Biology, Cell Biol 4001-001 Spring 2009 Exam 4
Name:_____________________________
Choose the best answer for the first 15 questions (3 points each): 1. Which of the following statements about Maturation Promoting Factor (MPF) is FALSE: a) It is a diffusible factor able to induce chromatin/chromosome condensation when injected into cells at any stage of the cell cycle. b) It is an enzyme with two subunits: M-cyclin and a cyclin-dependent kinase. c) Although originally found in Xenopus eggs, it is highly conserved in evolution from simple yeasts to mammalian cells. d) Its activity is controlled by phosphorylation and dephosphorylation of a cyclin subunit 2. One requirement for passage from G1 to S phase is polyubiquitination of which protein by the SCF-type E3 ubiquitin ligase: a) an inhibitor of S-Cdk b) M-cyclin (a B-type cyclin) c) DNA polymerase d) S-Cdk 3. DNA replication is restricted to once and only once during the cell cycle. To control this process, a pre-replication complex (pre-RC) forms at a replication origin: a) during early S phase prior to the start of replication b) immediately after DNA replication at the end of S-phase c) during M-phase when the chromosomes are still condensed d) during early G1 phase when no Cdk activity is present 4. Apoptosis induces all of the following changes in a cell except for : a) Phophatidylserine moves from the inner leaflet of the plasma membrane to the outer leaflet. b) The plasma membrane lyses due to osmotic pressure. c) The nuclear lamina is degraded, leading to blebbing of the nucleus. d) Nuclear DNA is cleaved into small fragments. e) The cell rounds up as the cytoskeleton is disrupted. 5. The TOR (target of rapamycin) kinase pathway is induced when a cell responds to peptide growth hormones or increased nutrient levels. The TOR pathway stimulates: a) entry into S phase of the cell cycle b) apoptosis by the intrinsic pathway c) an increase in protein synthesis and cell mass d) exit from the cell cycle into G0 6. In the polarized epithelial cells lining the intestinal lumen, the tight junctions function to: a) prevent solutes from crossing the epithelium between the cells b) provide mechanical strength to the layer of cells c) provide a mechanism for small signaling molecules to pass between the cells d) connect the intermediate filament network between adjacent cells
1
7. Which of the following are examples of a communicating junction that connect cells with small, aqueous channels? a) desmosomes b) hemidesmosomes c) tight junctions d) gap junctions 8. In some organisms the cell wall provides external support for the cell, but in cells (like those in vertebrates) that generally lack a cell wall the major source of support is: a) the cytoskeleton b) bone matrix c) osmotic pressure d) cholesterol 9. Which of the following is NOT a characteristic of a stem cell population: a) ability to divide indefinitely b) ability to maintain themselves through division c) ability to trigger an undifferentiated state in other cell types d) ability to remain mostly un-differentiated 10. Which of the following is NOT a type of structural fiber in the extracellular matrix? a) collagen b) cellulose c) glycosaminoglycans d) elastin 11. Most human cells in primary cultures undergo about 50 cell division cycles before entering a non-dividing state termed replicative cell senescence. This failure to continue proliferation is thought to result from: a) extensive chromosome translocations that occur in cultured cells b) a lack of telomerase activity that leads to shortened telomeres c) loss of organelles due to defective cytokinesis d) accumulation of a large number of mutations in genes that regulate the cell cycle 12. In its role as a cell-cycle brake in mammalian cells, retinoblastoma (Rb) protein: a) phosphorylates the E2F transcription factor b) binds to and inhibits the function of the E2F transcription factor c) is tagged with ubiquitin by the E3 ligase SCF d) binds to and inhibits the function of the S-Cyclin/Cdk 13. Which of the following conditions describe an active integrin protein: a) integrin is bound to GTP b) integrin is bound to a cytoskeletal anchoring protein and an extracellular matrix protein c) integrin is bound to nuclear DNA d) integrin is bound to to myosin motor domain
2
14. Why does the incidence of cancer increase as an organism gets older: a) Younger organisms are better able to detect and kill tumor cells b) The cells of younger organisms are less determined and differentiated c) The cells of older organisms have had longer to accumulate mutations d) The cells of older organisms stop expressing telomerase 15. Many unfortunate people inherit a predisposition to certain types of cancer. This is because: a) They inherited a defective protooncogene from one of their parents b) They inherited a defective tumor suppressor gene from one of their parents c) They inherited a defective DNA repair enzyme from one of their parents d) They inherited an oncogene from one of their parents 16. Mitochondria (as shown below) play an important role in metabolism and also several other cellular functions, in cluding the intrinsic pathway of apoptosis. a) What is the location of cytochrome c in a normal cell? What is the normal function of cytochrome c in these normal cells? (4 points) it is localized in the intermembrane space where it functions in the electron transport chain
b) Draw or explain the role of cytochrome c in the apoptosis pathway. Include in your answer the role of the adapter protein and effector protein (caspase) in the pathway. Note: You do not need to explain the role of regulator proteins. (9 points) In the intrinsic pathway for apoptosis, cytochrome c is released from the mitochondria in response to an apoptotic signal. It binds to an adapter protein (like Apaf1) which then undergoes a conformational change allowing its assembly into a heptamer complex called an apoptosome. Initiator caspases are recruited to the apoptosome where they undergo cleavage of the pro domain to become activated. The inititator caspases activate executioner caspases in a caspace cascade. The caspases are proteases that target cellular proteins like the nuclear lamins and cytoskeletal proteins leading to apoptosis.
c) How does the tumor supressor protein p53 affect the apoptosis pathway? (3 points) p53 is phosphorylated by kinases responding to DNA damage. The phosphorylated p53 is no longer recognized by Mdm2 E3 ubiquitinase so its levels build up in the cell. The p53 is a transcription factor that enters the nucleus and increases expression of pro-apoptotic proteins in the BH3-only class. The resulting proteins promote apoptosis by inhibiting the antiapoptotic proteins like Bcl2.
3
17. Fill in the chart below comparing replication, transcription and translation. (1 point per blank; 11 points total)
Replication Deoxynucleotides ACGT DNA Transcription Deoxyribonucleotides ACGU DNA Translation Amino acids
Smallest Unit (Building blocks) Template for synthesis
mRNA
Major Enzyme
DNA polymerase
RNA polymerase
ribosome
Start site on template
Origin of replication
promoter
AUG Initiation codon N-terminus to C-terminus
Direction of synthesis of product polymer
5' to 3'
5' to 3'
18. You have discovered a new very small unicellular organism in ocean sediments. The cells seem to have two methods of motility. In seawater, the cells propel themselves by the beating of a hair-like structure. Fixed samples were processed for transmission electron microscopy, yielding images like this one containing a cross-section of a hair-like structure. a. Does this image at right support the hypothesis that the new organism is a eukaryotic cell? Explain your answer (please be specific). (3 points) Yes, this image looks like a cross-section of a eukaryotic axoneme. 25 nm There are nine outer doublet microtubules and two central pair microtubules. Dynein arms are attached to the A tubule of outer oublets. b. You notice that when the cells are grown on an agar surface in a Petri dish, they lose the hairlike structure and begin to migrate on the surface with motility that resembles that of an amoeba. What cytoskeletal filament might be involved in this motility? (3 points) crawling of cells like amoeba is dependent on the actin filament cytoskeleton c. Briefly describe an experiment you could do to test whether the cytoskeletal filament you answered in "b" is involved in the amoeba-like motility? (4 points) 1) Treat cells with a drug that depolymerizes actin filaments. If motility is actin-based (and if the drug is able to enter the cells), the motility would be inhibited by the drug. 2) Carry out immunofluorescence with antibodies against actin or against Arp/2/3 proteins or localize with F-actin fluorescent phalloidin. If the cell crawling is actin-based, we would expect actin and Arp2/3 to be localized at the leading edge of the pseudopodium/ lammelipodium. 4
19. Bob was working with cultured rat fibroblast cells that divide every 20 hours. M-phase lasts for one hour; S-phase lasts for 6 hours; G1 phase lasts for 7 hours. Starting with a culture of cells that were dividing asynchronously, Bob subjected culture samples to three different treatments:
1. 20 hours with colcemid, a drug that depolymerizes microtubules 2. 20 hours with aphidicolin, an inhibitor of DNA polymerase 3. no drug treatment
After adding the drugs and placing the cultures in the incubator, Bob headed home and went to bed. In the night, he awoke in dismay, realizing that he had forgotten to label the cultures. After thinking awhile, he realized that there was a solution to his problem. The next day at the 20-hour time point, he collected and fixed cells from each culture, stained the cells with a fluorescent dye that binds DNA, and analyzed them on a flow cytometer. The results are shown below in 3 graphs.
a.
# of cells
b.
# of cells
c.
# of cells
1x 2x relative amount of DNA
1x 2x relative amount of DNA
1x 2x relative amount of DNA
Based on these results, identify which culture received which treatment and explain why you think this is the case: a. What treatment did the culture graphed in "a" receive? Why? (3 points) These cells uniformly have a 2X amount of DNA. The DNA has duplicated, but the cells must be piled up at the G2/M transition. They must have received the colcemid treatment which would prevent assembly of microtubules and the mitotic spindle.
b. What treatment did the culture graphed in "b" receive? Why? (3 points) These cells are distributed at different stages of the cell cycle so are growing asynchronously. They likely received no drug treatment.
c. What treatment did the culture graphed in "c" receive? Why? (3 points) Most cells have a 1X amount of DNA, indicating that DNA replication has not occurred. These cells probably received the aphidicolin treatment. Most cells are piled up at the G1/S border. Some cells that were in S phase at the time of the treatment, have DNA content somewhere between 1x and 2x.
5
20. Assume that you are provided with a cell that has just begun expressing a mRNA encoding an E-cadherin cell adhesion molecule. a. Following export of the processed mRNA from the nucleus, describe the next steps that will eventually lead to delivery of the E-cadherin protein to its final destination including: i) where the proteins will accumulate, ii) the organelles that the protein will pass through, and iii) any targeting signals that are necessary for the trafficking of the E-cadherin protein. (9 points) Ribosome begins translation, finds signal peptide, goes to ER translocon. From ER it will go through the Golgi (cis, medial, trans, etc.) to the PM where it will accumulate. It needs no signals past the ER since the PM is "default" for anterograde traffic.
b. What other proteins or protein complexes are necessary for the cell's newly synthesized and targeted E-cadherin to adhere to the adjacent cells to form a tissue layer? (5 points) Inside the cell, the cadherin needs actin or intermediate filaments, outside it needs a cadherin from the adjacent cell.
21. Some cells that are initially formed as part of an tissue layer held together by cadherins in adherens junctions must later migrate to elsewhere in response to developmental signals. Even though cell-cell junctions (like adherens junctions) can be disassembled, to efficiently migrate, the cells must remove the cadherins from the PM. Describe the steps involved in removing membrane proteins like cadherins from the PM including: i) the final destination for proteins that will follow this pathway, ii) the organelles that the proteins will pass through, and iii) any targeting signals that are necessary for the trafficking of such recycled proteins. (9 points) The cadherin will be endocytosed into a vesicle bound for the early endosome. The signal for this and subsequent internalization into an "internal vesicle" inside an endosome is monoubiquitination. From the early endosome it will go to the late endosome and then to the lysosome for degradation and recycling back to amino acids. Some people got confused about "recycling" thinking that I meant it would "recycle" back to the PM (but the point was to get it off the PM) or to the ER (though I can't imagine why). I generally didn't take much off for this if you had the correct answer above. There is no need to invoke a protease to cleave the extracellular domains since the cadherin binding can be adjusted by the Ca2+ level and by other means. Anyway, just cutting up the extracellular domains still leave a good bit of the protein in the PM that still needs to gotten rid of.
6
22. Many antibiotics target assembly of the cell wall/ECM of bacteria. How can these drugs be so effective against bacteria, and yet have little effect upon animals? (5 points): Bacterial walls are made of distinct components using different kinds of enzymes relative to animal cell walls, so antibio...