Lethal Mutation
Project Overview
Discipline: Science
Course: Genetics
RWLO Description:
Students will use the NCBI website (www.ncbi.nlm.nih.gov) to research a genetic disorder,
ALS, and the mutant protein, SOD1, which causes the disorder. Students will then download
the protein structures of both the normal and two mutant SOD1 proteins and view them in 3D
using downloadable viewing software called Cn3D. Students will describe the effect of the
mutations in terms of the effect on protein structure. Students will be able to correlate the
molecular mutation to the physiological symptoms of the disease.
The National Center for Biotechnology Information (NCBI) is a division of the National Library
of Medicine (NLM) at the National Institutes of Health (NIH). The NCBI maintains many
enormous databases, including GenBank, which stores all known public DNA sequences,
submitted from individual scientists and large genome centers around the world; OMIM, a
Web-based catalog that contains thousands of entries for genes and genetic disorders, which
is frequently updated by NCBI staff based on published work in the scientific community; the
Molecular Modeling Database (MMDB) of 3D protein structures; and many, many others. No
other source of up-to-date molecular biology information is even remotely of the size and
scope of this collection of resources.
Audience:
Undergraduate students in genetics (or other biology) courses studying the effects of gene
mutation on protein structure.
Context information:
Students should be knowledgeable on the four levels of protein structure (primary,
secondary, tertiary, and quaternary) including the types of bonds that stabilize or define each
level.
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Student Learning Objectives
For this RWLO, the student will be able to:

Use the NCBI website to research ALS and the protein SOD1.

Use a downloaded viewer program to view the 3D structure of the normal protein and
two mutant proteins.

Make comparisons of the normal structure with the mutant structure in terms of the
four levels of protein structure.

Explain how a specific gene mutation creates a protein with a mutant 3D structure.

Relate the mutant protein structure to the physiological symptoms of the disease.
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Procedure
Time: Approximately 45 minutes
Materials: Computer with internet connection
Prerequisites: Review of the central dogma of biology, gene mutation, and protein structure.
Implementation: This RWLO can be used as a homework assignment to reinforce and/or
demonstrate how a single nucleotide mutation in a gene affects the structure of the resulting
protein, and in turn how this altered structure creates a physiological malfunction (genetic
disease). This RWLO could also be used in the classroom as a demonstration by the
instructor.
Steps:
Download the 3D protein viewer:
1. Click here to begin.
2. Click on “Download Cn3D 4.1 for PC, Mac, and Unix” and follow the instructions.
(This will download the viewing software you will need to view a protein in 3D on your
screen.)
Now you are ready to begin the activity:
3. Go to OMIM. Search OMIM for “SOD1.”
Your results:
The first entry is *147450, Superoxide dismutase 1, SOD1
The second entry is #105400, Amyotrophic Lateral Sclerosis 1; ALS1
Start with the second entry first:
4. Click on the #105400 (second entry) to gather background information on ALS, the
disease caused by mutant SOD.
5. Have students focus on Paragraphs 1,2, 10, and 14 to get an overview of key points.
6. Students will answer the following questions:
A. 15 to 20% of cases of familial amyotrophic lateral sclerosis type 1 (ALS1)
are associated with a mutation in what gene? SOD1
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B. Familial forms of ALS follow what mode of transmission? Autosomal
dominant
C. What is another name for ALS? Lou Gehrig’s disease
D. What is the nature of the symptoms and the disease progression? Loss of
motor function with predominantly lower motor neuron manifestations. Wasting
of muscles of 1 hand and spreading of the disorder in a contiguous manner.
Lower motor neuron involvement is usually conspicuous, whereas involvement
of upper motor neurons is less marked.
E. Does the disease show incomplete penetrance and/or variable
expressivity? Highly variable in terms of age of onset and duration of the
disease. By age 85 years, about 80% of carriers have manifested the disorder.
F. Why is euthanasia an issue involved with ALS? The progressive paralysis
leads to increase of loss of function, culminating in complete dependence on
the help of others for all activities of daily living and, if life is sustained by
assisted ventilation, loss of the ability to communicate or swallow.
G. What is the title of the bestselling book by Mitch Albom, describing the
last year in the life of an ALS patient? Tuesdays with Morrie
7. Use the back arrow button to return to the OMIM results list.
8. Review entry *147450 (regarding SOD1) paragraphs 3, 13, and 16 and answer these
questions:
A. What reaction is catalyzed by superoxide dismutase? Disproportionation or
dismutation of superoxide radicals to molecular oxygen and hydrogen peroxide.
B. What scientist identified the first disease-producing mutations in SOD1?
Rosen in 1993
C. What type of mutations were found? Missense
D. Describe the two proposed mechanisms by which mutant SOD1 would
cause cellular damage in ALS patients.
i.
Hypoactivity of SOD1 could lead to a build-up of the toxic
superoxide radical.
ii.
Increased activity of SOD1 could lead to excessive levels of H2O2
and a highly toxic hydroxyl radical.
E. What specific mutation was found to be most frequent? Ala4val or A4V
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F. Examination of the crystal structure of human SOD established what?
“Examination of the crystal structure of human SOD established that all 12
observed sites of mutation causing ALS alter conserved interactions critical to
the beta-barrel fold and dimer contact, rather than catalysis.”
9. Click on “Links” for SOD1 (to the far right), and scroll down to “Structure” and click.
This takes us to the structure database. We will be using the following entries:
1UXM
A4V mutation of human SOD1
1UXL
I113T mutation of human SOD1
1SPD
Human superoxide dismutase 1 (SOD1)
10. First click on 1SPD, the normal structure of SOD1, and on the next screen click on
“View 3D Structure” (the gray button).
The viewing program should open up a new window with the structure of SOD1. Note
that you can click and drag on the image to rotate it in 3D.
Answer the following questions:
A. What secondary structures are visible? How many can you find of each
type? SOD1 is a dimer, so each subunit contains 10 beta sheets, although the
students may count 20 if they don’t yet notice it is a dimer. There are no alpha
helices.
B. What metal cofactors are present? Copper and zinc.
C. Locate the amino and carboxyl termini. What does this tell you about
quaternary structure? There are four “ends” which means there are 2 subunits
here, so it is a dimer.
11. Now click on 1UXM, view the structure and answer these questions:
A. Which level of protein structure is visibly altered by the A4V mutation,
compared to the normal SOD1 structure? Quaternary structure – the mutant
SOD1 is a polymer, not a dimer.
12. Now click on 1UXL, study the structure and answer these questions:
A. 1UXM is described as an “A4V mutation of human SOD1.” This means
that the fourth amino acid in the protein has been changed from adenine
to valine, due to a missense mutation in the gene. 1UXL is described as
an “I113T mutation of human SOD1.” What does this mean? The 113th
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amino acid has been changed by missense mutation from isoleucine to threonine.
B. Which level of protein structure is visibly altered by the I113T mutation,
compared to the normal SOD1 structure? Again, quaternary structure – the
mutant SOD1 is also a polymer, not a dimer.
13. Close the structure viewing windows and use the back arrow to return to the
structures list. On the right side of the 1UXM entry, click on “Links” and drag down to
“Full text in PMC.”
This will take you to a list of 2 journal articles which study the A4V and I113T
mutations. Click on the “Abstract” of each article, read through them quickly, and
answer these questions:
A. What can you find out about the location of the A4V and I113T mutations,
in terms of both tertiary and quaternary structure? They exist at the “dimer
interface” – the part of the subunit that interacts or bonds with the other subunit.
B. What effect do these mutations have on the dimer formation? They
destabilize the dimer and result in aggregation of the subunits.
Instructor: Be sure to emphasize that the aggregates cannot function properly, thus the
superoxide anions build up and become toxic to the cells. Because higher levels of SOD1
are produced in motor neurons, these cells show the effects first. The death of motor
neurons results in the symptoms of ALS described above.
14. Students complete the activity and all questions prior to the next class period. The
instructor might spend 10-15 minutes at the beginning of the next class answering
questions and giving any clarifications needed. Or, if preferred, the students can form
groups of 3-4 to discuss the activity.
15. To complete the project, students should use their answers to all the previous
questions to synthesize a 1-page written summary describing how mutation of the
SOD1 gene relates to the disease ALS. The summary should include a description of
ALS on both a physiological and cellular level and the mode of transmission in familial
cases; the normal structure and function of SOD1 protein in a motor neuron; the types
of SOD1 mutations associated with ALS; and an explanation of how these mutations
affect the structure of SOD1 and thus cause neuronal death. The summary should be
3/4 page typed, 250-300 words.
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Content Material
Student Directions:
Time Required: Approximately 45 minutes
Materials: Computer with internet connection
Prerequisites: Review of the central dogma of biology, gene mutation, and protein
structure.
Steps:
Download the 3D protein viewer:
1. Click here to begin.
2. Click on “Download Cn3D 4.1 for PC, Mac, and Unix” and follow the instructions.
(This will download the viewing software you will need to view a protein in 3D on your
screen.)
Now you are ready to begin the activity:
3. Go to OMIM. Search OMIM for “SOD1.”
Your results:
The first entry is *147450, Superoxide dismutase 1, SOD1
The second entry is #105400, Amyotrophic Lateral Sclerosis 1; ALS1
Start with the second entry first:
4. Click on the #105400 (second entry) to gather background information on ALS, the
disease caused by mutant SOD.
5. Focus on Paragraphs 1,2, 10, and 14 to get an overview of key points.
7
6. Answer the following questions:
a. 15 to 20% of cases of familial amyotrophic lateral sclerosis type 1 (ALS1)
are associated with a mutation in what gene?
________________________________________________________________
b. Familial forms of ALS follow what mode of transmission?
________________________________________________________________
c. What is another name for ALS?
________________________________________________________________
d. What is the nature of the symptoms and the disease progression?
________________________________________________________________
e. Does the disease show incomplete penetrance and/or variable
expressivity?
________________________________________________________________
f. Why is euthanasia an issue involved with ALS?
________________________________________________________________
________________________________________________________________
g. What is the title of the bestselling book by Mitch Albom, describing the
last year in the life of an ALS patient?
________________________________________________________________
7. Use the back arrow button to return to the OMIM results list.
8
8. Review entry *147450 (regarding SOD1) paragraphs 3, 13, and 16 and answer these
questions:
a. What reaction is catalyzed by superoxide dismutase?
________________________________________________________________
b. What scientist identified the first disease-producing mutations in SOD1?
________________________________________________________________
c. What type of mutations were found?
________________________________________________________________
d. Describe the two proposed mechanisms by which mutant SOD1 would
cause cellular damage in ALS patients.
________________________________________________________________
e. What specific mutation was found to be most frequent?
________________________________________________________________
f. Examination of the crystal structure of human SOD established what?
________________________________________________________________
9. Click on “Links” for SOD1 (to the far right), and scroll down to “Structure” and click.
10. This takes us to the structure database. We will be using the following entries:
1UXM
A4V mutation of human SOD1
1UXL
I113T mutation of human SOD1
1SPD
Human superoxide dismutase 1 (SOD1)
11. First click on 1SPD, the normal structure of SOD1, and on the next screen click on
“View 3D Structure” (the gray button).
The viewing program should open up a new window with the structure of SOD1.
Note that you can click and drag on the image to rotate it in 3D.
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12. Answer the following questions:
a. What secondary structures are visible? How many can you find of each
type?
________________________________________________________________
b. What metal cofactors are present?
________________________________________________________________
c. Locate the amino and carboxyl termini. What does this tell you about
quaternary structure?
________________________________________________________________
13. Now click on 1UXM, view the structure and answer these questions:
a. Which level of protein structure is visibly altered by the A4V mutation,
compared to the normal SOD1 structure?
________________________________________________________________
14. Now click on 1UXL, study the structure and answer these questions:
a. 1UXM is described as an “A4V mutation of human SOD1.” This means
that the fourth amino acid in the protein has been changed from adenine
to valine, due to a missense mutation in the gene. 1UXL is described as
an “I113T mutation of human SOD1.” What does this mean?
________________________________________________________________
b. Which level of protein structure is visibly altered by the I113T mutation,
compared to the normal SOD1 structure?
________________________________________________________________
________________________________________________________________
10
15. Close the structure viewing windows and use the back arrow to return to the structures
list. On the right side of the 1UXM entry, click on “Links” and drag down to “Full text in
PMC.”
This will take you to a list of 2 journal articles which study the A4V and I113T
mutations. Click on the “Abstract” of each article, read through them quickly, and
answer these questions:
a. What can you find out about the location of the A4V and I113T mutations,
in terms of both tertiary and quaternary structure?
________________________________________________________________
________________________________________________________________
b. What effect do these mutations have on the dimer formation?
________________________________________________________________
________________________________________________________________
16. Homework (check with your instructor for due date): Use your answers to all the
previous questions in this activity to synthesize a 1-page written summary describing
how mutation of the SOD1 gene relates to the disease ALS. The summary should
include a description of ALS on both a physiological and cellular level and the mode of
transmission in familial cases; the normal structure and function of SOD1 protein in a
motor neuron; the types of SOD1 mutations associated with ALS; and an explanation
of how these mutations affect the structure of SOD1 and thus cause neuronal death.
The summary should be 3/4 page typed, 250-300 words.
Referenced URLs:
 www.ncbi.nlm.nih.gov
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Assessment
Class Session 1: Instructor introduces the activity and distributes the content materials.
Instructor emphasizes that the last item (#16 “Homework”) will not be due at the next class
period. Students will complete the questions found in the activity as they perform the tasks.
Class Session 2: Students will bring the content materials with the completed questions to
class for clarifications. 50% of the total points are awarded for the student bringing the
completed questions and participating in the discussion group. Instructor assigns the #16
homework essay.
Class Session 3: The remaining 50% of points for this activity will be awarded for the essay.
Student essays will be graded based on content requirements as well as clarity, use of
scientific terms and language, and overall quality. A sample rubric is given on the following
page.
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Assessment
Grading Rubric for Student Essays
Assignment Criteria
5 Essay addresses all components of the assignment, with evidence of independent thought.
4 Essay addresses all components of the assignment.
3 Subject is clear—although essay misses some required components.
2 Subject is generally clear but not in keeping with assigned topic.
1 Essay is not in keeping with assigned topic.
Organization and Flow of Thought
5 Essay shows a logical relationship of related ideas in coherent, sequential paragraphs.
4 Essay shows good development of ideas in coherent, sequential paragraphs.
3 There is evidence of a sequence of ideas, but paragraph structure is faulty.
2 Essay relies on vague arguments and unsupported information.
1 Essay is incoherent, with little development of relationships among ideas.
Language Usage
5 Student has edited the essay, ensuring that sentences are clear and logical, with appropriate
language and no grammar or punctuation errors.
4 Student has edited the essay, but a few minor errors remain.
3 The style is generally weak and a moderate amount of errors remain.
2 Serious style and language errors remain.
1 The essay is stylistically inappropriate.
TOTAL POINTS
Assignment Criteria
Organization
Language Usage
Total Points
/15 pts
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Links to Course Competencies
This RWLO could be applied in the following courses: Introductory Biology, Genetics, and
others. Specifically, this RWLO meets the following course competencies:
Compliance with Core Curriculum Guidelines
Intellectual Competencies:
1. Reading: the ability to analyze and interpret a variety of printed materials-books, documents, and
articles- above the 12th grade level.
2. Writing: the ability to produce clear, correct and coherent prose adapted to purpose, occasion
and audience-above the 12th grade level.
3. Speaking: ability to communicate orally in clear, coherent, and persuasive language appropriate
to purpose, occasion, and audience –above the 12th grade level.
4. Listening: analyze and interpret various forms of spoken communication, possess sufficient
literacy skills of writing, reading- above the 12th grade level.
5. Critical Thinking: think and analyze at a critical level.
6. Computer Literacy: understand our technological society, use computer-based technology in
communication, solving problems, and acquiring information.
Exemplary Educational Objectives:
1. To understand and apply method and appropriate technology to the study of natural sciences.
2. To recognize scientific and quantitative methods and the differences between these approaches
and the other methods of inquiry, and to communicate findings, analyses, and interpretation
both orally and in writing.
3. To identify and recognize the differences among competing scientific theories.
4. To demonstrate knowledge of the major issues and problems facing modern science, including
issues that touch upon ethics, values, and public policies.
5. To demonstrate knowledge of the interdependence of science and technology, and their
influence on, and contributing to, modern culture.
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Supplementary Resources

ALS Association: http://www.alsa.org/

An online database of mutations in ALS-related genes, including SOD1:
http://alsod1.iop.kcl.ac.uk/
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Recommendations
Recommendations for Integration:
This activity is best integrated into the course with some components completed in the
classroom and some as homework assignments.
Alternatively, the instructor can use the entire RWLO as a demonstration for use in the
classroom.
This RWLO should be integrated into a series of lessons explaining the mechanisms of gene
expression and gene mutation. The RWLO provides students with a “case study” of the
SOD1 gene and how it, when mutated, causes a fatal disease. This reinforces the lessons
with a real-life example.
Back-up:
The Cn3D viewing software and the 3 protein structure files can be saved to hard drive or
disk ahead of time, in the case that internet access becomes unavailable.
The procedures for the instructor and content material for students could be made available
in hard copy in the case that RWLO files become corrupted or inaccessible.
Advice to the Instructor:
This RWLO could be adapted to other genes and genetic disorders. In order to do so, you
must locate the structure files for both the normal protein and at least one mutant protein.
However, a keyword search of the “Structure” database at the NCBI website tends to return
far too many structure files to be manageable. I recommend that you first search OMIM for
the gene name, then click and drag down the list under “Links” (to the right of the gene entry)
until you find “Structure.” This path will return a much smaller number of relevant structure
files.
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