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BLyS Sequence Analysis

I’ve been playing with some sequence analysis and phylogentic tree construction programs recently because I would like to introduce these sorts of data analysis into my biology classes. As a sample protein, I decided to use BLyS / BAFF, a protein important in regulating B Cell numbers. I’ve always wondered about the origin of this kind of molecule, since working on it in grad school, and this seemed like a decent way to get some ideas about where it might come from.

The first thing I did was go to the NIH’s National Library of Medicine website: http://www.ncbi.nlm.nih.gov

It’s easy to search for any protein / gene / whole genome you are interested in examining. Knowing that BLyS is vital in humans and mice, I chose to start with the human sequence. I retrieved it as the following:

>gi|20196464|dbj|BAB90856.1| BLyS [Homo sapiens]
MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCCLTVVSFYQVA
ALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAV

The easiest tool to find similar proteins in other animals is the Basic Local Alignment Search Tool for proteins, or BLASTp. Just using default settings, I pasted the sequence in the search field and hit go. (note, I actually just used the accession number, not the whole sequence)

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This retrieved tons of proteins with similar sequences from the vast database of sequence information, from which I chose several model species. One thing I wanted to do was to include several primates as a sort of internal calibration (assuming that they would all have very similar sequences compared to more distantly related species). I also wanted to get a few animals’ sequences who are quite distantly related to humans (frog and ground tit fir that bill)

Once I had a list, I put them all into a single text file and then used that in a second program. This time, I decided that the best ‘multiple alignment tool’ would be CLUSTALX. It’s been around for a while and can create data in a number of different forms. Besides, it’s free and versions are available for both mac and PC.

Again, for starters, I just accepted the default parameters and did a quick alignment:

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Obviously, there’s something odd about the canid familiars (dog) sequence, but before I did anything about that, I just wanted to see what a phylogenetic tree looked like. This is another thing that Clustal does well, it will export your sequence alignment as tree data in a number of formats, then I could plug that data into one final program. This last is a web based program that I access through a french site (but you can probably find it in a number of places). The program is called DRAWGRAM. It accepts alignment data and outputs a graphical tree representation of the alignment.

This is an important logical step… What I’m doing is asking for a family tree of sorts to be displayed that represents the relationship of the sequences I provided. We might want to assume that this also tells us how related the organisms that have these proteins are – and that’s not wrong, but it’s also not thorough as we’re only using ONE protein to make that assumption.

Here’s my first tree:

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Note how isolated Canis is on this representation.

Finally, I went back and truncated the Canis sequence to a place where I suspect the protein actually starts – my sequence from the NCBI gave me a string of Amino Acids at the front of the protein that I think are probably not there, but just got added by some computer algorithm without proper human oversight.

Once I did that Canis (by the way, I remained the sequence ‘DOG’ so I was sure it was the new one) fell in line with a sequence more similar to that seen in cats (felis):

ImageThat’s it for now. Although I expect that I will dig a little deeper with more animals to see if I can come closer to an ‘original BLyS’.

 References:

  1. Dereeper A., Audic S., Claverie J.M., Blanc G. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol Biol. 2010 Jan 12;10:8. (PubMed)
  2. Dereeper A.*, Guignon V.*, Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J.F., Guindon S., Lefort V., Lescot M., Claverie J.M., Gascuel O. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W465-9. Epub 2008 Apr 19. (PubMed) *: joint first authors
  3. Felsenstein J. PHYLIP – Phylogeny Inference Package (Version 3.2). 1989, Cladistics 5: 164-166
  4. Larkin,M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948.
  5. Thompson,J.D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25:4876-4882.
 
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Posted by on March 7, 2014 in Uncategorized

 

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Refining the Tree of Life

Image“Therefore I should infer from analogy that probably all the organic beings which have ever lived on this earth have descended from some one primordial form, into which life was first breathed.”

Darwin, Origin of the Species 

One of the basic ideas of biology is Darwin’s notion that all life on this planet is related through common ancestors at some time. It is only though the passage of ‘deep’ time, the infidelity of genetic machinery (among other processes) leading to variation and the separation of species by physical (or other) boundaries that has led to divergence into the great variety of species we see today. Constructing phylogenetic trees is a simple graphical way of communicating this concept.

The Animal Tree of Life, published in the 15 Feb 2013 Science magazine <LINK>discusses the construction of a phylogenetic tree over the past 25 years since molecular evidence was admitted as a means of establishing relationships with greater accuracy, and with less subjectivity than ever before. “  For the past century, the use of detailed descriptions of animal adult morphology and embryology has been at the heart of the study of evolutionary relationships among distant groups such as phyla. However the methodology can have both implicit problems and practical difficulties.”    1

This early paper used 18S rRNA sequences extensively to derive phylogenies because it was thought that this gene was required for life of many (all?) organisms and that sequence was highly critical to an organism’s survival, and would suffer fewer modifications over time than other, less crucial genes.

The earlier disagreements derived from varying interpretations of the morphological and embryological characteristics of animals. Many of these characters have evolved repeatedly in unrelated lineages as adaptations to similar selective pressures or have been lost from certain groups through disuse. Today’s strengthening consensus is almost entirely thanks to the use of molecular genetic data in reconstructing trees. Heritable changes in nucleotides and amino acids are abundant and generally much less prone to the problems of convergent evolution and loss than are morphological characters.2

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More recently, sequencing of mitochondrial and chloroplast DNA has revealed these organelles’ relationship to free-living bacteria, providing an interesting challenge for illustrators of these genetic trees and establishing solid data supporting the endosymbiotic theory.

Using the great wealth of DNA data we have today, relationships can be determined using similarities found in a number of different genes, providing a high degree of statistical assurance that the conclusions are unbiased and accurate.

Image

References

1. K. G. Fieldet al., Science 239, 748 (1988).

2. M. J. Telford, R. R. Copley, Trends Genet. 27, 186 (2011).

 
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Posted by on February 19, 2013 in Uncategorized

 

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Aside

A static view of the tree of life

Onezoom.org is an effort o display all of life and its relationship with one another in a single ‘zoomable’ phylogenetic tree. Currently one the mammalian branch of this tree is available, but it is still extraordinarily interesting and gives an idea of the grand scope of the project. Here’s an introductory video showing the intent of the project and why the organizers developed it in the way that they did.

Here’s a link to the site itself where you can try out the viewer. — By the way, +1 point on the upcoming chapter 5 quiz for any of my students who try this out and can email me a screenshot of where humans fit into this tree. I realize this requires you to go to the site, try out the viewer, find humans AND work out a way to visually document that -but there it is.

http://www.onezoom.org

I would like to thank nothinginbiology.org for pointing me toward this resource. I tried to reblog their post, but I ran into some silly technical problems and found it easier just to write up something myself.

OneZoom

 
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Posted by on October 20, 2012 in Education, Uncategorized

 

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