Transcript S1. Untangling the central dogma- Lecture

A clicker-based case study that untangles
student thinking about the processes in the
central dogma.
Karen N. Pelletreau1*, Tessa Andrews2, Norris Armstrong2, Mary A.
Bedell2, Farahad Dastoor1, Neta Dean3, Susan Erster3, Cori FataHartley4, Nancy Guild5, Hamish Greig 1, David Hall2, Jennifer K.
Knight5, Donna Koslowsky4, Paula P. Lemons6, Jennifer Martin5, Jill
McCourt6, John Merrill4, Rosa Moscarella7, Ross Nehm8, Robert
Northington1, Brian Olsen1, Luanna Prevost9, Jon Stoltzfus10, Mark
Urban-Lurain7, Michelle K. Smith1
Affiliations:
1School of Biology and Ecology, University of Maine.
2Department of Genetics, University of Georgia.
3Department of Biochemistry and Cell Biology, Stony Brook University.
4Department of Microbiology and Molecular Genetics, Michigan State University.
5Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder.
6Department of Biochemistry and Molecular Biology, University of Georgia.
7Collaborative Research in Education, Assessment, and Teaching Environments for the fields of Science, Technology,
Engineering and Mathematics (CREATE4STEM), Michigan State University.
8Center for Science and Mathematics Education and Department of Ecology and Evolution, Stony Brook University.
9Department of Integrative Biology, University of South Florida.
10Department of Biochemistry and Molecular Biology, Michigan State University.
*Correspondence to: School of Biology and Ecology, 5751 Murray Hall, Orono ME 04469. [email protected]
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What is Duchenne Muscular Dystrophy (DMD)?
100
Percent of Male Pa ents Using a Wheelchair
90
80
70
Percent
• A diseases that
manifests in
muscle weakness
and wasting
• Exhibits X-linked
recessive
inheritance
pattern
60
50
40
30
20
10
0
5-9
10-14
15-24
Age (Years)
2
www.cdc.gov
Mutations in the dystrophin gene
cause Muscular Dystrophy
The dystrophin gene:
-encodes for the protein
dystrophin
-is found on the X chromosome
Male
affected
Female
carrier
Mutations in the dystrophin
gene:
-result in different alleles
-can lead to the expressed
phenotype Muscular dystrophy
3
The dystrophin gene is large and can
have many different mutations along
the DNA.
X chromosome
Dystrophin gene
4
Case Study: Siblings living with
Duchenne Muscular Dystrophy (DMD)
• Liam: affected with DMD
• Elijah: unaffected
Liam
Elijah
affected
unaffected
Your goal: Use the information provided to
explore what nucleotide changes could result in
Liam developing DMD
5
X chromosome
79 exons encoding 3500 amino acids
1
2
3
4
5
There are five nucleotide differences between Elijah and Liam
in the dystrophin gene.
locations where
1
DNA differs
Elijah’s X
Liam’s X
affected
A
T
2
T
A
3
C
G
4
C
G
5
T
A
G
C
A
T
T
A
T
A
G
C
6
Dystrophin gene structure and the location of the five
different nucleotides.
affected
Gene structure can provide our first clue. Based solely
on these labeled features, discuss which of the DNA
differences would be more or less likely to result in the
muscular dystrophy phenotype?
7
✖
Dystrophin gene structure
affected
Difference #2:
Elijah’s mRNA
Liam’s mRNA
UCU
(ser)
UCA
(ser)
This type of substitution (a silent mutation) results in the
same amino acid, and therefore protein. The results from
other mutations are more challenging to predict…
8
Dystrophin gene structure
✖
✖
affected
Difference #5:
Elijah’s mRNA
Liam’s mRNA
GAU
(asp)
GAG
(glu)
Q1: Is difference #5 a possible cause of DMD in Liam?
A. Yes
B. No
9
What can we deduce about these two
amino acids and their possible function?
HINT: Your biochemistry
colleague reminds you that
glutamate and aspartate
are both structurally and
biochemically similar.
10
Dystrophin gene structure
✖
✖ ?
affected
Difference #1:
?
Q2: If difference #1 caused DMD, we would predict the mRNA
levels in Liam to be __________ the mRNA levels in Elijah?
A.
B.
C.
D.
the same as
higher than
lower than
different in some way from
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Dystrophin gene structure
✖
✖
✖ ?
affected
Difference #1:
Q3: You have now done the experiment and measured the mRNA levels in
both Elijah and Liam. If these are your results, would you conclude that
difference #1 a likely cause of DMD in Liam?
A. Yes
B. No
12
13
✖
Dystrophin gene structure
✖
✖ ?
affected
Difference #3:
Elijah’s mRNA
Liam’s mRNA
CAA
(gln)
UAA
(STOP)
Q4: When DNA polymerase reaches the nucleotides encoding the
premature stop codon it will…
A. stop when it reaches the first nucleotide encoding the premature stop
codon.
B. stop when it reaches the last nucleotide encoding the premature stop
codon.
C. not be affected by this base change and will continue to read through the
nucleotide difference.
14
The red boxes along this
DNA strand indicate
regions where the DNA
sequence could code for
stop codons.
If DNA polymerase
recognized these triplet
nucleotides as stop
codons, what would be
the consequence on DNA
replication?
15
✖
Dystrophin gene structure
✖
✖ ?
affected
Difference #3:
Elijah’s mRNA
Liam’s mRNA
CAA
(gln)
UAA
(STOP)
Q5: When DNA polymerase reaches the nucleotides encoding the
premature stop codon it will…
A. stop when it reaches the first nucleotide encoding the premature stop
codon.
B. stop when it reaches the last nucleotide encoding the premature stop
codon.
C. not be affected by this base change and will continue to read through the
nucleotide difference.
16
Polymerases read only one base at a time,
not triplet codons.
The DNA polymerase would not be affected by this base
change in Liam’s DNA and replication would proceed
normally.
17
NO EFFECT
?
?
18
Dystrophin gene structure
Difference #3:
?
Q6: What do you predict will be the effect of the premature stop
codon on mRNA size? It will result in:
A. a shorter mRNA in Liam.
B. a longer mRNA in Liam.
C. the same size mRNA in both Liam and Elijah.
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Liam’s DNA (affected)
coding strand
template strand
Q7: The above DNA sequence is being transcribed by an RNA
polymerase (red square). The premature stop codon mutation in
Liam’s DNA is indicated in blue. What will the RNA polymerase do
when it reaches the nucleotides encoding the premature stop
codon? It will:
A. stop when it reaches the first nucleotide encoding the
premature stop codon.
B. stop when it reaches the last nucleotide encoding the
premature stop codon.
C. not be affected by this base change and will continue to read
through nucleotide difference.
20
Dystrophin gene
structure
Difference #3:
Here are the results of your experiment. How do the
results above compare with your prediction?
21
Elijah’s DNA
coding strand
coding strand
Liam’s DNA (affected)
22
Elijah’s DNA
Liam’s DNA (affected)
Polymerases read only one base at a time,
not triplet codons.
The RNA polymerase would not be affected by this base
change in Liam’s DNA and transcription would proceed
normally.
23
NO EFFECT
NO EFFECT
?
24
Dystrophin gene
structure
Difference #3:
?
Q8: What do you predict will be the effect of the premature
stop codon on protein size? It will:
A. result in a smaller dystrophin protein in Liam.
B. result in a larger dystrophin protein in Liam.
C. result in the same size protein in both Liam and Elijah.
25
Dystrophin gene structure:
Difference #3:
Here are the results of your experiment. How do these
results compare with your prediction?
How can you explain the observed difference in the size
of the protein?
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Elijah’s mRNA
Liam’s mRNA (affected)
The ribosome does recognize triplet codons.
The ribosome will stop and fall off when it encounters
the premature stop codon that resulted from the base
change in Liam’s DNA. As a result, the process of
translation will stop, and the protein will be shorter. 27
What specifically recognizes stop
codons during translation?
Modified from Pearson
28
NO EFFECT
NO EFFECT
EARLY
TERMINATION
29
Dystrophin gene structure
affected
Elijah’s mRNA
Liam’s mRNA
UCU
(ser)
UCA
(ser)
CAA
(gln)
UAA
(STOP)
GAU
(asp)
GAG
(glu)
Q9: Which of the five nucleotide differences are mutations?
A. All five are mutations.
B. The three in the exons (difference #2, #3, #5)
C. The two in exons that change the amino acid sequence
(differences #3, #5)
D. The one in the exon that causes DMD (difference #5)
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Case Study Conclusions: Siblings living with
Duchenne Muscular Dystrophy (DMD)
UCU
(ser)
CAA
(gln)
GAU
(asp)
UCA
(ser)
UAA
(STOP)
GAG
(glu)
Liam
Elijah
affected
unaffected
Elijah’s mRNA
Liam’s mRNA
We can conclude that, in this case, the premature stop codon
(nonsense mutation) was the most likely mutation to lead to the
expressed phenotype of DMD in Liam because of the effect on
translation and the subsequent truncated protein produced.
31