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1. You study protein localization sequences. You look through protein sequence databases and find a handful of proteins that are interesting: They carry an N-terminal signal sequence that would localize them to the ER via the SRP (and be cleaved once the protein was translated), but also an internal nuclear localization signal. You look at these proteins one at a time in a well-studied cell line to determine where they localize to in the cell under normal conditions and following a handful of stimuli. You make two observations:
A) If a protein with these two signal sequences is soluble inside the ER lumen, it never enters the nucleus. These proteins are either released from the cell or enter the lysosome. Why? Speculate what would happen if you edited the protein and removed the ER signal sequence.
B If the protein is transmembrane, under particular conditions, it can be found in the nucleus. One example is ATF6, which is a major part of the unfolded protein response. ATF6 is normally present in the ER. If unfolded proteins accumulate, it travels to the Golgi and then can be found in the nucleus, where it acts as a transcription factor. Note that vesicles cannot pass through the NPC. Why/how does it go to the Golgi? What could happen there so that it can enter the nucleus?
2. Montana Molecular is a company in Bozeman that develops pathway sensor molecules. Go to their website and peruse their product offerings. Design an experiment using a Montana Molecular product (I recommend the cADDIS assay, but you might have fun playing with something else). It can be specific or broad, real or imaginary–the goal here is to get you to think about what these biosensors are used for. 1) What is your hypothesis? 2) Which product will you use to test that hypothesis? 3) If your hypothesis is true, what will you observe, using the MM products?
Give a VERY GENERAL description of how the Montana Molecular assay is performed and what the readout is (do not tell me buffers or step by step instructions). Mention a tool used to detect or quantify the readout.
3) Neurons are poised to release molecules like neurotransmitters into a synapse very quickly and suddenly to continue an action potential. Beginning with the moment when neurotransmitters enter the synapse, describe in detail 1) how the signal is read by the post-synaptic neuron 2) how that causes an action potential and what an action potential is, 3) how an action potential can lead to the rapid burst of pre-assembled neurotransmitters into the next synapse. Include how the molecules are held and released by the pre-synaptic neuron.
Some neurotransmitters like serotonin and acetylcholine are not proteins, and they are not assembled in secretory vesicles in the Golgi. The enzymes that create them are cytosolic. Explain how vesicles become packed with neurotransmitters.
4. You study a gene called YFG1 and its role in intestinal inflammation. Your collaborator send you a mouse colony that they engineered to carry a sequence in its genome that conceptually looks like the cartoon below (this would be an example of a transgene). This is valuable to you because mouse YFG1 functions differently than human YFG1, so using this mouse could allow you to make conclusions that are more relevant to human YFG1 and inflammation.
A) Describe what is produced, and under what conditions.
B) That IRES is stressing you out, perhaps it’s a BIOB425 emotional trigger. You suggest to your research team that you remove the segment between the two arrows above the smiley face, between the yellow boxes. Doing so would remove the segment and keep the rest of the sequence in-frame. A postdoc in your lab advises you not to do that because there’s a big advantage to engineering the transgene this way. What would happen if you remove that segment? What’s the advantage then to keeping it?
C) After understanding the value of this transgenic mouse, you want to make your own. Your collaborator is happy to share most of their techniques, but the IRES they claim is proprietary. The postdoc tells you that’s not a problem, and to find your own IRES. Where can you find a sequence to use? (hint: there’s several answers possible here).
Because of the villin promoter, this transgene is transcribed almost entirely in intestinal epithelial cells. If you saw no GFP signal with the IRES you chose, despite the fact that it seemed to be functional in another cell type, what could explain that?
5. This figure is a highly simplified diagram of how insulin is released from a cell, found in the pancreas. Use your words to describe how glucose uptake can lead to insulin release. Mention everything labeled here and what it does. Include a brief sentence about how [Ca2+]i is involved since it’s not part of the diagram.
2) Let’s say you engineer a cell to express PPO, from Journal Article #3. What could be the outcome of PPO stimulation with blue lig
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