; iron complexes with sulphur.  3 pumps in ETC; as electrons are passed along complex 1, 3 and 4 protons are forced into the intermembrane space.  The electron transport pathway: - Electrons drop in free energy as they go down the chain and are finally passed to O2, forming H2O. - The electron transport chain generate no ATP. - The chains function is to break the large free-energy drop from food to O2 into smaller steps that release energy in manageable amount. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  ETC basics; - A pair of electrons from NADH is passed through complex 1 to coenzyme Q. - At the same time a pair of proton is forced through to the intermembrane space. - Electrons cannot be passed unless protons are translocated. - Electrons are passed through complex 3 and 4, each time forcing a pair of protons through to the intermembrane space. - Electrons are passed into complex 2 from FADH2. - Complex 4 (cytochrome oxidase) uses the energy it receives along with an electron pair to reduced oxygen to water. - It takes 2 cytochrome oxidase complexes, two electrons pairs and four hydrogen ions to complete the reaction. - Oxygen needs to be replenished to keep electron chain going.  What about ATP generation? Chemiosmosis; the energy coupling mechanism: - Electron transfer in the ETC cause protein to pump H+ from the mitochondrial matrix to the intermembrane space. - H+ then moves back across the membrane, passing though channel in ATP synthase. - ATP synthase uses the exergonic flow of H+ to drive phosphorylation of ATP. - Chemiosmosis: the use of energy in a H+ gradient to drive cellular work. - ATP synthase = “molecular mill”. - The energy stored in H+ gradient across a membrane couple the redox reactions of the ECT to ATP. - The H+ gradient is referred to as a proton motive force, emphasizing its capacity to do work. - The flow of protons back into matrix provided the energy to run ATP synthesis.  An accounting of ATP production by cellular respiration: - During cellular respiration most energy flow in this sequence: Glucose -> NADH -> ETC -> proton-motive force -> ATP. - About 40% of the energy in a glucose molecule is transferred to ATP during cellular respiration, making about 38 ATP.  Inhibitors of the ETC: Rotenone, antimycin, cyanide or carbon monoxide. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  ETC does not proceed in absence of ATP synthesis: 1. ATP > ADP = reduced flux. 2. ADP > ATP = increased flux.  Uncoupler make the IMM leaky to H+ ions: ET continues but ATP cannot be made (i.e. DNP). Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 MEMBRANE POTENTIAL OF A CELL:  pH = -log(10)(c), c= the hydrogen ion concentration in moles per litre.  Each pH unit represents a tenfold change in the effective hydrogen-ion concentration.  Important considerations for diffusion across lipid membrane barriers: - Size of molecule. - Hydrophobic/ hydrophilic compatitibility (Partition coefficient). - Thickness of memebrane (5nm). - Existence of pores/transporters for charged particles. - Channel open probability/ transporter saturation. - Osmotic movement ofwater. - Chemical gradient. - Electrochemical gradients.  Fluid compartment sin the body: total body water (TBW) = 0.6xbody weight (42L for 70kg BW) -> 0.2xBW (14L) ECF + 0.4xBW (28L) ICF (cell membrane separates) -> ¾ of ECF (10.5L) Interstitial fluid + ¼ of ECF (3.5L) Plasma.  The smaller molecule and the less strongly it associates with H2O the more rapidly is will diffuse across the bilayer.  The plasma membrane arises when there is a difference in electrical charge on the two sides of the membrane.  Separation of charged ions is an electrical potential difference across the plasma membrane or Membrane Potential (Vm).  Measured in volts.  Intracellular side is negative with respect to the outside for K approx. -90mV. (High K+, some proteins, some CL-, some Na+ and some Pi- inside; High Na+, some K+ and some Cl- outside).  Ion channel mediate changes in membrane potential: - Ions diffuse across membrane through channel pore when channel is open. - Specific channels are opened (gated) in response to certain signals:  Change in membrane potential – Voltage gated.  Binding of an agonist to a receptor – Ligand gated.  Mechanical movement – Stretch/Volume gated. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  A lot of ion channel related diseases.  Resting membrane potential (Vr) – due to selective permeability to different ions – difference in conductance. - K+ leak channels: always open, greater membrane permeability to K+, drives efflux of K+. - Relatively low permeability to Na+. - Efflux of K+ counteracted by Na+-K+ APase pump; actively transports Na+ out and K+ in. Uses 20% of cells energy requirements.  The concentration gradient and the electrical gradient combine to form the electrochemical gradient.  At equilibrium potential the electrical gradient from influx and the chemical gradient for efflux are balance.  Chemical gradient: approx. 130mM K+ ICF and 5mM K+ ECF.  Electrical gradient at equilibrium: -90mV.  Equilibrium potential is calculated from the Nernst equation: Eion = (RT/zF)ln([ion_outside]/[ion_inside]). Eion = equilibrium potential for an ion. Z = valency. F = faraday constant.  The Nernst equation states that the equilibrium potential of anion is equal to a constant multiplied by the log to the base 10 of the ration of the external to internal concentrations of the ion.  The equilibrium potential determines the direction an ion will flow through open channels – this can determine the response of a cell to synaptic input i.e. depolarization or hyperpolarization.  Nernst can calculate equilibrium potential for a given ion, once the concentration inside and outside the cell are known.  Direction of ion flux in response to channel opening depends on concentration gradient and where the cell membrane potential is relative to the equilibrium potential for a given ion.  Potentiometer measures membrane potential.  Types of changes in membrane potential: upward deflection = decrease; downward deflection = increase.  Electrochemical driving force depends on the cell membrane potential anf the equilibrium potential for a given ion. i.e. equilibrium potential for K+ = Ek = -90mV: - Vm = -90mV; electrical gradient = chemical gradient; no net flux. - Vm = -100mV; electrical gradient > chemical gradient; K+ influx; ion flux drives Vm closer to the equilibrium potential for that particular ion. - Vm = -70mV; chemical gradient > electrical gradient; K+ efflux.  Why is membrane potential important? - Energy in potential differences can be used to regulate/drive cellular processes. - Action potentials – neurotransmitter release. - Muscle contraction. - Secretion e.g. insulin release.  Action potentials – Neuronal signalling: - Excitable cells – neurons and muscles – have a greater Vm than other types of cells. - Specialized use of this membrane potential – can undergo transient, rapid reversal of Vm known as action potentials. - Mediated by Na+ influx, followed by K+ efflux. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Action potentials are propagated along the length of a neuron, can travel long distances without loss of signal.  Trigger neurotransmitter release at junction between neurons (synapse).  Action potential to pre synaptic membrane; action potential arrives and triggers the entry of Ca2+; in response to Ca2+ synaptic vesicles fuse with presynaptic membrane then release neurotransmitter; ion channels open when neurotransmitter binds, ion flows cause change in postsynaptic cell potential.  Muscle contraction: - Action potential depolarizes the motor neuron. - Acetylcholine (Ach) is released by motor neuron axon terminals. - Binds to Ach receptors on skeletal muscle tissues. - Depolarization (positive changing in membrane potential) results in muscle contraction. - SEE 3RD YEAR BIO NOTES FOR EXCITATION CONTRACTION COUPLING.  Secretion – Insulin Release. - Pancreatic Beta cell: Glucose uptake increases ATP in pancreatic beta cells. - ATP closes a K+ leak channel. - The depolarizing force activates a calcium channel (voltage dependant, VDCC). - Ca2+ promotes fusion of insulin-containing granules with membrane (regulated exocytosis). - Released insulin is free to travel to target sites through the blood (endocrine action). Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 CELL IMPORT AND EXPORT MECHANISMS  Endo = into cell, exo = out of cell. 3 types of import (endocytosis); 2 types of export (exocytosis).  Why is assisted membrane transport required: size constraints and solubility issues.  Assisted membrane transport classification: - Channel-mediated; ions and water. - Carrier mediated; small water soluble molecules, ion exchange. - Vesicular transport; large molecules and multi-molecular aggregates.  Channels and carriers – characteristics: - Water channel; gated; up to 10^9 molecules/second. - Ion channel; gated; 10^6-10^8 molecules/second. - Solute carrier; cycle; 10^2-10^4 molecules/second. - ATP dependant; cycle; 10^2-10^4 molecules/second.  Chanel mediated transport: - Potential for gating, voltage, agonism and antagonism. - Selective – for specific ions. - Non-selective – for either cations or anions. - Variable conductance – may be greater in inward or outward directions i.e. Inwardly or outwardly rectifying.  3 main groups of solute carries: Uniporter (one in one direction), symporter (two in one direction) and antiporter (two in opposite directions).  Active or Passive.  Active = primary (directly ATP dependant); Secondary (co-transport driven by movement of one solute down gradient) or tertiary (relies on antiport activity).  Passive and active transport – solute carries bind and flip solute across the membrane: - Passive: Equal affinity for solute on both sides of membrane; solute-binding site interaction more probable on side of increased solute concentration; net effect is solute transfer down gradient. - Simple Active: ATP-dependant affinity for solute on the side of decreased solute concentration; carrier flips to release solute on side of increased solute concentration; net effect is solute transfer against gradient.  Solute carrier activity; - Specificity: carrier proteins specific for 1 or, at most, several, closely related molecules. - Saturation: transport limited by number of binding sites, maximal rate when all sites occupied. - Competition: closely related compounds can compete for binding and transport e.g. amino acids glycine and alanine. - Energy dependence: may be passive (no energy required, facilitated diffusion) or active (energy dependant, requires ATP, active transport).  Vesicular transport: - Endocytosis – 3 types: Pinocytosis (ECF); Receptor mediated (Large Molecules); Phagocytosis (Multi- molecular particles). - Exocytosis – 2 types: Constitutive and regulated.  Pinocytosis: - Cell drinking. - Non-selective. - Membrane indentation forms around ECF, membrane seals and is pinched off to form vesicle. - Brings ECF and extra plasma membrane into cell. - Microglia do this.  Phagocytosis: - Cell eating. - Specialized cells (e.g. immune cells). - Used to engulf large particles such as bacteria or damaged tissue. - Phagocytic vesicle fuses with lysosome, lysosomal enzymes degrade vesicle contents. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 - Chemotaxis and adherence or microbe to phagocyte -> ingestion of microbe by phagocyte -> formation of a phagosome -> fusion of the phagosome with a lysosome to form a phagolysosome -> digestion of ingested microbe by enzymes -> formation of residual body containing indigestible materials -> discharge of waste materials.  Receptor mediated endocytosis: - Selective uptake of specific molecules – large molecules. - Target molecule binds to surface membrane receptors. - Membrane indentation formed, lined with protein clathrin to form clathrin coated pits - Clathrin coat is removed and vesicle can fuse with lysosome.  Molecules transported into cells via endocytosis: - Cholesterol via binding to surface membrane ow density lipoprotein (LDL) receptors. - Iron via binding to surface transferring receptors.  Exocytosis: - Endocytosis in reverse. - Acts to:  Add plasma membrane.  Recycle and replace receptors removed from membrane by endocytosis.  Secrete substances e.g. hormones, neurotransmitters into ECF.  Constitutive secretion: vesicles continuously move to cell surface and fuse with membrane.  Regulated secretion: proteins (or other substances) stored in vesicles, only fuse with membrane in response to a signal e.g. neuronal input, hormonal.  Regulated exocytosis – pancreatic acinar cells: hormonals signals -> VIP + Secretin -> increase in cAMP - > vesicle fusion, enzyme releases or neural signals -> GRP + Ach -> increase in CA2+ -> vesicle fusion, enzyme releases. Acinar cells secrete pancreatic digestive enzymes into the GI tract.  Neurotransmitters are stored in vesicles and released by regulated exocytosis. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 CELL – CELL COMMUNICATION:  3 mechanisms of cell communication: - Gap junctions.  Direct communication through these.  Channels bridging cytoplasm of 2 cells.  Allow direct passage of ions and small molecules between cells. - Direct contact.  Specific cell-surface markers allow transient direct contact between 2 cells.  Important for immune cells (e.g. recognition of an infected or abnormal cells by phagocytes) - Extracellular chemical messengers.  3 classes: o Autocrine; cell releases a signalling molecule that binds to receptors on its own cell membrane. Local. o Paracrine; local diffusion of released chemical messenger, acts on nearby cells. Local. o Endocrine; signalling molecules released into the blood, long-range effects on cells remote from site of release.  Signalling is selective; depends on expression of the receptor on the target cell, non-target cells without the receptors are not affected.  Cell signalling involves 3 processes: 1. Receptor-ligand binding. RECEPTION 2. Activation of signalling cascade. TRANSDUCTANCE. In cytoplasm. Relay molecules form a signal transduction path. 3. Activation of a response. RESPONSE.  Classes of extracellular signalling molecules. 1. Hydrophilic.  Small molecules and ions (ion channels = site of signal transduction).  Peptide (plasma membrane receptors = site of signal transduction).  Amines (catecholamines) (plasma membrane receptors = site of signal transduction). 2. Lipophilic.  Steroid hormones (intracellular receptors = site of signal transduction).  Amines (thyroid hormones) (intracellular receptors = site of signal transduction).  Speed of transmission: paracrine > endocrine; however distance between initial stimulus and response not always determining factor.  Onset of effects: - Major determinant of speed is the class of response: 1. Modified – rapid, milliseconds to minute e.g. secretion or changes in metabolism. Ion channels, changes in membrane potential happen in milliseconds. 2. Transcriptional –hours. Involve gene expression, synthesis of new proteins e.g. growth or cell division.  Classes of Downstream effects: 1. Transport protein (altered ion transport). 2. Metabolism enzyme (altered metabolism). 3. Gene expression regulatory protein (altered gene expression). 4. Cell motility cytoskeletal protein (altered shape or movement). 5. Cell division cell cycle proteins (altered growth or division).  Plasma membrane receptor types: 1. Ligand-gated ion channels (ionotropic receptors); hyperpolarisation or depolarisation, cellular effects, milliseconds. 2. G-protein-couples receptors (metabolic), change in excitability, Ca2+ release and protein phosphorylation, cellular effects, seconds. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 3. Kinase-linked receptors; protein phosphorylation, gene transcription, protein synthesis, cellular effects, hours. 4. Nuclear receptors; gene transcription, protein synthesis, cellular effects, hours.  Ligand gated ion channel receptors: - Binding of ligand to the receptor causes conformational change -> channel opening. - Influx of ions changes membrane potential. - Current flow is terminated when ligand unbinds and channel closes - If the channel conducts Ca2+ this can act as a second messenger to activate various intracellular signalling pathways. - Channels conducting anion (Cl-) hyperpolarise the cell membrane, making a neuron less likely to fire an action potential. - Examples: neurotransmitter reports such as nicotinic acetylcholine receptor (nAChR) mediates skeletal muscle contractions and transient receptor potential (TRP) channels mediate sensation of pain.  Enzyme linked receptors: - The receptor itself is an enzyme – cytoplasmic domain. - Ligand binding causes conformational change and kinase activation. - Tyrosine kinase receptors phosphorylate tyrosine amino acids in target proteins, leading to protein activation and a cellular response. - Examples: insulin receptors; growth factor receptors e.g. epidermal growth factor, nerve growth factor essential for cell growth and division.  Nuclear receptors: - Lipophilic hormones e.g. steroids diffuse across plasma membrane and bind to intracellular receptor. - Hormone-receptor complex moves into nucleus and binds to DNA. - Binding of lipophilic hormone to receptor forms a hormone - receptor complex that binds specific site on DNA, the hormone response element (HRE). - This leads to activation of a specific gene and mRNA transcription. - mRNA is translated into a new protein. - New protein synthesis is relatively slow so effects of lipophilic hormones take time to develop and can persist for some time.  Termination of receptor mediated responses: - Ligand gated ion channels: rapid termination of ion flow, receptor unbinding and channel closure. - Enzyme linked receptors: ligand unbinding followed by response termination, response duration depends on signalling pathways activated. - Nuclear receptors: may have persistent effects, new protein synthesis.  Modulation of receptor mediated responses: - Upregulation: Increased expression of plasma membrane receptors and increased availability of receptor ligand. - Downregulation: reduced expression of receptors on plasma membrane (removed from membrane surface by endocytosis) and reduced availability of receptor ligand.  G-Protein coupled cell surface receptors: - In plasma membrane. - All Eukaryotes use GPCRs.  GPCR ligands: - Proteins. - Small peptides. - Amino acids. - Fatty acids. - Photons of lights. - Examples: bile acids, histamines and chemokines. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  GPCR pathway: - GPCR signal via 2 principal intracellular 2nd messenger signalling pathways: 1. The adenylate cyclase – cAMP – protein kinase A pathway: effector protein = adenylate cyclase (adenylyl cyclase); 2nd messenger = cAMP. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 2. Phospholipase C-Ca2+: Effector protein = phospholipase C; 2nd messenger = Ca2+. - Receptors couple to a class of protein called trimeric G-protein. - Alpha = 16 subtypes; beta = 5 subtypes; gamma = 11 subtypes. - Combinations permit signal diversity dependant on the combination of units linked to a particular receptor.  GPCR: - Ligand binding activities a G protein. - G proteins are bound to the cytoplasmic part of the receptor. - Consist of 3 subunits; alpha, beta and gamma. - Once activated the G protein splits off the receptor complex, moves along the membrane, binds to and activates an effecter protein. - Effector protein can be an enzyme or an ion channel. - Needs GTP to be activated.  GPCR activation causes activation of G proteins which in turn activate Adenylate cyclase. - In some cases G protein is bound to receptor, other binds only after receptor activation. - GTP bound, causes a large conformation change and activates the G protein. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  cAMP second messenger pathway: - G protein mediated activation of enzyme adenylate cyclase -> synthesis of cAMP. - cAMP activates protein kinase A (PKA). - PKA phosphorylates target protein to bring about cellular response. - Effect terminated by breakdown of cAMP by cAMP phosphodiesterase. - A rise in cAMP can alter gene transcription.  Phospholipase C –Ca2+ seconds messenger pathway: - G protein mediated activation of enzyme phospholipase C. - 2 intracellular signalling pathways activated: DAG and IP3. - Activated phospholipase C cleaves phosphatidylinositol bisphosphate (PIP2): 1. Diacylglycerol (DAG) -> PKC activation -> phosphorylation and activation of target proteins. 2. Inositol Triphosphate (IP3) -> Ca2+ release from intracellular stores -> activation of calmodulin - > phosphorylation and activation of various kinases. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  GPCRs facts: - >1% of all genes encode GPCRs. - GPCR is a gene superfamily – one of the largest families known, >800 homologous gene in humans – multiple subtypes in each family – usually given subscript numbers. - Effects of GPCR activation can be stimulatory or inhibitory, depending on subtypes of G protein coupled to a receptor. - GPCRs mediate effect of various neurotransmitters, hormones and are essential for sensory signal transduction such as vision and olfaction. - GPCRs are the targets for approx. 50% of all currently used drugs.  Signal amplification in 2nd messenger pathways: - One messenger binds to one receptor (one messenger molecule) -> several G proteins activated -> each G protein activates an adenylate cyclase -> each adenylate cyclase generate hundreds of cAMP molecules -> each cAMP molecule activates a protein kinase A -> each PKA phosphorylates hundreds of proteins (millions in total).  Vasopressin (Antidiuretic hormone, ADH): - Is secreted by the neurosensory cells of the hypothalamus (neuroendocrine)> - One of the targets is the V2 receptor in the collecting duct of the kidney (GPCR). - V2 receptor activation couples to the cAMP second messenger pathway. - Increases water reabsorption from formed urine by modification and transcriptional alterations in aquaporin-2 water channel expression. - AKA arginine vasopressin (AVP) or antidiuretic hormone (ADH).  GPCRS are targeted by bacterial toxins: cholera toxin and pertussis toxin (whopping cough). Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 Chromosomal Organisation – Composition and Packaging of DNA:  Structure of DNA and its organisation into functional units is known as genes.  The fundamental role of the heredity material (exception red blood cells and platelets, no nucleus, formed in bone marrow): - DNA -> gene transcription or replication of the cell (which requires gene transcription). - Gene transcription required for the function of the cell. - Replication of the cell and function of the cell required for tissue integrity/function.  In DNA we find nitrogen-containing bases: purines and pyridmidines.  Purines: consist of a six membered and a five membered nitrogen-containing ring, fused together.  Pyridmidines: have only a six membered nitrogen containing ring.  Purines: Adenine = 6-amino purine and Guanine = 2-amino-6-oxy purine.  Pyridmidines: Uracil = 2,4-dioxy pyrimidine (RNA), Thymine = 2,4-dioxy-5-methyl pyrimidine and Cytosine = 2-oxy-4-amino pyrimidine.  Base + sugar = nucleosides; nucleoside + phosphate = nucleotide.  DNA structure basics: - Deoxyribonucleic acid duplex (each strand is a linear polymer of nucleotides) 1. Deoxyribose sugar unit. 2. Phosphate. 3. Nitrogenous base (purine (adenine, guanine) or pyrimidine (cytosine, thymine).  Deoxy-ribose in DNA is ribose in RNA. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Forming the double helix model: - Bases on two separate single strand polymer forming specific base pairs which allow for the formation of a helix: o Watson-Crick Model:  Tw anti-parallel helical chains coil around a common axis.  Sugar-phosphate backbone runs on outside, bases inside.  Bases lie perpendicular to common axis.  A regular double helix is formed due to the base pairs occupying the same internal space (A- T=G-C in space across helix).  Helical stacking promoted by intra-base pair hydrogen bonding and stack-stack Van der Waal’s attraction. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Differences between DNA and RNA: - RNA = single filament, DNA = double - RNA sugar = ribose, DNA = deoxyribose. - RNA bases = cytosine, guanine, adenine and uracil, DNA = cytosine, guanine, adenine and thymine.  A structural gene is a segment of DNA involved in producing a polypeptide chain; it includes region following and preceding the coding region, as well as intervening sequences (introns) between individual coding segments (exons).  The nucleosome: - The nucleosome is the basic structural subunit of chromatin consisting of 200bp of DNA and an octamer of histone proteins. - The double helix is packages by proteins called histones. - Histones: o H2A, H2B, H3, H4 (core histones). Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 o H1 (linker histone)> o 25% arginine or lysine residues (basic amino acids). o Form a complex around DNA which can be:  Supercoiled.  Stacked. o Can be methylated-demethylated (transcription).  The histone octamer – basic constituent of chromatin: - 2 molecules of each H2 histone for unit. (H3)2(H4)2 tetramer (H2A-H2B dimer)2. - 146bp wound on per unit in 1.8 turns (left hand supercoil)> - 8-bp linker attached to H1 (nucleosome seal). - Repeat.  Solenoid helices: - Nucleosome formation compact DNA seven fold. - Helical stacking of solenoids provides up to 10^4 fold condensation. - Now DNA resembles packets = chromosomes. - Most prominent in duplicate form during mitotic metaphase. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Message on packing; - Clear implication is that packaging allows accommodation of a large amount of genetic information. - To access information (i.e. DNA) chromatin must be opened up.  Chromosomes: - Human DNA – 46 units of linear double stranded DNA polymer; 22pairs of autosomes (Ch 1-22); 2 sex chromosomes (XX/XY). - Total length of human DNA: Per chromosome: 1.5-8.5cm = 2m per cell. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  Definitions; - Gene (old): a segment of DNA involved in producing a polypeptide chain; it included regions following and preceding the coding region, as well as intervening sequences (introns) between individual coding segments (exons). - Gene (new): a unit of heredity composed of DNA occupying a fixed position on a chromosome (some viral genes are composed of RNA). A gene may determine a characteristic of an individual by specifying a polypeptide chain that forms a protein or part of a protein (structural gene); or encode an RNA molecule; or regulate the operation of other genes or repress such operation. - Chromosomes: discrete unit of the genome carrying many genes. Each consists of a very long DNA duplex and approximately equal mass of proteins. It is visible as a morphological entity only during cell division. - Chromatids: the copies of a chromosome produced by replication. - Genome: the complete set of genetic material of an organism, which corresponds to the haploid set of chromosomes of an organism.  The nucleus: - Largest organelle (>6μ diameter). - Surface: nuclear envelope (double membrane), ribosome studded. Nucleoporin formed channels. - Discernible features (inside): o Hetero chromatin. o Euchromatin. o Nucleolus. - Constituents: o DNA, RNA. o Nucleoporin (histone/non-histone). - Active cell: o Prominent nucleoli. o Euchromatin. o Dispersed basophilia. - Resting cell: o Small absent nucleoli. o Heterochromatin. o Dense basophilia. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230 DNA REPLICATION AND MITOSIS:  Chromosomes diploid (22 autosomes) and haploid (sex chromosomes) sets.  DNA replication problem 1 – access: - Nucleosome formation compact DNA seven fold, helical stacking of solenoids provides up to 10^4 fold condensation. - Now DNA resembles packets = chromosomes. - Most prominent in duplicated form during mitotic metaphase.  Accessing supercoiled DNA: - Topoisomerase enzymes (nuclease/ligase activity) -> double stranded or single stranded. - Single stranded -> relaxation of wound structure. - Double stranded -> passing of double strands out and through coils. - Passing of double strands out and through coils and/or relaxation of wound structure -> accessibility of chromatin to replication machinery.  Opening the helix: - Started by the activity of initiator proteins – helicase. - Bind DNA and break hydrogen bonds at replication origins. - These sites are rich in A-T repeats, as A-T has 2 rather than 3 hydrogen bonds. - Numerous such sites exist along the length of the chromosome. - Replication proceeds in both directions and copies each strand. - Above 2 points reduce total copying time.  DNA Polymerase: - It is really a large complex of distinct DNA synthesis enzymes. - Major feature is the synthesis of DNA in a 5’ to 3’ direction. - Function is to add deoxyribonucletide to 3’ end of nascent strand by the formation of a phosphodiester bond. - Requires a primer strand with free 3’ OH group. Downloaded by Caoimhe Rogers ([email protected])  lOMoARcPSD|49612230  RNA primer synthesis: - Catalysed by primase enzyme (RNA polymerase). - Uses si