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S294/A

Module Examination 2018

CELL BIOLOGY

PART 1

Short answer questions Answer all SIX questions in this first part of the exam. Write your answers in the answer book provided, limiting your answer for each part of a question to a few sentences or more. You are advised to spend no more than 1 hour 15 minutes on this Part A of the paper, which carries 30 of the total marks for the examination. Each question is worth 5 marks.

Question 1

(a) Identify the structures indicated by the labels A – D in Figure 1.    

Ans: 

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(b) Name the two types of endoplasmic reticulum (ER) present in eukaryotic cells, and indicate at least one main function of each.

Question 2

(a) Outline, with the aid of a diagram, the . Details of selection for recombinant plasmids are not required.

Question 3

(a) With the aid of a labelled diagram, describe the structure of a mitochondrion.

(b) Where is mitochondrial DNA located and what does it encode?

(c) In considering a family pedigree, what two observations allow you to predict that a disease is caused by mutation of a mitochondrial gene? Explain your answer.

Question 4

(a) Specific transcription factors regulate the level of transcription of genes in eukaryotic cells. In general terms, how do these factors influence initiation of transcription of a target gene?

(b) Identify three different types of RNA found in eukaryotic cells and indicate, in general terms, the function of each.

Question 5

Reversible phosphorylation of specific amino acid residues is an important mechanism by which protein activity is regulated in eukaryotic cells and is particularly important in regulating the activity of intracellular signalling pathways.

(a) What type of enzyme catalyzes (i) phosphorylation and (ii) dephosphorylation of proteins?

(b) Briefly describe how phosphorylation can affect the activity of a protein.

(c) Describe how protein phosphorylation can influence the activity of an intracellular signalling pathway with reference to one of the following:

• a receptor tyrosine kinase (RTK)
• the Ras-MAP kinase pathway
• an enzyme-associated receptor such as a cytokine receptor.

Question 6

(a) With the aid of a diagram, outline the key features (composition and structure) of the cell membrane.

(b) In what respects was the fluid-mosaic model (proposed by Singer and Nicolson) refined following the discovery of lipid rafts?

Question 7

Figure 2 is a representation of changes in the concentration and activity of two important cell cycle regulators, M cyclin and M cyclin Cdk complexes, during two rounds of the cell cycle. The different phases of the cell cycle are represented by the boxes and arrows (numbered 1-4) above the graph

(a) Identify each of the phases (1-4) and briefly describe the major events taking place in each phase.

(b) With reference to the changing levels of M cyclin and the changing activity of M cyclin-Cdk complexes, depicted in Figure 2, briefly describe how these mediators regulate cell division.

Question 8

(a) Describe the key characteristics of stem cells.

(b) Outline how and why stem cells may provide a long-term therapeutic solution for Parkinson’s disease.

PART 2

Answer ALL parts of the question in this Part. Write all your answers to this Part in the answer book provided. You are advised to spend 45 minutes on this Part of the paper. Your answers should be written concisely, but in proper sentences. You should include diagrams and calculations in your answer where appropriate.

This Part carries 25% of the marks for the examination.

Question 9

Overexpression of a protein called RCAN has been linked to mitochondrial dysfunction in Alzheimer’s disease.

Investigators were interested in whether the level of RCAN affects expression of the mitochondrial protein ANT1 and the activity of a transcription factor called NFAT, and, if so, which parts of the RCAN protein are responsible for these effects. They generated three plasmid expression vectors: one encoding the full length RCAN, and two other plasmids encoding either the N- or the C-terminal end of RCAN. These plasmids were introduced into a human cell line by a process known as transfection. Control cells were transfected with an empty vector. After the transfection, cells expressed the corresponding RCAN protein (full-length, C-terminal end or N-terminal end). ANT1 expression and NFAT activity were measured in the transfected cells after 48 hours and the results are shown in Figure 3

Figure 3 (a) Levels of ANT1 following transfection with the indicated plasmids. The ANT1 protein expression was measured by immunoblotting and the values are given as a percentage of the control expression. (b) NFAT activity levels following transfection with the indicated plasmids. NFAT activity was measured using a luminescence assay and the values are expressed in arbitrary units. All values are the mean of three experiments.

(a) Why was transfection with an empty plasmid chosen as a control?

(b) Describe how the expression of the different plasmids affects expression of the ANT1 protein.

(c) Describe how the expression of the different plasmids affects NFAT activity.

(d) The results for the NFAT activity are provided as absolute numbers. Calculate NFAT activity for each of the RCAN plasmids as a percentage of the control. Show your working.

(e) Based on Figure 3, which part of the RCAN protein is important for the effects on ANT1 expression and on NFAT activity? Are both processes affected by the same part of RCAN? Explain how you came to this conclusion.

To assess the role of RCAN in mitochondrial dysfunction, the investigators used cytochrome c release from mitochondria as an indicator of increased mitochondrial permeability and dysfunction. A human cell line was transfected with the control and full-length RCAN plasmids and incubated for 48 hours (as before) and the cells were then lysed. The cytosol and mitochondria were recovered from the cell lysate and analysed by immunoblotting to measure the expression levels of three proteins: cytochrome c (cyt c), SOD-2 and β-actin.

SOD-2 is a mitochondrial protein, and β-actin is part of the cytoskeleton. The data are shown in Figure 4. The level of protein in a sample can be gauged from the density/darkness of the bands: the darker a band, the more protein is present

Figure 4 Immunoblots showing levels of cyt c and SOD-2 in mitochondria, and cyt c, SOD-2 and β-actin in the cytosol, of human cells transfected with either a control plasmid or plasmid encoding the full-length RCAN. All samples were prepared from equivalent numbers of cells.

(f) From your studies of S294, suggest a method that the investigators might have used to separate cytosolic and mitochondrial components and describe briefly the basis on which the separation is achieved.

(g) Describe where the investigators found SOD-2 and β-actin, and whether this confirms the expected localisation of these proteins. Why did the investigators test for the presence of these two proteins in the different samples when they were in fact interested in the localisation of cytochrome c?

(h) (i) Based on Figure 4, in which part of the RCAN over-expressing cells is most of the cytochrome c localised? Explain how this is apparent from the data given.

(ii) Compare the cytochrome c distribution in control and RCAN overexpressing cells.

(i) From your studies in S294, what consequences might follow from the altered distribution of cytochrome c in RCAN over-expressing cells?

PART 3

Answer ONE of the questions in this Part. You are advised to spend about 1 hour on this question. You should follow any specific guidance in the question. Note that some questions specifically request inclusion of diagrams but in all cases you may include diagrams where appropriate. This Part carries 30% of the marks for the examination.

Question 8

You are supplied with a copy of an article entitled ‘Antibiotic tricks a switch’ by Thomas Hermann. [Hermann, T. (2015) Nature, vol. 526, pp. 650–651]. This article discusses the findings of a study from Howe and co-workers [Howe J. A. et al. (2015) ‘Selective small-molecule inhibition of an RNA structural element', Nature, vol. 526, pp. 672–677]. The study reports on the identification of a new potential antibiotic, which targets a bacterial ‘riboswitch’ that regulates translation of an enzyme that bacteria require to synthesise riboflavin.

The following questions are based largely on the article, but in some cases draw on relevant material from S294.

(a) From your studies of S294, what is meant by ‘selective toxicity’ in the context of antibiotics and on what does selective toxicity generally depend?

(b) From your studies of S294, identify one antibacterial agent and indicate what bacterial process or component it targets.

(c) From your studies of S294, explain one molecular mechanism that confers antibiotic resistance on bacteria.

(d) From the article, what is the nature of the riboswitch that is targeted by the ribocil compound identified by Howe and co-workers, and where is it located?

(e) Describe, in a few sentences, how the riboswitch referred to in the article regulates synthesis of riboflavin in bacteria.

(f) Hermann explains in the article that, in seeking to identify a new antibiotic, Howe and co-workers used a ‘phenotypic screening’ approach.

(i) Briefly describe their screening process.

(ii) How many different molecules did they screen?

(g) From what you have learned from the article, would you expect ribocil to have a direct effect on a human host if it were used to treat a bacterial infection? Explain your answer.

(h) What two different pieces of evidence, described in the article, help to identify the riboswitch as the target for ribocil? How was this evidence obtained?    

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Last updated: Sep 02, 2021 10:06 AM