Midterm Flashcards

Question

Active Transporters (also called “Pumps”)

Answer 1

Coupled pump uses the energy gradient of the “coupled” molecule * **ATP driven – uses energy released during the hydrolysis of ATP into ADP and phosphate*** - ATP driven pump is responsible for establishing ion gradients in excitable cells such as Na+ and K+ or Ca+ Light driven – direct use of light energy

Answer 2

passive selective transport of ions driven by electrochemical gradient Ion channels are integral proteins in the lipid bilayer, can be open or closed. Open channel is characterized by specific selectivity. Channels can be classified by selectivity (what ion they allow to go through) and gating (what makes them transfer from open to closed state and back). EXAMPLE IS NEURAL COMMUNICATION

Answer 3

• By gating Ligand gated Voltage gated Mechanosensitive ``` • By selectivity Sodium Potassium Calcium Chloride Cationic Anionic Large pores (technically not “channels”) ```

Answer 4

the force tending to drive an ion across a membrane is made up of two components: one due to the electrical membrane potential and one due to the electrical membrane potential and one due to the concentration gradient of the ion. At equilibrium, the two forces are balanced and satisfy a simple math relationship give by the Nerst Equation: V = 62 log10 (Co/Ci) ``` V= membrane potential in milivolts Co = concentration outside Ci = concentration inside ```

Answer 5

Na conductance shows a fast, transient increase in response to depolarization K conductance shows a slower, more sustained increase

Answer 6

can be performed using patch-clamp approach

Answer 7

using X-ray crystallography or cryoelectron microscopy

Answer 8

Ribosomes are targeted to the ER only after protein signal (targeting) sequence becomes synthesized. SO... There is a common pool of ribosomal subunits sitting in the cytosol, If there is no targeting sequence synthesized, protein synthesis will occur via the free ribosomal cycle. HOWEVER, if there is a targetting signal, the ribosomal subunits will bind to the rough ER for protein synthesis. Coordinated with protein translocation - mainly on the Ribosomes bound to rough ER

Answer 9

Following synthesis, the soluble proteins are released into the lumen of the ER Signal peptide (signal sequence) is cleaved by signal peptidase and remains in the ER membrane

Answer 10

Following synthesis proteins remain in the ER membrane*** Stop transfer sequence (hydrophobic sequence) keeps protein imbedded in membrane, seperating lumen and cytoplasmic sides of the protein

Answer 11

Posttranslational modification – chemical modification of the protein that is not coded by DNA-RNA. (COVALENT BONDING OF DIFFERENT MOLECULES AFTER PROTEIN IS ALREADY MADE) Many dozens exist and can involve many amino acids and many types of “attachments” ex. Glycosylation – attachment of a sugar residue like with the Glycocalyx

Answer 12

the main destination for the proteins made in ER Protein synthesized in ER, then they need to be processed in the golgi apparatus and get there via vesicles. Golgi vesicles then take finished proteins for either exocytosis or endocytosis

Answer 13

Golgi consists of many flatten membrane enclosed sacs: “cisternas” (3 to 20 cisternas in every stack of Golgi) Has two sides: cis (entry side) and trans (exit side) Cis – close to the ER; Trans – close to the plasma membrane

Answer 14

Proteins entering ER (cis) are either moved trough the stack or returned back to the ER (if they have ER retention signal) Proteins exiting from Golgi (trans) either go to the lysosomes or cell surface Golgi is the place where further sugar protein modification occurs SO...it can either undergo UNREGULATED exocytosis in which newly snythesized soluble proteins are transported via a transport vesicle made up of newly synthesized plasma mem. lipids/proteinsto the cell membrane for constitutive secretion OR... it can go through REGULATED exocytosis where the secretory where the proteins are transported into a SECRETORY vesicle - which stores the proteins until an extracellular signal (hormone or neurotransmitter) signals for the secretion of these proteins. thus, the signal transduction allows for the regulated secretion of the proteins out of the cell.

Answer 15

Primary cites of intracellular digestion - ``` Includes acid hydrolases nucleases proteases glycosidases lipases phosphatases sulfatases phosolipases ``` The acidic environment of the lysosome is maintained by ATP dependent proton pump There’s transporters on the membrane of lysosome that also transport metabolites out of the lysosome after the enzymes have broken them down. o Nearly all of these enzymes in the lysosome are protected from being broken down themselves because they got lots of glycosyl modification; the (mannose 6-phosphate) that protectthem from being broken down by other lysosomal enzymes.

Answer 16

Phagocytosis – digestion of bacteria Endocytosis – digestion of the extracellular compounds Autophagy – digestion of the intracellular compounds

Answer 17

* ER plays a critical role in calcium signalling during muscle contraction * ER in muscle is called SR (Sarcoplasmic Reticulum) * SR is highly specialised to perform calcium pumping and release cycles MUSCLE CONTRACTION CAN NOT PROCEED WITHOUT PARTICIPATION OF ER!!!!!!!! Also, essential role of ER in neuronal calcium signalling (TG-thapsigargin - drug that blocks calcium uptake in the ER)

Answer 18

• ~2000 proteins make mitochondrial proteome • Only 20-30 are coded by the mitochondrial DNA • Most of the mitochondrial proteins are made in cytoplasm and imported into the mitochondria Mitochondria have their own complete machinery for protein synthesis but only for vey few proteins Mitochondrial DNA is maternally inherited: it allows us to track different inheritance because there is almost no DNA material mixing. You can track by mother inheritance and parental information in terms of origin of different genomic lines.

Answer 19

The whole protein is made in the cytoplasm and then it gets imported into mitochondria in an unfolded form through the protein translocator in the outer membrane and protein translocator in inner membrane. • Proteins that are destined to the mitochondria generally have this signal sequence which can be recognized by this transporting protein. Then can be translocated and unfolded and get into the matrix where they complete protein folding and the signal peptide gets cleaved and recycled. • But what is not really well understood if you look at mitochondrial protein only a few proteins have this signal protein. By looking at the protein we cannot determine if its mitochondrial or not. If we have a signal sequence we can but if it doesn’t, you cannot be for sure whether its mitochondria targeted or not. * proteins targeted to the (other than matrix) mitochondrial compartments have different targeting sequences * many mitochondrial proteins do not have any obviously recognizable mito-targeting sequence.

Answer 20

structures within cells that convert the energy from food into a form that cells can use” (NIH-2009) - Mitochondria produce energy in form of ATP - Mitochondria and calcium signaling - Mitochondria – storage of pro-apoptotic molecules (they are key master regulator of cell death which is apoptosis.) -Mitochondria – major source of ROS (Reaction Oxygen Species) >>> Oxygen species are major signaling molecules that guide a lot of intracellular processes. Mitochondria are the place where most of these species are synthesized which can signal throughout the cell. Mitochondria are critical for that. Nobody knows how they do this.)

Answer 21

``` • Diabetes Type II • Cardiomyopathy • Aging • Cancer • Parkinson’s Disease • Alzheimer's • Huntington’s ```

Answer 22

Mitochondria are central in producing energy in eukaryotic cells especially in cells that rely on oxidative phosphorylation for energy. • Two major pathways: glucose utilization and pathway for fatty acid utilization. Those are two types of food our cells can receive and process. A majority of foods when it comes to the tissues come in the form of glucose or fat associated with proteins. • One pathway goes from glucose, which is sugar, through pyruvate and then into the mitochondria. Then is the critic acid cycle (TCA) producing NADH and then end up in ETC which utilizes oxygen and eventually leads to the production of ATP • The other pathway also eventually leads either to TCA, producing NADH or directly producing NADH and then to electron transport chain and again the synthesis of ATP. KEY METABOLITES • GLUCOSE and FATTY ACIDS are the starting points. * Then PYRUVATE is the central end product of glycolysis. * ACETYL CO A is the major intermediate entering the TCA cycle from either side. * NADH is major donor of protons and electrons to the electron transport chain and the end product is ATP.

Answer 23

Outer membrane is permeably to the ions and small molecules ******98% of the body’s oxygen is used******* 1) NADH is produced by TCA cycle 2) NADH donates electron and proton to the respiratory chain 3) Electrons travel through the respiratory chain towards complex IV and this is coordinated with proton exiting from the matrix 4) Membrane potential is generated as a result of proton efflux 5) ATP is produced in ATP synthase when protons flow back into the matrix

Answer 24

* Oxidation – movement of the electrons from NADH to the oxygen which results in generation of the membrane potential * Oxidation can be coupled to the Phosphorylation: Membrane potential is used for attachment of the phosphate to ADP and production of ATP * Membrane potential can be used for: 1) ion transport 2) heat generation 3) pathological and physiological “leak” of the membrane

Answer 25

Roles of the mitochondrial ion channels: • calcium uniporter – regulation of energy metabolism rates • Katp channel – (proposed) to be involved in mitochondrial volume regulation • PTP large pore – pathological channel involved in cell death • NCX – (Sodium-calcium exchanger) helps to recycle calcium • UCP – uncoupling protein – heat generation. • VDAC – passage for metabolites across the outer membrane • MAC – apoptosis channel

Answer 26

In most cases mitochondria needs to be intact since integrity of the membrane is required What can be measured in the intact mitochondria: • NADH levels (imaging) • Membrane potential (imaging) • ATP levels (imaging) • Oxygen consumption (respirometry) -Place cells (mitochondria in air tight chamber) and See how quickly oxygen is taken up (how the cells Are "breathing")

Answer 27

Addition of the flavonoid Quercetin to the neuronal culture leads to the increase of the mitochondrial membrane • So the drugs that we believed improved mirochondrial function also helps protect the brain. • Flavonoids (E+Q) improve mitochondrial function by increasing the spare respiratory capacity of the mitochondria E+Q protect brain from stroke damage

Answer 28

• Cardiac cells heavily rely on ATP, but as soon as you depolarize the mitochondria you will remove that membrane potential and ATP will no longer be produced. • Without ATP supply the cardiac cell is dead within a few minutes

Answer 29

(Initial control: rate they take up oxygen at first) Then you add Oligomycin and it drops then FCCP was added And then Rotenone test and Antimycin test * Oligomycin blocks ability of mitochondria to produce ATP (ATP synthase) * FCCP resents membrane potential (does not allow a gradient to be formed) leads to increase in Oxygen because the only step that is occurring is the oxidation NO PHOSPHORALIZATION * Rotenone stops complex I * Antimycin stops complex III

Answer 30

``` • What happens during stress, is that you have a lot of influx of calcium. Stress is for example, in case of heart attack, lack of oxygen. So mitochondria cannot function well. You have this gigantic influx of calcium which enters the mitochondria. ``` • This leads to the phenomenon called calcium induced permeability transition pore. Which is condition that occurs during cell death, stroke, or heart attack. * This porin resets membrane potential. The pore will open, it will depolarize and will cause massive tissue damage. * This pathological pore which opens and kills mitochondria. Apparently what is important about it is that if you block this channel during this condition like in stroke you can significantly protect and diminish the tissue damage. ***********• PTP – Permeability Transition Pore opens during calcium overload; leads to the loss of the membrane potential*************

Answer 31

* mitochondrial intermembrane space is not only space for energy metabolites but also space for pro apoptotic molecules like Cytochrome C, AIF; SMAC/DIABLO molecules * They are stored there and are harmless for the cell unless apoptosis signal comes • Then apoptosis signal leads to the formation of the mitochondrial apoptosis channel (MAC), which is large pore. Even larger than your traditional pore. The channel leads to the leak of these molecules into the cytoplasm which trigger apoptosis.

Answer 32

* Membrane bound organelles (single membrane) * Break down alcohol; toxins and fatty acids • Note the outcome of the fatty acid hydrolysis is production of energy rich molecules of FADH2 and NADH (same enzymes as the mitochondria are producing the same energy rich molecules except they are breaking down different things)

Answer 33

* Water (85%) = semi-gel * Proteins * Carbohydrates * Lipids * Nucleic acids * Buffers * Ions * Trace elements

Answer 34

Maintenance of cell shape - Every cell in the body or different types of cells in the body all have a different shape. That maintenance of shape comes from the cytoskeleton. Whole cell movement - Not all cells just sit there. Many of them move around. The ones that are moving around a lot are the white blood cells. They function usually in connective tissue not blood vessels. Contraction Changes in cell shape -There is a lot of places where cells can change shape and its important for them to change shape. Somewhat during development. Structural integrity of cell -The cytoskeleton places an important role in structural integrity; in many cases, especially the intermediate filaments. Organization and movement of organelles: - mitochondria and the secretory granules and other organelles are organized and they move because of the cytoskeleton on the inside.

Answer 35

three types of filaments in the cytosol • Mircofilaments – they are the smallest, 7-8 nm (will not ask numbers on the exam) * Microtubules – largest filaments, about 24 nm * Intermediate filaments – as electron microscopy got better, they discovered an intermediate filament • Plus accessory and regulatory proteins of each are also part of the cytoskeleton

Answer 36

• It’s the component of MICROFILAMENTS • It’s globular protein • The monomer of the globular actin is G ACTIN, it will polymerize to form a filamentous chain which is called F-ACTIN >The polymerization requires ATP and Magnesium. > Microtubules and microfilaments have a polarity to them. They form from a minus end towards the plus end. >If they’re not capped or maintained in some way, they disassemble. The disassembly takes place from the minus end of the filament in actin filament. > Bundles of F-actin are referred as microfilaments. > It’s really F-actin that is stabilized, it’s capped on both ends so that it’s not continuing to disassemble or to fall apart. It plays a role in whatever the function of that cell might be. • Different tissue in the body have different isoforms of actin. They’re basically the same but they have physiology functions that vary depending on the isoforms of actin. Different isoforms of actin (not going to talk a whole lot about these) * α-skeletal - skeletal muscle * α-cardiac - cardiac muscle * α-vascular - vascular smooth muscle * γ-enteric - visceral smooth muscle * β-cytoplasmic - non-muscle cells * γ-cytoplasmic - non-muscle cells

Answer 37

actin filaments are anchored in a very regimented way in skeletal or cardiac muscle into these areas called the Z DISC They interact with bundles of another cytoskeletal protein, MYOSIN They induce a sliding of the filament. The sliding filaments take place in the non-muscle cells as much as they do in the muscle cells.

Answer 38

They play the role in function related to cell shape and motility. * Most of the functions in the non-muscle cells involve attachment to the cell membrane -- they do that through interacting with proteins that are transmembrane proteins in the cell membrane. * There is a bunch of actin binding proteins that are necessary for the actin to function within the cell.

Answer 39

* Tropomyosin is important to skeletal and cardiac muscle. * Alpha actin is important in muscle and non-muscle cells. These two are important in bundling filaments and something called microvilli. * Spectrin is very important in crosslinking the membrane skeleton and something that is called terminal web. * Myosin is important in sliding filaments in muscle and non-muscle cells. * There are various Caps, the various capping proteins keep the filaments from depolarizing.

Answer 40

( 4 examples) * Epithilial cell – are polarized, have a base and an apex, they can have cell surface and lateral specializations, can have finger-like projections called MICROVILI. They increase cell surface area. So you really see that these cells are involved in absorption. They absorb area of the GI tract (small intestine mostly as well as salivary gland ducts and kidney) * Stress fibers - They position organelles and move organelles in very distinct patterns within the cell • Cell motility -- lamellipodium forms and you polymerize actin. Actin myosin interaction then pulls up the rear end of the cell leaving bits and pieces of cell membrane and extracellular matrix and maybe integrins behind. • Actin forms the contractile ring that separates the two daughter cells during Mitosis

Answer 41

Cells form these LAMELLIPODIUM/Filapodia. As the lamellipodium forms and you polymerize actin, it protrudes the front of the cell out further and further. It forms, because there are actin filaments in there and they are binding to integrins on the cell membrane, those integrins then bind to the extracellular matrix. Cells don’t get infinitely long and skinny. What ends up happening is that the rear part of the cell has a actin myosin interaction and it PULLS UP the rear end of the cell leaving bits and pieces of cell membrane and extracellular matrix and maybe integrins behind.

Answer 42

When cells go through mitosis and they divide, actin forms the contractile ring that separates the two daughter cells. The contractile ring and the cleavage furrow is very important actin component in mitosis. Microtubules play a role in chromosome movement and separation.

Answer 43

Actin has to attach to the cell membrane to effectuate any of those sorts of cell shape changes. ACTIN FILAMENTS are bundled together by protein ALPHA ACTININ It comes through an ADAPTER COMPLEX that is made up of VINCULIN, and TALIN and a bunch of other specific proteins. They BIND TO A TRANSMEMBRANE RECEPTORS. In this case it’s the INTEGRINS. Those integrins either bind to the EXTRACELLULAR MATRIX and something called the FOCAL ADHESION. Actin can also bind to the same structures embedded into the membrane. As the actin interacts with myosin and it contracts, it can cause a contractile ring.

Answer 44

In RBC, the terminal web includes GLYCOPROTEINS There is a series of proteins that are very well studied ANCHORING BAND 3, PROTEIN 4.1, that organize this altogether. What binds to ANKYRIN in this is transmembrane complex is a protein called SPECTRIN. Spectrin is a super family. Spectrin binds to the transmembrane protein at various spots all around this cell. ACTIN and protein 4.1 help crosslink and bind these together as a certain amount of tension is put on the system that gives you this biconcave disc. Every cell has a TERMINAL WEB.

Answer 45

* Spectrin I is in the erthrocytes (RBC) only * Spectrin II is in every other cell in the body just underneath the cell membrane in various spots. * Alpha Actinin - going to see it in skeletal muscle, see it in cardiac muscle and other muscles. * Dystrophin – found in skeletal muscles. It is called dystrophin because it was discovered, in mostly boys at the time, that had duchenne and becker muscular dystrophy. Dystrophin was missing. Muscular weakness results and eventually death because even diaphragm doesn’t work.

Answer 46

Bundled actin filament have a structural function within microvilli * Actin bundles are attached at their plus ends to the tip of the microvilli * The actin filaments are bundled by the proteins VILLIN and FIMBRIN * The actin bundles are attached to the sides walls of the microvilli plasma membrane via myosin I and calmodulin * Within the terminal web, nonerythroid specturin (SPECTRIN II) and MYOSIN II links adjacent actin bundles to eachother and to intermediate filaments.

Answer 47

MICROTUBULES * Microtubules are made up of monomers or dimers of alpha and beta tubulin. Independent gene family products. * They require GTP to polymerize. * They assemble from a minus end, which is where things tend to disassemble. * They have a growing end which is the plus end. * The minus is always bound at something called microtubule organizing center and there are bunch of them inside cells. • When the microtubules start to form, they start to form at an organizing center which is where the minus end tends to attach. It require GTP and the GTP is on the terminal dimer * But if you hydrolyze or dephosphorylate the terminal dimer, it leads to something called dynamic instability and the microtubule collaspes * Microtubules continually grow from the centrosome, added to a cell extract. Quite suddenly however, some microtubules stop growing and then shrink back rapidly. A behavior called DYNAMIC INSTABILITY. * Microtubules are the 13 protofilaments. • Outside diameter is about 24 nanometers . • Inside, it is really a hollow tubule, its about 14 nanomenters. • You add them on beta to alpha. They’re added on at the plus end and they peel off at the minus end unless you dephosphorylate these at the terminal end then the microtubules will collapse.

Answer 48

In the cell they all tend to startout at something called the CENTROSOME = when two centrioles line up perpendicular to each other. Then there is a cloud of associated proteins that from these things called microtubule organizing center, these microtubules pull chromsomes apart during cell division. There are other microtubules organizing center. The one that is very well known and studied is the CILIA (long projections off the cell surface) in epithilial cells. They form off of the basal body which binds the negative end. Cilia function to move material across the cell surface. (EX. particles trapped in mucous on the surface of the trachea and lung)

Answer 49

Cilia have a power stroke that takes place when the cilia are extended up into the mucus and moving into the direction they are organized to. They drop off the mucus down into the liquid layer, recoil back, come up again around, hit the mucus and move it forward. They do this very fast and in an organized fashion. CILIA DO NOT MOVE WHEREAS FLAGELLA DO MOVE ( one of the major differences between the two)

Answer 50

I. Contains two centrioles II. Centrioles composed of 9 triplets of microtubules in a circle III. Centrioles are perpendicular to each other IV. Surrounded by a cloud of microtubule binding proteins (microtubule organizing center that binds minus ends)

Answer 51

Vesicles in microtubules and there are a couple of different motors. There is a motor that moves organelles from the minus end of the microtubule to the positive end. That motor protein called KINESIN. There are those that move from positive end back to where the centrosome is or cell center called DYNEIN. • Where this is really magnified is in an axon on a motor neuron where these motor proteins dragging things • dynein moves things towards the minus end (towards the cell center) • kinesin goes towards the plus end (periphery of the cell)

Answer 52

Intermediate filaments are much more structural -- are holding the cell into its shape. They don’t move things, they don’t have motor proteins associated, they don’t polymerize. * Intermediate filaments have a number of gene products. * The proteins in every type of cell are different but the general function is the same. * EPETHIAL cells have keratins. * The gene family which is the vimentin and vimentin related genes are in CONNECTIVE TISSUES * Then there is a special class in nerves called neurofilaments in NERVE cells. * So although there are different proteins in different tissues, the general structure is the same. * It’s an alpha helical monomer, it’s got an amino terminus and a carboxy terminus. It forms a coiled dimer that work together >>> forms tetramer >>>> form two tetramers >>>> they pack together putting 8 tetramers together >>> that gets coiled into a rope like filament that is 10 nm across. * It’s a very structurally rigid, strong, filament. * Does not do dynamic instability like microtubules do. They don’t spontaneously disassemble if you remove a regulatory protein. * Intermediate filaments attach to desmisomes and hemidesmisomes

Answer 53

Cellular connective tissue of the CNS Astrocytes (Astroglia) - Pure connective tissue cells - Vimentin and GFAP Oligodendrocytes (Oligodendroglia) - Contractile and produce myelin - Vimentin only Microglia (Mesoglia) - Macrophages - Vimentin only

Answer 54

Microtubules are highly dynamic and will frequently grow and shrink at a rapid yet constant rate. During this phenomenon, known as ‘dynamic instability’, tubulin subunits will both associate and dissociate from the plus end of the protofilament the primary determinant of whether microtubules grow or shrink is the rate of GTP hydrolysis,

Answer 55

Have a transmembrane domain that anchors the protein in the cell membrane, and a cytoplasmic domain that mediates attachment to the cytoskeleton. Most cell adhesion molecules belong to one of fourmajor gene families: * Cadherin - Are Calcium-Dependent Cell-Cell Adhesion Molecules * Ig family (immunoglobulin) - The Immunoglobulin Family Contains Many Important Cell Adhesion Molecules * Selectin - Are Carbohydrate-Binding Adhesion Receptors * Integrin - Are Dimeric Receptors for Cell-Cell and Cell-Matrix Adhesion, heterodimers with alpha and beta subunits

Answer 56

Molecules can be either HOMOPHILIC or HETEROPHILIC BINDING. In homophilic adhesion a molecule on one cell surface binds to another identical molecule on the opposide surface. In heterophilic adhesion, the molecule of a different type of molecule on the other cell.

Answer 57

• Tight junction – seals neighboring cells together in an epithelial sheet to prevent leakage of molecules between them -At the cell surface prohibits things from leaving the cell/lipids from moving around * Adherens junction – joins an actin bundle in one cell to a similar bundle in a neighboring cell * Desmosome – joins the intermediate filaments in one cell to those in a neighbor * Gap junction – forms channels that allow small water-soluble molecules, including ions, to pass from cell to cell; communication between cells • Hemidesmosome – anchors intermediate filaments in a cell to the basal lamina -Receptors bind at the base of the cell (looks like half of the desmoosome)

Answer 58

each of those junctions have different type of morphology Zona (Zonula) – belt -Zona Occludens & Adherens Fascia - patches (mainly found in intercalated disk cardiac muscle) - they are not spots but they are not whole belts either - Occludens & Adherens - Isolated ``` Macula - spot - Adherens only > Desmosomes – between cells > Hemidesmosomes – between cells and extracellilar matrix ```

Answer 59

(Aka Tight junction) Physical barrier to passage of molecules between cells (important in areas like the GI tract) It’s Homophilic binding of Claudins to claudins (proteins) and Occludins to occludins (proteins) to form sealing strands Prevents diffusion of membrane proteins in the apex of cells. Plays a role cellular polarity Tight junctions are formed when you have homotypic binding between the claudins to themselves or the occludens to themselves. They form these structures called sealing strands. More sealing strands, the tighter the seal. BLOOD BRAIN BARRIER = TIGHEST ADHERENCE IN BODY

Answer 60

(Spot and Zonula) Cadhedrin mediated (transmembrane glycoprotein) – cadhedrins are homotypically bound Calcium-dependent binding Homophilic binding – two like molecules Cytoskeletal element – Actin filaments to cytoskeleton Potentially contractile may have a role in morphogenesis

Answer 61

embryology of glands – you get an invagination of an epithelial sheet and that can be just a permanent gland or just a deep invagination and nothing happens. But there are sometimes where the epithelial tube pinches off and gives you an independent epithelial tube that has its own independent function. Two common examples: In a developing embryo – this is the neural tube and eventually that neural tube fuses and buds off and gives rise to the spinal cord Another one would be the eye– would be developing corneal epithelium. But the epithelium here has budded off and totally encapsulates this structure through this mechanism.

Answer 62

(Anchoring junctions) Cadhedrin mediated (transmembrane glycoprotein) Calcium-dependent binding Homophilic binding – two like molecules Cytoskeletal element – intermediate filaments attached to an actin-spectrin intracellular plaque Many together impart great tensile strength to epithelial cells So in cells that get a lot of stress there are many of them and in cells that don’t get a lot there are few (generally referring to mechanical stress) There’s lots of stuff here that’s anchoring the intermediate filaments.These plaques are composed of a lot of different proteins. They play a role in anchoring the keratin filaments if it’s an epithelial cell. They’re also inmuscle and in the desmosome in the heart it would be vimentin that would be attaching into these intermediate filaments in cardiac muscle. Why you rarely get blisters unless you do a whole lot of mechanical force and friction to dislodge, and there you’re usually dislodging, if it’s a big blister, you’re dislodging from the basement membrane and not from the individual cells.

Answer 63

it attaches through integrins to LAMININ – which is your extracellular matrix protein that is located in the LAMINA DENSA. LAMINA LUCIDA AND LAMINA DENSA = BASAL LAMINA In the lamina densa we also have TYPE 4 COLLAGEN, which attaches to COLLAGEN TYPE 7. Those then interact with COLLAGEN TYPE 1 and 3 FILAMENTS. These are in what we call a RETICULAR LAYER. Why is that important? If cells separate here, or here, or here, it causes a serious breech of integrity, especially for skin. Your first line of defense from the world is your skin.

Answer 64

It’s not a single phenotype to this disease Epidermolysis bullosa simplex - keratin 5 or keratin 14 -Those keratin intermediate filaments aren’t quite attaching to the desmosomes; you’ll get cells to fall apart. .Junctional epidermolysis bullosa - blister formation within the lamina lucida of the basement membrane. Mutations in laminins or Col IV or A6/B4 integrin . Dystrophic epidermolysis bullosa – Mutation in Collagen VII . this is collagen Type 7. It rips off and you get huge tears down into the connective tissue. Here you still have a little bit of a barrier for healing.

Answer 65

Communication junctions Connexon – mediated = hexagonal array of connexins Homophilic binding Calcium independent Pass ions and small molecules (< 1500d) – such as intracellular messengers Gated channel (Calcium, pH or 2nd Messenger gated) NO ADHESION The main role is COMMUNICATION. protein in the gap junction is a protein called the connexin. You get a hexagonal array of those to make a half of a pore called a connexon. The connexon on one side binds to the connexon on the other side, that’s homotypic or hemophilic binding. It does not require calcium to bind. That forms a water- filled pore that lets small ions and molecules pass in between. example) you have the 6 connexins making half of a connexon. And a connexon binds to the connexon on the other side. There’s a small gap between the cells. That’s why they’re called gap junctions. And molecules can get between these, very important in the function.

Answer 66

Transmembrane Receptors Heterodimers of α and β Chains 18 Alpha and 8 Beta Chains. >24 αβ receptors with varied functions. Receptors are Tissue and Ligand Specific Interact with the Cytoskeleton.

Answer 67

Cell Adhesion to the ECM Cell-Cell Adhesion in some Cells Cell Motility Cell Proliferation Cell Differentiation Apoptosis

Answer 68

Cytoplasmic domain can then hook up with adapter proteins vanculin, tailin, and alpha-actin and they go into the actin cytoskeleton. Then in this case it’s showing how this binding to a domain on fibronectin can pull these two arms together. And fibronectin, which is a big molecule can then bind to collagen fibrils out inside the connective tissue. And so what this depicts is that mechanical forces pulling on collagen, moving things transmit information into the cell. And in the case of the heart it transmits through the integrin and can trigger hypertrophy, which is an increase in cell size, which is how cardiac muscle responds to increased load. If you uncouple that, and there’s no mechanical tension, many cell types go through apoptosis. And then they play an important role in organizing that cytoskeleton. Organizing the cytoskeleton is an important component in whatever the cell function is. And in many cell types that interaction between the ECM and the actin cytoskeleton is how cells maintain their function

Answer 69

Most cell move due to actin polymerization push out this little foot process and that grows by adding actin to polymerizing filament, or bundles of filaments in the perfusion, or lamelapodia or filapodia But in human cells, or mammalian cells, that crawl around during development to function, what happens is that those lamelopodia, or pseudopods, that go out, they push out due to the actin polymerization and when they touch down they make a focal adhesion, , and attach. What also happens is they don’t push out forever, they have to contract and pull up the rear of the cell. So the cells protrude out and they pull up the back end of the cell through usually an actin/myosin interaction. And many times the leave bits and pieces behind, and laying down the track as pioneer cells for all the cells coming behind. That’s important in neurogenesis and for cells to follow cells

Answer 70

Two reasons that cells move: one is a chemo attractant. Why do epithelial cells migrate? They migrate in wound healing. We’ve all skin a shin or something and the scab forms, but what’s happening underneath is epithelium is migrating across and covering that extracellular matrix. Some of it is the chemo attraction, it’s a chemical that causes the cell to go towards it, but lots of it is the extracellular matrix interaction. How did they go those huge distances? Some of it is chemo attraction, but the majority of it is pioneer cells and other cells following.

Answer 71

Program cell death (PCD) – cell suicide – PCD are orchestrated, programmed within each nucleated cell and ready to act, cells reach a point of no return after which they will die PCD PATHWAYS: I. Apoptosis: induced to commit suicide - the primary PCD pathway II. Autophagy: self consume III. Necrosis: death induced by injurious agents (can be programmed cell death – necroptosis or not programmed cell death - ordinary necrosis) IV. Pyroptosis: PCD as part of an inflammatory reaction.

Answer 72

• During development, to clear away unwanted cells (webs between toes) * To maintain tissue homeostasis * To eliminate damaged cells * To prevent cancer * To eliminat infected cell * To have control over the death process to either prevent or induce inflammatory responses Sometimes a cell dies quietly and contains it’s content = APOPTOSIS and AUTOPHAGY Sometimes a cell spills its guts and stimulates and inflammatory response = Necroptosis and pyroptosis and non-PCD necrosis

Answer 73

* Our tissues do not continually expand because there is a balance between cell division and death * 70 Billion cells die by apoptosis in the normal adult every day * T cells that would react against the body are eliminated in the thymus before they enter circulation. * Immune responses lead to a great expansion of antigen-specific T and B cells, which must die off after the infection is controlled. In the vertebrate immune system more than half of many types of nerve cells die soon after they are formed. (Positive selection for those that are chosen and negative by those that are destroyed (like for example, the autoimmune b-cells)

Answer 74

Cell death is the default setting for most cells, which require stimulation to prevent death, such as a cytokine or contact with neighboring cells – keep cells alive. • If a cell is deprived of this stimulus, it undergoes PCD. • Thus, maintaining tissue homeostasis requires constant intracellular communication involving “stay alive” signals received only when the right cell is in the right place. Cancers: • turn off these requirements for extracellular factors and contact, allowing cells to constantly grow indefinitely and spread to improper locations in the body. (so no PCD)

Answer 75

DNA damage caused by chemicals, radiation, etc. induces DNA repair mechanisms, or if repair is impossible, apoptosis. By inducing apoptosis, cells that cannot be saved are induced to die in a way that prevents them from damaging surrounding tissues and to be rapidly cleared away by phagocytic cells such as macrophages. Cancers: Cell death is a normal physiological thing, but it can also be something pathological. You don’t want too much apoptosis because this can cause disease. You also don’t want too little apoptosis – cancers. * Cancer turns off PCD in response to DNA damage and senescence * Suppress immune surveillance: suppress cytotoxic T cell induction of apoptosis -- cancers also turn off those sensing mechanisms in the body where a killer T-cell would normally come along and kill the cancer cell. Cancer therapy: many anti-cancer drugs turn apoptosis back on.

Answer 76

Ischemia: Heart attack, brain injury, stroke Cells die by necrosis or apoptosis Ischemia reperfusion injury: when the oxygen floods back into the area you can have a couple of things go wrong, 1) you can have too much oxygen causing free or reactive oxygen species, and 2) you can have too many white blood cells coming to the area. And these convulse and induce the cell to undergo necroptosis, or apoptosis and you can have a secondary injury. Drugs that block PCD reduce tissue injury in heart attacks and strokes So what they often will do is give you drugs that can reduce programmed cell death, prevent further injury after the incident.

Answer 77

Viruses • Virus infected cells often go into apoptosis as a defense mechanism. • Turning off apoptosis is a major goal of many viruses, which have genes encoding proteins that inhibit this process. Increase or mimic Bcl-2 (stay alive signal), degrade p53 (triggers cell death), stimulate anti-apoptotic kinases (stay alive signals), etc. • Necroptosis can act as a fail-safe mechanism for an infected cell to die, and this mechanism of cell death releases the contents of the cell into the local environment, stimulating an immune response. • If a cell can't undergo apoptosis and it is infected sometimes it will die by necrosis. Well it seems like one of the tricks that a cell has is when the apoptosis pathways have been turned off but it really wants to die it can undergo necroptosis as a backup plan for killing itself. And this can stimulate an immune response which can be good or bad depending on the situation, and then finally pyroptosis will come to at the very end. • Pyroptosis can be induced by HIV-1 when it infects resting T cells, resulting in release of inflammatory mediators.

Answer 78

it entails cell shrinkage, loss of membrane potential, the mitochondria which is important to the whole signaling pathway, and then the nucleus breaks up Most common PCD • Intrinsic and Extrinsic mechanisms of induction • Activation of caspases is a hallmark of apoptosis • Physical changes: • Loss of electrical potential across the inner mitochondrial membrane →release of cytochrome C from mitochondria to cytoplasm • Cell shrinks and condenses • Cytoskeleton collapses • Nuclear envelope disassembles • Nuclear chromatin condenses and fragments • Cell surface blebs • Large cells break into apoptotic bodies Surface of cell becomes chemically altered so that phagocytic cells such as macrophages can engulf them before they spill their contents. Phosphatidylserine moves from inner leaflet of plasma membrane to cell surface. ‘eat me signal’ Apoptotic bodies form and are engulfed. Rapid clearance to prevent an inflammatory reaction -- This is in contrast with necrosis, where the cell’s contents spill out and induce localized inflammatory responses.

Answer 79

your nuclear DNA is digested, and this is also a specific process triggered by caspases But the point is that at the lowest level you have the DNA wrapped around nucleosomes in a repeating pattern. Nucleases are triggered which cleave in between these nucleosomes randomly, not everyone is cleaved so you would have a single size, but at various spaces so what you end up if you run the DNA of an apoptotic cell on a gel it separates into a ladder. Whereas if a cell is dying by necrosis and all hell has broken loose you don’t have that ladder, it just got digested randomly. So this ladder is a hallmark of apoptosis and there are assay (tests) for apoptosis that look for this ladder or look for the ends of the DNA that have been exposed.

Answer 80

natural process where’s there’s always self-digestion to recycle things but it can get out of control and lead to cell death or it can just be part of cell death -- * A normal, non-pathological process by which a cell digests cell components (proteins, organelles) that are dysfunctional or otherwise unnecessary and recycles them. * Cytoplasmic constituents are incorporated into an autophagosome which then fuses with a lysosome for degradation of the components. * Is particularly active after cell injury as the cells tries to save itself and during times of starvation when resources are limiting. * Autophagy can also be a programmed cell death pathway - Autophagic PCD. However, it is not clear if cell death by autophagy is intentional or if the dead cell was trying save itself but failed.

Answer 81

Necrosis is when the membrane integrity is lost and fluids flood into the cell, swell it, and burst it. – * Conventional Necrosis - NOT programmed cell death - is killing of cells by an outside agent, toxins, trauma, loss of blood flow (Ischemia), etc. * The injury to the cell induces cell autolysis (self digestion) • Membranes become permeable, water enters, cells swell and burst, releasing cellular contents into the extracellular space – creates a big mess in the extracellular space * Triggers inflammatory responses * Is usually detrimental to the host blocks clearance of dead cells by phagocytes * Myocardial infarction (heart attack) * Ischemia-reperfusion injury

Answer 82

Pyroptosis • A PCD in which caspase 1 is the initiator. * Induces membrane rupture - cell lysis, release of IL-1β and inflammatory responses * Primarily exists to fight off pathogens * HIV infection of resting T cells in lymphoid tissues induces pyroptosis, while HIV infection of T cells in blood induces apoptosis. * Improper caspase 1 activity is associated with several diseases including myocardial infarction (heart attack), inflammatory bowel disease and endotoxic shock. * Inhibitors of caspase 1 can treat symptoms of these diseases.

Answer 83

Summary of different morphologies, mechanisms and outcomes of the 3 forms of cell death Charact. Apoptosis Pyroptosis Necrosis Morphology Cell lysis NO YES YES Cell swelling NO YES YES Pore formation NO YES YES Membrane blebbing YES NO NO DNA fragmentation YES YES YES Mechanism Caspase 1 NO YES NO Caspase 3 YES NO NO Cytochrome C release YES NO NO Outcome Inflammation NO (anti) YES YES Programmed cell death YES YES NO

Answer 84

Caspase (Cysteine and aspartic acid -ase (enzyme)) Synthesized as inactive pro-caspase precursors that are subsequently cleaved by other caspases. This results in a cascading reaction that is the commitment of a cell to death.

Answer 85

The intrinsic pathway is triggered by the p53 tumor suppressor in response to DNA damage and other types of severe cell stress Conventional anticancer therapies (chemo, radiotherapy) activate this pathway via p53 p53 activates the intrinsic pathway through transcriptional upregulation of pro-apoptotic members of the BCL2 family of proteins such as PUMA and BAX p53 is inactivated by mutations in more than half of human cancers

Answer 86

- Cell surface receptors on cells are part of the TNF family (tumor necrosis factor) o TNF receptor and Fas receptor are death receptors - If TNF is going to induce cell death, it binds the receptor and induces the activation of a death domain on that receptor - Death domain then activates an initiator caspase (caspase 8 or 10), which dimerizes (just like caspase 9 does), and activates through cleavage the executioner caspases Thus, both the intrinsic and extrinsic pathways have initiator caspases that are activated by dimerizing, and activate effector/executioner caspases by cleavage

Answer 87

Cell-intrinsic apoptosis — intrinsic apoptosis (mitochondrial pathway) DNA damage activates p53 - p53 is the guardian of the genome — decides if the cell can survive DNA damage (if the cell can repair itself properly or if the cell is too damaged and a risk to the body) Cell cycle is stopped and cell tries to repair DNA If this fails, p53 activates PUMA and NOXA - These are transcriptionally activated if p53 gives the signal for cell death PUMA and NOXA bind to and inhibit Bcl-2 and BCL-XL, and activate Bax and Bak - Bcl2 family proteins usually inhibit BAX and BAK from forming channels in mitochondria that lead to the release of CytC - Whether or not a cell dies is determined by the balance between Bcl2 holding BAX and BAK in check - When p53 decides the cell will die, it transcriptionally activates PUMA and NOXA, which then suppress the activity of Bcl2 and stimulate the activation of Bax and Bak Bax and Bak want to form channels in the outer mitochondrial membrane to release cytochrome C Guardians normally prevent Bax and Bak from forming channels - Bcl2 is regulated externally to maintain the supply of Bcl2 to a cell and keep Bax and Bak in check Sensors prevent Guardians from inhibiting Bax and Bak → release of cytochrome C → activation of APAF1 → formation of the apoptosome → activation of caspase 9 → activation of caspases 3, 6, 7 → caspase positive feedback cascade → cleavage of cell DNA and lamin → cell destruction by apoptosis - CytC combines with APAF1 protein to form an apoptosome - This structure then triggers the caspase cascade o Caspases are all zymogens — proteins that are inactive and need to be modified (usually cleaved or dimerized) to become active o Apoptosome binds to caspase 9, which is then activated o Once caspase 9 is in active form, it cleaves pro-caspases (3 or 7), creating active caspase 3 or 7 o Caspase 3/7 are the executioner caspases, and are the ones that do the real damage (the killer caspases) Survival signals — cytokines, growth factors, etc.

Answer 88

a lysosomal storage disease, due to a defect in ß-glucocerebrosidase enzyme - This disease demonstrates the relevance of the lysosomal system - Causes large holes/lesions in the alveolar bone, thus affecting roots of teeth - Affects Ashkenazi Jews (1 of 12)

Answer 89

Pinocytosis — “cell drinking” (like pino noir) - Small vesicles <150nm - Two types of pinocytosis > Indiscriminate pinocytosis where the cell invaginates its membrane and sucks up whatever is dissolved in the extracellular fluid. > Receptor-mediated endocytosis (1000x greater concentration than indiscriminate pinocytosis) Phagocytosis — “cell eating” - Large vesicles ≥250nm - Consume microorganisms and large cell debris Neutrophils and macrophages are the professional phagocytic cells in our bodies - Consume bacteria by putting out these pseudopods and then eventually wraps all the way around the bacterium and then brings it inside the cell, and it gets degraded. - Consume RBCs — turnover 1011¬ RBCs/day in our body; important function of macrophages Cells import through endocytosis, export through exocytosis

Answer 90

1) Vesicles bud off from the plasma membrane and fuse with an early endosome 2) Then early endosome matures into late endosome or fuses with existing late endosome 3) Then late endosome matures into lysosome or fuses with existing lysosome 4) Contents within lysosome are then degraded

Answer 91

Classical protein secretion Direct transit across the plasma membrane independently of a cell surface transporter Unconventional secretion pathways by organelle carriers - Secretory lysosome-mediated plasma membrane fusion - Exosomes generated in MVBs and discharged by fusion at the plasma membrane o Organelle between early and late endosome o Characterized by multi-vesicular bodies - Direct fusion of autophagosomes with the plasma membrane o Responsible for breakdown of intracellular organelles - Amphisome-mediated secretion - Both the autophagosome and MVB can fuse to form an amphisome or autolysosome - Direct fusion of autolysosomes with the plasma membrane - Plasma membrane budding or shedding

Answer 92

So the cells continuously pinocytose the plasma membrane. o Macrophages turn over 100% of their plasma membrane every 30 minutes. o Fibroblasts are slower but it’s balanced, so as much membrane added to cell surface by exocytosis is removed by endocytosis. o So two things are happening in a balance, otherwise you’d have too much of this membrane turned over or accumulating.

Answer 93

Phagocytosis — engulfs microorganisms and large cell debris - Makes large vesicles (phagosomes), which fuse with the lysosome Indiscriminate pinocytosis — traps whatever is in the extracellular space Receptor mediated endocytosis — molecules bind to receptors on the cell surface to become engulfed

Answer 94

E.g. cholesterol removing LDL particles - LDL binds to specific LDL receptors on the cell surface - Adaptin 2 then binds the receptor, and clathrin binds adaptin 2 - Then get invagination near this receptor binding - Invagination forms an invaginated pit that then buds off as a vesicle Type of coated vesicle: 1) Clathrin-coated with coating proteins Clathrin + adaptin 1 from the Golgi apparatus destined to the Lysosome (via endosomes) 2) Clathrin-coated with Clathrin + adaptin 2 from the Plasma membrane destined to go to the endosomes 3) COP-coated with COP proteins from the ER Golgi cisterna, Golgi apparatus, and Golgi apparatus destined to go to the Golgi cisterna and ER Clathrin coated vesicles have clathrin cage molecules surrounding the vesicles (making a cage) - Clathrin cage molecules are proteins with a heavy and light chain, which form a triskelion structure - These triskelion structures come together to form a cage around the vesicles

Answer 95

It is an example of Receptor Mediated Endocytosis - Extracellular cargo molecules bind their receptor - Receptor then binds adaptin 2 on the inner surface, which causes clathrin to bind - the receptors all start to aggregate and the membrane starts to invaginate, curve inwards. You now have this vesicle forming, This process starts to cause curvature of the membrane - So the vesicle forms with a clathrin coat - Dynamin wraps around the neck of this vesicle and that cleaves that, and now you have this clathrin-coated vesicle inside the cell - separate from the plasma membrane - Once it is inside the cell some enzyme reactions occur and the adaptin 2 and the clathrin fall off (the vesicle then gets uncoated) - Finally, you are left with a naked transport vesicle which can now fuse with the early endosome. When this vesicle fuses with the early endosome, that lower pH causes the LDL to dissociate from its receptor. Then the receptor buds off from tubular regions of the early endosome and recycles back to the cell surface, while the LDL gets transferred to the lysosome and gets degraded by the hydrolytic enzymes that are in the lysosome and the free cholesterol gets transported across the lysosomal membrane into the cytosol.

Answer 96

Specific receptors for specific macromolecules Associated with clatherin-coated vesicles 1000-fold concentration compared wth simple pinocytosis One example is cholesterol, others are vitamin B12, iron, and viruses (Influenza A) which use this as well, and there are many hormones as well that participate in thismechanism too, and growth factors.

Answer 97

- LDL with cholesterol binds LDL-R - LDLR binds adaptin 2, and clathrin binds adaptin 2, forming a clathrin coated vesicle - Uncoating results in a naked vesicle - Naked vesicle fuses with endosome o Endosome pH is lower than cytosol pH, so acidic environment causes LDL to dissociated from the receptor o Receptor buds off in vesicles and is transported back to the membrane, while the LDL is transported to the lysosome for degradation Antibodies that target PCSK9 (drug) prevent the rerouting and degradation of LDL receptors, thereby reducing plasma concentrations of LDL. EGF, epidermal growth factor

Answer 98

1. RECYCLING: like with LDL and its receptor, it comes in the cell with these transport vesicles, fuses with the early endosome and the LDL goes to the lysosome but the receptor can recycle back to the cell surface. 2. TRANSCYTOSIS – You can have vesicles that bud off from the early endosome and take whatever they are carrying across the cell and get expelled and then can be transferred to an adjacent cell. 3. DEGRADATION - you can then have all contents going through degradation in the lysosome.

Answer 99

Early endosome pH ~6.5 Early/late endosome pH ~4.5 LDL dissociates most easily from its receptor around pH 6 — thus dissociates in the endosome Iron is the next easiest - Dissociates from the transferrin receptor - Transferrin is the transport protein that carries iron - Iron binds the transferrin receptor at neutral pH - Once internalized, iron dissociates immediately - The transferrin receptor returns to the plasma membrane, and transferrin returns to the plasma Next, M6P (mannose-6-phosphate) dissociates from its receptor in the late endosome - M6P-R can bud off vesicles and be transported to the trans golgi network Last, EGF — epidermal growth factor - EGF binds very tightly to its receptor, so it needs a very low pH to dissociate - Thus, it is degraded in the lysosome along with its receptor - This is a way for the cell to control growth factor levels

Answer 100

The acid hydrolases are actually made in the cis-Golgi. They have mannose groups added to them, which are then phosphorylated They then go through the Golgi and bud off in clathrin-coated vesicles with a different adaptin than used in receptor-mediated endocytosis. The proteins associated with mannose 6-phosphate receptor. They are transported in these clathrin-coated vesicles. The clathrin dissociates and then these transport vesicles fuse with the late endosome where the phosphates are removed Now the enzyme gets transported to the lysosome and that forms the lysosome.

Answer 101

Occurs when a cell is dead, or the cell is under starvation and desperately needs amino acids for metabolism - First, a membrane made by the ER is formed and starts to surround the organelle, forming an early autophagic body The early autophagic body then fuses with a lysosome, and lysosomal hydrolases break down the organelle - This results in a tubular vesicular body that is broken down more into the late autophagic body, and then a residual body Types of autophagy Macroautophagy — membrane comes from ER and forms around fairly large organelles - Mitophagy — when autophagy is around mitochondria Autophagosome — autophagy around microorganisms or multiple organelles Multivesicular bodies can also undergo endosomal microautophagy Or, can just have a chaperone that takes a protein to the lysosome These all fuse with the lysosome where degradation occurs

Answer 102

Phagocytosis — form a large vesicle around microorganisms/large cell debris to form a phagosome that fuses with the lysosome, where contents are degraded Pinocytosis - indiscriminate — captures extracellular fluid - Receptor mediated — naked vesicles fuse with the early endosome, and then either fuse with a late endosome or mature into a late endosome, which then fuses with a lysosome or matures into a lysosome, where degradation of contents occurs Autophagy — ER membrane forms around cargo (e.g. mitochondria), forming an autophagosome that then fuses with the lysosome, where degradation occurs

Answer 103

* Most prominent organelle in a eukaryotic cell * Nuclear envelope, which has 2 concentric membranes they are continous with the ER * Contains chromosomes, nuclear DNA • 1-5 nucleoli per nucleus (cancer cells can sometimes contain more) – the site where rRNA gets synthesized -- so it is where 5 chromosomes that’s got the genes for ribosomal RNA are in the DNA, they cluster there, and that’s why you can see the nucleoli.

Answer 104

The nuclear envelope of the nucleus dissolves at mitosis -As the chrosomes condense, more of the nuclear envelope dissolves and you end up with no nuclear envelope or membrane and the cell can now now undergo mitosis It is thought that the nuclear envelope originated from invagination of the plasma membrane This membrane is made up of inner and outer membranes (outer mem. continuous with ER) -nuclear lamina attach to inner membrane You also have nuclear pores - To get things in and out of the nucleus you have nuclear pores. INTO NUCLEUS: newly made proteins from ctyosol OUT NUCLEUS: ribosomal subunits which are assembled in nucleus as well as polA tailed and 5'capped mRNA

Answer 105

To get proteins in and out of the nucleus the proteins have specific sequences. To get into the nucleus cells have nucleus-localization sequence and you can see they are very basic amino acids, positively-charged amino acids. Export from the nucleus is a different sequence with a number of hydrophobic amino acids.

Answer 106

• Have a very complex arrangement. • You can see on the cytosol the nuclear pore has cytosolic fibrils so it is made up of many proteins and a whole set of proteins in the actual pore crossing the nuclear envelope. • On the inside of the nucleus is the nuclear basket (circle at the pore's base that is thought to control Import and export of molecules in/outside nucleus) • When the proteins translocate into the nucleus they have the basic nuclear- localization signal and bind to and import to a receptor. The receptor binds to the cytosolic fibrils and is able to cross through the meshwork of fibrils through the basket and into the nucleus.

Answer 107

STEPS: Protein with nuclear-localization signal binds to receptor crosses the nuclear pore into the nucleus and in the nucleus Ran-GTP binding protein binds to the receptor. That causes dissociation of the nuclear protein from the receptor protein. Ran-GTP is bound to this receptor protein and that translocates out of the cell through a nuclear pore In the cytosol Ran-GTP has a GTPase activity and can hydrolyze its own GTP and give off a phosphate and is now Ran-GDP and can assist in the process again.

Answer 108

These are proteins in the intermediate filament family. and are associated with the inner nuclear membrane. Upon initiation of mitosis, you get phosphorylation of nulcear pore proteins and lamins >> and you get dissolution of the nuclear envelope >> after mitosis occurs they get dephosphorylated and start to reform lamins with some of the membrane proteins and lipids >>> the new nucleus starts to form and you have the nuclear envelope.

Answer 109

Disease associated with mutation of just one lamin, nuclear lamin protein, lamin A. • With this mutation they age really fast, they lose their hair, have osteoporosis, heart disease. • In patients with progeria, the nuclear lamina does not completely form. The heterochromatin is not associated with the nuclear lamina and starts to be transcribed when it shouldn’t be. • It seems that the more laminae you have, the more ridged the cell is. That seems to occur in bone. If you have less lamin A, you have less ridged nucleus or nuclear membrane. This ends up with the cell becoming really adipose. And that may be why with these patients they particularly have osteoporosis and problems with their craniofacial development and teeth development.

Answer 110

* It is made up of three domains, what’s called fibrillar centers of cores, dense, fibrillar region, and granular component or region. * The nucleolus also dissolves during mitosis, although there is still some of it seen, which is the nucleolar organizing region. • It is where all 5 chromosomes all aggregate and where the ribosomal RNA genes are associated there together. The nucleolus is the site of ribosomal RNA synthesis. * Fibrillar cores or centers are loaded with RNA polymerase I, which is responsible for transcribing ribosomal RNA. * The granular regions have a lot of the ribosomes and there are some modifications of the RNA.

Answer 111

The first ribosomal RNA is 45S ribosomal RNA. The 45S rRNA is processed to 30S rRNA and 18S rRNA. 30S is then further processed to 28S rRNA and 5.8S rRNA. Non-nucleolar 5S rRNA form the ribosome particles together with a lot of protein OUTSIDE to creat final form of ribosome used for translation OUT OF THE NUCLEUS mRNA made in the nucleus by the euchromatin and is transported out of the nuclear pores with nuclear transport receptors in Nucleolus you’re making rRNA. This is processed with proteins associated with the appropriate rRNA and you get the ribosomes, small and the large subunits. These also have to be transported out of the nuclear pore and into the cytoplasm and do so WITHOUT UNFOLDING and traveling by squeezing through pore.

Answer 112

Labile cells, for example skin, and oral mucosa. multiply constantly. Stable cells. they don’t normally divide, but when there is a need, they may divide. Rentry into the cell cycle. ex. liver cells Permanent cells they completely lose the prolifation and cell dividing capacity like neurons, and cardiac myocytes, these differentiated cells are incapable of dividing. CANCER, EXCESS SKIN - may need bariatric surgery = are both a result of cell proliferation Cyclosporine-induced gingival overgrowth – due to excessive proliferation in the gingiva of teeth, side affect of immunosupressent medication Leukemia results from the proliferation of a clone of abnormal hematopoietic cells with impaired differentiation, regulation, and programmed cell death (apoptosis).

Answer 113

Normal cells are not immortal and have a finite division numbers Telomere erosion- the Hayflick limit - roughly 200 base pairs lost per turn at the telomere until it is so short it signals cell stop TELOMERE EROSION AND ADDITION • Telomere length prevents age-related decline • Telomere- repetitive DNA that caps the end of each chromosome - With each cell division, some of the telomere is lost, but Telomerase- telomere terminal transferase, is an enzyme that elongates chromosomes (2009 Nobel Prize in Physiology or Medicine for Telomere Research)

Answer 114

• G1- Gap between M and S-phase increase in cell size RNA and protein synthesis • S- DNA Synthesis 2N to 4N DNA CONTENT duplication of centrosomes (centrioles) • G2- Gap between S and M Synthesis of proteins (like tubulin for mitotic spindles) Synthesis of maturation promoting factor (MPF) for inducing mitosis THE PHASES ABOVE ARE PART OF INTERPHASE • M- Mitotic phase - Mitosis and cytokinesis

Answer 115

To ensure that they replicate all their DNA and organelles, and divide in an orderly manner, eukaryotic cells possess a complex network of regulatory proteins known as Cell-cycle control system Guarantee a set sequence that each process has be completed before the next one begins Regulated at certain critical points of the cycle by feedback from the process currently been performed G0 PHASE- which is the cell is resting/dormant, and most of the stable cells are in this stage, the stable cells they don’t usually proliferate but they can come back when there is a need, the liver cells after hepatectomy there is a need to proliferate, so they go from G0 to cell cycle. CHECK POINTS G1 aka restriction point – between G1 and S1 phase "is the environment favorable?" G2 check point – between G2 and M phase "Is all the DNA replicated? Is there any damage? Has the damage been fixed?" M check point "Are all the chromosomes properly attached to mitotic spindle?" There are others but G1 and G2 are the most important

Answer 116

o Ligand receptor binding - Ligand binds to this receptor, then the receptor will make certain conformation change and recruit certain downstream signaling > Ex: growth factors o Enzymes - they can make modification, for example kinase or phosphorylase -- can be on and off signals o Transcription factor – DNA binding Proteins that bind to DNA regulate expression Rb Protein - tumor suppressor -binds E2F-E2F cannot bind DNA ans transcription is blocked - cell growth phosphorylates Rb and releases E2F; E2F binds DNA and turns on transcription advancing cell cycle o Signal transduction cascade these signal transduction cascade, they are small signals that triggers a network of signals - apoptosis

Answer 117

factors that guide the cells entering from one phase into the next. CYCLIN concentration varies through the cell cycle, so it rises, and goes down through the cell cycle, and during each phase of the cell cycle, different types of cyclins are involved. The cyclins don’t work alone, they need to cooperate with another group of proteins called cyclin dependant protein kinases, so it is a kinase, and the function of kinase is to put a phosphate, they are called Cdks. • Cyclin concentration varies through cell cycle – No enzymatic activity by itself • Cyclin dependent protein kinase (Cdk) concentration remains constant • Cyclin-cdk is active – Cyclin-dependent protein kinase G1-Cdk = Cyclin D (partnered with Cdk4, Cdk 6) G1/S = cyclin E (partnered with Cdk2) S-Cdk = cyclin A (Cdk 2) M-Cdk = cyclin B (Cdk 1)

Answer 118

there is an inhibitory phosphate site and there is an active phosphate site. So you put on phosphate here it turns on, is active, but also you need phosphatase to remove this inhibitory phosphate to eventually have an active complex.

Answer 119

* Control system monitors status and stops cell cycle progression if errors/problems * Phosphorylation-dephosphorylation of cyclin-Cdks * CDK inhibitor proteins

Answer 120

are a group of inhibitors that can block the activity of cyclin and Cdk complex, so this is why it is called inhibitors. P16 - Tumor Suppressor - often lost in oral/head and neck SCC loss of 9p21 (locus) - common in oral cancer results in activation of cyclin d-cdks phosphorylation of Rb amp of 11q13-CCND1 - common oral cancers P21 blocks cyclin-cdk blocks G1 to S and S Phase Progression P53 - Guardian of the genome Tumor Suprpressor Gene - commonly lost in cancers G1 to S Transition: checkpoint to stop dividing or commit apoptosis Activates Cdk Inhibitor Protein (p21) n DNA damage and p53 arrrest in G1 activate p53 transcript. factor or p21 - p21 inhibits cyclin-cdk cell repairs or dies p14 , p15, p19, p27, p57, cycle control failures lead to uncontrolled proliferation

Answer 121

``` Human papillomavirus (HPV) • it’s a known risk factor to oral cancer ``` • Infectious disease, is sexually transmitted • This is well studied and the reason why HPV leads to oral cancer is because it controls cell cycle, and the molecules this virus attacks include P53, Rb and P16. > One of the mechanisms that this HPV targets P53, to trick the cells to continue proliferation. So the virus just mimics the proteins that can bind with P53, and once it binds to P53 it can make the cells mistake. Makes it think that p53 is ready to get degraded, so it recruits the ubiquitination and then guides the p53 to proteasome and once p53 is degraded there will be no guardians in our genome to sense/detect DNA damage, so this is how smart the HPV virus can be to utilize this system and to trick the cells to make uncontrolled proliferation

Answer 122

* c-Myc is called an oncogene, so the more you have, the more the cells have a tendancy to proliferate * c-Myc transcripts for a transcription factor • A mutated version of Myc is found in many cancers, which causes Myc to be constitutively (persistently) expressed. • c-Myc drives cell proliferation through upregulation of cyclins, downregulation of p21

Answer 123

Prophase – Chromosomes condense – Centrosomes begin to separate and spindle assembles Prometaphase – Breakdown of nuclear envelope – Chromosomes attach to spindle via kinetochores Metaphase – Chromosomes aligned in equator (metaphase plate) – Homologous chromosomes line up independently – Sister chromatids of chromosome attached to spindle on opposite poles Anaphase – Sister chromatids separate and are pulled to opposite poles – Anaphase-promoting complex (APC)-triggers chromatid separation; degrades M-cyclins ``` Telophase – Chromosomes arrive at poles – Nuclear envelope reassembles – Chromosomes decondense – Division of cytoplasm with formation of contractile ring ``` Cytokinesis – cell divided in half by contractile ring

Answer 124

They all go through mitotic proliferation, so the germ-line cells from the zygote proliferate. • In female, these cells will immediately go into meiosis. >> Then they go into arrest, so they stop there, and some of the cells during this phase will go through apoptosis >> Some cells will be circled with some somatic cells >> they are here to protect and support the germ-line cells. (this is called a follicle. This is right after birth) >> after puberty, each month, usually one, occasionally multiple, eggs will become mature and ovulation happens >> this iis how germ-line cells develop into germ cells in females. • The males, they also start with mitotic proliferation, but they immediately go into mitotic arrest, so they stop in the cell cycle >>> after birth, they start to proliferate again. Upon puberty, they start the meiosis process Take home message: they both have zygote proliferation in the beginning but immediately in females the germ-line cells enter meiosis and meiotic arrest. In males, it is mitotic arrest then after birth is mitotic proliferation and upon puberty is start of meiosis.

Answer 125

All offspring from asexual reproduction are identical to the parents and identical to each other. They are simple and uniform. The advantage of sexual reproduction is mixing our genome. Each time the germ-line cells from parents mix. The offspring have much more diversity.

Answer 126

3 main competitive advantages * Reshuffling the genes to help a species survive in an unpredictably variable environment * Elimination of deleterious mutant alleles and help to prevent them from accumulating in the population * Selection of survivals from parasites

Answer 127

* Strong analogy to mitosis * Increases genetic variation * Human male- 24 days and happens after puberty * Human female- decades – whole proccess starts before birth and continues for decades until individualis depeleted of eggs • One doubling of DNA plus two cell divisions – Meiosis 1 – Meiosis 2 Haploid egg + haplois sperm = diploid zygote (fertilization) • This goes through mitosis to make more somatic cells and a small portion remain as germ-line cells. • 2N → 4N → 2N → N • Every gamete is genetically unique • Chromosomes, maternal vs paternal, randomly sorted due to independent segregation • Human: 23 pairs chromosomes lead to 2n genetically distinct gametes – 8.4 x 106 different gametes

Answer 128

Prophase 1 • chromatin condenses and there is a homologue chromatid pair. This happens in early prophase to mid prophase. Formation of Bivalent - structure formed when a duplicated chromosome pairs with its homolog at the beginning of meiosis; contains four sister chromatids. Metaphase 1 • The homologue pairs line in the middle. • Recall that in mitosis chromosomes line up at the metaphase plate independently WHEREAS in meiosis homologous chromsomes are paired at the metaphase plate – they only need to make sure daughter cell get one copy from each parent Anaphase and Telophase I • They are paired and there is separation and go through the first division, meiosis 1. MEIOSIS 2 – Prophase II • cells are still diploid because only one duplication and one division happened. Metaphase 2 and Anaphase 2 * The chromosomes always align in the middle. You can see here it is called equatorial plate. They line here in metaphase 2. * In anaphase 2, they start to separate. As you can see the chromosomes some of them are red with a little bit of blue. Telophase II • And then eventually in the second division, they become 4 haploid cells. In meiosis 2, the cohesin protein that used to glue the chromatids together is degraded and the sister chromatids can be separated in the second division and make haploid cells. So in this stage, in the females’ development of germ cells, there is meiotic arrest and the chromosomes are already paired. During the long rest, when the female reaches age 40-45, the glue between the chromatids may be less and not as strong, and this is one of the reasons why abnormal chromosome children happen more frequently in females who gave birth after 45 years of age.

Answer 129

• Exchange of DNA between maternal and paternal chromosomes- Crossing- Over – Increases genetic variation • Occurs in prophase 1***** • Recombination – Synaptonemal complex-protein machinery for recombination – Disassembled by end of prophase • Chiasma (χ) – Connection between two chromatids from crossing-over > Each gene on chromosome we have a way to locate. It is like zooming in an address. First we have a short arm we call “p” and a long arm we call “q” and in between these two is a place where centrosome stays and we have different band, a sub-band

Answer 130

• Bivalent chromosome >> chiasma >> Then they exchange certain genes and cross over. * Several crossing over can happen in one bivalent. * This crossing over structure has a special term, chiasmata. • The chiasmata is a physical structure that links the homologue chromosome during meiosis 1. The bivalent starts at prophase 1 and continues to metaphase 1 and during that phase, crossing over happens so that is when we can observe chiasmata under the microscope. GENES THAT ARE A GREATER DISTANCE AWAY FROM EACHOTHER ARE LESS LIKELY TO CROSS OVER THAN GENES THAT ARE CLOSER APART – less roam to cross over

Answer 131

• Nondisjunction- homologous chromosomes fail to separate – Gamete lacks a specific chromosome – Gamete has an extra chromosome • 10% meiosis in human oocytes have nondisjunction – Leads to aneuploidy – Increases with age Nondisjunction. • In normal meiosis you have two chromsoms in a cell, but in abnormal you end up with one with three and the other with one. And if normal gametes are fertilized with a gamete with extra or less, the individual will have extra or less chromosomes. • Trisomy chromosome 21: an example of non disjunction Different disease is monosomy, missing a chromosome. This is also called Turner Syndrome. (One X chromosome)

Answer 132

- 3.2x109 base pairs of DNA x 2 of each chromosome = 6.4x109 base pairs total in each cell o = 2 meters in length - 2 meters x 50 million million (trillion) cells in the human body = 100 billion km long! o Distance from the sun to Pluto is 6 billion km (average) Chromosomes = - 10,000:1 packing ratio DNA must be packed down to fit into the cell - DNA is organized first into nucleosomes (eukaryotes only) - The core of nucleosomes consists of 8 histone proteins (Positively charged (basic) to bind the negatively charged DNA (acidic) — 11nm fibers) - The DNA strand (147bp) wraps around the histone core, and then the nucleosomes assemble into helical coils called 30nm fibers - Nucleosome = beads on a string o Nucleosomes are connected by linker proteins (the string) o Linker DNA Is 10-90bp - ****The 11nm fiber is the most open form of the histone organization**** - Histone core is an octamer of 8 proteins plus DNA o H2A, H2B, H3, H4 (2 of each of these) - Histone H1 is a linker protein added to generate the 30nm fiber o Needed to pack down from the 11nm fiber to the 30nm fiber - Chromatin = DNA + proteins that produce chromosomes

Answer 133

structural, non-histone proteins that help bind together the higher order structure of chromosomes To form the chromosome, we then have to add scaffold proteins (Something for the DNA to hang onto)

Answer 134

Karyotyping Humans have 46 chromosomes — 22 pairs of autosomes, and 2 sex chromosomes (XX in females, XY in males) - Both autosomes are transcriptionally active, so sets/copies of genes are “on” on both chromosomes, with the notable exception of the Xi chromosome in females - The Y gene is much smaller than the X chromosome Karyotype = the full set of 46 human chromosomes G (Giemsa) banding of metaphase chromosomes is used to identify individual chromosomes, to assess the karyotype and identify genetic abnormalities - Banding pattern enables us to identify each chromosome o Dark bands are AT-rich regions We have 2 copies of each chromosome, and both are active - Thus we can compensate for a mutation in one gene (unless it’s a dominant gene)

Answer 135

Chromosome painting via FISH provides a way to specifically identify chromosomal regions DNA oligos can hybridize to target DNA, so we can tag the DNA with different colors - this allows us to identify chromosomes and their defects (more easily than karyotyping in just black and white) - More complex than Giemsa

Answer 136

1. Provide a way of organizing genes so that their expression can be regulated 2. Provide structures to assist DNA replication and partitioning during cell division - a. Origin of DNA replication (not strictly part of the chromosome, but is on the DNA) - b. Centromere for mitotic segregation  - c. Telomere allows replication of linear DNA without loss of ends 3. Allow occasional transfers of genetic information and duplications of genes — evolution vs. genetic diseases Although the genes in related species will be very similar, their arrangement on chromosomes and even the organization of the chromosomes themselves has a lot of plasticity - Two closely related species may have radically different sets of chromosomes - Thus karyotyping is not everything

Answer 137

p arm — short arm (petite) q arm — long arm Centromeres - Point of contact between sister chromatids - Site of kinetochore formation — where the spindle fibers connect to the sister chromatids o Composed of 2-4mb (mega-bases) of multiple repeats of simple sequences  171bp repeats, “alphoid DNA” which is bound by nucleosomes, or other types of highly repetitive DNA • Together called “satellite DNA” o Centromeric chromatin is in the form of constitutive heterochromatin  It is always heterochromatin and has little or no expressed genes on it Kinetochore assembles at centromeres and associates with mitotic spindles during mitosis and meiosis - Microtubule organizing center (MTOC) is the anchor that is used to pull the two cells apart

Answer 138

Found at each end of chromosomes Protect the ends of the chromosome from degradation by cellular nucleases Telomeres contain repetitive oligomers of high GC content for a few thousand bp: TTAGGG This must be replaced each cell division by telomerase in germ cells and stem cells, or shortening of the chromosome will eventually make the cell non-viable - shortening is a signal for the cell to go into senescence and apoptosis - Our cells have a finite number of divisions Mutant mice which cannot repair telomeres can only reproduce a few generations - Dolly, the first cloned sheep, had problems with this too Telomerase, a riboprotein (RNA-protein complex) uses RNA as a primer for DNA synthesis (thus it’s a reverse transcriptase) to extend the telomere and restore its length

Answer 139

Telomerase preserves chromosome ends - Telomerase is a reverse transcriptase — DNA synthesis using an RNA template Telomerase has an RNA template that ligates with a few bps to the end of the DNA, and is used to extend the DNA - Extend the RNA far enough so that when you back fill, the bit that’s lost is the bit that’s been added prior - Thus in total, you don’t lose anything, and your DNA is preserved Telomerase is ACTIVE in germ cells and many stem cells - Need stem cells to divide over and over again in order to develop a large repertoire of cells Telomerase is INACTIVE in cells that divide a limited number of times (most cells in the body), and in cells that never need to replicate again — post-mitotic cells But telomerase is turned back on again in cancers — thus telomerase is a possible target of treatment In chronic HIV infection, CD8+ T lymphocytes over-replicate and lose telomere length, contributing to immune dysfunction - If you’re constantly making killer T cells, they don’t have telomerase, so they keep getting shorter and shorter - Thus you can tell how well or bad off someone is by looking at the length of their telomeres

Answer 140

the inactive X chromosome which is highly methylated, condensed and located away from active transcription areas of the nucleus. • In females, one X in each cell is randomly turned off during the ~1000 cell stage of development, which keeps the number of expressed X-linked genes equal for males and females (Dosage compensation) Calico cats are not a separate species or breed. They are a variant where a gene for coat color is on the X chromosomes, so XY males have one gene and so are uniform in color, while the XX females are mosaics based on which X is active in which part of their coat.

Answer 141

Almost all cells have exactly the same DNA, but different cell types look and function very differently. This is result of expression of different sets of proteins Protein expression is controlled primarily by Regulation of transcription of mRNA by RNA polymerase II (pol II) Protein expression is also regulated by splicing, translation and posttranslational modifications (phosphorylation, degradation, etc.) *****Gene regulation determines cell fate during development/regulates how a cell functions and responds to the environment.*****

Answer 142

This is regulated by: • The sequence of the DNA (genetics) to which transcription factors bind • Which transcription factors are expressed in a cell • Modifications to DNA and to histones (epigenetics) • Whether the DNA is in a heterochromatin or euchromatin configuration All of these factors interact with each other to determine which genes are transcribed and which are not.

Answer 143

* Genetics: DNA sequence to which transcription factors bind. * Epigenetics: Chemical modifications to DNA or to histones that alter transcription rate but don’t modify the DNA sequence.

Answer 144

``` Euchromatin (light staining) Transcriptionally available 11 nM fibers Not complexed to histone H1 • Histones acetylated • Histones methylated in ways that favor transcription • DNA not methylated ``` ``` Heterochromatin (dark staining) Transcriptionally repressed condensed 30 nM strands Complexed to histone H1 • Histones deacetylated • Histones methylated in ways that repress transcription • DNA methylated ```

Answer 145

The location of a gene in the nucleus can change to a location in the nucleus that is more favorable to transcription. Heterochromatin – condensed chromatin • Usually at the periphery • Mostly transcriptionally inert Constitutive chromatin – never converted into euchromatin • Centromeres and telomeres, most of the Y chromosomes • Doesn’t have genes • Always compacted so no transcription • Contains several satellite sequences – repeat sequences Facultative heterochromatin – interconverted between euchromatin and heterochromatin

Answer 146

The differentiation of cells is the result of switching on of cell type-specific genes to make proteins needed in that cell type. The switches for differentiation are usually transcription factors. MyoD turns on many other genes that make muscle-specific proteins The response of a cell to environmental stimuli is controlled in part by pathways from the outside of the cell down to the level of transcriptional regulation

Answer 147

Initiation of transcription Many promoters have a TATA (or TATAA) box, this is what we focus on here. TATA binding protein (TBP) bind the TATA box. TFIID/TBP = A complex of proteins including TBP. 1. TFIID/TBP binds TATA 2. TFIIH binds 3. pol II is recruited 1-3 = Transcription Pre-initiation Complex (also includes other proteins not shown) 4. Mediator complex binds to the tail of pol II blocks transcription starting 5. TFIIH phosphorylates pol II on its tail this activates pol II, freeing it from Mediator, and it now can start transcription at a basal level. —> Other transcription factors, some general and some gene-specific, bind to DNA sequences upstream. Some are in the enhancer. Gene-specific transcription factors increase transcription above the basal level and determine overall transcription rate. Each cell type and each state (replicating or not, activated or not, stressed or not, etc.) expresses different set of transcription factors at varying levels. DNA folding allows transcription factors bound to enhancers to interact with the promoter

Answer 148

Example: MyoD: the master regulator for differentiation into muscle cells  turns on many other genes that make muscle-specific proteins  specialization into a muscle cell o MyoD binds to muscle-specific promoters to turn on muscle-specific genes  differentiation into a mature muscle cell (and stops replicating) o MyoD must be made all the time  it is involved in a feedback mechanism that allows for myoD to be produced and expressed all the time. HOW?  Induces p21  shuts down cell replication, as well as represses a kinase (CDK, cyclin dependent kinase) that would phosphorylate myoD and inhibit it (“repressing a repressor”) • The RESPONSE of a cell to environmental stimuli is controlled in part by pathways from the outside of the cell down to the level of transcriptional regulation Thus MyoD sustains its own expression in a positive feedback loop to maintain muscle cell differentiation

Answer 149

Epigenetics (“epi,” meaning “on” “upon” or “above”); chemical changes in the DNA or histones that regulate gene expression without altering of the DNA sequence * While the DNA sequence remains unchanged, how it will be read will be changed by epigenetics. * Epigenetic modifications are HERITABLE as cells divide, the daughter cells inherit these modifications • If epigenetic changes occur early in an organism’s life, they are not inherited

Answer 150

Histone modifications: are reversible and the result of a dynamic interplay between enzymes that install the modifications and those which remove them. Acetylation of the histone TAILS acetylation is favorable to transcription (remember: acetylation & activation) opens up nucleosome into free chromatin * Performed by histone acetyltransferases (HATs) that work in tandem with histone methylation * Removal of histone acetyl groups: histone deacetylases (HDACs) which repress transcription * HATs and HDACs are regulated RAPIDLY and their concentration regulates histone acetylation o NOTE: LYSINE is in high concentration on histone tails = target for methylation and acetylations changes the histone’s charge from positive to neutral, allowing their dissociation from DNA, the opening up of chromatin 30 nm fibers >>> 10 nm fibers, and transcription to begin Methylation  favorable or repressive to transcription, depending on the amino acid that is methylated (histone code) * Methylation of histones can involve single, double, or triple methyl groups * Performed by histone methyltransferases (HMT); histone methyl groups are removed by histone demethylases . • Sometimes, histone methylation contributes to chromatin condensation generate heterochromatin/inhibit transcription • Other times, histone methylation contributes to maintain euchromatin (open chromatin) and recruit transcription factors. Histone methylation provides longer term regulation than histone acetylation

Answer 151

Most common on cytosine units that are followed by a GCG or CpG Most stable of epigenetic modifications Is found throughout the genome and is essential for viability; 60-80% of CpG are methylated in the human genome (regions of genome are always repressed, such as at retrotransposons) Some promoters are highly methylated Rarely activated, whereas others are never methylated and thus are deemed metabolically active. • CpG Islands; regions in promoters where there is an unusually high frequency of CGs DNA methylation is highly repressive for transcription; characteristic of heterochromatin formation; inhibits binding of transcription factors Catalyzed by DNA methyltransferase CpG methylation is recognized by proteins that recruit histone deacetylases and other chromatin remodeling factors turn euchromatin into heterochromatin  repress gene expression. CpG methylation patterns are maintained during cell replication Tumor suppressor genes are turned off by DNA methylation DO NOT CONFUSE HISTONE METHYLATION WITH DNA METHYLATION DNA METHYLATION INHIBITS TRANSCRIPTION, BUT IT IS ALSO EPIGENETIC AND CAN BE INHERITED BY DAUGHTER CELLS

Answer 152

The HIV-1 promoter is repressed by nucleosomes and needs the HAT p300 to be recruited to the promoter to acetylate them Infected T cell is activated by an antigen this increases expression of transcription factors and HATs Nucleosomes that are in contact with DNA near promoters act as a physical barrier to pol II binding and moving along the DNA. Transcription factors bind the promoter and recruit the HAT p300 p300 banishes HDACs and recruits HATs that acetylate histones. Histone acetylation weakens the binding of nucleosomes to DNA and more transcription factors can bind and pol II can travel past the location of the nucleosomes.

Answer 153

In embryonic development the repression of gene expression must be removed so that zygotes regain the potential to express all tissue-specific genes - totipotency. This reset allows fertilized eggs to differentiate into all the tissues in the body There are two waves of CpG methylation removal: 1. During gametogenesis - formation of sperm and egg 2. Soon after fertilization during early embryogenesis

Answer 154

non-gene products are NON-CODING for proteins • Some have function (ex: centromeres, telomeres)  structural purposes • Some are non-functional (“junk DNA” and other immaterial, noncoding sequences; not called “junk” DNA anymore because they actually serve a purpose)

Answer 155

A gene determines a heritable trait - a “unit of selection” in evolution. A gene encodes a functional product - either protein or RNA. Variations in DNA sequence among copies of a gene are called alleles. The sequence of all the DNA is the genome. A gene is transcribed, but the modern definition includes all the DNA elements that go into regulating transcription - both the coding and the non-coding elements - promoters, enhancers, introns, etc. These elements are all subject to evolution and selection. A gene is found on DNA.

Answer 156

Protein Synthesis >>> Transcription (in the Nucleus) and Translation (at the ribosome between assembled subunits); in Eukaryotes, transcription and translation are physically separated from one another. • Protein Coding Genes >> first make messenger RNA (mRNA) via transcription by means of RNA polymerase II; synthesis of mRNA strand occurs in the 5’ >> 3’ direction. o Protein coding genes consist of:  Promoter: upstream from the initiation site of transcription; binds to the RNA polymerase II with the help of transcription factors.  Enhancers: can be far away from the promoter region  Coding regions: “ORFs” are OPEN READING FRAMES, AKA the CODING REGIONS; Coding regions are within EXONS and are not spliced out during alternative splicing  Introns: noncoding regions; oftentimes spliced out.  PolyA signals o Mature mRNA transcripts leave the nucleus and are brought to the ribosome, which subsequently assembles (small and large ribosomal subunits)  mRNA  tRNA  primary protein structure (amino acid sequence)

Answer 157

a simple transcription unit without the need for splicing; the gene is transcribed by RNA polymerase II and does not need to be spliced.

Answer 158

What most of our cells are have multiple exons (regions that code for subunits or bits of the final protein) and introns are between them (to be spliced out; splicing = internal processing). A typical complex gene has multiple exons and different splicing patterns provide variant mRNA transcripts that encode different proteins known as PROTEIN ISOFORMS • Ex: muscular proteins, such as fibroblasts and striated muscles that are all products of differential splicing ( and subsequent cleavage and polyadenylation) of ONE complex gene, alpha-tropomyosin. o Different gene structures  different functions

Answer 159

• Solitary Genes: genes that exist as ONLY 1 COPY  25-50% of all genes are single copy o You only need a certain amount of that gene to be expressed; one gene is sufficient for the desired amount of protein produced/ proper functioning. • Tandem Arrays: A series of copies of a gene arranged in tandem along a chromosome; exist for genes that code for products such as rRNA, for large quantities of rRNA are necessary for biological function. o DUPLICATED GENE o Tandem arrays are series of METABOLICALLY ACTIVE genes; still contain their own promoters, enhancers, etc. o Spacers separate the genes within a cluster to insulate them and prevent “read through” by polymerase enzymes (if there were no insulators, then there would be no separation between the gene copies and there could be more transcription than desired).

Answer 160

• Gene Familes: multiple copies of genes that are similar, but not identical; have >1 member. Reside on the same chromosome. o Similar to TANDEM ARRAYS  you still have duplication/crossovers of one gene, but evolution results in modified genes from that same ancestral line o Modified genes will have different functions. o There can also be non-functional family members within a gene family  PSEUDOGENES  Pseudogenes are nonfunctional genes that have been mutated over time; originated from functional genes but eventually usually nothing at all. “Ghosts of previous genes.” o Beta-globin: a part of the HEMOGLOBIN molecule; an example of GENE DUPLICATION to create a GENE FAMILY.  Hemoglobin: binds oxygen; embryonic and fetal forms of hemoglobin exist  in fetal blood, more oxygen must be bound more tightly.  In development, you first express embryonic form of hemoglobin, then a fetal form of hemoglobin, and FINALLY, a post-natal (adult) form of hemoglobin; earliest forms of hemoglobin are most avidly bound to oxygen. • The genes start out as identical (gene family) but diverge into variants that perform different tasks.

Answer 161

o hnRNA = heterogenous nuclear RNA: RNA before processing (polyA tails, methyl caps, splicing) o mRNA = RNA after processing; used for translation in to proteins o tRNA = transfer RNA; bring amino acids to ribosomes o rRNA = ribosomal RNA; part of the ribosome o snRNA = small nuclear RNA; a variety of RNA that are part of ribonuclear protein complexes that perform function such as RNA splicing and telomere preservation. o tRNA, rRNA, and snRNA are all from non-protein coding genes

Answer 162

o ***1.5% of all DNA in body are EXONS (coding for proteins) o Introns are much more prevalent; fill in large gaps between exons o RNA products (tRNAs, rRNAs) are a small portion of the genome o ***98.5% of the genome is non-coding (but not necessarily NONFUNCTIONING) o Simple sequences; microsatellites and satellite sequences are small repetitive sequences in the centromere. o ***Approximately 50% of the human genome is comprised of unique sequences; the other 50% is of repeating sequences o ***45% of the repeated sequences are made of TRANSPOSONS (jumping genes) that can move around the genome. ALL are coded on DNA sequence. DNA Transposons: a DNA sequence that can change its position within the genome; at low frequency, sequence is cut out of DNA and can be inserted somewhere else on the genome. The same number of bases/sequence quantity is the same. • "CUT and PASTE" = 1 copy, only ``` RNA Transposons (retrotransposons): transcribed into RNA, make a copy of that, and then go back to form a new DNA sequence that can be inserted into genome at a different location from the original sequence. • Retrotransposons require reverse transcriptase. 8% of our DNA is viral in origin (presence of endogenous retroviruses) • "COPY and PASTE " = 2 copies result • LTR: Long terminal repeats = sequences that are duplicated at each end of the retroransposon. ``` o Capable of making RNA and often also making proteins that are not useful to the cell; potentially harmful o DNA methylation at endogenous retrovisues keep them “off” Difference in the size of genomes of different species are due to the number of transposons that influence the quantity of DUPLICATED GENES, not that some species have more genes than others.

Answer 163

o Do not make useful proteins; are essentially parasitic DNA and their origin is retroviral o Transposition = moving around to different parts of the genome; relatively low frequency of transposition but they have an effect on evolution of organisms. o When transposition occurs in GERM LINE CELLS, the sequence change is HERETIBLE. o Some chromosomal transpositions/translations, etc cause various diseases, such as hemophilia and cancer, upon mutation. o The genome works to keep the transposable elements transcriptionally silent by means of DNA and histone methylation = represses transcription. *** Overall takeaway: *** Transposable DNA can inactivate genes or their products by disrupting them; they can activate genes or their products by inserting near them; some can make RNA and proteins that are disruptive. Hemophilia: caused by insertion into clotting factor IX Cancer: activation of oncogenes by insertational mutagenesis Transposable elements can evolve = duplicate genes and their products, modifying existing ones.

Answer 164

Different from Conventional Retroviruses in that they o Lack the structure of retroviruses o Most abundant mobile DNA elements in mammals (21% of human DNA)

Answer 165

identify individuals based on their unique pattern of satellite or microsatellite DNA; variable number tandem repeats. o Simple sequence repeats: • Microsatelite DNA = 1-13 bp repeats; some occur within coding regions of disease and can thus cause mutations (Trinucleotide Repeat Disorders) • Satellite DNA = mostly repeats of 14-500 bp; often at telomeres and centromeres

Answer 166

Region of DNA that controls a hereditary characteristic of an organism Humans = 25,000 genes Bacterium = 500 genes

Answer 167

Locus: specific position or location of a gene on a chromosome Ploidy: number of chromosomes present in cell nucleus Diploid: cell or organism containing 2 sets of chromosomes and hence 2 copies of each gene Haploid: cell or organism containing 1 set of chromosomes Genotype: Set of genes carried /inherited by an individual cell or organism Phenotype: The observable characteristics of an organism Genome: The total genetic information carried by a cell or an organism

Answer 168

Allele: an alternative form of the same gene Homozygous: An organism with identical alleles for a given gene Heterozygous: An organism with different alleles for a given gene Hemizygous: An organism with a single copy of a gene. Usually applies to X-linked genes in males

Answer 169

Alleles that mask or hide other alleles, are said to be dominant. An allele that is masked, or covered up, whenever the dominant allele is present is said to be a recessive allele. Symbol: Dominant alleles – upper case letters Recessive alleles - lower case letters Complete (Classic) Dominance: when the heterozygous is completely indistinguishable from the dominant homozygous Homozygous dominant Heterozygous dominant Homozygous recessive

Answer 170

When one allele is NOT completely dominant over another (they blend – genetic blending) Example: In flowers, the color red (R) is incompletely dominant over white (W). The hybrid color is pink

Answer 171

when the contributions of both alleles are visible in the phenotype example: In humans, blood type is determined by 3 alleles – A, B, and O A and B are expressed equally i.e. co-dominant blood group alleles Dominant – A and B (co-dominance) Recessive – O

Answer 172

is the frequency (proportion) of alleles in a population Allele frequency = (times an allele is observed in a population) / (total number of alleles in the population) >> expressed as a percentage, decimal or fraction Reflection of genetic diversity in a population changes in allele frequencies over time and can indicate that genetic drift is occurring or that new mutations have been introduced into the population Genetic drift = random variation in allele frequency from generation to generation Remains relatively constant unless there is a selective advantage (high frequency vs low frequency) – natural selection Example: high allele frequency when mutations in genes that promote survival or fertility will be favored by selection because it’s more likely that these mutants will survive and pass the mutations to their offspring OR low allele frequency when affected individuals die and their affected alleles are, over time, eliminated from the gene pool

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