Problem set            ...

Problem set                                                                                                                                                                                 
A Genome Browsing Problem Set: no more than 3 pages

1) Go to the UCSC Genome Browser and view a human gene of your choice. Describe how you found it. Below the graphics window, click on the "default tracks" and "default order" buttons. Drag and drop the RefSeq Gene Predictions and Repeating Elements tracks to the top of the window. Take a screen shot of the top half of these graphics, including the chromosome ideogram and base coordinates (but not the zoom buttons and above), and paste this shot into your report. Crop to a good size for presentation.
2) Find the human opsin 5 gene with the UCSC Genome Browser and describe how you found it.
3) Now you will customize the view of the human opsin 5 gene. Include the following rows of data, in this order, and hide any rows not listed here:
a) base position ("full" view)
b) NCBI RefSeq genes ("pack" view)
c) Multiz alignment of 100 Vertebrates (also called "Conservation." Use the "full" view and set the default view in the track settings, too)
d) Repeating Elements (Also known as "RepeatMasker." Use the "full" view)
4) Zoom out enough to see all the exons of opsin 5 plus a small amount of flanking sequence. Take a screen shot of the graphics window, including the ideogram and base coordinates (but not the zoom buttons and above) and paste into your report. Crop to a good size for presentation.
5) Click on the gray sidebar in the RefSeq Genes row and configure those datarows to show "RefSeq All," "full" view. You can now see 10 different alternatively spliced transcripts. Take a screenshot of these transcripts and paste it into your report.
6) The largest LTR repeat is found near the middle of the gene. Using the Multiz alignment rows and showing other species not shown in the defaults, when did this repeat insertion arise in the animal lineage? Are there any primates which do not have this LTR repeat? Include a convincing screen shot (or two) in your report, cropped to the proper size. Explain what you found.

Aquaporin-5: no more than 5 pages
In this exercise, you will be manipulating the structure of a protein pore which allows the passage of water. Your goal is to provide a convincing argument of how this is accomplished. Your argument will consist of carefully chosen screen shots and clear descriptions. All molecular views will be shown in JSMol viewer at the rcsb.org website.
1) Go to the CBS NetNGlyc website, determine and record the amino acid coordinate of NP_001642 most likely to be glycosylated. Show a screen shot and explain your answer. Below, this coordinate number will be referred to as "yournum" which will be used in step 7, below. (screenshot and explanation: no more than half a page)
2) Go to rcsb.org and search for the file of accession number 3d9s, the structural file for Aquaporin-5.
3) Click on the "3D View" tab and then select the "JSmol Viewer" for the rest of this problem. Rotate and change the "Display Options" of the protein. Based on what you see, where is the pore or pores? Show screen shot(s) to explain your answer. Keep this window open. (screenshot(s) and interpretation: no more than half a page)
4) In another browser window, use the CBS TMHMM website to determine the transmembrane domains of Aquaporin-5. Show a screen shot and interpretation. (no more than half a page)
5) Return to the 3D view of Aquaporin. Using the results of step 4, above, orient the exterior-facing amino acids at the top of the window, and the cytoplasmic-facing amino acids on the bottom of the window. Show a screen shot and explain your decisions. (no more than half a page)
6) Bring up the "console" of the 3d9s 3D window by right clicking on the window and selecting console.
For the following steps, you will be manipulating the view of aquaporin in the 3D viewer. Your answer should include all the commands you used to display aquaporin-5.
7) Map the glycosylation site onto the structure by entering the following command in the console and hit return (yournum from step 1, above)
select yournum.CA; label Asn; font label 20; set labelfront;

Commands/approaches you may need
• The "restrict" command in JSMol console will only display what you define, causing everything else to disappear. When multiple structures are shown in one view, you can limit the view to only one or more of these structures. For example, entering the following in the console limits the view to only two of the aquaporin structures (the "A" and "C" chains) which are each 266 amino acids long. Floating your mouse over structures allows you to identify their numbers. You may wish to use this command, or parts thereof, to answer the question in step 8.
restrict 1-266:A,1-266:C;
• The locations of water molecules and an immovable lipid ligand in the structure have also been determined but by default they are hidden. The following command makes them visible (water is gray, the lipid is white). They are assigned numbers, too, which can be seen by floating your mouse over them:

select hetero; spacefill;
• Should this be necessary, you can use "hide" to hide certain molecules, uncovering what is behind. For example

hide 1-10,15-20;
8) Using the 3D window, rotation, coloring, zooming, console commands you learned in class, this document, the book, or what you find on the web, demonstrate the most likely path of water molecules through the aquaporin structure. Use screenshots to support your claims. As usual, clear images are more valuable than pictures filled with unnecessary decorations or colors. Be sure that your evidence is clearly visible and explained. You will need to provide all the commands you use to display aquaporin-5.
9) An extra two points will be awarded to the student with the best and most convincing structural views of the class. If there is no clear winner, no extra points will be awarded.