MIT Biology Department 7.012: Introductory Biology-Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel 7.012 quiz practice Quiz 3 on Friday, November 12th 10-11 AM Review Session Wednesday 11/10 from 7-9 pm Tutoring Session Thurs. 11/11 from 4-6 pm Final Exam is on Monday, December 13th 9-noon
7.012 Quiz 3 practice Quiz 3 on Friday, November 12th 10 – 11 AM Review Session Wednesday 11/10 from 7-9 pm Tutoring Session Thurs. 11/11 from 4-6 pm MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel
Question 1 A)You infected mice with mouse mammary tumor virus(a retrovirus). After a period of time, most infected mice had developed breast tumors whereas uninfected mice did not. You isolated cell lines from over 50 independent tumors. You demonstrated that all of these lines had virus integrations in the same chromosomal location. Can one conclude that the virus integrates into cellular dNA at only one site? Explain. B)The ras oncogene is involved in a variety of human and animal cancers. DNA was isolated from a number of normal and cancerous tissues -Cellular dna was digested with ecori Digested DNA was separated by gel electrophoresis and transfered to a nitrocellulose membrane The membrane was probed with the radioactive labelled cloned ras DNA and then the membrane was exposed to x-ray film The resulting autoradiograph is shown below 1)white blood cells from a healthy human 2)human lymphoma cells(cancerous) 3)human bladder carcinoma cells(cancerous) 4)human sarcoma cells(cancerous) 5)blood from a healthy mouse 6)mouse myeloma cells(cancerous) 3 10 kb 5 kb 1 kb more than 2 copies/genome 2 copies/genome 1 copies/genome
2 Question 1 A) You infected mice with mouse mammary tumor virus (a retrovirus). After a period of time, most infected mice had developed breast tumors, whereas uninfected mice did not. You isolated cell lines from over 50 independent tumors. You demonstrated that all of these lines had virus integrations in the same chromosomal location. Can one conclude that the virus integrates into cellular DNA at only one site? Explain. B) The ras oncogene is involved in a variety of human and animal cancers. DNA was isolated from a number of normal and cancerous tissues. -Cellular DNA was digested with EcoRI. -Digested DNA was separated by gel electrophoresis and transfered to a nitrocellulose membrane. -The membrane was probed with the radioactive labelled cloned ras DNA and then the membrane was exposed to x-ray film. -The resulting autoradiograph is shown below. 1) white blood cells from a healthy human 2) human lymphoma cells (cancerous) 3) human bladder carcinoma cells (cancerous) 4) human sarcoma cells (cancerous) 5) blood from a healthy mouse 6) mouse myeloma cells (cancerous) 1 2 3 4 5 6 10 kb 5 kb 1 kb more than 2 copies/genome 2 copies/genome 1 copies/genome
Question 1 continued a) How do you explain the presence of sequences complementary to the oncogene in the DNA from healthy human and mouse samples? Why don' t they have cancer? b)Why is the hybridizing band from sample l a different size than that from sample 5? c)For each cancer examined above, based on the autoradiogram, choose the most likely mechanism of transformation and explain your choice 1)point mutation within the gene 2 )chromosomal rearrangement involving the gene 3)gene amplification 4)oncogenic retroviral insertion. Question 2 You are studying the cell cycle in haploid yeast cells. You isolate a cell that is a 25 C, but arrests at 36C at the point in the cell cycle where the expression of the at temperature-sensitive cell division cycle(cdc)mutant, cdcX-. cdcX-grows normal normal cdc gene is required To determine where in the cell cycle expression of cdkX is required, you design experiments based on the following facts 1)The drug nocodazole arrests, but does not kill yeast in mitosis(M phase) 2)Cell density can be measured to determine if the yeast cells have completed a cell division. For your experiments You incubate cdkX- cells at 25 C with nocodazole until all the cells are synchronized. You then shift the cells to 36C and remove the nocodazole The cells divide once and then arrest You incubate cdkX-cells at 36 C until all arrest. You then add nocodazole and shift the cells to 25 The cells arrest without dividing. a)Given these experiments you can assume that the protein encoded by the cdkX gene is not required at what phase(s)of the cell?
3 Question 1 continued a) How do you explain the presence of sequences complementary to the oncogene in the DNA from healthy human and mouse samples? Why don't they have cancer? b) Why is the hybridizing band from sample 1 a different size than that from sample 5? c) For each cancer examined above, based on the autoradiogram, choose the most likely mechanism of transformation and explain your choice: 1) point mutation within the gene 2) chromosomal rearrangement involving the gene 3) gene amplification 4) oncogenic retroviral insertion. Question 2 You are studying the cell cycle in haploid yeast cells. You isolate a cell that is a temperature-sensitive cell division cycle (cdc) mutant, cdcX-. cdcX- grows normally at 25 °C, but arrests at 36 °C at the point in the cell cycle where the expression of the normal cdcX gene is required. To determine where in the cell cycle expression of cdkX is required, you design experiments based on the following facts: 1) The drug nocodazole arrests, but does not kill yeast in mitosis (M phase). 2) Cell density can be measured to determine if the yeast cells have completed a cell division. For your experiments: • You incubate cdkX- cells at 25 °C with nocodazole until all the cells are synchronized. You then shift the cells to 36 °C and remove the nocodazole. The cells divide once and then arrest. • You incubate cdkX- cells at 36 °C until all arrest. You then add nocodazole and shift the cells to 25 °. The cells arrest without dividing. a) Given these experiments you can assume that the protein encoded by the cdkX gene is not required at what phase(s) of the cell?
Another mutant cdkY-is a cold-sensitive mutant that arrests at 18C, but grows normally at 25C. cdkY-cells arrest between the s and G2 phases of the cell make cdkX-cdkY-double mutants and perform the following experiments. cycle. You You incubate cdkX-cdkY- double mutants at 36C until all the cells are synchronized and then shift the cells to 18 The cells arrest without dividing You incubate cdkX-cdkY-double mutants at 18C until all until all the cells are synchronized and then shift the cells to 360 The cells divide once and then arrest b)given these experiments you can assume that the protein encoded by the cdk X gene is not required at what phase(s)of the cell? c)In what stage(s)of the cell cycle do you expect the protein encoded by the cdkx gene to act? d)A cell in the process of DNA replication(cell A)is fused with a cell in early G1(cell B).How does the timing of the cell cycle for the cell B nucleus in the fused cell compare to that expected for the cell B nucleus if the cells remained independent? e)What soluble factor(s)are found in cell a but not in cell B(check all that apply)? pRB protein a camP dependent kinase TGFβ a G2 cyclin Human papilloma virus an S-phase cyclin a cyclin dependent kinase an oncogene a g1 cyclin
4 Another mutant cdkY- is a cold-sensitive mutant that arrests at 18 °C, but grows normally at 25 °C. cdkY- cells arrest between the S and G2 phases of the cell cycle. You make cdkX- cdkY- double mutants and perform the following experiments. • You incubate cdkX- cdkY- double mutants at 36 °C until all the cells are synchronized and then shift the cells to 18 °. • The cells arrest without dividing. • You incubate cdkX- cdkY- double mutants at 18 °C until all until all the cells are synchronized and then shift the cells to 36 °C. • The cells divide once and then arrest. b) Given these experiments you can assume that the protein encoded by the cdkX gene is not required at what phase(s) of the cell? c) In what stage(s) of the cell cycle do you expect the protein encoded by the cdkX gene to act? d) A cell in the process of DNA replication (cell A) is fused with a cell in early G1 (cell B). How does the timing of the cell cycle for the cell B nucleus in the fused cell compare to that expected for the cell B nucleus if the cells remained independent? e) What soluble factor(s) are found in cell A but not in cell B (check all that apply)? ___ pRB protein ___ a cAMP dependent kinase ___ TGFβ ___ a G2 cyclin ___ Human papilloma virus ___ an S-phase cyclin ___ a cyclin dependent kinase ___ an oncogene ____ a G1 cyclin
Question 3 Part i As a premier cancer biologist, you often plate cells in dishes, feeding them serum with growth factors and allowing them to grow for 2 weeks. Sometimes after incubation of strains you observe the following when looking at the side of a culture dish Strain a Strain b a)Which plate shows abnormal cells? Explain b)Predict the behavior of these cell lines if grown without added growth factors by drawing what the plates will look like after incubation without growth factors. Simply modify the existing figure below for your answer.(Note: one cell from each strain is initially deposited in each dish. Strain a Strain b Part ll A fellow researcher gives you two cancerous cell lines to examine and determine possible mutations. The results are shown below Cell Line Mutation WT none(wild type DNA 2 at the same region on both copies of chromosome 4 lutation in a gene on only one copy of chromosome 7 c) Based on this data above identify the type of cancer gene that is mutated in each of the cell lines Cell Line Cancer gene (oncogene or tumor suppressor gene) WT none 2
5 Question 3 Part I As a premier cancer biologist, you often plate cells in dishes, feeding them serum with growth factors and allowing them to grow for 2 weeks. Sometimes after incubation of strains you observe the following when looking at the side of a culture dish. Strain A Strain B a) Which plate shows abnormal cells? Explain. b) Predict the behavior of these cell lines if grown without added growth factors by drawing what the plates will look like after incubation without growth factors. Simply modify the existing figure below for your answer. (Note: one cell from each strain is initially deposited in each dish.) Strain A Strain B Part II A fellow researcher gives you two cancerous cell lines to examine and determine possible mutations. The results are shown below. Cell Line Mutation WT none (wild type DNA) 1 a deletion at the same region on both copies of chromosome 4 2 a point mutation in a gene on only one copy of chromosome 7 c) Based on this data above, identify the type of cancer gene that is mutated in each of the cell lines. Cell Line Cancer Gene (oncogene or tumor suppressor gene) WT none 1 2