Question 1

 This question relates to Experimental Investigation 4 Research Skills EI 4: Analysing gene expression – Western blotting.

a. You are provided with three preparations of cellular protein at concentrations of: 390 μg ml 780 μg ml 1.12 mg ml . −1 −1 −14/5/2021 Tutor-marked assignment 04: View as single page https://learn2.open.ac.uk/mod/oucontent/view.php?id=1692927&printable=1 3/9

i. What volumes of each of the cellular protein preparations and diluent (to the nearest μl) would be required to prepare 250 μl of solution with a final concentration of 100 μg ml ? Show your answers in a table:

Protein preparation 390 μg ml 780 μg ml 1.12 mg ml Vol. protein/μl: Vol. diluent/μl: Final volume/μl: 250 250 250 −1 −1 −1

ii. If 20 μl of one of the diluted samples were to be loaded into a well of an SDS–PAGE gel, how much protein (μg) would be present?

iii. What volume of the diluted samples would be loaded in order to examine 1.5 μg of cellular protein?

iv. How can you demonstrate that approximately equal amounts of cellular protein are present on each lane of a Western blot?

b. In Experiment 1 of EI 4: Analysing gene expression – Western blotting, you used a Caco-2 cell lysate to determine which of three antibodies that recognise the protein ABCB1 is most appropriate for detection of ABCB1 by Western blot (Task 2 of Experiment 1). Write a laboratory report on this part of the experiment (Task 2 of Experiment 1). In writing your report, you should consider the following: the specific guidance given below the Model Report on EI4 the advice in the document ‘Writing scientific reports’, in particular Section 2.5 which has guidance on writing Methods for interactive onscreen experiments. Your report should include: an Introduction in which the aim of the experiment is clearly stated.

a Methods section which includes details of the experimental conditions.

a Results section which includes annotated pictures of Western blots that provide evidence to support your conclusions. You should have 3 blots, one for each of the three antibodies. (Note that4/5/2021 Tutor-marked assignment 04: View as single page https://learn2.open.ac.uk/mod/oucontent/view.php?id=1692927&printable=1 4/9 these should be of gels prepared with an appropriate acrylamide concentration.)

Conclusion.

Question 2

This question relates to Topic 5 (Part 3). View larger image Figure 1 Region of genomic DNA that is transcribed to form the plant miR171b primary RNA. Draw a schematic figure of the secondary structure for miR primary RNA transcribed from this gene. You should show the base sequence of the miR (blue) and the sequence to which it base-pairs (red), and indicate which bases are base-paired in the double-stranded paired region.

a. Briefly describe the ways in which the availability of a mature miRNA can be regulated in a eukaryotic cell.

b. The coding strand of the genomic DNA base sequence of a region that is transcribed to form the plant miR171b primary RNA is shown graphically in Figure 1. The bases that encode a mature 21 nucleotide miR are shown in blue and the corresponding region to which it pairs to form the pre-miR (pre-miRNA) is shown in red.4/5/2021 Tutor-marked assignment 04: View as single page https://learn2.open.ac.uk/mod/oucontent/view.php?id=1692927&printable=1 5/9

Question 3

A variety of research technologies that make use of exogenous non-coding RNAs (ncRNAs) are used in cell biology research. These involve the introduction of specific types of ncRNA in order to manipulate an aspect of the recipient cell. In each case, the ability to target a specific endogenous nucleic acid is crucial to the technique. Three different applications of such technologies that make use of ncRNAs are outlined below. For each, describe (in a few sentences) the approach that is involved and explain how target specificity is assured in its application.

a. Manipulation of cultured mammalian cells to down-regulate levels of a particular protein to investigate its function.

b. Manipulation of cells to suppress the actions of an endogenous miRNA to investigate its role in a cell.

c. Genome modification (genome editing) to make targeted changes to the genome of living cells.

Question 4

This question relates to Topic 6.

a. Pick a signalling pathway that you have studied in S317 and describe in no more than 200 words how it illustrates the key principles of cellular communication.

b. Explain briefly (in two or three sentences) how the MAP kinase Fus3 acts as a molecular switch.

c. Adenylyl cyclase can be stimulated or inhibited downstream of GPCRs.

i. Draw a single diagram that illustrates the regulation of adenylyl cyclase by both Gα and Gα proteins downstream of GPCRs.

s i4/5/2021 Tutor-marked assignment 04: View as single page https://learn2.open.ac.uk/mod/oucontent/view.php?id=1692927&printable=1 6/9

ii. What effect do cholera and pertussis toxins have on cytosolic cAMP production?

Question 5

This question relates to Topic 6.

The MAP kinases Fus3 and Kss1 are both phosphorylated and activated by the kinase Ste7 within S. cerevisiae. The phosphorylation of Fus3 is essential for mating, whereas phosphorylation of Kss1 is necessary for filamentous growth. Both Fus3 and Kss1 bind to Ste7 with equal affinity, but they are never concurrently phosphorylated by Ste7. This question is based on published results of experiments performed to understand how Ste7 is directed to phosphorylate either Fus3 or Kss1. The authors of this study used an in vitro assay in which they could monitor the activity of Fus3 or Kss1 over time by looking at changes in fluorescence emission. With their particular assay, a decline in fluorescence indicated activation of Fus3 or Kss1. To examine the activation of Fus3 and Kss1 they added purified, recombinant proteins. The purified proteins they used were: Fus3 Kss1 A mutated form of Ste7 (called Ste7EE) that is constitutively active Ste5. Some results from their study are shown in Figure 2. Figure 2 Change in fluorescence emission (arbitrary units) in in vitro assays of (a) Fus3 and (b) Kss1 activities. The proteins included in the assays are indicated on the appropriate plots. Examine the results presented in Figure 2 and answer the following questions.

a. The grey lines (Fus3 only and Kss1 only) are control experiments. What do they show?

b. How does Ste7EE affect activity of Fus3 and Kss1?

c. The authors of this study concluded that Fus3 is an intrinsically poor substrate for Ste7. Based on the data presented in Figure 2, would you agree or disagree with this statement? Justify your statement.

d. What effect did the addition of Ste5 have on the activation of Fus3 and Kss1 by Ste7EE?

e. The results from this study show that Fus3 activation has a dual requirement for Ste7 and Ste5. From what you have learnt about the mating pathway in S. cerevisiae in S317, explain briefly why this dual requirement is important and describe the role of Ste7 and Ste5 in activation of Fus3.

Question 6

This question relates to Research Skills activities Reading Scientific Literature 2 and 3 (RSL 2 and RSL 3), as well as Digital and Information Literacy 1 (DIL 1: Citation searching). You are supplied with a copy of a News and Views article taken from the journal Nature, entitled ‘Plant biology: Coding in non-coding RNAs’. This short paper, authored by Waterhouse and Hellens (2015), presents a summary of the findings of another paper, Lauressergues et al. (2015), that is published in the same issue of this journal. Waterhouse and Hellens also discuss the evolutionary implications of these authors’ findings. It isn’t necessary for you to read the original research article by Lauressergues et al. (2015). Note that pri-miRs as referred to in the Waterhouse and Hellens (2015) article are the primary miRNA transcripts produced by transcription of miRNA genes (see Figure 3.1 in Topic 5 Part 3); they are NOT the same as pre-miRs. From your reading of the paper by Waterhouse and Hellens (2015), answer, in your own words, the following questions.

a. What is the range of lengths of primary miRs (pri-miRs) found in maize?

b. Which plant protein is responsible for processing the pri-miR to generate a duplex miRNA?

c. From this article, what approach was used by Lauressergues et al. (2015) to determine whether the miPEPs predicted in pri-miRs existed in plant cells?

d. What are the reported observations of Lauressergues et al. (2015) as to where the miPEPs are found in plants?

e. What are the reported observations of Lauressergues et al. (2015) as to the effect of the miPEPs on miR transcription?

f. What is meant by ancient and recent miRNAs, and how do these differ in terms of the number of species in which they are present?

g. What is the association between miPEPs and the evolutionary age of the miRs that encode them, and what do the authors suggest is the reason for this observation?

h. What problem do miPEPs present for the automated annotation of genome and transcriptome sequences?

i. What do the authors claim is the origin of many plant miRs and which paper is referenced in support of this claim?

j. Identify this referenced paper in Web of Science. Carry out a citation analysis and read the paper’s

Abstract.

i. How many times has this referenced paper been cited? Include a screen shot that clearly shows the details of the paper and the number of times cited.

ii. From the Abstract, what do the authors suggest is the reason for the apparent lack of target mRNAs for many young plant miRs?