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(1)
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Using the standard curve you constructed determine the molecular weight of the polypeptides assigned by your teacher.
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Answers will vary depending on the polypeptides chosen.
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(2)
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Explain your rationale for using the banding pattern of the
electrophoresed samples to determine the genetic distance among the three species of fish.
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Answer: Genetic divergence and evolutionary relationship among animals as well as plants can be determined by gel electrophoresis. Individual polypeptides that differ by as little as a single amino acid can be resolved on a polyacrylamide gel based on their molecular weight. The banding pattern of proteins from one species of animal can be compared to another species to determine the genetic distance between the two species. Genetic distance is a measure of the time since two species were able to mate and produce viable offspring. The similarity of protein banding patterns between the two spiny-rayed fish indicates they have a shorter genetic distance than either has to the soft-rayed fish.
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(3)
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Observe the electrophoretic patterns from each of the three samples. Do you recognize similarities or differences? Describe. Which two fish species have a similar banding pattern and why?
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Answer: When gel results are analyzed, it should be clear that the yellow perch and walleye have a similar banding pattern, indicating they are both derived from a common ancestor only several million years ago. The salmon banding pattern is quite different from either the walleye or perch, indicating an earlier divergence.
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(4)
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Myosin is a very large protein molecule (500 kdal) that makes up the thick filaments of muscle tissue. It contains two identical major chains (200 kdal each) and four light chains (about 20 kdal each). During sample preparation, myosin breaks down to its heavy- and light-chain fractions.
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(a) Using your standard curve plot, locate the heavy-and light-chain bands on the gel. How far should the heavy- and light-chain bands have migrated?
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Answer: Answers may vary, but should approximate the following distances: The heavy-chain bands should have migrated about 5-6 mm, the light-chain bands about 50-51 mm.
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(b) How many bands do you expect to find for the heavy and light chains? Explain.
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Answer: There should be one heavy- and one light-chain band. Because the two heavy chains have the same molecular weight they will appear as one band. The light chains will appear as one band as well, for the same reason.
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(c) The average amino acid residue in a protein has a molecular weight of 120 Daltons. How many amino acid residues make up the heavy chains? the light chains?
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Answer: The heavy chain (200 kdal) is made up of 1667 amino acid residues (200,000 Daltons divided by 120 Daltons). The light chain is made up of about 176 amino acid residues.
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(5)
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Based on the protein banding pattern on your gel, what can you infer about the DNA relationship among the three species of fish?
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Answer: The protein banding pattern of the two spiny-rayed fish is similar because their DNA must be more similar than either would be to the DNA of the soft-rayed fish.
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