Final Exam Notes.txt

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  1. Does the nuclear envelope of a yeast cell break-down during the M phase?
    No, therefore the microtubule-based mitotic spindle forms in the nucleus
  2. What is the deal with fission yeast?
    They divide by forming a partition (cell plate, or septum) and have the typical progression of the eukaryotic cell cycle.
  3. What is the deal with budding yeast?
    They divide by budding and have normal G1 and S phases, but atypical (hazy) G2 phases.
  4. Describe the behavior of a temperature-sensitive Cdc mutant:
    At permissive low temperatures (the permissive condition), the cell appears normal and divides normally. But upon warming to the restrictive temperature (the restrictive condition), mutants stop progress at the specific step in the cell cycle they are unable to complete.
  5. Cdc genes:
    Cell-division-cycle genes; genes which encode a Cdc protein, controlling a specific step of set of steps in the eucaryotic cell cycle, originally identified in yeasts. Many mutations affecting these genes may cause the cell to arrest at a specific point in the cell-cycle.
  6. When can a Cdc mutant be selected and maintained?
    When their phenotype is conditional (temperature sensitive, etc)
  7. What would happen if the Cdc mutation was not conditional?
    We can't study them, because they would just die
  8. The power of yeast with respect to Cdcs:
    They allow us to look for mutants and allow us to clone genes from mutants
  9. What are cleavage divisions?
    Division of a cell without growing, as in with the Xenopus oocyte.
  10. What are the advantages to studying early embryonic cell cycles?
    They reveal the workings of the cell-cycle control system in a simplified manner to achieve duplication of the genome and segregation into daughter cells. Also they are very large, for easy observation. Also they are a synchronized system, same cell cycle starting point
  11. Disadvantage to studying cell-cycle control on cultured Mammalian cells?
    Under standard conditions, isolated, cultured cells stop dividing after a certain number of cycles. For this reason mutated cell-lines may be "immortalized", proliferating indefinitely and allow for an unlimited source of genetically homogenous cells.
  12. How can we tell what stage an animal cell has reached in the cell cycle?
    Look at the living cells in a microscope, staining with DNA-binding fluorescent dyes (chromosomes), staining with antibodies recognizing cellular components (such as microtubules, revealing mitotic spindles). S-phase cells can be identified by supplying them with BrdU, which is a thymidine analog that gets incorporated into the DNA, and then staining with anti-BrdU antibodies (and can estimate duration of phases).
  13. The General Cell-Cycle
    G1 (growth phase from which G0 can be entered), S phase (duplication of genetic material), G2 phase (growth phase 2), M phase (mitosis). From G1 only, cells can enter G0 and leave, as long as they haven't differentiated.
  14. What is the deal with cellular differentiation and the cell cycle?
    Any cell must first leave the cell cycle and enter g0, in order to differentiate. With the epithelial cells, they get more more differentiated towards the top layer. Once cells differentiate, they do not renter the cell-cycle. Exception: advanced cancer cells de-differentiate and go back into the cell cycle
  15. What two cells were fused in the Xenopus system and what did we find?
    People took a mitotic cell (no nuclear membrane, chromosomes) and fused it with a G1 cell (chromatin) (using drugs or viri), and found that the G1 nucleus to disintegrate and the chromatin to start condensing. therefore some factor in the mitotic cell that would induce mitotis
  16. Start checkpoint:
    • In late G1, where the cell commits to cell-cycle entry and chromosome duplication
    • (Even if extracellular signals that stimulate cell growth and division are removed); no entry into G0
  17. G2/M Checkpoint:
    Trigger of early mitotic events leading to chromosome alignment on mitotic spindle. Occurs with favorable environment and complete replication of DNA
  18. Metaphase-to-Anaphase Checkpoint:
    Stimulation of sister-chromatid separation. Occurs if all chromosomes are attached to the mitotic spindle. (any non-properly attached kinetochore sends out a signal blocking the Cdc20-APC/C activation).
  19. CDks:
    • Cyclin-dependent kinases which phosphorylate proteins that initiate or regulate the major events of the cell cycle. Their levels are constant in the simplest cell cycles, though their activity levels cyclically rise and fall with the control of a complex array of enzymes and other proteins that regulate these kinases, especially cyclin. Without being tightly bound to cyclin, whose levels cyclically fluctuate, CDKs have no kinase activity. This is because the CDK active site is partially obscured without cyclin…but it takes the conformational change caused by the phosphorylation of an amino acid near the active site by Cdk-activating kinase (CAK) to induce full activation, increased CDK activity.
    • They are the catalytic subunits
  20. What happens with an increase in Cdk activity at the G2/M checkpoint?
    Increased phosphorylation of proteins that control chromosome condensation, nuclear envelope breakdown, spindle assembly, and other events occurring at the onset of mitosis.
  21. The four classes of cyclins:
    • 1. G1/S-cyclins
    • 2. S-cyclins
    • 3. M-cyclins
    • 4. G1-cyclins
  22. Cyclins:
    • The most important Cdk regulator. Proteins upon which Cdks are dependent. They undergo a cycle of synthesis and degradation in each cell cycle. The cyclical changes in cyclin protein levels result in the cyclic assembly and activation of the cyclin–Cdk complexes; this activation in turn triggers cell-cycle events.
    • They are the regulatory subunits.
  23. G1/S-cyclins:
    Activate Cdks in late G1 and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase.
  24. S-cyclins:
    Bind Cdks soon after progression through Start and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events.
  25. M-cyclins:
    Activate Cdks that stimulate entry into mitosis at the G2/M
  26. G1-cyclins:
    Helps govern the activities of the G1/S cyclins, which control progression through Start in late G1, in most cells.
  27. Ubiquitin-Protein Ligases:
    • regulate transitions at critical cell-cycle points by regulated degradation The irreversible protein degradation assures unidirectional progression in cell cycle.
    • Feeds into accelerators and the brakes of the cell-cycle-control complex
  28. Checkpoint Mechanisms:
    • Molecular surveillance mechanisms to ensure that cell-cycle transitions are not initiated until the previous stage has been completed successfully.
    • How to identify a defective Cdc genes by using permissive mutants:
    • Take mutants and add library of all the genes of that species (DNA library--all DNA chopped up and put in vectors--plasmids). Every cell would only pick up one plasmid. If the plasmid is encoding a normal gene, the mutant will stay mutant, not form colonies (at higher temperature, for example). But if the defective gene is added, then the mutant becomes normal. Then you meet the condition of the conditional mutant (raise the temperature) and can form colonies. Now go back to the cells, and take out the plasmid, you sequence it and know what the gene was.
  29. What are the "accelerators" in Cell Cycle progression?
    • Mainly Cyclins, CDKs, (and Cyclin-CDK complexes)
    • Regulate protein phosphorylation, pushing the cell to go to the next level
  30. What are the protein degraders in the Cell Cycle Progression?
    Ubiquitin-Protein Ligaeses; regulate transitions at critical cell-cycle points by regulated degradation assuring unidirectional progression in cell cycle. Plays a role with the accelerators and the brakes.
  31. What are the "brakes" in cell cycle progression?
    Checkpoint Mechanisms: Molecular surveillance mechanisms to ensure that cell-cycle transitions are not initiated until the previous stage has been completed successfully
  32. CDK1:
    the only CDK complement for various cyclins in (budding) yeast, also the CDK complement for cyclin B in vertebrates forming M-CDK [also Cyclin A to form S-CDK, but CDK2 also is Cyclin A's complement)
  33. Maturation Promoting Factor:
    Purified from Xenopus eggs, able to induce Xenopus eggs into maturation and mitosis once sperm was added. Essentially the cyclin-CDK complex. Scientists knew from enzyme assays this was a kinase, made of two components (enzyme and regulatory subunit). They could see that with every round of cell division was preceded by a rise in regulatory protein levels, thus they termed this 'cyclin'.
  34. Experiment demonstrating dynamics of mitosis-regulation and how cyclin-CDKs are generically regulate:
    • Following the activity of MPF (phosphorylation of proteins) and the presence of cyclin B in early and late mitotic events (prior to and after the metaplate)
    • a. Untreated extract: Following fertilization, both MPF activity and cyclin B levels rise in and before early mitotic events, then sharply drop synchronously.
    • b. With addition of RNA-ase, nothing happened with MPF nor cyclin B, as well no division. Shows that these things and going through cell-division need new translation
    • c. Then they inactivated the RNAase and added a species of mRNA for cyclin B. Just that was sufficient to reinstate everything. So just cyclin B was necessary and dependent on new translation, and the kinase didn't need to be made anew.
    • d. Then they added a chemically-modified version of cyclin B mRNA that is non-degradable from the RNAase. Now the levels of the cyclin went up as well as the MPF activity, but they never came down, and the cell never proceeded to late mitotic events. So this shows that the cell not only has to make cyclin B, but it has to take it away to not undergo mitotic arrest. That is typical of all the cyclins.
  35. CAK:
    • Cdk-activating kinase; induces a conformational change by the phosphorylating an amino acid in the T-loop, near the active site, of the CDK in the cyclin-CDK complex. This induces full activation, increased CDK activity. Takes place at the
    • (serine/threonine 161) in Xenopus.
  36. Wee1 kinase:
    • Inhibits Cdk activity by phosphorylating two closely spaced sites above its active
    • site. In Xenopus, the inhibitory phosphorylation is at tyrosine 15
  37. Cdc25 Phosphatase:
    (Re)activates cyclin-Cdk complex by removal of the phosphates placed by Wee1 kinase. Dephosphorylation occurs at tyrosine 15 in Xenopus.
  38. The three levels of control of mitotic Cdk:
    Binding to cyclin, then a cascade of phosphorylation and dephosphorylation of Cdk, then the inhibitory protein p27 binding to Cdk
  39. Duo-Specific enzymes:
    Small group of enzymes (only 2 or 3 identified) that phosphorylate both tyrosines and serines/threonines
  40. CKI:
    Cdk Inhibitor Proteins; Inhibit the function of cyclin-Cdk complexes by capping, and after it has been phosphorylated, it is polyubiquitinated by SCF and can be degraded.
  41. p27:
    Inhibitory protein comprising the third level of regulation in which the cell temporarily inactivates the cyclin-Cdk complex by capping it with p27, until it can be used. Migrating at 27 K-dalton, hence its name
  42. APC/C:
    • Anaphase Promoting Complex or cyclosome, is a member of the ubiquitin ligase
    • family of enzymes (E3). Ubiquitinates two proteins, securin and the S-and M-Cyclins, leading to their destruction.
    • When bound to a non-phosphorylated Cdc20 or Cdh1, it is activated. M-cyclins had a domain for the polyubiquitination, a destruction box. once the cyclin is destroyed, the CDKs are inactivated, leading to the dephosphorylation of their substrates by phosphotases (required for the completion of the M phase).
    • Securin inactivates separate. When degraded, then separase is made active and can cleave cohesins, leading to anaphase.
  43. What are the enzymes responsible for the de/phosphorylation of the activating subunit of APC/C (Cdc20 or Cdh1)?
    Cdc14 phosphatase (dephosphorylates prior to metaphase, therefore promoting active APC), and G1 cyclin-Cdk (phosphorylates through late mitosis)
  44. Securin:
    A protein binding to and inhibiting separate, prior to anaphase. Active APC then polyubiquitinates securing such that it degrades and separate then can cleave the cohesins and anaphase is initiated.
  45. Cohesins:
    Proteins binding the sister chromatids together throughout the structure…must be broken down by separase, which means the attached inhibitory securin must be degraded first.
  46. Lysosomes:
    Degrades proteins, lipids, nucleic acids, etc.
  47. Proteosome:
    An open barrel lined with proteins with protease function, degrading entering proteins. Any protein with dozens of ubiquitin added in a chain fashion is a target. Polyubiquitination is therefore the signal for proteosome degradation.
  48. What are the enzymes responsible for polyubiquitination?
    E1, E2, E3. Activate ubiquitin, passes ubiquitin to the ubiquitin ligase, and the ligase adds the ubiquitin to the protein. And this repeats.
  49. Substrates of MPF or Mitotic cyclin-Cdk:
    Nuclear lamins, and phosphotase (CDC25, the more phosphorylated, the more active), and therefore is involved in a positive feedback loop, and wee1 kinase (M-Cdk inhibits this inhibitor somehow, another positive feedback).
  50. How does the nuclear membrane know to fall apart?
    Under the nuclear membrane is the meshwork known as the nuclear lamina. The nuclear lamins A, B, and C are the proteins (homologous to intermediate filaments) that make up the lamina; not phosphorylated, they come together forming long fibers. Lamin B is the one actually anchored to the membrane. With phosphorylation of the tetramer, the interactive complexes fall apart (creation of lamin dimers); (according to slides, cells with mutant human lamin A are not phosphorylated by MPF)
  51. Situation of S cyclin-Cdk:
    • Cyclin must bind CDK. There must be phosphorylation/dephosphorylation happening, an inhibitory protein, and protein degradation.
    • Promotes the S phase (DNA replication). CDK must be bound by the cyclin, and is in a complex already in G1, but isn't active due to the inhibitory protein until mid-late G1. At the coincidental peak of the G1 cyclin CDK, during G1, has Sic 1 as a substrate. That then gets hyperphosphorylated, and then is a target of polyubiquitination and protein degradation. Once that happens, the S1 cyclin CDK is then active. Leads to initiation of DNA replication.
  52. Organ and body size are determined by what fundamental processes?
    Cell growth, cell division, and cell death.
  53. What are the categories of the extracellular signal molecules regulating cell size and number?
    • 1. Mitogens, which stimulate cell division, primarily by triggering a wave of
    • G1/S-Cdk activity that relieves intracellular negative controls that otherwise block progress through the cell cycle.
    • 2. Growth factors, which stimulate cell growth (an increase in cell mass) by
    • promoting the synthesis of proteins and other macromolecules and by
    • inhibiting their degradation.
    • 3. Survival factors, which promote cell survival by suppressing the form of
    • programmed cell death known as apoptosis.
    • They are
    • generally soluble secreted proteins, proteins bound to the surface of cells, or
    • components of the extracellular matrix.
  54. Mitogens:
    • Extracellular signals (proteins) stimulating cellular proliferation. control the rate of cell division, acting in the G1 phase of the cell cycle, interact with cell-surface receptors to trigger multiple intracellular signaling pathways.
    • As we discuss in Chapter 15, mitogens interact with cell-surface receptors to
    • trigger multiple intracellular signaling pathways. One major pathway acts
    • through the small GTPase Ras, which leads to the activation of a MAP kinase cascade. This leads to an increase in the production of gene regulatory proteins,
    • including Myc. Myc is thought to promote cell-cycle entry by several mechanisms, one of which is to increase the expression of genes encoding G1 cyclins
    • (D cyclins), thereby increasing G1-Cdk (cyclin D–Cdk4) activity. As we discuss
    • later, Myc also has a major role in stimulating the transcription of genes that
    • increase cell growth.
    • The key function of G1-Cdk complexes in animal cells is to activate a group
    • of gene regulatory factors called the E2F proteins, which bind to specific DNA
    • sequences in the promoters of a wide variety of genes that encode proteins
    • required for S-phase entry, including G1/S-cyclins, S-cyclins, and proteins
    • involved in DNA synthesis and chromosome duplication. In the absence of
    • mitogenic stimulation, E2F-dependent gene expression is inhibited by an interaction between E2F and members of the retinoblastoma protein (Rb) family.
  55. START/Restriction Point
    • -point in cell cycle when cell "commits" to entering S phase and completing cell cycle
    • -happens late in G1 phase
    • -Before START point, E2F inactive. After START point, E2F is active!
  56. 3 checkpoints that detect state of chromosomes (not DNA damage)
    • 1. Intra-S-phase checkpoint
    • 2. Spindle assembly checkpoint
    • 3. Spindle position checkpoint
  57. E2F
    • -E2F is a transcription factor. It binds to DNA and causes transcription activation.
    • -transcribes genes encoding DNA replication and S phase cyclin/Cdks.
    • -is a "delayed response gene"
    • -activated by cFos
  58. Possible changes in E2F activation in Cancer cells
    • 1. Rb is inactivated by another mean (mutant Rb)
    • -retinoblastoma cancer has a mutant Rb...E2F is always active!
    • -Rb mutation is a loss of function mutation
    • 2. Ras is always active (mutant Ras)
    • -phosphorylation of Rb would no longer be dependent on mitogen
    • -gain of function mutation
    • -Ras mutation is 2nd most common cause of cancer
    • 3. Gain of function of Myc
    • -cause of many blood cancers
  59. Mechanism for activation of E2F
    • 1. Mitogen binds to mitogen receptor
    • 2. mitogen receptor is activated and activates Ras
    • 3. Ras triggers MAP kinase cascade
    • 4. MAP kinase cascade leads to transcription/translation of immediate early genes (one gene being Myc)
    • 5.Myc (a regulatory protein) binds to G1-Cdk and activates it indirectly
    • 6. activated G1-Cdk phosphorylates Rb protein
    • -Rb protein is an inhibitory protein. Before phosphorylation, Rb attaches to E2F and keeps it inactivated. Once phosphorylated, it unbinds from E2F.
    • 7. Rb protein then releases E2F...E2F is now free and active!
  60. Gain vs. loss of function
    • Oncogenes (gain of function that we se in cancerous cells)
    • tumor suppressor genes (loss of function)
    • and caretaker genes (loss of function)
    • colorectal cancer, progression, and the group of mutated genes in a progressive manner
  61. Therapies: three types:
    • conventional cancer therapy: (been around since late 60s), standard of firstline therapy today
    • new wave of chemotherapy: …more general… when you don't know something specific about the cancer, just attack all cancer
    • newest: most research: targeted cancer therapy. to develop this drug, need to know something about the molecular change
  62. Conventional therapy:
    generally includes chemotherapy and/or radiation therapy; oncologists needs to assess good candidacy and the specifics of the treatment; before knowing much about the cancer, they knew some drugs would cause cultured cancer cells to lyse and die. This was actually apoptosis, but it took a while to figure this out. Chemotherapy is a systemic approach. Some drugs are administered for 10 minutes, some for 3 hours. All your cells get exposed. But damage happened faster and was more drastic with the cancer cell lines. Radiation therapy can be targeted to a specific location, to where the growth is. Causes DNA damage. Blood cells most susceptible normal cells. Therefore ~10 yrs ago, if blood count low, they had to stop treatment. Nowadays, prescribe injections of growth factors
  63. HOw to start on therapy?
    Many cancers don't respond well to chemotherapy and radiation therapy (usually a daily thing). So they start you off for 6 weeks. But after metastasis, radiation therapy not done. Trying to see if it takes. Generally Cancer Cells not responding well generally had damage in p53, due to not going through apoptosis. Also since you are promoting damage, if you damage p53, it may make the tumor non-responsive to drugs, as they try to induce apoptosis and without p53, they can't.
  64. Chemotherapy:
    • Taxol:
    • inhibited the depolymerization of microtubules, didn't allow cells to depolymerize them, and repolymerizes them into spindle fibers during cell division
    • therefore chromosome separation was affected.
    • Nucleotide analogs, they look like nucleotides, something wrong with them, get incorporated and wouldn't replicate normally
  65. New Wave of non-conventional general chemotherapeutic agents:
    • DNA Replication inhibitor; their goal is to prevent the tumor from growing, not destroy.
    • For example: Alimta: discovered in a cell bio lab, Suppresses growth, believed to be p53 independent. These tumor cells sometimes do shrink (senescence). Enters cells using the folate (B12) transporter. Well tolerated for long term (>13 cycles, a few years) treatment: Adding daily folate and B12 shots minimize toxicity.
    • IN the beginning, people on this drug were becoming B-12 deficient. So they basically take extra B-12.
    • Considered a treatment, not a cure.
    • Complications: can lead to resistance due to high probability of mutations occurring rendering the cancer resistance and the drug ineffective.
  66. Targeted therapy:
    • custom designed anti-cancer drugs, specific to the cellular aberration in the specific type of cancer. Cost is an issue, small target audience, at the same time, hundreds of drugs can be developed. Another limitation can be needing to know about the particular aberration.
    • Example: Gleevec: Abl kinase inhibitor
    • small molecule drug, they fit into the active site/domain in alb kinase and inactivate it. A kinase that has a regulated expression, turned on at particular time. proliferation MAP kinase pathway. With common leukemia, alb gene is translocated onto another chromosome and into downstream from a highly active promoter. Suddenly a kinase gene normally under tight regulation is being made all the time and it's proliferation signal is on all the time. people don't know why these translocations occur, and the particular chromosome that results can be screened by karyotyping and is called a philadelphia chromosome. Suddenly an alb kinase is constitutively being made. Gleevec binds the (gain of function enzyme) kinase and inactivates it.
    • *doesn't go over slide 8
  67. Come up with inhibitors to the gain of function enzymes
    • Get inhibitors or molecules that inactivate receptors so they cannot transduce the signal
    • tamoxifin binds estrogen receptor and inactivates it. If the cancer is positive for that receptor, then the patient is put on this.
  68. using antibodies against antigens.
    • Proteins are antigens.  Mamallian systems make antibodies against foreign antigens. Purifying
    • the antibodies will be a tool against the antigen. To fish it out or
    • Sometimes when they are bound the antigen is inactivated as well.
  69. Avastin:
    : monoclonal antibody (recognizing one specific region of a protein) to VEGF (vascular endothelial Growth factor, a mitogen), used in treatment of kidney and colon cancer. Successful tumor cells secrete VEGF, they develop vasculature, and then their VEGF can be rendered inactive.
  70. Avstin also used for:
    • Macular degeneration, disease of aging where the retina degenerates and you go blind. The common form is by growth of blood vessels into the retina when they shouldn't. Then people applied VEGF to retina. and this is now a common practice.
    • very different field benefitting from the molecular basis of VEGF
  71. Ras is
    • the second most commonly mutated gene in a number of cancers, also it is
    • the G protein involved in MAP kinase signal transduction. Ras is
    • anchored to the membrane and must be for signal transduction, despite
    • active or in high levels. gain of function in RAS in cancers, inhibit
    • ras from being bound to membrane. Then we inhibit the enzyme binding the
    • ras to the membrane, so we inhibit ras farnesyl transferase: down
    • regulates MAP kinase cascades. inhibitors, for gain of function
  72. Gene replacement approach,
    • not limited to cancer, introduce good copies of the genes, used for the
    • loss of function mutation, like p53 introduction, explored with viral
    • vectors, for tumor suppressor genes/loss of function
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Final Exam Notes.txt
2013-05-16 15:26:12
BIo 340

bio 340
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