MIT Biology Department 7.012: Introductory Biology -Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel Name Section: 7.012 Problem set 8 Please print out this problem set and record your answers on the printed copy. answers to this problem set are to be turned in at the box outside by 4: 10 Wednesday, November 26. Problem sets will not be accepted late Solutions will be posted on the web November 27, 2003 "PLEASE NOTE Questions 1-3 cover material from lectures 23-25 and thus would be good practice for Quiz Il 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 5678 a)Circle the protein bands on the gel that would be good candidates for cyclin proteins? Explain your choices 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 7012fall2003
Name:___________________________________ Section:_____ 7.012 fall 2003 1 7.012 Problem Set 8 *PLEASE NOTE,: Questions 1 - 3 cover material from lectures 23-25 and thus would be good practice for Quiz III. 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. 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. 1 2 3 4 5 6 7 8 Please print out this problem set and record your answers on the printed copy. Answers to this problem set are to be turned in at the box outside by 4:10 Wednesday, November 26. Problem sets will not be accepted late. Solutions will be posted on the web November 27, 2003. MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
Name Section Question 1, continued c)What does synchronization do to the cells and why is it necessary for this experiment? d) Why does low serum or no serum synchronize your cells? 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? 7012fall2003
Name:___________________________________ Section:_____ 7.012 fall 2003 2 Question 1, continued c) What does synchronization do to the cells and why is it necessary for this experiment? d) Why does low serum or no serum synchronize your cells? 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?
Name Section 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 form one end Which end of the receptor protein is likely to have been deleted? Why would this deletion change the localization of the PDgF receptor b) 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 ii)What other kinds of proteins might be affected in Type 1 cells? i)Type 2 cells do not respond to PDGF because PDGF receptor proteins are secreted out of the cell. what kind of mutation would lead to this result? 7012fall2003
Name:___________________________________ Section:_____ 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 form one end. • Which end of the receptor protein is likely to have been deleted? • Why would this deletion change the localization of the PDGF receptor? b) 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. ii) What other kinds of proteins might be affected in Type 1 cells? iii) Type 2 cells do not respond to PDGF because PDGF receptor proteins are secreted out of the cell. What kind of mutation would lead to this result?
Name Section: 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? b)What is the most likely function of the genes that are downregulated in the tumor cells? c)What is the most likely function of the genes that are the same in tumor and normal cells? 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? 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 proliferate, but stop dividing. Give an explanation of for this outcome 7012fall2003
Name:___________________________________ Section:_____ 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? b) What is the most likely function of the genes that are downregulated in the tumor cells? c) What is the most likely function of the genes that are the same in tumor and normal cells? 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? 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
Name Section Question 4 Shown below is a schematic representation of an antibody made in B cells a) Label the following structures (i)-(v)on the antibody diagram i the light chains i the heavy chains i11)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 macron vi) If this were a membrane-bound antibody, indicate the membrane-spanning region of the antibod i) List two ways that anti body diversity is created ii)What are the interactions between the light and heavy chains that make them associate with each other? What kind of bonding is this? 7012fall2003
Name:___________________________________ Section:_____ 7.012 fall 2003 5 Question 4 Shown below is a schematic representation of an antibody made in B cells. 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. ii) What are the interactions between the light and heavy chains that make them associate with each other? What kind of bonding is this?