day 3: genetics selcen guzey and tamara moore university of minnesota
TRANSCRIPT
Day 3: Genetics Selcen Guzey and Tamara Moore
University of Minnesota
Agenda
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
Use the post-it notes to create a “Parking Lot Chart” to be addressed throughout the day.
Challenge: integrating engineeringMisconceptions: structure of DNAStrategies: how to include published scientific
data into my genetics unitQuestion: 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 Genomes vary in size, number of genes, and gene
density!
What genetic attributions allow humans to get by with no more genes than nematodes?
What makes humans and chimps so different?
Haploid genome sizebp: base (nucleotide) pairs
# of genes
E. coli 4.6 million bp 4,400
Caenorhabditis elegans (Nematode)
Chimpanzee
Human
100 million bp
3.3 billion bp
3 billion bp
20,100
19,700
<21,000
Human Genome
DNA is the genetic material!
What piece of history is missing in this picture?
If you would like to recreate this picture who would be in this picture?
OR
Rosalind Franklin
The structure of DNA
The Structure of a Gene
EnhancerPromoter Exon IntronExon Intron
Poly-A signal sequence
Upstream Downstream
Exon
TATA and CAAT boxes, CG sequences
Transcription start site
Beta Hemoglobin (HbB)
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
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 In this activity, you will examine the DNA
sequence of the beta-globin gene from five people.
The codes represent the “sense” for the DNA sequence- Sense strand looks like mRNA.
1)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)
2)Examine the gene sequence of Person C, D, E, and F. Changes shown in bold!
From DNA to mRNA3’- TTCAGTCGT - 5’ DNA template
strand5’- AAGTCAGCA- 3’ DNA sense strand Transcription
5’- AAGUCAGCA- 3’ mRNA codon
Translation
Lys Ser Ala Protein
The codon table for mRNA
Questions for Discussion
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
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 β-globin chains leading to severe disease.
Silence mutations are rare (Person C and E).
More on Mutations
Gene mutation- Point mutations/single base substitutions
a)Substitutions CCC CCAb)Frameshift mutations:
insertions and deletions 5’ CCC CCA GGG 3’ 5’CCC CCA AGA GGG 3’
Chromosomal Mutations
a)Deletionb)Duplicationc) Inversiond)Translocation
Point mutations can be also categorized as: nonsense mutations, missense mutations, and silent mutations
Testing for Sickle Cell AnemiaMst 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 factorsPosttransitional control: Protein activation
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 expressionCase 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? http://video.about.com/lymphoma/Non-Hodgki
n-s-Lymphoma.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
time (yrs)
# s
urv
ivors
type A
type B
1 2 3 4 5 6 7 8 9
Does Raymond have Type A or Type B?
http://emedicine.medscape.com/article/202677-media
Challenge Since there is no observable difference in
Type A and Type B tumors, how could you determine which type of DLBCL your patient has?
• Type A (GCB): good prognosis with chemotherapy
• Type B (ABC): poor prognosis with chemotherapy
http://emedicine.medscape.com/article/202677-media
Proteins! How could you find the protein differences
between cells of Type A and Type B DLBCL?
Stephen Friend et al. 2002 Sci. Am. 286:44-49http://bcs.whfreeman.com/lodish6e Chapter 5 animationhttp://www.bio.davidson.edu/courses/genomics/chip.chip.html
Differences in protein…Surrogate: differences in mRNA
DNA microarray http://www.genome.gov/10000533
DNA microarray analysis
Elevated mRNAin lymphoma
Decreased mRNAin lymphoma
Similarlevels of mRNA
Analyze genes expressed in tumors
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?
Compare your gene expression profile to others within your group, to identify those whose cancer cells share the most similarity to yours.
Organize all of the strips into a single panel, and stick them to a piece of paper using double-sided tape.
Can you classify the patients in your group into categories based on gene expression? If so, how many categories?
1 3 5 2 4 6
tumor sample
123456
1 3 5 2 4 6
tumor sample
123456
1 3 5 2 4 6
tumor sample
123456
Tumor Sample
Lymphoma Case Study
Prognosis: On the basis of gene expression patterns what is Raymond’s prognosis?
DLBCL subgroup 5-year survival rate
A 60-70%
B 25-35%
time (yrs)
# s
urv
ivors type A
type B
1 2 3 4 5 6 7 8 9
Raymond
Subgroup BSubgroup A
If you were a lymphoma patient, would you want to know your subgroup?
A. 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.
time (yrs)
# s
urv
ivors type A
type B
1 2 3 4 5 6 7 8 9
Genetic Engineering
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: 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?
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
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
Each plate contains a different C. elegans strand. Observe the worms and then write down your observations. Note any differences in morphology or behavior.
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.
Dmpy11: Short and fat
L1-L3 worm, and adult L4 in the center, and several adult hermaphrodites
Bli-1:blisters on the cuticle of the worms
Rol 6: Rollers
RNAi lab with C. elegans
Step 1: Grow E. coli overnight cultures
Step 2: Seed NGM-LITE and NGM-LITE/amp+IPTG plates with E. coli
Step 3: Transfer C. elegans to OP50-Seeded NGM-LITE plates
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 Mnhttp://www.cbs.umn.edu/CGC/ Carolina bio supplywww.carolina.com
