Pharm 207/Bio 207 Home Page Using Internet Resources in Molecular Biology - Lecture 6 Phylogenetic Trees

Introduction

Molecular evolution deals with two problems: (i) understanding evolution of macromolecules (DNA, RNA, proteins); (ii) reconstruction of evolutionary history of genes and organisms.

The most common principle in reconstruction of phylogenies is the so called principle of maximum parsimony or minimum evolution which implies search for a tree with minimum mutations to explain differences observed between sequences.

UPGMA = unweighted pair group method with arithmetic mean.

UPGMA example for 4 taxa:

(i) We start with a distance matrix with 4 OTUs (operational taxonomoc units) designated as A, B, C, D. In our case when we use information about protein sequences, each sequence (or what's represented by this sequence) will be one OTU. The distance matrix contains evolutionary distances for pairs of OTUs, i.e. measures how far those two sequences have diverged. Distance may be calculated, for instance, as the number of amino-acid differences between two sequences.

                           A      B      C
                       B  d(A,B) 
                       C  d(A,C) d(B,C)
                       D  d(A,D) d(B,D) d(C,D)

   d(X,Y) - is the evolutionary distance for two taxa X and Y.

(ii) Let's assume d(A,B) has the smallest value. We join them in the following
 way:

                           AB      C              d(A,B)/2 
                       C  d(AB,C)                ___________ A  
                       D  d(AB,D) d(C,D)        |
                                                |___________ B


         d(AB,C)=(d(A,C)+d(B,C))/2   d(AB,D)=(d(A,D)+d(B,D))/2

         AB - is a joint taxon of A and B.

(iii) Let's assume d(AB,C) is the smallest distance now.

                                                 ___________ A  
                                           _____|
                                          |     |___________ B
                                          |
                                          |_________________ C

                                               d(AB,C)/2

         d(ABC,D)=(d(A,D)+d(B,D)+d(C,D))/3


(iv) The last step is to connect D to the tree.

                                                 ___________ A  
                                           _____|
                                          |     |___________ B
                                     _____|
                                    |     |_________________ C
                                    |
                                    |_______________________ D              
                               
                                             d(ABC,D)/2

Reading Materials

1. Fitch W.M. (1977) On the problem of discovering the most parsimonious tree. Amer. Natur., 111, 223-257.
2. required Saitou N., Nei M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4, 406-425.
3. Czelusniak J. et al. (1990) Maximum parsimony approach to construction of evolutionary trees from aligned homologous sequences. Methods in Enzymology, 183, 601-615.
4. required Li W.-H., Gouy M. (1990) Statistical test of molecular phylogenies. Methods in Enzymology, 183, 645-659.
5. Sneath P.H., Sokal R.R. (1973) Numerical taxonomy. Freeman, San Francisco.
6. Kimura M. (1983) The neutral theory of molecular evolution. Cambridge University Press.
7. Nei M (1987) Molecular evolutionary genetics. Columbia University Press.
8. Li W.-H., Graur D. (1991) Fundamentals of molecular evolution. Sinauer Associates, Inc.

Software and information resources:

Lecture Goals & Assignment

To learn how to prepare data, construct and analyze phylogenetic trees.

Examples

General scenario:

Sequences could be obtained from the NCBI server by searching using textual description in Entrez or representative sequence in BLAST search (use blastp program).

Then sequences should be extracted in FASTA format and multiple alignment should be built using ClustalW program available at Biology WorkBench.

Obtained multiple alignment should be then used to constract phylogenetic tree again using ClustalW program and then visualized using PHYLIP ptogram at Biology WorkBench.

Phylogenetic tree then can be saved as gif files and included into your Web page.

Step-by-step example:

We use a set of a few cAMP-dependent protein kinases as a test.

1. If you have set of aligned sequences go to step 5, if you have set of sequences which are not aligned go to step 4, if you have less than 4 sequences go to step 2.

2. We start with a single sequence in FASTA format:

>gi|2408086|gnl|PID|e1168586 camp dependent protein kinase regulatory chain
MVAGPEAIGPDAKYVPELGGLKEMNVSYPQNYNLLRRQSVSTESMNPSAFALETKRTFPPKDPEDLKRLK
RSVAGNFLFKNLDEEHYNEVLNAMTEKRIGEAGVAVIVQGAVGDYFYIVEQGEFDVYKRPELNITPEEVL
SSGYGNYITTISPGEYFGELALMYNAPRAASVVSKTPNNVIYALDRTSFRRIVFENAYRQRMLYESLLEE
VPILSSLDKYQRQKIADALQTVVYQAGSIVIRQGDIGNQFYLIEDGEAEVVKNGKGVVVTLTKGDYFGEL
ALIHETVRNATVQAKTRLKLATFDKPTFNRLLGNAIDLMRNQPRARMGMDNEYGDQSLHRSPPSTKA

3. Search for similar sequences using NCBI BLAST search:

• Go to NCBI server
• Press button "BLAST".
• Select "Basic BLAST search".
• Select Program - blastp.
• Paste your sequence into the text input box.
• Press button "Search".
• Press button "Format results".

4. Calculate multiple alignment using ClustalW program at Biology WorkBench.

• Open another Netscape window and go to Biology WorkBench.
• Register using "Set up a free account" if not registed before.
• In Biology WorkBench use button "Protein Tools".
• Add several sequences (5 or more) from BLAST search results (or other place):
  • Use "Add New Protein Sequence" and button "Run".
  • If using BLAST click on sequence id and then "DISPLAY: FASTA" button/menu to get sequence in FASTA format.
  • Copy/Paste sequences in FASTA format from "BLAST search results" page (or other place) into ClustalW text input box.
  • Use button "Update".
• Check checkboxes for inputed sequences and use "CLUSTALW - Multiple Sequence Alignment" to calculate the alignment.
• Use "Run" and then "Submit" buttons.

5. Calculating the tree.

• Use "Import Alignment(s)" button to incorporate alignment for further analysis.
• Check checkbox for inputed alignment.
• Use "DRAWTREE - Draw Unrooted Phylogenetic Tree from Alignment" or "DRAWGRAM - Draw Rooted Phylogenetic Tree from Alignment" to calculate the unrooted or rooted trees, respectively.
• Use "Run" and then "Submit" buttons.

6. Processing the image.

• Use "Save Image As" option of the Browser to save images of phylogenetic trees.
• Add the images to your Web page and describe.