In this question you are required to interpret data from a study investigating expression of a protein called survivin and the effects of a drug called hedamycin on cancer cells.
Figure 2 An experiment to identify regulatory DNA sequences in the region upstream of the survivin gene. (a) The section of cloned DNA present in each of the plasmids. The number corresponds to the length of the DNA upstream of the survivin gene transcription start site (denoted +1). For example pLuc-6270 contains 6270 base pairs (bp) of the DNA upstream of the survivin gene transcription start site, while pLuc-649 contains 649 bp of the DNA upstream of the survivin gene transcription start site. (b) A bar chart showing the level of expression of the luciferase reporter gene measured as the level of light output in cell lysates containing each of the plasmids in (a).
Answer the following questions based on Figure 2.
(i) Describe how the level of reporter gene expression differs in cell lysates containing the different plasmids. Do not just list the values. (3 marks)
(ii) Using the luciferase activity measured in cells transfected with pLuc-6270 as a baseline, calculate the percentage change in luciferase activity in cells transfected with pLuc-2840. Show your working. (5 marks)
(iii) Consider the levels of luciferase expression observed with the different plasmids. Does the region of the DNA between –6270 and –2840 contain a binding site for a transcriptional activator or a transcriptional repressor? Explain your answer. (5 marks)
(iv) Quantitative PCR is a technique that can be used to accurately measure the level of gene expression in a cell. Briefly outline this technique. (4 marks)
Figure 3 shows the results of an experiment to determine whether hedamycin affects survival and/or proliferation of HeLa cells. Equal numbers of cells were seeded in culture dishes and treated with hedamycin at the doses indicated for 48 hours, after which time images of the cell cultures were taken on a microscope to observe cell morphology and cell number. Note that healthy HeLa cells are triangular and extend processes in culture, whilst dying cells tend to round up. Only living cells remain attached to the dish.
Figure 3 Equal numbers of HeLa cells were plated. The cells were incubated with hedamycin at the doses indicated for 48 hours.
Answer the following questions based on Figure 3.
(ii) Based on Figure 3, describe (in a few sentences) the effect of hedamycin on HeLa cells. In answering, you should consider the information that you have been given about HeLa cells and describe any differences in the response to the two hedamycin concentrations. (5 marks)