Searching for Mutant Prion Sequences

1. We now know that the wildtype prion protein contains a specific octapeptide repeat sequence and we are going to see if we can use this knowledge to try to find mutant prion sequences.

2. First, go to the bottom of the "View Protein Sequence(s)" window and click on the button that says "Return". This will return you to the Protein Tools homepage. (Note: If you are working on a Macintosh computer, the "View Protein Sequence(s)" window is a separate window. To get back to the homepage, simply close the window.)

3. Once you are back on the Protein Tools homepage, click on the box next to the wildtype prion sequence to remove the check in the box. Then, scroll down the menu and highlight the choice that says "PATTERNMATCHDB ¡V Search for Regular Expressions in a protein sequence DB". This tool will allow us to use the octapeptide repeat sequence to search a database for sequences that have this repeat in them. Click on the "Run" button.



4. A new screen will appear. First, we need to specify which database or databases we want the Workbench to search to find sequences containing the octapeptide repeat. Scroll down the page and highlight "GenBank Primate Sequences". We want to search this database because we are looking for a human mutant sequence (and humans are primates).



5. Now go to the input box titled "Regular expression to match" and type in the sequence "GGGWGQPH" (note: it does not matter if the sequence is typed in upper or lower case). Then click "Submit".




6. The results of the "patternmatch" search will appear on a new screen. But, which sequence is the mutant sequence that we are looking for? We are going to use the "Show Records" tool again to find the mutant sequence that causes Creutzfeldt-Jakob disease in humans. Highlight all of the sequences and click on the "Show Records" button at the bottom.



Look around for the mutant sequence that causes CJD. The correct sequence is shown below from within the "Show Records" tool.
Note: In order to get out of the Record page, you will need to press the "Back" button if you are using Internet Explorer or the "Return" button at the bottom of the page if you are using Netscape.



7. Import the sequence that reads "GBPRI: 1911643_1911644 prion protein [Homo sapiens]¡K ". Highlight this sequence, as it is the one that we want to import into the Protein Tools homepage. Once this sequence is highlighted, click on the "Import Sequence(s)" button.



8. Now we have both the wildtype prion sequence (containing 5 repeats of the octapeptide GGGWGQPH) and the mutant prion sequence that causes Creutzfeldt-Jakob disease. Before we move on, be aware that the text next to the mutant sequence that once said "human brain Ukrainian familial Creutzfeldt-Jakob" now says "PRNP=prion protein {intervening sequence, repetitive element}". The Workbench sometimes changes the desciption when a sequence is imported and that can be confusing. The best way to keep track of which sequence is wildtype and which sequence is mutant is to look at the numbers. The wildtype sequence is "190467" and the mutant sequence is "1911643_1911644". These numbers will never change so this is the best way to distinguish between mutant and wildtype should you become confused (see the figure below).




Next, let's see how the mutant sequence that causes CJD compares to the wild type prion sequence.