If you wanted to home in on the CF gene today, you would not need to chromosomal walk. You could use a process similar to what we will do. Point your web browser to the Genome Browser at the University of California at Santa Cruz. There are similar Genome Browser versions located on other campuses, such as the one located in England at the Sanger Center. Now follow these directions:
1) Click on “Browser” in top left menu and make sure “human” is selected from the pull down menu.
2) Enter into the blank box the RFLP maker “D7S8” and click on “Submit”.
3) You should see the D7S8 marker, as well as a few other more recently discovered markers that were not available when CF was being cloned for the first time.
4) Go to the very bottom where you can see a collection of pull down menus. Choose “Hide” for all features but modify these three settings:
Base Position: On
Chromosome Band: Dense
STS Markers: Full
5) Click on the “Refresh” button. This should clean up the view a bit. Make sure you can still see D7S8.
6) Scroll back up to the top of the browser window and enter
into the “position” box:
8) Click on “Jump”. “Jump” tells the browser to move to the range of DNA bases numbered 116,000,0000 through 117,5000,000 on chromosome 7 (or 116 Mb – 117.5 Mb). This is approximately the 1.5 Mb range of bases that is flanked by MET on one end and D7S8 on the other. Find both markers at this time to verify you are in the right section of the human genome. All we have done so far is electronically zoomed out from the single RFLP marker D7S8 to a wider perspective showing both RFLP markers.
9) Now, change the settings as follows:
STS Markers: Hide
GeneScan Genes: Full
10) Click on the “Refresh” button. You should see a series of brown horizontal lines with vertical tick marks that indicate all the predicted genes in this 1.5 MB region. The vertical marks are where a gene hunting computer program predicted exons might located.
11) Write down how many genes are predicted for this 1.5 Mb region. Which one is CF?!
12 ) Now change the settings as follows:
GeneScan Genes: Hide
13) Hit the “Refresh” button. How many different known genes are there (as of 2003)? Which region of chromosome 7 is CF located? Describe this region in three different ways using the information you have seen in the Genome Browser. Do the number of predicted “GeneScan” genes and “Known Genes” agree? Explain your answer.
14) Scan the known genes and find the gene called CFTR. When the CF gene was cloned and sequenced, the investigators wanted to call it something a bit more descriptive than just the CF gene. So they called it the Cystic Fibrosis Transmembrane Conductance regulator. As we continue our exploration of this human locus, try to figure out why they gave it this name.
15) Double click on the CFTR line and read what you have found.
16) Scroll down some and click on “AceView” and find answers to the following questions. The image on the right of the AceView page shows the length of the gene and the mRNAs produced from this gene.
How many different mRNAs are produced from this gene?
How long is the longest mRNA?
How many exons are there?
List some of this gene’s and the encoded protein’s features.
17) Leave this browser window as it is for now and open a new web browser window.
18) Go to GeneCards which was created in Israel but the version we will use is housed at one of the mirror sites in at Stanford University.
19) Perform a search for “CFTR”.
20) When you get the results page, click on the link in the top left corner that says “Display the complete GeneCard” for this gene (CFTR)
21) Note CFTR’s chromosomal position as a red line. Does CF’s postion match what you found in the Genome Browser? Determine the exact start and stop positions for CFTR. The term “pter” means the terminus of the p arm (p stands for petite which is French for small). How long is the CFTR gene? Which strand is the coding strand? This is indicated as “orientation” on this page. How long is the mRNA? What percentage of the gene is composed of exons?
Scroll down to the portion that shows a collection of colored bar graphs headed by “CFTR expression in normal human tissues based on proprietary W.I.S DNA array (GeneNoTE) results”. Which 5 tissues expresses the highest levels of CFTR? Rank them in order from highest to lowest. Any surprises? Now click on the button that says “Search PubMed” for CFTR. How many abstracts were returned with your search? What is the most recent publication date you found?
22) Go back to the GeneCards page and click on “SWISS-PROT: CFTR_HUMAN”. This database focuses on the human proteome instead of the human genome. A proteome is the protein equivalent to a geneome – the total protein content of an organism.
23) Click on this link at about the middle of the results page called “Feature table viewer”.
24) On this results page, scroll to the very bottom and view the predicted features of CFTR. Click on each of the colored features to determine how many transmembrane (TM) domains (one TM is shown as a pair of green rectangles close together, the location of the ATP binding sites (blue boxes).
25) If the N terminus of CFTR is in the cytoplasm, draw a picture of CFTR. At this time, just focus on the topology, or the number of times CFTR snakes across the plasma membrane. You might want to use a pencil in case you have to revise your predicted structure.
26) Add to your drawing the features of ATP binding sites, phosphorylation sites and glycosylation sites. To see these features clearly, drag the red lines (located on both sides of this view) by clicking on the rectangles at the bottom and dragging the lines to frame the area with all the little blue circles. Then click on the Zoom button. How many ATP-binding sites, phosphorylation sites and glycosylation sites are there? Add these to your picture. Note that the TM domains have gotten thicker (more green rectangles) since you have zoomed in. You can slide the slider bar at the bottom of the window to the left and right to see other areas of the protein structure at this magnification.
27) Reset the view of the entire CFTR protein. Now use the red bars again to zoom in for a higher resolution. Center the red bars on the first (located closer to the amino terminus) ATP binding site. Continue to zoom in until you can see the single letters representing the amino acids of CFTR.
28) Move the slider bar near the bottom over to the right until you can see amino acid 508 which is a phenylalanine (represented by the letter F). Click on the green symbol above F508 and read the text in the top left corner. What does it say? Mark this amino acid on your drawing.
29) How many transmembrane domains are in CFTR? How many ATP binding sites? Phosphorylation sites? Glycosylation sites? What feature of CFTR is closest to the amino acid F508?
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