Biology 200

Sample Final Exam

Question 1. (____/ 7 marks)

Chloroplasts, mitochondria, and nuclei are three intracellular organelles that are surrounded by two biological membranes. Although they possess some structural similarities, they have many differences in function and biogenesis. For statements 1 to 7 in the table below, indicate whether it applies to:

A. Chloroplasts only

B. Mitochondria only

C. Nuclei only

D. Both chloroplasts and mitochondria, but not nuclei

E. All three organelles

STATEMENT                                                                                     Fill letter A-E

1) These organelles contain remnant genomes carried on circular chromosomes, which reflect their evolutionary origins.

2) mRNA is transported through multi-protein complexes in the membranes surrounding this organelle to the cytoplasm for translation.

3) This organelle contains euchromatin and heterochromatin.

4) The outer envelope membrane is continuous with the endoplasmic reticulum.

5) The outer envelope membrane, but not the inner envelope membrane, is porous and allows molecules to diffuse across freely.

6) The inner envelope membrane of this organelle is highly folded increasing its surface area.

7) This organelle contains an additional internal membrane system, in addition to the two membranes surrounding the organelle.

Question 2. (____/ 12 marks)

For each of the observations below, write a statement that could explain these results, based on what the data shows and on your knowledge of cellular structure/ function (3 marks each)

A. If taxol is added to cells at metaphase, the chromosomes do not move towards the spindle poles in anaphase.

B. Electron micrographs show that mitochondria in heart muscle have a much higher density of cristae than mitochondria in skin cells.

C. Microtubules formed in vitro from tubulin that is bound to a non-hydrolyzable form. of GTP were found to be exceptionally stable.

D. Some membrane proteins can be readily extracted with 1M NaCl, whereas others require the use of a detergent.

Question 3. (____/15 marks)

On the domain maps provided, draw and label all targeting sequences that you predict would be found in the proteins, A - E. If you predict no targeting sequence is required, clearly indicate this in your answer – a blank will NOT be considered for marks. (2 marks each):

A. Soluble ER resident

B. Rubisco Small Subunit 2B – a nuclear-encoded chloroplast stromal protein

C. Transcription factor

D. α-tubulin

E. Glycophorin A (plasma membrane protein), 1 transmembrane domain, N-terminus outside the cell.

Match each of the proteins mentioned above with its cellular location, as identified in each micrograph (arrow) below. (1 mark each):

Micrograph                     Protein (A-E)

1

2

3

4

5

Question 4 (____/9 marks)

The epidermal growth factor receptor (EGFR) is a transmembrane protein localized to the plasma membrane in skin epithelial cells. When EGFR is activated by binding to the extracellular peptide EGF, it is internalized via vesicles. Ultimately, this internalization will lead to changes in cell growth and division. Researchers studied the relationship between EGFR internalization and cell growth by investigating wild type cells compared to cells with a non-functional mutant form. of clathrin.

A. The first experiment examined the amount of EGFR found in early endosomes in the wild type and mutant cells, in the absence (-EGF) or presence (+EGF) of the extracellular peptide. Describe the results shown, and explain what you can conclude based on these data. (3 marks)

B. In the next experiment, they examined the effect of EGFR internalization on cell cycle proteins, to see whether there were any changes. The SDS-PAGE data below shows the results for Mitotic Cyclin (M-Cyclin), a protein known to promote entry into mitosis, in the absence or presence of EGF. Describe the results, and explain what you can conclude based on the gel shown. (2 marks).

C. The researchers discovered that after the wild-type cells are exposed to EGF, a transcription factor called ERK1/2 moves into the nucleus. On the other hand, ERK1/2 remained localized in the cytoplasm when the clathrin mutants were exposed to EGF. Considering this and the rest of the data in this question, propose a model describing how EGF-binding to EGFR might affect the cell cycle progression. In your explanation, include the role of EGF and its receptor, clathrin, the transcription factor ERK1/2 and Mitotic Cyclin. (4 marks)

Question 5 (____/ 10 marks)

Actin Capping Protein (ACP) binds to the plus end of actin filaments, preventing the actin filaments from gaining or losing monomers. Its activity is blocked by regulatory proteins such as V-1. In this experiment, Takeda et al. (2010) examine the role of V-1 and ACP in the regulation of actin polymerization. In Panel A, the ratio of F-actin (actin filaments) to G-actin (actin monomers) were measured for WT (normal) and V1 (over-expresses V-1) cells. In Panel B, actin filaments and nuclei were stained with fluorescent dyes and cells were examined through fluorescence and light microscopy (red = actin; blue = DNA). Assume that both cell lines express the same amount of ACP.

A. Compare the bars in the chart in Panel A. What do they tell you about the amount of actin in each cell type? How might the change in expression levels of V1 be affecting ACP’s ability to function? (3 marks).

B. Panel B shows paired brightfield and fluorescence microscopy images of each of the cell types measured in Panel A. Compare the shape of the WT cells to the V1 cells, and explain how the actin shown in the fluorescence images is contributing to that shape. (4 marks)

C. Based on the data shown in Panels A and B, how might V-1 be influencing cell shape in the overexpressing cells? (3 marks)

Question 6 (____/ 8 Marks)

You generated a yeast strain with a temperature-sensitive mutated form. of M-cyclin that is unable to fold stably at a non-permissive temperature (37°C). To assess its role in the cell cycle, you grow these mutant cells in a test tube at the permissive temperature (25°C) then shift them to 37°C.

A. Explain what happens to M-cyclin concentration as the cell progresses through interphase at the permissive temperature. How does this compare at the non-permissive temperature? (2 marks)

B. What effect would this mutation have on the regulation of cell cycle at the non-permissive temperature (2 marks)?

C. You fluorescently labelled the DNA of your mutant yeast cells and used fluorescence activated cell sorting (FACS) to analyze the effect of this mutation on the cell cycle. Note that these cells complete a cell cycle in 90 minutes where interphase lasts for approximately 70 minutes. In your experiment, you have 2 tubes (labeled Tube 1 and Tube 2). In Tube 1, you incubate the yeast cells at the non-permissive temperature, 37°C, for 2 hours. In Tube 2, you incubate the cells for 2 hours at non permissive temperature, followed by 30 minutes at the permissive temperature. In the appropriate spaces below, draw the expected FACS readout from each Tube and label the graphs with the relevant phase of cell cycle expected.

Question 7 (____/ 9 Marks)

Nocodazole is used as an anti-cancer drug that binds to a/b tubulin subunits inside cells. Shown below are fluorescence images of cells in the presence or absence of nocodazole, by fluorescence-tagged immunolabeling for a-tubulin.

A. Describe the fluorescence pattern seen in Panels A and B. What does this tell us about the effect of nocodozole on microtubules? (3 marks)

                        Describe the Fluorescence                                        The Effect of Nocodozole

Panel A

Panel B

B. Based on your knowledge of microtubule polymerization at the plus end, propose a model for how Nocodazole might be leading to the results shown. (2 mark)

C. Predict what would happen to the cell in Panel B if the nocodazole was washed away and the cell was allowed to recover. (1 mark)

D. Predict the impact of nocodazole treatment on constitutive secretion in these cells? (1 mark) Explain why. (1 mark)

E. Propose a role for nocodazole in the M-phase which makes it useful as an anti-cancer drug. (1 mark)

Question 8 (____/ 9 Marks)

During normal cellular function, protein activity must be strictly controlled such that cellular processes can be turned ‘off’ or ‘on’ quickly. One strategy for such control involves chemical modification of proteins, through addition or removal of covalently bonded chemical groups.

Name THREE distinct examples/proteins by which protein activity can be turned “on” or “off” through three distinct types of chemical modification. For each example, describe: 1) general mechanism i.e. type of chemical modification (1 mark), 2) example of a protein whose function is regulated by this mechanism (1 mark), 3) how is the protein function turned “on” and “off” (3 marks total per example)

Note: All examples and explanations must come from BIOL 200 course material.