You are not sure how to interpret these results, but your colleague mentions that the phenotype observed under the microscope may be related to changes in membrane fluidity. To test this possibility, you add to the media a phospholipid that cells incorporate and integrate in their cellular membranes. This phospholipid is
modified with a green fluorophore so it can be
imaged under the fluorescent microscope to
measure FRAP (fluorescence recovery after
photobleaching). The graph shown is
representative of the results obtained after
photobleaching small areas in the cellular
membranes of wild type cells grown in media
lacking FT (black), cells double KO for SFT2 and
SFT3 grown in media lacking FT (blue) and cells
double KO for SFT2 and SFT3 which media have
been supplemented with FT (green)
D) How does the lack of SFT2 & SFT3 affect the recovery of fluorescence? Max 2senten
E) How does this relate to the function you stated in section B for the genes isolated in this screen and membrane fluidity? Max 4 sentences
F) How does the sample of the double KO cells supplemented with FT help you to reach a firmer conclusion about the role of SFT2 and SFT3? Max 4 sentences
Given the relevance of the phenotypes observed, you want to better understand the molecular mechanisms behind the function of these genes, so you proceed to analyze their sequences.
G) The nucleotide sequences of SFT2 and SFT3 show a very high percentage of similarity. Based on this fact, what term best describes the relationship between these two genes?
H) Based on the information provided so far (sequence similarity and KO phenotypes) you hypothesize SFT2 and SFT3 are functionally redundant. Briefly describe the process of thinking you could have followed to reach such a hypothesis.
Max 4 sentences
You decide to move forward and study one of the genes in more detail, SFT2. You start looking more carefully at its sequence, looking for features that could explain its function. Two of these catch your attention immediately: the hydrophobicity plot shown below and the presence of a region with similarity to transcription factors.
I) Based on this hydrophobicity plot, what can you say about SFT2’s topology? Just state your answer. Max 1 sentence
You then want to study SFT2’s subcellular localization, and for that purpose you decide to generate antibodies against the protein.
J) If you want to make sure your antibody only recognizes SFT2 and not SFT3, should you generate polyclonal or monoclonal antibodies? Justify your answer.
Max 3 sentences
Once you have your specific
antibody, you lyse for cell
fractionation yeast cells grown in
regular media (control) or in media
lacking FT (No FT). You collect the
fractions of interest and run them
on an SDS-PAGE followed by WB
against SFT2 and SFT1. This diagram
represents the results obtained.
Please notice that for clarity the
controls to ensure each of the
samples have been properly isolated
and processed have been omitted.
K) How does the SFT2 protein change in the absence of FT? Describe the change observed in the gel and state a possible explanation. Max 3 sentences
L) How does SFT2’s localization change in the absence of FT? Describe the change observed and state a possible explanation. Max 3
M) Given the presence of SFT2 in lane #2, what feature may you have missed when you analyzed the primary sequence of SFT2 that can explain this result? Justify your answer. Max 3 sentences
N) Knowing that the changes observed in SFT2 are induced by changes in membrane fluidity, write down in chronological order a numerical list of the events that will take place in wt cells when FT is low to return to a condition in which the amount of FT is back to normal. (Hint: be sure to use all the information provided, beginning with membrane fluidity and including how SFT1 may be regulated and its potential role; your list should have around 6 steps)