Tutorial 5 worksheet (2)

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Jan 9, 2024

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Participation Mark: _________ (1%) Name: _________________________ Tutorial: _________ BIOL200 Unit 3 Problem Solving Worksheet Problem 3.2.1 (tags: #gel electrophoresis, #DNA packing, #nuclease digestion, #experimental controls) Evidence for the packing of DNA in the eukaryotic nucleus originally came from experiments such as the ones described in the next few problems. Researchers first postulated that nucleosomes existed in the eukaryotic chromatin quite early, but then they had to prove it experimentally. One of the most powerful experiments conducted, which helped to prove the existence of nucleosomes was the nuclease digestion experiment. In this experiment, they used micrococcal nuclease to digest DNA. The micrococcal nuclease enzyme is a large bacterial enzyme that can only cut exposed DNA (i.e. ‘naked’ DNA). It cannot cut DNA that is tightly bound to proteins. Experimental Protocol: 0. Starting point: 30nm chromatin fibre. 1. Wash chromatin with weak salt solution to remove H1 histone. 2. Digest 11nm fiber with micrococcal nuclease (according to treatments listed below) 3. Remove all associated proteins with a strong salt solution. 4. Separate DNA fragments using gel electrophoresis 4 different treatments are used in Step 2 (Treatment A = Lane A): • Treatment A: No nuclease treatment. • Treatment B: Brief (30sec) nuclease digestion of naked DNA. • Treatment C: Extended (20min) nuclease digestion of chromatin • Treatment D: Brief (30sec) nuclease digestion of chromatin. a) Which treatment(s) are the controls? What do they tell you? Taylor Maynard T21 Treatments A and B are the controls. Treatment A shows what DNA looks like on a gel, without nuclease added. Treatment B shows what nuclease digestion looks like on “naked” DNA (no proteins bound to it)

b) What proteins remain associated with the chromatin after step 1? c) How will the length of the nuclease digestion impact the results? d) For each step of the experiment, draw what the chromatin should look like. e) Is there experimental evidence confirming the presence of nucleosomes? Explain using the information in the gel to support your answer. Treatment A. No nuclease digestion B. Brief (30sec) digestion of naked DNA C. Extended (20min) digestion of chromatin D. Brief (30sec) digestion of chromatin Steps in Experiment 0. Starting Chromatin 1. Wash with salts to remove H1.** ** All proteins are removed in this step. 2. Treat samples based on info in columns 3. Remove associated proteins with a salt solution All other histone proteins other than H1; so, two of H2A, H2B, H3, and H4. Extended digestions will result in more cutting of DNA than brief digestions. Enzymes do not work instantaneously, they take time to reach their full effect, so brief digestions will not result in extensive cutting; instead, the length of cutting will vary. 88888 00010 Ny 002020 0000 0000 III 0000 80 00 an yes Lane C has 1 band at approx 140 bp This band represents 140 bp of DNA surrounding

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Tutorial 5 worksheet (2)

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