BIOL5372M - Advanced Biomolecular Technologies - University of Leeds

Question 1. You want to amplify the DNA between the 2 stretches of sequence shown below. Of the listed primers, choose the pair that will allow you to amplify the DNA by the polymerase chain reaction (PCR).

DNA to be amplified

5'-GACCTGTGGAAGC----------------------------------CATACGGGATTGA-3'

Primers

A. 5'-GACCTGTGGAAGC-3'
B. 5'-GCTTCCACAGGTC-3'
C. 5'-CATACGGGATTGA-3'
D. 5'-GTATGCCCTAACT-3'
E. 5'-TCAATCCCGTATG-3'

Question 2. Name ONE method you could you use for the following:

A. To demonstrate a protein-protein interaction between two transcription factors

B. To demonstrate that a transcription factor binds to the promoter region of a gene

C. To silence gene activity

D. To determine whether a particular protein is expressed in the cell or not

E. To determine the mass of a protein

F. The determine the structure of a protein

Question 3. Below are a restriction map of plasmid and a plant gene of known sequence. Your task is to clone the cDNA for this gene so that the start codon is closer to the Eco RI site than the Pst I site of the polylinker, but get rid of the promoter+RBS+Hin insert already in the plasmid. Explain how you would accomplish the cloning (+ screening) using a series of numbered steps. Do NOT write in paragraph form, only a numbered list of steps!

Question 4. The Ets-1 transcription factor (MW 54Kda) can bind to the promoter/enhancer regions of the gene which codes for the protein MMP1 (matrix metalloproteinase 1) and induce its expression. The Ets-1 transcription factor has a DNA-binding domain at its C-terminus and a trans-activation domain at its N-terminus. Consider the following situations which attempts to highlight why the accumulation of truncated protein could be harmful to a cell:

A. If a cell makes substantial amounts of the C-terminal fragment of the Ets-1 transcription factor (truncated protein of MW 33Kda), containing the DNA-binding domain, what is likely to happen to the transcription of gene MMP-1? Explain your answer.

B. If, in addition to the C-terminal fragment of Ets-1 (truncated protein), a cell also makes an equal amount of the full-length protein, what is likely to happen to the transcription of gene MMP-1? Explain your answer.

C. Consider two cell lines or strains that each have a fully functional allele of the transcriptional factor Ets-1 gene on one copy of chromosome 11. On the second copy of chromosome 11, strain 1 lacks a second transcription factor Ets-1allele and strain 2 contains the Ets-1 allele that codes for the C-terminal fragment described in part A. If strain 1 and strain 2 have different amounts of MMP-1 mRNA, which strain do you expect to have more MMP-1 mRNA? Explain your answer.

D. What experiment could you carry out to show that both the truncated and the full length Ets-1 protein are present in a cell? Do NOT write in paragraph form, only a numbered list of steps.

Question 5. The purification of a protein usually requires multiple steps and often involves several types of column chromatography. A key component of any purification is an assay for the desired protein. The assay can be a band on a gel, a structure in the electron microscope, the ability to bind to another molecule or an enzymatic activity. Consider the purification of the plant enzyme oxalate oxidase, shown in Table 1. This enzyme is used in medical diagnosis to determine the concentration of oxalate ions in urine: kidney stones are formed when oxalate concentrations are high.

Table 1: Purification results for three-stage isolation of the plant enzyme, oxalate oxidase

A. For each step in the purification procedure, calculate the specific activity of the enzyme (units of activity per mg of protein). Add these values into the column "specific activity (units/mg) in the table above. How can you tell that purification of the enzyme has occurred at each step?

B. Which of the purification step was most effective? Which was the least effective?

C. If you were to carry out the purification through additional steps, how would the specific activity change? How could you tell from the specific enzyme activity that the enzyme was pure? How could you check on that conclusion?

D. If the enzyme is pure at the end of the purification scheme in Table 1, what proportion of the protein in the starting cell does it represent?

Question 6. Transcription of gene X is controlled by transcription factor A. Gene X is only transcribed when transcription factor A is phopshorylated. Data on the tissue distribution of transcription factor A and the activities of a protein kinase and a protein phosphatase specific for transcription factor A are presented in the table below.

Question 7. Real time PCR is a powerful method to accurately quantify levels of gene expression in an organism. Below is a melt curve taken immediately after the amplification process for 16 real time PCRs carried out using the DNA-binding fluorophore SYBR green. Two peaks are observed, one melting at 75 oC and one at 81 oC.

A. Explain what the two peaks indicate.

B. Given that the PCR fragment amplified is expected to melt at a temperature of around 79-80oC, explain the reason for two peaks observed. .

C. If the PCR was repeated using cDNA reverse transcribed from a freshly prepared RNA sample and found to result in a third peak melting at around 82 to 86oC, what would be the most likely reason for the third peak.

D. What could have been done in the preparation of the RNA sample to prevent the third peak?

E. What PCR controls could you run to prove your hypothesis about the identity of the third peak?

Question 8. You are investigating a novel signal transduction pathway, FLAGIT, that has been recently discovered. Your approach is to transfect cultured cells with three mutated forms of the gene, Flag1 (Mutantl, Mutant2 and Mutant3) to determine the effects of the mutations on FLAGIT signalling. The cultured cell line you are working with is difficult to transfect, so you will use a retroviral bicistronic vector system which will enable you to express both the Flag1 gene and GFP, and then use flow cytometry to sort transfected (GFP+) cells.

On their way to the cell surface, Flagl proteins are cleaved in the trans-Golgi network, giving rise to a heterodimer cell surface receptor comprised of an extracellular subunit, which contains the ligand binding domain, and an intracellular subunit, containing a transmembrane region. These two Flag1-derived proteins are non-covalently associated at the cell surface. Flagl signalling is activated when the extracellular domain binds ligand. Upon ligand binding a proteolytic cleavage is triggered which results in the release of extracellular Flagl still bound to ligand. Loss of the extracellular domain activates a second cleavage within the cell membrane which releases the intracellular portion of Flagl from its membrane tether. This latter cleavage is mediated by the enzyme Flagl secretase and can be inhibited by Flagl secretase inhibitors (FSIs). As a result of the second cleavage the intracellular section of the Flagl protein is released from the membrane and is free to translocate to the nucleus where it acts as a transcriptional activator for genes such as Fes.

You resuspend your sorted cells in l mL of tissue culture medium and then plate the cells under different conditions of ligand availability and FSI addition and find the pattern of Fes induction as assessed by PCR as shown in Table 1.

Table 1. Pattern of Fes induction assessed by PCR

A. Explain the pattern of response for each mutant and the wild type (use bullet points).

To investigate further the nature of the mutations, you perform a Western blot using a polyclonal antibody which recognises both Flagl subunits, and a diagrammatic representation of your results is shown in Figure 1.

Figure 1. Western blot to investigate the effect of mutations in Flag1

B. What can you deduce about the structure of the Flag1 gene and the nature of the three mutations from this blot?

C. How could such mutations in Flagl generate the altered patterns of Fes induction described earlier?

Attachment:- Advanced Biomolecular Technologies.rar