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| + | This page has been retired, current tutorial content is available [[Help:Tutorial|here]]. |
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| - | {{Template:TutorialNavigate|8}}
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| - | == The negative feedback loop ==
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| - | The final event we are going to add to the pathway is the negative feedback loop formed by the mPer/mCry dimer. In order to inhibit the expression of the Circadian clock genes, the mPer and mCry proteins need to dimerize and translocate to the nucleus. After dimerization, mPer is protected from degradation (see [[Help:Tutorial:Step 6|step 6]]). Once the dimer is in the nucleus, it will inhibit the activation of the E-box element by Clock/Bmal1, thereby acting as a negative feedback loop. After this step, the pathway will look like this:
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| - | {|class=prettytable
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| - | |[Image:negative_feedback.png]]
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| - | |}
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| - | ==== Copy the mCry and mPer DataNodes ====
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| - | {{TutorialVideo|kg8M0QfCfII|360|Drawing the negative feedback loop}}
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| - | The negative feedback loop will need two additional variations on the mCry and mPer proteins:
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| - | * The mCry/mPer dimer outside the nucleus
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| - | * The mCry/mPer dimer inside the nucleus
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| - | To add these DataNodes, you can copy the 4 mCry and mPer DataNodes under the ''Gene'' label you added in [[Help:Tutorial:Step 6|step 6.1]] twice and move them to right location (see image above).
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| - | ==== Draw the dimerization of mPer and mCry ====
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| - | Drawing this is similar to drawing the dimerization of Clock and Bmal1 in [[Help:Tutorial:Step 5|step 5.2]]. First, copy the 4 mCry and mPer DataNodes under the ''Gene'' label you added in [[Help:Tutorial:Step 6|step 6.1]]. Place the copied DataNodes above/right of the seperate mPer and mCry DataNodes, stack them and create a complex (select all four DataNodes and press CTRL-P). Connect two arrows from the individual mPer and mCry elements to the newly created mPer/mCry complex and set their line type to 'curved'.
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| - | ==== Draw the translocation of mPer/mCry ====
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| - | After dimerization, the mPer/mCry dimer translocates to the nucleus. There is no information about cellular location on the pathway yet, this is going to happen in [[Help:Tutorial:Step 8|step 8]], but we can already draw the translocation. Copy the mCry/mPer complex and move the copy next to the Clock/Bmal DataNodes. Draw and connect a dashed arrow between the original and copied dimer. You can set the line style of the dashed arrow to 'elbow'.
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| - | ==== Draw the inhibition of Clock/Bmal1 function by mPer/mCry ====
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| - | The mPer/mCry complex located in the nucleus, inhibits the activation of the E-box element by Clock/Bmal1. Inhibition can be represented by the T-bar line:
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| - | {|class=prettytable
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| - | |[[Image:tbar.png]]
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| - | |}
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| - | Draw and connect a T-bar line from the mPer/mCry complex in the nucleus to the Clock/Bmal1 dimer bound to the E-box using the [[Image:newtbar.gif]] ''T-bar button'' on the toolbar.
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| - | ==== Add colors to the degradation and dimerization events ====
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| - | To emphasize the two routes mPer can go (either degradation or dimerization with mCry), you can give each route a different color. Change the color of the arrow and degradation shape to red. Select the arrow and degradation shape and click on the ''Color'' property in the properties table. A dialog will appear where you can choose the color. Repeat the same steps for the arrows that represent the dimerization, but now set the color to blue.
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