Name: _______Answer Key________________ 1 Recitation Section or TA: 7.013 Exam Three May 2, 2007 Exam starts at 11:05 am and ends at 11:55 am. There are 9 pages including this cover page. Please write your name on each page. Only writing on the FRONT of every page will be graded. (You may use the backs, but only as scratch paper.) Questions that call for short answers should be limited to 15 words or fewer. Page 2 5 pts Page 3 12 pts Page 4 17 pts Page 5 17 pts Page 6 15 pts Page 7 16 pts Page 8 12 pts Page 9 6 pts TOTAL out of 100_______Name: _______Answer Key________________ 2Topic 1. Ectodermin is a transcription factor found in the 500-cell stage frog embryo. It is expressed in a graded fashion, and levels of expression correlate with future fate of the embryo as follows. Animal PoleVegetal PoleNeuronMuscleGutRelative EctoderminConcentrationLowNoneHighAnimal PoleVegetal PoleNeuronNeuronMuscleMuscleGutGutRelative EctoderminConcentrationRelative EctoderminConcentrationLowNoneHigh Mutants were isolated, which have the following distribution of the Ectodermin protein. 1a. (2 pts) For each, write next to the diagrams above what cell types you predict will form and where. Within the cells that make high levels of Ectodermin, Ectodermin activates expression of the Chordin and Noggin genes, both of which encode secreted proteins. 1b. (3 points) On the diagram of a normal embryo to the right, indicate on each line whether Noggin RNA is expressed, and whether Noggin protein would be detected. (As above, shading indicates relative Ectodermin concentration.) Animal PoleVegetal PoleNoggin RNA? Noggin Protein?Animal PoleVegetal PoleNoggin RNA? Noggin Protein?Animal PoleVegetal PoleAnimal PoleVegetal PoleNoneNoneNoneLowNoneNoneAnimal PoleVegetal PoleAnimal PoleVegetal PoleNoneNoneNoneNoneNoneNoneLowNoneNonemuscle gut gut gut gut gut yes yes maybe yes no noName: _______Answer Key________________ 3 You isolate a mutant that does not express either Chordin or Noggin. 1c. (3 pts) Which of the following scenario(s) would be most likely to explain this phenotype: Mutant Ectodermin protein, increased methylation of Noggin promoter, or failure of Noggin splicing? (Circle one and explain in 15 words or fewer.) Loss of Ectodermin function prevents expression of both Chordin and Noggin You isolate another mutant that fails to express Noggin, but expresses Chordin normally. 1d. (3 pts) Which of the following scenario(s) would be most likely to explain this phenotype: Mutant Ectodermin protein, increased methylation of Noggin promoter, or failure of Noggin splicing? (Circle one and explain in 15 words or fewer.) Failure to splice would prevent proper Noggin expression alone, not affecting Chordin; or promoter methylation (decreasing transcription), as this could be changed by mutation of a regulatory factor Noggin and Chordin are required for activation of the NeuroD gene, encoding a transcription factor that is required for development of neurons. 1e. (3 pts) Loss of NeuroD function leads to absence of all neurons as well as absence of the pancreas. Explain how NeuroD can direct formation of these two different cell fates. (Explain in 15 words or fewer) NeuroD works in combination with different factors in developing pancreas versus neurons. Treatment of early embryos with 5-azaC, an inhibitor of DNA methylation on cytosine, leads to a highly abnormal newborn, including increased numbers of neurons and a larger pancreas, but also abnormalities in almost every other organ and tissue. 1f. (3 pts) Explain the mechanism by which 5-azaC treatment leads to abnormalities? (15 words or fewer). Transcription of many genes will INCREASE abnormally, as DNA methylation inhibits transcription. Chromatin structure will change and gene expression will be abnormal. Transcription will INCREASE abnormally and so the animal will be abnormal.Name: _______Answer Key________________ 4In a separate experiment, you isolate a mutant with an altered NeuroD promoter. The mutation leads to an increased neuron number and a pancreas that is larger than normal, but in other respects the animal is normal. Wild type and mutant promoter regions have the following sequences (altered bases are underlined): wild type mutant 5’ TAACCTCGAATCGATTG 3’ 5’ TAACCTAAAATTTATTG 3’ 3’ ATTGGAGCTTAGCTAAC 5’ 3’ ATTGGATTTTAAATAAC 5’ 1g. (3 pts) What is a likely mechanism by which the change in the neuroD promoter causes the associated phenotype? (15 words or fewer) C removal will remove methylation and increase transcription of NeuroD. May have removed an inhibitor-binding site. Topic 2. Classical assays suggested that the brain contained no stem cells whatsoever. Recently, however, a seminal experiment indicated that cell division occurs in certain small regions of the brain. 2a. (2 pts) For researchers who had been trying to isolate neural stem cells, why was this finding so important? (15 words or fewer) A key property of stem cells is that they divide Most stem cells express the cell surface protein Sca-1, whereas non-stem cells do not. 2b. (2 pts) Assuming that neural stem cells express Sca-1, what piece of laboratory equipment would likely be used to isolate neural stem cells? FACS (Fluorescence activated cell sorter) Once a population of cells that might be neural stem cells (“putative stem cells”) had been isolated, researchers performed the following assays to ask whether the cells had stem cell properties. 2c. (4 pts) Putative stem cells were placed in culture for several days. If these were neural stem cells, list the two key properties of stem cells that you would observe. Self renewal and production of differentiated neurons 2d. (2 pts) It has been shown that some neural stem cells can produce several functional types of neurons. Cells with this ability are called pluripotent 2e. (2 pts) Other neural stem cells can produce only one kind of neuron. Cells with this ability are called unipotent/monopotent 2f. (2 pts) Are neural stem cells committed or differentiated? Explain in 15 words or fewer. Committed (determined), they have decided on their fate, but do not have final function.Name: _______Answer Key________________ 5 In another assay, researchers introduced putative neural stem cells isolated from a newborn mouse into the brain of another newborn mouse, which normally produces
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