MIT Biology Department 7.012: Introductory Biology -Fall 2004 Instructors: Professor Eric Lander, Professor Robert A Weinberg, Dr. Claudette Gardel Solutions to 7.012 Problem set 8 Question 1 You have isolated cells from an alien organism called Sacul Sinned. You want to know if these cells contain proteins that are similar to the cyclins found in eukaryotic cells. To this end, you radioactively label all the proteins in the cell to allow for visualization on a gel. You then synchronize the cells by placing them in low serum for 1 day. Finally, you stimulate the cells with PdgF and isolate protein from the cells at different time points. The proteins are then run out on a gel similar to a dna gel, such that the proteins are separated by size and you can visualize them through their radioactivit 6 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 as potential cyclin proteins because cyclin proteins are only present during the part of the cell cycle that they regulate b)How many cell cycles did your time points cover if each of the protein bands you selected above are actually cyclin proteins? Explain you answer The time points cover 2 cell cycles. Cyclins are proteins that bind to cyclin dependant kinases and activate them. The cyclin proteins are only present during the part of the cell cycle that they regulate. If the bands that you circled are cyclin proteins then the fact that each of them appears twice over your time course suggests that your cells have gone through two cell divisions 7012Fall2003
7.012 Fall 2003 1 Solutions to 7.012 Problem Set 8 Question 1 You have isolated cells from an alien organism called Sacul Sinned. You want to know if these cells contain proteins that are similar to the cyclins found in eukaryotic cells. To this end, you radioactively label all the proteins in the cell to allow for visualization on a gel. You then synchronize the cells by placing them in low serum for 1 day. Finally, you stimulate the cells with PDGF and isolate protein from the cells at different time points. The proteins are then run out on a gel similar to a DNA gel, such that the proteins are separated by size and you can visualize 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 as potential cyclin proteins because cyclin proteins are only present during the part of the cell cycle that they regulate. b) How many cell cycles did your time points cover if each of the protein bands you selected above are actually cyclin proteins? Explain you answer. The time points cover 2 cell cycles. Cyclins are proteins that bind to cyclin dependant kinases and activate them. The cyclin proteins are only present during the part of the cell cycle that they regulate. If the bands that you circled are cyclin proteins then the fact that each of them appears twice over your time course suggests that your cells have gone through two cell divisions. 1 2 3 4 5 6 7 8 MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
Question 1, continued Synchronization places all the cells in the same stage of the cell cycle, such that as the celles c)What does synchronization do to the cells and why is it necessary for this experimen progresses they enter the other phases at the saime time. It is necessary for the above experiment because if the cells were in asynchronous growth, then you would not be able to detect the disappearance and reappearance of individual cyclin proteins because the times at which this happened would be different for 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 receiving enough extracellular growth signals in low or no serun to start another cell cycle so they enter quiescence 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 assume the alien cells contain a protein similar to the PDGF receptor. How does PdG binding to the receptor cause the activation of the receptor and subsequent promotion of the cell cycle? PDGF causes the receptor subunits to dinerize, when they dimerize the cytoplasmic domains cross phosphorylate each other and this activates the receptor. The activation of the receptor and its downstream targets leads to an increase in cyclind protein levels among other things, which promotes cell cycle progression 7012Fall2003
7.012 Fall 2003 2 Question 1, continued c) 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 cycle progresses they enter the other phases at the same time. It is necessary for the above experiment because if the cells were in asynchronous growth, then you would not be able to detect the disappearance and reappearance of individual cyclin proteins because the times at which this happened would be different for 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 receiving enough extracellular growth signals in low or no serum to start another cell cycle so they enter quiescence 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 assume the alien cells contain a protein similar to the PDGF receptor. How does PDGF binding to the receptor 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 cross phosphorylate each other and this activates the receptor. The activation of the receptor and its downstream targets leads to an increase in cyclinD protein levels among other things, which promotes cell cycle progression
Question 2 Your professor Flow Enirehtac asks you to learn more about the PDGF receptor inside these alien cells. In order to discover the function of various regions in the protein you create cells that 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 cells the protein is located. You find that the pdgF receptor protein is found in the cytosol of all the cells 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 to the 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 that is 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 now found free in the cytoplasm. Which component(s)of the protein secretion pathway might be missing in type 1 cells The signal sequence or SRP ii)What other kinds of proteins might be affected in T ype 1 cells? Secreted proteins iii)Type 2 cells do not respond to PDGF because PDGF receptor proteins are found outside the cell. what kind of mutation would lead to this result? a mutation in the transmembrane domain 7012Fall2003
7.012 Fall 2003 3 Question 2 Your professor Flow Enirehtac asks you to learn more about the PDGF receptor inside these alien cells. In order to discover the function of various regions in the protein you create cells that 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 cells the protein is located. You find that the PDGF receptor protein is found in the cytosol of all the cells. 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 to the 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 that is 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 now found free in the cytoplasm. Which component(s) of the protein secretion pathway might be missing in Type 1 cells. The signal sequence or SRP ii) 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 found outside the cell. What kind of mutation would lead to this result? A mutation in the transmembrane domain
Question 3 In 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 find from this experiment is that some genes are upregulated in the tumor cells i. e. they have an increase in the mRNA levels of a certain gene and that some genes are downregulated in the tumor cells meaning that they have a decrease in the mRNA levels of certain genes. You also have 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 plays a 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 the signaling molecule to be constantly on)or how it is regulated at the translational level such that it has increased protein levels but not mRNA levels e)You clone the gene from(d)and you discover that it is p21 as. You then take normal fibroblasts and overexpress this allele of the Ras gene in them. To your surprise the cell do not oliferate, but stop dividing. Give an explanation of for this outcome Overexpression of Ras in normal fibroblast causes cells to stop dividing and enter a state called senescence. This result can be explained in two ways. Over expression of Ras in an otherwise norma cell causes molecules involved in growth inhibition to be activated. This suppresses the effects of ras Also, cells with tumorigenic ability are not produced by a mutation in one gene. It takes several deregulated genes to give a cell that will now grow uncontrollably. Only if you overepxressesed Ras in fibroblast that are missing rb and p53 function would you get cells that grow uncontrollably. You need to get rid of the brake while pressing on the gas pedal 7012Fall2003
7.012 Fall 2003 4 Question 3 In 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 find from this experiment is that some genes are upregulated in the tumor cells i.e. they have an increase in the mRNA levels of a certain gene and that some genes are downregulated in the tumor cells meaning that they have a decrease in the mRNA levels of certain genes. You also have 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 plays a 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 the signaling molecule to be constantly on) or how it is regulated at the translational level such that it has increased protein levels but not mRNA levels. e) You clone the gene from (d) and you discover that it is p21Ras. You then take normal fibroblasts and overexpress this allele of the Ras gene in them. To your surprise the cell do not proliferate, but stop dividing. Give an explanation of for this outcome. Overexpression of Ras in normal fibroblast causes cells to stop dividing and enter a state called senescence. This result can be explained in two ways. Over expression of Ras in an otherwise normal cell causes molecules involved in growth inhibition to be activated. This suppresses the effects of Ras. Also, cells with tumorigenic ability are not produced by a mutation in one gene. It takes several deregulated genes to give a cell that will now grow uncontrollably. Only if you overepxressesed Ras in fibroblast that are missing Rb and p53 function would you get cells that grow uncontrollably. You need to get rid of the brake while pressing on the gas pedal
Shown below is a schematic representation of an anti body made in b cells Antigen binding site Antigen binding site Variable Variable region region Light chain Light chain Heavy chain Heavy chain Membrane-spanning Membrane-spanning region of a membrane-bound region of a membrane-bound antibody antibody Region of secreted antibody that interacts with phagocyte a)Label the following structures(1)-(v)on the antibody diagram i1)the heavy chains 1)the antigen binding sites iv)the variable regions v) If this were a secreted antibody, indicate the region of the anti body that would interact with macrophages vi) If this were a membrane-bound antibody, indicate the membrane-spanning region of the antibod 1) List two ways that anti body diversity is created 1) Association of different light chains and different heavy chains 2)Recombination of different V(D)and segments on the immunoglobulin genes 3)Imprecise joining of nucleotides at the joining sites ii) What are the interactions between the light and heavy chains that make them associate with each other? what kind of bonding is this? Disulfide bridges covalent bond 7012Fall2003
7.012 Fall 2003 5 Question 4 Shown below is a schematic representation of an antibody made in B cells. Light chain Light chain Heavy chain Variable region Variable region Antigen binding site Antigen binding site Membrane-spanning region of a membrane-bound antibody Region of secreted antibody that interacts with phagocytes Heavy chain Membrane-spanning region of a membrane-bound antibody a) Label the following structures (i) – (v) on the antibody diagram. i) the light chains ii) the heavy chains iii) the antigen binding sites iv) the variable regions v) If this were a secreted antibody, indicate the region of the antibody that would interact with macrophages. vi) If this were a membrane-bound antibody, indicate the membrane-spanning region of the antibody. b) i) List two ways that antibody diversity is created. 1) Association of different light chains and different heavy chains 2) Recombination of different V (D) and J segments on the immunoglobulin genes. 3) Imprecise joining of nucleotides at the joining sites. ii) What are the interactions between the light and heavy chains that make them associate with each other? What kind of bonding is this? Disulfide bridges = covalent bonds