Search for FMRP in Drosophila

A couple of weeks ago you had the idea of studying Fragile X Syndrome in Drosophila. The plan was to use the known human FMRP protein and then use it to scan the Drosophila genome to find a similar gene. Then it's just a matter of mutating the gene in Drosophila, and investigating what effect such a mutation may have on fly mental function.

Then you got stuck. No Drosophila genome means nothing to scan. So for the past two weeks you have been sequencing the Drosophila genome. Now that's behind you, and you have a genome sequence in hand. The time has come to continue with the plan.

1.   From the course web site, click on Resources and Links and then on NCBI (National Center for Biotechnology Information, a repository of vast amounts of information).

2.   Click on the down arrow to expand the choices for the Search box from All Databases, and select Protein.

3.   Enter FMRP into the for box, and press Go (or press Enter). 

4.   You might expect to pull up an entry for the human FMRP gene, but no such luck. The search returned well over 100 entries (!), some from flies – which is interesting – some from mice... (This number may be different depending on when you're doing this search). Let's try to reduce the number to something more manageable.

5.   Click on Preview/Index. At the bottom of the resulting screen, you'll see a mechanism to add terms. Click on the down arrow to expand the choices from All fields and select Organism, then type human into the box to the right. Finally, click AND (to specify that you're looking for entries that contain the word FMRP AND the organism human), and click Go.

6.   I got 38 entries now. Certainly an improvement, but surely humans don't have 38 different FMRP proteins! The problem is that we're searching multiple overlapping databases and getting the same protein back multiple times. To cure that, click on Limits, expand the choices in the box labeled Only from, and select a single database (I chose GenBank). Press Go again.

7.   Now I'm down to 15 entries, but most don't look very interesting. The remaining are different from each other in ways that need not concern us now. Click on the entry with the accession number AAH86957.

8.   Lots of information here. One important item is a reference to a journal article that describes the work that led to the results you're looking at. Clicking on the PubMed number leads you to an abstract and a link to the full length article -- potentially very useful! But what we're after right now is the protein sequence, seen at the bottom of the page. The sequence is given according to the one-letter amino acid codes (see course web site, Links and Resources, Genetic Code, for a list of them).

SQ1. Is this the right sequence? At the top under ORGANISM you'll find a list of terms starting with "Eukaryota". I'm willing to admit I'm a eukaryote, but am I also a Haplorrhini and a Catarrhini? What does that mean?

SQ2. Under FEATURES, there are three Regions given. What might they mean?

SQ3. What amino acid begins the protein? Why?

9.   The display is good for some purposes -- the numbering makes it easy for humans to find what amino acid is at a specific positions -- but computers prefer straight sequence, without numbers or spaces. To get this, scroll back up to the top of the page, click on the down arrow to expand the Display box from GenPept, and select FastA. You probably already know what FastA format is, but if you've gotten this far, you're now looking at it: One line of documentation preceded by ">" and multiple lines of sequence. If you want to save this file, click on the down arrow to expand the box showing Send to, and select Text. Then use the browser to save the file. Alternatively, just copy the sequence, with or without the documentation line.

10. Now we're ready to do the search. Click on NCBI at the top of the screen to return to NCBI home page, and click on Blast in the horizontal toolbar near the top of the page. Blast (Basic Local Alignment Search Tool) is undoubtedly the most widely used bioinformatic tool in existence. We'll talk about what it does and how it does it, but for now, let's use it to find the fruit-fly gene we want. We have a protein sequence, and we want to find a fruit-fly protein, so look under Basic BLAST and click protein blast.

11. Paste the FMRP sequence into the Search box and click the Blast button near the bottom of the page. You'll probably get a page pretty quickly showing conserved domains in the protein. That's for another day. For now, wait a bit more to get the search results. These results may take tens of seconds to come up or more or less, depending on the time of day and alignment of the stars.

SQ4. (While you're waiting...) What is Blast doing?

12. Again, way too many hits! Each red line in the Graphic Summary Section is a very good hit, and each line in the Descriptions section gives a link to one of those hits. We'll cut them down in a moment, but first,, scroll down until you reach the Alignments section, which should look something like:

This shows the alignment of the protein we submitted (human FMR1 protein), i.e., the Query, to the protein that Blast found, the Subject. The best hit happens to be "AAH86957.1, FMR1 protein [Homo sapiens]". No surprise! It found itself! Continue to scroll down and you'll see similar proteins from other organisms: dog (Canis familiaris), pig (Sus scrofa), cow (Bos taurus), orangutan (Pongo pygmaeus), mouse, rat, chimp... the amazing thing is that this protein is nearly identical amongst mammals. The same is true of most protein. We share a common toolbox.

SQ5. About those thick red lines... What happens when you mouse over one of them?

SQ6. What sense can you make of the organisms associated with the hits?

13. Use the back arrow several times to go back to the Blast submission page (where you pasted in the protein sequence). You'll see at in the middle of the page a section called Choose Search Set. In the box marked organism, type Drosophila, and click Drosophila melanogaster. Then click the Blast button again.

14. In several seconds... much better! Now you have only a few hits, (scroll down) all from Drosophila. Here's the top hit:

SQ7. How good is this hit? There doesn't seem to be nearly as many identical amino acids as in the previous case. Perhaps it is just a random protein that happens to be a little bit similar?

There are 8 entries that have the identical sequence, from three different databases (emb=European Molecular Biology Laboratory; ref=RefSeq; and gb=GenBank). You'll notice that the degree of similarity between the human and fly FMRP protein is much less than between human and mammalian proteins. Only 44% of the amino acids are identical, according to the statistics above the alignment. Is this degree of significant? The Expect value (6e-99) gives you an indication. It says that a match this good or better would arise at random with a probability of 6 in 10^99 (1 with 99 zeros after it!). In other words, it couldn't possibly have arisen purely by chance. We'll return later to Expect values, since it is very important to understand what they mean and what they don't mean.

15. OK, we've got the protein. What about the gene? We need that in order to make a mutant fly that we can test for physiological function. Click on the GenBank entry AAF14639.1. This gets you to the protein sequence. To find the gene sequence, look at the DBSOURCE (database source) field. A protein sequence is almost never obtained by direct sequencing of the protein but rather by computer translation of the nucleotide sequence. The DBSOURCE is given as AF205596.1. Click on that link.

16. This brings you to a page called "Drosophila melanogaster clone LD09557 Fragile X related mRNA". The source of the protein sequence was virtual translation of a cloned piece of mRNA (since you can't clone RNA directly, it was first made into a DNA copy, or cDNA). Scroll down to the field called CDS (CoDing Sequence -- don't ask me why anyone would think this is a proper abbreviation!). It says that the coding sequence (some would say gene) extends from position 423 to position 2468 in the cDNA sequence.

SQ8. Test that. Scroll down to the cDNA sequence (under ORIGIN) and find position 423. Do you find the beginning of a start codon? Then find position 2468. Do you find the end of a stop codon?

17. This may seem a roundabout path to get to the gene. Why not start with the human gene (instead of the human protein) and use it to pick out the fly gene? OK, let's do it that way. Go back to step 2 and this time, instead of selecting Protein, select Nucleotide. Click Go and, as before, confine the search to humans and to GenBank and limit the search to GenBank. This time even after imposing limits, you still get a couple of dozen sequences.

18. Choose the first complete sequence (it will say "complete cds" in the descriptor line), get the sequence from it as before.

SQ9. Why does the sequence end with so many A's?

19. This time choose nucleotide blast. Find your way to Blast, and again paste the sequence into the window. This time you'll need to change the defaults, specifying that the database is not the human genome but rather Others. Under Program Selection, Optimize for, choose "Somewhat similar sequences". Now click the Blast button. When the results are returned (maybe a minute), you'll see that there are tons of hits, but this time there are many mammalian sequences with multiple differences with respect to the human sequence. But never mind that -- we're after flies. So go back to the submission page and specify Drosophila melanogaster and Blast again.

SQ10. How long is the best hit?

SQ11. How good (E-value) is the best hit? What does that mean?

SQ12. How can it be that the protein search gave five matches with extremely low expect values (definitely not merely by chance) but the nucleotide search found virtually nothing at all?