Day 3: Genetics
Selcen Guzey and Tamara Moore
University of Minnesota
Agenda
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Teaching Genetics- Challenges, misconceptions, strategies,
and questions
Content Assessment
Genetic Variation
Mutations
Gene expression
Lunch
Integrating Genetic Engineering: Plasmids and C. elegans lab
Teaching Genetics-Challenges,
misconceptions, strategies, and questions
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Use the post-it notes to create a “Parking Lot Chart” to
be addressed throughout the day.
Challenge: integrating engineering
Misconceptions: structure of DNA
Strategies: how to include published scientific data into my
genetics unit
Question: What is a plasmid?
Genetic Variation
How much variation do you think exists among humans?
How much variation do you think exists between a human
and a chimp?
How much variation do you think exists between a human
and a Caenorhabditis elegans (C. elegans) (Nematode)?
Genomes
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Genomes vary in size, number of genes, and gene density!
Haploid genome size
# of genes
bp: base (nucleotide) pairs
E. coli
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4.6 million bp
4,400
Caenorhabditis elegans 100 million bp
(Nematode)
20,100
Chimpanzee
3.3 billion bp
19,700
Human
3 billion bp
<21,000
What genetic attributions allow humans to get by with no more
genes than nematodes?
What makes humans and chimps so different?
Human Genome
DNA is the genetic material!
What piece of history is missing in
this picture?
OR
If you would like to recreate this
picture who would be in this
picture?
Rosalind Franklin
The structure of DNA
The Structure of a Gene
TATA and CAAT boxes, CG sequences
Enhancer Promoter
Exon
Intron
Exon
Intron
Exon
Poly-A
signal
sequence
Transcription start site
Upstream
Downstream
Beta Hemoglobin (HbB)
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Gene loci: 11p 15.5.
3 exons (coding regions) scattered
over 1600 base pairs
Yields a 626-bp mRNA transcript
Translated into a 147 amino acid
polypeptide
Beta Hemoglobin Gene and Sickle Cell
Anemia
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SCA is an autosomal
recessive disease caused by
a point mutation in the
hemoglobin beta gene (HBB).
The production of a
structurally abnormal
hemoglobin (Hb), called
HbS.
It occurs in 1 in 500
individuals of African
descent.
http://www.hhmi.org/biointeractive/media/DNAi_sicklecell-lg.mov
Beta-Globin Gene
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In this activity, you will examine the DNA sequence of
the beta-globin gene from five people.
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The codes represent the “sense” for the DNA
sequence- Sense strand looks like mRNA.
1)
2)
Examine the gene sequence of Person A and B and
find the difference in gene sequences of Person A
and B (Look at position 6)
Examine the gene sequence of Person C, D, E, and F.
Changes shown in bold!
From DNA to mRNA
3’- TTCAGTCGT - 5’ DNA template strand
5’- AAGTCAGCA- 3’ DNA sense strand
Transcription
5’- AAGUCAGCA- 3’
codon
mRNA
Translation
Lys
Ser
Ala
Protein
The codon table for mRNA
Questions for Discussion
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Would a person who has a sequence like person A’s and a
second sequence like person B’s have SC anemia?
Assume that a person has one allele with the sequence
shown for person B and a second allele with the
sequence shown for person F. Would the person have SC
anemia?
Would person D and E have SC anemia?
A person can have a mutation on beta-globin gene and do
not have SC. Propose a hypothesis to explain this.
HBB-mutations
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100 types of mutations affect HBB, and only one point
mutation causes SC Anemia. (Person B)
Splice mutations and mutations that occur in the
HBB gene promoter region tend to cause a reduction,
rather than a complete absence of β-globin chains and
so result in milder disease.
Nonsense mutations (Person D) and frameshift
mutations (Person F) tend to not produce any βglobin chains leading to severe disease.
Silence mutations are rare (Person C and E).
More on Mutations
Gene mutation- Point
 Chromosomal Mutations
mutations/single base substitutionsa) Deletion
a) Substitutions
b) Duplication
CCC
CCA
c) Inversion
b) Frameshift mutations: insertions d) Translocation
and deletions
5’ CCC CCA GGG 3’
5’CCC CCA AGA GGG 3’
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Point mutations can be also categorized as:
nonsense mutations, missense mutations, and
silent mutations
Testing for Sickle Cell Anemia
Mst II recognizes the sequence CCTNAGG and cut the DNA where N is.
DNA from normal homozygous individuals (AA), heterozygous carriers of the trait (AS),
and homozygous sickle cell patients (SS) produces different sizes of restriction fragments.
When the fragments are separated by gel electrophoresis the patterns result on the gel can
be used to identify people with sickle cell anemia.
1. Which family members have the sickle cell genotype (SS), carrier genotype
(AS), or the normal genotype (AA)?
2. Draw a pedigree showing inheritance of sickle cell anemia in the family.
3. Make a Punnett Square and explain the probabilities of various genotypes
and phenotypes for offspring of 2 carrier parents.
Mst II recognizes the
sequence CCTNAGG
Gene Expression: Switching genes on and off
Gene expression in humans is controlled by a variety of
mechanisms:
Transcriptional control that prevents mRNA from being synthesized:
Heterochromatin vs. Euchromatin, acetylation/deacetylation of
DNA, DNA methylation, transcription factors (activators and
inhibators)
Posttranscriptional control: Alternative mRNA splicing
Transitional control: Initiation factors
Posttransitional control: Protein activation
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Cancer results from genes that do not turn off properly. Cancer
cells have lost their ability to regulate mitosis, resulting in
uncontrolled cell division!
Transcriptional
Post transcriptional
Transitional
Post transitional
Analyzing gene expression
Case Study - What’s wrong with Raymond?
Presentation:
Raymond, a 43-year-old man, was admitted to the
hospital with a rapidly growing, painless mass in
his armpit. He reports that he has been
experiencing fever, weight loss, and night sweats
during the past four months.
Case Study - What’s wrong with Raymond?
Tests: Enlarged lymph nodes were removed and the
histology of the cells was examined, revealing abnormal B
cells. A bone marrow biopsy was done, along with
additional blood tests, X-rays, and CT scans.
Diagnosis:
Raymond has lymphoma.
What is lymphoma?
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http://video.about.com/lymphoma/Non-Hodgkin-sLymphoma.htm
Case Study - What’s wrong with
Raymond?
Tests: Enlarged lymph nodes were removed and the
histology of the cells was examined, revealing abnormal B
cells. A bone marrow biopsy was done, along with
additional blood tests, X-rays, and CT scans.
Diagnosis:
Raymond has diffuse large B-cell lymphoma (DLBCL).
Diffuse large B-cell lymphoma
What is it?
What is the prognosis?
Lymphoma = tumor of
white blood cells
# survivors
type A
type B
1
2
3
4
5
6
time (yrs)
7
8
9
Does Raymond have
Type A or Type B?
http://emedicine.medscape.com/article/202677-media
Challenge
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Since there is no observable difference in Type A and Type
B tumors, how could you determine which type of
DLBCL your patient has?
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Type A (GCB): good prognosis with chemotherapy
Type B (ABC): poor prognosis with chemotherapy
http://emedicine.medscape.com/article/202677-media
Proteins!
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How could you find the protein differences between
cells of Type A and Type B DLBCL?
Differences in protein…
Surrogate: differences in mRNA
Stephen Friend et al. 2002 Sci. Am. 286:44-49
http://bcs.whfreeman.com/lodish6e Chapter 5 animation
http://www.bio.davidson.edu/courses/genomics/chip.chip.html
DNA microarray
http://www.genome.gov/10000533
DNA microarray analysis
Analyze genes expressed in tumors
Decreased mRNA
in lymphoma
Elevated mRNA
in lymphoma
Similar
levels of
mRNA
http://llmpp.nih.gov/
http://lymphochip.nih.gov/signaturedb/
http://llmpp.nih.gov/lymphoma/explore_figure1.html
Gene 1
Gene 2
Gene 3
Gene 4
Gene 5
Gene 6
How do we organize microarray data?
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Compare your gene expression profile to others within your
group, to identify those whose cancer cells share the most
similarity to yours.
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Organize all of the strips into a single panel, and stick them to
a piece of paper using double-sided tape.
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Can you classify the patients in your group into categories
based on gene expression? If so, how many categories?
tumor sample
1 3 5 2 4 6
1
2
3
4
5
6
tumor sample
1 3 5 2 4 6
1
2
3
4
5
6
Tumor Sample
1 3 5 2 4 6
1
2
3
4
5
6
Gene
tumor sample
Lymphoma Case Study
Prognosis: On the basis of gene expression patterns
what is Raymond’s prognosis?
5-year survival rate
A
60-70%
B
25-35%
# survivors
DLBCL subgroup
Raymond
type A
type B
1 2 3 4 5 6 7 8 9
time (yrs)
Subgroup A
Subgroup B
If you were a lymphoma patient, would you want to
know your subgroup?
Yes, definitely. I want to know immediately.
B.
Maybe, but I want to have discussions with my family first.
C.
No, but I want my physician to do the testing to determine subgroup and
chemotherapeutic treatment.
D.
No.
E.
I’d rather not choose.
# survivors
A.
type A
type B
1 2 3 4 5 6 7 8 9
time (yrs)
Genetic Engineering
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What is biotechnology?
What is genetic engineering?
What can genetic engineering do?
What are some examples of genetically engineered
products?
Gene cloning and uses of cloned genes
DNA Insertion into a Plasmid
 Individually, read
the memo and the
article
 Observe the DNA sequence of plant A,
plasmid DNA, and restriction enzymes
 Develop your procedure for this process
(which is a model).
Additional Notes:
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You will make a physical paper model of the insertion
of the pesticide resistant gene into the plant DNA to help
the technicians “see” the process you are describing to
them.
Restriction site (This is the location where the
insertion of the gene will work.)
The technicians already know the process for adding the
Ampicillin resistant gene, so the plasmid DNA provided
for our model already includes the Ampicillin resistant
gene. However, in the lab, the insertion of the Ampicillin
resistant gene and the pesticide resistant gene will happen
at the same time. Therefore, you don’t need to worry
about the Ampicillin resistant gene insertion.
Caenorhabditis elegans: The worm
Why study C. elegans?
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It is eukaryotic just like us.
It has DNA as genetic materials. It produces RNA and protein as
well.
It is a multicellular organism.
It develops from an embryo (sperm + egg) to adult using
developmental processes
It grows, reproduces, gets old and dies.
It has a digestive system for eating, a nerve system with a "brain"
for "learning" and muscles to help it move.
Its genome size is small (~ 10 Megabase) with about 40%
homology to human (3.2 Gigabase).
Lab experiments with C. elegans
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Its size is small. The adult worm is only 1 mm. This makes it
possible to house large numbers in a laboratory setting.
The total number of somatic cells in an adult worm is about 959
cells comparing to human's trillions of cells.
Its life cycle/span is short (about 3 days/weeks respectively). This
shortens the amount of time needed for each experiment and
increases the speed of scientific progress.
It is transparent. This characteristic makes it an excellent tool for
studying biology questions like cell division and cell lineage (a
cell's family tree).
It is non-parasitic free-living that feeds on bacteria (e.g. E. coli).
This makes it safe for use in laboratory settings.
Life Cycle
Lab Activity
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Each plate contains a different C. elegans strand.
Observe the worms and then write down your
observations. Note any differences in morphology or
behavior.
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Why do you think some worms move different than
the others?
How do you silence the gene that you think has
mutation in it?
As a group, write down a hypothesis and then design
an experimental study to test your hypothesis.
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L1-L3 worm, and adult L4 in the center, and
several adult hermaphrodites
Dmpy11: Short and fat
Bli-1:blisters on the cuticle of the worms
Rol 6: Rollers
RNAi lab with C. elegans
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Step 1: Grow E. coli overnight cultures
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Step 2: Seed NGM-LITE and NGM-LITE/amp+IPTG plates
with E. coli
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Step 3: Transfer C. elegans to OP50-Seeded NGM-LITE
plates
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Step 4:Induce RNAi by feeding
More information about C. elegans
 http://www.wormbook.org
Where to find C. elegans?
 C. elegans center at the U of Mn
http://www.cbs.umn.edu/CGC/
 Carolina bio supply
www.carolina.com
Contact information
Selcen Guzey
kendi003@umn.edu
Tamara Moore
tamara@umn.edu