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 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 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 size bp: base (nucleotide) pairs # of genes E. coli4.6 million bp4,400 Caenorhabditis elegans (Nematode) Chimpanzee Human 100 million bp 3.3 billion bp 3 billion bp 20,100 19,700 <21,000
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 mRNA 3’- TTCAGTCGT - 5’ DNA template strand 5’- AAGTCAGCA- 3’ DNA sense strand Transcription 5’- AAGUCAGCA- 3’ mRNA codon Translation Lys Ser Ala Protein
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 CCA b) Frameshift mutations: insertions and deletions 5’ CCC CCA GGG 3’ 5’CCC CCA AGA GGG 3’ Chromosomal Mutations a) Deletion b) Duplication c) Inversion d) Translocation 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.
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 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? http://video.about.com/lymphoma/Non-Hodgkin-s- Lymphoma.htm http://video.about.com/lymphoma/Non-Hodgkin-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) # survivors 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 TypeB tumors, how could you determine which type ofDLBCL 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. 286:44-49 http://bcs.whfreeman.com/lodish6e Chapter 5 animation http://www.bio.davidson.edu/courses/genomics/chip.chip.html Differences in protein… Surrogate: differences in mRNA
DNA microarray analysis Elevated mRNA in lymphoma Decreased mRNA in lymphoma Similar levels of mRNA Analyze genes expressed in tumors http://llmpp.nih.gov/ http://lymphochip.nih.gov/signaturedb/ http://llmpp.nih.gov/lymphoma/explore_figure1.html
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?
Lymphoma Case Study Prognosis: On the basis of gene expression patterns what is Raymond’s prognosis? DLBCL subgroup5-year survival rate A60-70% B25-35% time (yrs) # survivors 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) # survivors 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?
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.life cycle/span 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.
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 Mn http://www.cbs.umn.edu/CGC/ Carolina bio supply www.carolina.com
Contact information Selcen Guzey firstname.lastname@example.org Tamara Moore email@example.com