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Molecular Biology

"The Cytochrome-C" Lab


Beth Kramer
ENSI 1992

Middle School Version by
Karin Westerling (ENSI 1992)





Revised 5-25-01
Middle School Version added


Amino acid sequences in cytochrome-c are compared for several different organisms, and the number of differences found are used to infer degrees of relationship. These data are also compared with a cladogram constructed for those same organisms from their anatomical features, providing an example of independent confirmation.


Independent confirmation strengthens scientific inferences.



1. Molecular structure of homologous molecules shows degrees of similarity.

2. Degrees of relationship are inferred from degrees of similarity

3. Relationship patterns derived from molecular structure are consistent with those derived from anatomical structure.


   Students will....

1. cite a valid example of independent confirmation.

2. explain why independent confirmation is important in science.

3. recognize what comparisons of molecular structure suggests about evolutionary relationships.


   CLICK HERE for PDF pages for easy printing.

1. Background/Procedure
2. Analysis questions (print on back of Background/Procedure sheet)
3. Chart of amino acid sequences of cytochrome-c in 20 organisms
4. (optional): sheet with two charts (perhaps on back of fig. 36.2 sheet):
----- Fig. 36.3: Nucleotide differences for cytochrome-c gene.
----- Fig. 36.4: Phylogenetic tree based on these nucleotide differences.


One 45-60 minute period.

   (see end of lesson for the formatted handouts).

1. Background and Procedure
2. Analysis questions (can be printed on back of "Background/Procedure")
3. Chart of amino acid sequences (adapted from Fig. 36.2 from Strahler, Science and Earth History, 1987, page 348.
4. Sheet with 2 charts: Figs 36.3 and 36.4 from pages 350, 351 respectively, same source. This sheet could be used to make an overhead transparency for class discussion, if preferred.





Background: Ideally, you have just completed the activity ("Making Cladograms") in which you constructed a cladogram (a type of phylogenetic tree) from comparative anatomy data for seven organisms. You will now examine another method biologists use to try to determine relationships -- that of comparing amino-acid sequences of homologous proteins.

The discovery of DNA and its role in coding for the synthesis of proteins brought with it a new and powerful tool for the study of phylogeny (evolutionary relationships). Through biochemistry, there has emerged (in only the last twenty years) a remarkable means for double checking the pattern of organic evolution through geologic time. In genetics we learn that the assembly of protein molecules in the cell is coded for by the DNA on the chromosomes. Comparisons of the similarities and differences between DNA in two groups of organisms serves to establish homologous relationships.

Amino-acid sequences have now been determined for a large number of proteins. If we can compare the sequence of the amino-acids in a protein that several organisms have, then we can make inferences about the section of DNA that coded for the protein in the organisms. Further, since DNA comes from an organism's ancestors, we can begin to establish the ancestral relationships of these organisms. As in other characteristics, we would expect those organisms with fewer differences in their protein amino-acid sequences to be derived from common ancestors more recently, and therefore to be more closely related. We can then use this molecular evidence to place organisms in their proper places in a cladogram.

An excellent resource is now available to you to find and compare amino acid sequences in a variety of proteins in a variety of organisms. It's called the Biology Workbench. Be sure to check this out. They have excellent tutorials to walk you through the process. In very little time, you will be creating your own data pages (or even better, your students can do this).

The nice thing about these two activities is that they can be used together to show independent confirmation of the relationships within the same group of organisms. The major point being that if there is independent confirmation, that makes the inferences drawn about the organisms' relationships stronger.




Give the students this activity to do in their groups of four. We suggest that you do not give a lot of background. The student directions are clearly spelled out in their handout.

Middle School Version (from Karin Westerling, added 12/2002):
Seriously consider using this variation, even at the high school level. Probably easier to do, but just as powerful. See it in the PDF files.

Post Lab Discussion: In the discussion afterward, get the students to come up with the conclusions discussed above -- it's much more powerful if they have drawn their own conclusions. Don't forget to discuss this important question mentioned in the comparative anatomy activity: "We assume that the more similar (fewer differences in) the sequence of amino acids, the more closely related they are. (Later in class, be prepared to discuss why we feel we can make this assumption)." Desired responses to the Analysis questions are available; just send an email request with your school site email address on it to the WEBMASTER.

A useful extension: You can show them the nucleotide data, and the phylogenetic tree constructed from that data, from pages 350-351 in Strahler to reinforce their conclusions. NOTE: if your students have studied DNA structure and protein synthesis, they should recognize the basis for the similar patterns from nucleotide differences to amino acid differences. Also note the phylogenetic tree from Strickberger's "Evolution" 1996 (p. 258) that is based on data from seven proteins. These sheets are attached as pdf files at the end of this lesson.


1. Observe active participation.
2. Build a written test which addresses the "Assessable Objectives"



Follow this lesson with the Chromosome Comparison lesson, which shows how degrees of similarity in banding patterns on chromosomes correlates with evolutionary relationships between apes and humans, based on anatomy and biochemical indicators.

New Middle School Version:
Try a recent version of this lesson developed for use in middle school Life Science classes, but probably just as useful in high school Biology. It was developed by ENSI-trained teacher Karin Westerling, and is somewhat easier to use. Besides the 4 page student handout, there are 9 pages of data tables (to distribute 1-2 per team). These are available for downloading on the PDF page. You can obtain the desired responses to the questions by emailing your request to the WEBMASTER using your school email address.

3. The UCMP (University of California Museum of Paleontology) has an excellent presentation of cladistics, phylogenies, and modern systematics (what, why, when, and how). Take a look at it: (<http://www.ucmp.berkeley.edu/exhibit/phylogeny.html>).

4. See the excellent online tutorial by the UCMP: "What did T. rex taste like?" It makes an excellent introduction to classification, phylogenetic trees, and cladistics. This could be given as a homework assignment (online).

5. For an excellent tutorial to introduce phylogenetic (evolutionary) trees, see our review of an article in the American Biology Teacher.

6. Consider following this lesson later with the Human-Chimp DNA reading-worksheet assignment, developed by ENSI-using teacher, Tom Mueller.


Useful article in Molecular Clues to Evolution briefly explains how cytochrome c comparisons can show degrees of relationship (page 29).

Strahler, Arthur. Science and Earth History, 1987.

The Biology Workbench: a rich source of amino acid sequences in a large variety of proteins and in a huge variety of organisms.

The Biology Workbench for Students: same type of resource, adapted for use by students.


Some of the ideas in this lesson may have been adapted from earlier, unacknowledged sources without our knowledge. If the reader believes this to be the case, please let us know, and appropriate corrections will be made. Thanks.

1. Original Source: Beth Kramer

2. Modified by:

3. Reviewed / Edited by: Pending

4. Edited / Revised for website by L. Flammer 10/27/98

5. Variation for Middle School, by Karin Westerling 12/2002

 The following is a useful worksheet for students to complete while reading the article, to help focus and direct their reading. A KEY follows, for teacher use, or for use by students as a self-check.

Name________________________________ S.N.____ Date________ Per____


BACKGROUND: You have just completed an activity in which you made a cladogram showing the evolutionary relationships between seven organisms. The data used to draw that cladogram was based on shared characteristics that were inherited from their ancestors.

Biochemical characteristics, like similarities in nuclear DNA, mitochondrial DNA, or protein structure, can be used to produce cladograms also. If there is strong agreement between the patterns produced using anatomical similarities and those produced by using biochemical structures, it provides what we call "independent confirmation" of the cladogram. Independent confirmation is where two or more sources of evidence that are not dependent on each other produce the same pattern. The more independent confirmation that is available, the more confidence we have that the evolutionary relationships shown in the cladogram are correct.

In this activity, we will examine and compare the amino acid sequence of a protein. All seven of the organisms used in the previous activity produce this protein (it is a homologous protein). The complete sequence of amino acids for this protein has been determined for many organisms, including six of those seven animals. We assume that if we find fewer differences in the amino acid sequences, those animals are more closely related. (Be prepared to discuss why we can make this assumption.)

PROCEDURES: You have already done and discussed the activity entitled "Making Cladograms". The final cladogram produced in that activity (using anatomical similarities) is shown below. You may be asked to work in groups of four, and share the following task with your partners.

You will be provided with a chart (Fig.36.2) showing the amino acid sequence in a protein that is homologous for the 20 organisms shown, including six of the seven organisms already studied (data on this protein is not available here for the lamprey). The name of the protein is Cytochrome-c. It is an enzyme that is important in the breakdown of food molecules by a cell. It helps release the energy in food molecules so that it can be used by the cell. Each amino acid is represented by a unique letter in the chart.

Compare the sequence of amino acids (letters) in human Cytochrome-c to the sequences for each of the 5 remaining animals (shown in the diagram below) by counting the number of differences. HINT: Highlight or underline the organism being checked, then circle each amino acid which is different from the one above it in the human sequence (use pencil). It helps to use a ruler or other straight edge. Notice that the amino acids showing no differences in any of the organisms are surrounded with vertical boxes, so you can just ignore them when you scan the line of letters. When an organism has a "-" instead of an amino acid, that means there is no amino acid there. When comparing a "-" to an organism that has an amino acid at the position, it should be considered as a difference. When both organisms have a "-" at that spot, it is not considered as a difference.

Record the number of differences next to each animal's name. Compare your numbers with those of your team members. If there is a discrepancy, repeat the scan and count.

Record the confirmed number of amino acid differences in the appropriate hexagon on the cladogram below (below the vertical arrow pointing to that animal). Answer the Analysis questions (overpage).


1. Does the data from the amino acid sequence generally agree with the anatomical data that was used to make this cladogram? (i.e., do organisms with fewer shared anatomical traits also have more amino acid differences?)_______

2. Based on the molecular data, make a general statement about the "human-monkey" relationship as compared to the "duck-chicken" relationship (which shows three differences in their amino acids).



3. If the molecular data, the structural similarities, and the fossil record all support the same pattern of relationships, can we be fairly confident that the pattern is correct?_______ Why?



4. A) Using the molecular data, make a general statement which compares the "human-kangaroo" relationship ("H-K")to the "human-frog" ("H-F") relationship.
B) Does the cladogram agree with this statement?______ C) Explain your reasoning.




5. The chicken and the turkey are both birds and have the same sequence of amino acids in their cytochrome-c protein. Explain how two different species can have identical cytochrome-c and still be different species.




6. Neurospora and (bread mold) and Saccharomycetes (bakers yeast) are both fungi. Chickens and turkeys are both birds. A) What can you say about the evolutionary relationships between the two birds compared to the relationship between the two fungi? B) Explain your reasoning (use of simple diagrams might help).




7. In a short paragraph, summarize what important information can be obtained from cladograms (NOT the info that was used to make the cladograms).
(Possible Alternative, if so directed: With your group, write two paragraphs in which you summarize what cladograms are all about, and draw conclusions from the data presented in the chart. The first paragraph should summarize how cladograms are made and what important information can be obtained from them. The second paragraph should be the conclusions that you make about the relationships of the seven organisms studied in this activity and the previous one. Think this conclusion through thoroughly first, and discuss with your group what should be included in it. Incomplete, sketchy, or sloppy conclusions will be given back to be re-done.)



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