READ THROUGH THE ENTIRE ASSIGNMENT BEFORE YOU START!!
This week, our gene of interest is KiSS1, which was isolated in a lab in Hershey, Pennsylvania, and named in honor of the chocolate kiss.
The protein is known to be involved in metastasis suppression, as well as puberty. We are interested in the puberty related function, which is due to its neuropeptide activity. The gene is known in mouse and human, but we're looking for a different model system. We decided to try to find homologs in fish, as its a good model system for studying puberty. * Note: neuropeptides are usually very short and very conserved, though the overall gene in which it sits may not be as conserved. The active peptides are produced by proteolytic cleavage. *
We decide to take our mouse protein and do a blast search.
After running Blast, a number of hits were found. Surprisingly, in zebrafish, there were two different hits, even in the neuropeptide part (10-14 amino acids, depending on which definition you use)! Then we notice there is a second hit in frog too. We decided to take a represntative sequence from various evolutionary groups to try to figure out whats going on. The hits we decided to use are:
Open a window to the ClustalW page.
Now we are going to run ClustalW:
Paste in your sequences, and use all of the default parameters. Click on the "Submit" button.
Scroll down to the "Result files (text):", click on the link "CLUSTALW" underneath it, and print it out. Look at the output file (or at the colored alignment on the previous page, whicever you find easier) and answer the following questions (Describe the alignment):
Open a window at EBI here
Paste or upload the same sequences, and run with the default parameters. Print the results here too, either directly from the page, or download and print (If it doesn't align when downloaded, make sure the font is Courier, and drop the size a bit if it doesn't fit nicely in lines).
Click on the "View result with Jalview" tab (it may take a bit of time to load). It will open a new window (it may show up behind your window - if you don't see it after about a minute, move your window, and it may be there).
Stretch the window so that you can see the whole alignment. Go to the "Colour" menu and choose "% Identity". Look at the results, and answer the following:
Go to the "color" menu (in the Jalview window, not the browser window) and choose "Above Identity Threshold" (NOT Modify) - you will get a slider that will let you control the percent identity of the coloration.
Play around with the slider a bit to answer the following:
Keep your ClustalW windows open. Now we are going to compare to Muscle.
Open the EBI page for Muscle.
Paste in the sequences and click "Submit". Print out the alignment file as before.
You can open Jalview or just use your printouts to answer the following:
Compare the results of Muscle and Clustal Omega:
Due to the mistake, and in attempt to understand the 'extra' sequences better, we decide to add more sequences, particularly of the second group, if we can find them. We find a lizard sequence, and another fish with two sequences, add add them:
Run Clustalw and Muscle on this list. (here are the links again to make this easier ClustalW and Muscle) Print out the alignment files, and answer the following (once again, feel free to use jalview, the plain alignment, the color option, whatever you find easier to deal with):
First we'll compare ClustalW:
Now for Muscle: