Exercise 8

Ex.
no:8 PHYLOGENETIC ANALYSIS

Our system of taxonomy is based on phylogeny. That is, we classify organisms together because they have a common evolutionary ancestor. In most cases, we cannot determine ancestry directly because the fossil record is poor for most organisms. Instead we rely on shared, homologous features,and we say that organisms that share many features are closely related. Organisms that share manyfeatures probably had a relatively recent common ancestor.For example, chimpanzees and humans share about 98% of their DNA because the common ancestor of chimps and humans lived only about 6 million years ago. In 6 million years, there has not been enough time for very much divergence to take place. However, the DNA of humans and yeast is more dissimilar because humans and yeast shared an early eukaryotic ancestor no more recently than about 1.2 billion years ago. AIM: To Analyze the Evolutionary relatedness between two species is determined by the recency of their common ancestor. Recency of the ancestor can usually be determined by the two species degree of similarity. Classifying Based on Molecular Similarity Comparison of the sequence differences in either nucleic acids or proteins can indicate relatedness.This method has the advantage that it is relatively easy to understand. Highly related organisms are expected to have many sequence similarities; distantly related ones are expected to have low similarity.There are two possible exercises here: Do Ex. A without using internet resource, and Ex. B if you do have computers with Internet access. In both cases, we are going to draw conclusions about the relationships of humans (Homo sapiens), rhesus monkeys (Macaca mulatta), mice (Mus musculus), rats (Rattus norvegicus), domestic ducks (Anas platyrhynchos), Canada geese (Branta canadensis), Nile crocodiles (Crocodylus niloticus), and alligators (Alligator mississippiensis). Obviously, the eight species consist of four seemingly related pairs. What do you predict about the molecular similarity of organisms within each pair? In both exercises A and B, we will use single-letter codes of the International Union of Pure and Applied Chemistry (IUPAC) to represent amino acids. Table 1. Single-letter IUPAC codes for the 20 standard amino acids. A alanine G glycine M methionine C cysteine H histidine N asparagine D aspartic acid I isoleucine P proline E glutamic acid K lysine Q glutamine S serine T threonine V valine W tryptophan
F phenylalanine L leucine R arginine Y tyrosine Exercise A Table on the shows the amino acid sequences for the first 60 amino acids of the beta chain of hemoglobin for the species above. Not all of the amino acids in the beta chain are shown because some of them are the same for all the species. The first 60 amino acids in the beta chain of hemoglobin in eight species.
1. The first step is a laborious one: working in groups of four, you must find the number of
differences in amino acid sequence between each pair of species. There are 28 pairs to be analyzed, as shown by the unshaded cells in Table 3 below:
You should divide the comparisons up among your group members so that each person is responsible for certain comparisons. To make sure youre doing the comparisons correctly, the human-rhesus monkey number should be 4 , and the crocodile-alligator number should be 17.
2. According to the data, which two species in Table 3 are most closely related? Why do we
think these two lineages diverged the shortest time ago?

3. Which species is most closely related to humans? Which is most distantly related to
humans?
4. To humans, alligators and crocodiles look very similar. However, which species
difference in Table comes closest to the difference between alligators and crocodiles? How can we explain this? Exercise B (if computers with Internet access are available) With modern bioinformatics tools, it is possible to do a more sophisticated analysis than is possible by hand. Also, protein databases have many organisms in them, so you can examine a great variety of evolutionary relationships. Lets attempt to look at the relationships of the beta chain of hemoglobin for our eight species using these tools.
Procedure B 1. Swiss-Prot is one of the worlds leading protein databases. Go to the Swiss-Prot database at http://us.expasy.org/sprot/:
Figure 1. The Swiss-Prot protein database.
1. Using the text box at the upper right, enter the protein you want to find and the organism. For example, for human hemoglobin, we might put in hemoglobin human in the search box. Press the Go button. Do not use any ANDs or ORs in a Swiss-Prot search box. 1. You will get a page that lists several possible proteins, one of which is HBB_HUMAN, the beta chain of human hemoglobin. Click on that link and youll be taken to a page that gives you the proteins official name, its gene name, many references on how it was sequenced, a summary of its function, and many other pieces of information.
1. However, now we just want to know the amino acid sequence. At the very bottom is information on the length (146 amino acids), molecular mass and amino acid sequence of the hemoglobin beta chain. The sequence is presented this way:
Figure 2. The hemoglobin beta chain sequence as presented by Swiss-Prot. At the lower right is a link that refers to FASTA Format. FASTA is a very common format for presenting either DNA or protein sequences to bioinformatics programs. If you click on the FASTA link, the sequence above changes to:
>sp|P02023|HBB_HUMAN Hemoglobin beta chain - Homo sapiens (Human), Pan troglodytes(Chimpanzee), and Pan paniscus (Pygmy chimpanzee) (Bonobo). VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGN PKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLA HHFGKEFTPPVQAAYQKVVAGVANALAHKYH
We could copy this amino acid sequence (starting with VHLT onto our computers clipboard and then enter it into bioinformatics programs. We would then do a search for the
hemoglobin beta chain of another organism, and so forth. For this exercise, well use some hemoglobin sequences that have already been compiled, so dont copy the human sequence above.
Using the Back function of your browser, go back to the main HBB_HUMAN page of Swiss-Prot, go to the top, and enter only HBB into the search box. Press Go. This asks the database to present all hemoglobin beta chains it has (about 500). This is an impressive diversity, representing organisms from stingrays to penguins to humans. This gives an idea of the variety of organisms that could be incorporated into your phylogenetic study using the hemoglobin beta chain. However, the sequences we need are already compiled on a Web site. Go to http://biology.clemson.edu/bpc/bp/Lab/111/hemo.htm and click on the Hemoglobin Filelink. A Microsoft Word file will be downloaded to your desktop. It contains hemoglobin beta chain sequences for humans, rhesus monkeys, mice, rats, domestic ducks, Canada geese, Nile crocodiles,and alligators. Open the hemoglobin file. Copy the human sequence (from >human to the end) onto your clipboard. Go to a popular bioinformatics site: ClustalW at the European Bioinformatics Institute: http://www.ebi.ac.uk/clustalw/index.html. The W in this name stands for Weights.
ClustalW performs multiple alignments (it aligns more than two sequences at the same time so corresponding sections are being compared), it determines the relationships between them, and it finally clusters them on a tree-like structure called a cladogram or phylogram. We hope this structure shows the degree of relatedness between the protein or nucleotide sequences. Go to your downloaded Hemo file, select all the text, and paste it in the text box on the submission form:
The only option to change is that Output Order (just above the text box) should be set to Input rather than to Aligned. Then press Run. After a short pause, you will get a screen that starts with a table of differences between the different organisms. For the hemoglobin beta chain, this table starts:
The scores in this table show that the hemoglobin beta chain sequences were 94% identical between humans and rhesus monkeys, 81% identical between humans and rats and so forth.
At the bottom of the output, there is a cladogram, but this is something called a guide tree that has more to do with how ClustalW handled the problem than with evolution. So ignore that and go back up to the beginning of the multiple alignment, which starts as follows:
This section aligns corresponding amino acids with one another so we may get an accurate estimate of how many amino acids have changed from one species to another. A * under a column means that all species had an identical amino acid in that spot, a : means all the amino acids were similar, and a . means they were less similar, and a means one or more amino acids were markedly different. Were now going to use these aligned sequences to do our evolutionary comparison.
Select all the text starting with CLUSTAL W (1.82) multiple sequence alignment and going down to the last line of the alignment that mentions amino acid 146.
Use the back key on your browser to go back to the ClustalW page where you entered your protein sequences. Clear all that text and paste in the contents of your clipboard. Then change the ClustalW parameters (last line above the text box) like this:
Press Run. When your output is delivered, scroll down to the bottom and find the treelike cladogram. Press the button labeled, Show as a Phylogram Tree. To understand
the phylogram structure, refer to the phylogram below, which was done as an example using different organisms and triosephosphate isomerase, an enzyme in glycolysis:
The branches here group together related sequences, and the lengths of the branches indicate degree of difference between sequences. This phylogram indicates that the triosephosphate isomerase of humans, chimps, mice, and rats is rather similar, but this mammalian sequence is quite different from that of fruit flies, mosquitoes, and yeasts. Also , the two yeasts (with their long branches) differ from each other more than the fruit fly and mosquito differ from each other. Result: Now look at your hemoglobin output for our eight species. Which two species are most closely related? Why do we think these two lineages diverged the shortest time ago? Which species is most closely related to humans? To humans, alligators and crocodiles look very similar (much more similar than humans and rhesus monkeys, for example). However, according to the data, did the alligator and crocodile lineages separate before or after the human and rhesus monkey lineages separated? Why do we say this?

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Ex.

no:8 PHYLOGENETIC ANALYSIS

think these two lineages diverged the shortest time ago?

Figure 1. The Swiss-Prot protein database.

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