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Department
Biology
Course
BIOLOGY 1A03
Professor
Lovaye Kajiura
Semester
N/A

Description
Proteins Functions: Protein Type Role in Cell or Organism Defence Antibodies/complement proteins attack & destroy virus and bacteria Contractile and motor proteins Movement – actin & myosin Enzymes Catalyze chemical reactions Hormones Act as signals that can coordinate activities. Receptor proteins Receive chemical signals from outside and initiate response Structural proteins Provides support for cells and tissues (hair, feathers) Transport proteins Moves substances across cell membrane and throughout body Stanley Miller’s Experiment  wanted to know if complex organic materials can be synthesized by simple molecules.  Designed to produce ancient Earth. 1) A glass contained gases that had high free energy (atmosphere) 2) It was then connected to a small flask containing H2O, which was constantly boiled, so that water vapour could mix with gases in large flask 3) When the vapour cooled and condensed, it would return to the small flask, where it would be boiled again. 4) Nothing happened, until Miller sent ELECTRICAL discharges across electrodes in the “atmosphere”. These sparks represented electrical energy (lightning). 5) The solution in the boiling flask began to turn pink, then deep red and cloudy. Found were carbon complex molecules. Recreated start of chemical evolution. Other hypotheses: Hydrothermal vents: geyser on the seafloor. Hot mineral rich water, life may have begun here. Murchison Meteorite: contained millions of different chemicals. Basis of life came from outer space or thorough contamination from atmosphere. Monomer – amino acid; Polymer – peptides (proteins) Amino acids can be ionized or non-ionized. Made of an amino group, a central carbon, a carboxyl functional group, and a R-group. R-groups: hydrophobic; Amino acids: hydrophilic. 20 different amino acids. Isomers: Structural – differ in the order in which the atoms are attached. Geometric – differ in arrangement around a double bond. Can be trans: different side; Cis: same side. Optical – mirror images of each other. Carbon is asymmetrical. Non-superimposable (think of hands); most amino acids have optical isomers Optical Isomers & Parkinson’s Disease: L-DOPA and D-DOPA: treatments. L-DOPA is effective, but D-DOPA is biologically inactive. Structure relates to function. Proteins: Peptide bond forms between 2 amino acids in a condensation (dehydration) reaction  Water is removed Level Structure Stabilized by Primary Sequence of amino acids Peptide bonding Secondary Formation of α-helix and β- Hydrogen bonding between pleated sheets groups. Depends on primary Tertiary Interactions between R- Contributes to the 3D groups. H-bonds, van der structure of the polypeptide. walls, covalent bonds, Depends on primary & disulphide bonds, ionic bonds. secondary. Quaternary Interactions among R-groups. Depends on primary & Same types of interactions as tertiary, hierarchal. tertiary Sickle cell disease – single change in primary amino structure will affect red blood cell shape (structure + function) – causes clogging, and lack of oxygen transport. Chaperone proteins – help facilitate in the folding of proteins (speeds refolding of proteins in their normal shape after denaturing) Prions – improperly folded proteins, leads to infectious particles. Ex. Mad cow disease. Thalidomide tragedy – in the 1950s, women used thalidomide as a sedative against morning sickness. It contained optical isomers (mirror images). 1 was a sedative, while the other caused truncated limbs and had interfered with blood vessel development.  One of the thalidomide optical isomers disrupted blood vessel development in the fetus, BUT, thalidomide is now being used as an anti-tumour agent because it cuts off blood vessels from cancer cells. Nucleic Acids: Nucleic acids – polymers made out of monomers of nucleotides (pentose sugar, phosphate group, nitrogenous base). Nitrogenous base is attached at C #1, phosphate at C #5, OH for bonding C #3. Sugar: deoxyribose (no O on the second C); ribose (OH on the second carbon) Bases: Purines (Adenine & Guanine); Pyrimidines (Cytosine & Thymine/Uracil) Phosphodiester linkage: -C-O-P-O-C- (3C of one sugar to the 5C of another sugar) (condensation reaction = water is released) Genetic code – one specific sequence of base pairs forms one gene (expressed in phenotypes etc.) DNA: info containing molecule (required for growth & reproduction); RNA: contains info used to make proteins (transcripted from DNA) Double Helix: - antiparallel strands - nitrogenous bases point to the center of the double helix, sugar phosphate backbone is on the outside (hydrophilic & hydrophobic) - A & T; G & C (Chargaff’s complementary base pairing) - Held together w/ H-bonds. - Length of 1 turn: 3.4 nm; distance between bases: 0.34 nm; width of helix: 2.0 nm - DNA is in this shape because it takes less space, sugar/phosphates are outside because they are hydrophilic. Nitrogenous bases are hydrophobic. Overall, it is water soluble. - Contains a major groove (large distance between strands) and minor groove (small distance between strands) Replication of DNA:  Hydrogen bonds break between complementary base pairs, and DNA strands separate.  Both strands of DNA can serve as templates. Free nucleotides are added with energy to where the other strand used to be. 2 double helices are formed. Human Side of Research:  Most important event was the use of Franklin’s X-ray crystallography pictures that showed it was a double helix, also allowed for them to make measurements. Carbohydrates: Functions: Energy source – glucose is rapidly metabolized Energy storage – starch (plants) & glycogen (humans) Structural support – cellulose, chitin, cartilage Transport of energy source – sucrose, lactose Cell surface signals – cell communication and cell-cell recognition. Monosaccharides – simple sugars. Disaccharide – 2 monosacharrides. Glucose + glucose => maltose; Glucose + Fructose => sucrose; glucose + galactose => lactose Polysaccharide – polymers that form when monomers are linked together. Glucose – starch, cellulose, glycogen; Galactose – cartilage (glucose can be in linear form-very rare or ringed form) Condensation reaction – glycosidic linkage (the two OH groups, which C depends on the monosaccharide) α-glucose: OH on C1 is below plane; β-glucose: OH on C1 is above plane. Polysaccharide Chemical Structure Three-Dimensional Structure Starch – energy storage in α-1,4-glycosidic linkage Unbranched – amylose; plant cells Branched – amylopectin Glycogen – energy storage in α-1,4-glycosidic linkage Highly branched helices animal cells Cellulose – structural support Β-1,4-glycosidic linkage Parallel strands joined by in cell walls of plants/algae hydrogen bonds Chitin – structural support in Β-1,4-glycosidic linkage Parallel strands joined by the cell walls of fungi and hydrogen bonds. exoskeletons Peptidoglycan – structural Β-1,4-glycosidic linkage Parallel strands joined by support in bacterial cell walls peptide bonds Lactose Intolerance: - Pepto-Bismol symptoms (nausea, cramps, gas, diarrhea) - Inability to produce lactase (enzyme) which splits lactose into glucose & galactose. - Needs to use lactaid pills/milk (provides the lactase enzyme) - Testing: stool acidity/hydrogen breath test to diagnose for lactose intolerance Galactosemia: - enlarged livers, renal/kidney failure, developing cataracts, - not treated, can lead to damage - lacks enzyme that converts galactose into glucose. Genetic testing needed for diagnosis Lipids: Lipids – hydrophobic, different from the other macromolecules. Fats cannot be polymers, so no monomers and are made of hydrocarbon chains. Major Classes & Functions of Lipids: Fats (triglycerides) – 3 fatty acids + glycerol Steroids – ring shaped, isoprene subunits tails, hormones (testosterone), membrane structure (cholesterol) Phospholipids – glycerol + phosphate – plasma membrane. Waxes – bee hives & plant leaf surfaces (protection against H2O loss) Glycerol and fatty acids are bonded together via condensation reactions, creating an ester linkage (HO-C=O) Amphipathic molecule – has both polar (hydrophilic) regions and nonpolar (hydrophobic regions). In phospholipid bilayer, polar heads, nonpolar tails. Micelles. Bangham’s Experiment – when lipid bilayers are agitated, layers break and re-form as micelles. Water on the inside and on the outside. Why are membranes important? - allows for boundary, compartimization, allows for greater complexity/metabolic activity due to the different chemicals/enzymes - compartments: many contain different enzymes, products. - permeability – ability to allow substances to pass (selective permeability in membranes) Functions of the plasma membrane: - functions as a boundary, which separates a living cell’s internal environment from its external nonliving environment. Separates life from nonlife. - acts as a selective barrier allowing only certain molecules to pass through and controls the movement of materials in & out of the cell. - the enclosed volume serves to increase reaction efficiency and concentrates reactants. Why is membrane fluidity important? - ability to be dynamic/ever changing - allows for compartimization. - permits useful substances to be transported in, and harmful wastes to be transported out. Membrane Fluidity is related to: 1. Presence of unsaturated or saturated fatty acids Fluid: - unsaturated hydrocarbon tails have one or more double bond (causes kink), which results in the fatty acid tails spaced apart and polar heads loosely packed. Inside of membrane opens up, causes greater permeability. - this DECREASES the strength of hydrophobic interactions between hydrocarbon tails, results in “fluid” state Viscous: - saturated hydrocarbon tails have NO double bonds, no kinks. - closely packed allowing for interactions extending along their lengths. - polar heads are tightly packed, resulting in an increase in the hydrophobic interactions resulting in a “viscous” state. Low permeability 2. Number of Carbon atoms. - more carbon atoms = increase in van der waals interactions. More hydrophobic interactions, thus it is more viscous and less permeable. 3. Absence of presence of cholesterol - cholesterol is a steroid, common in the plasma membranes of animals - modulates membrane fluidity. At moderate temperature – cholesterol restrains phospholipids movement, which reduces membrane fluidity. At low temperatures – cholesterol prevents the close packing of phospholipids, which ENHANCES membrane fluidity and prevents solidification. 4. Ambient Temperature - higher temperatures generally increase fluidity & permeability (exceptions with cholesterol) - as temperature decreases, strong hydrophobic reactions between saturated hydrocarbon tails causes them to solidify more readily in comparison to unsaturated hydrocarbon tails Types of fatty acids: - steric acid (saturated 18 C) - oleic acid (unsaturated 18 C) - palmitic Acid (saturated 16 C) - linoleic acid (unsaturated 18 C) Diffusion (think of keys): - passive process - net movement of a substance (solute) is down a concentration gradient, from a region of high concentration to a region of low concentration - movement may or may not be through a membrane - process continues until a dynamic equilibrium is reached. Osmosis: - passive process - diffusion of water across a selectively permeable membrane - H2O diffuses from less concentrated solution (hypotonic) to more concentrated solution (hypertonic) - H2O diffusions from a region with a high concentration of free water molecules to a region with a low concentration of free water molecules. Hypotonic – a solution with a lower solute concentration than that inside the cell Hypertonic – a solution with a greater solute concentration than that inside the cell Isotonic – a solution with equal solute concentration compared to that inside the cell (net movement is zero) Real World Examples: RBC: iso – no change; hypo – swell (lyse, no cell wall, which causes cells to burst); hyper – shrink (crenate) [Gatorade helps to stop swelling of RBCs because it contains ions] Begonia plants: hypo – great situation (turgid); iso – wilting (flaccid); hyper – pulls away from cell wall (plasmolysis) Contractile Vacuole of paramecium – inside of cells is more concentrated than outside (hypotonic solution). Water will move in, but it does not swell do to contractile vacuole which will released water to the outside. Use of Liposomal Nanomedicines: - phospholipids and therapeutic agents are mixed together, causes the drugs to be surrounded by either a lipid bilayer or monolayer. (hydrophobic space inside) - used to deliver cancer cell-killing drugs into tumours. These LNs are introduced into the organism’s/patient’s circulatory system. - The LN’s exist the blood vessel where it is damaged (where there is a tumour). - Liposomes enter tumours, and either the drugs are leaked out of the LN and into the tumour, or the LN fuses with the cell membrane of the tumours. - Advantages: allow the medicine to accumulate in the desired location rather than in healthy tissues, and protect the drugs from being broken down/modified. - Disadvantages: challenging to design because they must be large enough to hold the drugs and small enough to leave the blood vessels. Must also be stable. Davison-Danielli Sandwich => Polar heads point toward membrane protein layers (hydrophilic), nonpolar tails in the middle (hydrophobic). Incorrect due to freeze-fracture technique Singer & Nicolson Fluid Mosaic Model => Membrane proteins are individual embedded in the phospholipid bilayer. A membrane consists of a changing mosaic pattern of membrane proteins “bobbing” in a fluid bilayer of phospholipids. Accounts for amphipathic nature of membrane proteins (some are in hydrophobic area, some are in hydrophilic area) Freeze Fracture & Freeze-Etch: - membrane is frozen, then fractured at point of least resistance. - Using electron microscopy, pits and mounds are observed in the membrane interior. - Mounds and pits are found in the middle of the lipid bilayer. (nose strips) Integral proteins – transmembrane proteins that are embedded in the membrane (goes through membrane) Peripheral proteins – are not embedded in membrane, bind to the hydrophilic ends of the integral proteins, does not go across. (can be found inside or outside hydrophilic area) Integrins: - transmembrane proteins that connect inside the cell, across the membrane, to the extracellular matrix (fibronectins). Allows for communication. Facilitated Diffusion: - passive transport - assisted by a specific transport protein (faster than diffusion) - concentration gradient dependent - examples include glucose, ions, water soluble substances Sodium Potassium Pump: - requires energy (such as ATP) - transport proteins pump molecules AGAINST gradient (low to high) - 3 Na+ bind to membrane protein. Protein kinase uses ATP and phosphorylates the protein, phosphate group attaches to protein, and it changes confirmation - Na+ leave the protein, and protein now has strong affinity for K+. - 2 K+ bind to protein, and causes the protein phosphatase to remove phosphate group - K+ are released by conformational change. Protein now has high affinity for Na+. Functions of membrane proteins: - transport - enzymatic activity - signal transduction -> translate hormone to message - intercellular junction (or intercellular joining) -> cells are joined together to form tissues - cell-cell recognition -> glycoproteins on cell surface - attachment to cytoskeleton and the ECM -> integrin How is facilitated diffusion different/similar from active transport? - facilitated is passive transport, but active transport is not. One needs energy and goes against the gradient - both use transmembrane (transport) proteins The Molecular basis of Cystic Fibrosis: - CFTR (chlorfluoric transport) - Accumulation of mucous in the lungs - Abnormal type of CFTR results in Cl- not being moved properly. (lack of H2O movement, no osmosis) H2O stays within cells - Diagnosis – sweat patch test - Symptoms – difficulty breathing, lung infections, sinus infections, poor growth, diarrhea, infertility Trans Fats: Why are they so bad for us? - partially hydrogenated oils - triglycerides, naturally unsaturated fatty acid tails have been chemically altered to be more saturated by adding H-bonds - maintain semi-solid texture and flavour longer. - Unhealthy because they are harder to break down due to semisolid state – builds up in blood vessels. - Cardiovascular disease, membrane integrity, free radical damage. - LDL Cholesterol Uptake: - cholesterol circulates in blood, mainly as particles (LDL) - normal liver cells remove excess cholesterol from blood receptor – mediated endocytosis Inside the Cell: Prokaryotic Cells: - archaea, bacteria - few/no substructures - smaller, simpler design - no membrane bound nucleus, has nucleoid - plasma membrane surrounds cytoplasm Eukaryotic Cells: - membrane bound nucleus - relatively larger than prokaryotic cells - more organelles. Nucleus: - surrounded by nuclear envelope, contains chromosomes (DNA, cell’s genetic info) and nucleolus (site of rRNA synthesis and ribosome assembly) - inside surface has nuclear lamina (stiffens structure and maintains shape) Nuclear envelope – double membrane, studded with pore like openings. Physical barrier between nucleus & cytoplasm (facilitates exchange) Nuclear pore complex – greater than 50 different proteins that form the openings in the nuclear envelope (connects inner nucleus to cytoplasm) Ribosomes: - some are present in cytosol or on RER. Comprised of RNA and protein - consist of small and large subunits and are involved in protein synthesis Rough Endoplasmic Reticulum (RER) - continuous with nuclear envelope. - Consists of membrane-bound tubules and sacs - Ribosomes studded into the cytoplasm - Function: Protein synthesis & processing in the lumen of the RER (lumen contains enzymes) Golgi Apparatus: - closer to the RER (will fuse with plasma membrane) - consist of cisternae - has cis face (receives products from RER) and trans face (sends products to their destinations) - Protein processing Smooth Endoplasmic Reticulum (SER) - lacks ribosomes on the cytoplasmic surface of the membranes - contains enzymes which synthesizes lipids Peroxisomes: - single-membrane bound organelle - locations of oxidative reactions (processing of fatty acids) - contains catalase, which converts hydrogen peroxide into water and oxygen Lysosomes: - single-membrane bound organelles - participate in solid-waste processing and storage of materials - acidic interior - used to breakdown macromolecules and recycle organelles - phagocytosis (food particles come in and are engulfed by lysosome) - autophagy – damaged organelle is digested - receptor mediated endocytosis – macromolecules outside the cell bind to membrane proteins that act as receptors. Plasma membrane pinches to form a vesicle called an early endosome. Will be processed and have a lowered pH. Early endosome becomes late endosome and receives digestive enzymes from golgi. Eventually becomes lysosome. Vacuoles: - found in fungi and plants - occupy large portion inside the cell and serves as storage depots (water, ions, proteins, pigments) Mitochondria: - sites of ATP production - possess two membranes (inner membrane has folds/cristae, outer membrane is smooth) - possess their own mitochondria DNA and ribosomes - capable of independent division - endosymbiosis (mitochondria used to be separate cell) Chloroplasts: - have a double membrane (both inner & outer membrane are smooth) - inner membrane houses thylakoids (sacs stacked in grana) - sites of photosynthesis Cell Walls: - stiff to protect the cells (can occur in plants, bacteria & fungi) Tools used in the Examination of cells and their organelles: Light Microscopy & Electron Microscopy (SEMS and TEMS) - helps identify cells - micrograph (help distinguish cell shape) - proximity, location, size Differential Centrifugation - isolation of certain components, such as organelles. - Centrifuge solution at low speed. After low speed, pellet contains large components. Supernatant, and then use medium speed. After medium-speed, medium components are found in pellet. Supernatant again, and spin at high speed. Pellet now only contains small components. Density Centrifugation - Add sample to tube of variable-density solution - Run centrifuge. Cell components separate by density into distinct bands. To extract specific cell components for analysis, poke tube with needle and withdraw a specific band. Confocal Microscopy - cell could be alive - use of fluorescent dyes to see living cell processes - interacting live cells. Molecules that move into and out of the nucleus through the nuclear pores: rRNA and ribosomal subunits – nucleolus and into cytoplasm mRNA and tRNA – nucleus and into cytoplasm enzymes associated with DNA replication, transcription, nucleotide triphosphates – synthesized in the cytoplasm and enter nucleus Importing Molecules in the nucleus: - proteins that are directed to the nucleus possess a molecular “zip code” called the NLS (different for each cell). Info needed inside nucleus was found in the tail - NLS will bind to importins (delivery truck) and is moved into nucleus. ATP is the fuel. Ran protein unloads the protein with the hep of GTP. After this, NLS will detach Exporting Molecules in the nucleus: - protein exported contains NES. NES attaches to exportins, which requires Ran, GTP and fuel to help it exit the nucleus Experiment that proved NLS was in tail region: - protease cleaved tails off nucleoplasmin protein core. Attach radioactive labels to both the tails and cores. - Both are injected into the cytoplasm of the cell. - Tail fragments were located in the nucleus, the core fragments stayed in the cytoplasm. The Shipping of Proteins: 1. Protein enters RER while being synthesized by ribosome 2. Protein leaves RER and travels to the cis face of the Golgi 3. Protein enters Golgi’s cis face and is processed as it moves towards the trans face 4. Protein exits golgi at trans and moves to the plasma membrane 5. There, it will leave the cell . Pulse Chase Experiment: - technique involves marking molecules at specific intervals and examining their position over time - reveals that proteins move form the RER to the Golgi apparatus, to the secretory vesicles and then they are eventually secreted to the exterior of the cell Transport Vesicle – delivers contents to its specific destination. Example: proteins bound for the lysosomes are given a carbohydrate tag. Each tag is different, and places it with a specific vesicle. This tag attaches to the membrane protein in a vesicle. When the vesicle reaches its destination, the proteins on the vesicle surface will interact with the receptors. Vesicle then delivers content. The Cytoskeleton: - structural support, and is dynamic, so it can alter to change the shape of cells, to move materials within the cell, to move the whole cell itself. Actin Filaments Intermediate Microtubules (microfilaments) Filaments Protein Subunits Actin Keratin, vimentin, α-tubulin, β-tubulin lamin etc dimers Structure Strands in double Fibrous proteins are Hollow tubes (25 nm) helix, intertwined (7 wound to form thick nm) cables (10 nm) Function - maintenance of cell - maintenance of Cell motility (flagella shape, cell motility cell’s shape, anchors and cilia), movement (cell crawling), muscle the nucleus and other of chromosomes contraction, cell organelles, during mitosis, division in animals participates in nuclear maintenance of cell (cytokinesis), lamina formation shape, organelle cytoplasmic movement streaming, organelle movement Actin molecules – movement due to interactions with myosin - cell crawling (amoeba) – actin filaments pushes the plasma membrane into bunches called pseudopodia, adherence to a solid substrate and then a myosin-driven contraction of filaments at the other end. Causes whole cell to move - Cytokinesis – actin filaments slide past one another, causing the cell to pinch into 2 - Cytoplasmic streaming – directed flow of cytosol and organelles.  Head region of attaches to actin on the actin filament, while still bound to the actin, the myosin head flexes causing the filament to be pulled with it. Actin filament slides. ATP causes the myosin head to unflex and detach from actin. The myosin head can now attach to a new actin molecule farther up the actin filament. A Motor protein moves a vesicle along a microtubule: 1) kinesin (motor protein) consists of 3 regions: head region (acts as feet), tail region and stalk. 2) The two head pieces possess binding sites, one for binding to the microtubule and the other for binding to ATP 3) Tail region bind to vesicle. 4) As each head alternatively binds to ATP, kinesin takes step forward on microtubule track. Structure of Cilia and Flagella: - cross-sections of cilia and flagella reveal 9+2 arrangement (axoneme) - 9 microtubule pairs (doublets) are attached to dynein motor proteins - 2 central microtubules. - Microtubules are joined by protein spokes and bridges - Surrounded by plasma membrane How do flagella bend? 1) ATP is hydrolyzed by dynein 2) Confirmation changes occur 3) Walking proceeds as microtubules slide past each other within flagella/cilia 4) Since the entire structure is fixed/attached by the protein spokes to the 2 central microtubules, microtubule sliding is limited. This restriction eventually results in the bending of the flagella or cilia. Basal Body: - cross sections of the basla body (the structure which attaches the axoneme to the cell) has a 9+0 microtubule arrangement, 9 triplets of microtubules (no central pair) - has the same structure as a centriole Centrioles (90 to each other, only found in animal cells because plant cells have MTOC) Chapter 6 – Study Question: Common Fatty Acids: Palmitic Acid – Saturated Steric Acid – saturated Oleic – unsaturated Linoleic – unsaturated Stanley Miller’s experiment – building blocks of life and their origin (not life). Created a stimulating of the atmosphere. No ozone layer, able to find samples of the building blocks of macromolecules. Murchison meteorite – found monosaccharides etc on the meteorite in Australia – may have come from outer space (or was contamination) Hydrothermal vents – at high pressure, but does not boil. Able to collect extreme types of macromolecules and their subunits. (may have come together inside the ocean) Optical isomers – mirror images, have the same molecular formula, but they are non superimposable (handedness). L-DOPA (effective) /D-DOPA (not effective)(Parkinson’s) = D-DOPA (beam will be shot to the right); L-DOPA (beam will be shot to the left). Amino Acids (needs to know 20): Non-ionized form of amion acid (carboxyl group not charged) Ionized form (carboxyl group is charged (O- instead of OH) Charged amino acids, and non charged amino acids Quaternary – haemoglobin (find more) Nucleic Acid: Nucleotide (phosphate, pentose sugar, nitrogenous base) Phosphodiester bond Major groove – allows for the enzymes to come in during DNA replication. Carbohydrates: provides for energy Glucose – straight chains or circular structure. In a ring when in solution Membrane Structure: Fluid structure (proteins are embedded in the phospholipid bilayer, and are dynamic = Freeze/fracture technique) - Transproteins (throughout the membrane); Peripheral (on the side) Lipid bilayer with no unsaturated fatty acids = lower permeability Lipid bilayer with many unsaturated acids = higher permeability due to the kinks. Cholesterol on membrane integrity: Cold – membranes are allowed to open up due to cholesterol, higher permeability Higher – cholesterol will stick to fatty acids, causing lower permeability - Bad cholesterols & Good cholesterols Transports: Simple diffusion: area of high to low concentration to create equilibrium (can involve membrane or not) Osmosis: diffusion of water. (solute concentration of free water molecule concentration) Facilitated diffusion: use of protein Active transport: low to high concentration, use of energy Hypertonic solution – more concentration outside than inside, shrinking and crenate Hypotonic solution – more concentration inside, swelling and lyse Sodium-Potassium pump – neuron potential - conformational change with ATP and low to high concentration Secretory Pathway: RER – contains ribosomes. Allows for the formation of vesicles, which will attach to cis-face of the golgi, can change the protein, and then leaves golgi via the trans face. Pulse Chase: - allows for experimenters to track direction of movement of a target molecule and the direction where it is going. - Label it with radioactive isotope, look at it at various time intervals, and see where it is. - RER – continuous with nuclear envelope; vesicle; golgi; vesicle (smooth ER has no ribosomes) Animal cells have centrioles, Plant cells have MTOCs.  be able to differentiate between animal and plant cells. Cytoskeleton: Actin filaments; intermediate; microtubules. - not responsible for myosin, only kinesin & dynein Cell crawling – amoeba Kinesin – walking across microtubule w/ vesicle, using ATP. Centrioles – perpendicular, help to organize microtubules for cell division. (9 triplets of microtubules) Axoneme – 9 doublets + 2 central pairs. Flagella movement: - between each microtubule has spokes. Dynein will go and attach/detach on the length of the flagella. Because it is secure on the bottom, it will move and bend. GTP – helps with the unloading of protein in nucleus. Cell Cycle: All cells arise from pre-existing cells by the process of cell division = Virchow’s hypothesis. - Occurs in somatic cells - daughter cells are genetically identical to parent cell Functions of Mitotic Cell Division: (IPPMATC) - Reproduction in asexually reproducing species - growth & repair in multicellular species - bacteria undergo the process of binary fission for replication. 4 Cell cycle phases: 1. G1 phase (interphase) 2. S phase (interphase) 3. G2 phase (interphase) 4. Mitosis phase G1 phase - cell growth & duplication of organelles S-phase – DNA synthesis occurs (chromosomes replicate) G2 phase – cell growth & duplication of organelles continues to build the protein “machinery” Why do gap phases exist? – Before mitosis occurs, the parent cell must be large enough in size and must have made the required to organelles, so that daughter cells will operate properly. G0 phase – non dividing state. Examples include nerve cells (matured neurons), mature muscle cells. Liver cells can go from G0 phase back to the cell cycle due to external cues. Mitosis: - represents only a small portion of the cell cycle Prophase (preparation phase) - nucleoli began to disappear, chromatin fibres contract by tightly coiling (condensed DNA) - chromosomes are visible and consist of two identical sister chromatids joined together at the centromere. - mitotic spindle forms (microtubules polymerize) - assembly of microtubules begins in the centrosome (animals) or MTOC (plants) - in animals, the centrioles begin to move apart to opposite sides of nucleus (2 poles) Prometaphase: - chromosomes do not appear completely aligned or organized - nucleoli disappear and nuclear envelope breaks down - a specialized structure called a kinetochore (the structure on sister chromatids where spindle fibres attach) is formed near the centromere. - Spindle fibres attach to sister chromatids at the kinetochore regions - Kinetochore microtubules are polymerized, randomly oriented at first, then they become aligned parallel with microtubules - Kinetochore microtubules begin moving the chromosomes toward the middle of the cell. Kinetochore microtubules – extend from the poles to the kinetochores, attach to the kinetochores (outside, adjacent of the centromere, centromeres join the sister chromatids together). They assist in the migration of chromosomes. Nonkinetochore microtubules – radiate from each centrosome towards the metaphase plate without attaching to chromosomes. May overlap with those from the opposite pole. - Its function is assist in the elongation of the entire cell during anaphase and forms a cage-like network which facilitates the activities of the cell cycle components. Metaphase: - chromosomes line up along the metaphase plate - centromeres are aligned on the metaphase plate, which is located equidistant from the two poles - Each chromosome is held by kinetochore spindle fibres to opposite poles - centrosomes complete their migration to the opposite poles of the cell. Anaphase: - binding proteins between the sister chromatids break down due to shortening of microtubules - centromeres of sister chromatids disjoin and segregate. Called Disjunction Segregation - chromosomes move centromere first (appear in Vshape) - towards the end of anaphase, the two poles have identical numbers of chromosomes - cell elongates Telophase: - nonkintochore microtubules further elongate the cell - two daughter nuclei form - formation of nuclear envelopes around each set of chromosomes. Cytokinesis (cytoplasm division): - telophase is OFTEN, but NOT ALWAYS, followed by cytokinesis - Animals, fungi and slime moulds have cleavage furrow - Slime moulds => no cytokinesis may lead to multinuclei organism - Plants have cell plate How do chromosomes move during the process of mitosis? - tubulin subunits of the kinetochore microtubules are depolymerized (hydrolyzed) from the kinetochore ends. - Motor proteins (dyneins) attach & detach along the kinetochore - Microtubules’ length does not play a role in the shortening - Chromosomes move - Experiment: Use fluorescent labels to make the metaphase chromosomes fluoresce blue and the microtubules fluoresce yellow. At the start of anaphase, a beam of laser light will photobleach a section of microtubules to mark them without changing their function. The result is that the photobleached region remained stationary and the spindle fibres got shorter between the phorobleached region and the kinetochore. Cell Control: Heterokaryon – “different nuclei” taking cells from different stages and fusing them together either by using electrical or chemical methods Cell Fusion Experiments – Johnson & Rao (INSTANT CHANGE ONLY) To show whether there are any molecules that affect cell control Only shows results of G phase cells Mphase fused into G1 phase cell - G1 phase enters mitosis, chromosomes will thicken/condense S phase is fused to G1 phase cell - G1 phase will enter S phase, replication of DNA S phase is fused to G2 phase cell - G2 phase will not enter S phase (because DNA is already replicated) Microinjection Experiment – Markert & Masui M phase cytoplasm is injected into G2 phase cell - G2 phase begins mitosis Interphase cytoplasm is injected into G2 phase cell? - nothing. Remains in G2 phase This experiment showed that regulatory molecules control entry into M and S phases. Molecular signals in cytoplasm of M phase cells initiate mitosis Cell Cycle Enzymes: Cyclins – proteins that oscillate in concentration during the cell cycle and regulate cyclin dependent kinase activitiy. Mitosis promoting factor (MPS) (Cyclin + Cdk) – present in the cytoplasm and consists of the protein kinase (cdk) which catalyzes the phosphorylation of a protein and a cyclin. Cdk (Cyclin dependent kinase) - protein kinase, which uses ATP - adds phosphate group to protein - induces a conformational change - dependent on cyclin, could not work by itself Phosphatase – removes phosphate group from the kinase target protein, REVERSES the conformational change Protease – degrades proteins (proteolytic enzymes) Functions of Cell Cycle Checkpoints (control) - under normal conditions, cell checkpoints ensure that DNA replication (S phase of interphase) and mitosis take place only when conditions are favourable and the related processes are functioning properly - checkpoints regulate the correct sequence of events in time and enforce synchrony of different complexes - involve rapid and reversible alterations. For example, when functional groups change or when proteins are broken down Three main checkpoints: G1 checkpoint – checks if nutrients are sufficient, growth factors are present, if DNA is damaged. Also ensures that cell is large enough to divide * If fell fails G1 checkpoint, cell would fail to divide G2 checkpoint – ensures that DNA replication is successful. Checks if it is replicated correctly and if there are any MPS * If G2 checkpoint failed, daughter cell would not have COMPLEMENTARY DNA. Metaphase Checkpoint – ensures that all chromosomes are attached to spindle fibres via the kinetochores. * If Metaphase checkpoint failed, daughter cells will not have correct number of chromosomes. Cancer: - uncontrolled cell division - cancel cells divide faster than normal cells - cell cycle is not regulated - cell cycle checkpoints fail Metastasis – spread (movement) of cancerous cells from their original growth site to other organs. Examples: G1 checkpoint not functioning properly: Rentionblastoma children - dysfunctional Rb protein (tumour suppressor) - G1 checkpoint is disrupted - Cell division is constant & unregulated - Leads to malignant tumours in the retina Chemotherapy: - anticancer drugs that are applied to the whole body via injection - mode of action => kills rapidly dividing cancer cells or cause them to stop in G1 phase - Side effects -> hair loss, nausea, weight loss, weakness. This is because some chemo drugs kill normal cells such as hair follicle + intestinal cells - Current research => attachement of chemo drugs that bind to specific membranes on cancer cells - Focused upon delivery of chemo drugs via liposomes (only affects cancer not other cells) Meiosis Gamete – a haploid (n) reproductive cell that has one set of chromosomes Fertilization – occurs during the fusion of two gametes (one from each parent) Sexual Reproduction in Humans: - brings 2 gametes (1 from male, 1 form female) together - 2 parents produce offspring (progeny) - parents have 2n (diploid nuclei) - gametes n (haploid) and differ one from another in gene composition - gametes have half the DNA content of each parent nucleus (n is ½ of 2n) - each parent passes on half of its genes to its offspring - offspring inherit a unique combination (mixture) of genes from both parents => this is called genetic variation Haploid number n indicates the number of distinct type of chromosomes present. Ploidy – the number of each type of chromosome present. Chromosomes: - composed of DNA and proteins - carry genes (hereditary information) - chromosomes of the same type, size and with the same genes at identical locations are called homologs or homologous chromosomes Genes: - units of heredity - made of DNA (nucleic acid) located on chromosomes - have specific sequences of nucleotides (monomers) most genes program cells to synthesize proteins - the actions of these proteins produce the organism’s inherited traits Alleles – are the different version of a specific gene Sex chromosomes – chromosomes that determine an individual’s sex Autosomes – non-sex chromosomes Karyotype – a display of an individual’s chromosomes that is organized in terms of chromosome number, size and type  modern times, classified by number, but in older publications, chromosomes were organized by length  karyotypes are useful for genetic screening to identify chromosome defects in their number size & type Examples of genetic disorders: Klinefelter Syndrome - XXY karyotype (1/1000). Occurs in males only. Can occur due to duplication of X chromosome during meiosis and/or lack of division between the pairs. - Leads to breast development, narrow shoulders, wide hips, frontal baldness, small testes (sterile), feminine contours, lack of chest hair, normal intelligence CML (Chronic Myelogenous Leukemia) - portion of chromosome 22 stitches with a small piece from chromosome 9 (Philadelphia chromosome) - affects cells that give rise to white blood cells Cri du Chat syndrome (1/50000) - deletion of a little bit on chromosome 5 - abnormal glottis & larynx (mewing crying sound) - size deletion of chromosome 5 influences physical & mental skills - severely mentally challenged. Locus – position of a gene along a chromosome (or along a DNA double helix). Makes up the banding patterns 1. Prior to Meiosis: - during Interphase, each chromosome in the 2n parent cell replicates. Meiosis: - occurs in sexually reproducing individuals - consists of two consecutive cell divisions, Meiosis I and Meiosis II - Meiosis I: homologs from each pair of homologous chromosomes, separate and move to 2 different daughter cells - Meiosis II (most similar to mitosis): sister chromatids of each chromosome separate and move to each of the two daughter cells. At the end of meiosis Ii and cytokinesis, there are 4 haploid gametes each with one copy of each chromosome produced. Meiosis I Early Prophase I: - nuclear envelope breaks down and chromosome condense - replicated homologous chromosomes pair to form tetrads, this process is called synapsis - Synapsis: is precise; homologous chromosomes align gene by gene; the exact mechanism which causes synapsis is still unknown, but it may involve a protein complex called the synaptonemal complex. Late Prophase I: - Crossing over (recombination) occurs between two non-sister chromosomes - Chiasma – one crossing over - Chiasmata – several crossing overs Creighton & McClintock Experiment Strain 1: - long chromosome 9, with knob, kernels are coloured & waxy Strain 2: - short chromosome 9, no knob, kernels are colourless & starchy Strain 1 x Strain 2: - chiasmata form between non-sister chromosomes - wants different chromosomes to get different characteristics Results: 1) long chromosome 9, no knob, kernels are colourless & waxy 2) short chromosome 9, with knob, kernels are coloured & starchy 3) same as parental types Conclusion: - physical exchange of DNA (knob) took place - they are displaced in phenotypes, crossing over occurred Metaphase I: - tetrads line up along the metaphase plate - pairs are still next to each other, exchanged DNA already, connected to microtubules - Alignment of tetrads is random, and moves independently of the other tetrads Anaphase I: - homologs separate and start migrating the opposite poles of the cell - disjunctional segregation occurs during Anaphase I - homologs separate and begin moving to opposite sides of the cell. - Sister chromatids remain attached Telophase I and cytokinesis: - Chromosmes finish their movement to opposite sides of the cell - Cytokinesis divides the cytoplasm to form 2 haploid cell (n chromosomes) - Cleavage furrow formation, reformation of nuclear envelope Meiosis II: Prophase II: - spindle apparatus forms and centrosomes are replicated Metaphase II: - chromosomes line up along the metaphase phase Anaphase II: - when sister chromatids detach, are no longer called chromatids, are now full fledged chromosomes - separate at centromeres and start to move to the opposite poles - disjunctional segregation Telophase II: - chromosomes finish movement to opposite poles - nuclear envelopes form around each haploid set of chromosomes Cytokinesis: - cytoplasm is divided - at the end of the meiosis II, each cell has divided and four haploid daughter cells are formed - 2 chromosomes Reduction Division: - reduces chromosome number from 2n (diploid) to n (haploid) - daughter cells have half the number of parent chromosomes - union of two haploid (n) gametes during fertilization restores the diploid (2n) number Genetic Variation: - daughter cells are genetically different from the parent cells - 1. Crossing over between homologous chromosomes (prophase I, meiosis I) - 2. Independent assortment of homologous pairs (metaphase I, meiosis II) - Random fusion of gametes during fertilization Approximate number of different combinations of chromosomes in gametes: # of combs = 2^n Ex. Humans n = 23. # = 2^23 - not including crossing over & mutations Asexual Reproduction: - all individuals that reproduce asexually can produce offspring that are genetically identical (one parent is needed only) Sexual Reproduction: - two parents needed, but only females can give birth to offspring, fewer offspring are produced - role: genetic variation for adaptation. Purifying Selection Hypothesis: - in species that reproduce via sexual reproduction, only ½ of offspring will inherit a damaged allele (compared to the species that asexually reproduce which pass on damaged genes to ALL offspring) - Natural selection will favour the success of offspring that do not inherit damaged gene, decrease in these individuals Changing Environment Hypothesis: - a population comprised of genetically diverse individuals will be more likely to survive and successfully reproduce than a population, which is composed of genetically identical individuals - this genetic variation is very important for the maintenance of biodiversity. Sexual reproduction produces more genetic variation for adaptation - Sex is important due to significant variation and mutations are rare in asexual reproduction Experiment: - Lively and et. Studied species of snails that are susceptible to infection due to trematode worms (symbiotic relationship). These worms eat their reproductive organs and are rare in some habitats but common in others - If Changing environment hypothesis is correct, frequency of sexual reproducing individuals should be much higher in habitats where parasites are common than it is in habitats where parasites are rare. - Worms would rather attack organisms that sexually reproduce because of genetic variability. Future is in doubt if it laches on to asexual host, since asexual host might die. Errors in Meiosis: - nondisjunction may occur during meiosis I, if the homologous pairs of chromosomes do not separate properly. Nondisjunctions may occur during meiosis II, if the sister chromatids do not separate properly - lack of crossing over of the genes - Other mistakes involve chromosome structure alterations : deletions and duplications Aneuploidy – abnormal number of certain chromosomes. Individuals have too many or too few of specific chromosomes. Trisomy – individuals have three copies of a certain chromosome Examples: Down Syndrome – trisomy 21. Large foreheads, shorter in stature, special needs, very pronounce crease in their hands, their tongue is different – very large Patau Syndrome – trisomy 13. Severely mentally challenged, cleft palate, deaf, malformed organs Edwards Syndrome – trisomy 18. Small newborns, low set ears, webbed neck, receding chin, organ malformations Turner’s syndrome – XO female. Only 1 X chromosome (monosomy). Short stature, webbed neck, shield like chest, internal sex organs do not mature, females are sterile. Triple X syndrome – XXX females. Usually normal phenotype and fertile, others have variable expression Jacob’s Syndrome – XYY males. Normal male, but much taller than average, normal fertility and intelligence, very competitive Polyploidy - chromosome number is greater than two complete chromosome sets (ex. Triploidy 3n) - three complete sets of chromosomes (occurs in plants, important for plant evolution, seedless watermelon) Why do mistakes occur: - meiotic errors seem to be accidental in their occurrence. Environmental causes (radiation, chemicals) - generally mistakes occur without any genetic predisposition - alterations : deletions, duplications. Also, crossing over errors may occur  patterns of occurrence strongly suggest that sex chromosomes and chromosome 21 may be more prone than other chromosomes to aneuploidy  maternal errors seem to occur in greater frequency when compared to paternal errors  the older the mother, the higher the chances of having a child with down syndrome (80% are spontaneously aborted, 20% survive to birth). Tests such as amniocentesis & CVS can be used  eggs become older as mother becomes older Know the Features of amniocentesis & CVS Amniocentesis: - sample of amniotic fluid taken at 14 – 16 week of pregnancy. Are centrifuged to separate fluid from fetal cells. Fetal cells are cultured until a sufficient number is used for karyotyping Chorionic villus sampling (CVS): - Taken as early as 8 to 10 week of pregnancy - Suction tube inserted through cervix to get fetal cells - Used to create karyotypes. More expensive and used for high risk pregnancies. MENDEL AND THE GENE Gregor Mendel (1865) – rules of inheritance due to experiments on garden peas. Sutton/Boveri – chromosome theory of inheritance. Meiosis causes the patterns of inheritance that Mendel observed Heredity – transmission of traits from parents to their offspring. Trait – any characteristic of an individual 2 Hypotheses: blending inheritance (traits observed in both parents are blended together to form the traits observed in their offspring) and inheritance of acquired characteristics (traits present in parents are modified, through use, and passed on to their offspring). Garden Peas: - used this because it was easy to grow, short reproductive cycle, lots of seeds - Genetics – model organisms because conclusions may apply to many other species as well Mating: - peas normally pollinate themselves through self-fertilization, but Mendel prevented this by removing the male reproduction organs containing pollen from each flower. This pollen was used to fertilize the female reproductive organs of flowers on different plants, thus creating cross pollination Mendel studied seven traits (phenotypes): - seed shape - seed colour - pod shape - pod colour - flower colour - flower and pod position - stem length Used pure lines (homozygous) plants that produced identical offspring. Used these plants to create hybrids by mating 2 different pure lines. These offspring are the F1 generation Pure: Round + Wrinkled => all round seeds (F1 generation) F1 + F1 => 3 round: 1 wrinkled. (F2 generation) Round is dominant, wrinkled is recessive. Particulate Inheritance Hypothesis – hereditary determinants (now called genes) maintain their integrity from generation to generation, directly contradicting the blending inheritance hypothesis. Different versions of a gene are called alleles = different alleles are responsible for the variation in the traits. Alleles found in an individual are called its genotype. Genotype affects phenotype (traits we can see). Principle of Segregation: alleles segregate into different gametes during gamete formation, then come back together when an egg is fertilized by a sperm to form a zygote. R- dominant; r- recessive. RR – homozygous; Rr – heteroxygous Punnett squares Two Traits: - dihybrid crosses (mating between parents that are both heterozygous for two traits) - Results supported the principle of independent assortment = alleles of different genes are transmitted independently of each other Hypothesis of dependent assortment = alleles of different genes stay together when gametes form. RY and ry stay together. - RRYY, RrYy, RrYy, rryy. - F2 phenotype results are consistent with predictions of independent assortment The Chromosome Theory of Inheritance Sutton & Boveri’s observations of meiosis: states that chromosomes are composed of genes - principle of segregation can be explained by the independent alignment and separation of homologous chromosomes containing alleles for a gene at meiosis I. Each gamete carries only one allele for seed shape, because the alleles have segregated during meiosis. - Alleles for two diff. genes located on different nonhomologous chromosomes assort independently of one another at meiosis I X-Linked Inheritance: - inheritance patterns can occur when genes are carried on the sex chromosomes - non-sex chromosomes = autosomes. Genes can be located on the same chromosome. Physical association of 2+ genes found on the same chromosome is called linkage. Linkage and sex-linkage are different. Sex-linked = found on a sex chromosome. - Linked genes are predicted to always be transmitted together (violates principle of independent assortment) Crosses cannot be done
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