BIOC212FINAL Flashcards
(210 cards)
1
Q
What are the pros and cons of the confocal microscopy?
A
Pros:
1) 3-dimensional rendering of cells
2) live cell imaging
3) multiple snapshots per second
Cons:
1) Limitation of specificity markers
2) “co-localization” only implies co-function
3) limited by resolution ability, wavelength of light is larger than that of the protein and the wavelengths can be bleached which causes overexposure.
2
Q
Define resolution
A
Ability to see if there are two separate objects. In electron microscopy, provides means to evaluate quality of macromolecular structures computed from sets of their two-dimensional line projections.
3
Q
How does bacteria generated energy vs animals/plants?
A
- Bacteria uses their 2 membranes (inner and outer), and create a potential that separates and concentrates things to create a gradient. Either PE or KE to generate chemical energy source.
- Animals use specialized organelle mitochondrion and plants use chloroplasts. Need to be multiple membranes.
4
Q
What is the difference between respiration and photosynthesis?
A
In respiration, you take energy from food and you derive electrons from it. You pump the protons into separate compartments and create a gradient.
Photosynthesis is the reverse. You take energy from sunlight and create an electrochemical gradient.
5
Q
What is the process of respiration in the mitochondrion?
A
At the mitochondrion, there is fat, carbohydrate and protein degradation. Once it is degraded, we can create a substrate and break it down further. This is done in the citric acid cycle, releasing C02. Then Redox reactions take place, and 2 electrons release, then NADH, NAD+ and 2 electrons are in the electron transfer chain. H+ gets pumped and oxygen is the terminal acceptor as well as H20 being a product of respiration.
6
Q
What is the process of respiration in the chloroplast?
A
The opposite takes place than what is happening in the mitochondrion. CO2 and H20 is coming in, and glucose (carbohydrate molecules) and 02 is coming out. This is the product of the photosynthesis - the purpose is to fix carbons and make sugars.
7
Q
Where are two places that respiration happens more?
A
1) Cardiac muscle - needs energy all of the time therefore it is filled with many mitochondria.
2) Sperm tail (lots of mitochondria there)
8
Q
What is cristae?
A
- Inner membrane of mitochondria folds up into little loops called cristae. Important regions with a lot happening. They have a lot of surface area for chemical reactions to take place on.
9
Q
Why do mitochondria undergo fission and fusion events?
A
1) If they undergo oxidative stress and are not doing well, they need fission events to throw out all the garbage in one part of the mitochondria, then they can cut it out and bring to lysosome for degradation (autophagy)
2) Sometimes they fuse, during the cell cycle this happens many times, they want to share recourses between mitochondria and there are specific proteins needed in this process.
10
Q
What is an example of mitochondrion undergoing fission events?
A
- Protein (Dynamin - GTP-dependent protein) that forms polymer that wraps around the centre of mitochondria, eventually constricts it so it pops apart. This is GTP dependent constriction of microtubules mediated by a dynamin protein.
11
Q
What is an example of mitochondrion undergoing fusion events?
A
- Myocytes, fuse to form long muscle fibres of arms and legs. They need to get rid of nuclei, mitochondria also needs to fuse.
12
Q
Define NADH.
A
NADH is an electron carrier, it donates electrons to the electron transport chain. Passed through complexes, pump 1 protein each time from matrix to inter-membrane space.
Use protons to derive formation of ATP.
- From NADH, donate electrons to transport chain, passes complex 2 and goes from 1 –> 3 –> 4.
13
Q
What are ketone bodies?
A
They are a carbon source used if you are in a starved state, diabetes uses this, it is for energy for your brain, and RBC’s.
14
Q
What is the process of generating ATP by eating food?
A
- There are food derived molecules from cytosol: amino acids, fatty acids, pyruvate, ketone bodies.
- They go into the mitochondrial matrix where the main business is happening and derive Acetyl CoA.
- Then they get put into the citric acid cycle.
- There is an enzyme called citrate synthase that combines with acetyl coA (2 carbons) and oxaloacetate (4 carbons). Now we have a 6 carbon compound, citrate.
- Citrate undergoes enzymatic steps, looses 2 carbons (regenerate oxaloacetate - cycle). 2 carbons that came in as acetyl CoA come out as C02.
- Generated 1 GTP, 2 NADH, 1 FADH2,
- For every NADH, we have 3 ATP< every FADH2 we have 2 ATP.
15
Q
Is Acetyl CoA glucogenic?
A
It is not glucogenic, it cannot be turned back into glucose. There has been a mechanism developed to maximize the energy, and in return it sacrifices the ability to regenerate as a result.
16
Q
How is pyruvate regulated and what happens if it is degenerate?
A
- Pyruvate is regulated by many enzymes: mitochondria pyruvate carrier, pyruvate dehydrogenase, and pyruvate carboxylase.
- If it is degenerated, there is a rare disorder that will happen so it is important to keep this regulated.
17
Q
What are 7 important functions the mitochondria has?
A
1) Production of ATP
- oxidative phosphorylation in mitochondria, produces most of the ATP used by eukaryotic cells.
2) Regeneration of NAD+
- NAD+ is required for glycolysis and other reactions under aerobic conditions, NAD+ is regulated when NADH donates electrons via oxygen via the respiratory chain.
3) Provision of precursors for biosynthesis of amino acids, nucleotides, fatty acids.
4) Participation of synthesis of heme and iron-sulfer clusters.
- Plays central role in respiration and other cellular processes.
5) Cell signalling
- Buffer [Ca2+], ion that plays important role (i.e. muscle contraction)
6) Generation of reactive oxygen species.
- They could damage macromolecules, but also involved in signalling.
7) Regulation of apoptosis.
- Molecules released from mitochondria trigger a proteolytic cascade that leads to cell death.
- If the telomere is too short.
- In retina, when you wake up there is a layer of apoptosis, thats why you can’t see straight when you wake up, fresh cells are coming in.
- During development, looks like 5 fingers, there is innervation and skin is removed.
18
Q
What is an example of a specialized phospholipid?
A
Cardiolipin
- It takes a much broader position, usually cylinder shaped. This is because two phospholipids joined via the second glycerol, bigger tail than head group, so it spreads out.
- Actively binds to one or more complex of the electron transport chain, if cardiolipin is not there, they do not stick together and you will not get proper electron transport chain.
- Essential for efficient mitochondrial electron transport function.
19
Q
What is the function of ubiquinone and cytochrome C?
A
They are transport electrons.
20
Q
Why is glycerol a useful molecule?
A
It is useful because it has 3 hands.
- 3 hydroxyls that can attach to 3 different things. You can make triglyceride, phospholipids (useful for making membranes)
21
Q
What is the difference in electron transport chain in terms of utilizing energy vs explosive combustion?
A
- If we took hydrogen and oxygen and provides a spark, there would be an explosion, all the energy that was contained in them was expended at once. We can use that to generate energy but it is hard to trap, therefore a lot of this energy is not utilized.
- With our carbon sources, instead of letting it all explode and burn instantly, we trap it into ATP, and we do this w/ Redox Pairs. There needs to be an electron donor and acceptor, otherwise the electrons cannot be passes. The main way to do this is to create a proton-motive force. —> When we pass electrons, we move proton from matrix to inter-membrane space, concentrate them to create a gradient, and harness that to form ATP.
22
Q
What is the function of Iron-Sulphur clusters?
A
Carry electrons down the electron transport chain, attached to iron at the middle of the cluster.
23
Q
Describe (diagram) of the electron transport chain:
A
1) Complex 1
- NADH donates electrons (2), within complex 1, one proton is pumped out to inter-mitochondrial space.
- Bypasses complex 2, and goes through ubiquinone and citric acid cycle.
2) Passed to complex 3. Passes cytochrome C and complex 4.
- each time it goes through complex it pumps out 1 proton.
- Electron finally given up by cytochrome c to where electron terminal acceptor = oxygen and generates water.
24
Q
Describe the fate of the breakdown of one molecule of glucose
A
- Glucose broken to pyruvate, to acetyl CoA, into citric acid cycle, then electrons transported, oxygen into water and the carbons make C02.
25
What is the difference between NADH and NADH2?
- NAD+ is NADH (It accepts two electrons and one proton)
- NAD2 is used to account for the second hydrogen that gets removed from the substrate being oxidized.
26
When does FADH2 come in?
FADH2 comes in from the citric acid cycle, it does not join/share the electrons at complex I, it bypasses and shares w/ complex 2, then goes to complex 3, pumps proteins, at complex 4, gets 6 protons and can get 2 ATP from this. (bypass makes it less)
27
What is the net product from oxidation of one molecule of glucose?
(cytosol - glycolysis)
1 glucose --> 2 pyruvate + 2 NADH + 2 ATP
28
What is the net product from mitochondrion (pyruvate dehydrogenase and citric acid cycle)
2 pyruvate ---> 8 NADH + 2FADH2 + 2ATP
29
What is the net product from oxidation of one molecule of Palmitoyl CoA (activated form of palmitate, a fatty acid)
1 palmitoyl coA --> 31 NADH + 15 FADH2 + 8 GTP
30
Describe Glycolysis
- It is the breakdown of glucose to pyruvate.
31
What is the concentration of glucose inside the cell?
[glucose] is almost zero. It almost immediately gets transferred to glucose 6 phosphate. This is helpful to trap glucose into the cell.
32
What is an example of substrate level phosphorylation?
- Add two phosphate groups, break fructose up into two carbon free carbons, add NADH, get pyruvate. Results in 3 carbons, and you get 2 NADh and 2 ATP.
- directly take phosphate off can now add phosphate back to ADP to make ATP.
- anaerobic, most steps are reversible, except for hexokinase.
- Now pyruvate is a 3 carbon compound, this only goes one way. Commit carbon that comes from glucose to citric acid cycle, loose C02, 3 carbon becomes 2 carbon compound.
Conversion of PEP to pyruvate in glycolysis. The enzyme, pyruvate kinase catalyses the transfer of a phosphate group from PEP to ADP, producing pyruvate and ATP as products. This reaction involves substrate PEP donating phosphate group directly to ADP to form ATP, without the need for electron transport or protein gradient.
33
Define allosteric
Product binds to enzyme and causes activity, which is not the binding site.
34
What is the rate limiting step?
This is when Acetyl CoA is in the citric acid cycle. We need to regulate this step, it is important because sometimes we might want to stop the process.
ex) Do not want to get rid of all the pyruvate so we turn some of it into Acetyl CoA.
ex) do not turn into glucose, we can store as fatty acid and use if we need it, this is importance for the brain and RBC's.
35
What is the function of Insulin?
- Strongly controls pyruvate dehydrogenase (PDH), you have tons of glucose but you need to pump it down.
- Can store it as glucagon (happens mostly in muscle and liver)
- when you start to loose weight, glycogen will go first, has water that holds it there, so you will loose water weight first.
- Glucagon --> increased levels of glucose.
36
What is Rubisco?
Rubisco is very unique and fixes carbon, adds carbon dioxide and puts it into a molecule that we can degenerate ATP from and put it into storage etc. (valuable)
- enzyme present in plant chloroplasts.
37
What is a ligand?
It is an extracellular signalling molecule which is what binds to the receptor on the plasma membrane outside of the cell.
38
How do we send signals across the membrane? What are a few exmaples.
- Signal transduction cascade (intra-cellular signalling molecules), then you get different effector proteins and perform specific functions.
ex.) metabolic enzyme - altered metabolism
ex.) transcriptional regulatory protein - altere gene expression
ex.) Cytoskeletal protein - alter shape or movement.
39
What are four different kinds of signalling?
1) Contact-dependent (one cell physically interacts w/ another cell)
2) Paracrine (cells release a signalling molecule that attack/go to the target cells around it)
3) Synaptic (never cells are unique cells. They are very long, send signals to the axon to the dendrite. Message from previous neuron to other end of the body, where it reaches the neurotransmitter to send message at synapse)
4) Endocrine (signal goes through blood, from kidney to liver, send a hormone to send the message, communicate either locally at paracrine level or far away at endocrine level)
40
What are two types of receptors?
1) Cell surface receptors (signals)
- Cell surface ligands that bind ligands, most cases they are hydrophilic molecules, they cannot do it themselves
2) Intracellular receptors (carrier protein)
- Small hydrophobic molecules, it can go right through membrane, sometimes with help of a transporter, they can get in and bind to receptor inside, they are soluble.
- Applies to steroid hormones, receptor is also a transcription factor.
41
Different cells react to different signals, different cells types will react differently to the same signal. What is an example of this happening on the same cell? If these don't happen, what is the consequence?
1) Ligands bind to respective receptor, promoting survival of the cell.
2) Ligands bind to respective receptor, making the cell grow and divide.
3) ligands bind to respective receptor, making the cell differentiate.
- If this does not happen, and they are lacking specific signals, then the cell will go through apoptosis.
42
Acetylcholine is a signalling molecule. What are some examples of its function?
- It has an acetyl group at the bottom, top part is a choline, and it has a positive charge quaternary amine.
- heart pacemaker cell (binds to the receptor and helps stimulate heartbeat)
- Salivary gland cell (helps secrete saliva)
- Skeletal muscle cell (causes contraction)
- same molecule in different cells/locations causing different results.
43
What is the function of ion-channel coupled receptors?
- pore that can help bring ions into the cell
- to concentrate them, it depends on which ion you are talking about, often done in combination of other molecules or with help of ATP.
44
What are G-Protein Coupled Receptors?
- 7 transmembrane domain G protein
- When it is activated by a binding signalling molecule, it recruits three subunits, alpha, beta, gamma.
- They bind and get activated, the signalling event depends on which G-protein you are talking about.
45
What are enzyme-coupled receptors?
- Two receptors that are separate and when they bind ligand, they homodimerize.
- In the intracellular area, they have enzymatic activity, when they come in contact with its buddy, it transphosphorylates, both activated, and another signalling event can occur downstream.
- Binding of ligand promotes this and binds them together.
- They tend to be in solitary/inactive, by binding the ligand it brings together the two monomers and once together, two active enzymatic catalytic domains that can act on eachother.
- Kinase can phosphorylate together and promote signalling.
46
What are GTP binding proteins?
- Small molecule GTP binding proteins or small molecule GTPase.
- Turn off by GEF and turn on by GAP.
47
What are two types of signalling?
1) Signalling by phosphorylation
- turned on by protein kinase, turned off by protein phosphotase
- phosphorylation can either activate or inactive a protein
2) Signalling by GTP protein
a) monomeric GTPase determines turning GTP binding proteins on or off by GAP/GEF.
48
What happens if you do not have he GDP/GTP GAP/GEF cycle?
Yo can have mutations, this cycle needs to be regulated and you need to have control.
49
What are some effects that the signalling can have?
- Signal coming down from membrane, activation of protein kinase, inactivation which also effects cascade of proteins, which then affects gene expression,
- Can also directly phosphorylate an inhibitor protein which regulates transcription factors, that go promote transcription.
50
What is a Scaffold Protein?
-Proteins are not able to create actions themselves, bring lots of proteins together in one place by recruiting them.
- This is done by activating a receptor with a ligand, then many proteins bind.
This is a signalling complex.
51
What are adaptor proteins?
They are an intermediate - between receptors and what is downstream of it, so they have to connect.
52
Different domains bind different things, each has its own pH domain to recruit its own protein, to create a critical mass and to bring proteins together to promote signalling events.
What are two types of domains?
1) PTB domains - bind tyrosines that are phosphorylated but in specific motifs. PTB domain containing proteins can bind to phosphotyrosine on the signalling receptor. (NPX motif, asparagine proline, tyrosine, tyrosine gets phosphorylated)
2) pH domain - domain binds all different kinds of lipids that have diff pH domains.
ex. SH2 domain --> bind to proline rich regions. (happens when we need to recruit them)
53
What is Protein MCAK?
It is an example of clustering receptors together to get important signalling happening.
Two downstream signals are coming together to either inactive or activate.
54
Wha is cyclic AMP?
It is important to control it.
It is an important signalling molecule.
- You can destroy it with phosphodiesterase
- temporarily make it/destroy it, once you get the desired number of signalling events, you kill it and it is important that we turn it off.
55
Describing an example of signalling.
1) Binding of G protein coupled receptor, activates the G protein, alpha beta gamma, formation of PI(4,5)P2, recruit phospholipase C-beta, which cuts leaving diacyglycerol, activates protein kinase C, phosphorylates and regulates, inositol trisphosphate released, opens up calcium channel, and protein kinase C is calcium dependent kinase, double activated protein kinase C, to phosphorylate downstream targets and mediates the whole event.
56
What is GPCR?
G protein coupled receptors
57
There is major signalling happening for G proteins, it keeps going until it is deactivated. What is a way to deactivate them? and what would this process look like?
- one way to do this is phosphorylating
1) Activated GPCR stimulates GRK to phosphorylate the GPCR on multiple sites
2) Leads to binding of other proteins --> arrestin (arrestin binds and then inactivates receptor = desensitized GPCR)
3) Blocked which prevents further activation of trimeric G proteins.
58
This is an important mechanism:
receptor by itself, when it binds to ligand, it dimerizes and transphosphorylate, creates binding site and has other signalling events that occur downstream of this.
Describe an 2 examples of this:
1) EGF binds to EGF receptor, dimerize, transphosphorylates, Now there are multiple signalling events.
- Two monomers, homodimerization, two domain that contain the kinase domains are now in close proximity, they each contain a substrate for tyrosine kinase, each time it phosphorylates, becomes a binding site for another protein.
- Protein 1,2,3 via SH2 or PTB domain.
2) PDGF receptor, 5 different tyrosines can be phosphorylated, and they can create binding sites for different enzymes. Proteins = PI-3 kinase, GAP, PIL-4, have specific activaiton of PDGF, homodimerization, phosphorylation, we can create different signalling complexes.
59
What four main things do monomeric GTPases bind to?
1) GTP
2) GEFs
3) GAPs
4) Effectors: downstream proteins interact with monomeric GTPase to mediate their cellular function.
Members of the family: Ras, Rho, Rab, Ran, ARF
60
Describe an example of the Ras signalling pathway:
1) Ras protein is downstream of RTK (receptor tyrosine kinase)
2) binds to receptor, transphosphorylaton occurs.
3) Protein --> Grb2 binds to phosphotyrosine via SH2 domain.
4) binds to proline-rich domain via SH3.
5) recruits second protein: sos
- sos has a GEF binding domain, promotes the binding of GTP, and removal of GDP, ras has now been activated, it goes and does many important functions.
61
How can PI (phosphoinositol) species act as an important signalling molecule?
- left chain is diaclyglycerol, then phosphate, then inositol. It has 6 positions.
- One position is bound to glycerol
- 2-6 positions are available to be phosphorylated, we continue to phosphorylate them.
- if we have PI-3 kinase, we can generate 3 options, and get all kinds of PI-species, each one is specific for a certain area of the cell.
- Proteins bind specifically to the species, when we don't want them anymore, we cut them off, fall off, job done.
- if we want them back again, we need to make PI 4 phosphate again.
- true for all PI species, we control them.
62
Describe the stimulation of growth by the mTOR complex 1:
- mTOR is a major metabolic regulator in the cell, and this is one way that we know it is regulated.
- breaks in the car, stops a lot of metabolic processes from happening based on different cues.
- on surface of lysosome.
- it allows the cell to undergo cell division of mTor, controlled by growth factors, which is what activates mTor.
First thing that it responds to is the amount of amino acids. Reaches into lumen of cytosol and says how many amino acids, if enough build protein, if not enough, stop building new protein.
- Also controlling sugar and lipid metabolism.
1) Growth factor. "lets grow" a lot of protein.
2) Receptor binds growth factor, homodimerizes, transphosphorylates, activates two seperate signalling cascades, one via Ras (Erk MAPK) and one via PI 3 Kinase (AkT)
3) Those are major kinase involved. Interact with protein: TSC. A Rheb GAP. ERK and AKT inhibit GAP to turn into GEF. Small molecule GTP binding proteins on surface of lysosome. Need this to activate mTOR. First needs to
4) Rheb activates mTOR and stimulates growth controlled by mtor complex.
5) mTOR attacks Rag. (Ragulator - regulates RAG GEF activates). Lysosomal amino acids tell Rag to grow and activates it if it has enough which activates mtor and you get growth.
6) Cytosolic amino acids, bind to amino acid receptor, inhibitor Gator2, inhibits Rag GAP, which inhibits Rag. Promoting the activator, and inhibitor the inhibitor, which activates Rag that activates mTOR.
63
Steroids are important major signalling pathways. How do they signal?
1) There is a sterioid hormone
2) There is a hormone receptor complex formed with a receptor in the cytolpasm
3) The complex moves into the nucleus, and binds to sites on the chromatin, activating mRNA transcription and having different cellular responses.
- can promote or inhibit transcription
- might be a cotransporter to help
64
What is the function of the cell cycle?
Decision making at the molecular level.
65
What is the function of the Cdk enzyme?
-Cdk enzyme is needed and it is a protein.
- When it is done its job , it is destroyed and thrown away, there are many different kinds.
- at points where we have to make decisions cyclin increases, and then the choice is made, the amount goes down and it is destroyed, this is regulating the cell.
- There are some that you need input from that are regulating major transition events in the cell cycle.
66
What is the function of M-Cdk? What is it composes of? What happens if it is not regulated?
It regulates the cell cycle during mitosis, and it regulates transition from metaphase to anaphase. It is a critical step in ensuring the accurate segregation of chromosomes during cell division,
- if M-Cdk is not regulated, then it leads to various diseases, cancer etc.
- Composed of 2 subunits: cyclin B and CdkI, during G2 phase of the cell, levels of cyclin B and CdkI increase, and they peak during mitosis.
67
What is CAK?
Cdk-activating kinase
- CAK activates cdk and we need cdk for cell regulation.
68
Describe the first phosphorylation cycle: (Cdk) and the second phosphorylation cycle.
First:
(A) Inactive - Cdk is just there with some ATP.
(B) Partly Active - Cyclin comes along and binds to cdk, changes conformation to a T-loop.
(C) Fully Active - cdk - activating kinase comes and phosphorylates cdk on a certain location and makes it active.
Second:
1) At this point, the Cdk + cyclin is fully active with the activating phosphate.
2) Second kinase comes along --> Wee 1 kinase
3) Phosphorylates Cdk at a second site, inhibits it and inactivates it.
4) Cdc25 phosphotase dephosphorylares this specific phosphate and makes it activates again.
- Cdc25 and Wee1 kinase are in competition.
69
What is the function of protein p27?
it Inactivates cdk-cyclin.
70
Describe the pathway to regulate mitosis.
1) M-Cdk (Active) activates the Greatwall Kinase. Which activates ENSA. Which inactivates PP2A-B55.
- ENSA is an inhibitory protein
- PP2A comes and dephosphorylates and prevents mitosis from happening, to stop this from happening, activate Cdk, which activates greatwall kinase, which activates ENSA< which makes PP2A inactive and it cannot prevent mitosis anymore.
2) M-Cdk (active) phosphorylates Cdk substrates which results in mitosis!
3) PP2A-B55 (active) would inhibit mitosis but this gets stopped by activating the greatwall kinase.
71
Describe the pathway involving CAK, Wee1, Mcdk, Cdc25 etc.
1) You have inactive M-Cdk. CAK ativates it and Wee1 inactives it. You still have an inactive M-Cdk, it has an activating phosphate but it also has an inhibitory phosphate.
2) Cdc25 coimes in and activates the M-Cdk. Two positive feedback looops come from this.
3) Active M-cdk activates PP2A-B55 which activates Wee1 which inactivates M-Cdk.
4) Active M-cdk inactives PP2A-B55 which actives Cdc25 which then activates M-cdk.
72
Describe the "tug of war" pathway:
- M-Cdk phosphorylates substrates, activates them, and promotes mitosis.
- PP2A-B55, dephosphorylates substrates, prevents mitosis.
- phosphorylation of greatwall kinase activates PP22A which dephosphorylises Cdc25 and Wee1, which activates positive feedback at the top.
73
After regulating mitosis, with M-Cdk, what is the next pathway/step that happens? Describe it:
- The next step is the Fuse, at some point we want to turn the process off.
1) The M-Cdk is active. Then there is the APC/C inactive, and then the Cdc20 comes in and activates the APC/C to then form APC/C-Cdc20 active complex. This is anaphase onset. And it inactivates M-Cdk.
2) When the M-Cdk is active, there is Cdh1 inactive, but when the M-cdk is inactivated, the Cdh1 is dephosphorylated and it becomes active.
3) After there is the anaphase onset, there APC/C is dephosphorylates and the Cdc20 is released - this is done by the M-Cdk inactivation. And then the APC/C and Cdh1 active join together to form a complex, which is active, and goes to perform other functions in the cell.
The idea behind it:
- Cdk lights the fuse and it knows when the job is done. as soon as the job is done, APC/C-Cdc20 dephosphorylates and turns into APC/C-Cdh1 which mediates next step of the cell cycle and promotes regulation.
- Protein complex APC/C is E3 ubiquitin ligase of Cdk cyclin, once activated it acts on E3 ligase of cdk cyclin, with E1/E2 and it leads to degradation of M-cyclin in proteasome, and inactivation of complex.
- Cdk phosphorylares APC/C, why would it want to promote activity of its executioner? Because it is a timer.
74
What happens in Prophase?
-Replicated chromosomes, each consisting of two closely associated sister chromatids, condense.
- Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have replicated and moved apart.
75
What happens in Prometaphase?
- Starts abruptly with the breakdown of the nuclear envelope
- Chromosomes can now attach to spindle microtubules via the kinetochores and undergo active movement.
76
What happens in Metaphase?
- Chromosomes aligned at the equator of the spindle, midway between the spindle poles
- The kinetochore microtubules attach sister chromatids to opposite poles at the spindle.
77
What happens in anaphase?
- Sister chromatids synchronously separate to form two daughter chromosomes, each pulled slowly toward spindle pole it faces.
- Kinetochore microtubules get shorter, spindle poles move apart, both processes contribute to chromosome segregation.
78
What happens in Telophase?
- Two sets of daughter chromosomes arrive at the poles of the spindle and decondense
- Nuclear envelope reassembles around each set, completing formation of two nuclei and marking the end of mitosis.
79
What happens in cytokinesis?
Cytoplasm divides into two by contractile ring of actin and myosin filaments, pinches the cell in two to create two daughters, each w/ one nucleus.
80
What is cohesin?
- It is a protein
- Composed of a number of proteins that wrap around DNA to keep sister chromatids in place
- Process of cohesin wrapping around is essential for proper alignment and seperation of chromosomes during mitosis and meiosis, any defects in cohesin function can lead to chromosomal abnormalities and genetic disorders
81
What is the function of Securin?
Body guard/inhibitor that prevents enzyme seperase.
82
Describe the process with APC/C, Cohesin, Securin etc.
1) You have inactive APC/C and then Cdc20 activates it.
2) Securin and inactive seperase have formed a complex. Active APC/C comes in and there is ubiquitylation and degradation of securin and the seperase has disattached and is now active.
3) M-Cdk lights the fuse on APC/C to find transition from metaphase to anaphase.
4) Cohesin G2 interacts with M-Cdk and goes to mitotic spindle (metaphase) and then the active seperase comes and it cleaves and dissociates cohesins to anaphase.
- seperase seperates things, protease that cleaves cohesin. Inactive when bound to securin, when APC/C comes and degrades securin, you have active seperase comes and clicks away all the cohesin, that once kepts the 2 sister chromatids together and now can actively do final seperation in anaphase.
83
Describe the signalling pathway that includes RhoA.
1) You have inactive RhoA that gets turned on by GEF, and turned off by GAP.
- GEF is activated by Aurora-B centralspindin.
2) Now you have Active RhoA that forms two things; Formin, and Rho-associated kinases (including ROCK)
3) Formin forms actin filaments. Which form the assembly and contraction of myosin ring.
4) Rho-associated kinases, inhibite myosin phosphptase, and regulates myosin light-chain phosphorylation, which activates myosin II. This forms the assembly and contraction of myosin ring.
84
MTORC1 is an important protein complex. How does its pathway work/how does it accomplish the outcomes that it does.
1) There is a growth factor (promotes cell division), attached to a tyrosine receptor kinase, that activates another growth factor receptor. And there are also amino acids that is what regulates the MTORC1 complex.
2) Now that the complex is formed and regulated it can go perform different tasks.
- 4EBP inhibits binding with EIF4E so then protein synthesis can occur.
- S6 Kinase 1 activates EIF4B which leads to protein synthesis. And also S6 Kinase 1 leads to lipid synthesis.
- Lipin inhibits SREBP which leads to Lipid Synthesis.
- Inhibits UIK1 which leads to Protein turnover.
All of these are mediated and regulated by the mTORC1 complex.
85
How is cancer formed?
- normally cells divide to a certain point and then stop, if they just keep dividing, they cause tumours.
- formed from altered DNA/damages which causes damage in genes, gene expression etc.
- patients die when cancer has left the memory tissue and moved along, it isnt the primary tissue that is dangerous it is the metastatic tissue.
86
What is RSV?
- It is Rous Sarcoma Virus
- Carcinogenic agent was virus
- it is a retrovirus that codes for its own reverse transcriptase, integrate into chromosomes of cells that infects, people use retroviruses as a tool for genetic manipulation.
- RSV is a retrovirus that encoded enzyme (reverse transcriptase) to reverse transcribe their RNA genomes into complementary DNA (cDNA) which can integrate into cellular genomes.
87
What are 7 characteristics/cellular transformations of cancer cells?
1) Immortalization
2) Altered morphology (round shape)
3) Loss of contact inhibition (ability to grow over one another)
4) Anchorage- Independent growth (growing without attachment to solid substrate)
5) Reduced requirement for growth factor
6) Increased transport of glucose
7) Tumorgenicity
88
What makes RSV different compared to avian retroviruses?
- It has an additional gene: src that has a role in triggering formation of sarcomas.
- without src could not do its job
89
What was an important discovery that they found in src DNA sequences?
They found that there were both RSV infected cells and uninfected cells, and realized that cellular genes may have a role in cancer.
90
What is the difference between c-src and v-src?
- c-src is a cellular src gene: proto-oncogene
- v-src is a viral src gene: oncogene
91
How do src proteins function as a tyrosine kinase?
Kinase: enzyme that removes high-energy phosphate group from ATP and transfers it to a suitable protein substrate.
- phosphorylates specific tyrosine amino acids in substrates.
92
Describe the action of protein kinases and signalling:
- kinases generally phosphorylate and thereby modify the functional state of substrate proteins
- e.g) Akt/PKB kinase influences multiple biological processes by phosphorylating a number of downstream substrates.
- processes such as proliferation, angiogenesis, apoptosis, protein synthesis.
- signalling cascades by kinases and then effect on gene transcription.
93
What is "selective growth advantage"?
The difference between birth and death in cell population. Allows cancer cells to outgrow the surrounding 'normal' cells.
94
Define oncogene
A gene that increases the selective growth advantage of the cell in which it resides.
95
Define proto-oncogene
A normal gene that can become an oncogene as a result of mutations or increased expression.
96
Tumor-supressor:
A gene when inactivated or lost, leads to an increase in selective growth advantage of the cell in which it resides.
97
Normal cell cycle:
Balance between proto-oncogene (gas) and tumor-supressor gene (brakes)
98
How do mutations lead to cancer?
- There is too much proto-oncogene --> oncogene
- too much gas!
and the tumour supressor gene is inactivated (no brake)
99
How do you identify nonviral oncogenes?
- you have speculated carcinogens = mutagens that turn proto-oncogenes into oncogenes
- to test this= perform transfection
transfection: introduce DNA of cancer cells into normal recipient cells by process.
Example:
Used NIH3T3 cells, immortalized fibroblasts that are great at taking up DNA.
Treated mouse cell line w/ (3-MC), potent carcinogen that is component of coal tars.
Tumor formed.
100
What are 6 ways to turn a proto-oncogene into oncogene?
1) Amplification: genetic alteration producing a large number of copies of a small segment of the genome.
2) Insertion/deletion (indel): insertion or deletion of a few nucleotides.
3) Translocation: a specific type of rearrangment where regions of two non-homologous chromosomes are joined. (e.g. Philadelphia Chromosomes)
4) Point mutations: single nucleotide substitutions (e.g. A to G)
5) Driver mutation: a mutation that directly or indirectly confers a selective growth advantage to a cell.
6) Passenger mutation: a mutation that does not confer a selective growth advantage ('along for the ride')
Tumour mutation burdens can vary.
101
What are cytogenetics? What is an example?
- Study of inheritance in relation to the structure and function of chromosomes
- Karyotyping: process of pairing and ordering a cells chromosomes.
102
What event happens to result in the Philadelphia chromosome?
- There is translocation between chromosome 9 and 22
- This translocation event leads to cancer
- it is effectively hooking up two genes that should have nothing to do with each other
103
What is BCR-ABl? Describe it:
- it is a cellular oncogene
- translocation leads to a 'new' gene in CML (Chronic Myeloid Leukemia)
- ABl: tyropsine kinase (proto-oncogene)
- Bcr: Breakpoint cluster region
- fusion leads to Abl kinase constitute fusion activity with emitting strong growth-promoting signals in dysregulated manner.
- ABL (cellular kinase) has a job in signalling cascade, in this case it is not oncogenic yet, once it looses its regulation, it is always on, and then it is proto-oncogenic.
104
What are RTKs?
Cell surface receptors.
- examples: epidermal growth factor receptor, EGFR.
- ectodomain protrudes in extracellular space to recognize and bind ligand.
- hydrophobic transmembrane domain threads through plasma membrane domain lipid bilayer.
- kinase domain sits inside cell.
- Involved in RAS signalling pathway, PIT kinase pathway, growth + survival pathway.
- once binds, activate dimerization and phosphorylation of downstream targets.
1) amplification
2) being in proximity
- Growth factor binding normally leads to RKT dimerization and activation of kinase domains
- mutations can result in ligand-independent activation
- RTK overexpression (e.g. ERBB2) may cause cell to become hyper responsive to low growth factor concentrations and/or cause ligand independent RTK dimerization due to mass action effect.
105
Describe the impact and targeting BCR/ABL: Imatinib (Gleeve)
They used personalized medicine to find out that BCR/ABL is inhibited by Imatinib. They found this out by screening a bunch of libraries. This was a transformation event, it was a win!
106
What is erbB2/HER2?
It is oncogene. It leads to breast cancer.
- erbB2/HER2 is a receptor tyrosine kinase. It responds to epidermal growth factor.
Aka. neuro/glioblastoma derived oncogene homolog (neu).
Aka. human epidermal growth factor receptor 2 (HER2)
- dimerization activates proliferation and survival gene expression signalling pathways.
107
What is the difference if ErbB2/HER2/Neu is amplified or not?
- If it is overexpressed then 30% if them are breast carcinomas.
- If not amplified, chances of having breast cancer is significantly lower.
108
How did the fish analysis of ERBB2?HER2+ of Breast cancel cells work?
- Use probe @ DNA level to see all the cancer cells. Personalized medicine approach.
109
What is Traztuzumab (Herceptin)?
Binds to extracellular domain of this receptor and inhibits HER2 homodimerization, thereby preventing HER2 - mediated signalling.
110
Describe Ras (cellular oncogene)
- GTPase, downstream of receptor tyrosine kinase.
Ras signalling cycle:
- GEF = on
- GAP = off
- three Ras genes (K-Ras, H-Ras, N-Ras) in human cels that act in similar fashion.
- Problem in cancer, if there is a point mutation and cannot be turned off properly. There are multiple pathways that Ras can intercept, when it is on, effects signalling cascade, protein synthesis, growth, survival, proliferation.
111
How can a missense mutation yield a mutant Ras protein?
- Ras is a GTP (G) protein that is activated when bound to GTP
- uses GTPase function to hydrolyze (cleave) GTP --> GDP, thereby inactivating itself.
- Missense mutations in G12 and 61 turn Ras from a proto-oncogene to a oncogene
- lead to GTPase negative-feedback mechanism being inactived
- Mutated Ras genes (KRAS, HRAS, NRAS) are found in many cancers, 50% of colorectal cancers and 95% of pancreatic cancers.
112
Define tumor supressor genes.
- gene that when inactivated or lost leads to an increase in the selective growth advantage of the cell in which it resides (e.g. Rb and p53)
- can be sporadic or familial form.
113
What is the connection between Retinoblastoma and tumour supressor genes?
- can be diagnosed in children with no family history (sporadic form), can have single tumour in one eye, w/ radiation and surgery being effective.
- diagnosed in children with family history (familial form) often have multiple loci of tumors in both eyes (bilateral retinoblastoma). These children have higher risk of bone cancers (osteosarcomas) as well as other tumours later in life.
114
What is a "Hit" when talking about cancer?
It is a mutation event that would be effecting DNA, there is a 2-Hit hypothesis for tumour surpressor genes. Familial needs one hit, and sporadic needs 2 hits.
115
What is a mitogenic pathway?
pathways that regulate/stimulate mitosis.
116
What are transcription factors?
proteins involved in converting/transcribing DNA into RNA.
117
The cell asks a question "to divide or to not divide?" when is this question asked?
- This happens in the G0 or quiescent phase. It is the stage where the cells remain metabolically active but do not proliferate unless called to do so.
- Once in the "cell cycle clock" divide to enter G0, or enter the active cell cycle, to then enter G1 -> S -> G2 -> M.
118
What is the function of Rb?
- Rb is a gatekeeper
- nuclear protein that can be phosphorylated in a number of ways. mitogenic signalling pathways.
- Acts as a transcriptional repressor that determines if the cell will enter S phase.
- If Rb is hypophosphorylated, then the cell cycle can proceed.
- If it is not phosphorylated, then it will prevent to go through the cell cycle, because it will bind them and repress the ability to transcribe genes and shut down the cell cycle.
119
What is the Restriction Point (R)?
- denotes the point in time when the cell must make the commitment to advance through the remainder of the cell cycle, through the M phase, to remain in G1, or to retreat from the active cell cycle in G0.
120
How does the Rb gene fit with the knudson 2-hit hypothesis?
- Rb gene as a tumour supressor fits perfectly with the 2-hit hypothesis
- If it is sporadic, then 1 hit, loose the second allele thought LOH, mutant Rb allele, unilateral. (so needs two things)
- If it is familial, it is already one hit behind the curve, all need is one additional hit, tumours pop up faster - bilateral.
- higher chancer outside of eye, born behind curve, already with a mutant, so thats why you only need one hit.
121
What does LOH mean?
Loss of Heterozygosity.
122
What is epigenetic silencing?
It is a way to loose tumour surpressor genes.
123
How do you loose tumour surpressor genes?
- Many familial cancers explained by the inheritance of mutant tumour suppressor genes
- Early stage cancer cells find ways to eliminate wild-type copies of TSGs. (e.g. mitotic recombination can lead to LOH)
- can occur during G2 phase of the cell cycle
- Subsequent segregation of chromatids may yield a pair of daughter cells that have undergone LOH.
124
Describe the mechanism of LOH and Inactivation of WT copy of tumour suppressor gene:
- If it is inherited or acquired, then 'first hit' but if it is not, then a second hit is required.
- for the second hit, there are many different events that could take place.
- terminal deletions, mitotic recombination, loss of reduplication, point mutation, interstitial deletion, disruptive translocation, gene conversion, epigenetic silencing.
125
What does epigenetics mean?
Alteration in gene expression, modifying DNA, but not what it codes for.
126
Where and what are nucleotide analogs used for?
They are used in clinic to prevent cytosines being methylated.
127
Describe the structure of Chromatin:
- Tightly packed DNA, wrapped around histone proteins, allows long DNA to be compacted.
- Way DNA is bound, dictates how well it can be transcribed.
- DNA is tightly packed = heterochromatin.
- DNA is loosely packed = transcription factors, euchromatin (this is tightly controlled)
128
When is chromatin activated and when is it silenced?
- Heterochromatin= transcriptionally silent, DNA itself can be methylated. Prevents TF's from binding and transcribing DNA + modifications on histone proteins.
- Euchromatin= not a lot of methylation, allows TF's to bind.
129
How does chromatin being activated or not, impact tumour suppressor genes?
- Promoter methylation can lead to TSG inactivation without having a mutation.
- if it is methylated, DNA sequence around it can still be transcriptionally silent, even though DNA sequence looks normal.
130
What are some enzymes that could methylate DNA? And what impact would this have?
- DNMT1 and DNMT3b
- cooperate to silence genes (TSG's) in cancer cell genes.
- so the more you have of it, the more cancer.
131
What is the function of p53?
- P53 plays an important role in regulating cell division and preventing formation of cancer.
- DNA damage and dysregulated growth signals lead to p53 stabilization.
- p53 forms a homotetramer to function.
- functions as a transcription factor that halts cell cycle
- p53 target genes include growth arrest genes, DNA repair genes, regulators of apoptosis.
132
How do p53 genes get activated/how does the process work?
- Many things could cause p53 genes to activate: lack of nucleotides, UV radiation, ionizing radiation, hypoxia, oncogene signalling, blockage of transcription.
-then p53 (tetramer) goes and performs its functions: cell cycle arrest (senescence/return of proliferation), DNA repair, block of angiogenesis and apoptosis.
133
What happens if we get a mutation that blocks p53 gene?
If it prevents p53 from doing its job as a tetramer, it will poison all of the tetramers, and prevent p53 from working and doing its job.
134
Is one gene firing enough for Rb to do its job?
Yes
135
TSG Haploinsuffiency happens when one gene is not enough. What gene follows this model?
- p53
- half amount of the protein is not sufficient to do the job, the threshold is 100% for p53 to function.
- one mutation is enough to have the tumore supressor inactivated, so it does not follow the 2 hit hypothesis because it only needs one hit.
- it is dominant negative (bad apple ruining the batch)
- p53 acts as a tetramer, if mutation in one allele, poison all of them. Any one will have mutated subunits, p53 will not act as a tumour suppressor gene anymore.
136
What does "dominant negative" mean?
- A mutation whose gene product adversly (bad) affects the normal, wild-type gene product within the same cell. This usually occurs if the product can still interact with the same elements as the wild-type product, but still blocks some aspects of its function.
137
How do pharmacologically target 'untargetable' tumour supressor genes? mutants in cancer cells?
Synthetic lethal approaches to killing cancer cells.
138
What is the concept of synthetic lethality? describe it in the context of cancer:
What are some benefits?
- the combine effects of two alleles, each of which is non-lethal but when acting in combination, results in lethality. This was developed during genetic studies.
In the context of cancer:
Normal cell: Gene A (X) and Gene B = cell survival
Normal cell: Gene A and Gene B (X) = cell survival
Cancer cells: Gene A and Gene B (cancer mutation) = cellular death)!
Benefits:
- selective for cancer-cell specific genetic mutations
- strategy can be applied to any type of cancer mutation, including tumour suppressors and mutations deemed 'undrugable'
139
How would you translate synthetic lethality into the clinic PARP inhibitors and DNA damage repair?
- first synthetic lethal therapy using PARP inhibitors for patients with BRCA-1 mutant or BRCA-2 mutant (ovarian + breast cancers)
- by interfacing with PARP enzyme, traps PARP1 on DNA, prevents it from being removed, now cells are really dependent on BRCA.
- for tumour cells that have lost BRCA (cause of mutation) adding PARP inhibitor pushes them over the edge, they can't be repaired, and it results in cell death.
- could also use CRSPR to knock out gene int the cell.
- use small molecule library to screen for synthetic lethal ineractors.
140
What are two major obstacles to cellular immortalization?
1) Replicative senescence: an irreversible halt in cell proliferation with retention of cell viability over extended periods of time.
2) Crisis: 'genetic catastrophe' that leads to death by apoptosis.
141
What is replicative senescence?
It is the process of growing old, cells that remain metabolically active but have lost the ability to re-enter the cell cycle, this happens after around 50 years.
Hayflick limit.
142
What are the different roads to senescene?
1) cell culture
2) oxidative stress
3) DNA damage
4) Cytotoxic drugs
5) oncogene activation
6) Telomere dysfunction
- Senescence is triggered by telomere shortening, which can be bypasses by disruption of tumour supressive, (i.e. p53 and Rb pathways)
- After Senescence bypass, cells undergo crisis, during which time chromosomes fuse leading to apoptosis (p53 - independent)
- asynchronous (loose p53 or Rb can bypass this, remove break keep going, telomers short -> crisis, chromosomes ends fuse together leading to apoptosis)
143
How would cancer cells escape crisis?
- cancer cells can escape crisis by expressing telomerase enzyme to keep long telomers (85%-90% of human tumours are telomerase positive)
- at this point, cells that have escaped crisis can then proliferate indefinitely and are said to be immortilized.
- HRTR and telomerase are involved in maintaining length/stability of telomers. It is often upreguated in cancer cells, tumour growth dependent on telomerase levels.
144
Define hypertrophy:
Increase in cell size in reaction to a physiological or pathological stimulus. Occurs in cells unable to divide.
Ex. Skeletal muscle hypertrophy w/ training.
Ex. Increase in cell size in cardiac muscle.
145
Define hyperplasia:
Increase in cell number in response to a physiological or pathological stimuli. Occurs in cells with a capacity to divide.
Ex. Hyperplasia of epithelial cells in female breast during pregnancy.
Ex. Liver regeneration.
- reversible, usually ends when stimulus ends.
146
What is Neoplasia/Neoplasm?
Neoplasm: new growth.
- pathological disturbance of growth characterized by excessive and unceasing proliferation of cells, independent of normal regulatory controls and therefore are irreversible.
- arise from genetic alterations
- neoplasms may be benign (not cancer) or malignant (cancer)
- precursor to cancer
147
What are benign tumours?
- growth that is confined to a specific site within a tissue and gives no evidence of invading adjacent tissue.
- refers to epithelial growth that has no penetrated through basement membrane
- most harmless, can cause issue if they release high levels of hormones or in specific locations (brain)
148
What are Malignant Tumours?
- Aggressive growth, locally invasive, possible metastatic
- 90% of cancer related deaths are the result of metastasis spawned from original primary tumours
149
Every time a cancer cell divides in tumour, what should you assume?
You can assume that the cell population as a whole, doubles in size.
150
How do actual tumour growth models work?
- Human cancers develop many decades of time later
- Age = large factor
- smoking effects only seen 20 years later
151
Describe the documentation on the Intestinal Tract:
- well documented in the epithelial of intestine (high rate of tumours)
- underlying these epithelial is basement membrane (basal lamina) to which cells are anchored
- epithelial layers is site of most of pathological changes associated with development of colon carcinomas
152
Describe the pathway to metastatic colorectal cancer:
1) Normal Intestinal epithelium
-> inactivation of APC tumour suppressor gene
2) Dyplastic epithelium
3) Early adenoma
-> Activation of K-Ras oncogene
4) Intermediate adinoma
-> Inactivation of tumour suppressor gene on 18
5) Late adenoma
-> Inactivation of TP53 tumour suppressor gene
6) Carcinoma
-> Inactivation of other tumour suppressor genes
7) Metastatic colorectal cancer
153
What is EMT?
It is a pathway in cancer biology.
- Epithelial to mesenchymal transition
- EMT is cellular process during which epithelial cells acquire mesenchymal phenotypes and behaviour following the down regulation of epithelial features.
- cells that display fibroblast like morphology, as well as increase migratory capacity.
154
Describe when the carcinoma is in site:
- does not have characteristics of invasive cancer yet
- has not passed through membrane
- not benign neoplasm, but carcinoma (stage 0) - 'pre-cancer'
- as long as completely removed/treated it will be fine
- if left alone, can grow and be dangerous
155
What are some core EMT changes?
1) cytoskeletal remodelling
2) cell-cell adhesion weakening
3) Acquisition of cell motility
4) Basement membrane invasion
156
Describe the Invasion of the Metastasis Cascade:
1) Localized invasiveness enables i site carcinoma cells to breach the basement membrane
2) Intravasation into lymphatic or blood vessels.
3) blood vessels can transport cancer cells to distant sites.
4) cancer cells colonize to form metastatic tumours.
157
What is MET?
- It is the reverse process of EMT
- Mesenchymal to epithelial formation
- Mesenchymal-like cells may acquire apical-based polarity, recognize their cytoskeleton and exhibit increased cell-cell adhesion, resulting in organized epithelium.
158
What do we use model organisms for?
- Testing compounds
- Genetic models -> want to show that gene does (Reverse genetics)
- Genetic screens -> find genes whose loss or gain produces a phenotype (Forward genetics)
159
What are some eukaryotic model organisms?
1) S. Cerevisiae (yeast)
2) C. elegans (nematode)
3) D. Melanogaster (fruit fly)
4) Danio Rerior (zebrafish)
5) Mus musculus (house mouse)
160
Describe some characteristics of S. Cerevisiae:
- eukaryotic, unicellular fungi
- generation time, 2-3 hours
- can exist as haploid or diploid
- can reproduce asexually or sexually
- can be frozen and revived
- replicate by budding, clearly a mother and daughter cell, as you get older there are bud scars, see how many yeast spawned off.
- can exists as haploid cycle, have lines, mutants, cross them, dominant, recessive etc. genetic screening.
- can mate, survive as diploid, be used to test for complementation.
161
Describe characteristics of C.elegans:
- animal, multicellular
- generation time: 3 days, 3000 progeny (time from conception until they are capable of producing progeny, limiting factor in lab environment)
- very simple, translucent, can trace fate of each, (1090 total) cell.
- two sexes, male and hermaphodite, can self fertilize and be crosses.
- can be frozen and revived.
- can freeze dauer worms and preserve them, this has a lot of advantages.
162
Describe D. Melanogaster characteristics:
- animal, multicellular
- generation time: 10 days> 100 progeny
- more complex than C elegans
- extremely well studied, numerous genetic tools
163
Describe Danio Nevio
- Vertebrate animal
- generation time: 2-3 months, 200-300 eggs
- Mid development relatively similar to mammal
- eggs are transparent
- can target genes with morpholinos
164
Are stem cells model organims?
not really
165
Describe the characteristics of Mus Musculus? (Mice)
- Generation time: 3 months, 2-12 progeny
- very small as mammals, easy to house
- Inbred strains available
- closer to humans then most mammals
166
What are some things to consider about larger mammalian model organisms?
- larger in size better for toxicology, pharmacology research.
- closer to humans (primates), long generation times, small litter sizes, expensive, some ethical considerations
167
What are some characteristics about Aradidopsis thaliana?
- model organism of the plant world
- short generation time around 2 months
- normally self-fertilize, but can be crossed.
- dry seeds can be easily stored and shipped.
168
What are organoids?
- three-dimensional structures derived from stem cells and capable of mimicking structure and function of organs in human body.
169
What is the basis of screening?
1) Perturb lots of genes (randomly or systemically)
- most common in mutant genes
2) Look for phenotype
- organism dies, changes etc. in some obvious way
3) Figure out what gene you mutated.
170
How would you screen temperature sensitive mutants?
- to screen them, everything starts the same. place at low and high temp, look for mutants that survive at low but die at high, isolate them and study them.
171
What is an example of finding cell division cycle (cdc) mutants?
- transferred yeast from permissive (23c) to restrictive (36c) temp. Looked for mutants that paused at one point in the cell cycle. Figure out roles of different CDC genes.
172
What is a strategy to identify mutant genes?
Forward genetics: In forward genetics, researchers start with a phenotype of interest and work backwards to identify the gene responsible for the phenotype. This typically involves generating a large population of mutants using chemical mutagenesis or other methods, screening for mutants with the desired phenotype, and then identifying the gene that is mutated in those mutants. This strategy can be used in many organisms, including plants, animals, and microorganisms.
Reverse genetics: In reverse genetics, researchers start with a known gene and work forwards to understand its function. This typically involves creating a mutant of the gene of interest using gene editing techniques like CRISPR/Cas9, and then studying the phenotype of the mutant. This strategy can be used in many organisms, including plants, animals, and microorganisms.
Positional cloning: In positional cloning, researchers use genetic markers to identify the genomic region associated with a phenotype of interest, and then narrow down the region to identify the specific gene responsible for the phenotype. This strategy is often used in organisms with well-characterized genomes, such as mice and humans.
Complementation analysis: In complementation analysis, researchers cross two mutants with the same phenotype to determine if they have mutations in the same gene or in different genes. If the mutants have mutations in the same gene, they will not complement each other and the offspring of the cross will have the same phenotype as the parents. If the mutants have mutations in different genes, they will complement each other and the offspring of the cross will have a wild-type phenotype. This strategy can be used in many organisms, including plants, animals, and microorganisms.
173
How did they find cdc28?
- took library of human cDNA, infected in S. Prombe, temp. sensitive, got 5 colonies.
- human cdk1 can substitue for S. prombe cdk.
- identified many genes that controlled the cell cycle.
174
Describe C. elegans and the discovery of cell death:
- look for mutations uncoordinating mutants, corresponded to nervous system of muscle mutants.
- C. elegans can tolerate a lot of mutations to the nervous system.
- 1090 cells -> form in development, long predictable pattern. Can generate complete lineage of what happens in cell during division, figure out the fate.
You have a wealth of mutant worms with defined phenotypes (unc, lin) and you know exactly what cells are suppose to form and when. You can look at any mutant and determine what cells failed to form. Can complement these mutants w/ cDNA or genomic DNA, then figure out the sequence of the gene.
They saw that 959 cells exists in adults (cell death mutants are discovered) and 131 cells die during development.
They discovered that the cell mutant was Ced-1 (-1)
Last stage of apoptosis, when cell dies, gets swallowed by adjacent cell, ground up lysosome and dissolve.
175
After doing the first screen in discovering cell death they found ced-1 (-1) What was ced-1 (-1) used for? When they did the second screen, what did they discover?
- when ced-1 (-1) is there dead cells do not get swallowed and recycled, they just sit there.
- they are really noticeable on microscope.
- used as a tool to study apoptopic pathway.
- when they did the second screen, they found a second mutation, to rescue phenotype and make it look normal. They got a gene that is Ced-3 (-1). Virtually all the 131 apoptopic cells had this.
- Ced-3. It proves cell death. and showed that it was an active process "apoptosis". This is essential.
176
What is the Egl-1 mutant?
- promotes cell death
- got more Ced3
- got Ced4
- when Ced9 is on, cell survives
- When ced9 is off, you have cell death
Using BLAST, map Ced9 and find similarity to Bcl-2 which is an oncogene.
- The Egl-1 mutant refers to a strain of C. elegans that carries a mutation in the Egl-1 gene, which leads to a reduction or loss of its function. As a result, the mutant worms exhibit defects in programmed cell death, which can affect their development, behavior, and survival.
177
What is BAK/BAX?
- it is similar to Egl-1
- it is inappropriately expressed and it is a pre-apoptotic protein
178
What happens when you express Bcl-2 in humans? What was the process to figuring this out?
- When you express Bcl-2 in humans, you inhibit apoptosis in C.elegans. Both Ced9 and Bcl2 promote cell survival.
1) Introduce vector w/ promoter. For Hsp-16 turns on if you heat shock the C. elegans.
2) Clone cDNA for Ced9.
3) Clone in cDNA for bcl-2 cDNA.
4) Introduce vector and heat shock, look at nervous system around.
Control= nothing happens
Ced9= any extra cells that normally die in development (block apoptosis)
Bcl2= not as effective as Ced9, but same effect, anti-apoptic protein.
179
What is the current understanding of the apoptosis pathway?
1) death stimulus signal, activates proteins (BAK, BAX) cause leakage of cytochrome C from mitochondria.
2) Blc2 function, anatagonize BAK ad BAX. (homologous to Ced9)
3) If death signal is too strong, BAK and BAX keeps going.
4) Ced4/APAF1 binds, cytochrome C leaks, APAF binds it, activates Casp9, cuts itself, cuts and activates Ced-3/Casp3, this is exoctioner and causes apoptosis.
180
Wild type
Cell divides and stops at appropriate time.
181
Loss of function Lin14 mutant:
division of cells shuts off early.
182
Gain of function Lin14 mutant
protein develops too much, just keeps repeating early-stage larvae.
183
What normally happens to Lin 14 proteins?
- They are normally suppose to go away, but mutants occur so transcript is always present and lin14 just keeps going.
184
What is the difference between Lin4 and Lin14?
Lin4= opposite of Lin14.
Lin4 LOH = Lin14 Gain of function
- Lin4 only purpose is to block expression of Lin 14.
- lin14 and lin4 are two opposing hetero-chronic mutants
- lin14 does not code for a protein, they did northern blot to see what it produced.
- The function of Lin-14 is to repress the expression of certain genes that promote the transition from the early to the later stages of development, particularly in the first two larval stages. This repression is necessary to ensure that developmental events occur in the correct sequence and at the appropriate time.
- so you want it to be regulated and have the correct amount.
- Lin-4 is considered "good" because it plays a critical role in ensuring that developmental events occur in the correct sequence and at the appropriate time during the development of C. elegans. Without Lin-4, the timing of developmental events is disrupted, and the worms may not develop properly.
185
How did they figure out what Lin4 binds Lin14 3' UTR and inhibits translation?
- WT, Lin4 was a little 21 base transcript (had some 60 but at a lower level)
- In Lin4 mutant, it goes away
- Over express Lin4, 21 base comes back and see bigger 60 bases.
186
What is the function of Let7?
- it is another 21 base RNA w/heterochrony phenotype
- does not protein code etc.
- Let-7 is a family of small RNA molecules that regulate gene expression. It is complementary to specific mRNA sequences and functions as a post-transcriptional regulator of gene expression by binding to the 3' untranslated region (UTR) of target mRNAs, which can lead to mRNA degradation or inhibition of protein translation.
- They cloned 21 base area, and saw micro-RNAs everywhere. Could then figure out the machinery involved in making them, and the reason they are the same size.
- conserved from worms to humans. Human tumour suppressor. In lung cancer, let7 is down regulated, upregulation of gene Ras.
187
Describe miRNA processing and activity
1) Transcription of primary transcript (microRNAs)
2) Drosha
3) Pe-microRNA
4) exorted --> Dicer, chops 21 base form
5) Protein argonaute comes, binds, guide to find RNA transcripts in genome silence. Chops, promotes deadnylation, blocking translation.
- miRNAs often recognize complementary sequence --> 3' TUR
- beginning and end need good homology but inside does not need to be perfect
The miRNA processing pathway can be broadly divided into two steps: (1) the maturation of primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs) in the nucleus, and (2) the processing of pre-miRNAs into mature miRNAs in the cytoplasm.
In the nucleus, pri-miRNAs are transcribed from genomic DNA by RNA polymerase II or III as long, single-stranded RNA molecules with a stem-loop structure. The stem-loop structure contains the miRNA sequence and a complementary sequence known as the miRNA* or passenger strand. The pri-miRNA is first processed by the Drosha/DGCR8 complex, which recognizes the stem-loop structure and cleaves the RNA at a specific site to generate a pre-miRNA hairpin structure of approximately 70 nucleotides in length. The pre-miRNA is then transported to the cytoplasm by the nuclear export protein Exportin-5.
In the cytoplasm, the pre-miRNA is further processed by the RNase III enzyme Dicer, which recognizes and cleaves the stem-loop structure of the pre-miRNA to release a miRNA duplex. The miRNA duplex consists of the mature miRNA and the miRNA* strands, which are both approximately 22 nucleotides in length. The duplex is then loaded onto an Argonaute (AGO) protein to form the miRNA-induced silencing complex (miRISC).
188
Describe the Drosophilia experiement in discovering sevenless: finding modulators of a pathway.
In Drosophilia, there are eye repeating units a few hundred times, and H photo receptors R1-R8. They noticed a mutation --> sevenless. This was when there was a missing photo receptor that was R7. Sevenless is a receptor tyrosine kinase, essential only in gene R7. It binds adaptor protein, recruits exchange factors, Induces RAS to let go of GDP, and take up GTP, which activates RAS and downstream signalling. Ras-GEF (Sos) - son of sevelnless.
They also discovered that if you knowck out BoSS in R8, sevenless never gets stimulated, and R7 does not survive.
189
What is the difference in discovery from a suppressor screen approach to an enhancer screen approach?
Suppressor gene screen approach:
If you have AAAA --> B B (X) --> C C, too much A gets counterbalanced by loss of B, reducing of B eliminates phenotype.
Enhancer screen approach:
A -> B B (X) -> C C Reduction of B creates or alleviates phenotypes.
190
How did they find downstream components for Drosophilia/Sevenless (Suppressor approach example)
Wild type: Normal eyes
Sevenless: weird eyes
overactive sevenless: messed up eye
overactive sevenless + downstream mutation (such as son of sevenless - Sos) -> counterbalance having overactive sevenless for mutants that have male normal looking eye with one copy of sevenless.
191
How did they find downstream components for Drosophilia/Sevenless (Enhancer approach example)
1) Use temp. sensitive mutant of sevenless gene itself.
-> at restrictive temp, they don't have normal activity/fold.
- as you raise temp, its not all of nothing, there is a range where some protein is folding correctly and some is not.
- 24.3 degrees, sevenless phenotype.
- 22.7 degrees, wild type phenotype.
this helped find downstream components
192
What are transposons?
- Jumping genes
- Parasitic DNA elements that are capable of making copies of themselves and integrating into new parts of genome.
193
What are the two broad families of transposons?
1) Retrotransposons (copy and paste)
2) DNA transposons (cut and paste)
194
Describe retrotransposons
1) Retrotransposons (ERV and LTR)
- copy and past
- transcribed, reverse transcribed to DNA, take DNA, cut genome, integrate new copy of DNA.
2) Non-LTR Retrotransposons
- do not have long terminal repeats
195
Describe DNA transposons. what is a bad result or an advantage that could occur.
- never go through RNA intermediate
- transposon codes for some protein, cuts out transposon, helps integrate into new part of genome
- if transposon cuts in the middle of that gene, can destroy the function of that gene (inheritantly mutagenic) - this is why transposons are bad.
Advantage: A way to make mutations. Mutated genome w/ transposon, have method to figure out what gene, you just mutated. --> PCR to figure out gene w/ transposon landing in it is what got mutated. Sequence DNA around the landing site.
Transposons used as mutagen to conduct genetic screens.
196
Drosophilia field have DNA transposons called P-elements. What are things that a modified P-element can do?
1) Insertional mutagenesis: P-element lands in gene, disrupts its function.
2) "Gene Trap": same as above, with reported that is expressed with gene.
- when in development a given gene is expressed
3) "Enhancer trap": P-element lands near enhancer, contains reporter that is expressed when enhancer is active.
- When in development a particular regulatory element that controls the expression of genes is expressed.
197
What is a big advantage of the P-element?
You can use PCR to figure out where your element landed, and what gene/enhancer is disrupted.
198
Describe mammalian embryonic development:
- sperm fertilizes egg = zygote
- mouse and human quite similar at this stage of development, human develops a bit slower.
- then division "cleavage stage of embryo", still finite size, bounded to membrane, cells getting smaller.
- ZGA occurs. Transcription starts occurring from the actual embryo. Maternal transcripts start being degraded at the same time. (important when study mouse -> cut early stages, mRNA is not coming from mouse it is coming from mother)
- Then 16 cell stage and you start to see diversions. Distinct inner and outer cells. Outer cells epithelial form layer, inner cells apolar.
- embryo begins to fill with fluid, forms blastocyst (ball of cells)
- outer cells --> trophoblast
- inner cells -> inner cell mass, divergent fate, some epiblast (apolar), some primitive endoderm (hypoblast)
- eventually three lineages form
- implantation in the uterus
- baby umbilical cord = epiblast
- placenta = Trophoblast
199
What is the function of the placenta?
It mediates nutrient and gas exchange with the mother, it exists because we do not lay eggs.
200
What happens after implantation?
- more complex structures form around epiblast, ball cells, layer cells, differentiation, gastrulation.
- gastrulation = primitive streak, groove in disc. Where cell differentiation occurs.
- cell of epiblast form different lineages, cells migrating, endoderm, mesoderm. The epiblast that do not migrate, become last of three germ layers = ectoderm.
1) endoderm (internal layer)
2) mesoderm (middle layer)
3) ectoderm (external layer)
- posterior is the back where the primitive streak is, and anterior is the front.
201
What happens after Gastrulation?
- notochord (critical cord of signalling molecules to guide development - day 17)
- neural plate (day 19)
- neural groove etc. Somites -> give rise to muscle tissue (Day 20)
- day or two after, heart starts beating.
- Cells special functions and organs form.
202
What was a subsequent main concept discovered after gastrulation?
- Hox genes expressed along anterior-posterior axis
- critical in guiding anterior/posterior development m
- in humans and mice, 4 clusters of Hox genes (A, B,C, D) all same pattern.
- anterior activated earlier than posterior.
- pattern on 2D -> 3D.
203
What are morphogens? What is an example?
- Morphogens are signaling molecules that play a critical role in controlling the development and patterning of tissues and organs in multicellular organisms.
1) field of cells
2) morphogen gradient forms (exposure to secreted protein)
3) cellular response to gradient
- the distance away from the morphogen determines the fate of the cell
- closer more signalling
ex. Neural tube. BMP morphogen. Each different section expresses different gene regulatory proteins expressed etc.
204
What is BMP pathway?
- a lot of signalling pathways
- receptors, phosphorylated, mediate transcription
- BMP-4: ectroderm (epithelial stem cells), mesoderm (stem cells), endoderm (hepatocyte progenitors)
205
There are hundreds of cell types from a handful of pathways, how is this possible?
1) Combinatorial signalling
- get signals from different things at any one time
- TFs
2) Cell memory
- if two cells are not the same to start, the way they would respond to different signals will be different. Different sets of Transcription factors and chromatin openess.
206
Describe the "cellular memory" at the level of chromatin
1) Euchromatin: can bind to TFs
2) Heterochromatin: bind less, cause more tightly bound.
207
Explain the 4 different kinds of domains:
PTB domain: (phospholoryated tyrosine domain binds but in a specific motif (specific sequences in a certain location- guarantee protein-protein interactions.)
* Have NPX motif
* Proline X tyrosine
Can physically bind to tyrosine to the signalling receptor.
PH domain:
* Binds all kinds of different lipids
* Many types of PH domains - but all have the same name
◦ Bind to molecule with 3 phosphates on it - PI(4,5)2. Generate molecule on plasma membrane and the IRS1 adaptor protein of this domain binds on to it. Green protein can also be phosphorylated and recreuit a different domain, SH2 domain. Can be phosphotyrosines or phoshoserines. Does not have same binding ability as PTB domain, but both bind to phosphorylated residues.
SH2 domain:
Bind other phosphorylated residue.
SH3 domain:
* Binds to proline-rich regions. Have certain domain on protein that has multiple prolines. Now will specficially bind to them and recruit them when you need them.
* Sos can make a complex of proteins, and mediate a downstream complex with effects.
208
How is cyclic AMP turned on/regulated?
Activated GPCR (G-protein coupled receptor) . Alpha subunit is activated by GTP.
connected is adenylyl cyclase. Now we generate active cycle AMP. because it is binding to adenylyl cyclase.
Cyclic AMP binds to regulatory subunits of PKA (protein kinase A) . Releasing catalytic domains, which go into nucleus, phosphorylate
CREB, activates it, and promote transcription of whatever genes
we want.
209
What are some events that signalling G proteins and Cyclic AMP leads to?
Liver: Vasopressin: Glycogen breakdown
Pancreas: Acetylcholine: Amylase secretion
Smooth muscle: Acetylcholine: Muscle contraction
Blood platelets: Thrombin: Platelets: Platelet aggregation
210
How does Cytokenesis actually happen?
Have the two spindle poles. One on the left and one on the right.
This is where the microtubules are originating from and spreading out towards the central plane from both sides.
Get different kinds of microtubules (big strong, bind to kinetochore, which goes on both sides)
- when both bound, they can start pulling.
- there are also less strong excess microtubules, so these are going to help pull apart.
- Motor proteins that are binding to mitotubules called Kinesins which pulls in the positive direction.
-Microtubule is grounded at the centrosome, this is the negative end. Polarized polymer of tubulin subunits, negative end in centrosome, positive attaches to chromosomes. Kinesins move from negative end to positive end.
- Dyaneins move to negative direction.
They work together everything does to help pull apart.
- Need to line them up properly for this to work properly, on the plate, this is what the motor proteins are doing they are joselin them around and getting them in the right position.
- When it is all in the right spot then you proceed to the seperation!
