Using Amino Acid Sequences
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Using Amino Acid Sequences to Show Evolutionary Relationships

Gwynn Pealer and Steve Ferris

Target age or ability group: High school.
Class time required: 1-2 class periods
Materials and equipment: Student worksheet
Summary of activity: Certain globular proteins (e.g., myoglobin, hemoglobin) are used in animals to transport and bind oxygen in the bloodstream and throughout the body. Myoglobin contains one polypeptide chain (153 amino acids long) twisted around a coenzyme called heme. Hemoglobin contains four polypeptides--two each of two polypeptide chains: a hemoglobin (141 amino acids) and b hemoglobin (146 amino acids)--united by the iron-containing coenzyme heme. While the hemoglobin molecules in two different species are identical in function they are not identical in their amino acid sequences.

In this activity, the student will compare the amino acid sequences from fragments of five different globin molecules. Each fragment is shown as a series of one-letter amino acid symbols. To compare the sequences, the student will count the letter differences between each species pair. The number of differences should then be recorded in the matrix provided. The student will then construct a graphic representation of the evolutionary relationships among the five molecules--a kind of phylogenetic tree. Any phylogenetic tree is necessarily an hypothesis; most are definitively neither right nor wrong. Supporting arguments and evidences are important in the construction of any phylogeny--these data should not be used to the exclusion of data from other sources such as geography, morphology, physiology, etc.

Prior knowledge, concepts or vocabulary necessary to complete activity: Students should have studied proteins and understand that protein synthesis is determined by DNA nucleotide sequences. Students should be familiar with the concept of a phylogenetic tree. Commonly they will have seen evolutionary trees based on morphology. This activity will result in a phylogeny based on molecular structure.
Teacher instructions: Biologists search for evidence to group organisms into "relatedness" categories. The categories are used to construct phylogenetic trees. The "family trees" commonly found in textbooks are based primarily on morphology--these often do not show anything but similar structure and questions of analogy and homology arise. Today molecular studies help to complete the picture of relationships between species.

It should be emphasized that this activity involves counting differences in amino acid sequences in fragments of proteins, not the entire protein. Counting the differences in the sequences of complete proteins would require a computer. This activity however will give students a sense of how scientists acquire these data and how they are used while, at the same time, minimizing the repetitive tedium of the process. Both DNA and RNA sequences from many genes belonging to many organisms and the amino acid sequences of many proteins can be found at several sites on the World Wide Web. We found the following site to be particularly useful:

http://www.Kyoto-center.genome.cd.jp/

Teacher's Guide

Using Amino Acid Sequences To Show Evolutionary Relationships

Introduction: In different organisms the amino acid sequences of proteins with the same function are similar, but not identical. These differences can be used to indicate how closely organisms are related. This activity uses the amino acid sequences from certain globin proteins.

Goal: The students will determine the number of differences in fragments of several globin molecules and will construct a phylogeny showing which polypeptides are most similar and which are most different. The students will be able to see the chronological order in which the proteins diverged from each other.

Directions:

  1. Count the number of differences in amino acids between protein fragment #1 and protein fragment #2 (a blank will be scored as a difference). The box below shows you how to score the differences.

    The circled letters indicate differences in each protein fragment; thus, there are 3 differences between the amino acid sequences in polypeptides #1 and #2.

    These are different globin molecule fragments. The letters are symbols for the amino acids in the sequence of the molecule. (See Table 1.)

    #1 VNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR--

    #2 VNFKLLSHCLLVTLACHHPTEFTPAVHASLDKFFLAAVTVLTSKYR--

    #3 ENFRLLGNVLVCVLAHHFGKFFTPPVQAAYQKVVAGVANALAHKYH--

    #4 ENFKLLGNVLVCVLAHHFGKFFTPOVQAAYQKVGVAGVANALAKYH--

    #5 KYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG

    Table 1. Amino Acid Symbols
    A = alanine G = glycineM = methionineS = serine
    C = cysteineH = histidineN = asparagineT = threonine
    D = aspartic acidI = isoleucine P = prolineV = valine
    E = glutamic acidK = lysineQ = glutamineW = tryptophan
    F = phenylalanineL = leucineR = arginineY = tyrosine

  2. In the same way as step one, count the number of differences in the fragments making comparisons between each pair (1<-->2, 3, 4, 5; 2<-->3, 4, 5; 3<-->4, 5; 4<-->5) and write the number of differences in the following chart.

    Assume that these five proteins diverged from a common ancestor-protein sometime in the past; they diverged--changed but did not replace--this ancestor-protein over time. It is most likely that the genes which held the recipes for these proteins have been duplicated several times to allow for these changes. Using this concept, place the protein fragments in the "tree" on the next page.

  1. Describe the reason you put each fragment in its particular location on the "tree."

  2. Species X and Y have 25 amino acid differences. Species X and B have 10 amino acid differences in the same protein. Species Y and B have 27 amino acid differences.

    a) Which organism (X, Y, or B) diverged from the common ancestor first? ____________ Explain your reasoning.

    b) Which pair of organisms--X & Y, X & B, or Y & B--are likely to share more characteristics than the other two pairs. ______________ Again, explain your reasoning.


Using Amino Acid Sequences To Show Evolutionary Relationships

NamePeriod Class:

Introduction: In different organisms the amino acid sequences of proteins with the same function are similar, but not identical. These differences can be used to indicate how closely organisms are related. This activity uses the amino acid sequences from certain globin proteins.

Goal: The students will determine the number of differences in fragments of several globin molecules and will construct a phylogeny showing which polypeptides are most similar and which are most different. The students will be able to see the chronological order in which the proteins diverged from each other.

Directions:

  1. Count the number of differences in amino acids between protein fragment #1 and protein fragment #2 (a blank will be scored as a difference). The box below shows you how to score the differences.

    The circled letters indicate differences in each protein fragment; thus, there are 3 differences between the amino acid sequences in polypeptides #1 and #2.

    These are different globin molecule fragments. The letters are symbols for the amino acids in the sequence of the molecule. (See Table 1.)

    #1 VNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR--

    #2 VNFKLLSHCLLVTLACHHPTEFTPAVHASLDKFFLAAVTVLTSKYR--

    #3 ENFRLLGNVLVCVLAHHFGKFFTPPVQAAYQKVVAGVANALAHKYH--

    #4 ENFKLLGNVLVCVLAHHFGKFFTPOVQAAYQKVGVAGVANALAKYH--

    #5 KYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG

    Table 1. Amino Acid Symbols
    A = alanine G = glycineM = methionineS = serine
    C = cysteineH = histidineN = asparagineT = threonine
    D = aspartic acidI = isoleucine P = prolineV = valine
    E = glutamic acidK = lysineQ = glutamineW = tryptophan
    F = phenylalanineL = leucineR = arginineY = tyrosine

  2. In the same way as step one, count the number of differences in the fragments making comparisons between each pair (1<-->2, 3, 4, 5; 2<-->3, 4, 5; 3<-->4, 5; 4<-->5) and write the number of differences in the following chart.

    We can assume that these five proteins diverged from a common ancestor-protein sometime in the past; they diverged‹changed but did not replace‹this ancestor-protein over time. It is most likely that the genes which held the recipes for these proteins have been duplicated several times to allow for these changes. Using this concept, place the protein fragments in the "tree" on the next page.

  1. Describe the reason you put each fragment in its particular location on the "tree."

  2. Species X and Y have 25 amino acid differences. Species X and B have 10 amino acid differences in the same protein. Species Y and B have 27 amino acid differences.

    a) Which organism (X, Y, or B) diverged from the common ancestor first? ____________ Explain your reasoning.

    b) Which pair of organisms--X & Y, X & B, or Y & B--are likely to share more characteristics than the other two pairs. ______________ Again, explain your reasoning.

On to Molecular Biology and
Primate Phylogenetics

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