2005_pset5_ans - 7.06 Problem Set #5, Spring 2005 1. You...

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7.06 Problem Set #5, Spring 2005 1. You are working as a researcher for a biotechnology firm, developing orange trees that are more resistant to cold weather. There is a class of cold-response proteins called COR proteins that you have been studying. Wheat, which grows under widely different climates, expresses a particular COR protein (WheCOR) that is not expressed in orange trees. The WheCor gene is nuclear-encoded, and the WheCOR protein is targeted to the chloroplast stroma. You hypothesize that making a transgenic orange tree that expresses WheCor might make it more resistant to cold weather. a. First you decide to determine if the WheCOR protein can be transported into orange tree chloroplasts. A colleague suggests that you do an in vitro post-translational uptake assay. In this assay he suggests adding purified radiolabeled WheCOR made in a cell- free system to orange tree chloroplasts and some cytoplasmic extract. As a negative control, he suggests repeating that same experiment, but adding cyanide prior to adding the WheCOR protein. Then he says you can add the protease trypsin to both the cyanide-treated and untreated chloroplasts. Finally you would run an SDS-PAGE gel of your reaction mixture and ascertain if your protein is protected by the chloroplasts by looking for signal or absence of signal in the resulting autoradiographic film. You explain to your colleague that he is confused and that this experiment wouldn’t proceed as he anticipates it would. What mistake is your colleague making? You cannot use cyanide-treated chloroplasts as a negative control. Cyanide poisons the oxidative phosphorylation pathway by dissipating the proton-motive force. In mitochondria, the dissipation of the proton-motive force would cause proteins to be unable to enter the organelle. However, this force only drives protein import into mitochondria, and not into chloroplasts. b. After talking to your confused colleague, you decide to design your own experiment. Therefore you introduce the WheCor gene into orange tree plant cells, using a vector that integrates into the nuclear DNA. You observe through fluorescence microscopy that the WheCOR protein is not being transported into the transgenic orange tree chloroplasts, although the endogenous orange tree chloroplast proteins are. To determine what is wrong, you do the following experiment. You use three experimental groups: 1) wild-type wheat, 2) mutant wheat that contains a point mutation in the WheCOR stromal-import sequence, and 3) the transgenic orange tree expressing WheCOR. You separate the chloroplasts from the rest of the cell components (cytosol, other organelles and membranes) for each experimental group. You then make a lysate from both the chloroplasts and the non-chloroplast cell components, and perform Western blotting using an anti-WheCOR primary antibody. You see the following result:
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Wheat Mutant Wheat Transgenic Orange Tree Give a possible explanation for this result. These results confirm that WheCOR is not being transported into the orange tree
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2005_pset5_ans - 7.06 Problem Set #5, Spring 2005 1. You...

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