7.012 Fall 2003 1Solutions to 7.012 Problem Set 8Question 1You have isolated cells from an alien organism called Sacul Sinned. You want to know if thesecells contain proteins that are similar to the cyclins found in eukaryotic cells. To this end, youradioactively label all the proteins in the cell to allow for visualization on a gel. You thensynchronize the cells by placing them in low serum for 1 day. Finally, you stimulate the cellswith PDGF and isolate protein from the cells at different time points. The proteins are thenrun out on a gel similar to a DNA gel, such that the proteins are separated by size and you canvisualize them through their radioactivity.a) Circle the protein bands on the gel that would be good candidates for cyclin proteins?Explain your choices.Any of the bands that appear and then disappear during your time course would be good choices aspotential cyclin proteins because cyclin proteins are only present during the part of the cell cycle thatthey regulate.b) How many cell cycles did your time points cover if each of the protein bands you selectedabove are actually cyclin proteins? Explain you answer.The time points cover 2 cell cycles. Cyclins are proteins that bind to cyclin dependant kinases andactivate them. The cyclin proteins are only present during the part of the cell cycle that they regulate. Ifthe bands that you circled are cyclin proteins then the fact that each of them appears twice over yourtime course suggests that your cells have gone through two cell divisions.12345876MIT Biology Department7.012: Introductory Biology - Fall 2004Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel7.012 Fall 2003 2Question 1, continuedc) What does synchronization do to the cells and why is it necessary for this experiment?Synchronization places all the cells in the same stage of the cell cycle, such that as the cell cycleprogresses they enter the other phases at the same time. It is necessary for the above experiment becauseif the cells were in asynchronous growth, then you would not be able to detect the disappearance andreappearance of individual cyclin proteins because the times at which this happened would be differentfor each of the cells on your tissue culture plate.d) Why does low serum or no serum synchronize your cells?Low or no serum causes all the cells to exit the cell cycle and enter Go . The cells are not receivingenough extracellular growth signals in low or no serum to start another cell cycle so they enterquiescence or Go. Thus, when serum is added all the cells begin the cell cycle together.e) Since you used PDGF in your experiment and it stimulates your cells to divide, you assumethe alien cells contain a protein similar to the PDGF receptor. How does PDGF binding to thereceptor cause the activation of the receptor and subsequent promotion of the cell cycle?PDGF causes the receptor subunits to dimerize, when they dimerize the cytoplasmic domains crossphosphorylate each other and this activates the receptor. The activation of the receptor and itsdownstream targets leads to an increase in cyclinD protein levels among other things, which promotescell cycle progression.7.012 Fall 2003 3Question 2Your professor Flow Enirehtac asks you to learn more about the PDGF receptor inside thesealien cells. In order to discover the function of various regions in the protein you create cellsthat no longer express the wild type form of the receptor but various truncated forms.You then use an antibody against the PDGF receptor in order to visualize where in these cellsthe protein is located. You find that the PDGF receptor protein is found in the cytosol of all thecells.a) All the truncated forms of the PDGF receptor are missing sequence from one end.• Which end of the receptor protein is likely to have been deleted?The N-terminus. This deletion most likely removes the signal sequence which directs this protein tothe endoplasmic reticulum.• Why would this deletion change the localization of the PDGF receptor?Without this sequence the protein is produced in the cytosol.b) You also have cells that have a 30 amino acid deletion at the other end of the PDGF receptor.Would you expect this type of receptor to function in the presence of PDGF? Why or why not?You may expect that this receptor would not function as it is missing 30 amino acids of the region thatis phosphorylated.c) You look closely at many other cell lines that fail to respond to PDGF.i) Type 1 cells have a single gene mutation and fail to respond to all growth factors.Antibody staining of these cells shows that many different membrane proteins are nowfound free in the cytoplasm. Which component(s) of the protein secretion pathwaymight be missing in Type 1 cells.The signal sequence or SRPii) What other kinds of proteins might be affected in Type 1 cells?Secreted proteins.iii) Type 2 cells do not respond to PDGF because PDGF receptor proteins are foundoutside the cell. What kind of mutation would lead to this result?A mutation in the transmembrane domain.7.012 Fall 2003 4Question 3In order to study genes involved in the progression of cancer you do gene expression analysis.To do this you compare the mRNA levels of normal cells versus tumor cells. What you findfrom this experiment is that some genes are upregulated in the tumor cells i.e. they have anincrease in the mRNA levels of a certain gene and that some genes are downregulated in thetumor cells meaning that they have a decrease in the mRNA levels of certain genes. You alsohave some genes whose mRNA levels stay the same.a) What is the most likely function of the genes that are upregulated in the tumor cells?These genes are most likely involved in the promotion of the cell cycle.b) What is the most likely function of the genes that are downregulated in the tumor cells?These genes are most likely involved in the inhibition of the cell cycle.c) What is the most likely function of the genes that are the same in tumor and normal cells?Genes that stay the same may not be involved directly with the regulation of cellular proliferation.d) You find out later that one of the genes whose mRNA levels stayed the same actually playsa critical role in the progression of cancer. How can this be?Either this gene obtained a mutation which changes how the protein acts (a mutation that causes thesignaling molecule to be constantly on) or how it is regulated at the translational level such that it
View Full Document
Unlocking...