Mammal Evolutionary RelationshipsLab Protocol

PART A

  1. First you need to get the sequence data for the hemoglobin protein from our marine and land animals: seals, whales, dogs, and cows.

Go to or click on the protein link on the class website

  1. In the search box, type in HBB (signifying the hemoglobin protein we are comparing) and then the name of the first species in the following list:

Harbor seal, Phoca vitulinaMinke whale, Balaenoptera acutorostrata

Dog,Canis familiaris Cow,Bos taurus

Then Click “Search”

  1. If the screen that pops up contains a list of choices, like a google search, choose the listing with an Accession “P0####”. Or else you will be brought to this page automatically. The page should look like this:
  1. Click on the “FASTA” link near the top of the page to see the actual amino sequence for this protein in a coded format using single letters to represent each of the 20 amino acids (A=alanine, M=methionine, P=proline, etc.)

You should see a screen similar to this:

  1. Copy the entire sequence, from before (>) to the last letter. You must include the header line, starting from the greater than symbol (>). THIS IS IMPORTANT.
  1. Paste this sequence into a word document that you will save as “FASTA text file.”
  1. Now search for the HBB (hemoglobin) sequences for the rest of the species in your list (#2 in these instructions). Paste these amino acid sequences into the same FASTA text file.
  1. EDIT your file by deleting the header of each sequence and adding the species name instead. You MUST preserve the “>” symbol.

For example:

The harbor seal sequence begins something like this:

>gi|122664|sp|P09909.1|HBB_PHOVI RecName: Full=Hemoglobin subunit beta

This can be edited to simply say this: > Harbor seal

VHLTGEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFDSFGDLSSADAIMGNPKVKAHGKKVLNSFSDGLKNLDNLKGTFAKLSELHCDKLHVDPENFKLLGNVLVCVLAHHFGKEFTPQVQAAYQKVVAGVANALAHKYH

Also, make sure there is a blank line left in between each sequence.

Minke whale…….

Be sure and save this file for the four mammals

ALIGNING AND ANALYZING YOUR SEQUENCES

  1. Go to or click on the click on the class website to go to the Clustal Omega so the sequences you saved can be analyzed for similarities.
  1. Copy and paste your saved file into Clustal

  1. Click on the submit button and let the fun begin. The program will check the amino acid sequences for similarities and differences and return the results to you.

  1. Click on the “results summary” tab under the results heading.
  1. Click on “Percent Identity Matrix”. The grid you see shows the percent similarities between the four different animals

These might not show but this is the set up

Bos_taurus Minke_whale Harbor_seal Canis_familiaris

1: Bos_taurus 00.00 00.0000.0000.00

2: Minke_whale 00.00 00.0000.0000.00

3: Harbor_seal 00.00 00.0000.0000.00

4: Canis_familiaris 00.00 00.0000.0000.00

  1. Click on the back button to get to the results page again and click on the “Phylogenetic Tree” tab to view the tree for the four mammals.
  1. Complete part A with the information from above.

PART B

  1. Below are the representative marine mammals for your new cladogram:

 Minke whale, Balaenoptera acutorostrata Harbor seal, Phoca vitulina

 Dolphin,Lagenorhynchus obliquidens River otter, Lontra canadensis

 Walrus, Odobenus rosmarus Manatee, Trichechus manatus

  1. In this exercise, we will compare the hemoglobin proteins of these marine mammals to some representatives of the major taxa of land mammals:

 Dog, Canis familiaris  Rat, Rattus norvegicus

 Cow, Bos Taurus  Human,Homo sapiens

 African elephant, Loxodonta africana  Red kangaroo, Macropus rufus

  1. Reminder: We are testing the hypothesis that all marine mammals have a common land mammal ancestor.
  1. Once again, use the hemoglobin beta protein sequence to test this hypothesis. You will be discovering a tree with all the marine mammals and all the land mammals listed above.
  1. Follow the complete process we used in Part A to obtain your amino acid sequences from GenBank, align them in Clustal Omeqa, and build the evolutionary tree.
  1. Then use your phylogenetic tree to determine the evolutionary relationships amongst marine mammals and the representative land mammals. Determine whether your hypothesis was supported by the molecular data as you complete part B of the activity.

1 / AP Biology-Krabath