DNA Alignment, A Jar of Teeth, a Model Great White and a Whole Bunch of Other Shark and Trout Stuff.

Now that I have my office space at UCC, I thought I’d decorate it, however when look20170124_151745ing around at the things the other PhD students have
around their desks, I must say I could do a lot better. Plus I need some trout things! At least I am up and running here as a PhD student, well maybe not running, its more of a leisurely strolling pace if I am honest. My colleagues are telling me to enjoy this quiet time while it lasts. I have been enjoying reading the papers I have been given, along with the ones I am finding.

Additionally I have been playing around with some software I used for my undergraduate dissertation while looking into genes and regions of DNA that thought to be of importance regarding anadromy in Salmonids.


I found an interesting paper by McCormick et al. (2008) on mineralocoricoid receptors, the study looked at the effects of in vivo cortisol, 11-deoxycorticosterone (DOC) and aldosterone on salinity tolerance, gill Na(=),K(+)-ATPase(NKA) activity and mRNA levels of NKA in atlantic salmon (Salmo salar). This study was undertaken in response to other recent studies that have shown that fish express a gene with a high sequence similarity to the mammalian mineralcorticoid receptor (MR). Which suggests that there could be a possibility that other hormones, other than cortisol could carry out some mineralocorticoid functions in fish.  The McCormick et al.(2008) study found that after cortisol exposure, physiological levels of cortisol increased, there was increased gill NKA activity and improved salinity tolerance. Where as DOC and aldosterone had no effect on gill NKA or salinity tolerance. Levels of NKA mRNA increased in response to freshwater and saltwater acclimation, it was observed that these increases were up-regulated by cortisol. Overall the results support the notion that cortisol and not DOC and aldosterone, is involved in regulating the mineralocorticoid functions of ion uptake and salt secretion in teleost fish.

So why am I finding this interesting?

Well, I thought it’d be interesting to see the ‘highly similar sequencing’ for my self so I found the Genbank reference, and compared it to the mammalian model, a mouse. I used blast to align the sequences and then downloaded them in FASTA format to look at the sequence similarity in Clustal X, sure enough it was pretty similar. So that got me thinking, what if I blast the fish sequence and look for similarities of around 80-100% matches. Scrolling though the results I started to notice something cool,  a lot of fish that exhibit anadromy where in the 97-99% similarity range. Now this isn’t too surprising as many of these fish belong to the same family so you would expect to see a degree of similarity. So I went looking for some strictly marine species and strictly freshwater species from different families to see how there sequences would compare and after a bit of adjustment sure enough the sequences aligned. Again high similarity, however there was much more variance in the sequence when considering these marine and freshwater fish.

Fig.1 Clustal X output of the MR sequence for only Andaromous fish species, stars indicate nucleotide matches across all species.
Fig.2 Clustal X output of the MR sequence for all fish species (anadromy,marine and freshwater), stars indicate nucleotide matches across all species.

As can be seen from Figures 1 and 2 above this region of the gene is similar across all the species, however similarity rates are greater in Fig.1 than in Fig.2 (even when excluding the gap caused by the 5th species down in Fig.2). The increased number of stars in Fig.1 has got me interested. As this is just the result of me playing around with genbank and these alignment software I have no idea what it could mean, if it means anything at all.

So it inspired me to look a little further into genes and regions of the genome associated with anadromy, and I didn’t have to look to far as in the same paper I noted the sodium/potassium ATPase mRNA alpha sub unit 1 isoform a1a, or NKAa1a for short, was linked with changing gill morphology which increases salt tolerance in fish. Which is very important if you’re a fish practicing anadromy.

So back to Genbank I go! I found a sequence for good old brown trout (Salmo trutta) and put the FASTA sequence into blast and got a lot of matches and 99% similarity results. So I downloaded them and found the same thing, Salmo salar,  Oncorhynchus masou, Oncorhynchus mykiss, Oncorhynchus nerka and a bunch more salmonids. These results are not surprising as all of these fish are closely related, however it is interesting that these genes are very highly conserved across the anadromous species and less so when looking at marine and strictly freshwater species.

Fig.3 Clustal X output of the NKAa1a sequence for only anadromous fish species, stars indicate nucleotide matches across all species.
Fig.4 Clustal X output of the NKAa1a sequence for all fish species (anadromous, fresh water and marine), stars indicate nucleotide matches across all species.

Fig.3 shows a much higher match rate than Fig.4, this may be a result of all the anadromous fish being salmonids. However, this data does represent species from different genera: OncorhynchusSalmo and Salvelinus. Additionally, it is believed that the split between Oncorhynchus and Salmo occurred well before the Pliocene some even suggest it may have gone back as far as the early Miocene about 20 mya.

Again I must that I am just playing with data and the software therefore these results don’t particularly show anything at this current time. I do plan to look at a few other regions that I have on my list and to scan though the current literature and scan for other regions to add to that list. Eventually I would like to run some stats to see if there is any significance between the differences in the number of single nucleotide polymorphisms (SNPS), represented by the absence of stars in Fig.1,2,3 and 4, in anadromous species and marine and freshwater species.


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