MIT Department of Biology 7.013: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. Claudette Gardel 7.013 Practice Quiz 3 2005 1Question 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 rasoncogene 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 rasDNA 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 2Question 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 Erythropoietin (EPO), a protein growth factor secreted by the kidneys, is essential for the terminal differentiation of red blood cells (erythrocytes) in the bone marrow. EPO binds to a transmembrane EPO receptor found on erythroid precursor cells. The following homozygous mutations were made only in the hematopoietic stem cell lineage. How would these mutations affect (increase, decrease, or not change) the number of red blood cells formed, compared to the wild-type situation? Briefly explain your reasoning for each mutation. Consider each mutation independently. a) A mutation in the EPO gene that resulted in the deletion of only the signal sequence of the EPO protein. b) A mutation in the EPO receptor gene that resulted in the deletion of only the transmembrane domain of the EPO receptor. c) A mutation in the EPO receptor gene that resulted in the deletion of only the cytoplasmic domain of the EPO receptor. 3Question 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. Cancer GeneCell Line (oncogene or tumor suppressor gene) WT none 1 2 4Question 3 continued You learn that cell line 1 is a skin cancer cell line. The region you identified as deleted on chromosome 4 in these cells normally contains a gene called p16. d) What is the role of the p16gene product in the normal cell based on the information above? You obtain another cell line (cell line 3) that has one wild-type copy of chromosome 4 and one mutant copy of chromosome 4 (as described above in cell line 1). e) Will cell line 3 display a cancerous phenotype when grown in the presence of growth factors? Yes/No (Circle one.) Explain briefly. f) Will cell line 3 display a cancerous phenotype when grown in the absence of growth factors? Yes/No (Circle one.) Explain briefly. Part III g) Cell line 2 is a breast cancer cell line that expresses a mutant version of a receptor protein called KIT. Choose from the following options to explain the role of KIT in normal cells. Circle one. • Activation of KIT causes cells to undergo apoptosis. • Activation of KIT promotes progression through the cell cycle. • Activation of KIT has no effect on the cell cycle. • Activation of KIT causes cells to enter G0. h) Specifically how could a point mutation in the gene encoding the KIT receptor cause the abnormal behavior depicted in Part I. 5Question 4 The receptors Robo and DCC are important for axon’s ability to responding to guidance cues from Netrin and Slit proteins expressed near the brain midline. The mammalian model is summarized below. • Netrin is secreted from the midline and bound by DCC on the neuron. This attracts the neuron toward the midline. • Robo is expressed on neurons once they have crossed the midline. • Slit is secreted from the midline and bound by Robo on the neuron. This repulses the neuron from the midline. • DCC receptors on the neurons expressing Robo become non-responsive to Netrin binding. Figure by MIT OCW. a) In this model of axon guidance it is important that Netrin is… a (short/long) range attractant and a (short/long) range repellant 6 +++++++++++++---------------------------------------------+1. Attraction to Midline: netrin activation of DCCMidlineSlitNetrinDCCDCCRobo2. Crossing & Moving from the Midline:Event 1: Upregulation of Robo expression, repulsion by SlitEvent 2: Loss of netrin responsiveness, despite maintained DCC expressionb) You’re interested in studying this interaction so you use a convenient model organism, the beloved fruit fly. You make mutants in fruit flies that have the robo or slitgenes
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