Comparing the Cruetzfeldt- Jakob Mutant Prion Sequence with the Wildtype Sequence

1. Check on the boxes next to both sequences. Then, scroll down the menu and highlight the tool that says "CLUSTALW ¡V Multiple Sequence Alignment". The CLUSTALW tool will allow us to view the sequences stacked one on top of the other so that we can see where and how they differ. Once this tool is highlighted, click on the "Run" button below the window to see the alignment.




2. We are not going to concern ourselves with the next screen for the purposes of this tutorial. Simply click on the button that says "Submit".



3. The results of the sequence alignment will appear.



4. There is a lot to look at in this alignment, so let's start at the beginning. Above the alignment, there is a consensus key that explains what the colors mean. Royal blue means that the two amino acids in that position of the protein sequence are 100% homologous, or exactly the same (also designated by an asterisk below the alignment). The green and navy blue colors designate some level of homology, but for the purposes of this tutorial, we will not concern ourselves with these colors in this alignment.

5. Also, note that the mutant sequence located on top is much shorter than the wildtype sequence located on the bottom. The dashed lines that flank the mutant sequence simply indicate that the corresponding sequence is not represented because the mutant sequence that was imported was shorter than the wildtype sequence. Also, towards the end of the alignment there is a dashed line that interrupts the mutant sequence and then the mutant sequence begins again. This represents the Workbench's attempt to find as much homology as possible between the two sequences (to do this, it had to add some gaps to the mutant sequence). IT DOES NOT MEAN THAT THE MUTANT SEQUENCE IS IN TWO PARTS!! The mutant sequence is continuous, so don't let the dashed lines confuse you.

6. Now that we know what everything means, look at the alignment. If you look at the long string of amino acids that have 100% homology, designated by the royal blue color, you will notice that the 5 repeats of the octapeptide "GGGWGQPH" match up with 5 octapeptide repeats in the mutant sequence. On the contrary, very little homology is seen on either side of the identical octapeptide repeat region. However, take a closer look at the mutant sequence. Starting at the beginning of the mutant sequence, look for the octapeptide repeat, GGGWGQPH. What you should notice is that the mutant sequence contains an additional 3 copies of the octapeptide repeat upstream of the 5 octapeptide repeats that match up perfectly with the wildtype sequence. Similarly, if you survey the mutant sequence that follows the region of perfect homology, you will find another 2 copies of the octapeptide repeat (the last repeat is incomplete, "GGGWGQ" because the mutant sequence ends). To find these last 2 copies of the octapeptide repeat, it helps to ignore the gaps that the Workbench placed in the alignment in an attempt to find the best homology:

In other words, the mutant prion sequence that causes CJD in humans contains 10 repeats of the octapeptide sequence "GGGWGQPH" compared to the 5 repeats found in the wildtype prion sequence.

7. Scroll back up to the top of the page and click on "Import Alignment(s)". This will import the alignment into the Alignment Tools homepage for further analysis of the alignment.